| Avian influenza is a highly contagious disease caused by the avian influenza virus,which spreads quickly and cause great damage,and there is no specific treatment for it.Once infected,birds can only be culled,posing a serious threat to food safety as well as to society and the economy.At present,the effective control method for avian influenza is mainly inactivated vaccine,however,after vaccination,there are still problems of low antibody qualification rate of some immunized poultry groups and poor immunization effect.Insufficient amount of antigen contained in the vaccine is an important cause of the phenomenon.Therefore,accurate detection of the antigen content in inactivated avian influenza virus vaccines is essential.This thesis focuses on the establishing of purification processes and determination methods for intact virus particle content in inactivated avian influenza viral antigen samples of H5N8 and H7N9 subtypes.The primary research objectives are outlined below:(1)The H5N8 inactivated avian influenza virus antigen was studied and characterized and analyzed using high performance size exclusion chromatography(HPSEC).After treatment with specific positive serum antibodies,the characteristic peaks of the virus particles were determined by the change of antigen peaks on the column,and the suitable gel chromatographic column and mobile phase were screened.However,when the detection method was established,impurities were found to interfere with the characteristic peaks.For accurate quantification,the impurity interference at the characteristic peak was first removed by pretreatment of the virus stock solution.The ability of various pretreatment methods such as precipitation,nuclease digestion and chromatography to remove impurities from the inactivated avian influenza virus antigen stock solution was investigated.It was verified by SDS-PAGE and hemagglutination tests that the ion-exchange medium DEAE FF had the best pretreatment effect for purification and had the advantages of fast processing speed,simple operation and not affecting the virus structure.The optimized ion exchange chromatography pretreatment method effectively removed impurity interference at the characteristic peaks of HPSEC detection,and the recovery of virus particles reached 100%.The pretreatment of inactivated avian influenza virus antigen stock solution using a gravimetric column loaded with DEAE FF media under the same purification conditions,and the media still had a good purification effect after 30 repetitions,indicating that the pretreatment method has industrial application prospects.(2)The HPSEC-Multi-angle laser scattering technique(MALLs)rapid detection method was used to study H5N8 inactivated avian influenza virus.The number of intact virus particles in the sample could be accurately obtained by establishing a linear correlation curve after the sample was purified by DEAE FF,diluted in gradient and detected on the HPSEC-MALLs.The number of virus particles showed a good linear relationship with the peak area(R~2=0.997),which can be used for quantitative analysis of H5N8 inactivated virus antigen.The particle size and molecular weight of the sample were analyzed,and the particle diameter of 127.7 nm was detected by dynamic light scattering(DLS),which was consistent with the TEM results;the molecular weight of the virus was2.96×10~5 k Da(±3.74%).The reproducibility and specificity of the established assay were also verified,and the reproducibility of the assay was good(RSD<5%,n=3),and the number of virus particles correlated well with the hemagglutination titer.(3)The established method for quantification of avian influenza virus particle pretreatment was successfully applied in the quantification of H7N9inactivated virus particles,using the method for DEAE FF ion exchange pretreatment of H7N9 inactivated avian influenza virus as well as HPSEC-MALLs quantification.the number of H7N9 inactivated avian influenza virus particles also showed a good linear relationship with the chromatographic peak area(R~2=0.998),indicating that HPSEC-MALLs quantification of inactivated avian influenza intact particle content can be applied to different serotypes of vaccine antigens. |
