豬肌肉生長抑制素前胜肽cDNA之選殖與表現載體之構築

本試驗目的在進行肌肉生長抑制素前胜肽（myostatin propeptide）cDNA之選殖與其表現載體之構築，並將其轉形至酵母菌宿主細胞，藉以表現外源性之肌肉生長抑制素前胜肽產物。試驗採用已選殖1128 bp肌肉生長抑制素互補去氧核糖核酸（cDNA）作為模板，利用巢式聚合瓷連鎖反應（Nest PCR）選殖，合成828 bp之肌肉生長抑制素前胜肽PCR產物，並用限制瓷截切，將肌肉生長抑制素前胜肽cDNA序列，再將之接入pGAPZ α A中，完成表現載體（pGAPZ αA Myostatin-propeptide）之建構。同時利用瓊脂電泳與限制酶截切、核啟酸序列分析與比對，確認肌肉生長抑制素前胜肽之開放譯讀框架沒有被破壞。並將此構築完成之表現載體轉形至酵母細胞，再經SDS-PAGE與西方墨點法分析，確認有2株具有較高表現外源性重組肌肉生長抑制素前胜肽之酵母菌轉形株。

關鍵字

質體；前胜肽；表現載體

並列摘要

The objectives of this study were to clone porcine myostatin propeptide cDNA and to construct its expression vector, so as to transfer the expression vector into yeast cells to generate recombinant myostatin propeptide. The 1128 bp of myostatin cDNA being used as DNA template were used for performing nest- PCR to amplify the 828 bp of the myostatin propeptide cDNA. The amplified cDNA and pGEM-T Easy vector were ligated by T4 ligase, which transformed those vectors into competent cells afterward. The transformed competent cells of plasmid DNA and pGAPZαA expression vector were cleaved by using EcoR I and Xba I restriction enzymes, and both of them were ligated by T4 ligase to achieve construction of pGAPZαAMyostatin-propeptide expression vector. Those constructed vectors further were transferred to competent cells, and the transferred competent cells were cultivated in LB broth and their DNA was extracted to determine nucleotide sequencing. The above-mentioned results demonstrated that open reading frame of myostatin propeptide cDNA in pGAPZαAMyostatin-propeptide vector was complete. Those confirmed expression vectors were further transformed into yeast cells by using the electroporator. We picked out 2 yeast transformed-clones because both had shown higher secretion of recombinant myostatin propeptide into supernatants, which was confirmed by using SDS-PAGE and western blot. From the results mentioned above, we were certain that these yeast transformants could be used to yield recombinant myostatin peptide.

並列關鍵字

Plasmid ； Propeptide ； Expression vector

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