猪囊尾蚴排泄分泌抗原M13h基因的真核表达质粒的构建及表达分析  

Construction and expression of eukaryotic expression vector containing M13h ES antigen gene of Cysticercus cellulosae

作　　者：赵光辉[1,2,3] 鲁琨[1,2] 张改平[2] 宁长申[1] 王选年[2,4] 张龙现[1] 菅复春[1] 宋海涛[1,2] 

机构地区：[1]河南农业大学牧医工程学院,河南郑州450002 [2]河南省农业科学院河南省动物免疫重点实验室,河南郑州450002 [3]华南农业大学兽医学院,广东广州510642 [4]河南科技学院动物科学学院,河南新乡453003

出　　处：《河南农业大学学报》2009年第2期168-172,共5页Journal of Henan Agricultural University

基　　金：国家“863”计划项目(2003AA249030)

摘　　要：根据GenBank中的参考序列设计并合成引物序列，从pMDM13h中扩增带酶切住点和His—tag的基因序列；用HindⅢ和XhoⅠ双酶切PCR扩增产物和pcDNA3．0载体，连接构建真核表达重组质粒pc3M13h，利用PCR，双酶切和测序鉴定其正确性；重组质粒pc3M13h转染COS-7细胞，瞬时表达的转染细胞（96孔板）在转染36．48h后，应用双抗体夹心酶联免疫吸附试验和免疫细胞化学，分别检测pc3M13h在COS-7细胞培养上清液和细胞内的表达．结果表明，设计合成的引物成功扩增了带有酶切位点和His—tag的基因序列；成功构建了真核表达重组质粒pc3M13，双抗体夹心酶联免疫吸附试验和免疫细胞化学检测显示，pc3M13h重组质粒不但能够在COS-7细胞中进行表达，而且能够进行微量的分泌表达．One pair of primers sequence was designed and synthesized according to reference sequences in GenBank and used to amplify gene with restriction enzyme sites and His-tag from pMDM13h. Amplified products of PCR and pcDNA3.0 vector were digested by Hind Ⅲ and Xho Ⅰ and the digested products were linked to construct eukaryotic expression vector pc3M13h which was identi- fied by double enzymes digested, PCR and sequencing. The recombinant plasmid was then transfected into COS-7 cell to do transient expression. The transfected cells 36 - 48 h after the transfection were detected by immuocytochemistry （ICC） and its cultural supernatant was detected by two antibodies sandwich ELISA. The results indicate that gene sequence with enzyme sites and His-tag was amplified by the designed primers. The eukaryotic expressing plasmid pc3M13h was successfully constructed through identification of PCR, enzyme digested and sequencing. Detection of pc3M13h in culutural supernatant and in cell of COS-7 cell using two antibodies sandwich ELISA and ICC indicates that the recombinant pc3m13h not only can express in COS-7 cell, but also can microcrystallinely express and secrete into cultural supernatant.

关 键 词：猪囊尾蚴 排泄分泌抗原 基因表达 

分 类 号：S852.4[农业科学—基础兽医学]