【摘要】 目的:利用谷胱甘肽S-转移酶(GST)融合蛋白表达系统在大肠杆菌中表达纯化Myostatin成熟肽。方法:应用RT-PCR从大鼠的腓肠肌mRNA中逆转录扩增Myostatin成熟肽编码序列,克隆入原核表达载体pGEX-4T1中,IPTG诱导其在大肠杆菌BL21(DE3)中表达,采用Western-blot用抗-GST抗体验证融合蛋白表达。融合蛋白经Glutathione-agarose色谱纯化。结果:PCR扩增的Myostatin基因序列与GenBank数据库中的序列一致;30°C,0.1mmol/LIPTG诱导16小时,GST-Myostatin融合蛋白获得优化表达,表达效率为25%;Western-blot证实融合蛋白的特异性表达。结论:成功构建融合蛋白GST-Myostatin重组基因,并在大肠杆菌中获得有效表达,为Myostatin抗体的制备及进一步研究该蛋白的功能奠定基础。更多还原

【Abstract】 Objective: To express the rat Mature Peptide of Myostatin in Escherichia coli and purify Mature Peptide of Myostatin by using Glutathione S-transferase (GST) expression system. Methods: The Rat Mature Peptide of Myostatin cDNA, obtained from Rat gastrocnemius muscle via RT-PCR, was inserted into the prokaryotic expression vector pGEX-4T1 to express GST-Myostatin fusion protein in Escherichia col (E. coli) BL21(DE3) strain under the optimized induction of isopropyl-β-D-thiogalactopyranoside (IPTG). Western-blot analysis was performed using anti-GST antibody to confirm the fusion protein, which was then affinity purified by Glutathione-agarose chromatography. Results: DNA sequencing showed that Rat Mature Peptide of Myostatin gene nucleotide sequence obtained from RT-PCR was identical with that of Myostatin cDNA recorded in the GenBank. The maximum quantity of the fusion protein was produced at 16h after induction with 0.1mmol/L IPTG at 30°C. Western-blot analysis indicated that the recombinant protein could react specifically with anti-GST antibody. Conclusion: The recombinant gene of GST-Myostatin was constructed successfully and the fusion protein was expressed effectively in E. coli cells. These results suggest that the reported method would be useful in obtaining a large quantity of Rat Mature Peptide of Myostatin, which would contribute to the further study on Rat Mature Peptide of Myostatin antibody production.更多还原