高尔基体糖蛋白GP73基因的构建、表达及鉴定  

Construction,expression and dientification of GP73 fusion protein

作　　者：李新禹[1] 李淑珍[1] 陈莉[1] 单瑞辉[1] 唐晓波[1] 

机构地区：[1]哈尔滨医科大学药学院生物制药学教研室,黑龙江哈尔滨150081

出　　处：《哈尔滨医科大学学报》2011年第6期510-512,515,共4页Journal of Harbin Medical University

基　　金：哈尔滨市科技创新人才研究专项资金项目(2008RFXXS018)

摘　　要：目的重组及表达人高尔基体糖蛋白73(GP73)的融合蛋白,为建立一种新的肝癌诊断方法奠定基础。方法提取转染腺病毒E1A基因的人肾上皮细胞(293细胞)中的总RNA,应用RT-PCR、PCR等技术扩增出GP73基因,将其分别与带有HIS标签的PET-32a及GST标签的PGEX-4T-1载体连接,转化入DH5α菌中进行克隆并测序鉴定。进一步构建表达体系,用异丙基β-D-硫代半乳糖苷(IPTG)诱导,最终在BL21菌中表达GP73融合蛋白(分别含有HIS,GST-Tag),并进行SDS-PAGE及Western blot鉴定。结果 293细胞株提取的RNA经RT-PCR、PCR扩增后得到一条255 bp的DNA片段,经测序鉴定为GP73基因序列;并在大肠杆菌中实现了可溶性高表达,Western blot鉴定为GP73融合蛋白。结论采用原核表达方法可获得高纯度的GP73融合蛋白,为进一步开发GP73诊断试剂打下基础,为肝癌的诊断开辟新途径。Objective To clone and express human Golgi glycoprotein73 （GP73）fusion pro- tein, and for establishment of an early diagnostic method of hepatocellular carcinoma （HCC）. Methods The gene encoding GP73 was cloned using RT-PCR and PCR techniques from total RNA of HEK293 cell, and the amplified gene was inserted into vectors PET-32a of HIS tag and PGEX4T-1 of GST tag, and subcloned into DH5et. The recombinant plasmids were constructed and transfoIlned into E. coli BL21 cells, and protein expression （including His-Tag and GST- Tag, respectively ） were induced by [PTG and identified by SDS-PAGE and Western blot. Re- suits A band of DNA about 255 bp by DNA sequencing was confirmed as GP73 gene, the ex- pression system was constructed, and soluble expression of GP73 fusion protein was achieved successfully, which was identified by Western blot. Conclusion Fusion protein GP73 with high purity could be expressed in prokaryotic system. The study lays a foundation for opening up new ways for early diagnosis of liver cancer.

关 键 词：肝癌 GP73基因 克隆 

分 类 号：R735.7[医药卫生—肿瘤] Q78[医药卫生—临床医学]