Eukaryotic Expression and Polyclonal Antibody Preparation of Porcine NLRC5
[Objective]Nod-like receptor family member C5(NLRC5)is widely expressed in mammals and is involved in the regulation of immune response and antigen presentation processes.The objective of this experiment was to express the recombinant expression products of porcine NLRC5 gene through eukaryotic expression system,and prepare porcine NLRC5 polyclonal antibody,so as to provide basic support for the subsequent study of the molecular mechanism of porcine NLRC5.[Method]The transmembrane structure and signal peptide of NLRC5 protein was analyzed by bioinformatics online.The porcine NLRC5 gene was amplified by PCR,and the eukaryotic expression vector pCAGGS-HA-NLRC5 was constructed and transfected into HEK-293F cells.Western blotting was used to detect the expression of recombinant protein,and polyclonal antibodies were prepared by immunizing New Zealand White rabbits.The titer of polyclonal antibody was determined by indirect ELISA,and the specificity of the antibody was detected.Indirect immunofluorescence(IFA)and Western blotting were used to detect the reactivity of NLRC5 polyclonal antibody with different cells.Using NLRC5 polyclonal antibody as the primary antibody,the expression of NLRC5 protein in ST cells treated with siRNA was detected by Western blotting.[Result]The results of bioinformatics analysis showed that NLRC5 protein had no transmembrane domain and contained 1 signal peptide.The eukaryotic expression plasmid pCAGGS-HA-NLRC5 was successfully constructed.Western blotting results showed that NLRC5 protein was successfully expressed with molecular weight of about 203 ku.The prepared NLRC5 polyclonal antibody had a titer of 1∶25 600 and good specificity.IFA and Western blotting results showed that the antibody could react with a variety of cell lines.siRNA validation showed that it could be used to detect the expression of NLRC5 in cells.[Conclusion]The recombinant porcine NLRC5 protein was successfully obtained by eukaryotic expression system and rabbit derived polyclonal antibody was prepared,which provided experimental materials for further investigation of the defense effect of NLRC5 on foreign infection in innate immune response.
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维普
