| Avian influenza(AI)is a common disease caused by avian influenza virus(AIV)that endangers poultry,causing serious economic losses to the poultry industry and even threatening human life and health,H7N9 AIV had been prevalent in many parts of the country since 2013,and the prevalence of H7N9 AIV had been greatly reduced since the vaccine was used in 2017.Therefore,it is very important to detect the level of serum antibodies produced after immunization to evaluate the immune efficacy of vaccine strains,which can provide a reference for further prevention of H7N9 AIV.This study will establish a blocking ELISA method for the detection of N9 antibody levels in serum by expressing the reactive AIV N9 protein as a coating antigen using an insect cell baculovirus expression system.It has important application value for the distribution of other N9 subtype AIV infections.1 Expression and identification of N9 subtype avian influenza virus NA protein in insect cellsThe NA gene of DK/HuN/S 11670/2015(H1 1N9)was cloned into the baculovirus transfer vector pFastBac HT A,and the recombinant transfer vector pFastBac HT A-N9 was constructed.After identification by PCR and sequencing,it was transformed into DH1OBac competent cells with blue and white spots.After screening and extracting the recombinant bacmid-N9,which was identified correctly by PCR and sequencing,it was transfected into sf21 insect cells to obtain recombinant baculovirus.After infecting sf21 cells with recombinant baculovirus,the reactivity of recombinant N9 protein was identified by IFA,Western blot and indirect ELISA.The results showed that the expressed recombinant N9 protein could react specifically with the mouse N9 protein monoclonal antibody,and the indirect ELISA method established with the expressed recombinant N9 protein as the coating antigen could distinguish the N9 protein antibody positive serum and the non-N9 protein antibody serum.The above results showed that the recombinant N9 protein had good reactivity,which laid the foundation for the establishment of a blocking ELISA method for detecting serum N9 antibodies.2 Establishment of AIV N9 protein blocking ELISA antibody detection methodThe optimal working conditions of the blocking ELISA method were determined by using the total protein of the cell membrane of the recombinant baculovirus-infected insect cells as the coating antigen.The optimal concentration of protein coating was 1:5000(5.75 ?g/mL)and the optimal concentration of mouse N9 protein monoclonal antibody was 1:30000(0.92?g/mL)by checkerboard titration.Based on the optimal antigen coating concentration and the optimal concentration of mouse-derived N9 protein monoclonal antibody,the remaining optimal reaction conditions were further determined by changing the single factor step by step:the optimal coating conditions were overnight at 4? for 12 h;the optimal blocking conditions 5%skim milk was incubated at 37? for 1h;the optimal dilution and incubation time of the serum to be tested were 1:20 and incubated at 37? for 1.5 h;the optimal incubation time for monoclonal antibodies was 37? for 1 h;The optimal dilution and incubation time of HRPlabeled goat anti-mouse IgG(H+L)secondary antibody is 1:5000,and incubated at 37? for 1 h;the optimal color development time was 10 min.Blocking ELISA method was used to calculate the inhibition rate of 193 negative serum samples under the best working conditions.After calculating the average inhibition rate and standard deviation of 140 serum samples with the inhibition rate greater than 0,the critical value was obtained according to the critical value formula,and the negative result was obtained.The mean inhibition rate of serum was 8.67%,the standard deviation was 4.06%,the negative cut-off value was 16.79%,and the positive cut-off value was 20.85%.The specificity test results showed that the blocking ELISA method can only specifically identify the N9 subtype AIV serum,with good specificity.H11N9,H1N9,H7N9-Re2 and H7N9-Re3 positive sera were double diluted with SPF chicken serum,and the diluted sera with HI titers of 20?27 were tested by blocking ELISA.All values greater than or equal to 24 were judged as positive,indicating that the blocking ELISA method had better sensitivity.32 samples of H7N9,H1N9 and H11N9 subtype-positive chicken serum with clear laboratory preservation background were selected for blocking ELISA detection.The results showed that 32 serum samples were judged to be positive,indicating that the blocking ELISA method had a good broad spectrum.3 N9 subtype positive serum samples and 2 non-N9 subtype serum samples were selected for repeatability test,the results showed that the coefficient of variation within and between plates were lower than 12%,indicating that the blocking ELISA method had good repeatability.478 chicken serum samples,378 duck serum samples and 40 goose serum samples that were inoculated with H7N9-Re3 vaccine strain in the farm were tested for HI and then tested by blocking ELISA.The results of H7N9-Re3 serum sensitivity test showed that when the HI titer was less than 23,after the blocking ELISA method,the serum was judged to be negative,so the HI titer was equal to 23 as the detection limit critical point.The clinical sample test results showed that the chicken serum negative coincidence rate was 92.31%when the HI titer was equal to 20,and the HI titer was greater than 20.When it was equal to 23,the chicken seropositive coincidence rate was 98.47%,and the total coincidence rate was 97.91%;when the HI titer was equal to 20,the duck seronegative coincidence rate was 91.50%,and when the HI titer was greater than or equal to 23,the duck seropositive coincidence rate was 94.86%.The total coincidence rate was 93.29%;when the HI titer was equal to 20,the seronegative coincidence rate of the goose was 90.00%,and when the HI titer was greater than or equal to 23,the seropositive coincidence rate of the goose was 92.00%,and the total coincidence rate was 91.43%.For chickens,the coincidence rate was higher,indicating that this method was more suitable for evaluating the level of N9 antibody in chicken serum.In conclusion,the blocking ELISA antibody detection method established in this study could be used to preliminarily evaluate the immune efficacy of the H7N9-Re3 vaccine strain in farms. |
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