摘要
通过生物信息学分析确定水稻(Oryza sativa L.)Os VDAC5的可溶性肽段,将相应的编码区克隆于p GEX-4T-1原核表达载体中,进行IPTG诱导表达和GST亲和层析纯化,利用SDS-PAGE和Western Blot检测目的蛋白。结果表明,成功构建的p GEX-4T-1-Os VDAC5(1-75aa)原核表达载体,经过体外IPTG诱导,在35 k Da处可检测到可溶性目的蛋白,并获得外源蛋白表达的最适温度为15℃和最佳诱导时间为8 h;经过GST亲和层析,SDS-PAGE可检测到较为单一的蛋白条带,得到纯化的GST-Os VDAC5(1-75aa)融合蛋白。
The soluble peptide of Oryza sativa L. OsVDAC5 was predicted by bioinformatics analysis and the coding region of this part was cloned into p GEX-4T-1 prokaryotic expression vector. Then the GST-Os VDAC5（1-75aa） fusion protein was induced by IPTG and purified by GST affinity chromatography. SDS-PAGE and Western Blot were used to detect the target protein. The p GEX-4T-1-Os VDAC5（1-75aa） prokaryotic expression vector was constructed. The results from SDS-PAGE and Western Blot showed that a soluble fusion protein expressed at about 35 k Da after being induced by IPTG in vitro. And the optimum temperature was 15 ℃ and the best induction time was 8 h. In addition, a single target protein band was detected and the purified GST-Os VDAC5（1-75aa） fusion protein was obtained by GST affinity chromatography.
出处
《湖北农业科学》
2016年第11期2921-2925,共5页
Hubei Agricultural Sciences
基金
国家自然科学基金项目(31170226)
