农业生物技术学报2012，Vol.20Issue(4)：389-396,8.DOI:10.3969/j.issn.1674-7968.2012.04.007
猪肌肉生长抑制素(MSTN)前肽定点突变载体的构建及其原核表达
Plasmid Construction and Prokaryotic Expression of a Site-directed mutant of Porcine Myostatin Propeptide-coding Gene
摘要
Abstract
Myostatin (MSTN) can inhibit the growth of skeletal muscle, while the mutated myostatin propeptide (Pro-MSTN-D75A) can inhibit myostatin activity. Pig is a very economically important livestock in China. It will play an important role in live pig production of China to increase the volume of lean meat of pig by injection of mutated myostatin propeptide. In this study, we have developed a system and optimal conditions to express myostatin propeptide. Total RNA was extracted from longissimus of 3-month embryo of Tongcheng pig and the MSTN propeptide cDNA (exclusive the signal peptide) was amplified by RT-PCR. Simultaneously, a site-directed MSTN cDNA mutant (the 75* aspartic acid changed to alanine, D75A) was also generated. Prokaryotic expression vectors of pGEX-ProM (SP-) and pGEX-ProM (SP-)-D75A were subsequently constructed and transformed into Eschenchia coli cells (BL21) to express GST-fusion proteins of the wild type and mutant. The optimal expression was observed after 5h induction with 0.6 mmol/L IPTG for the wild-type MSTN gene but after 6 h induction with lmmol/L IPTG for the D75A mutant. The fusion proteins were purified by GST-Trap system with expected molecular size (~51 kD consist of 26 kD GST tag and 25 kD myostatin propeptide). Concentrations of the purified wid-type (ProM(SP-)) and mutatant (ProM (SP-)-D75A) were 1.87 mg/mL and 1.21 mg/mL, respectively. This work has provided us a basis to prepare anti-MSTN antibody and to further investigate the functions of porcine MSTN propeptide.关键词
猪/肌肉生长抑制素/前肽/突变/原核表达Key words
Pig/ Myostatin/ Propeptide/ Mutate/ Prokaryotic expression引用本文复制引用
姜声旺,张晓玮,王在贵,崔文涛,李奎..猪肌肉生长抑制素(MSTN)前肽定点突变载体的构建及其原核表达[J].农业生物技术学报,2012,20(4):389-396,8.基金项目
国家自然科学基金(NO.30901022) （NO.30901022）
