FIV gp40七肽重复区基因的克隆表达及其产物的纯化

Cloning and Expression of Heptad Repeat Region of the Feline immunodeficiency virus and Product Purification

本研究利用LearnCoil-VMF程序预测到FIVEnv蛋白gp40存在两个七肽重复区(Heptadrepeat,HR1和HR2),对包括HR1和HR2在内的部分gp40胞外区基因(称为HR1-HR2)进行了人工合成,以此基因为模板获得了HR1和HR2基因的扩增产物,同时构建了用氨基酸连接子SGGRGG将HR1和HR2连接起来的串联基因(即HR1linkerHR2,命名为2-Helix),采用大肠杆菌GST融合表达系统对HR1、HR2和2-Helix蛋白进行了表达,并对2-Helix进行了纯化。同时利用凝胶过滤层析证明2-Helix在PBS缓冲系统中以寡聚体的形式存在。

By using a computational program, Learn Coil-VMF, we determined the two conserved heptad repeat regions (HR1 and HR2) of FIV gp40 protein. The gene of gp40 subdomain including HR1 and HR2 was constructed with PCR method. The HR1 and HR2 were subsequently expressed and purified as a single chain (named 2-Helix) connected by a six aminoacid linker in E. coli GST fusion expression system. The single HR1 and HR2 were also cloned and expressed in the same way. Subsequently the 2-Helix′s formation of oligomer was tested. The results showed that the HR1 and HR2 interacted with each other and formed an oligomer.