Screening And Identification Of Glycoprotein B Epitopes And Eukaryotic Expression Of Glycoprotein D Derived From Herpes Simplex Virus-2

Herpes simplex virus 2 (HSV-2) infection may result in a common and ancient of sexually transmitted diseases - genital herpes. The incidence of HSV-2 infection is following gonorrhea and syphilis among sexually transmitted diseases. Thus, HSV-2 infection caused wide concern.HSV infection is a major health concern worldwide. Evidence obtained from both animals and humans indicated that B- and T-cell responses contribute to protective immunity against HSV-2 infection. Glycoprotein B is a transmembrane envelope component of herpes simplex virus 1 (HSV-1) and HSV-2, which plays an important role in virion morphogenesis and the penetration into host cells, and can induce neutralizing antibodies and protective T cell response successfully when both human and animals were immunized. Thus, the HSV-2 gB sequence was screened using B- and T-cell epitope prediction systems, the B-cell epitopes and the HLA-A*0201-restricted epitopes were identified. These B-cell epitopes elicited high IgG antibody titers in Balb/c mice, with a predominantly IgG1 subclass distribution indicating Th2 bias. Further, specfic IgGs induced by these two epitopes were evaluated as the neutralizing antibodies for virus neutralization. Using several immune parameters, these evaluated T-cell epitopes can indude stabilization of HLA-A*0201 molecules on T2 cells by gB peptides, in vitro functional IFN-γELISPOT and cytotoxic CD8+ T cell assays. The results indicated that the predicted T-cell epitopes could stabilize the HLA-A*0201 molecules on T2 cells, and stimulate IFN-γ-secreting and the formation of cytotoxic CD8+ T cells.HSV-2 DNA was extracted and used as template in polymerase chain reactions to amplify gD gene. The PCR product was cloned into the donor plasmid pFastBacⅠ. After the transformation and selection, the recombinant plasmid was sequenced. Then, the pFastBacⅠ-gD was introduced into the competent cells E. coli DH10Bac, which containing a shuttel vector, bacmid. The recombinant bacmid DNA was isolated and transfected into the insect cells Sf9 to produce the first generation recombinant virus. The insect Sf9 cells were infected with the recombinant virus to express the target protein. The expressed protein was detected by SDS-PAGE and Western blot. The gD gene was successfully expressed by Bac-to-Bac? Baculovirus Expression System. This work made a base for the further study in the future.