Expression Of Recombinant Human Cytomegalovirus Gene Of Tegument Protein

Human cytomegalovirus (HCMV) infection is common all over the world most with the performance of subclinical infection or latent infection. It usually brings serious damages for pregnant women, fetus and people with low immunity. HCMV drugs can partly improve correlation disease, but at the same time they have side effects and drug resistance problems. Therefore anti HCMV vaccine is in urgent need. An ideal HCMV vaccine should contain as much as possible with the immune protective antigen composition which can induce broad-spectrum neutralizing antibody(NAb) response and cytotoxic T lymphocyte(CTL) response, and also can prevent primary infection and reduce the morbidity of HCMV infection and related disease, then has good tolerance and long-term security. HCMV is an intracellular infection virus, and it can be completely removed only by cellular immunity. Regular preventive vaccine for infected individuals cannot induce effective immune responses, so new therapeutic vaccines need to be researched and developed to remove pathogens and abnormal cells to cure diseases. In respect of HCMV-mediated infection and viral replication, glycoprotein B (gB) and phosphoprotein65(pp65) are two important proteins. gB is the major antigen to induce neutralizing antibodies, while pp65is the major antigen to induce cellular immunity.In this study, we fused pp65362-504and gB607-621effectively by overlap PCR and cloned the fusion gene into the expression vector pFLAG-ATS for generating pFLAG-ATS-pp65362-504-gB607-621-Fusion gene was not expressed by several different induction conditions in the E.coli. In order to achieve the purpose of the soluble expression of fusion protein, Bac-to-Bac expression system was used. A vector was constructed, which carries a reporter gene encoding the enhanced green fluorescent protein (EGFP) and a sequence encoding the fusion gene pp65362-504-gB607-62i with six-histidine tag, under control of the p1O and PH promoter respectively, and the expression of pp65362-504-gB607-621was investigated in insect cells. The fusion gene pp65362-504-gB607-621was obtained by PCR, and recombined into donor vector pFastBacTM ual-EGFP to generate pFastBacTM ual-EGFP-pp65362.504-gB607-621. The orientation and sequence of the recombinant plasmid were confirmed by restriction enzyme double digestion and sequencing. The recombinant plasmid was transformed into E. coil DH10Bac generating recombinant bacmid-EGFP-pp65362-504-gB607-621verified by PCR. Then the recombinant vector was transfected into the insect cell line Spodoptera frugiperda9(Sf9) by lipofection and the expression of pp65362.504-gB607-621was detected by SDS-PAGE and Western Blot. The results demonstrated that the gene of HCMV pp65362-504-gB607-621can be soluble expressed in Sf9cells with reactogenicity.