Prokaryotic Expression Of Canine Distemper Virus F Protein And Preparation Of Monoclonal Antibody

Canine distemper(CD)is a highly contagious disease caused by Canine distemper virus(CDV).It has a wide range of hosts and can infect a variety of animals including Canidae,Procyonidae,Mustelidae,Felidae,Ailuridae and even primates of the macaqidae.Due to the high incidence and mortality of CD,it poses a major threat to the dog breeding industry,fur animal breeding industry and wildlife protection in China.At present,there is no better treatment for CD,and the most effective method for the disease is through vaccination.Therefore,it is important to establish a rapid and sensitive detection method for the prevention and control of this disease.F protein is a relatively conserved envelope protein in CDV,and it is also one of the important antigens that induce the host to produce neutralizing antibodies.In this study,the F gene sequence of CDV wild virus LN(10)strain was analyzed,the F protein extracellular region gene was cloned by PCR,and the PET-28A-CDV-F recombinant plasmid was constructed by linking with the prokaryotic expression vector pET-28a.It was transformed into Escherichia coli BL21(DE3)competent cells,and the recombinant bacteria were induced by IPTG under optimized conditions.After ultrasonication,the protein expression form and specificity were identified by SDS-PAGE and Western blot.The results showed that the recombinant F protein had the highest expression level at 37°C,0.2 mmol/L IPTG,and 6 h after induction.It was mainly expressed in the form of inclusion bodies,with a size of about 33 k Da.The recombinant F protein was successfully purified by KCl staining and protein gel cutting gel purification,and the concentration of protein was 1.66mg/m L.The result of Western blot showed the protein could react with anti-His tag monoclonal antibody and CDV positive serum with good specificity,respectively.In order to obtain the monoclonal antibody against CDV F protein,the purified recombinant F protein was emulsified with Freund’s adjuvant in the same amount as the immunogen to immunize BALB/c mice by intraperitoneal injection.After three times of routine immunization and one time of booster immunization,the spleen cells of mice were taken and fused with SP2/0cells.The positive hybridoma cells were screened by limited dilution method and indirect ELISA,and the cell lines stably secreting antibodies were injected into the abdominal cavity of mice to prepare ascites.Finally,two monoclonal antibodies were obtained,named 1B6 and 1G8.The two monoclonal antibodies were identified by antibody subclass kit,and the results showed that the two monoclonal antibodies belonged to Ig M,κtype.The titers of 1B6 supernatants were 1:1 600and 1G8 supernatants were 1:3 200,and the titers of ascites reached 1:10~6 by indirect ELISA using the prokaryotically expressed CDV recombinant F protein as coating antigen.The two hybridoma cells were cultured continuously,and the titer of the two monoclonal antibodies were still consistent with those before freezing by indirect ELISA,indicating that the two hybridoma cells could secrete antibodies continuously and stably.The number of chromosomes of 1B6 and 1G8hybridoma cells was 98 and 102,respectively.The two monoclonal antibodies can specifically recognize to the CDV vaccine strain CDV3and the wild strain LN(10)1 infected Vero cells expressing CDV receptor dog SLAM(Vero-dog SLAM,VDS)by indirect immunofluorescence assay.Western blot analysis of the specificity of two monoclonal antibodies showed that the two monoclonal antibodies specifically recognized recombinant CDV F protein.Virus neutralization test showed that the two monoclonal antibodies could not effectively neutralize CDV LN(10)1 strain.In conclusion,CDV F protein was successfully expressed by prokaryotic expression system,and two monoclonal antibodies against CDV F protein were successfully prepared by using this protein.The two monoclonal antibodies had good reactivity with CDV wild-type strain and vaccine strain,which laid a foundation for the establishment of CDV detection method or immunological technology for the diagnosis of CD.