Construction And Expression Of HSV-1 GC Glycoprotein Eukaryotic Expression Vectors

The prevalence of HSV infection is considerably worldwide. HSV-1 is a common cause of mucosal herpes, herpetic keratitis and encephalitis. Herpetic keratitis is the leading infectious cause of blindness. Encephalitis is often accompanied by severe neurologic sequelae. Herpes genitalis is a sexually transmitted infection caused primarily by HSV-2. HSV infection increases the rate of human immunodeficiency virus (HIV) infection and transmission, which can cause acquired immune deficiency syndrome (AIDS). Until now, no treatment has show obvious efficacy to HSV infection. Drugs which can restrain the viral DNA replication used in the clinical, can not eradicate HSV infection and easy to produce drug resistance. Vaccination has remained the best method for preventing virus spread, but HSV vaccine is still under preclinical phase. One reason is the virus can provide a survival advantage in vivo by immune evasion. HSV gC is a C3b binding complement regulatory protein while gE/gI form a complex that binds the Fc domain of IgG, inhibiting activation of complement and ADCC. Recent studies show that combined immunization with gC-1 and gD-1 conferred higher protection in mice than gD-1 alone, suggesting gC-1 may b e u s e d t o e n h a n c e t h e i m m u n e e f f e c t o f t h e v a c c i n e . 1. Construction of eukaryotic expression vector PCI-neo-MARs-mCMV-gC1 -IRES-DHFR-L22RWe constituted plasmid PCI-neo-MARs-mCMV using MARs-mCMV to replace the prior promoter hCMV. The DHFR-L22R gene was constructed (the code CTT was substituted for the code CGG which encoded amino acid from Leu to Arg). HSV-1 gC gene, IRES gene, DHFR-L22R gene were cloned into eukaryotic expression vector PCI-neo-MARs-mCMV. The recombinant plasmid PCI-neo-MARs-mCMV-gC1-IRES-DHFR-L22R was got and digested with restriction endonucleases. A large amount of the recombinant plasmid was prepared and purified.2. Stable and high level expression of the herpes simplex virus type 1 (HSV-1) glycoprotein C (gC) gene in CHO-K1 and Sp2/0 cells.The recombinant plasmid was transfected into CHO-K1 and SP2/0 cells by the instruction of Lipofectamine^TM 2000 for stable expression. The stable expression cell strains were selected by G418 and methotrexate (MTX). CHO-K1 and SP2/0 cells stably expressing HSV-1 gC protein were established and detected by Slot blot.3. Abundant expression and identification of HSV gC-1 proteinStably transfected cells were cultivated abundantly, and the cultured supernatants was collected and concentrated. The expressed gC-1 protein was purified with His-Ni Sepharose column. After purification, gC-1 protein exhibited a major Coomassie blue-stained band on SDS-polyacrylamide gels and was detected by Western analysis with anti-His mAb. The results showed that the gC-1 protein was expressed and it has a specific binding bioactivity.