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.
出处
《中国生物工程杂志》
CAS
CSCD
北大核心
2013年第3期28-33,共6页
China Biotechnology
基金
广州市科技计划项目(2011J4300107)
关键词
PACAP
重组蛋白
基因表达
蛋白纯化
PACAP
Recombinant protein
Gene expression
Protein purification
