Purification, Identification, And Immunogenicity Analysis Of Three Herpes Simplex Virus Glycoproteins Expressed In Mammalian Cell Lines

Herpes simplex virus (HSV) infections have become increasingly common worldwide, and can happen in any stage of lifetime. HSV I mainly results in oral surface and ocular infection as well as herpes encephalitis while HSV II represents the major cause of genital ulcerations. In addition to the pain and discomfort associated with the disease, genital herpes causes psychological morbidity, may increase the risk of acquiring human immunode?ciency infections, and can be spread to susceptible sex partners, the fetus or newborn infants. Although antiviral agents as acyclovir have been effective in limiting the severity of genital lesions, they do not, however, prevent the establishment of latent infection, nor prevent recurrent herpetic disease. Therefore, the development of alternative or complementary therapies for preventing or treating this disease would be a major public health advance. One of these alternative approaches could be the development of a safe and effective HSV vaccine.Envelope proteins of HSV play a vital role not only in the infection process of adsorption and invasion but also in the stimulation to the organism that gives rise to immune response. Among these proteins, glycoprotein B (gB) and glycoprotein D (gD), with important epitopes, which can induce specific immune response, are the primary targets of humoral and cellular immunity of the host. Therefore, gB and gD are the ideal target proteins in the development of HSV vaccine.In this research, antigenic epitopes of gB1, gD1 and gD2 were analyzed to screen epitope mass region, and meanwhile, RNA structure of the genes and codon preference of eukaryotic cell were optimized. The optimized genes were chemically synthesized and cloned into eukaryotic expression vector pCEP4. CHO cells were transfected with the recombinant vectors (pCEP4–gB1, pCEP4–gD1, and pCEP4–gD2) with the purpose of recombinant proteins expression. 72 hours after transfection, the recombinant proteins secreted into cell supernatant were detected by using Western blotting method. The purification of the proteins was conducted by using immobilized metal affinity chromatography. ELISA was used to detect the antigenicity of the proteins. Kunming mice were immunized with the recombinant proteins to assay the immunogenicity. Antiserum was used for neutralization assays. Furthermore, CHO stable cell line was transfected with the recombinant vectors (pCEP4–gB1, pCEP4–gD1, and pCEP4–gD2) with the purpose of continuously expressing recombinant proteins. Sub clone was carried out to screen the cell lines which have a high productivity.Gene sequencing analysis demonstrated that the recombinant plasmid vectors (pCEP4–gB1, pCEP4–gD1, and pCEP4–gD2) were constructed successfully. Western blotting analysis indicated the molecular weights of the proteins are approximate 85kDa, 46kDa, and 46kDa respectively. The concentrations of the proteins were 95 ug/mL, 95 ug/mL, and 50 ug/mL respectively. In addition, ELISA assays showed that the recombinant proteins have good antigenicity and immunogenicity. Neutralization assays showed that the specific antibodies can protect host cells against the attack of HSV. After the screening of CHO stable cell lines, six high productivity cell lines were established.