Prokaryotic Expression Of Equine Influenza Virus NP Gene And Establishment Of Indirect ELISA Detection Method

Equine influenza virus(EIV)causes a respiratory infection disease that.spreads in horse.Its main clinical symptoms are fever,cough,nasal discharge,hard breath,listlessness and loss of appetite.NP is highly homologous and is the most conserved protein of influenza virus.The NP of Newcastle disease virus,rabies virus,measles virus and other RNA viruses is widely used as the coating antigen in ELISA diagnostic kit.Therefore,it is of great significance to develop an ELISA test for detecting the EIV antibody in serum by recombinant NP.The RNA of A/equine/Heilongjiang /13(H3N8)EIV was extracted from allantoic fluid in chicken embryo,and the NP gene was amplified with RT-PCR,and then cloned into pET-32a(+)to construct expression plasmid pET-NP.After sequencing,the correct plasmid was transformed into E.coli Rosetta(DE3)and induced by IPTG.The recombinant NP was obtained by Ni-NTA purification system and the antigen characteristics of recombinant NP was analyzed by western-blot.After that,the recombinant NP as the coating antigen was used to establish an indirect ELISA method to detect the EIV antibody in equine serum.In order to investigate the epidemiology of EI in Guangdong Province,a total of 95 horse serum samples collected from racehorse was detected by the ELISA method developed in this study.The results showed that the recombinant NP had good immunogenicity.Under the optimum induced condition,the protein was soluble in the supernatant and had a high expression leve.Using recombinant NP as the coating antigen to establish an indirect ELISA method,the optimal reaction conditions: the concentration of coating antigen was 100ng/mL,blocked with 5% Non-fat milk powder,serum diluted with the proportion of 1:400,the second antibody of ELISA diluted with the proportion of 1:20,000.The ELISA method established above has the advantages of high specificity,high sensitivity and good repeat ability.The total positive rate of EIV antibody in the 95 serum samples is 42.1%.The positive rate of EI antibody in serum collected from mare horse was higher than that of male horse;The positive rate of EI antibody in serum collected from horse of unknown breeds was higher than that of horse of the other varieties.There is no significant correlation in susceptibility to EIV when studing variety,gender and age in statistical analysis..In this study,the recombinant NP obtained by prokaryotic expression has good antigenicity.The established ELISA method can provide technical support for the prevention and control of EI in China.The results of EIV antibody detection and analysis can provide data supporting for the prevention and control of EI in Guangdong Province.