中文摘要：

目的研究人免疫缺陷病毒1型（HIV-1）的vpr基因和不同变异株对宿主细胞周期和凋亡的影响，以及其致细胞周期变化和致细胞凋亡机制的两者间的可能关系。方法将14个带有不同变异位点的中国感染者HIV-1 vpr片段分别连入pcDNA3．1（＋）真核表达载体，构建重组质粒。将这些重组质粒电转染Jurkat细胞，并设立保守株vpr基因转染细胞、突变株vpr—Fs基因转染细胞、空载体转染细胞和未转染细胞作为对照。经G418选择培养及RT—PCR检测目的基因转染成功后，PI染色，流式细胞仪检测被转染细胞的细胞周期分布和细胞凋亡。结果流式细胞仪检测上述14个带有不同变异位点的HIV-1 vpr基因片段的Jurkat细胞，发现转染保守片段HIV-1 vpr的Jurkat细胞，其细胞周期出现G2期阻滞和细胞凋亡率明显升高，但转染vpr C端截断的vpr—Fs片段的细胞、空载体pcD-NA3．1（＋）转染细胞和未转染的Jurkat细胞无此现象。转染了HIV-1 vpr基因序列相对应的Vpr蛋白中含有70V、85P、86G、94G突变的片段，较vpr保守片段其致感染细胞G2期阻滞和凋亡的能力明显下降，且Vpr蛋白的AE亚型致细胞周期阻滞和致凋亡能力较其他亚型普遍为低。初步发现vpr诱导G2期阻滞百分比越高其所致凋亡率越高。结论HIV-1 vpr基因有明显的致感染细胞G2周期阻滞和致细胞凋亡的作用，但vpr C端截断的vpr—Fs片段无此功能。首次发现中国感染者HIV-1 vpr基因表达蛋白的70V、85P、86G、94G位点突变能使其致感染细胞G2期阻滞和凋亡的能力下降，Vpr的AE亚型致细胞周期阻滞和凋亡能力较其他亚型普遍为低。对14个变异片段的分析显示vpr诱导G2期阻滞的程度与其致凋亡水平可能相关，提示两者的发生机制可能有一定的关联。本研究为进一步探讨HIV-1致病机制和探索可能的基因干预措施打下基础。

英文摘要：

Objective To explore ability of the vpr gene of human immunodeficiency virus type 1 （HIV-1 vpr） to induce cell G2 arrest and apoptosis, and the influence when it mutated, the relationship between Vpr-induced G2 arrest and apoptosis inductions. Methods Fourteen mutant vpr fragments selected from Chinese patients with HIV. Both eukaryotic expression vector pcDNA3.1 （ ＋ ） and PCR products purified, double-cut by Hind Ⅲ and BamH Ⅰ and the cut products legated and transformed into competent ceils JM109. The 14 reconstructed plasmids electronically transfected into Jurkat-cells, and established cells with peDNA3.1-vpr , pcDNA3. 1-vpr-Fs and pcDNA3. 1 blank cells, and without pcDNA3. 1 cell. Cells were harvested after 24 h. mRNA expression was detected by RT-PCR, the DNA content and percentage of apoptosis were monitored by flow cytometry. Results Transfected with 14 mutant HIV-1 Vpr protein, cells display different G2 percentage and apoptosis ratio. HIV-1 vpr induce cell cycle G2 arrest and apoptosis, wherase Vpr Fs with a C-terminal end truncation, vector pcDNA3.1 （ ＋ ） and the blank cells can not. The G2 percentage and apoptosis ratio reduced when transfected with vpr expressing mutating of 70V, 85P, 86G, 94G compared to the wild type. Subtype AE has a weaker potential to induce cell cycle G2 arrest andapoptosis. Preliminary, we find that the higher G2 percentage followed the higher ratio of apoptosis. Conclusion HIV-1 vpr can induce cell cycle G1 arrest and apoptosis, wherase Vpr Fs with a C-terminal end truncation can not. We firstly found that mutated sites of 70V, 85P, 86G, 94G may reduce the ability of Vpr to induce cell cycle G2 arrest and apoptosis, subtype AE of vpr in Chinese HIV-1 patients has a weaker potential to induce cell cycle G2 arrest and apoptosis. Analysis of various mutations in the vpr gene revealed that the extent of Vpr-induced G2 arrest correlated with the levels of apoptosis. And investigate the pathegenesis of HIV vpr. This can also make a good foundation for further stu