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| Prokaryotic expression of human procalcitonin and preparation of its monoclonal antibody
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| LIU Qing， WANG Hui， LU Ye， WU Liang， CHEN Sheng-xia， JIANG Xu-gan |
| （School of Medicine，Jiangsu University，Zhenjiang Jiangsu 212013，China） |
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Abstract Objective: To express human procalcitonin(PCT) recombinant protein in prokaryocytes and prepare high purity PCT monoclonal antibody. Methods: The constructed PCT-pET22b expression vector was transfected into Escherichia coli BL21 to induce expression of PCT protein, which was purified by Ni-NTA affinity chromatography and identified by Western blotting and colloidal gold method. The obtained PCT recombinant protein was subcutaneously injected into Balb/c mice by long-term immunization, and then spleen cells and myeloma cells(SP2/0) were fused, the positive hybridoma cell lines were screened and subcloned 4 times. The cell strain was injected into the peritoneal cavity of the mouse to collect the peritoneal effusion to obtain monoclonal antibody, which was purified by ammonium octoate ammonium sulfate and Protein G affinity chromatography. Results: SDS-PAGE analysis showed a significant band at a relative molecular weight of approximately 14 kDa, which was consistent with the size of the target protein. Western blotting and colloidal gold methods demonstrated that the soluble protein was PCT. Seven positive hybridoma cells were successfully screened by cell fusion technique and named as B3, C4, C7, E9, F8, F10, G2. B3 and C4 cell strains were selected to prepare monoclonal antibodies by in vivo induction. After purification, the purity of antibody was over 95%. Indirect ELISA showed that the titers of antibodies B3 and C4 were up to 107, the affinity constants were 5.06×108 and 7.3×108, respectively. Conclusion: High purity PCT recombinant protein and its monoclonal antibody were obtained, which provided support for the further preparation of PCT reagent kit.
［Key words］procalcitonin； cell fusion； monoclonal antibody
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Received: 05 March 2019
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2019, Vol. 29 
