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Tuberculosis is a serious, infectious, zoonotic disease caused by Mycobacterium tuberculosis. Infections are transmitted in humans and livestock via aerosols. Rv1048c is a hypothetical unknown protein in the standard strain of Mycobacterium tuberculosis H37Rv. Rv1048c exists only in pathogenic Mycobacterium tuberculosis and is highly conserved; however its function is still unclear. The recombinant Mycobacterium smegmatis strain Ms_Rv1048c, with heterologous expression of the Rv1048c gene, was constructed by using the pMV261 expression plasmid. The biological characteristics of the recombinant bacteria were studied, such as their growth pattern, drug resistance, and virulence. Expression of Rv1048c significantly reduced the growth rate of the strain, enhanced its ability to form a biofilm, and reduced its tolerance to sodium dodecyl sulfate, H2O2, and various anti-tuberculosis drugs, and reduced the viability of infected RAW264.7 macrophages. Rv1048c also significantly reduced the level of early pro-inflammatory factors in infected RAW264.7 cells. Rv1048c protein is considered to be a virulence protein that might regulate the growth of M. tuberculosis strains. The results of the present study indicate that Rv1048c plays an important role in Mycobacterial infection. [ABSTRACT FROM AUTHOR]

Abstract Tuberculosis is a serious, infectious, zoonotic disease caused by Mycobacterium tuberculosis. Infections are transmitted in humans and livestock via aerosols. Rv1048c is a hypothetical unknown protein in the standard strain of Mycobacterium tuberculosis H37Rv. Rv1048c exists only in pathogenic Mycobacterium tuberculosis and is highly conserved; however its function is still unclear. The recombinant Mycobacterium smegmatis strain Ms_Rv1048c, with heterologous expression of the Rv1048c gene, was constructed by using the pMV261 expression plasmid. The biological characteristics of the recombinant bacteria were studied, such as their growth pattern, drug resistance, and virulence. Expression of Rv1048c significantly reduced the growth rate of the strain, enhanced its ability to form a biofilm, and reduced its tolerance to sodium dodecyl sulfate, H2O2, and various anti-tuberculosis drugs, and reduced the viability of infected RAW264.7 macrophages. Rv1048c also significantly reduced the level of early pro-inflammatory factors in infected RAW264.7 cells. Rv1048c protein is considered to be a virulence protein that might regulate the growth of M. tuberculosis strains. The results of the present study indicate that Rv1048c plays an important role in Mycobacterial infection.

Coronavirus Disease 2019 (COVID-19), yang disebabkan oleh Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), telah menyebabkan lebih dari 7 juta kematian di seluruh dunia. Strategi komprehensif diperlukan untuk mengatasi pandemi ini, terutama vaksinasi untuk mengurangi infeksi lebih lanjut. Pengembangan vaksin bergantung pada pemanfaatan protein imunogenik dari virus SARS-CoV-2, khususnya pada Receptor-binding Domain (RBD) yang terletak dalam Subunit 1 Spike Glycoprotein. Protein ini berperan penting dalam interaksi virus dengan sel inang melalui reseptor ACE2 untuk menginfeksi inang bersama dengan Subunit 2 untuk memediasi fusi pada membran sel. Meskipun beberapa vaksin telah tersedia, kebutuhan terhadap alternatif vaksin yang lebih terjangkau dan mudah diakses masih terus dikembangkan, terutama untuk negara-negara berkembang dan berpenghasilan rendah. Salah satu alternatif yang dapat dikembangkan dapat berupa vaksin berbasis protein rekombinan. Dalam penelitian ini, urutan gen RBD dioptimasi untuk digunakan pada konstruksi plasmid rekombinan pada ekspresi protein heterolog dalam sistem ekspresi prokaryot yaitu Escherichia coli BL21(DE3). Kodon yang telah dioptimasi kemudian disisipkan ke dalam vektor plasmid pET-15b dan dikonstruksikan menjadi plasmid rekombinan untuk transformasi ke dalam sel inang dengan metode heat-shock. Penelitian ini dievaluasi dan divalidasi melalui PCR koloni, isolasi plasmid, dan sekuensing. Hasil pada penelitian ini dapat menjadi dasar bagi penelitian lanjutan pada ekspresi dan purifikasi protein RBD SARS-CoV-2 pada E. coli BL21(DE3) sebagai kandidat vaksin protein subunit rekombinan atau uji diagnostik pada penelitian terkait COVID-19.

Terpenoid indole alkaloids (TIAs) vincristine and vinblastine are two essential anticancer drugs used in cancer treatments. These compounds are naturally available only in Catharanthus roseus in low accumulation, leading to efforts to upregulate TIAs substances through other expression organisms. The combination of vindoline and catharanthine into α-3~,4~- anhydrovinblastine (AVLB) is an important step for the biosynthesis of vinca alkaloids, which serves as a precursor to vinblastine and vincristine. Vascular class III peroxidase (PRX1) plays a crucial role in catalyzing the dimerization reaction. In this study, we created a recombinant plasmid consisting of the PRX1 gene with the aim to transform and express it stably in fungi. The full-length 1092 bp C. roseus PRX1 coding sequence was optimized to be suitable for a fungal translation system while keeping its amino acid sequence unchanged. The pGreen3 vector was used as the backbone to harbor the PRX1 and hygromycin-resistant gene (HYG)- which enabled fungi cells to survive in the HYG selection medium. This HYG gene was cut from the pGreen2 vector and inserted into the pGreen3 vector. Both PRX1 and HYG genes are located between the gpdA promoter and TrpC terminator structure. The recombinant plasmids were screened and amplified by transformation into Escherichia coli DH10B, and cultured in the LB medium containing kanamycin antibiotic. Our recombinant vector is suitable for further expression into endophytic fungi. [ABSTRACT FROM AUTHOR]

Copyright of Journal of South China University of Technology (Natural Science Edition) is the property of South China University of Technology and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

PTEN is a suppressor gene that methylation in its promoter is potentially used for diagnosis and prognosis for breast cancer and other cancer. We aim to develop a simple, fast, and economical Multiple Methylation-Specific PCR (MMSP) PTEN, recognizing a PTEN promoter gene for methylation and un-methylation developed in one single closed tube. This trueexperimental study uses a recombinant plasmid prepared with a TA cloning system. For the development of PTEN with Multiple MSPs, two formulations for MMSP PTEN were designed and compared, in which the best was to study specificity, limit of detection, reproducibility, and repeatability. The methods were validated with a clinical sample that identified its methylation status using Methylation-Specific PCR (MSP). As a result, the best formulation for MMSP PTEN was formulation 2, which comprises of 1:3 methylation and un-methylation primer, with only high specificity for detecting its target and high sensitivity of 1.5625%, while its limit detection was 0.00032 ng/µl. It has high reproducibility and repeatability, which showed consistent data in the experiments with clinical samples in this activity. This simple molecular evidence aids in detecting biomarkers that are potentially used for the diagnosis and prognosis of cancer and the epidemiological study of methylated PTEN promoters.

l-lysine-coated magnetite nanoparticles (L-lys@Fe3O4) were employed for the magnetic immobilization of recombinant Escherichia coli cells producing extracellular l-asparaginase II. Impact of magnetic immobilization on the plasmid instability and development of a plasmid-free cell community was investigated. Also, undesirable interactions of nanoparticles with the produced enzyme leading to enzyme inactivation or enzyme entrapment were evaluated. Employed l-lys@Fe3O4 nanoparticles were ~ 12.5–22 nm in size with 42 emu/g saturation magnetization. Immobilized cells were magnetically sensitive and completely harvested by applying an external magnetic field (immobilization efficiency equal to 100%). l-asparaginase activity analysis determined that L-lys@Fe3O4 nanoparticles have no inactivation effect on the produced recombinant l-asparaginase II. But a little fraction of the enzyme can be expected to be entrapped within the nanoparticles. However, in contrast to total amount of enzyme the entrapped enzyme was negligible. Due to magnetic immobilization, a small (4.3%) reduction in the community of plasmid-bearing cells was observed but this value was not statistically significant. These findings can pave the way for further applications of magnetic immobilization to immobilize recombinant bacterial cells. [ABSTRACT FROM AUTHOR]

The present study aims to investigate the genetic stability of the plasmid expressing chicken interleukin-2 (ChIL-2) and chicken interleukin-4 (ChIL-4) fusion genes carried by the DH5α/pUC57 recombinant strain during the large-scale production process, and to explore its potential as an immune adjuvant for live chicken coccidiosis vaccine in commercial batch production. The laboratory genetic stability identification of the recombinant DH5α/pUC57 was carried out by subculturing the recombinant plasmid for 30 consecutive generations. The results showed that after the recombinant strain being cultured for 30 consecutive generations on the lysic broth (LB) plates containing ampicillin (Amp+), the recombinant bacterias remained Gram-negative (G-), and could ferment glucose and mannitol to result in specific phenomena, whereas none fermented lactose was found. The homology of the 16S rDNA fragments between the recombinant bacteria and E. coli was greater than 95%, the supercoil ratio of the recombinant plasmids of each generation was greater than 90%, and the plasmid loss rate was 0. The expression product of the recombinant plasmid could stimulate the differentiation of spleen lymphocytes of SPF chicks. The above results indicate that there were essentially no changes in the structure of the 30 generations of continuous passages for the ChIL-2-ChIL-4 fusion gene recombinant bacteria and recombinant plasmids, and the plasmids can be isolated stably, which can provide material support for the expansion of production. [ABSTRACT FROM AUTHOR]

Copyright of Chinese Journal of Preventive Veterinary Medicine / Zhongguo Yufang Shouyi Xuebao is the property of Chinese Journal of Preventive Veterinary Medicine Editorial Office and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Copyright of Journal of China Medical University is the property of Journal of China Medical University Editorial Office and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Abstract Background To develop a new 18 high-risk human papillomavirus (HR HPV) detection and genotyping assay, which is important to evaluate the risk degree of HR HPV for causing cancers. Methods All 18 HR HPV and β-globin relative DNA fragments were synthesized and cloned to a plasmid pUC57 to obtain their recombinant plasmids. Based on the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) platform, each of the 18 HR HPV genotypes were investigated using their constructed recombinant plasmids. The new 18 HR HPV genotyping assay was tested using 356 clinical specimens and the results were compared to ones detected by the Roche Cobas 4800 HPV assay (Cobas). The discrepant results between two assays were resolved by sequencing and genotyping methods. Results The new 18 HR HPV MALDI-TOF MS genotyping assay was developed using HPV recombination plasmids. The sensitivity was 103 to 102 copies/reaction for the all 18 HR HPV. This new developed HR HPV genotyping test was used to detect the clinical specimens. When the results on clinical samples detected by the new MALDI-TOF MS HPV test were compared with ones detected by the Roche Cobas 4800 HPV assay in terms of 14 HR HPV, the concordance was 80.1% (kappa coefficient, 0.60; 95% confidence interval [CI], 0.52–0.69). The discrepant results were resolved by sequencing and genotyping and suggests that the developed HR HPV assay is more sensitive and specific. Conclusions The new developed 18 HR HPV detection method based on MALDI-TOF MS platform is a high-throughput assay for the all 18 HR HPV genotypes and a powerful complement to current detection methods.

Objective of this work was cloning of the Vibrio cholerae nanH gene as part of a plasmid vector, providing expression of foreign genes under the control of T5 promoter, and construction of a E. coli strain – producer of V. cholerae recombinant neuraminidase.Materials and methods. V. cholerae о1 strain served as a DNA donor, pQE30 – as a vector plasmid. The gene was PCR-amplifi ed, the cloning was carried out by means of conventional methods, performance of recombinants and localization of the required protein was determined based on the results of electrophoresis of cell lysates. Neuraminidase activity was identifi ed by fl uorescence in ultraviolet light after incubation with specifi c substrate (4-methylumbelliferyl-N-acetylneuraminic acid).Results and discussion. Recombinant plasmid pNanH, containing the cloned gene nanH V. cholerae, has been constructed. The gene is inserted into BamHI-PstI sites of the polylinker of pQE30. Expression of the cloned gene in the producer strain E. coli JM103pNanH occurs under the control of T5 promoter after isopropyl-1-thio-β-D-galactopyranoside (IPTG) induction. The strain shows neuraminidase activity. The recombinant NanH protein is accumulated in the producer’s cells in two forms. The fi rst form, with molecular mass (MM) of 89.5 kDa, is an unprocessed protein with the hexahistidine block (6His-tag) at its N-terminus, it is located in inclusion bodies. Its percentage is 5.6–6.6 % of the total cell proteins. The second one, with MM of 83 kDa, is found in the periplasmic space and corresponds to the mature NanH, its percentage being 3.4–3.8 %. The total percentage of both forms is 9–10 % of total cell proteins, which allows to consider the strain E. coli JM103pNanH to be a super-producer of the required protein. The strain may be used for purifi cation of NanH preparations for construction of specifi c diagnostic, therapeutic and pharmaceutical preparations as well as for investigation of the protein as a virulence/persistence factor of the pathogen.

Copyright of Shipin Kexue/ Food Science is the property of Food Science Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Objective(s): Anti-tumor effects of Lactobacilli as normal flora have been described. In a previous study, we identified a protein isolated from the bacterium Lactobacillus casei ATCC 39392 in acidic pH conditions named metallopeptidase. Therefore, we decided to evaluate the effect of the recombinant plasmid coding metallopeptidase protein on the inhibition, proliferation, or apoptosis of the colorectal and breast cancer cell lines. Materials and Methods: Identified metallopeptidase gene of L. casei under the specific colon cancer promoter was transferred to the Human SW480 and MDA-MB231 cells. Cell viability was evaluated in these two cancer cell lines via MTT assay, apoptotic changes, and expression level of p53 and MAP2K1 genes in comparison with healthy blood cells as a control group. Results: Viability of SW480 and MDA-MB231 cells was identified at 25% and 7%, respectively. An increase in apoptotic cell death in the SW480 cell line was observed as revealed by Tunnel staining. The expression assay of TP53 and MAP2K1 genes showed that MPL protein altered gene expression in a cell type-specific manner. Tunnel analyses showed that the pronounced cytotoxic effect of pEGFP-C2/ MPL plasmid on SW480 cells was mediated through apoptosis. Conclusion: These results suggest that endogenous recombinant MPL under colon specific promoter inhibits the proliferation of SW480 colorectal cancer cells by increase in MAP2K1 and P53 activation. L. casei metallopeptidase under the same circumstances could not affect the growth rate and viability of MDA-MB231 breast cancer cells in vitro. [ABSTRACT FROM AUTHOR]

Objective of this work was the cloning of Vibrio cholerae hlyA gene in a plasmid vector providing expression of foreign genes under the control of T5 promoter, and construction of E. coli strain – super-producer of Vibrio cholerae recombinant hemolysin. Materials and methods. V. cholerae о1 strain served as a DNA donor, pQE30 – as a vector plasmid. The gene was PCR-amplified, cloning was carried out by means of conventional methods, productivity of recombinants and localization of the required protein was determined based on the results of electrophoresis of cell lysates. Results and conclusions. A recombinant plasmid pHlyA, expressing the cloned hlyA gene of Vibrio cholerae El Tor under the control of T5 promoter after IPTG induction, has been constructed. Carrying this plasmid strain E. coli M15[pREP4]pHlyA is the super-producer of hemolysin: the content of the product in whole cell lysates is up to 13 %, and in inclusion bodies – up to 17 % of the total cell proteins. The product of the cloned gene, in spite of the absence of proteolytic processing and presence of the hexahistidine block (6His-tag) at its N-terminus, possesses hemolytic activity towards sheep erythrocytes. 6His-tag will provide for obtaining a purified preparation on specific sorbents with a view to create diagnosticums as well as to study the significance of hemolysin as a pathogenicity/persistence factor. The advantages of this producer are the high output of the required protein, inability of synthesis of any accessory biologically active substances, short-term period of biomass growing (4–6 h including induction) and possibility of culturing without sticking to the guidelines for work with the agents of particularly dangerous infections.

Hepatitis B causes major deathly infections in developing countries. The surface antigen preS1, hepatocytes attachment site, plays a crucial role in development and progression of disease. The present study was, therefore, an effort to develop an efficient expression system for PreS1 gene in Esherishia coli. PreS1 gene was amplified and cloned in pTZ57R/T vector. Following confirmation through restriction enzyme digestion and sequencing, it was subcloned in pET22b(+). E. coli BL21 (DE3) CodonPlus cells were transformed with the recombinant plasmid. PreS1 gene was induced using IPTG, the protein was expressed and quantified with Bradford assay and analyzed on SDS-PAGE. Enzyme Linked Immunosorbent Assay and Western Blot analysis were performed for protein integrity and conformation. [ABSTRACT FROM AUTHOR]

Objective of the study is to construct recombinant E. coli strain – producer of TcpA protein of cholera agent El Tor, carrying tcpACIRS gene in its genome, and use the strains for antigen production. Materials and methods. Utilized was non-toxigenic genovariant strain of Vibrio cholerae biovar El Tor from the “State Collection of Pathogenic Bacteria” at the premises of RusRAPI “Microbe”, as well as commercial E. coli strains and plasmids for cloning (Invitrogen, USA). Chromosomal DNA from V. cholerae cells was extracted using Charge Smitch gDNA Mini Bacteria Kit applying nucleo-sorption. To extract plasmid DNA from E. coli cells PureLink Quick Plasmid DNA MiniprepKits were used. The presence of tcpACIRS gene was assayed by PCR, using designed through our own efforts primers. DNA fragments were isolated from agarose gel with the help of PCR Clear-Up-System panel. SDS-PAGE was performed according to U.K.Laemmli method. Protein content of samples was measured by M.M.Bradford method. The panel for affinity chromatography was applied for recombinant TcpA protein purification. Results and conclusions. Constructed safe strain of E. coli is the producer of recombinant TcpA protein, basic subunit of toxin-coregulated pilus of adhesion of cholera agent biovar El Tor. The region of tcpA gene of Vibrio cholerae biovar El Tor was cloned as part of vector plasmid pET302 by the restriction sites XhoI-BamHI in E. coli strain BL21(DE3)Star. In the stated design protein biosynthesis is under transcriptional control of phage promoter T7 and induced by isopropyl-ß-thiogalactoside (IPTG). Tested were the conditions for optimum TcpA protein production and the layout of its purification using affinity chromatography. It was demonstrated that TcpA is present in cells of intestinal bacterium, both in native form and as inclusion bodies. Overall TcpA protein production amounted to 60 mcg/ml. Obtained purified TcpA protein can be used for studies of its immunogenic and physical-chemical properties, as well as development of immune-diagnostic preparations to evaluate the level of TcpA production in various V. cholerae strains, and identification of antigen composition of cholera vaccine preparations.

AIM: To construct a recombinant eukaryotic expression vector of rat beta defensin-2(rBD-2), transfect it into the rat corneal epithelial cells with lipofection, determine the expression of target gene in the transfected cells, and discuss the potentiality of recombinant plasmid expressed in corneal epithelial cells, hoping to provide an experimental foundation for further study on the antimicrobial activity of rBD-2 in vitro and in vivo and to assess the probability of defensins as a new application for infectious corneal diseases in the future. METHODS: The synthetic rBD-2 DNA fragment was inserted between the XhoI and BamHI restriction enzyme cutting sites of eukaryotic expression vector pIRES2-ZsGreen1 to construct the recombinant plasmid pIRES2-ZsGreen1-rBD-2, then transformed it into E.coli DH5α, positive clones were screened by kanamycin and identified with restriction endonucleases and sequencing analysis. Transfection into the rat corneal epithelial cells was performed by lipofection. Then the experiment was divided into three groups: rat corneal epithelial cell was transfected with the recombinant plasmid pIRES2- ZsGreen1-rBD-2, rat corneal epithelial cell was transfected with the empty plasmid pIRES2-ZsGreen1 and the non-transfected group. The inverted fluorescence microscope was used to observe the transfection process. At last, the level of rBD-2 mRNA expressed in the transfected cells and the control groups are compared by the real-time fluoresence relative quantitative PCR. RESULTS: The recombinant eukaryotic expression vector of pIRES2-ZsGreen1-rBD-2 was successfully constructed. The level of rBD-2 mRNA in transfected cells was significantly higher than that in control groups through the real-time fluorescence relative quantitative PCR. CONCLUSION: The recombinant eukaryotic expression vector pIRES2-ZsGreen1-rBD-2 could be transfected into rat corneal epithelial cells, and exogenous rBD-2 gene could be transcripted into mRNA in the transfected cells.

Objective To construct the recombinant plasmid of long non-coding RNA-uc. 412(lncRNA-uc. 412 ) and to obverse its expression in rat mesangial cells after transfection. Methods Total RNA was extracted from rat mesangial cells. Upstream and downstream primers were designed according to the gene information of lncRNA-uc. 412. The lncRNA-uc. 412 gene fragment isolated and purified after amplification was obtained by PCR and agarose gel electrophoresis. The plasmid vector pRP[Exp] and lncRNA-uc. 412 gene fragment were digested by same enzyme, and then were amplified by PCR and purified by agar gel electrophoresis, and then we obtained the recombinant plasmid after adding T4 ligase. The ln-cRNA-uc. 412 recombinant plasmid was identified by restriction enzy me digestion and sequencing. Rat mesangial cells were transfected with the recombinant plasmid as the experimental group and with the blank plasmid as the control group by lipo-fection. The relative expression of lncRNA-uc. 412 in the rat mesangial cells was detected by RT-qPCR. Results The double enzyme digestion indicated that the length of resulting band was consistent with the length of lncRNA-uc. 412 , which was about 200-300 bp. The base pairs of the lncRNA-uc. 412 recombinant plasmid were correct by sequencing. The relative expression of the lncRNA-uc. 412 in the control group was 1.00 ±0.00, and the relative expression in the experimental group was 8.01 ±0.97, with statistically significant difference (P <0. 05 ). Conclusion The lncRNA-uc. 412 recombinant plasmid is constructed and transfected into rat mesangial cells successfully. [ABSTRACT FROM AUTHOR]

Copyright of Journal of Food Safety & Quality is the property of Journal of Food Safety & Quality Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Abstract Background Silent information regulator 2 (SIR2) proteins are a family of NAD + -dependent protein deacetylases that are considered potential targets for anti-parasitic agents. In this study, we cloned and characterized SIR2A of the protozoan parasite Eimeria tenella (EtSIR2A) and investigated its protective efficacy as a DNA vaccine. Methods The EtSIR2A gene encoding 33.37 kDa protein from E. tenella second-generation merozoites was cloned, and recombinant EtSIR2A protein (rEtSIR2A) was produced in an Escherichia coli expression system. The rEtSIR2A was used to immunize rabbits. Anti-rEtSIR2A antibodies were used to determine the immunolocolization of EtSIR2A in the parasite by immunofluorescence assay (IFA). Transcript and protein expression of EtSIR2A in different development stages of E. tenella were observed by quantitative real-time PCR (qPCR) and western blot (WB) analysis, respectively. The recombinant plasmid pCAGGS-EtSIR2A was constructed and its efficacy against E. tenella infection in chickens was evaluated. Results qPCR and WB analysis revealed EtSIR2A expression was developmentally regulated at both the mRNA and protein levels. EtSIR2A mRNA levels were higher in unsporulated oocysts than at other developmental stages, including sporulated oocysts, sporozoites and second-generation merozoites. In contrast, EtSIR2A protein expression levels were highest in second-generation merozoites, moderate in unsporulated oocysts and sporulated oocysts and lowest in sporozoites. Immunostaining with anti-rEtSIR2A antibody indicated that EtSIR2A was mainly located in the cytoplasm of sporozoites and second-generation merozoites, and was strongly expressed during first stage schizogony. Animal-challenge experiments demonstrated that immunization with pCAGGS-EtSIR2A significantly increased average body-weight gain, and decreased mean lesion score and oocyst output in chickens. Conclusions These results suggest that EtSIR2A may play an important role in parasite cell survival and may be an effective candidate for the development of new vaccines against E. tenella infection in chickens.

Objectives: Campylobacters are a major cause of gastroenteritis worldwide. These are fastidious in culture and false negative results are seen in many clinical laboratories. Among molecular methods, the dot-blot technique is widely used for a variety of purposes, especially diagnostics. So, the authors aimed to detect C. jejuni and C. coli simultaneously using a dot-blot assay., Methods: After evaluating the bioinformatics studies, a cadF -conserved fragment was selected for the design of primers and probe. DNAs from standard strains and a recombinant plasmid, prepared in this study, were used to assess the technique. The specificity of the method was also surveyed using DNAs from other enteric bacteria. The limit of detection was evaluated by recombinant plasmid and different concentrations of the designed probe., Results: A 95-bp fragment of cadF was selected, and in silico analysis studies showed that it is conserved between both species. Also, the non-specific annealing of the primers and probe with other bacteria was not seen theoretically. The technique with recombinant plasmid as well as DNAs of standard strains created black spots on the membrane, confirming that the probe was correctly synthesized. No non-specific reactions with other bacterial species were observed (specificity=100%). The limit of detection of the test was determined to be 50 µg/ml., Conclusions: This is the first study to simultaneously detect two important pathogens in the Campylobacter genus and was able to detect C. jejuni and C. coli with acceptable sensitivity and specificity., Competing Interests: The authors declare no conflicts of interest.Sponsorships or competing interests that may be relevant to content are disclosed at the end of this article., (Copyright © 2023 The Author(s). Published by Wolters Kluwer Health, Inc.)

The NS1protein, a nonstructural protein of Tembusu virus, plays a key role in the pathogenesis of TMUV. To research host proteins that interact with NS1 protein, the cDNA library of duck embryo fibroblasts (DEF) was successfully constructed. The recombinant plasmid, pGBKT7-NS1, was transformed into the yeast Y2H to be cloned and tested for autoactivation and toxicity. The autoactivation and toxicity test of bait showed that the yeast two hybrid test could be carried out normally. A total of 7 clones from the library were got by Yeast Two-Hybrid System, and 4 proteins, including RPS7, MORC3, GABARAPL1 and MTSS1, may be interacted with DTMUV NS1 after sequencing and blast. Then we chose the host protein of RPS7 for GST pull down assay and the recombinant plasmid of pGEX-6p-1-NS1and pEGFP-RPS7 were constructed. Then the proteins of GST-NS1 and GFP-RPS7 were successfully expressed in vitro for GST pull down assay. The results showed that there was a real interaction between the two proteins when the protein of GST-NS1-GFP-RPS7 was obtained eventually. The Real-time RT-PCR was used to detect the expression level of RPS7, MDM2 and P53 mRNA after the recombinant plasmid of pEGFP-NS1 was expressed in 293 T cells. It is showed that the expression of NS1 protein causes the low expression of RPS7 and MDM2 mRNA and eventually causes the high expression of P53 mRNA. This research lays the foundation for clarifying the pathogenic mechanism of Tembusu virus and the function of NS1 protein in virus propagation process. [ABSTRACT FROM AUTHOR]

The phosphatidylinositol 3-kinases (PIK3s) are lipid kinases. Mutation in the exon 9 and exon 20 determined as a predictive factor in anti-HER-2 therapy. In some countries, such as Singapore, China, and Peru, PIK3CA exon 9 E545A was reported to produce the highest rate of mutation. In this research, we developed and optimized PIK3CA exon 9 E545A detection methods with intercalating dye SYBR Green I based on the Tm Shift approach by using prepared recombinant plasmid pGEMT-easy PIK3CA exon 9 and PIK3CA exon 9 E545A. Recombinant plasmid was used due to the limited number of samples. Methods Recombinant plasmid was prepared based on manufactured procedures, and this process was then followed by Tm prediction with Poland software, Tm Shift SYBR Green I development, and its characterization (reproducibility, repeatability, sensitivity, qPCR efficiency, and qPCR amplification), respectively. Result A method for PIK3CA E545A detection based on TM shift SYBR Green I has been successfully developed. The melting temperature for PIK3CA exon 9 was 78.1 ± 0.1 °C, while that for PIK3CA exon E545A was 80.20 °C. The Tm of mutant was the same as that predicted using Polland Software. The reproducibility of the methods was high, with the coefficient values for inter and intra assays were below 10% with a high sensitivity at 1%, while R 2 0.99 and PCR efficiency was 97.75%. Conclusion The results presented here demonstrate that the PIK3CA exon 9 E545A detection method has a good sensitivity and efficacy assay, which proves that the method has a high diagnostic accuracy in breast cancer. [ABSTRACT FROM AUTHOR]

Adherent cells such as mouse RAW cells or human cancer U2OS cells are beneficial to DNA transfection, with 20%–60% transfection efficiency. However, this DNA transfection is rarely used on suspension cells due to its low transfection efficiency (≤5%). We recently found a new DNA transfection method to increase the efficiency up to 13.5% in suspension cells without PMA treatment. We also found that DNA transfection of human TNFAIP1 or CXCL1 recombinant plasmid DNA in THP-1 cells induces a high level of TNF-α protein. Overall, this new method is simple yet efficient and can be used for the overexpression of DNA in suspension cells. [ABSTRACT FROM AUTHOR]

Background: Cystic echinococcosis (CE), as a zoonotic disease cause to health threat and economic losses. Despite implemented control programs, few countries have been able to decrease or eliminate this infection. Vaccination of the intermediate host offers an additional strategy to control the parasite transmission and EG95 antigen is considered more than the others in the vaccine issue. According to the high protection induced by the EG95 recombinant vaccine, this study was designed to construct recombinant plasmid formulation of EG95 antigen. Methods: In 2015, the Echinococcus granulosus eggs were recovered from an infected dog in Parasitological laboratory of Tarbiat Modares University in Tehran, Iran. Following hatching, the oncospheres of E. granulosus were activated to increase the presence of the desired mRNA. The extracted mRNA was transcribed to the cDNA which used as template in RT-PCR. Then the EG95 gene cloned into pET28a vector and the recombinant plasmids expression was investigated in prokaryotic and eukaryotic cells. Results: The recombinant plasmid encoding EG95 antigen was successfully constructed and identified by PCR, restriction enzyme digestion and sequencing. In vitro expression of the EG95 antigen was confirmed in prokary­otic and eukaryotic systems by SDS-PAGE and western blotting analysis. Conclusion: Because of potential advantages of DNA vaccines, including ability to induce long-term immune responses, low production cost and stability in different temperatures, this study carried out to construct the EG95 gene into a vector. This recombinant vector can be evaluated in further studies as a DNA vaccine may provide new prospects for the development of a vaccine against cystic hydatid disease.

The results of the construction of an avirulent strain of V. cholerae genovariant of the O1 serogroup of the El Tor biovar with efficient production of the cholera toxin (CT) B-subunit, which causes the formation of antitoxic immunity during cholera infection, are presented. In the beginning of the study, a modified P18899ΔCTXϕchr:TnphoA (KmRHly-) strain was obtained via nondirectional transposon mutagenesis on the basis of a nontoxigenic strain of a P18899ΔCTXϕHly+ genovariant. It lost the ability to produce thermolabile hemolysin, which is an additional toxin. The ctxB gene, which encodes biosynthesis of the CT B-subunit, was further introduced into cells of this strain. To accomplish this, the cointegrative recombinant plasmid pIEM3 was used. It was generated in the process of the fusion of a conjugative plasmid pIEM1 and a nonconjugative one, pCTΔ27, which is a derivative of pBR322 (which carries the cloned gene ctxB). Via restriction analysis, it was established that disjunction of a cointegrate occurred in vibrio cholera cells, followed by the preservation of a multicopy plasmid (pCTΔ27) only. As a result, avirulent clones of KmRTcR with a high production level (5-6 μg/mL) of secreted CT B-subunit were obtained, one of which (E99) was selected for further studies. Via real-time PCR, it was discovered that the expression of the ctxB plasmid gene in the cells of a designed strain of V. cholerae, E99, doesn't depend on the activity of the key regulatory gene toxT, which located on the chromosome. Efficient expression of the ctxB gene in E99 cells allows their use to obtain CT B-subunit in order to produce cholera immunobiological drugs. [ABSTRACT FROM AUTHOR]

Haemorrhagic septicaemia (HS) an acute septicaemic disease caused by the bacteria Pasteurella multocida, is an almost neglected disease although it had been significantly caused high mortality in infected buffaloes and cattles. A great effort has been made to search for vaccine candidates for HS using different approaches and methodologies, as the vaccine that is currently available only confer short-term protection with side effects. Thus, this study was conducted to construct a recombinant protein expression system expressing Murine polyomavirus-like particles and the fimbrial protein of Pasteurella multocida for future application as vaccine for HS in livestock. Virus-like particles (VLPs) are viral subunit that mimics the conformation of authentic viruses without requirement of containing infectious genetic material. In this study, polymerase chain reaction (PCR) was used to isolate and amplify fimbrial protein gene of P. multocida B:2 strain. To introduce restriction enzyme (RE) site at the fimbrial gene sequence, PCR was employed using the upstream primer and downstream primer with NotI and XhoI site. The ~435 bp of amplified fimbrial protein gene was cloned into an expression plasmid containing Murine polyomavirus major protein 1 (VP1), pGEX-4T-1 plasmid, between XhoI and NotI site and was then transformed into E. coli JM109 strain. The putative transformants were verified by RE digestion and sequencing. The plasmid (pGEX-VP1) and recombinant plasmid (pGEX-VP1-fimbrial) were transformed into BL21 (DE3) cells for protein expression. Control (GST-VP1) and recombinant (GST-VP1-fimbrial) protein was optimized under various IPTG concentrations (0.4 mM to 1.0 mM) in order to produce the highest protein yield. The highest expression level of control protein (GST-VP1) was observed at 0.8 mM IPTG (~66 kDa), while the IPTG concentration yielded the highest expression level for GST-VP1-fimbrial protein is 0.6 mM which showed the size ~82 kDa. Further analysis will be conducted in order to produce the ultimate protein product which is the purified VP1-fimbrial protein in order to elucidate the potential of the purified protein as a vaccine candidate. [ABSTRACT FROM AUTHOR]

Objective: To determine the targeted regu1αting role of HIF-1α on the expression of HE4 based on a constructed lu-ciferase reporter HE4 gene vector and the transfection ofpcDNA-HEF-1α. and investigate the mechanisms of HE4 mediated renal fibrosis. Methods: The sequence of was chemically synthesized and directionally cloned into pGL3-BASIC. The recombinant pGL3-HE4 lu-ciferase reporter vector was identified by PCR. enzyme digestion. Then both pGL3-HE4 and pcDNA3.1-HIF-1α transfected into HK2 cells to detect the activity of luciferase. Western blot qRT-PC'R was used to analyzed the re1αtionship between hif-1α and HE4 after HK2 cells transfected with pcDNA3.1-HEF-1α. The mechanisms of HE4 mediated renal fibrosis was analyzed by western blot. qRT-PC'R after HK2 cells was transfected with pcDNA3.1-HE4 or added the recombinant protein of EE4. Results: The pGL3-HE4 luciferase reorter vector had been identified by enzyme digestion as expectably. The hif-1α could bind to the region of HE4 promoter to promote the transcription of E1E4. The expression of HE4 was significantly increased after transfected with pcDNA3.1-HIF-1α in HK2 cells (P<0.05). After transfected with pcDNA3.1-HE4 in HK2 cells, the expression of COL4A1. COL1A1 was also significantly increased (P<0.05). Conclusion: The luciferase reporter vector of HE4 was successfully constructed and provided a tool for further studying. EE4 was targeted by HIF-1α and overexpression of HE4 could promote the accumu1αtion of ECM involved in the regu1αtion of renal fibrosis by upregu1αt-ing the expression of COL4A1 and COL1A1. [ABSTRACT FROM AUTHOR]

To detect the copy number of ammonia oxidizing bacteria ( AOB) of activated sludge rapidly and accurately, and to explore an easy preparation method of absolute quantitative PGR standard, this study used purified PGR product and preparative recombinant plasmid, respectively, to conduct absolute quantification PGR ( AQ-PCR ). The melting curve, amplification curve, standard curve and differences of absolute copy number were compared, the advantages of the two standard preparation method were analyzed. The results showed that the two melting curves of preparation standard were a single peak, the index phases of the amplification curves were parallel, R2 values of standard curves were more than 99% , the AOB absolute copy number of PGR product as a standard was less than the results of recombinant plasmid standard (P < 0. 05). It indicates the purification of PGR products as a standard is more convenient, more economic, and lower personnel professional requirements than the recombinant plasmid standard. In the case of the high purity, PGR product has the same application effect with recombinant plasmid standard. [ABSTRACT FROM AUTHOR]

Background: Cystic echinococcosis (CE), as a zoonotic disease cause to health threat and economic losses. Despite implemented control programs, few countries have been able to decrease or eliminate this infection. Vaccination of the intermediate host offers an additional strategy to control the parasite transmission and EG95 antigen is considered more than the others in the vaccine issue. According to the high protection induced by the EG95 recombinant vaccine, this study was designed to construct recombinant plasmid formulation of EG95 antigen. Methods: In 2015, the Echinococcus granulosus eggs were recovered from an infected dog in Parasitological laboratory of Tarbiat Modares University in Tehran, Iran. Following hatching, the oncospheres of E. granulosus were activated to increase the presence of the desired mRNA. The extracted mRNA was transcribed to the cDNA which used as template in RTPCR. Then the EG95 gene cloned into pET28a vector and the recombinant plasmids expression was investigated in prokaryotic and eukaryotic cells. Results: The recombinant plasmid encoding EG95 antigen was successfully constructed and identified by PCR, restriction enzyme digestion and sequencing. In vitro expression of the EG95 antigen was confirmed in prokaryotic and eukaryotic systems by SDS-PAGE and western blotting analysis. Conclusion: Because of potential advantages of DNA vaccines, including ability to induce long-term immune responses, low production cost and stability in different temperatures, this study carried out to construct the EG95 gene into a vector. This recombinant vector can be evaluated in further studies as a DNA vaccine may provide new prospects for the development of a vaccine against cystic hydatid disease. [ABSTRACT FROM AUTHOR]

Objective: To establish pc DNA3.1-snail eukaryotic recombinant expression plasmid. Methods: Human Snail gene c DNA was amplified by reverse transcription polymerase chain reaction(RT-PCR). After digested by Bam H I,Eco R I and ligation,Snail was inserted into pc DNA3. 1(+)eukaryotic expression vector. The positive colonies were screened and identified by single,double enzyme digest,agarose gel electrophoresis and DNA sequence analysis. pc DNA3. 1-snail plasmid was then transfected into siha cell line with Turbo Fect Transfection Reagent. Western-blot was used to detect the protein expression of snail. Results: The pc DNA3.1-snail recombinant plasmid was verified by enzyme digestion electrophoresis showed the expected fragment. The inserted sequence in pc DNA3.1(+)-snail was the same as the sequence of snail c DNA published in NCBI Gen Bank. Western blot results validated the recombinant plasmid expressed in siha cell line efficiently. Conclusion: The eukaryotic recombinant expression plasmid of pc DNA3.1(+)-snail was successfully constructed. [ABSTRACT FROM AUTHOR]

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Accessory cholera enterotoxin (ace) gene of Vibrio cholerae, amplified by PCR, is cloned into the BamHI-HindIII sites using plasmid pQE30. Expression of ace gene is under the control of T5 promotor. The Escherichia coli strain M15[pREP4]pAce90 (KM 194), containing recombinant plasmid, is an active producer of 6His-Ace protein, which possesses biological activity in the models of suckling mice and causes accumulation of fluid in the intestine. The product is contained in inclusion bodies found in the cells of coliform bacterium. The engineered producer strain can be used to obtain Ace preparations with a view to study its significance as pathogenicity factor in cholera vibrio.

Experimental studies have shown that dental pulp tissue has potential to regenerate dentine in response to adverse stimuli, such as caries and associated operative procedures. However, the potential of dental pulp regeneration seems to be limited by regenerative capacity of the cell involved. In this study, we report the effect of transfection of a recombinant plasmid containing human BMP-2 gene in proliferation and differentiation of dental pulp tissue in vitro. The regenerative capacity was analyzed by ALP production and calcium content. Results showed that the transfected dental pulp cell was able to differentiate into the odontoblast phenotype, indicating the presence of odontoblast progentitor cells in dental pulp tissue.

QepA is one of the genes that confer quinolone resistance in bacteria. The aim of this study was to analyze the genetic structures of plasmids that carry a qepA3, a recently discovered allele of qepA in Enterobacteriaceae clinical isolates. 656 non-redundant Enterobacteriaceae clinical isolates were screened for the qepA3 gene and five isolated were identified to carry the gene. Plasmids were isolated from these isolates and were found to increase antibiotic resistance once the plasmids were transferred to Escherichia coli. These plasmids were subcloned and sequenced to analyze the genetic structures surrounding the the qepA3 gene. The results showed that the five plasmids had different genetic structures; one of qepA3-containning isolates had either the blaCTX-M-14 or blaTEM-12 gene in place of the blaTEM-1 gene. The structures of both pKP3764 and pECL3786 have not been previously described. In comparison with pHPA, there were a number of changes in DNA sequences up- and down-stream the qepA3 gene. These findings provide better understanding of the genetic variations in qepA3 and would be useful for diagnosis and control of quinolone resistance in clinical settings.

Objective: To clone the scaffold guide RNA expression vectors targeting Wnt pathway and to verify the efficacy of these plasmids in fine-tuning Wnt pathway using an HEK293 cell model. Methods: The negative regulators of Wnt pathway were selected in this study. Guide RNAs targeting the promoter region of these genes were designed and corresponding DNA oligos were synthesized for cloning into lenti-sgRNA-Ms2-zeo. The plasmid construction were done by annealing the two complementary DNA sequences before insertion into lenti-sgRNA-Ms2-zeo vectors with the enzyme BsmBI. The acquired clones are confirmed by sequencing. After confirmation, the plasmids were co-transfected with lenti-Ms2-P65-HSF1-Hygro and lenti-dcas9-VP64-blastine into 293 cells. Expression of the target genes were analyzed by qPCR assay. Results: Four genes were selected in this study, all of which were well-established negative regulators of Wnt pathway. Two guide RNAs recognizing the promoter of each gene were designed and corresponding plasmids expressing the gRNA fused with a MS2 scaffold were constructed. Sequencing results indicated that all the clones were correctly done. 4 kinds of plasmids were randomly selected for further gene activation efficiency analysis. As expected, the gRNA expression vectors could significantly increase the target genes when co-transfected with lenti-Ms2-P65-HSF1-Hygro and lenti-dcas9-VP64-blastine. Conclusions: The plasmids that expressed gRNAs targeting the known Wnt negative regulators were successfully constructed, which could fine-tune the Wnt pathway by increase the target gene expression. [ABSTRACT FROM AUTHOR]

A total of ~38.6 million mortalities occur due to liver cancer annually, worldwide. Although a variety of therapeutic methods are available, the efficacy of treatment at present is extremely limited due to an increased risk of malignancy and inherently poor prognosis of liver cancer. Gene therapy is considered a promising option, and has shown notable potential for the comprehensive therapy of liver cancer, in keeping with advances that have been made in the development of cancer molecular biology. The present study aimed to investigate the synergistic effects of the abilities of the hemagglutinin neuraminidase protein of Newcastle disease virus (NDV), the pro-apoptotic factor apoptin from chicken anaemia virus, and the interferon-γ inducer interleukin-18 (IL-18) in antagonizing liver cancer. Therefore, a recombinant DNA plasmid expressing the three exogenous genes, VP3, IL-18 and hemagglutinin neuraminidase (HN), was constructed. Flow cytometry, acridine orange/ethidium bromide staining and analysis of caspase-3 activity were performed in H22 cell lines transfected with the recombinant DNA plasmid. In addition, 6-week-old C57BL/6 mice were used to establish a H22 hepatoma-bearing mouse model. Mice tumor tissue was analyzed by immunohistochemistry and scanning electron microscopy. The results of the present study revealed that the recombinant DNA vaccine containing the VP3, IL-18 and HN genes inhibited cell proliferation and induced autophagy via the mitochondrial pathway in vivo and in vitro. [ABSTRACT FROM AUTHOR]

Verticillium wilt of olive, caused by the soil-borne fungus Verticillium dahliae, is one of the most serious diseases of olive tree. In this study, a SYBR Green-based quantitative polymerase chain reaction (Q-PCR) assay targeting the intergenic spacer (IGS) region of the ribosomal DNA (rDNA) was developed to quantify V. dahliae microsclerotia (MS) in soils cropped with olive tree. In order to make the assay quantitative, the number of rDNA units in the genome was estimated using Q-PCR and fixed at 25 copies/genome. The assay was highly specific for V. dahliae, with no cross-amplification with other soil-borne pathogens. The sensitivity analysis showed similar slopes and efficiency, from both fungal DNA (slope = −3.405, r = 0.976, E = 96.64 %) and the positive recombinant plasmid ( y = −3.36, r = 0.989, E = 98.43 %), thus indicating a high accuracy of the assay. The assay exhibits a high intra- and inter-run reproducibility at a very low concentration of 10 copies/μL (CV% ≈ 1 %). When the real-time PCR assay was applied to quantify MS in five naturally infested soil samples, it was able to detect V. dahliae in as few as two MS g of soil. Q-PCR estimates of pathogen DNA were significantly correlated with disease severity (r = 0.944) and with the soil plating method (r = 0.845). This new assay will be a valuable tool and can be applied for disease risk prediction before installing new plantations, and provides a more complete and rapid examination for soils subjected to such a treatment program. [ABSTRACT FROM AUTHOR]

Objective To explore the effect and mechanism of fragile site WWOX gene on regulating proliferation of gallbladder cancer cells in vitro. Methods The pcDNA3.0 - WWOX recombinant plasmid which was previous successfully built was transfected to GBC-SD cells and empty carrier by liposome medium. Liposome and GBC-SD were served as the negative control and the blank control， respectively. After 48 hours transfection， inverted microscope was used to observe the changes of gallbladder cancer cells' morphology， MTT and BrdU were used to detect the proliferation level of gallbladder cancer cells，and flow cytometry instrument was used to detect the change of the cell proliferation cycle. Results The results of inverted microscope shown: the number of GBC-SD cells in pcDNA3.0-WWOX group decreased significantly，the suspension cells and cell debris increased，while cells in the vector control，NC and Mock groups were in normal proliferation state. MTT test showed the proliferation levels of GBC-SD cells in pcDNA3.0-WWOX group was lower than those in the control group in 24 h，48 h，72 h， 96 h and 120 h， and the differences were statistically significant（P < 0.05）. The cell proliferation activity in the pcDNA3.0-WWOX group was obviously inhibited over time. BrdU detection results showed the cell proliferation rate of pcDNA3.0 - WWOX group was（0.44±0.03）， while that in the three control groups was（0.78±0.02）， （0.81±0.01）and（0.85±0.01）， respectively. It showed that cell proliferation activity in pcDNA3.0-WWOX group was lower than the control groups， and the difference was statistically significant （P < 0.05）. Cell cycle detection showed the cells increased in G0/G1 phase and decreased in G2/M and S phases of pcDNA3.0-WWOX group. The cell apoptosis rate was significantly higher and the proliferation index was significantly lower than those of the control groups （P < 0.05）. However， there were no significant differences among the three control groups（P > 0.05）. Conclusion The overexpression of WWOX gene in vitro could effectively inhibit the proliferation activity of gallbladder cancer cells. WWOX might participate in the development of the malignant biological behavior of gallbladder cancer cells. It is expected to become a new potential target for the gene therapy to gallbladder cancer. [ABSTRACT FROM AUTHOR]

Objective To construct and identify the recombinant plasmid pmCherry-C1-Argonaute1 and to provide the basis for further research on the biological role of Argonaute1 protein in the field of cell stress and its related diseases. Methods The total RNA was extracted from Hela cells and c DNA containing Argonaute1 was inverse transcribed. Argonaute1 fragment containing EcoRI and Bam HI site was amplified by PCR from purified tamplate from He La cell line and inserted into pmCherry-C1 fluorescent expressing vector. The recombinant pmCherry-C1-Argonaute1 plasmid was transfected into He La cells and the expression of red fluorescent fusion proteins was examined by Western blotting and the co-localization in stress condition of Argonaute1 and G3BP( biomarker in stress granules) and DCP1α ( biomarker in processing bodies) was detected by fluorescence microscope. Results The recombinant plasmid was sequenced correctly which was identified by EcoRI,Bam HI,the restriction single / double enzyme digestion and gene sequencing. The red fluorescent fusion protein was also detected in transfected He La cell by Western blotting. The result of three-color immunofluorescence co-localization indicated that Cherry-red labeled Argonaute1 was co-localized with G3 BP and DCP1α in cells under stress condition. Conclusions The recombinant eukaryotic plasmid of pmCherry-C1-Argonaute1 is constructed successfully and expresses Cherry-red labeled Argonaute1 fusion protein effectively. These results will help to study the biological role of Argonaute1 in cell stress and related diseases. [ABSTRACT FROM AUTHOR]

There have been only a few antigen genes of Eimeria brunetti reported up to now. In this study, the gene encoding the microneme protein 2 (EbMIC2) was isolated from oocysts of E. brunetti by RT-PCR and the immunogenicity of recombinant EbMIC2 was observed. The EbMIC2 was cloned into vector pMD19-T for sequencing. The sequence was compared with the published EbMIC2 gene from GenBank revealed homology of the nucleotide sequence and amino acids sequence were 99.43 and 98.63%, respectively. The correct recombinant pMD-EbMIC2 plasmid was inserted into the pET-28a (+) expressing vector and transformed into competent Escherichia coli BL21 cells for expression. The expressed product was analyzed using SDS-PAGE and Western-blot. The results indicated that the recombinant EbMIC2 protein was recognized strongly by serum from naturally infected chicken with E. brunetti . Rat rcEbMIC2 antisera bound to bands of about 36 kDa in the somatic extract of E. brunetti sporozoites. The recombinant plasmid pVAX1-EbMIC2 was constructed and then the efficacies of recombinant plasmid and recombinant protein were evaluated. The results of IgG antibody level and cytokines concentration suggested that recombinant EbMIC2 could increase the IgG antibody level and induce the expressions of cytokines. Animal challenge experiments demonstrated that the recombinant EbMIC2 protein and recombinant plasmid pVAX1-EbMIC2 could significantly increase the average body weight gains, decrease the mean lesion scores and the oocyst outputs of the immunized chickens and presented high anti-coccidial index. All results suggested that EbMIC2 could become an effective candidate for the development of new vaccine against E. brunetti infection. [ABSTRACT FROM AUTHOR]

Objective To construct the recombinant eukaryotic expression plasmids of human adenylyl cyclase-associated protein 1 (CAP1) and to explore its intracellular location and functions. Methods By using Hela cDNA as the template, the cDNAs encoding CAP1 was amplified by PCR and inserted into pCMV-Myc vector to construct the recombinant plasmid. The recombinant plasmid was transfected into 293 cells using lipofectamine 2000. The protein expression and the intracellular location of the inserted gene were confirmed by Western blotting and immunofluorescence, respectively. Scratch-repair experiment was used to detect the cancer cells' migration ability. Results The recombinant eukaryotic expression plasmid of human CAP1 was successfully constructed and transfected into eukaryote cells. The recombinant plasmid was successfully expressed in eukaryote cells. CAP1 was located in the cytoplasm. The results of scratch-repair experiment showed that the overexpression of CAP1 could significantly inhibit the cells' migration. Conclusion CAP1 recombinant plasmid was successfully expressed in eukaryotic cells. CAP1 protein was located in the cytoplasm. The overexpression of CAP1 inhibited cell migration. The present study provides important experimental evidence for further study on CAP1. [ABSTRACT FROM AUTHOR]

Objective To detect the transfection efficiency of recombinant plasmid pEGFP-N1-apoJ in rat bone marrow mesenchymal stem cells (BMSCs) at different DNA concentrations and time points and to detect the expression of apoJ in target cells. Methods We used bone marrow adherent culture method to culture rat BMSCs. LipofectaminTM 2000 mediated respectively 0.8, 1.2, 1.6 and 2μg of pEGFP-N1-apoJ recombinant plasmid to transfect BMSCs. The transfection results were observed under the fluorescence microscope. We determined the transient transfection efficiency at 24h, 48h and 72h. Western blot was used to detect the expression of ApoJ in pEGFP-N1-apoJ plasmid transfection group, empty pEGFP-N1 plasmid transfection group and control group. Results In different DNA concentrations, when 1.6μg of pEGFP-N1-apoJ plasmid was transfected with 3.0μL of liposome in rat BMSCs, the transient transfection efficiency was the highest; it reached 27.8%, 34.3% and 28.2% at 24h, 48h and 72h. Western blot results showed that pEGFP-N1-apoJ recombinant plasmid expressed large amounts of ApoJ; small amounts of protein expression of ApoJ was found in the empty plasmid and the control groups. The expression of ApoJ was higher in PEGFP-N1-apoJ group than in the empty carrier and the control groups (P<0.05). Conclusion Recombinant plasmid PEGFP-N1-apoJ can be transfected into rat BMSCs successfully via liposome. ApoJ is expressed in large amounts in rat BMSCs. [ABSTRACT FROM AUTHOR]

Thirty clinical isolates of H. parasuis from pig farms in eastern China were screened for antimicrobial susceptibility. A novel plasmid, designated pHPSGC, was extracted from one isolate with evidence of resistance (elevated minimum inhibitory concentration) to florfenicol. DNA sequencing demonstrated that pHPSGC (5297 base pairs) contains three open reading frames (ORFs), corresponding to the genes rep , floR and lysR . The rep gene of pHPSGC shared 99% sequence identity with the rep gene of pHPS1019. In addition, the region containing floR and lysR in pHPSGC shared 99% similarity with the corresponding region of pCCK381. pHPSGC may be derived from a recombination event between pHPS1019 and pCCK381. A florfenicol resistance gene in H. parasuis may have been transferred via recombination between different plasmids. [ABSTRACT FROM AUTHOR]

Background: Molecular cloning is an essential step in biological engineering. Methods involving megaprimerbased PCR of a whole plasmid are promising alternatives to the traditional restriction-ligation-based molecular cloning. Their widespread use, however, is hampered by some of their inherent characteristics, e.g., linear amplification, use of self-annealing megaprimers and difficulty with performing point insertion of DNA. These limitations result in low product yield and reduced flexibility in the design of a genetic construct. Result: Here, we present a novel technique of directional cloning, which overcomes these problems yet retaining the simplicity of whole-plasmid amplification. QuickStep-Cloning utilizes asymmetric PCRs to create a megaprimer pair with 3'-overhangs, and hence, facilitates the subsequent exponential whole-plasmid amplification. QuickStep- Cloning generates nicked-circular plasmids, thereby permitting direct bacterial transformation without DNA ligation. It allows DNA fragment integration into any plasmid at any position, in an efficient, time- and cost-effective manner, without tedious intermediate DNA gel purification, modified oligonucleotides, specialty enzymes and ultra-competent cells. The method is compatible with competent E. coli cells prepared using the conventional calcium chloride method. Conclusion: QuickStep-Cloning expands the versatility of megaprimer-based cloning. It is an excellent addition to the cloning toolbox, for the benefit of protein engineers, metabolic engineers and synthetic biologists. [ABSTRACT FROM AUTHOR]

Recent data suggest that tumor persistence and recurrence could be caused by the presence of cancer stem cells (CSCs). Aldehyde dehydrogenase 1 (ALDH1) has been implicated in cancer pathogenesis and used as a CSC marker. We previously reported that cervical carcinoma contains a small subpopulation of cells expressing ALDH1 []. In this study, we used small interfering RNA to suppress ALDH1 expression and introduced an ALDH1 reporting vector into HeLa cells followed by various in vitro assays. We showed that knockdown of ALDH1 expression reduced the cell migration ability of HeLa cells, whereas augmented expression of ALDH1 increased cell migration. However, there was no difference in the cellular proliferation, apoptosis, cell cycle, and invasion. These results indicate that ALDH1 is directly involved in HeLa migration. [ABSTRACT FROM AUTHOR]