使用一种新策略在大肠杆菌中高效表达hbFGF(英文)

Cloning and High Expression of hbFGF with a New Strategy

翻译起始区 (TIR)的二级结构是影响翻译效率的决定性因素 ,同时密码子的偏好性问题也是个至关重要的方面。基于以上两点考虑 ,对hbFGF 5’末端 35个碱基进行了改造 ,对其中 4个位点进行了定点突变 ,另有 4个位点进行了随机点突变。这些突变都可能造成TIR二级结构变化。这 4个随机突变共有 32种组合 ,使用RNA结构预测软件DNASISv2 .5对这 32种序列分别模拟其二级结构且计算其自由能 ,并选取了 10条自由能最高的序列。根据这10条序列 ,分别设计引物引入突变 ,克隆至表达载体pET 3c上 ,然后转化宿主菌E .coli,通过诱导表达纯化及生物测活等常规实验方法 ,最后确定有两株为高表达菌株 ,从某种程度上证明了用计算机辅助设计定点突变的方法来优化外源基因在E .coli中的表达是有效且很有潜力的。

Computer program DNASIS v2.5 was used to help designing the site directed mutations for optimizing the expression of hbFGF in E.coli. The secondary structure of the translation initiation region (TIR) is a determinant factor for translation initiation rate, meanwhile, codon preference plays an important role, too. According to the two principles, 4 sites in 5'end of hbFGF cDNA were definitely changed, and another 4 sites randomly changed. These mutations will lead to potential variation in the secondary structure of TIR. Then computer program DNASIS v2.5 was utilized to analyse the total 32 TIR sequences resulted from the combination of the 4 randomly mutated sites. Ten sequences with highest free formation energy ( Δ G 0) were chosen for subsequent cloning. By PCR using synthetic primers containing the 8 changed sites described above, ten hbFGF cDNA were amplified and cloned to pET 3c respectively. E.coli strain BL21(DE3) was transformed and induced to express recombinant hbFGF. Two high expression clones were obtained by SDS PAGE and MTT assay, indicating that computer program aided design for optimizing expression of foreign genes in E.coli is useful.