水稻源禽流感病毒H9N2亚型HA蛋白两种纯化方法的建立  

Establishment of Two Purification Methods of Avian influenza virus H9N2 Subtype HA Protein from Rice(Oryza sativa)

作　　者：牛香香 李雪洋 许倩茹 李青梅[4] 王雅楠[5] 张申立 马强[6] 张二芹 张改平[1,2] NIU Xiang-Xiang;LI Xue-Yang;XU Qian-Ru;LI Qing-Mei;WANG Ya-Nan;ZHANG Shen-Li;MA Qiang;ZHANG Er-Qin;ZHANG Gai-Ping(College of Veterinary Medicine,Henan Agricultural University,Zhengzhou 450046,China;International Associated Research Center of National Animal Immunology,Zhengzhou 450046,China;College of Veterinary Medicine,Northwest A&F University,Yangling 712100,China;Henan Provincial Key Laboratory of Animal Immunology,Henan Academy of Agricultural Sciences,Zhengzhou 450002,China;College of Veterinary Medicine,Jilin University,Changchun 130062,China;Institution of Animal Science&Veterinary Medicine,Henan Academy of Agricultural Sciences,Zhengzhou 450002,China)

机构地区：[1]河南农业大学动物医学院,郑州450046 [2]国家动物免疫学国际联合研究中心,郑州450046 [3]西北农林科技大学动物医学院,杨凌712100 [4]河南省农业科学院动物免疫学重点实验室,郑州450002 [5]吉林大学动物医学院,长春130062 [6]河南省农业科学院畜牧兽医研究所,郑州450002

出　　处：《农业生物技术学报》2021年第7期1237-1247,共11页Journal of Agricultural Biotechnology

基　　金：转基因生物新品种培育重大专项(2016ZX08001006-10)。

摘　　要：血凝素(hemagglutinin,HA)蛋白是禽流感病毒(Avian influenza virus,AIV)表面的一种糖蛋白,能诱导机体产生中和抗体,也是AIV主要的保护性抗原。已在水稻(Oryza sativa)胚乳中成功表达禽流感病毒H9N2 HA蛋白。为了建立水稻源禽流感病毒H9N2亚型HA蛋白的纯化方法,本研究分别采用两种方法进行HA蛋白的纯化。方法一:将HA蛋白水稻粉末按照质量与体积1∶5比例加入到提取液中,室温下搅拌,离心、取上清液,过滤后,首先经Q阴离子层析捕获到HA粗提蛋白,其次利用HA蛋白抗体亲和层析精细纯化HA蛋白,收集洗脱液,检测目的蛋白;方法二:用与方法一同样的方法获得HA粗提蛋白,根据Butyl疏水填料特性,HA粗提蛋白液中加入硫酸铵,搅拌、离心和过滤,进行第2步纯化,将收集到的洗脱液浓缩、换液,再经过SP阳离子层析纯化,收集洗脱液,获得HA蛋白。将两种方法纯化的HA蛋白经过SDS-PAGE和Western blot检测蛋白的纯度,并将HA蛋白送公司测序。结果表明,方法一:Q阴离子层析与HA免疫抗体亲和层析两种组合纯化HA蛋白的纯度约为90%左右。该组合纯化蛋白的方法相对简单,但需要培养和纯化单克隆抗体,此方法适合实验室快速小规模的蛋白纯化;方法二:Q阴离子层析、Butyl疏水层析和SP阳离子层析3种组合纯化的HA蛋白纯度达到90%左右。该方法适合建立HA蛋白的纯化工艺,用于大规模纯化HA蛋白。这两种方法获得的HA蛋白经测序与已知的氨基酸序列一致。本研究首次建立2种从水稻中获得高纯度重组H9N2亚型HA蛋白的方法,为今后制备禽流感H9N2亚型亚单位疫苗提供基础资料。Hemagglutinin(HA) protein is a kind of glycoprotein on the surface of Avian influenza virus, which is a main protective antigen inducing neutralizing antibody. Avian influenza virus H9N2 HA gene was successfully expressed in rice(Oryza sativa) endosperm. In order to establish a purification method for H9N2 Avian influenza virus HA protein expressing in rice, two methods were used in this study to purify HA protein.Method 1: Rice powder expressing HA protein was added into the extract in a ratio of 1∶5 mass to volume,stirred at room temperature, centrifuged and supernatant was taken. After filtration, crude HA protein was firstly captured by Q anion chromatography. Subsequently, HA protein was purified by HA protein antibody affinity chromatography, eluent was collected and the target protein was detected. Method 2: Crude HA protein solution was obtained in the same way as method 1. According to packing property of the Butyl hydrophobic, ammonium sulfate was added to HA protein solution, which was then stirred, centrifuged and filtered for a second stage of purification. The collected eluent was concentrated and changed, and purified by SP-cationic chromatography, and the eluent was collected to obtain the HA protein. The HA protein purified by the 2 methods was tested by SDS-PAGE and Western blot, and the HA protein was sent to the company for sequencing. The results showed that the purity of HA protein was about 90% by Q anion chromatography combined with HA affinity chromatography. The method of purification was relatively simple, but monoclonal antibodies need to be cultured and purified. This method was suitable for rapid and small-scale protein purification in laboratory. The purity of HA protein was about 90% by Q anion chromatography, Butyl hydrophobic chromatography and SP cationic chromatography. This method is suitable for establishing the purification process of HA protein and is used to purify HA protein on a large scale. The HA protein obtained by these two methods was sequenced and the result was

关 键 词：转基因水稻 HA蛋白 离子交换层析 疏水层析 亲和层析 

分 类 号：S851.3[农业科学—预防兽医学]