Contruction And Detecting Method Establishment Of SIVmac239 Provirus And A SIVmac239/HIV-1 B(Thai) Chimeric Virus

SHIV is a kind of chimeric virus,which is constructed by gene recombination technicque. SHIV can be used in the developement of HIV vaccines and in the mechanism research of HIV pathogenesis. SHIVs have been constructed by foreign scientists since 1980s, several simian/human chimeric immunodeficiency viruses have been constructed up to now. Some highly pathogenic isolates have been used in the evaluation of McAbs' neutralizing activity and in the experiments of virus challenge.But there are no such reports in our country.The purpose of this work is to regenerate the provirus of SIVmac239 and try to construct a chimeric virus between SIVmac239 and HIV-1B(isolated from HENAN province,CHINA). In order to establish ELISA and Western-Blot methods for the detection of SIVmac239 and the chimeric virus, we expressed the full Gag of SIVmac239 in the pET-30a(+)/BL21(DE3) system and prepared anti-Gag McAbs by hybridoma technique.In the first section,two plasmids, p239SpSp5' (contains 5' half of SIVmac239 proviral genome plus 256 bp of flanking celluar DNA) and p239SpE3'(contains 3' half of SIVmac239 proviral genome plus 2533 bp of 3' flanking celluar DNA) were used to regenerate the provirus of SIVmac239. The two plasmids were digested by SphI ,then the provirus was obtained through ligating the mixture of the digestion products of two plasmids. The tat/rev/env gene were amplified by nested-PCR. The purified PCR products were digested with NcoI and SphI, then the countpart of SIVmac239 was substituted. Thus we acquired the provirus of SIVmac239 and the SHIV molecular clone, both of them were confirmed by PCR and restriction endonuclease digestion analysis. After transfecting MT-4 cells with the two plasmids...