| Feline coronavirus(FCo V)is a common cat disease pathogen.It can be divided into two biotypes,feline intestinal coronavirus(FECV)that infects cat intestinal epithelial cells and feline infectious peritonitis virus(FIPV)that causes feline infectious peritonitis(FIP).FIP is one of the most important infectious diseases in cats.It is a chronic,progressive and fatal infectious disease,which can cause fever,appetite loss,weight loss,dyspnea,diarrhea and other symptoms of infected cats.At present,there is no effective vaccine and treatment method forFIP.Therefore,this research plans to prepare monoclonal antibodies against FIPV N protein,And on this basis,establish a highly sensitive and specific antibody competitive ELISA detection method,providing a foundation for the detection of FIPV.According to the FIPV N gene sequence published on Genbank(Genbank number:MK840958.1),primers were designed to truncate and amplify the main antigen region(486 bp)of FIPV N gene.Purification of the amplified FIPV N gene,After purification,they were cloned into the expression vector p ET-30 a to construct a recombinant plasmid p ET-30a-N.The prokaryotic expression system was used to express FIPV truncated N protein(rFIPV N(17-172aa)),SDSPAGE identification shows,which was mainly expressed in soluble form.The expressed protein was purified to obtain the target protein with a concentration of 0.5 mg/m L.Western blotting showed that the recombinant protein had good reactivity.The spleen cells of BALB/c mice immunized with the purified protein for four times were fused with SP2/0 cells,and the positive hybridoma cells were screened by indirect ELISA,and the screened positive hybridoma cells were subcloned for three times.Finally,three monoclonal hybridoma cell lines were obtained,named5C5 、 1F8 、 4A10.Western blotting and IFA test results showed that 5C5 、 1F8 、 4A10 monoclonal antibodies could react specifically with FIPV.Using the obtained monoclonal antibody and FIPV N protein as antigen,a competitive ELISA method was preliminarily established.The matrix method was used to optimize and screen the best antigen dilution and coating conditions,the best blocking conditions,the best antibody dilution and reaction time,the best enzyme labeled secondary antibody reaction time,and the best color development time.It is determined that the best antigen coating condition is 12 h at 4 ℃with the concentration of 1.25 μg/m L,the best blocking method is 2 h at 37 ℃ with the concentration of 5% BSA,the best antibody action condition is 2 h at 37 ℃ with the concentration of 1:128 000,and the best secondary antibody action condition is 1 h at 37 ℃ with the concentration of 1:8 000,The best reaction time of color developing solution is room temperature10 min.After the optimal reaction conditions were determined,94 FIPV negative sera kept in our laboratory were tested and it was determined that the antibody competition ELISA method established in this study was judged to be antibody positive when the sample PI was ≥20.1% and antibody negative when the sample blockage rate was ≤15.9%.The reproducibility and specificity tests showed that the method has good intra-and inter-batch reproducibility and good specificity.The results of the comparison between the antibody competition ELISA method and the method developed in our laboratory for the detection of anti-FIPV S1 protein in 62 cat serum samples showed a high compliance rate of 85.5%.In conclusion,the method based on the monoclonal antibody developed in this study can be used for the clinical detection of FIPV antibodies,laying the foundation for the clinical diagnosis of FIP. |
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