重组人宋内痢疾杆菌脂多糖抗独特型抗体重链可变区基因的高效表达及纯化  

High level expression and purification of recombinant heavy chain variable region of human anti idiotypic antibody against LPS of Shigella sonnei

作　　者：刘怀田[1,2] 黄策[1,2] 王海涛[1,2] 俞晓峰 叶棋浓[1,2] 

机构地区：[1]军事医学科学院微生物流行病研究所 [2]生物工程研究所

出　　处：《中华微生物学和免疫学杂志》1997年第6期453-456,共4页Chinese Journal of Microbiology and Immunology

基　　金：国家自然科学基金

摘　　要：用聚合酶链反应（ＰＣＲ）从人源噬菌体抗体库中筛选出的宋内痢疾杆菌脂多糖抗独特型抗体基因扩增了重链可变区基因，将其克隆到表达载体ｐＧＥＸ２Ｔ内，转化大肠杆菌ＤＨ５α，获得高效表达，表达量占全菌总蛋白质的４０％左右，表达出的融合蛋白分子量为４×１０４，以包涵体形式存在。将此融合蛋白包涵体变性、复性后通过谷胱甘肽亲和层析柱（ＧＳＴＳｅｐｈａｒｏｓｅ４Ｂ柱），得到电泳纯的融合蛋白，每升培养物能纯化到２０～３０ｍｇ融合蛋白。Ｗｅｓｔｅｒｎｂｌｏｔ分析证明宋内痢疾杆菌脂多糖鼠单抗（５Ｂ１２）能特异地结合到分子量４×１０４的融合蛋白的带上。ＥＬＩＳＡ试验显示融合蛋白与宋内痢疾杆菌脂多糖单抗（５Ｂ１２）有结合活性，而与无关单抗，如大肠杆菌Ｊ５株单抗（Ｅ３）、炭疽杆菌单抗（Ｆ８ＦＡ）、鼠伤寒沙门氏菌单抗（Ｍ４６７）及乙肝病毒单抗（Ｍ１１）不结合。The gene of heavy chain variable region(Hv) of human anti idiotypic antibody against Shigella sonnei biopanned from phage antibody libraries was amplified by PCR.The Hv gene was cloned into an expression vector pGEX2T and transformed into E.coli DH5α.Clones harboring plasmid pGEX Hv produced a 40 kDa of fusion protein in E.coli at a level of 40% of the total cellular protein.After denatured and renatured,the fusion protein from inclusion body was purified to be electrophoretically pure by Glutathione Sepharose 4B affinity chromatography with yields of between 20￣30μg/ml of culture.Western blot analysis of the fusion protein showed that 5B12 McAb(anti LPS of Shigella sonnei )specifically bound to the 40kDa band.The expressed products in E.coli demonstrated significant binding activity with 5B12 McAb(anti LPS of Shigella sonnei )and non binding activity with E3 McAb(anti E.coli J5),F8FA McAb(anti B.anthracis) ,M467 McAb(anti S.typhimurium) and M11 McAb(anti HBs) in ELISA.

关 键 词：宋内痢疾杆菌 脂多糖 抗独特型抗体 基因表达 

分 类 号：R378.25[医药卫生—病原生物学] R392.13[医药卫生—基础医学]