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The variable domain of heavy-chain antibody (VHH) has been developed widely in drug therapy, diagnosis, and research. Escherichia coli is the most popular expression system for VHH production, whereas low bioactivity occurs sometimes. Mammalian cells are one of the most ideal hosts for VHH expression at present. To improve the yield of VHH in Expi293F cells, we optimized the signal peptide (SP) and codons of VHH. Firstly, the fusion protein VHH1-Fc was used to screen SPs. The SP IFN-α2 showed the highest secretion as quantified by enzyme-linked immunosorbent assay (ELISA). Subsequently, codon optimization by improving GC3 and GC content doubled the yield of VHH1 and kept its binding activity to Senecavirus A (SVA). Finally, the mean yields of other 5 VHHs that fused with SP IFN-α2 and codon-optimized were over 191.6 mg/L, and these VHHs had high recovery and high purity in the culture supernatant. This study confirms that SP IFN-α2 and codon optimization could produce VHHs in Expi293F cells efficiently, which provides a reference for the large-scale production of VHHs.

Metallothionein (MT) plays a significant role in heavy metal removal, antioxidant defense, and immune regulation. The current predominant approach for obtaining natural MT is extraction from tissue, which often entails complex procedures resulting in limited yields. In recent years, researchers have adopted the strategy of fusing labels such as GST or His for the heterologous expression of MT. However, a challenge in industrial production arises from the subsequent removal of these labels, which often leads to a significant reduction in the yield. The fusion with elastin-like polypeptides (ELPs) offers a promising solution for achieving soluble expression of the target protein, while providing a simple and fast purification process. In this study, ELP was fused with MT, which significantly up-regulated the soluble expression of MT. The fusion protein ELP-MT with the purity above 97% was obtained efficiently and simply by inverse transition cycling (ITC). ELP-MT exhibited a remarkable 2,2'-azinobis(3-ethylbenzothiazoline-6- sulfonic acid) ammonium salt (ABTS) scavenging activity, with the half maximal inhibitory concentration ( IC 50 ) of 0.77 μmol/L, which was 53.7 times that of the vitamin E derivative Trolox. In addition, the fusion protein demonstrated strong 1,1-diphenyl-2-trinitrohydrazine (DPPH) scavenging ability. Furthermore, ELP-MT had no toxicity to the proliferation and promoted the adhesion and migration of NIH/3T3 cells. All these results indicated that ELP-MT had good biocompatibility. We constructed the fusion protein ELP-MT combining the unique properties of MT and elastin, laying a technical foundation for the large-scale production of recombinant MT and facilitating the applications in food, health supplement, and cosmetic industries.

Human epidermal growth factor (hEGF) can be applied in the treatment of surgical trauma (burns, scalds), tissue repair, skin moisturizing, beauty, skincare, etc. However, the low expression and high cost limit the application of hEGF. In order to improve the expression level of hEGF and reduce the production cost, considering the high expression of polyhedrin, this study fused a partial sequence of polyhedrin with hEGF and expressed the fused sequence by using a silkworm baculovirus expression vector system. In view of the small molecular weight of hEGF, we connected hEGF genes in series and optimized the codons to construct multiple fusion expression vectors by fusing different partial sequences of polyhedrin at the N-terminus. The results showed that through the above strategy, the protein expression level of hEGF was significantly increased. The expression vector containing three concatenated hEGF genes with optimized codons and fused with the sequence encoding 25 or 35 residues at the N-terminus of polyhedrin showed the highest expression level.

The spike (S) protein plays a crucial role in the entry of SARS-CoV-2 into host cells. The S protein contains two subunits, S1 and S2. The receptor-binding domain (RBD) of the S1 subunit binds to the receptor angiotensin-converting enzyme 2 (ACE2) to enter the host cells. Therefore, degrading S1 is one of the feasible strategies to inhibit SARS-CoV-2 infection. The purpose of this study is to develop a degradation tool targeting S1. First, we constructed a HEK 293 cell line stably expressing S1 by using a three-plasmid lentivirus system. The overexpression of the mitochondrial E3 ubiquitin protein ligase 1 (MUL1) in this cell line promoted the ubiquitination of S1 and accelerated its proteasomal degradation. Further research showed the polyubiquitination of S1 catalyzed by MUL1 mainly occurred via the addition of K48-linked chains. Moreover, the specific peptide LCB1, which targets and recognizes S1, was combined with MUL1 to create the chimeric E3 ubiquitin ligase LCB1-MUL1. In comparison to MUL1, this chimeric enzyme demonstrated improved catalytic efficiency, resulting in a reduction of S1's half-life from 12 h to 9 h. In summary, this study elucidated the mechanism by which MUL1 promotes the ubiquitination modification of S1 and facilitates its degradation through the proteasome, and preliminarily validated the effectiveness of targeted degradation of S1 by chimeric enzyme LCB1-MUL1.

Polyethylene terephthalate (PET) is a largely-produced polymer worldwide. However, its extensive waste generation and resistance to degradation pose significant environmental concerns. Consequently, there is considerable interest in researching enzymatic degradation of PET. Relevant studies have shown that the addition of a carbohydrate binding module (CBM) can increase the affinity between the enzyme and the substrate, enhancing the enzyme's degradation ability. In order to develop more efficient PET hydrolytic enzymes, this study introduced carbohydrate binding domains (CBMs) from different families with different substrate affinities into the PET-degrading enzyme LCC-ICCG. High crystallinity PET powder and amorphous PET film were used as substrates to characterize the degradation efficiency of the modified enzymes, aiming to explore the enzyme with the optimal degradation ability. The results showed that the fusion of type B CBM reduced the degradation rate and T m value of the enzyme towards PET, while the introduction of type A and type C CBMs significantly improved the degradation rate of the enzyme towards the film-like substrate. The degradation rate and T m value of PET were also enhanced, especially with the fusion enzyme LCC-ICCG-CBM9-2, which showed an over 10-fold increase in the degradation rate compared to the original enzyme LCC-ICCG. Therefore, this study demonstrates that by introducing type A and type C CBMs, the degradation rate and thermal stability of LCC-ICCG towards film-like PET can be improved, addressing the issue of its low activity and enabling more effective PET degradation. This research provides support for plastic degradation technology and contributes to environmental conservation efforts.

Abscisic acid insensitive 5 (ABI5) is a basic leucine zipper transcription factor regulating ABA-mediated seed germination, and TaABI5 is closely related to the pre-harvest sprouting in wheat. Studies have shown that TaABI5-D3 , one of multiple copies of TaABI5 gene, encodes the intact TaABI5 protein. In this study, we constructed the prokaryotic expression vector pET-28a- TaABI5-D3 for expression of TaABI5-D3 in Escherichia coli and obtained the purified recombinant protein His-TaABI5-D3. This protein existed in the form of inclusion bodies, with the best expression induced by incubation with 0.6 mmol/L IPTG at 16 ℃, 150 r/min overnight (for 12 h). Subsequently, the purified His-TaABI5-D3 was injected into Balb/C mice for the preparation of polyclonal antibodies. Western blotting analysis indicated that the polyclonal antibody had relatively high specificity, laying foundations for clarification of the function of TaABI5 protein in wheat.

Algae has been proven to have the potential to be efficient biosorbents in the detection and remediation of heavy metal pollution such as cadmium in the environment. This study aims to enhance the cadmium adsorption capacity of Chlamydomonas reinhardtii by expressing the cadmium-binding protein CADR on the cell wall by the surface display technology. Firstly, the golden gate technique was employed to construct the transformation vector PET-X- CADR , which anchored CADR to the cell wall with the cell wall protein GP1. The high-throughput screening with the fluorescence signal of the fusion tag YFP resulted in three engineered algal strains with high expression of CADR on the cell wall. Physiological experiments demonstrated that the CADR displayed on the cell wall did not affect the growth of the engineered algal strains exposed to cadmium with the concentration below 200 μmol/L. In the presence of 200 μmol/L cadmium, the growth rates of CADR-engineered algal strains were three times as that of the wild-type, indicating stronger tolerance of the CADR-engineered algae to cadmium. The protein lyase GLE released during the mating of Chlamydomonas was used to isolate the cell walls of wild-type and engineered strains, the cadmium content of which was compared. The results showed that the cell wall of the engineered strain exhibited an increase of 33% in cadmium adsorption capacity. This study gives insights into the application of algae in the management of cadmium pollution in the environment, especially in the recycling of heavy metals from the environment.

Objective To prepare a Fc-silent chimeric antibody against human CD36 and analyse its bioactivity and phagocytic effect on CD36(+) platelets. Methods The genes encoding V H and V L fragments of the monoclonal antibody (mAb) 32-106 were obtained through RNA extraction from hybridoma cell lines, PCR amplification and sequence analysis. By using gene synthesis and recombination techniques, the vectors expressing light and heavy chains of the chimeric antibody against human CD36 were constructed. Stable cell lines secreting the chimeric antibody were established by selective culture using Zeocin and Blasticidin. Antibodies were purified by an affinity chromatography column. The purity and molecular weight of the chimeric antibody were detected by SDS-PAGE. The activity of the antibody binding to CD36 antigen was determined by ELISA and flow cytometry. The ability of the chimeric antibody to participate in CD36(+) platelet phagocytosis was analyzed by a platelet phagocytosis assay. Results The vectors expressing light and heavy chains of chimeric antibody were successfully constructed. After co-transfection into HEK293 cells, stable cell lines secreting chimeric antibody were obtained by screening and cloning. The high purity and correct molecular weight of the chimeric antibody were confirmed by protein silver staining. The results of Flow cytometry and ELISA showed that the chimeric antibody had the activity of binding to human CD36 antigen. Platelet phagocytosis assay showed that the Fc-silent chimeric antibody against human CD36 basically lost the ability of mediating monocyte phagocytosis of CD36(+) platelets. Conclusion In this study, the Fc-silent chimeric antibody against human CD36 was successfully prepared, and its loss of ability to mediate CD36(+) platelet clearance was confirmed in vitro, which provides a preliminary basis for the study of modified antibodies for the therapy of CD36-antibody-mediated fetal and neonatal alloimmune thrombocytopenia(FNAIT).

The ubiquitin/proteasome system (UPS) plays a crucial role in maintaining cellular protein homeostasis. The catalytic activity of proteasome in the UPS is regulated by β1 (PSMB6), β2 (PSMB7), and β5 (PSMB5) subunits. Interferon (IFN)-γ, tumor necrosis factor (TNF)-α, inflammation, and oxidative stress can induce the replacement of β1, β2, and β5 with their respective immuno-subunits β1i (PSMB9), β2i (PSMB10), and β5i (PSMB8), which can be assembled into the immunoproteasome. Compared with the standard proteasome, the immunoproteasome exerts enhanced regulatory effects on immune responses, such as processing and presenting MHC class Ⅰ antigens, production of pro-inflammatory cytokines, and T cell differentiation and proliferation. Abnormal aggregation of immunoproteasomes can cause neurodegenerative diseases like Parkinson's disease, Alzheimer's disease, and amyotrophic lateral sclerosis. To explore the function of PSMB9 after bacterial infection, we constructed a lentivirus plasmid overexpressing PSMB9-eGFP-His and transfected the plasmid into HEK293T cells for packaging by using a triple-plasmid system in this study. After screening with puromycin, we obtained a stable human leukemia monocytic THP-1 cell line expressing the fusion protein of PSMB9. Western blotting (WB) and fluorescence microscopy verified the expression of the fusion protein in the stable THP-1 cells. Quantitative PCR (qPCR) was employed to measure the copies of PSMB9 - eGFP in THP-1 cells. Immunofluorescence results found that eGFP-His did not affect the subcellular localization of PSMB9. The purification with nickel affinity chromatography confirmed that the fusion protein could be assembled into the 20S immunoproteasome and exhibited cleaving activity for fluorescent peptide substrates. These results indicated that the PSMB9 - eGFP fusion gene was integrated into the chromosome, and could be stably expressed in the constructed THP-1 cell line. This cell line can be utilized for the research on subcellular localization, dynamic expression, and activity of PSMB9 in live cells at different infection conditions and disease stages. It also provides a model for the stable cell lines construction of other immunoproteasome subunits PSMB8 and PSMB10.

This study aims to prepare bacterial outer membrane vesicles (OMVs) with anti-glypican-3 (GPC3) single-chain antibody and analyze their targeting effects on Hep G2 hepatocellular carcinoma (HCC) cells and tissue. The recombinant plasmid pET28a-Hbp-hGC 33-scFv was constructed by ligating Hbp-hGC 33-scFv to pET28a. Western blotting was employed to determine the prokaryotic expression of the fusion protein Hbp-hGC 33-scFv, on the basis of which the optimal induction conditions were determined. Hbp-hGC 33-OMVs secreted from the recombinant expressing strains were collected by ultrafiltration concentration and then characterized. The localization of Hbp-hGC 33-scFv in bacteria and Hbp-hGC 33-OMVs was analyzed by immune electron microscopy. The binding of Hbp-hGC 33-scFv to Hep G2 cells was observed by immunofluorescence. The Hep G2 tumor-bearing mouse model was established, and the targeted retention of Hbp-hGC 33-OMVs in the tumor site of mice was observed by a fluorescence imaging system in vivo . The results showed that the actual molecular weight of the fusion protein was 175.3 kDa, and the optimal induction conditions were as follows: OD 600 =0.5, IPTG added at a final concentration of 0.5 mmol/L, and overnight induction at 16 ℃. The prepared Hbp-hGC 33-OMVs were irregular spherical structures with an average particle size of (112.3±4.6) nm, expressing OmpC, OmpA, and the fusion protein Hbp-hGC 33-scFv. The Hbp-hGC 33-OMVs prepared in this study demonstrated stronger ability of binding to Hep G2 cells than the wild-type OMVs ( P =0.008). All the data indicated that Hbp-hGC 33-OMVs with anti-GPC3 single-chain antibody were successfully prepared and could be used for research on the targeted therapy of hepatocellular carcinoma.

This study aims to develop an effective bivalent subunit vaccine that is promising to prevent both porcine deltacoronavirus (PDCoV) and porcine epidemic diarrhea virus (PEDV). The receptor-binding domains (RBDs) of PDCoV and PEDV were fused and cloned into the eukaryotic expression vector pCDNA3.1(+). The fusion protein PDCoV-RBD-PEDV-RBD (pdRBD-peRBD) was expressed by the ExpiCHO TM expression system and purified. Mice were immunized with the fusion protein at three different doses (10, 20, and 30 μg). The humoral immune response and cellular immune response induced by the fusion protein were evaluated by ELISA and flow cytometry. The neutralization titers of the serum of immunized mice against PDCoV and PEDV were determined by the microneutralization test. The results showed that high levels of IgG antibodies were induced in the three different dose groups after booster immunization, and there was no significant difference in the antibody level between different dose groups, indicating that the immunization dose of 10 μg could achieve the fine immune effect. The results of flow cytometry showed that the immunization groups demonstrated increased proportion of CD 3 + CD 4 + T cells and decreased proportion of CD 3 + CD 8 + T cells, which was consistent with the expectation about the humoral immune response induced by the subunit vaccine. At the same time, the levels of interleukin (IL)-2, IL-4, and interferon (IFN)-γ in the serum were determined. The results showed that the fusion protein induced both humoral immune effect and cellular immune response. The results of the neutralization test showed that the antibody induced by 10 μg fusion protein neutralized both PDCoV and PEDV in vitro , with the titers of 1:179.25 and 1:141.21, respectively. The above results suggested that the pdRBD-peRBD could induce a high level of humoral immune response at a dose of 10 μg, and the induced antibody could neutralize both PDCoV and PEDV. Therefore, the fusion protein pdRBD-peRBD is expected to be an effective subunit vaccine that can simultaneously prevent PDCoV and PEDV.

To investigate the role of recombinant mussel mucin in wound healing, we aimed to prepare this mucin using Pichia pastoris as the host microbe. Our method involved constructing a genetically engineered strain of P . pastoris that expressed a fusion protein consisting of Mfp-3 and preCol-P peptide segments of mussel. After fermentation and purification, we obtained a pure recombinant mussel mucin product. We then conducted experiments to evaluate its effect at both the cellular and animal levels. At the cellular level, we examined its impact on the proliferation and migration of mouse fibroblast L929. At the animal level, we assessed its ability to promote wound healing after full-layer skin resection in rats. Our results showed that the recombinant mussel mucin protein has a content of 90.28% and a purity of 96.49%. The content of 3,4-dihydroxyphenylalanine (DOPA) was 0.73 wt%, and the endotoxin content was less than 0.5 EU/mg. Importantly, the recombinant mussel mucin protein significantly promoted both the migration and proliferation of mouse fibroblast, as well as the wound healing in rat skin. In conclusion, our findings demonstrate that recombinant mussel mucin has the potential to promote wound healing and can be considered a promising medical biomaterial.

Objective To investigate the preventive therapeutic effect and possible mechanism of single chain variable fragments chimeric protein (SD) of ovalbumin epitopes internalizing receptor DEC-205 antibody on food allergy in mice. Methods Mice were randomly divided to five groups (control, PBS, scFv DEC 100 μg, SD 50 μg, SD 100 μg) and treated for 24 hours before OVA administration. After challenge, the serum level of OVA-specific IgE, IgG1, IgG2a and IL-4 were detected by ELISA. Infiltration of eosinophils and mast cells in the jejunum was observed by HE staining and toluidine blue staining respectively. The bone marrow of tibia and femur was isolated and cultured to obtain immature dendritic cells(BMDCs), which were further treated with LPS (10 ng/mL), TSLP (50 ng/mL), scFv DEC protein (1000 ng/mL) and SD protein (10,100,1000)ng/mL for 24 hours, and the IL-10 level of supernatant was assayed by ELISA. Results Compared with PBS group, the number of SD-treated mice with diarrhea was markedly reduced. The difference in rectal temperature and the levels of serum OVA-specific IgE, IgG1, IgG2a and IL-4 decreased significantly after prophylactic administration of SD; The number of eosinophils and mast cells in jejunum also decreased significantly while the IL-10 level in the supernatant of BMDCs increased significantly after SD intervention. Conclusion SD mitigates experimental FA response by fosters the immune tolerance property of dendritic cells.

DLP4 (defensin-like peptide 4) is a novel insect defensin, which has strong antibacterial activity against Gram-positive bacteria and is not susceptible to develop drug resistance. In this study, an elastin-like polypeptide (ELP) and an intein fusion system were used for production and purification of DLP4, which combined the characteristics of the phase transition of ELP and the C-cleavage of the intein. A recombinant expression plasmid pET-ELP-I-DLP4 was constructed and transformed into Escherichia coli . Subsequently, DLP4 was purified by simple centrifugation, alternation of pH and temperature. However, the C-cleavage of the intein occurred unexpectedly during the process of expression and purification. To solve this problem, the intein was split into N-intein (I0 N ) and C-intein (I0 C ), and fused with ELP or DLP4 to construct two recombinant expression plasmids pET-ELP-I0 N and pET-ELP-I0 C -DLP4, respectively. These two plasmids were transformed into E . coli separately. The mixture of the two cultures of E . coli strains restored the C-cleavage activity of the intein. This operation yielded DLP4 of about 1.49 mg/L. Antibacterial test confirmed that the purified DLP4 exhibited expected activity. Thus, this approach can be used as an effective way for DLP4 expression and purification in the prokaryotic system.

The influence of different affinity tags on enzyme characteristics varies. The (S)-carbonyl reductase 2 (SCR2) from Candida parapsilosis can reduce 2-hydroxyacetophenone, which is a valuable prochiral ketones. Different affinity tags, i.e. his-tag, strep-tag and MBP-tag, were attached to the N terminus of SCR2. These tagged SCR2 enzymes, i.e. his6-SCR2, strep-SCR2 and MBP-SCR2, were heterologously expressed in Escherichia coli and purified to study their characteristics towards 2-hydroxyacetophenone reduction. Affinity tags did affect the characteristics of the recombinant SCR2 enzymes. Specifically, affinity tags affect the stability of recombinant SCR2 enzymes: 1) At pH 6.0, the remaining enzyme activities of his6-SCR2 and strep-SCR2 were only 95.2% and 90.0% of the untagged SCR2, while that of MBP-SCR2 was 1.2 times of the untagged SCR2 after incubating for 13 h at 30 °C. 2) The half-life of MBP-SCR2 at 50 °C was 26.6%-48.8% longer than those of strep-SCR2, his6-SCR2 and untagged SCR2. 3) The kcat of MBP-SCR2 was about 1.25-1.45 times of that of small affinity-tagged and untagged SCR2 after storing at -80 °C for 60 d. Structural informatics indicated that the α-helices at the C terminus of MBP-SCR2 contributed to the stability of the N terminus of fusion protein of SCR2. Data from circular dichroism showed that the MBP-tag has some influence on the secondary structure of SCR2, while melting temperature analysis demonstrated that the Tm of the recombinant MBP-SCR2 was about 5 °C higher than that of the untagged SCR2. This study obtained an efficient and stable recombinant SCR2, i.e. the MBP-SCR2. Moreover, this study could serve as a reference for other researchers to evaluate and select appropriate affinity tags for their research.

Antimicrobial peptides are the most promising alternatives to antibiotics. However, the strategy of producing antimicrobial peptides by recombinant technology is complicated and expensive, which is not conducive to the large-scale production. Oxysterlin 1 is a novel type of cecropin antimicrobial peptide mainly targeting on Gram-negative bacteria and is of low cytotoxicity. In this study, a simple and cost-effective method was developed to produce Oxysterlin 1 in Escherichia coli. The Oxysterlin 1 gene was cloned into a plasmid containing elastin-like polypeptide (ELP) and protein splicing elements (intein) to construct the recombinant expression plasmid (pET-ELP-I-Oxysterlin 1). The recombinant protein was mainly expressed in soluble form in E. coli, and then the target peptide can be purified with a simple salting out method followed by pH changing. The final yield of Oxysterlin 1 was about 1.2 mg/L, and the subsequent antimicrobial experiment showed the expected antimicrobial activity. This study holds promise for large-scale production of antimicrobial peptides and the in-depth study of its antimicrobial mechanism.

Tissue inhibitor of metalloproteinases-2 (TIMP-2) inhibits tumor migration and invasion. Obtaining TIMP-2 protein is conducive to a comprehensive and in-depth study of its function and mechanism in tumorigenesis and development. We collected human TIMP-2 protein through prokaryotic expression in vitro. We expressed, purified and characterized human TIMP-2 protein. First, the human TIMP-2 gene was cloned from the cDNA obtained by reverse transcription of total RNA of human lung cancer A549 cells, and constructed to pET28a vector. The recombinant plasmid pET28a-TIMP-2 was transformed into Escherichia coli BL21(DE3) after restriction endonuclease digestion and sequencing analysis. The expression of TIMP-2 protein was induced by isopropyl-β-D-thiogalactoside (IPTG), and the expression conditions were optimized. After purification by nickel affinity column, the fusion protein His-TIMP-2 was identified by Western blotting method and its biological activity was detected by gelatin zymography. The fusion protein His-TIMP-2 existed in the form of inclusion body in E. coli. In a certain range, the concentration of IPTG had no significant effect on the expression amount of His-TIMP-2. But in this expression system, induction temperature and time were the key parameters, and the expression amount of His-TIMP-2 in E. coli increased with the increase of induction temperature. The purified and refolded fusion protein could effectively inhibit the activity of matrix metalloproteinases expressed by human lung cancer A549 cells. The acquisition of active fusion protein lays a foundation for further study of the function and mechanism of human TIMP-2, and is of great significance for tumor therapy.

Drugs targeting immune checkpoint are used for cancer treatment, but resistance to single drug may occur. Combination therapy blocking multiple checkpoints simultaneously can improve clinical outcome. Therefore, we designed a recombinant protein rPC to block multiple targets, which consists of extracellular domains of programmed cell death protein 1 (PD-1) and cytotoxic T lymphocyte-associated antigen 4 (CTLA-4). The coding sequence was inserted into expression vector and stably transfected into HEK293 cells. The culture supernatant was collected and rPC was affinity-purified. Real-time quantitative PCR was used to evaluate the expression levels of ligands for PD-1 and CTLA-4 in several human cancer cell lines. The binding of rPC with cancer cells was examined by immunofluorescence cell staining, the influence of rPC on cancer cell growth was assayed by CCK-8. The results showed that rPC could be expressed and secreted by stably transfected HEK293 cells, the purified rPC could bind to lung cancer NCI-H226 cells which have high levels of ligands for PD-1 and CTLA-4, no direct impact on cancer cell growth could be observed by rPC treatment. The recombinant protein rPC can be functionally assayed further for developing novel immunotherapeutic drugs for cancer.

By using an RAD peptide display system derived from the ATPase domain of recombinase RadA of Pyrococcus furiosus, an anti-hCG antibody-like molecule was prepared by grafting an hCG-binding peptide to the RAD scaffold. After linking to sfGFP gene, a gene of hCG peptide-grafted RAD was synthesized and cloned into a bacterial expression vector (pET30a-RAD/hCGBP-sfGFP). The vector was transformed into Escherichia coli, and expression of the fusion protein was induced. After isolation and purification of the fusion protein, its binding affinity and specificity to hCG were determined by using a process of immunoabsorption followed by GFP fluorescence measurement. A comparison of hCG-binding activity with a similarly grafted single-domain antibody based on a universal scaffold was performed. The measurement of hCG-binding affinity and specificity revealed that the grafted RAD has an optimally high binding affinity and specificity to hCG, which are better than the grafted single-domain antibody. Moreover, the affinity and specificity of grafted RAD molecule are comparable to those of a commercial monoclonal antibody. In addition, the hCG-binding peptide-grafted RAD molecule has a relatively high biochemical stability, making it a good substitute for antibody with potential application.

Objective To study the role of mouse extracellular segment protein of signal-regulatory protein α gene (mSIRPα ext ) of mice in tumor immune regulation by cloning mSIRPα ext , constructing its prokaryotic expression vector and achieving the soluble expression of SIRPα. Methods The mSIRPα ext gene was amplified from mouse lymph node and the prokaryotic expression vector of pET32a-SIRPα ext was further constructed. After the soluble expression of recombinant Trx-mSIRPα ext fusion protein containing thioredoxin (Trx) tag was achieved, mouse bone marrow-derived monocytes were cultured and induced to differentiate into macrophages. Then the macrophages were co-cultured with L1210 leukemia cells labeled with 5(6)-carboxyfluorescein diacetate succinimide ester (CFSE). Trx-mSIRPα ext protein and Trx control protein were added into the co-culture system. The role of recombinant protein in the macrophage phagocytosis of tumor cells was observed by immunofluorescence cytochemical staining and confocal microscopy. Results Purified soluble Trx-mSIRPα ext protein was obtained and it was showed that it could enhance the phagocytosis of macrophages in mouse L1210 leukemia cells in vitro phagocytosis experiments. Conclusion Prokaryotic expression of Trx-mSIRPα ext protein can effectively enhance the phagocytosis of macrophages in leukemia cells, thus playing the role of anti-tumor immunotherapy.

Objective: To investigate the role of bone marrow mesenchymal stem cells (BMSCs) with CTLA4Ig and CD40LIg gene modification in rejection reaction after liver transplantation in rats and possible mechanisms. Methods: The modified Kamada's two-cuff technique was used to establish a Lewis-BN rat model of orthotopic liver transplantation, and a total of 75 rats were randomly divided into groups A, B, C, D, and E, with 15 rats in each group. The rats in group A (control group) were given infusion of isotonic saline via the portal vein during liver transplantation, those in group B (BMSC group) were given infusion of BMSCs via the portal vein during liver transplantation, those in group C (BMSCs with CTLA4Ig gene modification) were given infusion of BMSCs carrying the CTLA4Ig gene via the portal vein during liver transplantation, those in group D (BMSCs with CD40LIg gene modification) were given infusion of BMSCs carrying the CD40LIg gene via the portal vein during liver transplantation, and those in group E (BMSCs with CTLA4Ig and CD40LIg gene modification) were given infusion of BMSCs carrying CTLA4Ig and CD40LIg gene modification via the portal vein during liver transplantation. Postoperative survival and change in liver function were observed. HE staining was used to observe the pathomorphological changes of the graft liver, and ELISA was used to measure the levels of interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-10 (IL-10), and interferon-γ (IFN-γ) in peripheral blood. A one-way analysis of variance was used for comparison of means of multiple samples, and the Kaplan-Meier survival curve analysis was used for comparison of survival rates between multiple groups. Results: Group E had a significantly longer survival time after surgery than groups A, B, C, and D ( P < 0.05), groups C and D had a significantly longer survival time than groups A and B ( P < 0.05), and there was no significant difference between groups C and D ( P > 0.05). On day 10 after surgery, group A had significantly higher levels of alanine aminotransferase and total bilirubin than the other four groups ( P < 0.05). HE staining showed severe rejection reaction in group A, moderate rejection reaction in group B, and mild rejection reaction in groups C and D; pathological examination showed no marked rejection reaction in group E. Group A had significant increases in the levels of IL-2 and IFN-γ and significant reductions in the levels of IL-4 and IL-10 after surgery compared with the other four groups (all P < 0.05). Conclusion: Infusion of BMSCs with modification of both CTLA4Ig and CD40LIg genes can significantly inhibit acute rejection reaction after liver transplantation in rats and effectively prolong the survival time of the graft liver, with a better effect than infusion of BMSCs alone or BMSCs with modification of CTLA4Ig or CD40LIg gene.

Objective To construct the expression vector of the fusion protein of apolipoprotain B mRNA editing enzyme catalytic polypeptide-like 3A (APOBEC3A) and hepatitis B virus core proteins (HBc) with different sequences, and identify its expression, intracellular localization and cytosine deamination activity. Methods The APOBEC3A gene and the coding sequence of HBc and four kinds of truncated HBc containing C-termimal domain were amplified by PCR. The APOBEC3A with full-length HBc antigen or four kinds of truncated HBc were cloned into a eukaryotic expression vector pcDNA3.0 by In-Fusion method, and confirmed by DNA sequencing. The recombinants were then transfected into HEK293T cells. The expression and localization of the fusion proteins were detected by Western blotting and immunofluorescence cytochemistry. The cytosine deamination activity was analyzed by electrophoresis on a urea denaturing polyacrylamide gel. Results Five kinds of fusion protein were expressed in HEK293T cells successfully, and the expression of the vectors containing the truncated HBc were higher than that of the APOBEC3A-HBc vector containing the full-length HBc. The fusion protein of APOBEC3A-HBc was mainly expressed in the cytoplasm of HEK293T cells, and the fusion proteins of APOBEC3A-HBc144S, APOBEC3A-HBc144E, APOBEC3A-HBc144AAA, APOBEC3A-HBc144A were expressed mainly in the nuclei of HEK293T cells. The cytosine deamination activity of the fusion proteins containing the truncated HBc was higher than that of the fusion protein APOBEC3A-HBc. Conclusion The fusion protein expression vectors of APOBEC3A and HBc with different lengths and sequences have been constructed successfully. The expressing ability, intracellular localization and cytosine deamination activity are obviously different between the fusion protein APOBEC3A-HBc and the fusion proteins containing the truncated HBc.

Although there are 125 predicted DNase Ⅱ-like family genes in the Trichinella spiralis genome, plancitoxin-1-like (Ts-Pt) contains the HKD motif, a typical conserved region of DNase Ⅱ, in N- and C-terminal. It is generally believed that histidine is the active site in DNase Ⅱ. To study the nuclease activity of recombinant Ts-Pt with mutations in the active site from T. spiralis, different fragments of the mutated Ts-Pt genes were cloned using overlap PCR technique and inserted into the expressing vector pET-28a(+), and transformed into Escherichia coli Rosseta (DE3). The fusion proteins were purified by Ni-NTA affinity chromatography and SDS-PAGE. Nuclease activity of the recombinant proteins was detected by agarose gel electrophoresis and nuclease-zymography. The recombinant plasmids harboring the mutated Ts-Pt genes were constructed and expressed as inclusive body in a prokaryotic expression system. After renaturation in vitro, the recombinant proteins had no nuclease activity according to agarose gel electrophoresis. However, the expressed proteins as inclusive body displayed the ability to degrade DNA after renaturation in gel. And the nuclease activity was not affected after subjected to mutation of active site in N- and C-termini of Ts-Pt. These results provide the basis to study the relationship between DNase Ⅱ-like protein family and infection of T. spiralis.

The fusion of cell permeable peptide TAT and bifunctional antioxidant enzymes, GST (Glutathione sulfur transferase)-TAT-SOD1 (Cu, Zn superoxide dismutase), is an intracellular superoxide scavenger. Compared with SOD1-TAT, GST-TAT-SOD1 has better protective effect on oxidative damage but less transduction efficiency. A novel cell permeable bifunctional antioxidant enzymes with the fusion of GST, SOD1 and polyarginine R9 was constructed for higher transduction efficiency. The full nucleotide sequence of SOD1-R9 was synthesized and inserted into the prokaryotic expression vector pGEX-4T-1 with the GST tag. After the successful construction of the prokaryotic expression vectors of GST-SOD1-R9, the recombinant vector was then transformed into Escherichia coli BL21 (DE3) and the GST-SOD1-R9 fusion protein was produced with the induction of IPTG. The soluble expression of GST-SOD1-R9 fusion protein was combining with the induction temperature and time. The best soluble expression was obtained with the induction temperature of 25 ℃ and the induction time of 11 h. The fusion protein was purified through the combination of 80% ammonium sulfate precipitation and affinity chromatography using glutathione agarose, and verified by SDS-PAGE and special enzymatic activity. The thermal and pH stability of GST-SOD1-R9 fusion protein were analyzed and the SOD and GST activity of fusion protein were proved to be well maintained under physiological conditions. Finally, the transduction efficiency of GST-SOD1-R9 fusion protein was proved to be better than GST-TAT-SOD1 fusion protein (P<0.05). These works establish a foundation for further study of the protective effect of GST-SOD1-R9 fusion protein against oxidative damage.

Objective: To express and purify polyphosphate kinase (PPK) from Proteus mirabilis and prepare the polyclonal antibody against PPK., Methods: The antigenicity and hydrophobicity of PPK were analyzed using software. The N-terminal conservative sequence containing 309 amino acids was selected as the target peptide, and its corresponding gene sequence with modification based on prokaryotic cells-preferred codon was synthesized and inserted into plasmid pET28b(+). The constructed recombinant plasmid was transformed into Escherichia coli BL21 (DE3) and induced with IPTG. The expressed fusion protein was purified using Ni-affinity chromatography. The purified protein was injected along with adjuvant in rabbits to prepare the polyclonal antibodies against PPK., Results and Conclusion: PPK fusion protein expressed by E. coli was purified successfully using Ni-affinity chromatography. ELISA result demonstrated that the harvested rabbit anti-sera against PPK had a high titer of 1:512 000, and Western blotting showed a good specificity of the antibody, which can be used further study of the role of PPK in the pathogenesis of Proteus mirabilis infection.

Objective To investigate the role of mitoch-ondrial signal peptide in guiding enhanced green fluorescent protein-glucose-regulated protein 75 fusion proteins (EGFP-GRP75) into mitochondria. Methods At first, the signal peptide gene and GRP75 domain genes were spliced by overlap extension PCR. Unphosphorylatable mutant T62A/S65A, phospho-mimic mutant T62D/S65D and substrate binding-defective mutant V482F were further created through site-directed mutagenesis PCR. The fusion gene fragments were ligated into pEGFPC1 expression plasmid, respectively. The expressions of EGFP-GRP75 fusion constructs in HeLa cells were examined directly with Western blotting and laser scanning confocal microscopy. Results All the fusion proteins were highly expressed. Signal peptide remarkably reduced the expression of EGFP-GRP75 fusions compared with recombinant plasmids without signal peptide. Fluorescence was seen exclusively located in mitochondria of the cells transfected by signal peptide-contained plasmids, whereas signal peptide-absent EGFP-GRP75 fusion proteins were homogeneously distributed in the whole cell body. In addition, no changes were observed in the subcellular localization of EGFP-GRP75 fusion proteins that contained double or triple mutations at Thr 62 residues, Ser 65 residues and Val 482 residues. Conclusion Signal peptide is essential for targeting EGFP-GRP75 fusion proteins into mitochondria, and 62 threonine/65 serine/482 valine residues contribute to phosphorylation or substrate-binding characteristics are dispensable in the mitochondrial targeting function.

Objective: The fluorescent protein and gD envelope protein of equine herpes virus type 1 (EHV-1) were used to study the impact of tags on gD protein subcellular localization in BHK-21 cells., Methods: With the EHV-1 genome as a template, the gD complete gene was amplified by PCR technique. The product of PCR was cloned to pAcGFP1-C1 and pDsRed2-N1 plasmids. The recombinant plasmids were designated as pAc-GFP-gD (GFP-gD) and pDs-gD-Red (gD-Red). The GFP gene was inserted into the posterior position of gD gene signal peptide sequence. The modified gD gene signal peptide sequence was cloned to pVAX-1 plasmid, so that pVAX-S-GFP-gD' (S-GFPgD') recombinant plasmid was constructed. Meanwhile, the flag tag was added to N-terminal of gD sequence and they were cloned to pVAX-1 expression vector for constructing pVAX-Flag-gD recombinant plasmid. The BHK-21 cells were transfected with the 4 different recombinant plasmids and the subcellular localizations of fusion proteins were determined by lasar confocal scan microscopy., Results: Four eukaryotic expression vectors were constructed successfully. In BHK-21 cells, the vast majority of gD envelope proteins was localized in Golgi, and a small amount of gD was localized in the nucleus., Conclusion: Our finding reveals that the fluorescent protein of different insertion sites has no significant effects on the subcellular localization of gD, and provides a useful reference for other researchers.

To study the expression and subcellular localization of recombinant dammarenediol-Ⅱ synthase (DS) in Saccharomyces cerevisiae, the dammarenediol-Ⅱ synthase gene ds was cloned from Panax ginseng, and the gene ds was fused with the gene of green fluorescent protein to obtain the fusion gene ds-gfp. The recombinant expression plasmids pESC-HIS-DS and pESC-HIS-DS-GFP were constructed and transformed into S. cerevisiae INVSc1 to obtain recombinant strains INVSc1-DS and INVSc1-DS-GFP. Microsomes of recombinant strains were prepared by differential centrifugation and observed by fluorescence microscope. The green fluorescence was only detected in INVSc1-DS-GFP microsomes, which indicated that DS was a membrane protein. It was also proved that dammarenediol-Ⅱ was produced from the cyclization of 2,3-oxidosqualene catalyzed by DS through in vitro enzymatic reaction. In addition, our results revealed that the fusion expression of ds with gfp significantly improved the production of dammarenediol-Ⅱ from 7.53 mg·g(-1) to 12.24 mg·g(-1). This study provides a new strategy in the optimization of the pathway of ginsenosides biosynthesis in S.cerevisiae.

Two vectors were used to construct the recombinant gene yeast cell that can be used to bioassay of the pollution of tetracycline antibiotics in the environment.In the expression vector,the GPD(glyceraldehyde-3-phosphate dehydrogenase)promoter was used to drive the gene expression of tetracycline repressor protein(TR)fused with V5 antigen epitope gene,while in the reporter vector,the tetracycline response element(TRE)was used to regulate Lac Z report gene expression.The specificity and the sensitivity of the recombinant gene yeast cell were evaluated respectively by different concentrations of tetracycline antibiotics and non-tetracycline antibiotics.The results showed that there were significant dose effect relationships between the tetracycline antibiotics and the yeast cells,while non-tetracycline antibiotics showed no dose effect relationships with this biosensor.It is illustrated that the recombinant yeast cells can be used to monitor the tetracycline antibiotic pollution on the environment.

Exosomes, a population of extracellular membrane vesicles of 30-100 nm in diameter, play important roles in cell biological functions, intercellular signal transduction and especially in cancer diagnosis and therapy. To better apply exosomes in mechanistic study of breast cancer signal transduction, we constructed recombinant eukaryotic expression vector expressing the near-infrared fluorescence protein and CD63 fusion protein through cloning iRFP682 gene and exosomal marker protein CD63 gene into plasmid containing the ITR of AAV. The constructed plasmids were co-transfected with helper plasmid in AAV-293 cell lines and were packaged into rAAV. After titer measurement, the recombinant plasmids were transfected into breast cancer cell lines. The cell lines that stably expressing near-infrared fluorescence protein were selected by fluorescence. Through isolation, purification and identification, we finally obtained a new biomarker: iRFP682 labeled exosomes secreted by breast cancer cell lines, which could be used in further studies of the distribution and signal transduction of exosomes in breast cancer microenvironment.

This study was designed to prepare a monoclonal antibody against Orf virus 118 protein and explore the biological properties of ORFV118 using this antibody. We constructed a recombinant plasmid pET33b-ORFV118 that contained a full-length ORFV118 gene. The plasmid was transformed into E.coli BL21,and the expression of ORFV118 was induced by isopropyl-β-d-thiogalactoside (IPTG).Prokaryotic ORFV118 was purified via Ni-NTA affinity chromatography and was subsequently used as an antigen to immunize mice. An anti-ORFV118 antibody was prepared using hybridoma technology. The titer and specificity of this antibody were tested by an indirect ELISA and Western blot/immunohistochemistry, respectively. We successfully obtained three antibody-secreting hybridomas,1A2,3B5,and 5D10.The titers of the three hybridomas were 1:10000,1:6400,and 1:8000.The monoclonal antibody (mAb),1A2 and the highest titer was selected for further research. The mAb 1A2,an IgG1 type antibody was bonded to its immunizing antigen, both the eukaryotic and natural ORFV118 with high specificity. The immunohistochemical analysis showed that the focal specific staining was restricted to the epidermal layer and subcutaneous tissue, which conformed to the characteristics of an ORFV infection. The mAb 1A2 recognized ORFV118with high specificity. Further study of mAb 1A2 will facilitate our understanding of ORFV118 and provide potentially novel methods for the diagnosis, prevention, and treatment of Orf.

Objective: To clone and express the thioredoxin (Trx) from RH strain tachyzoites of Toxoplasma gondii , establish the prokaryotic expression vector and purify the recombinant protein, then produce the polyclonal anti-Trx antibody in rabbits., Methods: Trx fragment was amplified by PCR and cloned into the pET-28a (+) vector, and the recombinant protein was induced with IPTG and purified by Ni-NTA affinity chromatography. The polyclonal antibody specificity was detected by Western blotting., Results: The trx gene was amplified from T. gondii cDNA by PCR. The recombinant plasmid trx /pET-28a (+) was usefully constructed, and the recombinant TRX protein was expressed and purified. The TRX polyclonal antibody was also obtained. The specific band of TRX was detected by Western blotting., Conclusions: Western blotting can detect the specificity of polyclonal anti-Trx antibody, which will facilitate the biological functions of Trx.

Objective: To construct the eukaryotic expression vector of monocyte chemokine protein 1 (MCP-1) driven by the cytomegalovirus (CMV) promoter, transfect the vector into HEK293T cells, and detect its chemotaxis to macrophages., Methods: MCP-1 promoter was obtained from mouse genome by PCR, and inserted into the vector named pFLAG-CMV1. We validated it by double-enzymes digestion and sequencing. Then HEK293T cells were transfected with pFLAG-CMV1-MCP-1. The expression of MCP-1 was detected by Western blotting. Finally, we identified the chemotaxis of the recombinant vector to macrophage by Transwell(TM) assay., Results: The recombinant vector could generate target fragment by double enzyme digestion. HEK293T cells expressed MCP-1 after they were transfected with the recombinant vector, which increased the migration of macrophages., Conclusion: MCP-1 expressed by HEK293T cells could apparently increase the migration of macrophages.

Objective: To prepare the rabbit polyclonal antibody against canine bocavirus (CBV) structural protein VP2 and identify its specificity., Methods: The target sequence of gene fragment encoding VP2 C-terminal region (300 AA) was amplified from CBV infection clone. After the restriction enzyme digestion and nucleotide sequencing, the target gene was successfully inserted into pET32a(+) prokaryotic expression vector to form recombinant plasmid pET-32a(+)-VP2. Then pET-32a(+)-VP2 was transferred into E.coli (BL21), and VP2-his fusion protein was induced under the optimized induction of isopropyl β-D-thiogalactopyranoside (IPTG). The products were identified and analyzed by SDS-PAGE. The purified fusion protein was inoculated into adult rabbits to develop polyclonal antibody. After the titer of the antiserum was detected by ELISA, Western blotting and immunofluorescence staining were performed to evaluate the features of the prepared antiserum., Results: Restriction enzyme digestion and sequencing showed that prokaryotic expression vector pET-32a(+)-VP2 was successfully constructed. The soluble recombinant protein was highly expressed in E.coli BL21 as expected. After purification and inoculation into adult rabbits, the high-titer polyclonal antibody was prepared and the titer was 1:6 400 000. Western blotting and immunohistochemistry demonstrated that the specificity of the prepared polyclonal antibody was perfect., Conclusion: The polyclonal antibody against CBV structural protein VP2 has been successfully prepared.

Objective: To construct an eukaryotic expression vector of mannose-binding lectin associated protein 19 (MAp19) and further express MAp19 fusion proteins., Methods: MAp19 gene fragment was amplified by PCR. The eukaryotic expression vector pcDNA3.1/Myc-His A-MAp19 was constructed by gene cloning technology, identified by restriction enzyme digestion and further confirmed by sequencing. The recombined plasmid was then transfected into HeLa cells by liposome-mediated method, and the positive clones with vector pcDNA3.1/Myc-His A-MAp19 were selected by G418. Location of MAp19 fusion proteins in the transfected HeLa cells was observed under the fluorescence microscope, and the expression levels of MAp19 fusion proteins were detected by Western blotting., Results: The recombined vector pcDNA3.1/Myc-His A-MAp19 was successfully constructed. The result of DNA sequencing was in accordance with NCBI data bank. By G418 selecting, we obtained the HeLa cells which could stably express exogenous MAp19 fusion proteins. The protein was located in the cytoplasm. Western blotting also suggested that the MAp19 was secretory protein., Conclusion: The vector expressing MAp19 has been prepared successfully, and can express the target protein (MAp19) in the eukaryotic cells (HeLa cells).

Objective: To generate and express fusion vector with mip/flaA advantages epitope genes of Legionella pneumophila by select mip and flaA advantages epitope genes for future research on Legionella pneumophila protein vaccine., Methods: Following analysis of secondary structure and surface properties such as: physical and chemical properties, hydropathy, plasticity, antigen index and extracellular domain of Mip and FlaA proteins by bioinformatics methods, the region which active epitope may exist was selected as advantages epitope region. Then, the recombinant plasmid pET-mip, pET-flaA and pET-mip/flaA with advantages epitope genes were constructed by PCR amplification and T4 ligase connection, and induced the expression in E. coli., Results: Many potential antigenic epitopes in Mip and FlaA were identified, and the selected advantages epitope regions were cloned and expressed successfully. Moreover, the mip/flaA two advantages associated epitope fusion proteins were also successfully expressed., Conclusion: DNA Star software and Expasy online analysis system can successfully predict antigenic epitopes for Legionella pneumophila Mip and FlaA. And prokaryotic expression vector pET-mip/flaA with advantages epitope genes has been successfully constructed and efficiently expressed.

Objective: To construct podoplanin (PDPN) eukaryotic expression plasmid PDPN-pEGFP-N1, establish Chinese hamster ovary (CHO) cell line stably expressing recombinant human PDPN and investigate its biological activity., Methods: PDPN cDNA was cloned from HEK293 cells by reverse transcription PCR and recombinant DNA technology and inserted into plasmid pEGFP-N1 labeled by enhanced green fluorescent protein (EGFP). The recombinant vector was identified by PCR, restriction enzyme digestion and DNA sequencing, and then transfected into CHO cells. Recombinant PDPN-EGFP was observed by fluorescent microscopy and CHO cell line with the high expression of PDPN-EGFP was selected by flow cytometry. Recombinant PDPN was detected by Western blotting and the biological activity of the cell line was determined by platelet aggregation assay., Results: DNA sequencing and restriction enzyme digestion proved that the gene of PDPN was inserted successfully into pEGFP-N1 plasmid. After stable transfection of the recombinant plasmid into CHO cells, CHO with EGFP could be seen under a fluorescent microscope. The CHO cell line with the high expression of recombinant PDPN-EGFP was obtained after sorting by flow cytometry. Western blotting showed that the recombinant PDPN was expressed on the cell surface. The over-expressing PDPN-EGFP CHO cells were able to induce human platelet aggregation., Conclusion: The CHO cell line with the stable and high expression of recombinant PDPN-EGFP has been constructed successfully, and it could induce platelet aggregation.

Objective: To construct prokaryotic expression vector pET-28a(+)-human sucrase (hSUC) and express hSUC fusion protein in E.coli., Methods: The hSUC gene fragment was amplified by reverse transciption PCR (RT-PCR) and cloned into pET-28a(+) vector to construct the prokaryotic expression vector pET-28a(+)-hSUC. The recombinant plasmid was then transformed into E.coli BL21. Hisdidine (His)-tagged fusion proteins were induced by isopropyl-beta-D-thiogalactopyranoside (IPTG) and purified by nitrilotriacetic acid (Ni-NTA) agarose resin. The purified fusion proteins were identified by SDS-PAGE and Western blotting., Results: RT-PCR showed that sub-clone of hSUC was about 1482 bp. The recombinant plasmid was correctly constructed as demonstrated by sequencing and restriction enzyme analysis. The molecular mass of the fusion protein was about 61 240. Western blotting showed that the fusion proteins bound specifically to hSUC antibody., Conclusion: The hSUC protein has been successfully expressed and purified in E.coli.

To prepare virus-like particles containing FluA/B mRNA as RNA standard and control in Influenza RNA detection, the genes coding the coat protein and maturase of E. coli bacteriophage MS2 were amplified and cloned into D-pET32a vector. Then we inserted 6 histidines to MS2 coat protein by QuikChange Site-Directed Mutagenesis Kit to construct the universal expressing vector D-pET32a-CP-His. In addition, the partial gene fragments of FluA and FluB were cloned to the down-stream of expressing vector. The recombinant plasmid D-pET32a-CP-His-FluA/B was transformed to BL21 with induction by IPTG. The virus-like particles were purified by Ni+ chromatography. The virus-like particles can be detected by RT-PCR, but not PCR. They can be conserved stably for at least 3 months at both 4 degrees C and -20 degrees C. His-tagged virus-like particles are more stable and easier to purification. It can be used as RNA standard and control in Influenza virus RNA detection.

Objective: To observe the co-localization of human immunodeficiency virus (HIV) capsid protein p24 and tripartite motif containing 22 (TRIM22) in HEK293T cells., Methods: The retroviral packaging vector pLP1 was used as the template of p24. After the amplification by PCR, the sequence of p24 was cloned into the eukaryotic expression vector pDsRed-Monomer-N1. The recombinant vector was confirmed by colony PCR, double restriction enzyme digestion and DNA sequencing. HEK293T cells were co-transfected with the vector pDsRed-Monomer-N1-p24 together with pEGFP-N3-TRIM22. After 24 hours, the co-localization of p24-DsRed-Monomer and TRIM22-EGFP was detected under a fluorescence microscope., Results: Colony PCR, double restriction enzyme digestion and DNA sequencing confirmed that the eukaryotic expression vector pDsRed-Monomer-N1-p24 was constructed successfully. Fluorescence microscope showed that p24-DsRed-Monomer was co-localized with TRIM22-EGFP in HEK293T cells., Conclusion: HIV capsid protein p24 is co-localized with TRIM22 in HEK293T cells.

Objective: To obtain HEK293T cell line which expresses deformed epidermal autoregulatory factor-1 (DEAF1) highly and stably, and to screen candidate proteins which can interact with DEAF1 by co-immunoprecipitation (Co-IP) and mass spectrometry (MS)., Methods: Plasmids pEGFP-mDEAF1 and pEGFP-N1 were transfected into HEK293T and 2F-2B cell lines with TurboFect transfection reagent, respectively. The localization of DEAF1 was observed in the two kinds of cell lines by a fluorescence microscope. Plasmid p3×FLAG-mDEAF1 was transfected into HEK293T and 2F-2B cell lines, respectively. Non-transfected cells were used as a control group. The expression levels of DEAF1 mRNA and protein were detected by reverse transcription PCR, real-time quantitative PCR (qRT-PCR) and Western blotting, respectively. Plasmid p3×FLAG-mDEAF1 was transfected into HEK293T cells, and then HEK293T cell line expressing stably DEAF1 was selected with G418 and identified by Western blotting, immunofluorescence and flow cytometry. Nuclear proteins of HEK293T-DEAF1 and HEK293T cell lines were extracted and detected using Co-IP and SDS-PAGE with EZview(TM) Red ANTI-FLAG M2 affinity gel. Bands, which were obviously enriched and did not exist in the control lanes, were analyzed with MS., Results: DEAF1 was located in the nucleus under the fluorescence microscope. Reverse transcription PCR and qRT-PCR showed that the mRNA level of DEAF1 in DEAF1-expressed HEK293T cells was higher than that in control cell line. Western blotting revealed the expression of mDEAF1-FLAG fusion protein in the transfected cells. HEK293T-DEAF1 cells which expressed DEAF1 highly and stably were obtained successfully. Some candidate proteins which might interact with DEAF1 were screened by Co-IP and MS, such as cytokines and enzymes required for pre-mRNA processing, postinitiation RNA polymerase, and other transcription factors involved in peptide bond breaking and spatial structure formatting, and so on., Conclusion: The study obtained HEK293T-DEAF1 cells which expressed DEAF1 highly and stably as well as a large set of candidate proteins which might interact with DEAF1 using Co-IP and MS.

Objective: To explore the effect of human sorting nexin 3 (hSNX3) on the morphology and migration of colorectal cancer cells through over-expression of hSNX3 in SW620 cells., Methods: A lentiviral vector pEZ-Lv201 (pEZ-SV40-eGFP-IRES-Puro) inserted with hSNX3 gene was transferred into SW620 cells to construct and package recombinant lentiviral expression vector pEZ-hSNX3-Lv201. The cell clones stably over-expressing hSNX3 (SW620hSNX3) were screened to observe cell morphology and migration ability using Transwell(TM) assay and wound scratch assay. E-cadherin was detected by Western blotting., Results: Sequence analysis demonstrated that the lentiviral expression vector pEZ-hSNX3-Lv201 was successfully constructed. The packaging titers of the recombinant lentivirus and control virus were 2.12×10(9) copies/mL and 7.9×10(10) copies/mL, respectively. After SW620 cells were transfected with the lentivirus, we got stable hSNX3-overexpressed SW620hSNX3 cells. Most of SW620hSNX3 cells (88%) became oval instead of the original spindle-like shape. However, cell migration ability and expression of E-cadherin showed no significant differences between SW620hSNX3 cells and SW620 cells., Conclusion: Over-expression of hSNX3 alters the morphology of SW620 cells, but hardly affects cell migration.

Heat-shock protein (Hsp) 70 potentiates specific immune responses to some antigenic peptides fused to it. Here, the prokaryotic plasmids harboring the envelope glycoprotein E0 gene of classical swine fever virus (CSFV) and/or the Hsp70 gene of Haemophilus parasuis were constructed and expressed in Escherichia coli Rosseta 2(R2). The fusion proteins were then purified. Groups of Balb/c mice were immunized with these fusion proteins, respectively, and sera collected 7 days after the third immunization. Immune effects were determined via an enzyme-linked immunosorbent assay and flow cytometric analyses. E0-Hsp70 fusion protein and E0+Hsp70 mixture significantly improved the titer of E-specific antibody, levels of CD4+ and CD8+ T cells, and release of interferon-γ. These findings suggested that Hsp70 can significantly enhance the immune effects of the envelope glycoprotein E0 of CSFV, thereby laying the foundation of further application in pigs.

Objective: To construct a prokaryotic expression vector of human neutrophil gelatinase associated lipocalin (NGAL) and identify the bioactivity of the fusion protein., Methods: The cDNA of human NGAL obtained from GenBank was linked to a cloning vector to construct the prokaryotic expression vector pCold-NGAL. Then the vector was transformed into E.coli BL21(DE3) plysS. Under the optimal induction condition, the recombinant NGAL (rNGAL) was expressed and purified by Ni Sepharose 6 Fast Flow affinity chromatography. The purity and activity of the rNGAL were respectively identified by SDS-PAGE and Western blotting combined with NGAL reagent (Latex enhanced immunoturbidimetry)., Results: Restriction enzyme digestion and nucleotide sequencing proved that the expression vector pCold-NGAL was successfully constructed. Under the optimal induction condition that we determined, the rNGAL was expressed in soluble form in E.coli BL21(DE3) plysS. The relative molecular mass of the rNGAL was 25 000, and its purity was more than 98.0%. Furthermore, Western blotting and immunoturbidimetry indicated that the rNGAL reacted with NGAL mAb specifically., Conclusion: Human rNGAL of high purity and bioactivity was successfully constructed in E.coli BL21(DE3) plysS using the expression vector pCold-NGAL.

Objective: To construct a eukaryotic expression vector of human autophagy maker protein P62 labeled with myc tag, and detect its biological function., Methods: Human P62 gene was amplified from human breast DNA library by PCR and cloned into pXJ-40-myc vector. HEK293T cells were transfected with the recombinant plasmid myc-p62. Western blotting was conducted to detect the fusion protein expression and the effect on the phosphorylation of extracellular signal-regulated kinase (ERK)., Results: Double enzyme identification and sequencing result showed that P62 eukaryotic expression vector labeled with myc tag was successfully constructed and the inserted fragment was correct. Western blotting indicated that the fusion protein was successfully expressed and was able to inhibit ERK phosphorylation., Conclusion: The eukaryotic expression vector of myc-p62 was successfully constructed and proved to have an inhibitory effect on ERK phosphorylation.

Objective: To express CUB1 and CUB2 functional domain proteins of mannan-binding lectin-associated serine protease-2 (MASP-2) by prokaryotic expression system, and further prepare and identify the polyclonal antibodies against these two domains., Methods: The pGEX-6P-2 prokaryotic vector carrying the target gene CUB1 and CUB2 was used to prepare the recombinant protein GST-CUB1 and GST-CUB2. These two fusion proteins were purified with Glutathione SepharoseTM4B beads and then combined with Freund's adjuvant as antigens to immunize the BALB/c female mice aged 5 weeks for generating polyclonal antibodies. Subsequently, the specificity of the polyclonal antibodies was detected by Western blotting, and the titer was determined by indirect ELISA., Results: The fusion proteins GST-CUB1 and GST-CUB2 were successfully expressed and purified. Their polyclonal antibodies were gained from the BALB/c mice immunized with these two proteins. The polyclonal antibodies showed high specificity with no cross-reaction with other proteins. Indirect ELISA indicated that the titer of anti-CUB1 antibody was more than 1:32 000 and anti-CUB2 antibody was more than 1:16,000., Conclusion: The polyclonal antibodies against GST-CUB1 and GST-CUB2 fusion proteins were obtained respectively with high titers and strong specificity.

To construct a bait expression vector containing the duck circovirus Cap gene for use in the yeast two-hybrid system, the whole cap codon-optimized gene was inserted into pGBKT7 vector and confirmed by PCR, restriction enzyme digestion, and sequence analysis. After transformation into a Y2HGold yeast strain, the expression of Cap protein was analyzed by Western blotting. Toxicity and self-activation of the bait protein were detected using different dropout minimal base. PCR reaction, restriction enzyme digestion, and sequencing analyses indicated that the duck circovirus Cap gene was correctly inserted into pG- BKT7. Western blotting showed that the whole Cap protein was expressed. The recombinant bait protein had no toxicity and self-activation. Therefore, the bait vector with the Cap gene was constructed successfully, providing a foundation for future screening for interacting proteins in the yeast two-hybrid system.

To develop a recombinant lentivirus co-expressing structural protein of hepatitis C virus (HCV) and secreted Gaussia Luciferase (Gluc), we first constructed an expression vector that encoded HCV structural protein (C, E1, E2) and GLuc named pCSGluc2aCE1E2. The expression of HCV proteins and Gluc was confirmed by an immunofluorescence assay (IFA) and the detection of luciferase activity. Recombinant lentivirus (VSVpp-HCV) was developed by the co-transfection of pCSGluc2aCE1E2 into 293T cells with pHR'CMVA8.2 and pVSVG. The infectivity of VSVpp-HCV was confirmed by luciferase activity detection, IFA and western blotting. Virus-like particles were identified using electron microscopy after concentration. The results showed that the level of luciferase activity correlated with the expression of HCV protein after the infection of cells with lentivirus VSVpp-HCV. Therefore, the expression level of HCV proteins could be evaluated by detecting the luciferase activity of Gluc. In conclusion, this research pave a way for the development of transgenic mice that express HCV proteins and Gluc, which enable the evaluation of anti-HCV therapy and vaccine in vivo.

Objective: To construct the prokaryotic expression vector of human autophagy-related protein 4 homolog B (ATG4B) labeled with His-tag, obtain the purified His-ATG4B protein, and identify its activity preliminarily., Methods: ATG4B coding region was amplified from human mammary gland cDNA library by PCR, and was cloned into the prokaryotic expression vector pET-28a⁺. After verification by enzyme digestion, the correct recombinant plasmid His-ATG4B was introduced into E.coli Rossate. The expressed recombinant plasmid was purified by Ni-NTA beads and identified by SDS-PAGE and Western blot analysis. The function of the purified protein His-ATG4B was detected by GST pull-down assay., Results: The DNA fragment of about 1100 bp was successfully amplified from human mammary gland cDNA by PCR, and inserted into pET-28a vector correctly. The results of double digestion and sequencing suggested that the His-ATG4B recombinant plasmid was successfully obtained. His-ATG4B protein of about Mr 47 000 was induced and identified by SDS-PAGE analysis. GST pull-down assay showed that His-ATG4B could interact with LC3B in vitro, suggesting that it has a good biological function., Conclusion: The prokaryotic expression protein of His-ATG4B has been obtained successfully, which lays a foundation for further research on the function of ATG4B in autophagy.

Objective: To construct, express and purify human fusion proteins composed of a single-chain antibody fragment scFv that recognizes the prostate specific membrane antigen (PSMA) protein, Fdt, HA2 and tp, and to analyze the binding activity of the expressed fusion proteins., Methods: The fusion protein genes scFv, scFv-tp, and scFv-Fdt-HA2-tp were amplified by PCR, and the genes obtained were then cloned into the expression vector pET28 and expressed in E. coli BL21. The expressed products were identified by SDS-PAGE and Western blot and purified with Ni(2+)-NTA chelating agarose. The antigen-binding activity of the fusion proteins was determined by ELISA., Results: The human anti-PSMA fusion gene was successfully constructed and expressed in M15 as the inclusion body after induced with IPTG. All the target proteins expressed could bind the PSMA antigen., Conclusion: Fusion proteins can specifically bind the PSMA antigen. This finding contributes to the study of the targeted delivery of siRNA.