禽流感病毒H5N1亚型HA蛋白在昆虫细胞中的表达、纯化及反应原性分析  被引量:2

Expression,purification and reactivity analysis of HA protein of Avian influenza virus H5N1 subtype in insect cells

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作  者:仝晓丹 王玉娟 杨仕标 盛东北 赵文华 黄晓静 谭菲菲 王同燕 田克恭 TONG Xiaodan;WANG Yujuan;YANG Shibiao;SHENG Dongbei;ZHAO Wenhua;HUANG Xiaojing;TAN Feifei;WANG Tongyan;TIAN Kegong(National Research Center of Veterinary Medicine,Luoyang 471003.China;Pulike Biological Engineering,Inc,Luoyang 471000,China;Yunnan Animal Science and Veterinary Institute,Kunming 650224,China;Luoyang Putai Biotechnology Co.,Ltd.,Luoyang 471003.China)

机构地区:[1]国家兽用药品工程技术研究中心,河南洛阳471003 [2]普莱柯生物工程股份有限公司,河南洛阳471000 [3]云南省畜牧兽医科学院,昆明650224 [4]洛阳普泰生物技术有限公司,河南洛阳471003

出  处:《黑龙江畜牧兽医》2021年第10期72-76,共5页Heilongjiang Animal Science And veterinary Medicine

基  金:郑洛新国家自主创新示范区创新引领型产业集群项目“畜禽用安全高效新型疫苗研发及产业化”(181200211700);洛阳市河洛英才计划项目(新型兽用生物制品研发及产业化)。

摘  要:为了获得可溶性的禽流感病毒H5N1亚型HA蛋白,并将其用于单克隆抗体的制备和筛选,试验利用Bac-to-Bac杆状病毒表达系统进行HA蛋白的表达和纯化,以含全长HA基因的重组质粒为模板,经PCR扩增得到HA片段并克隆至pFastBac1载体,将鉴定正确的供体质粒转化至DH10Bac感受态细胞,获得Bacmid重组质粒,转染Sf 9细胞后获得重组杆状病毒。通过Western-blot和间接ELISA验证HA蛋白的表达情况和反应原性。结果表明:成功获得含有禽流感病毒H5N1亚型HA基因重组杆状病毒,HA蛋白可以分泌表达,大小约为63 ku,与预期大小一致,且能与H5N1标准阳性血清发生特异性反应;纯化的HA蛋白与免疫小鼠血清反应性良好。说明本研究成功表达并纯化获得了禽流感病毒H5N1亚型HA蛋白,并且具有良好的反应原性。In order to achieve soluble expression of HA protein of Avian influenza virus H5 N1 subtype,which was used for the preparation and screening of monoclonal antibody,the Bac-to-Bac baculovirus expression system was used to express HA protein of Avian influenza virus H5 N1 subtype in this experiment,and the purification was carried out.The recombinant plasmid containing the full-length HA gene was used as the template,HA fragments were amplified by PCR and were cloned into pFastBac1 vector.The correct donor plasmid was identified and was transformed into DH10 Bac competent cells to obtain recombinant plasmid Bacmid.The Bacmid plasmid was transfected into Sf 9 cells for the preparation of recombinant baculovirus.The HA recombinant protein expression and its reactivity were verified by Western-blot and indirect ELISA.The results showed that recombinant baculovirus containing H5 N1 HA was successfully obtained.The HA protein can be expressed in secreted form,with a size of about 63 ku,which was consistent with the expected size,and can react specifically with the standard positive H5 N1 serum.The purified HA protein reacted well with the serum of immunized mice.The results suggested that H5 N1 HA protein was successfully expressed and purified,and had good reactivity.

关 键 词:禽流感病毒 血凝素蛋白 杆状病毒表达系统 蛋白纯化 免疫印迹分析 反应原性分析 Sf 9细胞 

分 类 号:S852.62[农业科学—基础兽医学] Q78[农业科学—兽医学]

 

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