Purification And Monoclonal Antibody Peparation Of H9N2 HA Protein Expressed In Rice And Immunoassay Method Establishment

H9 subtype avian influenza virus was widely spread worldwide and had evolved multiple genotypes in poultry.In China,according to the National Influenza Center’s surveillance of avian influenza viruses from 2014 to 2019: H9N2 had replaced H5N6 and H7N9 as the dominant avian influenza virus subtype in chicken and duck populations.When secondary infections with other pathogenic bacteria caused by low immunity in poultry,it leading to a decrease in production performance and an increase in mortality,causing huge economic losses to the poultry farming industry.In addition,human infections with H9 subtype avian influenza virus had occurred,posing a potential threat to public health.The monitoring of antibody levels is an effective method to evaluate the effectiveness of vaccine immunization and to prevent morbidity.The monoclonal antibody to AIV H9 is a material to study the pathogenic mechanism of the virus and to establish the basis of virus detection method.Therefore,the research on the preparation of antibody test strips and monoclonal antibody to AIV subtype H9 is of great significance.In this study,we used rice seeds expressed H9N2 HA protein as the material and initially developed the purification process of recombinant HA protein.On this basis,purified HA protein was immunized with 6-8 weeks old Balb/c mice and the monoclonal antibodies of H9 AIV were prepared by hybridoma technology.Secondly we used colloidal gold-labeled recombinant HA protein as the probe,rabbit antichicken IgG and H9N2 subtype of multiple antibodies as the detection line and quality control line,respectively.Combining with HI detection method of highly pathogenic avian influenza,the test paper for AIV H9 antibody was established.1.Purification process of recombinant H9N2 HA protein in riceRice powder containing r HA was extracted with optimum extraction buffer by ratio of 1:5 and Stirred for 1.5 h.The HA crude extract was obtained after centrifugation and filter filtration.After screening by anionic column,hydrophobic column,cationic column,different order,finally the high-purity HA protein was obtained by three-step chromatography.27% elution by Q column,HA protein was enriched and concentrated;100% elution by Butyl column,most of the heteroproteins were removed;12% elution by S column,the high-purity HA protein was finely purified.SDS-PAGE and Western-blot showed that the purity of HA protein was above 85% after the three-step chromatography.2.Preparation of AIV H9 monoclonal antibodyThe purified r HA was emulsified with ISA 50 V adjuvant at ratio of 1:1 and immunized at a dose of 10 μg per mouse in 6-8 week old Balb/c mice.Immunization every two week,mice with the highest potency were superimmunized and fused after the serum were detected by indirect ELISA.6 cell lines were obtained after multiple subcloning.All six antibody light chains were Kappa;Specificity identification assay showed that the ascites of the six strains did not react with H5,NDV and negative control;Antibody isotype identification tests indicated that 5A5 isotype was IgG2 a,the isotype of 3A5 4E8 8G8 11E3 12E5 were IgG1 and all m Abs light chain type are Kappa.Cell neutralization assay showed that 3A5,4E8 and 8G8 had neutralizing activity;all monoclonal antibodies had hemagglutination inhibition;IPMA showed that all antibodies showed specific reaction to H9N2 virus.3.Development antibody test strips for H9 AIVA test strip method was established for detecting H9 AIV antibody with using colloidal gold labeled HA protein as probe,rabbit anti-chicken IgG and HA polyclonal antibody as detection line and quality control line respectively,and according to the HI diagnostic technical standard.A modified test suggested the content of colloidal gold labeled HA protein was 30 mg·L^-1.The optimal coating concentrations of rabbit anti-chicken IgG and HA polyclonal antibody were 1 g·L^-1and 1.2 g·L^-1 respectively.The test strip was detected for the specificity,sensitivity,stability and coincidence rate.The results showed that test strip only reacted with H9 positive serum,but did not cross react with H5 AIV,Newcastle disease virus（NDV）,infectious bursa disease virus（IBDV）,Newcastle disease virus（NDV）,Marek’s disease virus（MDV）positive serum and H9 negative serum,The minimum limit on the test strip was 1:12 800,while HI was 10log2 for H9N2 subtype positive serum,It can be stored at room temperature for at least 6 months.The total coincidence rate was 90.6% between the test paper and HI test,and the Kappa value was 0.759.In conclusion,it is a simple,rapid,accurate and specific semi-quantitative immunochromatographic strip,which can be preliminarily used for the clinical detection of H9 subtype antibodies.