The Preparation And Identification Of Polyclonal Antibody Of FMDV 3C Protein

FMD is a febrile,acute and highly contagious disease caused by FMDV.It mainly infects pig,cattle and sheep and other cloven hoofed animal.FMD is a global epidemic disease,which spreads quickly and covers a wide range.The outbreak of FMD brings nearcatastrophic economic losses to the local animal husbandry.It is one of OIE(Office international des épizooties)listed diseases and one of Class A diseases in China.3C protease is a common lyase of picornavirus,which plays a very important role in the process of polyprotein maturation.Studies have shown that 3C protease can destroy the protein complex that maintain normal physiological activity in host cells,also it has certain research value in antiviral drugs design.FMDV 3C protease plays an important role in the pathogenic process,which not only can cut self-coded polyprotein and host protein,but also regulates transcription and translation of host protein.FMDV 3C can inhibit the host immunity and is an important target for the research of FMDV.In this research,we cloned FMDV 3C gene and constructed prokaryotic expression plasmid.FMDV 3C protein could be successfully expressed and purified,and then rabbits were immunized using the purified recombinant protein to produce polyclonal antibodies.ELISA was used to detect the titer and Western blotting was used to analyze the specificity of the antibody.The results showed that the titer and the specificity were high.The polyclonal antibody was further applied to the detection of eukaryotic expressed 3C protein by IFA and Western blotting.The successful preparation of FMDV 3C polyclonal antibody provides an important test tool for relevant study of FMDV and its 3C protein.Construction of expression vector of pET-28a-3C: Based on 3C gene sequence of FMDV type O,strain Tibet/CHA/99(GenBank accession number: AJ539138),specific primers with enzyme digestion sites was designed.FMDV 3C gene was amplified by PCR using PXJ-3C plasmid kept in our laboratory as template and cloned into PET-28 a with BamH ? and Xho? digestion.Prokaryotic expression plasmid pET-28a-3C was successfully constructed.Inducible expression of FMDV 3C protein: pET-28a-3C plasmid was transformed into BL21 competent cells of Escherichia Coli.IPTG was used to induce expression.The result of SDS-PAGE and Western blotting analysis showed that the recombinant protein of FMDV 3C was expressed in the form of soluble protein.Finally,the purity and concentration of FMDV3 C protein reached the immune requirements using Ni-NTA agarose affinity chromatograpHy column and Millipore ultrafiltration.Preparation and identification of polyclonal antibody: We used purified 3C protein as antigen together with FCA to immunize New Zealand white rabbits via subcutaneous immunization.The blood of the heart was collected after two times of immunization.Then serum was separated and purified by recombinant protein G agarose gel FF.ELISA was used to detect the titer and Western blotting was used to analyze the specificity of the polyclonal antibody.The antibody could react with prokaryotic expression protein of FMDV 3C.At the same time,the antibody could react with prokaryotic expression protein in the dilution ratio of1:128000 and 1:256000.The polyclonal antibody with high reactivity and specificity was successfully prepared.Finally,it was found that the polyclonal antibody could react with 3C protein expressed by eukaryotic plasmid of PXJ-3C by IFA and Western blotting.