抗肠炎沙门氏菌单链抗体制备及其特异性分析  

Preparation and Specificity Analysis of scFv Aganinst Salmonella enteritidis

作　　者：王报贵[1] 武晓丽[2] 董素琴[1] 甘敏[1] 陈星星[1] 陈飞[1] 明星[1] 徐锋[1] 

机构地区：[1]南昌大学生命科学与食品工程学院中德联合研究院,南昌330047 [2]江西中医学院基础医学部,南昌330004

出　　处：《中国生物工程杂志》2013年第5期62-67,共6页China Biotechnology

基　　金："十二五"国家科技支撑计划(2011BAK10B06;2011BAK10B01;2011BAK10B02);国家自然科学基金(31000048;31170091;31260363;81160494);食品科学与技术国家重点实验室目标导向课题(SKLF-MB-201002);自由探索课题(SKLF-TS-200916);赣鄱英才555工程人才计划(2012年)资助项目

摘　　要：目的:利用基因工程技术制备抗肠炎沙门氏菌的单链抗体。方法:从抗肠炎沙门氏菌单克隆抗体的杂交瘤细胞中纯化RNA,反转录后扩增出抗体的重链可变区(VH)和轻链可变区(VL)基因片段,采用重叠延伸的方法,用柔性多肽Linker接头(Gly4Ser)3按VL-Linker-VH方式将VH基因和VL基因拼接成单链抗体基因片段后,连接到pGEX-4T-1载体上,进行重组转化。挑取阳性克隆,经IPTG诱导后,通过GST柱进行亲和层析,最后利用ELISA检测抗体的活性。结果:成功构建了表达抗肠炎沙门氏菌单链抗体的基因工程菌株,经SDS-PAGE和ELISA检测结果表明,诱导表达的单链抗体scFv分子量约为60 kDa,其能特异与肠炎沙门氏菌结合,但与副甲伤寒沙门氏菌、鸭沙门氏菌、鼠伤寒沙门氏菌有轻度交叉反应。结论:成功构建了抗肠炎沙门氏菌单链抗体的表达菌株,表达的单链抗体scFv可作为沙门氏菌的检测的候选抗体分子。Objective： To obtain the specific scFv against Salmonella enteritidis using genetic engineering technology. Methods： VH and VL genes were amplified by Reverse Transcription （RT） PCR from hybridoma cells secreting anti-Salmonella enteritidis monoclonal antibody, scFv gene （VL-Linker-VH） was obtained by Splice Overlap Extension （SOE） PCR with flexible polypeptide Linker connector （Gly4Ser） 3. Subsequently the scFv-pGEX-4T-1 recombinant plasmid was constructed and transformed into E. coli BL21 for expression using IPTG as an inducer. The expressed recombinant protein was purified by GST chromatography and identified by SDS-PAGE and ELISA. Results： DNA sequencing demonstrated that scFv was successfully inserted into pGEX- 4T-1. SDS-PAGE demonstrated that the molecular weight of the expressed scFv was about 60 kDa, and ELISA results confirmed that the obtained scFv can be recognized by not only Salmonella enteritidis but also S. enterica subsp enterzca, S. anatum and S. typhimurium. Conclusion： Specific scFv against S. enteritidis was obtained and can be used as candidate antibody molecules in detection of Salmonella.

关 键 词：沙门氏菌 单链抗体 重链可变区 轻链可变区 

分 类 号：Q511[生物学—生物化学]