H1N1型流感病毒血凝素蛋白胞外段的真核表达及免疫原性分析  

Eukaryotic expression and immunogenicity of extracellular segment of hemagglutinin protein of influenza H1N1 virus

作　　者：刘京 年悬悬 龚铮 吕传硕 邓涛 马宁 周蓉 张国梅 乐洋 张哲罡 刘博 张家友[1,2] 李妍玲 王鹏飞 杨晓明 LIU Jing;NIAN Xue-xue;GONG Zheng;LV Chuan-shuo;DENG Tao;MA Ning;ZHOU Rong;ZHANG Guo-mei;LE Yang;ZHANG Zhe-gang;LIU Bo;ZHANG Jia-you;LI Yan-ling;WANG Peng-fei;YANG Xiao-ming(Second Laboratory of Viral Vaccine Research,Wuhan Institute of Biological Products,Wuhan 430207,Hubei Province,China;不详)

机构地区：[1]武汉生物制品研究所病毒性疫苗研究二室,湖北武汉430207 [2]国家联合疫苗工程技术研究中心,湖北武汉430207 [3]山东省青岛市即墨区中医医院,山东青岛266220 [4]山东省青岛市即墨区七级卫生院,山东青岛266220 [5]中国生物技术股份有限公司,北京100029

出　　处：《中国生物制品学杂志》2021年第12期1401-1405,1411,共6页Chinese Journal of Biologicals

基　　金：“重大新药创新”科技重大专项(2016ZX09106003-008)。

摘　　要：目的真核表达H1N1型流感病毒血凝素(hemagglutinin,HA)蛋白胞外段,并分析其免疫原性。方法以H1N1型流感毒株基因组为参考序列,将H1N1 HA的胞外段氨基酸序列进行密码子优化后基因合成,将合成的目的基因片段与KS001载体连接,构建重组质粒KS001/HA,转染至Expi293F真核细胞,收集表达产物,进行12%SDSPAGE、Western blot及N-糖基化鉴定;将表达产物经Capto Q离子交换层析柱纯化,收集纯化产物进行12%SDSPAGE鉴定,BCA法测定蛋白浓度;用不同浓度的HA胞外段蛋白辅以佐剂免疫小鼠,通过病毒微量中和试验(microneutralization test,MNT)检测小鼠血清抗体效价。结果经菌液PCR、双酶切及测序鉴定证明质粒构建正确;表达的重组蛋白以单体形式存在,相对分子质量约70000,N-糖基化丰富;重组蛋白与佐剂配伍后的各剂量组均产生针对H1N1型病毒特异性抗体,且阳转率≥90%。结论成功构建了重组质粒KS001/HA,并于真核细胞中表达,表达产物联合佐剂免疫小鼠后具有较好的免疫原性。Objective To express the extracellular segment of hemagglutinin(HA)protein of influenza H1 N1 virus in eukaryotes cells and analyze its immunogenicity.Methods Using the genome of H1 N1 influenza strain as the reference sequence,the target gene was synthesized by codon optimization of amino acid sequence of extracellular segment of HA,and cloned into vector KS001.The constructed recombinant plasmid KS001/HA was transfected to eukaryotic Expi293 F cells,and the expressed product was harvested and analyzed by 12%SDS-PAGE,Western blot and N-glycosylation,then purified by Capto Q ion exchange column.The purified product was collected,identified by 12%SDS-PAGE,and determined for concentration by BCA method.Mice were immunized with different concentrations of HA extracellular proteins supplemented with adjuvant,of which the serum antibody titer was determined by microneutralization test(MNT).Results PCR,restriction analysis and sequencing proved that recombinant plasmid KS001/HA was constructed correctly.The recombinant protein existed in a form of monomer with a relative molecular mass of about 70000 and abundant N-glycosylation.The recombinant protein at various dosages supplemented with adjuvant induced specific antibodies against H1 N1 virus,with a positive conversion rate of not less than 90%.Conclusion Recombinant plasmid KS001/HA was successfully constructed and expressed in eukaryotic cells.The recombinant protein supplemented with adjuvant showed good immunogenicity in mice.

关 键 词：流感病毒 血凝素 胞外段 真核表达 免疫原性 

分 类 号：Q786[生物学—分子生物学] R392[医药卫生—免疫学]