Myostatin基因真核表达载体的构建及其在CHO细胞中的表达

Construction of Eucaryotic Expression Vector of Myostatin and Expression in CHO Cell

目的:构建人Myostatin基因的真核表达载体pVAC-Ms,并在真核细胞中进行表达,为后续研究Myostatin蛋白的检测方法、强力药物的筛选及废用性肌肉萎缩的治疗提供实验依据。方法:化学耦联方法合成Myostatin C-末端区(330bp)基因序列,利用PCR技术扩增5′端和3′端酶切位点为BamH I和EcoR I,然后克隆至真核表达载体pVAC1-cms,得到重组质粒pVAC-Ms。质粒纯化后采用脂质体介导转染CHO细胞,瞬时转染后收集CHO细胞,利用RT-PCR技术和细胞荧光免疫技术检测Myostatin在CHO细胞中的转录和蛋白表达。结果:酶切和测序结果表明,Myostatin真核表达载体pVAC-Ms构建成功,RT-PCR和细胞荧光免疫检测结果表明,瞬时转染的CHO细胞中Myostatin基因明显转录,蛋白明显表达。结论:实验成功构建了在真核细胞中能有效表达人Myostatin重组蛋白的真核表达载体,为运动员Myostatin蛋白表达的检测、强力药物筛选、训练监控及废用性肌肉萎缩治疗等方面的后续研究提供实验基础。

Objective： To further study the method of detection, drug screening and therapy of wasting muscle from myostatin as a new target. The eucaryotic expression vector of myostatin was constructed and the recombinant human myostatin was expressed in CHO cell. Method： We synthesized C-terminal domain （330bp） of myostatin and amplified the gene with 5＇ containing BamH I and 3＇ containing EcoR I restriction sites by PCR. The gene was cloned into the expression vector pVAC1-cms and the eucaryotic expression vector of myostatin was constructed. The CHO cells were infected with the recombinant plasmid pVAC-Ms by liposome transfection. After 36h, the transcription of myostatin mRNA was detected by RT-PCR and the myostatin expression was tested by cell immunofluorescence technique in transfected CHO. Result：The eucaryotic expression vector of myostatin pVAC-Ms was constructed successfully through the restriction analysis and sequencing. The transcription of myostatin mRNA and the myostatin expression was detected obviously in transfected CHO. Conclusion：The construction of eucaryotic expression vector of myostatin is successful in expression for recombinant human mature peptide of myostatin. The study will provide an experimental ground in the field of sports medicine such as detection of myostatin, drug screening, training monitoring and therapy of wasting muscle.