H9N2亚型禽流感病毒纯化及病毒HA蛋白定量方法研究

为实现H9N2亚型禽流感病毒（avian influenza virus，AIV）疫苗上下游过程的控制，本研究结合蔗糖密度梯度离心和SDS-PAGE灰度分析建立了H9N2亚型AIV纯化和病毒HA蛋白定量的方法。首先将收获的病毒原液经差速离心、PEG6000沉淀和蔗糖密度梯度离心分别进行澄清、浓缩和分离纯化；其次，采用改装过的高效液相色谱仪系统（HPLC）准确定位和收集病毒离心区带，并对该区带进行SDS-PAGE和Western blotting分析，同时考察该收集方式的线性和重复性；然后，在此基础上优化病毒液的澄清工艺以提高病毒回收率；最后，观察还原SDS-PAGE分离的H9N2亚型AIV蛋白条带的共迁移情况并确定糖苷酶PNGase F脱糖基处理的最佳条件，采用Image J软件分析SDS-PAGE图谱中4个主要病毒蛋白条带（NP、HA1、M1和HA2）的灰度以确定流感病毒血凝素的含量。结果表明，HPLC收集的病毒离心区带的蛋白浓度与PEG6000浓缩的上清体积在8~32 mL范围内具有良好的线性关系（R²=0.994），且该收集方式重复性好，批内和批间变异系数分别为1.29%和4.11%。病毒液经过澄清、浓缩和分离纯化后，最终的病毒回收率为79.55%。纯化的H9N2亚型AIV的蛋白浓度为1 000 μg/mL时，经糖苷酶PNGase F脱糖基处理后便能得到条带清晰平整且分离良好的SDS-PAGE图谱。经灰度分析，HA含量占总病毒蛋白含量的46.18%。本研究初步建立了H9N2亚型AIV纯化和病毒HA蛋白定量的方法，为H9N2亚型AIV全病毒灭活疫苗的研发和生产提供了简单、准确的检测手段。

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

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.