Expression Of Np Protein Of Canine Influenza Virus And Development Of An Indirect Elisa For Antibody Detection

Canine influenza(CI)is an respiratory disease caused by canine influenza virus(CIV).Typical signs in dogs include respiratory symptoms,fever,loss of appetite and so on.The epidemic spread of CIV could be a concern for public health because of the close relationship between humans and companion animals like dogs and cats and it may increase the potential for transmission of influenza viruses to human beingsIn this study,CIV were propagated in 10-day-old specific-pathogen free(SPF)embryonated chicken eggs,and virus RNA was extracted and transcribed into cDNA.The primers were designed according to the NP gene sequence(JN247620.1)registered in GenBank.The NP fragment was amplified by PCR and ligated to pMD18-T plasmid.The recombinant plasmid was ligated to prokaryotic expression vectors pGEX-4T-1,pET-28a and eukaryotic expression vector pFastBac-HTA.The recombinant plasmids NP-pGEX-4T-1 and NP-pET28a were transformed into host strains BL21(DE3)and Rosetta(DE3)respectively.After IPTG induction,based on SDS polyacrylamide gel electrophoresis,the specific band of 82.82kD and 59.98kD were observed.The expression level of NP-pET28a in Rosetta(DE3)was the highest.the best concentration of IPTG was 0.8mM,and the best induction time was 5h.The inculsion bodies were collected in ultrasonic cells,after denaturation and renatured,purified by HisTrap TMHP column.Western blot analysis showed the recombinant protein NP had good reactivity to the rabbit antibody against NP.The recombinant donor plasmid NP-pFastBacHTA was transformed to competent Escherichia coli DHlOBac with Bacmid DNA and Helper plasmid.Then the recombinant bacmid NP-DHIOBac was identified by PCR and transfected to sf-9 cells and thus obtained the recombinant baculovirus Pl-NP.After virus transmission to the third generation,Western blot and IFA analysis confirmed that the recombinant protein NP expressed in infecetd insect cells could be recognized by anti-NP antibody and mouse sera against CIV.The recombinant protein expressed both in prokaryotic and eukaryotic cells was purified and used to establish an indirect ELISA method for antibody detection.The protein expressed in eukaryotic cells has more specificity than that from prokaryotic cells.The optimum concentration of protein was 2?g/ml,the best dilution of serum was 1:100,the best dilution of secondary antibody was 1:5000,and the cut-off value was 0.229.A total of 136 serum samples were detected by indirect ELISA and hemagglutination inhibition(HI).129 positive serum samples were detected by ELISA and 120 were detected by HI.The positive rate of ELISA was 94.85%and higher than that of HI(93.38%).The results showed that the indirect ELISA method for antibody detection has specificity,high reproducibility and can be used for the diagnosis and epidemiological investigation of canine influenza.