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霍乱毒素由5个B亚基(CTB)和l个A亚基(CTA)(包括CTA1和CTA2)组成。该蛋白结构可帮助有毒的CTA1分子进入细胞。本研究拟利用原核不相容双质粒共表达系统获得霍乱毒素类似嵌合蛋白,用于大分子蛋白质黏膜给药的载体研究。将CTB基因片段克隆至载体p ET-28a中,获得重组质粒p ET-28a-CTB;以增强型绿色荧光蛋白(EGFP)代替有毒的CTA1,在CTA2序列N端融合穿膜肽(TAT),将EGFP-CTA2-TAT基因克隆至载体p ET-22b(+)中,获得重组质粒p ET-22b-EGFP-CTA2-TAT。利用p ET-28a-CTB和p ET-22b-EGFP-CTA2-TAT二者不同抗性,将双质粒分步转化到大肠杆菌BL21中。表达条件为0.75 mmol/L IPTG、20℃、200 r/min诱导20 h,重组嵌合蛋白能以可溶形式表达。经过Ni-NTA、Sephadex G-75纯化,Western blotting对蛋白质特异性进行鉴定,确定获得(CTB)5/EGFP-CTA2-TAT嵌合蛋白。
Abstract：Cholera toxin is composed by five units of cholera toxin B(CTB) and one unit of cholera toxin A(CTA)(composed by CTA1 and CTA2). With the help of the protein structure, toxin subunit(CTA1) can enter cells. This study intends to use the incompatible plasmids expression system to obtain a chimeric protein similar to the cholera toxin, using as a carrier for macromolecular protein entering cells. CTB gene was cloned to the plasmid p ET-28 a to obtain the recombinant plasmids p ET-28a-CTB. Enhanced green fluorescent protein(EGFP) was used to replace the toxic CTA1. TAT was fused to the N-terminal of the CTA2 protein. The gene EGFP-CTA2-TAT was cloned to the plasmid PET-22b(+) to obtain the recombinant plasmids p ET-22b-EGFP-CTA2-TAT. By taking advantage of different resistance of p ET-28a-CTB and p ET-22b-EGFP-CTA2-TAT, both plasmids were transmitted into the E. coli(BL21). Expression conditions were fixed as 0.75 mmol/L IPTG, 20℃ and induction 20 h by 200 r/min.Recombinant protein was expressed in soluble form. After purification by Ni-NTA and Sephadex G-75, specificity determined by western blotting assay, chimeric protein(CTB)5/EGFP-CTA2-TAT was obtained.
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基本信息:
DOI：10.13417/j.gab.034.001398
中图分类号:Q78
引用信息:
[1]周小芬,林卫萍,何华红,等.不相容双质粒共表达霍乱毒素类似嵌合蛋白[J].基因组学与应用生物学,2015,34(07):1398-1405.DOI:10.13417/j.gab.034.001398.
基金信息:
广东省自然科学基金博士启动项目(S2012040007488)资助
2015-07-25
2015-07-25