重组蛋白NtA-PACAP在大肠杆菌中的表达纯化及活性测定  

Expression of Fusion Protein NtA-PACAP in E.coli and Its Purification and Biological Analysis

作　　者：王晶[1] 吴陆生[1] 陈小佳[1] 董濠鋆[1] 沈巍[1] 张敏仪[1] 洪岸[1] 

机构地区：[1]暨南大学生物医药研究院生物工程药物广东省重点实验室基因工程药物国家工程研究中心,广州510632

出　　处：《中国生物工程杂志》2013年第3期28-33,共6页China Biotechnology

基　　金：广州市科技计划项目(2011J4300107)

摘　　要：目的:通过构建PACAP38与Agrin的N端结构域(NtA)相连的重组基因的原核表达载体,获得重组NtA-PACAP蛋白,并验证其生物学活性,为未来的规模化制备奠定基础。方法:将PACAP38与层粘连蛋白的受体蛋白Agrin的N端通过连接肽相连,合成重组蛋白的基因片段,并在此基因片段末端连上6个组氨酸标签密码子,然后将此重组基因片段克隆至大肠杆菌表达载体pET-3c中,转化E.coli BL21(DE3),获得重组工程菌并摸索发酵和纯化条件,IPTG诱导表达后收集菌体破碎离心,收集上清液,经阴离子交换层析和Ni-NTA树脂亲和层析两步纯化法获得目的蛋白;通过PC12细胞饥饿损伤后修复实验模型来检测NtA-PACAP38的生物学活性。结果:成功表达并获得纯度90%以上的NtA-PACAP38蛋白,生物学活性实验证明NtA-PACAP38具有促饥饿损伤后的PC12细胞增殖能力。结论:获得的重组蛋白NtA-PACAP38具有生物学活性,表达系统和纯化工艺可以用于下一步的规模化制备。Objective：To obtain a fusion protein NtA-PACAP, which PACAP38 links with the N-terminal domain of Agrin （NtA）, in a recombinant prokaryotic system and to analysis its bioactivity. Methods： The nucleotide sequence encoding fusion NtA-PACAP protein were designed and synthesized according E.coli codon bias, and His-tag was added to the C-terminal, then the sequence cloned into vector pET-3c. And this recombinant plasmids were transformed to E.coli BL21（DE3） for expression by IPTG induction. The targeted protein was got by two-step purification methods, which Anion Exchange Chromatography and then Ni-NTA affinity chromatography. Finally its bioactivity was evaluated by the cell injury repair experiments in vitro. Results：NtA-PACAP protein is obtained successfully. Bioactivity assay result shows that NtA-PACAP could promote PC12 cells proliferation after starvation injury. Conclusion： The fusion NtA-PACAP38 protein is worth of developing to a candidate drug.

关 键 词：PACAP 重组蛋白 基因表达 蛋白纯化 

分 类 号：Q782[生物学—分子生物学]