丝瓜籽核糖体失活蛋白基因Luffin-a原核表达载体的构建

Construction of prokaryotic expression vector for ribosome inactivating protein gene Luffin-a of Luffa seeds

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 method of construction of prokaryotic expression vector for ribosome inactivating protein gene Luffin-a of Luffa seeds. Methods Whole mRNA sequence of Luffin-a was found from Pubmed database, Luffin-a gene from immature Luffa seeds was cloned by RT-PCR method, and the sequence of nucleotides was identified after T-A clone, then the prokaryotic expression vector of pET-15 b （＋）-Luffin-a was constructed, the vec tor was transfected into E. coli BL21 （DE3）, and the expression was inducted by IPTG. Luffin-a expression product was identified by SDS-page. Results Luffin-a gene was cloned. Luffin-a expression product was detected in the supernatant of E. coli culture solution and ultrasonic broken bacteria after IPTG induction. Conclusion This study successfully constructs prokaryotic expression vector of Luffin-a.