| Prokaryotic expression and identification of human-mouse chimeric monoclonal antibody against Pseudomonas aeruginosa VgrG1a protein |
| Received:March 27, 2021 Revised:May 08, 2021 Click here to download the full text |
| Citation of this paper:HU Xin-yu,SONG Li-jun,TAN Li,LI Qi,LI Fu-long.Prokaryotic expression and identification of human-mouse chimeric monoclonal antibody against Pseudomonas aeruginosa VgrG1a protein[J].Chinese Journal of Clinical Medicine,2022,29(2):166-174 |
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| Author Name | Affiliation | E-mail | | HU Xin-yu | Hebei North University, Zhangjiakou 075000, Hebei, China
Department of Anaesthesiology, Changzheng Hospital, the Second Affiliated Hospital of Naval Medical University, Shanghai 200003, China | | | SONG Li-jun | Hebei North University, Zhangjiakou 075000, Hebei, China
Department of Anaesthesiology, Changzheng Hospital, the Second Affiliated Hospital of Naval Medical University, Shanghai 200003, China | | | TAN Li | Hebei North University, Zhangjiakou 075000, Hebei, China
Department of Anaesthesiology, Changzheng Hospital, the Second Affiliated Hospital of Naval Medical University, Shanghai 200003, China | | | LI Qi | Hebei North University, Zhangjiakou 075000, Hebei, China
Department of Anaesthesiology, Changzheng Hospital, the Second Affiliated Hospital of Naval Medical University, Shanghai 200003, China | | | LI Fu-long | Hebei North University, Zhangjiakou 075000, Hebei, China
The First Affiliated Hospital of Hebei North University, Zhangjiakou 075000, Hebei, China | lifulong8915833@163.com |
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| Abstract:Objective To explore the prokaryotic expression method of VgrG1a protein in type Ⅵ secretion system of Pseudomonas aeruginosa, and prepare murine and human-mouse chimeric monoclonal antibodies. Methods Sequences of the VgrG1a genes were retrieved from Genbank in National Center for Biotechnology Information (NCBI) website to generate recombinant pET-21a-VgrG1a plasmid. The plasmid was transformed to E. coli competent cells BL21 (DE3) plysS for induction. The induced recombinant protein was purified and identified by immunoprecipitation and enzyme-linked immunosorbent assay (ELISA). BALB/c mice were immunized with purified VgrG1a recombinant protein, and mouse polyclonal antibodies were prepared. The titer and specificity of mouse serum antibodies were determined by ELISA. Hybridoma cells were fused of mouse B-cells with myeloma tumour cells (P3X63Ag8.653). Then monoclonal cells were obtained by limited dilution method. Stable cell lines were selected harboring and expressing specific antibodies screened by ELISA. Gene of the antibody was acquired by sequencing. The plasmid of human-mouse chimeric gene was transfected into Expi293 cells to prepare human monoclonal antibodies. Results The recombinant plasmid of VgrG1a was successfully constructed. Next, the VgrG1a protein was over expressed and purified in E. coli,and the best induction conditions were 16 h at 16℃ with 0.5 mmol/L IPTG. The results of SDS-PAGE gel electrophoresis showed that the recombinant protein concentration was the highest at the relative molecular mass of 72 000.The titer of the prepared mouse antibody was still high after diluted to 1/640 000 with specifically recognition and binding to the target protein. The affinity of human-mouse chimeric monoclonal antibody 8A4F5 against antigen VgrG1a was higher than that of mouse monoclonal antibody. Conclusion The recombinant protein has good immunogenicity, and the monoclonal antibody prepared by recombinant protein has high antibody titer and specificity, which provides biomaterials for further study of protein structure and function, contributing to the development of diagnostic reagents and vaccines. |
| keywords:Pseudomonas aeruginosa infection type Ⅵ secretion system VgrG1a protein prokaryotic expression human-mouse chimeric monoclonal antibody |
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