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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 75th 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 Escherichia 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 1mmol/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.
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Received: 05 December 2011
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ZHU Xiao-Feng, XU Hou-Qiang, CHEN Wei, CHEN Ying-Lian, NI Kai, WU Xiao-Min, NI Meng-Meng, LU Xian-Jun. Cloning, Expression and Bioinformatics Analysis of IGF-1 and IGF-2 Genes in Congjiangxiang Pigs (Sus scrofa)[J]. 农业生物技术学报, 2019, 27(8): 1382-1391. |
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