| Acquired Immunodeficiency Syndrome caused by Human immunodeficiency virus has become not only a severe public threat to human beings, but also a hard burden to all the countries' economy. Despite international efforts to control the HIV/AIDS pandemic through behavioural modification, antiviral therapy and other interventions, the spread of HIV is continued. What's more, current antiviral drugs cannot completely remove the virus and because of their side effect, many patients cannot tolerate drug treatment or become resistant. Therefore, controlling the prevalence of HIV/AIDS has to rely on a preventive vaccine that is safe, highly effective and affordable.Because of high frequency mutation of HIV genes during spread, HIV genes exhibit extensive sequence heterogeneity. This heterogeneity has been used to classify HIV type 1 (HIV-1) strains into some groups and subtypes. HIV subtypes have unequal geographical distributions and they are stable in some period of time. The development of HIV/AIDS vaccine must be based on the genetic variability of HIV. At present, there are two major popular HIV-1 subtypes in China: subtype B and C, especially the recombinant strain of subtype B'/C. This recombinant subtype( B'/C ) is showing tendency of increase. In order to develop a specific vaccine for Chinese popular HIV strains, we used subtype B'/C CN54 strain as the target strain to study HIV-1 candidate vaccine.Selection of antigens is very important for inducing a broad range of immune responses against the viruses. We know now that protein Env encoded by gene, env is the most variable component of the virus and can induced only limited neutralizing antibodies. The proteins encoded by other genes of the virus can induce both humoral response and cell-mediated immunity response . So the selection of antigen is now focueds on inner proteins encoded by other genes of the virus. In order to induce broader anti-HIV immune responses, HIV/AIDS vaccines containing multi-antigen are designed no.Vaccinia virus is an ideal vector to construct poly-valent vaccine due to anumber of unique biological attributes including large DNA capacity, versatile non-essential sequences, safe to use and the potential of inducing both humoral and cellular immunities. In this study, vaccinia virus vector pVI75 containing double marker genes was constructed which can delete the marker genes in the process of screening recombinant vaccinia virus. Then the synthetic gene of gag/pol/nef(syngpnef) based on Chinese popular HIV-1 strain CN54(B'/C) was cloned into the downstream of synthetic early/later pE/L promoter of pVI75 and co-transfected primary chicken embryo fibroblasts(CEF) with Chinese vaccinia virus Tiantan strain to construct recombinant vaccinia virus expressing HIV-1 syngpnef genes by homogeny recombination.Transfer vector pVI75 was constructed by added a neor gene with a vaccinia virus promoter pH6 and a 200bp sequence lacZ' which homologous to lacZ into the vaccinia virus vector pSC65 which has a single marker gene lacZ to make the vector pVI75 having two marker genes(neor and lacZ). The construction of the pVI75 vector with two marker genes could make the result of screening more reliable and avoid of screening the false-positive recombinant virus. At the same time, the tandem repeated DNA sequence in the vector was designed by adding lacZ' sequence homologous to marker gene lacZ and this design has the ability of reducing unsafe character when adding tandem repeated DNA sequence into the vector. In the process of constructing recombinant vaccinia virus using vector pVI75, the foreign target gene was integrated into the genomic DNA of vaccinia virus and the marker genes were deleted by homologous recombination twice. Additionally, the vector pVI75 is also fit for constructing different recombinant vaccinia vaccine strains by using different antigens.Recombinant vaccinia vaccine strain expressing HIV-1 syngpnef gene was constructed by digesting plasmid PCR-Script-c-clade-syngpnef with restriction enzyme and cloning the part of syngpnef... |
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