人促甲状腺激素受体A亚基在大肠杆菌中的表达及免疫活性  

Expression of human thyroid stimulating hormone receptor A subunit in Escherichia coli and analysis of its immune activity

作　　者：刘玉盒 姜倩 张丽[1] 陈慧[1] Liu Yu-he;Jiang Qian;Zhang Li;Chen Hui(Department of Endocrinology and Metabolism,The Second Hospital of Lanzhou University,Lanzhou 730030,China)

机构地区：[1]兰州大学第二医院内分泌与代谢科,甘肃兰州730030

出　　处：《兰州大学学报（医学版）》2020年第4期14-19,共6页Journal of Lanzhou University（Medical Sciences）

基　　金：国家自然科学基金资助项目(81241029);兰州大学第二医院院内课题(bdkyjj-05)。

摘　　要：目的构建人促甲状腺激素受体(TSHR) A亚基的原核表达载体pET102/D-TOPO/TSHR A,在大肠杆菌中表达并分析其免疫活性。方法采用逆转录—聚合酶链式反应扩增TSHR A基因编码序列,定向连接到载体pET102/D-TOPO中,经聚合酶链式反应、酶切、测序鉴定后转化大肠杆菌BL21 StarTM(DE3);异丙基硫代-β-D-半乳糖苷诱导表达TSHR A亚基融合蛋白,超声裂解细菌,SDS聚丙烯酰胺凝胶电泳、Western blotting鉴定,在变性、复性、镍柱纯化后,目的蛋白与Graves患者血清结合鉴定其免疫活性。结果成功构建pET102/D-TOPO/TSHR A亚基表达载体,优化融合蛋白表达条件,SDS聚丙烯酰胺凝胶电泳鉴定重组质粒可表达47 kD大小的特异性蛋白,Western blotting鉴定其被His抗体、鼠源性抗人促甲状腺激素受体多克隆抗体识别,复性后融合蛋白与Graves患者混合血清呈强阳性结合,正常人血清未有任何形式的结合。结论成功构建人促甲状腺激素受体A亚基原核表达载体,并在大肠杆菌中表达了其亚基融合蛋白,该蛋白与Graves患者血清呈强阳性特异性结合。Objective The prokaryotic expression vector pET102/D-TOPO/TSHR A of the human thyroid stimulating hormone receptor(TSHR) A subunit was constructed and expressed in Escherichia coli, and analyzed for its immunological activity. Methods Amplification of TSHR A by RT-PCR and directional ligation into vector p ET102/D-TOPO, transformed by Escherichia coli BL21 StarTM(DE3) after identification by PCR, restriction enzyme digestion and sequencing. Expression of TSHR A fusion protein induced by isopropylthio-β-D-galactoside, bacteria pyrolysised by an ultrasonic crusher, identification by SDS polyacrylamide gel electrophoresis and Western blotting. After denaturing, renaturing and nickel column purification, the target protein was combined with Graves patient’s serum to identify its immune activity. Results pET102/DTOPO/TSHR A expression vector was constructed, fusion protein expression conditions optimized and recombinant plasmids identified by SDS polyacrylamide gel electrophoresis to express specific proteins of 47 kD;Western bloting confirmed that it could be recognized by His antibody, murine anti-TSHR polyclonal antibody and repeated experimental renaturation. After renaturation, the fusion protein showed a strong positive binding to the mixed serum of Graves patients, whereas normal human serum did not bind in any form. Conclusion The TSHR A subunit prokaryotic expression vector was successfully constructed and the TSHR A subunit fusion protein was expressed in Escherichia coli. The protein was strongly positively specific to the serum of patients with Graves disease.

关 键 词：人促甲状腺激素受体A亚基 大肠杆菌 免疫活性 

分 类 号：R593.22[医药卫生—内科学]