人类维甲酸受体α的克隆表达

Clone and expression of recombinant retinoid X receptor α

目的:构建人类维甲酸受体α基因的高效表达系统。方法:提取人肝组织总RNA,RT-PCR扩增Retinoid X receptorα(RXRα)的cDNA。将其克隆入原核表达载体pETDuet-1,构建重组表达载体pETDuet-1/RXRα。将已构建好的表达载体pETDuet-1/RXRα重组质粒转化大肠杆菌BL21,异丙基硫代-8-D-半乳糖苷(IPTG)诱导表达,Western Blot鉴定表达产物。结果:重组载体pETDuet-1/RXRα经限制性核酸内切酶BglⅡ和AatⅡ双酶切鉴定,得到符合预期大小的核酸片段。SDS-PAGE可见与预期大小相符的蛋白条带,Western Blot证实该条带即为RXRα。并对重组质粒pETDuet-1/RXRα在BL21菌株的表达效率进行评价,目的蛋白占菌体总蛋白的百分比为65.8%。结论:成功建立了pETDuet-1/RXRα的高效表达系统。

Objective：To construct efficient expression system of recombinant human retinoid X receptor α. Methods： Human total RNA was extracted from human liver tissue . The cDNA encoding the RXRα was amplified by RT - PCR, then was inserted into expression vector pETDuet - 1. The constructed expression vector pETDuet - 1/RXRα recombinant plasmid was transformed into E. coli BL21. The RXRα was induced to express by IPTG. The expression product was verified by Western blot. Results： The recombinant plasmid pETDuet - 1 /RXRα was identified by restriction enzyme Bgl Ⅱ and Aat Ⅱ, then the fragment was observed. The anticipated protein strap which was confirmed to be RXRα by Western blot was observed by SDS -PAGE. The interest protein amounted to 65.8% of the total protein. Conclusion： Recombinant human retinoid X receptor α can be expressed successfully.