| Acquired Immunodeficiency Syndrome (AIDS) is caused by Human immunodeficiency virus(HIV).Presently, the prevalence of AIDS has become one of most serous threats to Human's health and development .It is proved that a safe and effective vaccine is probably the only way to control and prevent infectious diseases.As is known, the live-vectored vaccine based on vaccinia virus, which can promote endogenous synthesis, presentation of antigens and induce strong both cellular and humoral immune responses, has been tested on clinical trials and exerted good effects. Considering safety, AIDS recombinant vaccinia virus vaccine candidates must be free of selection marker gene. Optimized codon usage of HIV-1 structural genes has been shown to increase the expression of antigens and immunogenicity. The objective of this study is to construct a recombinant vaccinia virus expressing polyvalent HIV antigens of Chinese prevalent strain CN54 and study on the immune responses elicited by priming with DNA vaccine and boosting with recombinant vaccinia virus. At the same time, this study compared the differences of cellular and humoral immune responses induced by vaccines encoding HIV-1 wild type antigens genes and synthesis antigens genes.Firstly, a novel shuttle vector pVI75 was constructed, which consists of three selection marker genes (ampr,neo,lacZ),two homologous TK sequences, promoter(pE/L), MCS, Ori and a small repeating fragment lacZ'(about 200bp and homologous to the ending sequence of lacZ gene). Secondly, the recombinant plasmids pVI75-gagpol and pVI75-syngpnef-syngp!20 were constructed. To make the rVV-syngpnef-syngp!20 (recombinant vaccinia virus with syngpnef & syngp!20 gene) express two target genes well, the two genes are under two promotes and not share one. Thirdly, one hour after infected with Vaccinia Tian Tan strain (VTT), CEF was transfected by the recombinant plasmids pVI75-Gagpol and pVI75-syngpnef-syngp!20. Because the recombinant plasmids pVI75-gagpol and pVI75-syngpnef-syngp!20 have two short TK sequences that are homologous to TK region on VTT genome, so the first recombination will happen between them. Under the pressure of G418, several blue plaques could be picked through covering the admixture, which includes low melting point agar, neutral red, and X-gal. At this time, the 'Blue virus' includes both target genes and the selection marker genes (neo & lacZ). After three cycles of plaque purification with G418 pressure (the wild VTT was suppressed to grow because of G418), re-culture the 'Blue virus' without G418 pressure and the second recombination will happen in the process of virus propagation between the ending sequence of lacZ gene and the small repeating fragment of lacZ'(they are homologous to each other). In this recombination, both lacZ and neo gene would be lost but the target genes still existed on the genome of VTT. About 48 hours later, whiteplaques were picked and additionally screened with antibody staining, then confirmed by PCR. 108 white plaques were picked for rW-gagpol and 256 white plaques were picked for rW-syngpnef-syngp!20. Through three cycles of plaque purification without G418 pressure, White virus with target genes but without selection marker genes was acquired. PCR and Dot blot assay showed that the recombinant vaccinia virus rW-gagpol and rVV-syngpnef-syngp!20 had lost neo gene and lacZ gene. HIV target genes on the genome of recombinant vaccinia virus could be detected by PCR. The results of Western blot indicated the recombinant vaccinia virus could successfully express HIV target proteins.Immune regime is one of the important elements affecting the immune response. This study chose the strategy of priming with DNA vaccine and then boosting with recombinant vaccinia virus. DNA vaccine immunized BALB/c mice intramuscularly three times at two weeks interval, and recombinant vaccinia virus boosted intramuscularly two weeks later. Two weeks after the last immunization, immunized mice were sacrificed for immune response assay. The specific IgG responses and IgG2a /IgGl...
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