B型流感病毒血凝素的表达及免疫原性测定  

Expression of influenza B virus hemagglutinin and its immunogenicity determination

作　　者：杨晨 耿小宇 原恺 张娟琨[1] 校海霞[2] YANG Chen;GENG Xiaoyu;YUAN Kai;ZHANG Juankun;XIAO Haixia(College of Biotechnology,Tianjin University of Science and Technology,Tianjin 300457,China;Tianjin Institute of Industrial Biotechnology,Chinese Academy of Sciences,Tianjin 300308,China;Shanxi Academy of Advanced Research and Innovation,Taiyuan 030032,Shanxi,China)

机构地区：[1]天津科技大学生物工程学院,天津300457 [2]中国科学院天津工业生物技术研究所,天津300308 [3]山西高等创新研究院,山西太原030032

出　　处：《生物工程学报》2022年第3期1112-1123,共12页Chinese Journal of Biotechnology

基　　金：国家重点研发计划(2018YFC1200603)。

摘　　要：B型流感病毒是引起季节性流感的原因之一,严重时会造成重大疾病或死亡。为了检测B型流感病毒2个疫苗候选毒株的血凝素(hemagglutinin,HA)蛋白胞外段在哺乳动物细胞中的表达及在小鼠体内的免疫原性,本研究将带有三聚体标签的HA胞外段(HA-ectodomain,HA-ecto)序列及神经氨酸酶(neuraminidase,NA)全长编码框经密码子优化后构建至pCAGGS载体中,通过线性聚乙烯亚胺将pCAGGS-HA-ecto与pCAGGS-NA共转染293T细胞。收集转染后96h的上清,通过镍离子亲和层析及分子筛层析获得三聚体形式的HA-ecto蛋白,然后将HA-ecto三聚体蛋白免疫小鼠,进行酶联免疫吸附试验(enzyme linked immunosorbent assay,ELISA)及血凝抑制实验(hemagglutination inhibition,HAI)检测HA-ecto蛋白诱导小鼠后产生的抗体水平。纯化结果显示,通过哺乳动物细胞表达系统能够得到分泌型表达的三聚体HA-ecto蛋白。ELISA及HAI结果显示,三聚体HA-ecto蛋白二次免疫小鼠后,能诱导小鼠产生较高水平的同源和异源交叉抗体。以上结果表明,哺乳动物细胞表达的B型流感病毒HA蛋白可作为亚单位重组流感疫苗的候选。Influenza B virus is one of the causes for seasonal influenza,which can account for serious illness or even death in some cases.We tested the expression of extracellular domain of hemagglutinin(HA-ecto)of influenza B viruses in mammalian cells,and then determined the immunogenicity of HA-ecto in mice.The gene sequence encoding influenza B virus HA-ecto,foldon sequence,and HIS tag was optimized and inserted into pCAGGS vector.The opening reading frame(ORF)of neuraminidase was also cloned into pCAGGS.The pCAGGS-HA-ecto and pCAGGS-NA were co-transfected into 293 T cells using linear polyethylenimine.Cell supernatant after transfection was collected after 96 h,and the secreted trimmeric HA-ecto protein was purified by nickel ion affinity chromatography and size exclusion chromatography.Subsequently,the mice were immunized with HA-ecto protein,and the corresponding antibody titers were detected by ELISA and hemagglutination inhibition(HAI)assays.The results showed that soluble trimeric HA-ecto protein could be obtained using mammalian cell expression system.Moreover,trimeric HA-ecto protein,in combination with the adjuvant,induced high levels of ELISA and HAI antibodies against homogenous and heterologous antigens in mice.Thus,the soluble HA-ecto protein expressed in mammalian cells could be used as a recombinant subunit vaccine candidate for influenza B virus.

关 键 词：B型流感病毒 血凝素蛋白 哺乳动物细胞表达系统 疫苗候选毒株 

分 类 号：R392-33[医药卫生—免疫学]