Prokaryotic Expression of Porcine Secretory Leukocyte Protease Inhibitor and Preparation of Its Monoclonal Antibody
[Objective]The purpose of this experiment was to prepare porcine derived secretory leukocyte protease inhibitor(SLPI)and its monoclonal antibody,laying the foundation for the establishment of porcine SLPI detection method and function research.[Method]Series connection of porcine SLPI gene was cloned into pET-28a(+)and pGEX-6P-1 vectors to construct the expression vector.The recombinant protein was expressed in Escherichia coli expression system,and the induced expression of recombinant proteins were analyzed by SDS-PAGE.After recombinant bacteria were ultrasonicated,the purified recombinant proteins were identified by SDS-PAGE and Western blotting.BALB/c mice were immunized with the purified recombinant protein,and hybridoma cells were prepared by cell fusion.The monoclonal antibody and subclass were screened and detected by indirect ELISA,and the specificity of monoclonal antibody were identified using Western blotting and indirect immunofluorescence assay(IFA).[Result]The pGEX-6P-1-SLPI and pET-28a-SLPI recombinant plasmids were successfully constructed,and high purity His-SLPI and GST-SLPI recombinant proteins were obtained with molecular weights of 27 and 50 ku,respectively.His-SLPI was expressed in inclusion body,while GST-SLPI was expressed in soluble form.Three hybridoma cell lines that could stably secrete monoclonal antibodies against porcine SLPI were acquired,namely 9D10 as the IgG3 subclass,9G11 and 10E2 as the IgG2b subclass.All three monoclonal antibodies could bind to SLPI in porcine intestinal epithelial cells.9D10 showed strong fluorescence signal in nucleus,while 9G11 and 10E2 showed fluorescence signals in both cytoplasm and nucleus.[Conclusion]Recombinant His-SLPI and GST-SLPI proteins were successfully prepared,and three stable secretion monoclonal antibodies against porcine SLPI with good specificity were acquired.The results would provide a technical platform for further studying the characteristics of porcine SLPI and its role in mucosal immunity.
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