| Herpers simplex virus(HSV)is one of the common viruses which can infect human.According to serotype,HSV can be divided into two types,type ? and type?.Type ? is mainly transmitted orofacially,while sexual transmission also exists.Type ?,is mainly transmitted sexually.HSV is prevalent all over the world.The characteristics of HSV,which are latent infection,recurrent attack and lifelong carrier,are related to its mechanism of immune evasion.HSV achieves latent and recurrent infection by avoiding the attack of human immune system.Therefore,once sufferers are infected with HSV,they will be tortured by long-term disease,since HSV is hard to get rid of.Glycoproteins(gp)is the antigenic determinant on the surface of virions.The glycoprotein of gD has already been selected as a candidate vaccine,for its participation in the adsorption and fusion process on the virus entry into host cells.According to research findings in recent years,envelope glycoprotein of gC2 is related to immune evasion.This research mainly focuses on the immune effects of three recombinant glycoproteins of gC2,gD1 and gD2.This research analyzed the protein antigen of HSV-2 gC2,the epitope-rich extracellular fragment of HSV gC2 was selected,the gene was synthesized and cloned into eukaryotic expression vector pMCE5,the recombinant plasmid pMCE5-gC2 was transfected into CHO cells.The expressed protein gC2 was characterized by Western-Blots and purified with His TrapTM FF crude column.As immunogen,the purified gC2 protein,as well as recombinant protein gD1,gD2,was synergized with freund's adjuvant and Al(OH)3 adjuvant respectively for sole vaccination and combined vaccination.PBS was set as the control group.Groups were as follows:group A1(gC2+ freund's adjuvant);group A2(gC2+ Al(OH)3);group B1(gD1+freund's adjuvant);group B2(gDl+Al(OH)3);group C1(gD2+freund's adjuvant);group C2(gD2+Al(OH)3);group D1(gC2+gD1+gD2+freund's adjuvant);group D2(gC2+gDl+gD2+Al(OH)3);group E1(PBS+freund's adjuvant);group E2(PBS+Al(OH)3).Immunized mice three times,anti-gC2 IgG antibody in serum was detection by ELISA,and the frequencies of Interferon-?(IFN-?)and Interleukin-4(IL-4)secreting cells were assessed by enzyme-linked immunospot(ELISPOT).The result showed that the synthesized gene fragment gC2 was cloned into vector pMCE5 and expressed in CHO cells successfully,the expressed and purified gC2 protein was detected with an estimated molecular weight of 80kDa on SDS-PAGE and Western-Blots(Concentration:40?g/ml).Immunized three kinds of recombinant proteins—antigen—three times,obtained serum and detected the antibody titer of serum in different groups.Analysis showed that all the antibody titer of serum in groups A-D got high titers.the antibody titer of serum in groups with adjuvant Al(OH)3 was higher than that in groups with freund's adjuvant.among groups with freund's adjuvant and adjuvant Al(OH)3,the antibody titer produced by recombinant protein gC2 was higher than that produced by gDl and gD2;the antibody titers of sole vaccination group were higher than combined vaccination group.The results indicated that the expressed recombinant protein HSV gC2,gDl,gD2 in CHO cells could induce strong humoral immune response.With 3 kinds of recombinant proteins working as stimulus,the frequencies of IL-4 was higher than that of IFN-y,and the effect of 3 different proteins on cellular immunity was that gDl>gD2>gC2.Moreover,among groups with freund's adjuvant,the effect of sole vaccination was better than that of combined vaccination;among groups with adjuvant Al(OH)3,the effect of combined vaccination was better than that of sole vaccination.By means of two different vaccinations,and two adjuvants formulation,this research analyzed the immune effect of recombinant proteins of gC2,gD1 and gD2,which laid a foundation for serological diagnosis and vaccine study of HSV. |
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