LTB融合禽流感病毒M2e基因在毕氏酵母中表达.docVIP

LTB融合禽流感病毒M2e基因在毕氏酵母中表达 　　摘要: 禽流感病毒一直是家禽和家畜健康养殖的巨大威胁，之前的研究表明，原核系统中表达的LTB和M2eHBc+融合基因的产物能有效诱导免疫动物对禽流感病毒M2e多肽产生特异性的粘膜免疫应答.酵母作为生物反应器应用于生物制品生产具有独特的优点.本研究构建了pPIC9K-LBM2eHBc+ 酵母表达载体并对酵母GS115进行了遗传转化，甲醇诱导表达结果表明LBM2eHBc+融合基因在酵母细胞中得到表达，表达出的融合蛋白能够被M2抗体识别. 　　关键词: 禽流感病毒；LTB；M2e表位；毕氏酵母;表达 　　中图分类号:Q786 　　 文献标识码:A文章编号:1672-8513(2010)03-0193-05 　　Expression and Construction of Yeast Expression Vector of 　　LBM2eHBc+ gene in ?P.pastoris? 　　ZHANG Guo?guang1，2，ZHANG Hui?huang?1，DONG Mei?1，CHEN Liang?1 　　 (1. Key Laboratory of Ministry of Education for Cell Biology and Tumor 　　Cell Engineering，School of Life Sciences，Xiamen University，Xiamen 361005，China; 2.Biology Department，Zhangzhou Normal University，Zhangzhou 363000，China) 　　Abstract: 　　Avian influenza virus (AIV) has been a constant threat to the healthy development of livestock and poultry breeding industry. Previous research has shown that the products of LTB and M2eHBc+ fused gene expressed in prokaryotic cells can effectively induce mice mucosal immune responses against M2e epitope. ?P.pastoris? yeast has a unique advantage as a bioreactor used in the production of biological products. LBM2eHBc+ fused gene fragment was obtained by PCR，then inserted into pPIC9K plasmid which contains methanol promoter and yeast signal peptide to construct yeast expression vector pPIC9K-LBM2eHBc+. The recombinant vector was transformed into ?P.pastoris? GS115. The methanol induction result has indicated that the LBM2eHBc+ fusion gene can be efficiently expressed in GS115 cell and the expressing protein has the specific binding activity to M2 antibody by Western blotting analysis. 　　Key words: avian influenza virus; LTB; M2e epitope; ?P.pastoris;? expression 　　禽流感（Avian Influenza，AI）是由正粘病毒科A型流感病毒引起的一种以侵害呼吸系统为主的接触性传染病，广泛分布于世界各地，每次爆发都给疫区养禽业带来毁灭性打击，而且1997年还发现一些高致病性的亚型可以传染人［1］.目前接种疫???是控制AIV流行的主要措施之一，异源表达的高度保守的M2蛋白能为动物提供很好的免疫保护［2-3］.M2蛋白N端24个氨基酸残基构成囊膜外区域（M2e），M2e是M2蛋白的中和抗体诱导表位，基于M2e的单克隆抗体能有效抑制流感病毒在体外培养细胞和小鼠体内的复制［4-5］，但是M2e只有24个氨基酸，不能有效刺激机