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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 Recombinant osteoporosis candidate gene matrix Gla protein (MGP) gene is highly expressed in Escherichia coli. Methods The cDNA sequence of MGP gene was amplified from total RNA of normal human lung tissue by reverse transcription polymerase chain reaction (RTPCR), cloned into the pGEMTeasy vector and sequenced as a complete coding sequence. The recombinant plasmid was constructed with the expression vector pTrcHisB, Into E. coli Top10 after induction with IPTG, Westernbloting confirmed protein expression. Results The cDNA sequence of MGP gene cloned into pGEMTeasy vector and pTrcHisB vector was completely consistent with that of gene bank. After transfection into E. coli, the expression of protein was induced by IPTG. Western blotting confirmed the significant increase of 2,3,4h protein expression after induction. Conclusion The successfully recombinant human MGP gene and recombinant can be successfully and efficiently expressed in E. coli. The protein expression induced by IPTG was time-dependent.