摘要
目的重组骨质疏松候选基因基质Gla蛋白(MGP)基因使其蛋白在大肠杆菌中高效表达。方法利用反转录聚合酶链反应(RTPCR)从正常人肺组织总RNA中扩增出MGP基因cDNA序列,与克隆pGEMTeasy载体相连,测序为完整的编码序列后与表达载体pTrcHisB构建重组体,转化入大肠杆菌Top10后用IPTG诱导,Westernbloting证实蛋白表达。结果克隆至pGEMTeasy载体及pTrcHisB载体中的MGP基因cDNA序列与基因库完全一致。转入大肠杆菌后经IPTG诱导有蛋白的表达,Westernbloting证实诱导后2、3、4h蛋白的表达量显著增加。结论成功重组的人MGP基因,重组体在大肠杆菌内能成功高效地表达。IPTG诱导后蛋白的表达为时间依赖性。
Objective To recombinate the human Matrix Gla Protein（ MGP）, the candidate gene for osteoporosis, and make MGP protein to express in E. Coli Top10.Methods Total RNA was abstracted from normal human lung tissue and human MGP eDNA was amplified by RT-PCR, then linked to the cloned pGEM-Teasy vector. Those with completed coding sequences were incised and recombinated with expression vector pTrcHisB after which were transfected to E. coli Top10. After the reconfirmation of the cDNA without mutation/absence, the expression of MGP protein was induced by IPTG, sedimented by centrifugation and retested using Western blot. Results The sequence of the target eDNA linked to pGEM-Teasy and pTrcHisB vector was the same as that reported in gene bank and no mutation or absence of the recombinated pTrcHisB-hMGP was found. After induced by IPTG, the MGP （target） protein with the same molecular weight as wild MGP was expressed in E. coli Top10 which was confirmed by Western blot. The levels of MGP protein expression at 2, 3, and 4 h after induced by IPTG were significantly higher than that of other time points suggesting the production of MGP was in a time-dependent manner. Conclusions Human MGP genes can be successfully cloned and the recombinated MGP protein can also be effectively expressed in E. Coli in a time-dependent manner which can provide a better way for further study of MGP.
出处
《中国骨质疏松杂志》
CAS
CSCD
2006年第4期354-356,共3页
Chinese Journal of Osteoporosis
关键词
MGP
基因重组
蛋白表达
MGP
Gene recombinate
Protein expression
