Prokarytic Expression Of H Gene Of Canine Distemper Virus Yanbian Strain And The Establishment Of Indirect ELISA

Canine distemper(CD), which is caused by canine distemper virus(CDV), is an highly contagious and fatal disease. The characteristics of the disease are associated with strong infectivity, high morbidity and mortality. It is seriously damage to the canine health, and influence the economic development.The H gene of CDV was amplified by reverse transcription polymerase chain reaction(RT-PCR). The amplified fragment was cloned into pMD-18T simple vector and get recombinant plasmid pMD18T-CDV-H. Then digested pMD18T-CDV-H and pGEX-4T-1 plasmid by restriction endonuclease. The recombinant expression plasmid pGEX-CDV-H was constructed and transfected into E.coli BL21(DE3), H gene was expressed at different temperatures, or with different IPTG concentrations and with different induction time, SDS-PAGE was used to analyze expression and antigenicity of H protein. The recombinant expression protein was purified and preparation of polyclonal antibody by immune mice, measured the result by ELISA. The results showed that the H gene was about 1824 bp identified by RT-PCR, contains 607 amino acids, and the homologous analysis of CDV-H nuclear and amino acid sequences was 88.9-99.8%,85.8-99.5% respectively within samples and references strains. The recombinant plasmid pGEX-CDV-H was constructed successfully, the recombinant protein with a relative molecular weight of 103 ku was identified by SDS-PAGE and Western-blot analysis indicated that fusion protein was specifically reached with CDV positive serum, investigated that the recombinant protein shown good reactionogenicity and biological activity, which could be used to identified as antigen in the diagnosic of CDV.We established the ELISA method of CDV with recombinant protein that purified by GST-tag resin as the identified antigen. By the condition of the ELISA screened, the 6.88μg/mL is the best package concentration of the antigen. The 4℃ for 12 h is the best condition for coating of the recombinant protein. Sealed 3% skimmed milk reacted at 37℃ for 3 h. The detected serum was 1:100 dilution and HRP-labeled Rabbit Anti-dog IgG was 1:5000 dilution, There are all at 37℃ for 1 h. The optimal reactive time of the OPD was 15 min at 37℃. The indirect ELISA method was proved to be effective and can be used in clinical detection through the analysis of specific test, repeatability test, contrast test and clinical sample.This experiment laid the foundation for the diagnosis, prevention and control of the Canine distemper virus.