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人绒毛膜促性腺激素 亚基在大肠杆菌中的表达 |
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张乐鸣 刘东海 董虹 张顺
摘 要 目的：以该室克隆的PCRⅡ/hCGβ载体为模板，亚克隆到表达载体PG5中使hCGβ基因在大肠杆菌中进行高效表达。方法：改变部分hCGβ序列，得到重组PG5/hCGβ载体，经序列分析证明，再转化到表达菌BL21中表达，所得包涵体经过柱纯化，复性，免疫发光分析和动物试验测定其活性。结果：经SDS-PAGE电泳在18 kD左右有一条明显的新增蛋白带，与预期的分子量相符，并用Western印迹证实，rhCGβ约占菌体总蛋白的10%，活性为7.8 U/mg。结论：重组hCGβ在大肠杆菌中获得了较高水平的表达。且经复性后有较高的免疫活性和生物活性。
关键词 人绒毛膜促性腺激素β亚基 序列分析 基因表达 复性
中国图书分类号 R392.11
Expession in E.coli of human chorionic gonadotropin subunit β cDNA
ZHANG Le-Ming，LIU Dong-Hai，DONG Hong et al.
Laboratory of Tumor Molecular Biology，Ningbo No.2 Hospital，Ningbo 315010
Abstract Objective:Using the previously cloned vector PCRⅡ/hCGβ as the template，subclone the aimed gene into expression vector PG5，make hCGβ expressed efficiently in E.coli.Methods:Several neucleotides of hCGβ in recombinant PCRⅡ/hCGβ were altered through PCR method，then the modified hCGβ was inserted into PG5，proved by sequencing analysis，and transformed E.coli host strain BL21.The expressed inclusion body was extracted，dissolved，and refolded.Its immunological and physiological activities were measured by immunoflurescent assay and animal experiments respectively.Results:The results of SDS-PAGE electrophoresis and western blot demonstrated that the IPTG induced bacteria expressed an extra band of 18 kD in comparison with the uninduced ones.The expression level was more than 10% of the total cell lysate，and the immunological activity，7.8 U/ml.Conclusion:The recombinant PG5/hCGβ was effeciently expressed in E.coli，and the recombinant protein has high immunological and physiological activities.
Key words hCGβ Sequencing analysis Gene expression Refolding
人绒毛膜促性腺激素(Human chorionic gonadotropin，hCG)是由胎盘滋养层合体细胞合成的激素，促进胚泡发育，维持妊娠。hCG与TSH(促甲状腺激素)、FSH(促卵泡激素)、LH(黄体生成素)等同为糖蛋白激素家庭一员［1］。这些激素都具有相同的α亚基和各自独特的β亚基。α亚基与信息传导有关，而β亚基则产生其激素效应［2］。作为抗原，β亚基比整个hCG分子更具有特异[1] [2] 下一页
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