Expression And Purification Of Recombinant N Protein And Development Of Monoclonal Antibody Of TGEV

Transmissible gastroenteritis of swine (TGE) is an acute, highly contagiousdisease induced by transmissible gastroenteritis virus of swine (TGEV). In the present,vaccination is the best way to control its prevalence in swine production.Part one: A pair of specific primer was designed and synthesized according to thesequence of nucleoprotein (N) gene of TGEV published in GenBank. The viralsubgenome mRNA was amplified by RT-PCR with the primer which was designedaccording to the reported reference. The prokaryotic expression vector of pET-32a (+)and the target genes were double digested, respectively and the product was insertedinto prokaryotic expression vector, positive plasmids, pET-32a (+)-N, were screened.The recombinant pET-32a (+)-N plasmid, which includes the N gene of TGEV, wastransformed into Escherichia coli BL21(DE3) and expressed by0.1mmol/L IPTG at37℃. With one-stop His labeled protein purification kit and N-protein of TGEV waspurified and obtained.Part two: With the purified recombinant N-protein, a coated antigen, an indirectELISA method was successfully developed to determine antibody of TGEV. Theoptional working conditions for the ELISA are as follows: antigen concentration is10μg/mL, serum dilution is1:40, blocking solution and serum dilution is diluted by10%FBS, concentration of HRP-SPA is1:40000. The result showed that the coincidentrate is of75%compared to Svanova TGEV/PRCV antibody diagnosis kit．Part three: The monoclonal antibody (McAb) against TGEV was prepared byimmunizing the Balb/c mice with purified recombinant N-protein of TGEV. Cell fusionwas carried out by standard method. Three hybridoma cells,1-27,2-7, and2-15wereexamined by indirect ELISA and were re-determined by3times of subclone withlimited dilution. The murine ascites was collected and monoclonal antibodies (McAbs)were determined after the mice were incubated with the hybridoma cells. Themonoclonal antibody titers of3hybridoma were106tested by indirect ELISA. The three McAbs were determined to be IgG1and they were obviously reacted to PK cellsinfected with TGEV, presenting yellow and green fluorescent in cytoplasm and cellmembrane.Conclusions:(1) We purified and obtained N-protein of TGEV.(2) Withthe purified recombinant N-protein, a coated antigen, an indirect ELISA method wassuccessfully developed to determine antibody of TGE.(3) The monoclonal antibody(McAb) against TGEV was prepared. The result will be useful for development of newimmunoassay of TGEV.