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[Objectives] Porcine CD205, as a receptor specific to porcine dendritic cells (DC), played a key role in the antigen presentation. In this study, nanobodies against porcine CD205 CysR-FN Ⅱ protein were screened by phage display technology, which laid a foundation for the research of porcine CD205-targeted antigen presentation. [Methods] CysR-FN Ⅱ protein of porcine CD205 molecule prepared in the previous research was used for the immunization of alpacas. After subcutaneous immunization for 6 times, peripheral blood lymphocytes of alpaca were collected, their total RNA was extracted and cDNA was obtained by reverse transcription. The gene fragments encoding nanobodies were obtained by PCR amplification, and the porcine CD205 phage display nanobody library was constructed. Bio-spannings were performed by phage display technology and monoclonal colonies were randomly selected to screen specific nanobodies against porcine CD205 CysR-FN Ⅱ protein by Phage-ELISA. [Results] After 4 rounds of bio-spanning, a total of 12 porcine CD205 CysR-FN Ⅱ protein specific nanobodies were selected by Phage-ELISA and sequence analysis with their molecular weight about 17×10³. [Conclusions] This study successfully selected and obtained porcine CD205 CysR-FN Ⅱ protein specific nanobodies. [ABSTRACT FROM AUTHOR]

ObjectiveTo prepare nanobodies against N protein of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and apply them to the detection of N protein antigen of SARS-COV-2. MethodsThe alpaca was immunized with N protein to construct a gene library of nanobody to N protein. The nanobody with affinity to N protein was screened with phage display technology; nanobody genes were cloned to yeast plasmids; positive clone strains were picked to induce expression; the secreted proteins were purified with affinity chromatography, desalted with dialysis and concentrated, then screened with enzyme-linked immunosorbent assay (ELISA) for the collection of high affinity nanobodies to N protein. A pair of nanobodies was screened with cross pairing experiment for the preparation of colloidal gold immunochromatography detection cards, and N-protein solution was dropwise added to the sample wells to observe the detection line and quality control line. ResultsTotally 46 nanobodies with different sequences were screened out and 6 of them were selected to express nanobodies with the concentration > 1.0 mg/ml and high affinity to N protein after purification, desalting and enrichment. Finally, the selected pair of antibodies was assembled into colloidal gold N protein immunodetection card to detect N protein with sandwich method. Obvious detection line and quality control line were observed in the test of SARS-CoV-2 N protein antigen using the detection card and the detection limit was ≥ 10 μg/mL for the test. ConclusionThe prepared nanobody can be used in the colloidal gold nanobody immunochromatography detection card to detect SARS-CoV-2 N protein.

Objective To screen and verify a peptide ligand specific for CD44v9. Methods A 12-mer phage peptide library was screened on CD44v9 coated on solid phase. Candidate sequences emerged after sequencing. Candidate phages were selected using enzyme-linked immunosorbent assay. The best sequence was chosen for further study. Binding of C9-3 to CD44v overexpressed HEK-293 cells was determined using immunofluorescence. Binding affinity and specificity were verified on gastric cancer tissues with immunohistochemistry. Results Phages significantly were enriched during panning process. After sequencing, nine individual sequences occurred in 30 selected clones. Among the 9 candidate sequences, C9-3 exhibited the highest frequency. Results of ELISA showed that C9-3 had the highest OD value and selectivity. Thus, C9-3 was chosen for peptide probe synthesis. C9-3 probe stained CD44v overexpressed HEK-293 cells, but not empty vector transfected HEK-293 cells. Immunohistochemistry scores of C9-3 were significantly different between gastric cancer and paracancer tissues (t=3.953, P＜0.01). A linear positive correlation was observed between C9-3 binding and CD44v9 expression (r=0.823, P＜0.01). Conclusion In this study, peptide ligand of CD44v9 was successfully screened. The peptide can bind to cells and cancer tissues via CD44v9. It has potential for gastric targeting probes. [ABSTRACT FROM AUTHOR]

Copyright of Journal of Hydrobiology / Shuisheng Shengwu Xuebao is the property of Editorial Department of Journal of Hydrobiology 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 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.)

Objective To establish a real-time fluorescent quantitative immuno-PCR (PDRT-IPCR) method for detecting B-cell maturation antigen (BCMA) based on phage display single chain antibody (scFv). Methods The antibody ANTI-BCMA fragment was ligated into phage M13KO7 by seamless cloning technique, and the recombinant phage plasmid M13KO7-ANTI-BCMA was constructed and stored in E. coli DH5α competent cells. The recombinant phage M13KO7-ANTI-BCMA was obtained after low temperature sedimentation of PEG/NaCl solution. Western blotting was used to identify whether the recombinant phage M13KO7-ANTI-BCMA displayed anti-BCMA scFv. The 96-well high adsorption enzyme plate was coated with different concentrations of BCMA, and the recombinant phage M13KO7-ANTI-BCMA was added. After pyrolysis, the DNA of the lysed phage was used as the amplification template for real-time fluorescence quantitative PCR, and the real-time fluorescence quantitative PCR amplification curves of different concentrations of BCMA were observed. The four-parameter Logistic fitting curve was carried out by using OriginPro2021 software, the linear range was determined according to the correlation coefficient R2, and the lowest detection limit was calculated. The specificity of PDRT-IPCR method for the detection of BCMA was verified with recombinant phage M13KO7-ANTI-CD19 and phage M13KO7 as controls. Results Anti-BCMA scFv was successfully displayed on the surface of recombinant phage M13KO7-ANTI-BCMA. With the decrease of BCMA coating concentration, the CT value of the corresponding amplification curve gradually increased. The linear range of BCMA detected by PDRT-IPCR was 0. 1 – 1000 ng/mL, R² was 0. 9993, and the lowest detection limit was 0. 077 ng/mL. The binding of recombinant phage M13KO7-ANTI-BCMA to BCMA was specific. Conclusion In this study, a PDRT-IPCR method for the detection of BCMA is established, which has the characteristics of sensitive detection limit, wide linear range and high specificity. [ABSTRACT FROM AUTHOR]

Copyright of Modern Food Science & Technology is the property of Editorial Office of Modern Food Science & 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.)

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.)

To prepare and characterize the nanobody against urease B（UreB） of Helicobacter pylori. The alpaca was immunized with the lysate of inactivated Hp.Using the recombinant protein UreB as target molecule, nanobodies that can specifically recognize recombinant protein UreB were screened from phage displayed immune libraries. Prokaryotic expression vector was constructed and induced by IPTG. Specificity of the nanobodies was confirmed by ELISA.A 5×107 phage display library was obtained. The screened nanobodies HU2-9 and HU2-36 with high OD450 in phage-ELISA and different sequences were recombinantly expressed. The SDS-PAGE analysis showed that the production was soluble and the production yield were 92 mg·L-1 and 32 mg·L-1,respectively.ELISA showed that production which was recombinantly expressed could bind to the lysate of Hp. The production could inhibit urease activity. nanobodies against urease B of Helicobacter pylori were successfully obtained. [ABSTRACT FROM AUTHOR]

The peripheral blood of eGFP immunized alpaca was used to construct a nanobody immune library, and the eGFP nanobody was solid-phase panned. The E.coli Rosetta expression vector was constructed and expressed nanobody. The binding activity and specificity of nanobody were identified. The working concentration and stability of the enzyme-labeled nanobody was verified after HRP enzyme-labeled nanobody. The nanobody and nanomagnetic beads were coupled to obtain the B13-EGFP fusion protein by immunoprecipitation. After four immunizations, the antiserum titer exceeded 1.28×106 and the eGFP nanobody library with a storage capacity of 1.85×108 cfu was obtained. Nanobodies of ten unique gene sequences were expressed in prokaryotic cells, and they were all soluble intracellularly. Nanobodies 4-28 have the best affinity constant（KD） with eGFP by ForteBio Octet, reaching 9.67×10-11 M; Nanobody 4-28 specifically binds with eGFP and GFP. The detection range of the HRP enzyme-labeled nanobody 4-28 was 1:10 000～1:25 000(100～40 ng·mL-1),which is higher than existing detection secondary antibodies on the market（dilution range 1:5 000～1:20 000）.Nanobody 4-28 was coupled with carboxylated magnetic nanobeads to immunoprecipitate the B13-eGFP fusion protein, leading to a simple and rapid separation of the target protein B13-eGFP. [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.)

Objective: The aim of this study was to acquire the mimic peptides of A/B antigen and combined it with sandwich chemiluminescence immunoassay （CLIA）to develop more effective way to measure the titer of A/B antibody. Methods: Three-round of biopanning was performed on the phage 12 peptide library with NaM87-1F6 and HEB-29 as targets，respectively. Positive plaques determined by enzyme-linked immunosorbent assay (ELISA) were then sequenced, and the mimetic peptides were synthesized according to their amino acid sequences. Competitive ELISA and agglutination inhibition were carried out to confirm the interaction between anti-A/B and the synthesized peptides. The mimetic peptides was then combined with CLIA to detect A and B antibody titers. Standard curves were prepared and the results obtained by the method based on CLIA and the micro-column agglutination technique (CAT) method were compared and the correlation between them was analyzed. Results: After three rounds of panning and related identification, 13 and 19 phage clones that specifically interacted with anti-A and anti-B antibodies were obtained, of which 9(MTRIQLRMMRLH) and 12 (PQMSRHRMRMLP) showed the same amino acid sequences, respectively. The synthesized peptides were named MTR and PQM, respectively. The results of competitive ELISA indicated that MTR can specifically antagonize the binding of anti- A and the replacement polypeptide of the A antigen; PQM can specifically antagonize the immunological binding of anti-B and the replacement polypeptide of the B antigen. Agglutination inhibition experiments showed that MTR/PQM could specifically inhibit the interaction between group A/B red blood cells and anti-A/B. The synthesized peptides combined with CLIA method proposed here had a broad linear range (2-2048) and a good linear correlation coefficient (for A antibody R2=0.9566, for B antibody R2=0.9762). The results obtained by the method based on CLIA were highly correlated with the results obtained by the CAT method (P<0.001). Conclusions: MTR and PQM have good A or B antigenicity and have the potential to be the artificial replacements of them. The method based on the combination of mimic peptides and the CLIA can quantitatively detect the titer of A and B antibodies, and the results are accurate and reliable. [ABSTRACT FROM AUTHOR]

Copyright of China Sciencepaper is the property of China Sciencepaper 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.)

Background and objective Currently, there are only limited number of diagnostic tumor markers for non-small-cell lung cancer (NSCLC). The aim of this study is to screen and identify non-small-cell lung cancer associated antigens, by using of cancer sera containing antibodies which react with autologous cellular antigens (tumor-associated antigens TAAs), which can be used in NSCLC early screening or target treatment. Methods Two T7 phage display cDNA libraries were panning against the sera from 24 patients with NSCLC. Then the enriched libraries were screened with the pool of sera from NSCLC patients, the positive clones encoding antigenic genes were obtained after screening, and the nucleotide sequences of positive were identified and analyzed with BLAST software in GenBank. Results After immunoscreen two T7 phage display cDNA libraries with serum from patients with NSCLC, thirty-one positive clones were obtained and sequence results showed they were derive from 15 genes, 12 of 15 genes were homologous to the genes known in GenBank, such as RHOA, ITGB4, and HNRNPA2/B1 et al . The other three genes expressed sequence tags (ESTs) were not found in GenBank. Conclusion Fifteen NSCLC associated antigen genes and three candiated gene were obtained by SEREX from T7 phage display cDNA libraries.

Background and objective Currently, only a limited numbers of tumor markers for non small lung cancer (NSCLC) diagnosis, new biomarker, such as serum autoantibody may improve the early detection of lung cancer. Our objective is construction human lung squamous carcinoma and adenocarcinoma T7 phage display cDNA library from the tissues of NSCLC patients. Methods mRNA was isolated from a pool of total RNA extract from NSCLC tissues obtained from 5 adenocarcinomas and 5 squamous carcinomas, and then mRNA was reverse transcribed into double stranded cDNA. After digestion, the cDNA was inserted into T7Select 10-3 vector. The phage display cDNA library was constructed by package reaction in vitro and plate proliferation. Plaque assay and PCR were used to evaluate the library.Results Two T7 phage display cDNA library were established. Plaque assay show the titer of lung squamas carcinoma library was 1.8×106 pfu, and the adenocarcinoma library was 5×106 pfu. The phage titer of the amplified library were 3.2×1010 pfu/mL and 2.5×1010 pfu/mL. PCR amplification of random plaque show insert ratio were 100% (24/24) in adenocarcinoma library and 95.8% in human lung squamas carcinoma library (23/24). Insert range from 300 bp to 1 500 bp. Conclusion Two phage display cDNA library from NSCLC were constructed.