Establishment And Purification Of High Expression Cell Lines In Serum-free Suspension Of HSV-2(gD)

Herpes simplex virus (HSV) as the first discovered human herpes virus has ability to establish latency infection and later reactivate to cause disease intermittently. HSV is divided into two types of HSV-1and HSV-2. HSV-1mainly results in oral surface and ocular infection as well as herpes encephalitis, while HSV-2mainly results in genital ulcerations often accompanying urethritis, cystitis or cervicitis which can happen in any stage of lifetime. As parasitic viruses, HSV establish lifelong latency in human ganglion cells and it is difficult to be removed effectively from hosts because they can escape the host immune response. Antiviral drugs such as acyclovir, can partially inhibit replication and shorten the course of infection, but they can’t fundamentally control the virus latent, spread and recurrence. Therefore, the ideal way to prevent HSV virus infection is developing a safe and effective HSV vaccine which can induce strong humoral immune response and specific cellular immune response.HSV-2encodes at least12glycoproteins. gD, gB and gC are the most abundantly expressed proteins on the surfaces of the infected cells and constitute the viral envelope. Among these proteins, gD plays an important role in virus adsorption and entrance through interacting with the cellular surface protein receptors. gD with important epitopes, is the major target for humoral and cellular immune response against HSV infection. Therefore, the glycoprotein gD is an ideal candidate antigen for HSV vaccine.In this study, the extracellular amino acid sequence of the gD protein of HSV-2 was screened for epitope-rich regions. After selecting the codons which are favoured by both eukaryotic and prokaryotic cells, DNA encoding the selected gD protein fragment was synthesized and cloned into eukaryotic expression vector pMD902. The recombinant plasmid pMD902-gD were transfected into CHO-DG44cells and gradually increased the concentration of MTX (methotrexate) to screen cell lines of high expression of the gD glycoprotein detected by Dot blot and SDS-PAGE. Cultivation the cell line selected before in large-scale, purified cell culture supernatant containing the recombinant proteins using ion exchange chromatography and gel filtration chromatography. The purity and the concentration of the purified protein was determined by high performance liquid chromatography (HPLC) and the Bradford method respectively. The antigenicity and immunogenicity of the purified gD protein were measured by ELISA and by immunizing BALB/C mice with it.The experimental results show that cell lines capable of expressing high levels of the gD extracellular fragment was established after a series of optimization and MTX pressure screening. Importantly, only one band about44kDa in size corresponding to the gD extracellular fragment was observed by Western blot analysis, indicating that glycosylation of the recombinant gD protein was uniform. The established purification process was very convenient and involved just one step of anion exchange column and desalting. The purity of the purified gD protein was as high as95%, the concentration approximately1.9mg/mL and the yield was up to57mg from1L of serum-free culture medium. The purified gD protein not only reacted with the anti-HSV-2positive sera but also induced strong, specific humoral immune response. The serum antibody titer of immune mice was≥1:5000, shown that the expression of recombinant gD has good immunogenicity. Our results lay the foundation for preparation of large amounts of the gD protein that are necessary for HSV-2vaccine development.