人巨细胞病毒pp65蛋白的原核表达及活性鉴定

Expression and Immunoreactivity of Human Cytomegalovirus pp65 Protein in E. coli

以病毒全基因组为模板,通过PCR技术扩增HCMV pp65基因编码序列,其产物连接到pMD18-T质粒,酶切并回收目的片段后克隆到表达载体pTO-T7,然后将重组质粒pTO-T7-pp65转化大肠杆菌ER2566,大量表达后将重组蛋白进行质谱法分析鉴定,再利用Western blot以及间接ELISA进行重组蛋白的活性及抗原性鉴定.结果显示:通过SDS-PAGE鉴定可知,大肠杆菌可能表达出了重组蛋白,质谱分析结果说明了重组蛋白为pp65蛋白,具有极高可信度,其相对分子质量约为63 kD,从Western blot和间接ELISA(enzyme linked immunosorbent assay)的结果可知,pp65蛋白作为抗原具有良好的免疫反应性与免疫原性,为制备相应的抗原诊断单克隆抗体打下了基础,同时也为开发HCMV IgG快速诊断ELISA试剂盒提供了可选原料.

HCMV pp65 protein coding sequence was amplified by PCR using HCMV genome DNA as tem- plate. The PCR products were ligated to pMD18-T vector, and then cut off by restriction enzyme and further ligated into the same enzyme digested multiple cloning site of pTO-TV expression vector. The recombinant expression vectors were transformed into E. coli ER2566 strains. After induction, recombinant protein expres- sion was confirmed by the mass spectrometry. The activity and immunoreactivity of pp65 protein were as- sayed by Western blot and indirect ELISA. The SDS-PAGE indicated the possible expression of recombinant pp65 protein in E. coli, and mass spectrometry examination further confirmed that the expressed recombi- nant protein was HCMV pp65 protein. The molecular weight of recombinant pp65 protein is 63 kD. Western blot and indirect ELISA results revealed that the recombinant pp65 protein had significant immunoreactivity and immunogenicity as antigen, which provided a basis for further developing corresponding monoelonal antibody for antigen diagnostic and supplying raw materials for rapid HCMV IgG ELISA kit.