人血脑屏障通透性相关基因Caveolin-1的克隆及其蛋白表达、纯化

Cloning, expression, purification of human blood-brain barrier permeability related gene Caveolin-1

目的克隆与人血脑屏障通透性相关基因Caveolin-1,并在大肠埃希菌中表达,最后进行蛋白纯化、鉴定。方法根据基因库中收录的Caveolin-1基因序列,设计其上下游引物,经过PCR反应合成Caveolin-1双链,然后与pET28a(+)载体重组,筛选阳性克隆并对DNA序列分析鉴定。将已构建好的重组质粒pET28a-Caveolin-1转入化大肠埃希菌BL21(DE3),异丙基硫代半乳糖苷诱导融合蛋白表达,表达产物经镍离子亲和层析(Ni^(2+)-NTA)纯化,SDS-PAGE检测和Western blot鉴定。结果 PCR扩增的Caveolin-1 DNA片段经鉴定确认为人Caveolin-1基因。经异丙基硫代半乳糖苷诱导的含有pET28a-Caveolin-1重组质粒的DE3菌表达出重组人Caveolin-1融合蛋白。重组蛋白经Ni^(2+)-NTA亲和层析纯化,获得了较高纯度的融合蛋白。结论本研究成功克隆了人血脑屏障通透性相关基因Caveolin-1,并对其进行蛋白表达、纯化,获得了较高纯度融合蛋白,为进一步的相关临床研究奠定了基础。

Objective To clone Caveolin-1, a gene related to human blood-brain barrier permeability, and express it in Escherichia coli. and to purify and identify the protein finally. Methods According to the sequence of Caveolin-1 gene contained in the gene bank, the upstream and downstream primers were designed. The Caveolin-1 double strand was synthesized by PCR and then recombined with pET28a （＋） vector. Positive clones were screened and identified by DNA sequence analysis. The constructed recombinant plasmid pET28a-Caveolin-1 was transfected into E. coli BL21 （DE3）, and the expression of the fusion protein was induced by IPTG. The expressed product was purified by Ni2＋-NTA affinity chromatography, identified by SDS-PAGE and Western blot. Results The Caveolin-1 DNA fragment amplified by PCR was identified as human Caveolin-1 gene. The recombinant human Caveolin-1 fusion protein was expressed by IPTG-induced DE3 strain containing the recombinant plasmid pET28a-Caveolin-1. The recombinant protein was purified by Ni2＋-NTA affinity chromatography to obtain a fusion protein of higher purity. Conclusion This study successfully cloned the blood-brain barrier permeability-related gene Caveolin-1, and expressed and purified the protein to obtain a higher purity fusion protein, which laid a foundation for further relevant clinical research.