Establishment And Purification Of HSV-2 GC Protein Serum-free Suspension High Expression Cell Line

Herpes simplex virus type II is a sexually transmitted pathogen that can cause genital herpes,neonatal herpes encephalitis,and pregnant women's premature delivery or abortion.It has high prevalence rate,and can cause subclinical infection and latent infection.Vaccination is the best way to prevent HSV-2 infection.HSV-2 is composed of core,capsid,envelop,and tegument.The envelop contains at least 11 glycoproteins,including gB,gC,gD,gE,and so on.gB and gD,which are the most abundant,have been tested their protective efficacy as vaccines in clinical trials.However,the results were regrettable.The third abundant glycoprotein gC₂,associated with immune evasion of HSV2,is thought to be a hopeful vaccine candidate.Establishment of a stable eukaryotic cell line to overexpress gC₂ and an efficient purification process is essential for the development of subunit vaccine against HSV-2.The DNA sequence of extracellular region of gC₂ was optimized,chemically synthesized,and cloned into plasmid pMCE5 or pcDNA3.1?+?.The recombinant plasmid pMCE5-gC₂ was transiently transfected into CHO-DG44 cells to evaluate whether it can be expressed as a secretory protein.The stable cells expressing gC₂ protein was screened by dot blot,and the expression level was enhanced by gradually increasing the concentration of MTX.Expression-stablity analysis and fed-batch cultivation were used to determine the cell strain with the highest yield and quality.The cell culture from the screened cells were collected and purified with different methods.The yield of the purified gC₂ protein was determined by BCA kit,the purity was determined by HPLC,SDS-PAGE and western blot.By electrophoresis analysis of PCR products and restriction enzyme digestion of the two recombinant plasmid pMCE5-gC₂ and pcDNA3.1?+?-gC₂,the two recombinant plasmids harboring gC₂ gene were constructed successfully.The recombinant plasmid pMCE5-gC₂ was transfected into CHO-DG44 cells.The expressed gC₂ protein as a secretory preotein was confirmed by Western blot analysis,its molecular weight is about 70 kDa.The stable expression cell lines was screened by gradually increasing the concentration of MTX and limited dilution method continually.Fed-batch cultivation and stability assay showed 3 clones,?including clone 8,clone 16,and clone 24?could express gC protein efficiently and stablely.The expressed gC protein was purified with 3 different methods,including cation exchange chromatography?buffer: 25 mM Tris-HCl,pH9.0,or buffer: 20 mM PB,pH7.0?,and anion exchange chromatography?buffer: 25 M Tris-HCl,pH8.5?.The anion exchange chromatography was proved to be the best purification method.In order to prepare a lot of gC₂ protein,the gC₂ protein was purified by anion exchange column?buffer: 25 mM Tris-HCl,pH8.5?,followed by an cation exchange column?buffer:25mM Tris-HCl,pH8.0?.SDS-PAGE and western blot analysis of the purified protein revealed two bands of gC₂,one in 70 kDa,and another in 50 kDa.Two batch of gC₂ protein were purified,one with a purity of 98.4%,and total of 90 mg,and the other with a purity of 96.8%,and total of 30 mg.In this study,the CHO-DG44 cell lines expressing HSV-2 gC protein stablely and effccienly was constructed successfully,and the gC₂ preotin with high purity was obtained by optimiaing the purification process.This study laid a foundation for developing HSV subunit vaccine and detection reagent.