单链抗体A7基因真核表达载体的构建  

Construction of eukaryotic expression vector of gene of single chain variable fragment A7

作　　者：李强[1] 魏晶[1] 韩晓敏[1] 李辉[1] 穆永佳[1] 李红花[1] 李英信[1] 孟繁平[1] 

机构地区：[1]延边大学基础医学院免疫学与病原生物学教研部,吉林延吉133000

出　　处：《延边大学医学学报》2008年第1期5-9,共5页Journal of Medical Science Yanbian University

基　　金：国家自然科学基金3046012830760234

摘　　要：[目的]构建抗乙酰胆碱受体主要免疫原区单链抗体A 7基因的真核表达载体,为单链抗体A 7基因的真核表达及基因治疗重症肌无力奠定基础.[方法]应用PCR技术,从已构建的含有抗乙酰胆碱受体主要免疫原区单链抗体A 7基因的重组原核表达载体pHEN 2单链抗体A 7上扩增单链抗体A 7基因并纯化,将纯化后的PCR产物经EcoRⅠ和AvrⅡ双酶切后,用低熔点琼脂糖凝胶电泳回收并纯化,再与经同样酶切并纯化的真核表达载体pPIC 9 K连接,将其产物转化至大肠杆菌DH 5α后扩增,再用AvrⅡ和EcoRⅠ酶切和测定序列检查ScFv A 7基因插入的准确性.[结果]构建pPIC 9 K-ScFv A 7重组载体,经序列测定检查核苷酸序列正确,且ScFv A 7基因准确地克隆至载体开放读码框架内.[结论]成功地构建了抗乙酰胆碱受体主要免疫原区单链抗体A 7基因的真核表达载体.OBJECTIVE To construct a recombinant eukaryoic expression vector of the single chain variable fragment（ScFv） A7 of antibody against the main immunogenic region（MIR） of acetylcholine receptor（AChR） for the further eukaryoic expression and the gene therapy in myasthenia gravis（MG）.METHODSThe ScFv A7 gene was amplified by PCR from the recombinant prokaryotic expression vector pHEN2-ScFvA7,then purified and digested with EcoRⅠand AvrⅡ.The digested products were run in low melting point agarose gel eletrophoresis and purified by Wizard PCR Preps DNA Purification System,then ligated into eukaryotic expression vector pPIC 9K digested and purified by the same method.The recombinant vector pPIC 9K-ScFvA7 was transformed into E.coli DH5α for amplification and isolated and digested with AvrⅡ and EcoRⅠ again.The pPIC 9K-ScFvA7 gene was analysed for an insert of the right size by digestion with AvrⅡ and EcoRⅠ.RESULTSThe sequencing showed that the nucleotide sequence of constructed ScFvA7 was correct and cloned into the open reading frame（ORF） in pPIC9K.CONCLUSIONThe recombinant eukaryoic expression vector pPIC9K-ScFvA7 has been successfully constructed.

关 键 词：重症肌无力 抗体 真核表达载体 

分 类 号：R392.1[医药卫生—免疫学]