抗人红细胞单链抗体基因的构建及表达  

Construction and Expression of Anti-hRBC ScFv

作　　者：操敏[1] 唐家琪[1] 李光富[1] 王长军[1] 潘秀珍[1] 李先富[1] 

机构地区：[1]南京军区军事医学研究所,南京210002

出　　处：《南京师大学报（自然科学版）》2000年第2期81-85,共5页Journal of Nanjing Normal University(Natural Science Edition)

基　　金：国家自然科学基金!资助项目 (396 70 6 45 )

摘　　要：从分泌抗人红细胞单克隆抗体的杂交瘤细胞 3F5中提取总 RNA,逆转录成 c DNA,用合成的寡核苷酸引物通过 PCR扩增和克隆单抗的轻、重链可变区基因 ,用双脱氧核苷酸链终止法分析核苷酸序列 ,采用 PCR拼接法构建单链抗体基因 ,并插入原核表达载体 PEt- 2 8a,转化大肠杆菌BL2 1(DE3) ,经 IPTG诱导后表达 .序列分析表明所克隆的 VL、VH分别属于鼠 Ig Kappa轻链 IV亚组和 Ig重链 亚组 .VH、VL基因均含正确的框架结构 ,构建的单链抗体基因全长 750 bp,在原核系统中的表达产物经 SDS- PAGE分析相对分子量约为 2 80 0 0 .Total RNA of 3F5 hybridoma cell which can secret anti RBC monoclonal antibody was used as template to isolate the variable heavy chain(VH) and variable light chain(VL) with primers through RT PCR. The sequence of VH and VL was analysed by Sanger's methods. The 3F5 ScFv was constructed by linking the VH and VL genes using splicing PCR method and was inserted into the plasmid PEt 28a ,then, the recombinant plasmid transformed the bacteria BL21(DE3) and obtained it's expression after induced by IPTG. The sequence analysis demonstrated that the VL and VH belong to Ig kappa light chain Ⅳ subgroup and heavy chain Ⅱ subgroup. The frame of VH and VL match the murine Ig fuctional framework.The successfully obtained ScFv was 750 bp and the molecure weight of it's expression product was 28 000 analysed by SDS PAGE.

关 键 词：3F5单克隆抗体 单链抗体基因 基因工程 RBC 

分 类 号：R392.11[医药卫生—免疫学] Q78[医药卫生—基础医学]