蜂毒肽与变构hIL-2融合基因原核表达载体的构建

Construction of prokaryotic expression vector of melittin fused with a hIL-2 mutant

目的构建蜂毒肽与人变构白细胞介素-2原核表达载体，为进一步研究该融合基因而奠定基础。方法以本室构建质粒pGEX-4T-2／Melittin-IL-2（^88Arg）为模板，设计引物，用PCR定点突变的方法将IL-2部分的^125Cys突变为Ala，PCR产物与pMD18-T连接，转化大肠杆菌，小提质粒，DNA测序后再将目的片段连接于pET-15b，最后酶切鉴定。结果PCR产物542bp，构建质粒双酶切、DNA测序鉴定如预期。结论成功构建蜂毒肽与人变构白细胞介素-2原核表达载体。

Objective To construct the prokaryotic expression vector of melittin fused with an hIL-2 mutant. Methods Using pGEX-4T-2/Melittin-IL-2 （^88Arg） as the template, a mutant M-IL-2 （^88Arg, ^125Ala） was generated by PCR with point mutations of 125Cys to Ala. PCR products were inserted into pMD18-T vector and sequenced, then M-IL-2 （^88Arg, ^125Ala） was transferred from pMD18-T to pET-15b to generate pET-15b/M-IL-2 （^88Arg, ^125Ala）. Results 542bp long fragments were generated by PCR and the sequence was confirmed by sequencing, the electrophoresis displayed the expected bands for pET-15b/M-IL-2. Conclusions the prokaryotic expression vector of melittin fused with a hIL-2 mutant was successfully constructed.