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目的:克隆人肌肉生长抑素(myostatin)基因并在大肠埃希菌中表达,纯化目的蛋白后制成冻干粉剂。方法:通过RT-PCR方法得到人myostatin基因的cDNA序列,构建重组克隆质粒pGEM5zf(+)myostatin-s及表达质粒pET32a(+)-myostatin-s,在合适条件下通过大肠埃希菌表达系统诱导表达并纯化该蛋白(myostatin-s),最终制成冻干粉剂。结果:成功构建重组质粒,并经测序与GeneBank资料Myostatin(GI:2623581)序列完全一致。经异丙基-β-D-硫代吡喃半乳糖苷(IPTG)诱导及纯化获得相对分子质量为33 000融合蛋白,并经Western blot证实后制成冻干粉剂。结论:通过重组基因片段可以获得纯化myostatin的成熟肽片段-融合蛋白myostatin-s,制成的冻干粉剂将为进一步人肌肉生长抑素免疫学研究奠定基础。
OBJECTIVE: To clone human myostatin gene and express it in Escherichia coli. Purify the target protein and freeze-dry it. METHODS: The cDNA sequence of human myostatin gene was obtained by RT-PCR. The recombinant plasmid pGEM5zf (+) myostatin-s and the expression plasmid pET32a (+) - myostatin-s were constructed and induced by E. coli expression system under appropriate conditions The protein is expressed and purified (myostatin-s) and finally made into lyophilized powder. Results: The recombinant plasmid was successfully constructed and sequenced exactly in accordance with the sequence of GeneBank Myostatin (GI: 2623581). The relative molecular mass of 33 000 fusion protein was induced and purified by isopropyl-β-D-thiogalactopyranoside (IPTG) and confirmed by Western blotting to make lyophilized powder. Conclusion: The purified myostatin gene fragment can be purified myostatin mature peptide fragment - fusion protein myostatin-s, made of freeze-dried powder will further human myostatin immunoassay lay the foundation.