pGEX-4T-1-Neurexin 1β原核表达载体构建及表达条件的优化  

Construction and optimal expression of the prokaryotic expressing recombinant pGEX-4T-1-Neurexin 1 β

作　　者：徐岩[1] 王梦林[1] 侯筱宇[1] 

机构地区：[1]徐州医学院,江苏省脑病生物信息重点实验室,生物化学与分子生物学研究中心,江苏徐州221004

出　　处：《中国科技纵横》2015年第24期181-182,184,共3页China Science & Technology Overview

基　　金：国家自然科学基金项目“脑缺血PsD-95酪氨酸磷酸化对突触后src信号网络的调控”,编号：81173030）;江苏省高校优势学科建设工程资助项目（PAPD）.

摘　　要：目的：构建Neurexin1p（Nrx1p）原核表达重组体pGEX-4T-1-Neurexin 1β；方法：pGEX-4T-1-Neurexin 1β真核表达载体为模板，经PCR扩增Nnc1βcDNA，然后将其克隆入pGEx-4T-1原核表这载体，筛选阳性质粒后转化入宿主菌BL21，选用ⅢTG进行诱导表达，优化表达条件，SDS—PAGE检测Nrx1B的蛋白表达。结果：琼脂糖凝胶电泳显示Nrx1β的cDNA扩增成功，重组体酶切结果及测序结果与预期结果一致。转化入BL21后在1PTG的诱导TNrx1B成功表达，且在24℃时，IPTG浓度为0.2mmol／L诱导12h后Nrx1p的蛋白表达量最高。结论：该研究成功构建了pGEX-4T-Nrx113原核表达载体，并优化了Nrx1B的蛋白表达条件，为下一步研究该蛋白质的功能奠定基础。Objective： To construct the prokaryotic expressing recombinantpGEX-4T-1-Nrx 1β codingfor Neurexin 1 β （Nrx 1 β ）.Method： Nrx 1 β cDNA was obtained by PCP,. using pcDNA3.1-Myc-Nrx l β template, and cloned into the pGEX-4T-1 vector. The positive prokaryotic expression recombinant was nansformed into Eschefichia coli BL21. After induced by IPTG with optical condition, Nrx 1 β expression was detected by SDS-PAGE. Resuk： Agarose gel dectmphoresis showed cDNAofNrx 1 β was successfully amplified. The cDNA was confirmed by restrictive enzyme digestion and DNA sequencing. After transforming into BL21, the protein expression was successfully induced by IPTGwith the optical condition of 0.2 mmol/L IPTG at 24℃ for 12 h. Conclusion： The prokaryotic expression recombinant ofNrx 1β was successfully constructed and the expression condition ofNrx 1β was optimized, which provide the foundation for studying the role of Nrx 1 β in nervous system.

关 键 词：NEUREXIN 1β 原核表达载体表达条件 

分 类 号：Q78[生物学—分子生物学]