| Herpes simplex virus type II is the principal cause of genital herpes. In recent years, morbidity of genital herpes has been rising heavely, and its impairment becomes severe increasingly. But now there are no workable medicine to control its infection and recurrence. It is crucial to develop vaccine to prevent genital herpes. Although immune responses induced by DNA vaccine are lower, DNA vaccine is the most favorable among the developed vaccines, for it is safe and simple to make. Furthermore, DNA vaccine can effectively stimulate cell-mediated and humoral immune responses, and can coexpress with cytokines to induce the most appropriate immune responses on the basis of need. Recently, foreign scholars have been working for enhancing immune responses of DNA vaccine by using molecular adjuvant, and had got some achievement. In order to construct effective DNA vaccine to prevent HSV-II infection and explore modulation mechanisms of immunity of molecular adjuvant, we have made the following works in this study: 1. Construction of DNA Vaccine of Herpes Simplex Virus Type II gD:According to HSV-II strain HG52 gD gene sequence in GenBank, a pair of primers were designed and the whole encoding sequence of HSV-II strain Sav gDwas amplified by PCR. The PCR product was inserted into pUCm-T vector. Subsequently it was digested by restriction endonucleases HindIII and XbaI, and inserted orientally into mammlian vector pcDNA3 at corresponding sites. Thus the recombinant was transformed into E.coli DH5 a . Identified by PCR, restriction enzymes analysis and sequencing, recombinant eukaryotic expression plasmid was named Pg. When Pg was transiently transfected into COS-7 cell, specific recombinant protein gD was demonstrated by in situ ELISA with specific gD McAb. It indicated that Pg was able to express in mammalian cells.2. Construction of recombinant eukaryotic expression plasmid of murine chemokine MIP-1αThe total RNA was extracted from LPS-stimulated RAW264.7 cell before the whole encoding sequence of MIP-1 a was amplified by RT-PCR. The RT-PCR product was inserted into pUCm-T vector. After RT-PCR product was digested by restriction endonucleases Hindlll and Xbal in pUCm-T vector, it was inserted orientally into mammlian vector pcDNA3 at corresponding sites. Then the recombinant was transformed into E.coli DH5 a . Identified by PCR, restriction enzymes analysis and sequencing , recombinant eukaryotic expression plasmid was named Pm. Specific recombinant protein rMIP-1α transiently expressed in COS-7 cell was demonstrated by RT-PCR and boyden chemotaxis chamber. It suggested that Pm be able to express in mammalian cells.3. Assessment for protective effects of Pg +Pm DNA vaccine on miceMice were injected in the quadriceps with 200ug Pg, Pg+Pm in a final volume of 200ul of NS separately. The control mice were immunized with pcDNA3 and NS, respectively. Immunization was made for three times. One month after the last immunization, mice were challenged intravaginally with 100LD50 of HSV-II strainSav. Mice were then examined daily to evaluate pathological conditions and survival rates within 14 days. The results showed that Pg+Pm DNA vaccine improved, the survival rate of mice and reduced both the number of mice with lesions and severity of herpetic lesions significantly. 4. Evaluation of the immune responses induced by Pg +Pm DNA vaccine:To detect the specific antibody titers by ELISA and microneutralization test, we collected serum at the second week and the fouth week after the last immunization. Lymphocyte transformation test (LTT) was made using MTT in the first month after the last immunization. Pg+Pm DNA vaccine resulted in a moderate but not significant enhancement of gD-specific IgG levels and induced significant enhancement of Th-cell proliferation responses. These results illustrated that Pg+Pm DNA vaccine induced higher antigen-specific cell-mediated immune responses than Pg DNA vaccine alone.In sum, we have successfully constructed the DNA vaccine of HSV-II strain Sav gD with MIP-1α...
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