H9N2亚型禽流感病毒纯化及病毒HA蛋白定量方法研究

doi:10.16431/j.cnki.1671-7236.2019.07.023

Abstract

Abstract:

In order to control the upstream and downstream processes of H9N2 subtype avian influenza virus (AIV) vaccine,the methods for purification of H9N2 subtype AIV and quantification of viral HA protein were established by combining sucrose density gradient centrifugation and SDS-PAGE gray analysis.Firstly,the harvested viruses were clarified,concentrated and purified by differential centrifugation,PEG6000 precipitation and sucrose density gradient centrifugation,respectively.Subsequently,the virus centrifugal zone formed after centrifugation was collected by HPLC,and identified by SDS-PAGE and Western blotting.The linearity and repeatability of the collection method were also investigated.On this basis,the clarification process of viruses was optimized to improve the virus recovery.Finally,the degree of co-migration of viral protein bands of purified H9N2 subtype AIV isolated by reduced SDS-PAGE was observed and the optimal condition for PNGase F deglycosylation treatment was decided.The gray of four main viral protein bands (NP,HA1,M1 and HA2) in SDS-PAGE was analyzed by Image J software to determine the content of hemagglutinin.The results showed that the protein concentration of the virus centrifugal zone collected by HPLC had a good linear relationship with the volume of virus supernatant in the range of 8 to 32 mL (R²=0.994).The intra-batch and inter-batch coefficients of variation were 1.29% and 4.11% respectively,which indicated a good repeatability of this collection method.After the optimization of clarification process,the final recovery of virus was 79.55%.When the protein concentration of H9N2 subtype AIV was 1 000 μg/mL,the SDS-PAGE showed clear and well-separated viral bands after deglycosylation treatment with PNGase F.The calculated total HA accounted for 46.18% of total viral proteins by SDS-PAGE gray analysis.This study established methods for purification of H9N2 subtype AIV and quantification of viral HA protein,providing a simple and accurate detection method for the development and production of H9N2 vaccine in the future.

Key words: H9N2 subtype AIV; hemagglutinin; sucrose density gradient centrifugation; purification; SDS-PAGE; quantification

CLC Number:

S855.3

PENG Chengcheng, WU Yixiao, LIU Xuping, TAN Wensong. Study on Methods for Purification of H9N2 Subtype Avian Influenza Virus and Quantification of Viral HA Protein[J]. , 2019, 46(7): 2069-2078.

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Study on Methods for Purification of H9N2 Subtype Avian Influenza Virus and Quantification of Viral HA Protein

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