牛乳铁蛋白素基因及其突变基因在大肠埃希菌中的融合表达

Fusion expression of lactoferricin bovine gene and its mutant gene in Escherichia coli

目的构建牛乳铁蛋白素基因(LfcinB)及牛乳铁蛋白素突变基因(mLfcinB)的表达载体,建立在大肠埃希菌中融合表达的方法。方法依据大肠埃希菌偏爱密码子设计LfcinB和mLfcinB,进行克隆扩增并测序,构建LfcinB和mLfcinB的融合表达载体并转化入大肠埃希菌,经异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,产物经谷胱甘肽-琼脂糖凝胶法纯化。结果成功构建了融合表达载体pGEX-4T-1-LfcinB和pGEX-4T-1-mLfcinB,表达载体转化大肠埃希菌BL21,经IPTG诱导3 h后LfcinB和mLfcinB融合蛋白表达率约为30%,洗脱纯化后产物约为60%。聚丙烯酰氨凝胶电泳显示融合蛋白为29 kDa,主要以包涵体形式存在,谷胱甘肽-琼脂糖凝胶纯化后得到3.1 kDa的目的蛋白。结论LfcinB及其突变基因在大肠埃希菌中的融合表达可提高LfcinB的表达率。

Objective To construct the expression vector for lactoferricin bovine gene （LfcinB） and lactoferricin bovine mutant gene （mLfcinB） and establish its methods for fusion expression in the Escherichia coli BL21. Methods LfcinB and mLfcinB gene were designed according to the Escherichia coli codon preference and then cloned and sequenced. LfcinB and mLfcinB fusion gene expression vector were built and transformed into Escherichia coli BL21, then, by isopropyl-β-D- thiogalactopyranoside （IPTG） induced expression, and the products were purified by glutathioneagarose gel purification. Results Fusion expression vectors pGEX-4T-1-LfcinB and pGEX-4T- 1-mLfcinB were constructed successfully, and then were transformed into Eseherichia coli BL21. Fusion protein of LfcinB and mLfcinB had the highest yield induced by IPTG after 3 h. Expression rate was about 30%, and the eluted purified product was about 60%. Poly-acrylic amide gel electrophoresis showed the weight of fusion protein were 29 kDa, and mainly existed in the form of inclusion bodies. Finally, the target protein of 3.1 kDa was obtained after glutathione-agarose gel purification. Conclusion LfcinB and mLfcinB fusion gene expression in Escherichia coli can increase the expression rate of LfcinB.