抗大田软海绵酸单链抗体基因的克隆、序列分析与表达  

Cloning sequence analysis and expression of ScFv against okadaic acid

作　　者：卢士英[1] 孟宪梅[1] 周玉[1] 宫彬彬[1] 任洪林[1] 柳增善[1] 

机构地区：[1]吉林大学人兽共患病研究所人兽共患病教育部重点实验室,长春130062

出　　处：《食品科技》2009年第6期2-7,共6页Food Science and Technology

基　　金：国家自然基金项目(30671762);国家质检总局项目(2003IK044;2008IK003)

摘　　要：为利用大田软海绵酸单链抗体建立检测方法,提取分泌抗大田软海绵酸单克隆抗体的3C5杂交瘤细胞株总RNA,用鼠源单链抗体可变区引物RT-PCR法扩增单克隆抗体可变区基因VH和VL,用一段柔性肽链-(Gly4Ser)3将其连接成单链抗体基因ScFv。克隆后测序,NCBI Igblast及ProtParam进行分析;结果获得一开放读框为726 bp的ScFv基因的克隆子,包括366 bp的VH、45 bp的Linker和315 bp的VL,序列中有明确的框架区(FR)和互补决定区(CDR);共编码242个氨基酸,在重链的第24、98位和轻链的23、87位氨基酸为抗体可变区特征性的半胱氨酸残基;经检索分析,所获序列与多种鼠源单链抗体基因同源性很高,具有重组功能性鼠抗体可变区基因的特征,申请GenBank登录号为EU574932,经原核表达其产物经ELISA检测具有与OA结合的活性。OA单链抗体基因的获得为开发OA等海生物毒素检测方法提供了新的思路,并为利用融合单链抗体同时快速筛检多种海洋生物毒素的检测方法提供重要的实验材料。In order to establish the detection assay for okadaic acid based on the single chain antibody, the total RNA from the hybridoma cell 3C5 which secrete anti-okadaic acid antibody was abstracted. The V. and VL were amplified by RT-PCR with the primer which desighed by the variable region of monoclonal antibody from mouse and were connected by a flexible linker-（GGGGS）3 with the technique of SOE-PCR. The ScFv was sequenced after being cloned in the pMD-18T and compared with those published genes in national center for biotechnology information（NCBI） by Igblast. The results showed that one clone of 726 bp was obtained including 366 bp VH, 45bp linker and 31 5bp VL, and they were capable of encoding 242 amino acids. The deduced amino acid sequences contained definite FRs and CDRs. There were two cysteine residues of the VH and VL respectively necessary for the maintenance of the antibody structure. This indicate that they were functionally rearranged mouse antibody. The GenBank accession is EU574932. The prokaryotic expression product of the gene has the binding activity with OA by ELISA. The preparation of OA-ScFv will develop a original detection assay for Diarrhetic shellfish toxin and provid subject for using the fusion ScFv to establish the rapid detection assay which can screen the three shellfishtoxins simultaneously.

关 键 词：大田软海绵酸 单链抗体 基因克隆 序列分析 原核表达 

分 类 号：Q78[生物学—分子生物学]