蛋白Ⅲ-α-银环蛇毒素融合蛋白表达质粒的构建及表达  

Construction of Expression Vector for Protein Ⅲ-α-Bungarotoxins Fusion Protein

作　　者：高荣凯[1] 王欲晓[1] 张海荣[1] 周丽君[1] 曲佳[1] 

机构地区：[1]海军总医院中心实验科,北京100048

出　　处：《中国生物制品学杂志》2011年第10期1162-1164,1173,共4页Chinese Journal of Biologicals

基　　金：全军医药卫生基金(06MA016)

摘　　要：目的构建适合选择感染性噬菌体(Selectivelyinfectivephage,SIP)技术筛选的蛋白Ⅲ-α-银环蛇毒素(α-Bungarotox-ins,α-BTXs)融合蛋白表达质粒。方法从银环蛇毒腺中提取总RNA,RT-PCR扩增α-BTXs基因,插入表达载体pTTMIN,转化E.coli XL1-Blue,IPTG诱导表达。经SDS-PAGE、Western blot及竞争抑制ELISA法检测融合蛋白的表达。结果获得的α-BTXs基因序列与相关报道完全一致;融合蛋白表达质粒构建正确;融合蛋白的表达量可达菌体总蛋白的20%,可与抗蛇毒素血清发生特异性反应,且保持了α-BTXs的抗原性。结论成功构建了蛋白Ⅲ-α-BTXs融合蛋白表达质粒,并在大肠杆菌中表达了融合蛋白,为利用SIP技术筛选抗α-BTXs人源抗体奠定了基础。Objective To construct the expression vector for protein Ⅲ-α-Bungarotoxins（BTX） fusion protein suitable for selectively infective phage（SIP） screening.Methods Total RNA was extracted from the venom gland of Bungarus multicinctus ioterium,with which α-BTXs gene was amplified by RT-PCR and inserted into expression vector pTTMIN for expression under induction of IPTG.The expression of fusion protein was determined by SDS-PAGE,Western blot and competitive inhibition ELISA.Results The sequence of obtained α-BTXs gene was completely consistent with that published.The expression vector for fusion protein was constructed correctly.The expressed fusion protein contained 20% of total somatic protein,showed specific reaction with antivenomous serum and maintained the antigenicity of α-BTXs.Conclusion The expression vector for protein Ⅲ-α-BTX fusion protein was successfully constructed,and the fusion protein was expressed,which laid a foundation of screening human antibody against α-BTXs by SIP technique.

关 键 词：Α-银环蛇毒素 SIP技术 重组融合蛋白质类 

分 类 号：Q78[生物学—分子生物学]