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目的探讨丝瓜籽核糖体失活蛋白基因Luffin-a原核表达载体的构建方法。方法从Pubmed中查到Luffin-a的mRNA全序列,用RT-PCR的方法从未成熟的丝瓜种子中克隆Luffin-a基因,T-A克隆后鉴定核苷酸序列,构建原核表达载体pET-15b(+)-Luffin-a,转化大肠杆菌BL21(DE3),并经IPTG诱导表达;SDS-PAGE鉴定Luffin-a表达产物。结果克隆出Luffin-a基因,IPTG诱导后大肠杆菌培养液和超声破碎的菌体沉淀中都有Luffin-a表达产物。结论本研究成功构建了Luffin-a原核表达载体。
Objective To investigate the construction of prokaryotic expression vector of Luffin-a gene of loofah ribosome inactivation protein. Methods The full-length mRNA of Luffin-a was obtained from Pubmed. The Luffin-a gene was cloned from immature loofah seeds by RT-PCR. The TAffin was identified by TA cloning. The prokaryotic expression vector pET-15b +) - Luffin-a was transformed into E.coli BL21 (DE3) and induced by IPTG. The expression product of Luffin-a was identified by SDS-PAGE. Results The Luffin-a gene was cloned. The expression of Luffin-a was detected in both Escherichia coli culture medium and sonicated cell pellet after induced by IPTG. Conclusion This study successfully constructed Luffin-a prokaryotic expression vector.