以Rev依赖性凋亡增强HIV-1 gp160的抗原性

Augment of the antigenicity of HIV-1 gp160 by Rev dependent apoptosis

目的 检测Rev(HIV的调控基因 )依赖性肿瘤坏死因子受体 (TNFR 1)和gp16 0双重表达质粒pDM12 8 TNFR 1(pT12 8)的凋亡诱导功能。方法 采用基因枪转导及流式细胞仪检测新质粒的表达功能。结果 新结构具有特异的选择性表达作用。当Rev存在时 ,能间接表达TNFR 1,明显诱导HeLa细胞凋亡 ,使绿荧光细胞百分率非常显著地低于阴性对照 (P <0 .0 1)。等质量转染时 ,TNFR 1表达量少于Hup6 0TNFR 1的pDC30 2 (pT6 0 ) ,故间接表达不及单纯pT6 0的直接表达 ,绿荧光细胞百分率显著高于pT6 0转染组 (P <0 .0 1)。培养 40h ,才有明显杀伤功能并接近单纯pT6 0 ,差异无显著性 (P>0 .0 1)。单纯pT12 8不能直接表达TNFR 1,绿荧光细胞百分率非常显著地高于单纯pT6 0转染组 (P<0 .0 1) ,接近阴性对照 ,培养 40h时差异无显著性 (P >0 .0 1)。当AD8或pMD +pRnv存在并表达Rev时 ,pT12 8均能表达TNFR 1,杀伤HeLa细胞 ,绿荧光细胞百分率非常显著地低于阴性对照 (P <0 .0 1)。pT12 8与pRev或AD8转染人正常的角质生成细胞时 ,能表达TNFR 1,诱导细胞凋亡。培养 72h后 ,阴性对照和单纯pT12 8组的绿荧光角质生成细胞数皆显著地超过pT12 8+pRev和pT12 8+pAD8组 (P <0 .0 1)。结论 pT12

Objective To detect the function of Rev dependent apoptosis induction by pDM128-TNFR-1 (pT128) that can express both TNFR-1 and gp160 at same time. Methods Gene gun and flow-cytometry technique were used to detect the expression of the plasmid. Results The new plasmid has the function of specific and selective expression that is indirect expression of TNFR-1 when Rev exists. It markedly induced apoptosis of HeLa cells and significantly decreased the percentage of green fluorescent cell than the negative control (P<0.01). As the expression amount of TNFR-1 from pT128 is not as much as that from Hu p60 TNFR-1 in pDC302 (pT60) while same amount of DNA is transfected, the indirect expression is not as much as the direct expression and its percentage of green fluorescent cell is significantly higher than that of pT60′s (P<0.01). After a 40-hour culture, the apoptosis induction of pT128 is close to that of pT60 and the difference is not significant (P>0.01). Simply pT128 does not direct express TNFR-1, its percentage of green fluorescent cell is significantly higher than that of pT60′s (P<0.01) and close to negative controls. The difference is not significant after 40 hours culture (P>0.01). When AD8 or pMD+pRnv exists and expresses Rev,pT128 also can express TNFR-1 and kills HeLa and the percentage of green fluorescent cell is significantly lower than that of pT60′s (P<0.01). When pT128 transfects normal human keratinocytes with pRev or AD8, it can also express TNFR-1 and induce apoptosis. After a 72-hour culture, its percentages of green fluorescent keratinocytes from negative controls and simply pT128 are both significantly higher than those of pT128+pRev and pT128+pAD8(P<0.01). Conclusion Manipulated apoptosis induction of pT128 ensures sufficient antigen expression of HIV DNA vaccination, formation of apoptosis body, activation of APC epitope and development of specific immunity memory of anti-apoptotic body. Simply pT128 does not cause injury in human body. The new construct is also adapted to different HIV antigen expression system and can kill HIV infected cells. It can expediently express antigen in human skin and induce apoptosis. This study can be a candidate strategy in HIV DNA vaccine research. [