ABSTRACT
Objective To understand the rule of vpr gene variance of HIV-1 strains.Methods RT-PCR was used to amplify vpr gene of HIV-1 strains in Shenzhen. PCR products were sequenced and used for gene phylogenetic analysis and the 32-46 amino acids of Vpr protein were compared. The difference of 77 amino acid polymorphism distribution between domestic region and foreign region was analyzed. Results 01_AE was the major HIV-1 subtype in Shenzhen. The gene distance among subtype B was larger than in other subtypes. 77-amino acid of Vpr protein had three polymorphism forms as Arginin, Glutamine and Histidine, with Glutamine as the wild form. There were no significant differences in the three amino acid distributions between HIV-1 strains from domestic region and foreign region. Conclusion vpr genes of different HIV-1 strains belonged to 01_AE subtype. There was polymorphism seen in the vpr gene which was consistent with both domestic and international HIV-1 strains.
ABSTRACT
Objective To establish cell strain expressing the genes of HIV vpr and mutant HIV vpr-FS, and to explore cell apoptosis ability by HIV Vpr and Vpr-FS. Methods The recombinant plasmids were constructed by cloning HIV vpr and HIV vpr-FS genes into the eukaryotic expression vector pcDNA3.1respectively. To determine the primary structures of HIV vpr and HIV vpr-FS, plasmids were cleaved by restriction enzymes. After the plasmids were transfected into HeLa cells by liposome, the HeLa cells were selected with G418 selective medium, mRNA expression of HIV vpr or HIV vpr-FS of transfected cells was detected by RT-PCR, and Vpr and Vpr-FS protein expression were detected by Western blot assay respectively. The DNA content and the percentage of apoptosis in HeLa HIV vpr cell, HeLa HIV vpr-FS cell and HeLa pcDNA3.1 cell were monitored by flow cytometry and the DNA fragmentation was analyzed by agarose gel electrophoresis. Results BamH Ⅰ and Hind Ⅲ cleavaged products of pcDNA3.1-vpr and pcDNA3.1-vpr-Fincluded 342 bp length fragments suggesting that the length of DNA sequence containing HIV vpr (HIV vpr-FS) within pcDNA3.1 was the same as theoretical length. The HeLa cells transfected by pcDNA3.1-vpr or pcDNA3, l-vpr-FS and selected with G418 could express HIV vpr or HIV vpr-FS by RT-PCR, and express HIV Vpr or HIV Vpr-FS protein by Western blot. The results of flow cytometry and DNA fragmentation showed that there was significant different in the number of apoptotic cells between HeLa HIV vpr cell and HeLa HIV vpr-FS cell, but the difference between HeLa HIV vpr-FS cell and control group was not obvious. Conclusion Recombinant plasmids pcDNA3.1-vpr and pcDNA3. 1-vpr-FS were constructed successfully, and the cell strain expressing HIV Vpr and HIV Vpr-FS proteins was established. The HIV Vpr could induce host cell apoptosis, while the mutant of Vpr did not or weakened this ability. This study provides foundation for further study on HIV vpr gene.