抗重组志贺样毒素ⅡA亚单位单链抗体的构建及表达  

Construction and expression of anti-SLT 2A scFv gene in E. coli

作　　者：毛旭虎[1] 马颖[1] 陈洪章[1] 邹全明[1] 

机构地区：[1]第三军医大学医学检验系临床微生物学及免疫学教研室重庆市生物制药工程技术研究中心,重庆400038

出　　处：《免疫学杂志》2006年第4期374-376,380,共4页Immunological Journal

基　　金：军队杰出人才基金资助项目(04J009)

摘　　要：目的构建重组志贺样毒素ⅡA亚单位(SLT2A)单链抗体基因(scFv),并在大肠杆菌中进行分泌表达。方法通过连接肽(Gly4Ser)3将已成功扩增的抗SLT2A单克隆抗体(mAb)5F3的VL和VH连接成scFv基因VHLinkerVL,并在VH基因5′端和VL基因3′端引入SfiⅠ和NotⅠ的酶切位点,克隆至经改造的分泌性表达载体pCANTAB6His中,转化EcoliHB2151,IPTG诱导表达。表达产物经亲和层析纯化后,SDSPAGE和Westernblot分析鉴定。结果经限制性酶切鉴定及DNA测序分析证实,基因构建正确。SDSPAGE和Westernblot分析表明scFv基因在大肠杆菌中得到表达,表达产物的相对分子质量为27000,与理论预期值相符,且可与相应抗原特异结合。结论成功地实现了抗SLT2AscFv基因的原核分泌表达,为探讨O157菌感染的机制及防治研究奠定了基础。Objective To construct and express the gene of scFv against recombinant SLT 2A in E. coli. Methods The V, and VL genes cloned from mAb 5F3 were ligated with a flexible linker （Gly4Ser）3 to construct scFv gene, and then corresponding restriction endonuclease digestion site was introduced into 5′and 3′ ends of the constructed gene. The scFv gene was cloned into an expression vector pCANT-AB6His and expressed in E. coli HB2151. Expressed protein was purified by affinity chromatography and detected by SDS-PAGE and Western blotting. Results DNA sequence analysis proved that scFv gene was correctly cloned into the expression vector. SDS-PAGE and Western blot analysis showed that scFv was successfully expressed in Ecoli HB2151 and the expressed protein had specific antigen-binding activity. The relative molecular mass （M1） of the protein was 27 000, which was corresponding to prediction. Conclution The successful expression of antiSLT2A scFv gene in E. coli lays a solid foundation for its further application in diagnosis and therapy of 0157 infection.

关 键 词：志贺样毒素 单链抗体 分泌表达 

分 类 号：Q78[生物学—分子生物学]