雄激素受体基因各功能域片段的原核表达与功能鉴定  

Prokaryotic expression and functional identification of androgen receptor functional domains

作　　者：张雨琪 丁明珠 吴金凤 郑海香 张磊 王星 Zhang Yuqi;Ding Mingzhu;Wu Jinfeng;Zheng Haixiang;Zhang Lei;Wang Xing(School of Basic Medical Sciences,Fujian Medical University,Fuzhou 350122)

机构地区：[1]福建医科大学基础医学院,福州350122

出　　处：《安徽医科大学学报》2023年第2期219-224,共6页Acta Universitatis Medicinalis Anhui

基　　金：国家自然科学基金(编号:81873966);福建医科大学高层次人才启动经费计划(编号:XRCZX2019016);病毒学国家重点实验室开放研究基金(编号:2020KF007);福建省自然科学基金项目(编号:2021J01663)。

摘　　要：目的系统构建雄激素受体(AR)野生型全长和4个功能域截短体的原核表达质粒,Western blot与凝胶迁移(EMSA)实验鉴定各融合蛋白及其功能。方法基于pGEX-4T-1载体,构建AR全长及各功能域的谷胱甘肽巯基转移酶(GST)融合表达重组质粒。应用异丙基-β-D-硫代半乳糖苷(IPTG)对重组质粒进行诱导表达,确定各重组蛋白的最佳参数。分别用AR内源性抗体和GST标签抗体进行Western blot鉴定,分析诱导前、诱导后细菌裂解液中的目的蛋白,并利用谷胱甘肽琼脂糖树脂进一步纯化。将纯化后蛋白与病毒荧光探针进行孵育,复合物于非变性凝胶中电泳检测纯化蛋白功能。结果成功构建了AR全长和3个功能域截短体的原核表达质粒。PCR和双酶切鉴定均显示在各插入片段大小相符处出现阳性条带,且测序结果与NCBI GenBank标准株比对一致。其中,96 ku GST-AR-NTD+DBD与86 ku GST-AR-NTD两个融合蛋白得以成功表达及后续纯化。纯化蛋白可与病毒基因组DNA直接结合。结论来源同一基因序列的AR截短体重组质粒原核表达条件不同,纯化后的AR蛋白可用于深入了解AR各功能域与其他分子间的直接相互作用机制。Objective To construct the full-length prokaryotic expression plasmid of the wild type of androgen receptor(AR)and the truncated body of four functional domains,and to identify the fusion protein by Western blot and electrophoretic mobility shift assay(EMSA).Methods Based on the pGEX-4T-1 vector,the recombinant plasmids were constructed to express the full-length and functional domains of AR.IPTG was used to induce the expression of the recombinant proteins,which were isolated and purified by glutathione sepharose 4B beads under the optimized condition.The specific protein expression in the bacterial lysate and the purified protein isolated with glutathione sepharose 4B beads was identified by Western blot with AR antibody and GST labeled antibody.The purified protein was incubated with a fluorescent probe of the virus,and the complex was detected by electrophoresis in a non-denaturing gel.Results The prokaryotic recombinant plasmids of full length and three functional domain truncated AR were successfully constructed.The recombinant clones were identified by using bacterial culture as a template,and further verified by double enzyme digestion.It showed that there were identical bands in the same sizes as the inserted fragments.The nucleotide and the amino acid sequences were aligned to the reference sequence in NCBI GenBank.The GST fusion protein,GST-AR-NTD+DBD(96 ku)and GST-AR-NTD(86 ku)were successfully induced and verified.The purified protein could be directly combined with the viral genome DNA.Conclusion The prokaryotic expression conditions of truncated AR plasmid from the same gene sequence are different.The purified AR protein can be used to understand the direct interaction mechanism between functional domains of AR and other molecules.

关 键 词：雄激素受体 原核表达 GST融合蛋白 功能域突变体 

分 类 号：Q786[生物学—分子生物学]