白念珠菌MP65和SAP2蛋白原核表达质粒的构建与表达

Construction and expression of prokaryotic expression plasmid of MP65 and SAP2 in Candida albicans

目的构建以白念珠菌基因MP65和SAP2为目的基因的原核表达质粒,IPTG诱导其表达融合蛋白,并对其免疫原性进行分析。方法PCR法自白念珠菌标准菌株获取MP65和SAP2基因,分别插入至原核表达载体pGEX-4T-2和pET32a中;将重组表达质粒转染感受态E.coliBL-21,经IPTG诱导表达、纯化后,SDS-PAGE和Western blot分析。结果PCR法克隆出全长为1 140 bp的MP65和1 197 bp的SAP2基因,构建的原核表达质粒pGEX-4T-2-MP65及pET32a-SAP2,可分别表达出66 KD左右的GST融合蛋白和His融合蛋白。结论成功获取了白念珠菌基因MP65和SAP2,所构建的原核表达质粒在BL-21中成功表达;两种蛋白均有免疫原性。

Objective To construct prokaryotic expression plasmids of MP 65 and SAP 2 genes in Candida albicans and to exam- ine the immunogenicity of the fusion prolein expressed by the plasmids. Methods Using PCR, MP 65 and SAP 2 were amplified from a standard strain of Candida albicans , then inserted into prokaryotic expression vector pGEX4T-2 and pET32a separately. GST- MP65 and His-SAP2 were expressed in E. coli BL-21 following induction by IPTG. The protein was analyzed by SDS-PAGE and West- ern blotting. Results The genes of MP 65 （1 140 bp） and SAP 2 （1 197 bp） were amplified by PCR. The pGEX-4T-2-MP65 and pET32a-SAP2 had been constructed and successfully expressed GST-MP65 and His-SAP2 separately in E. coli BL-21. Conclusions MP 65 and SAP 2 in Candida albicans were successfully amplified and the prokaryotie expression peasmids were expressed in E. coli BL-21. Both of the two proteins had immunogenicity.