Construction Of Herpes Simplex Virus Ⅱ DNA Vaccine And Study Of Its Immunological Characteristics

Herpes Simplex Virus II (HSV-2) mainly causes genital tract infection at the mucosal surface and is. called genital herpes (GH) in clinic. In recent years, Occurrence of GH has increased greatly, and GH has become critical sex transimitted disease (STD) in many countries and districts. It is also one of eight STD in China. Following the initial infection, HSV-2 becomes latent in the proximal ganglion and can reactivate for life. HSV-2 can be transmitted easily through sex. It can form subclinical, latent and recurrent infection. There are no any anti-virus drugs, which can efficiently control the occurrence and recrudescence of HSV-2. Vaccine is the most promising approach to prevention and treatment against GH. DNA vaccine, which can fully induce immune response, is the one of most optimising vaccines against HSV-2.In the research, the protective antigen coding gene gD2 (Arg92-Ala302) of HSV-2 wild strain was cloned, which had been isolated from clinic specimen. The recombinant eucaryotic expression plasmid, pVAX-gD, was constructed. Expression of pVAX-gD in vitro was investigated. The ability of pVAX-gD to induce immune response in animals and the protective effect to animals were studied.A HSV-2 wild strain from clinic specimen was isolated and identified with tissue culture methods and diagnosis of transmission electron microscope, which caused serious recrudescence. PCR primers were designed according to DNA sequence of HSV-2 glycoprotein D (gD2) offered by Gene Bank. The full length gD gene of wild strain was specially amplified and cloned into pUCm-T vector. Sequencing results showed that the homology of nucleotide and amino acid between the gD of wild strain and HSV-1 gD were 84.6% and 82.7%, and that the homology of nucleotide and amino acid between the gD of wild strain and HSV-2 gD were 98.4% and 95.7%, respectively.BIMAS and SYFPEITH were used to predict antigen epitope of gD2 protein. Antigen epitopes recognized by MHC I , MHC II allele were mainly located in Arg92-Ala302 fragment of gD2 protein. gD2 (Arg92-Ala302) gene fragment was amplified by PCR and ligated into the eucaryotic expression vector pVAXl. Identified by enzyme digestion, the recombinant plasmid pVAX-gD was obtained.The recombinant plasmid pVAX-gD was transfected into mammalian cell COS-7, mRNA of gD2 gene fragment could be detected by RT-PCR in transfected cell. The results of SDS-PAGE and RT-PCR showed that there was a protein band about 23kD in...