人类免疫缺陷病毒感染细胞的Rer蛋白依赖性凋亡引导

Rev dependent apoptosis induction of HIV infected cells

目的:以人类免疫缺陷病毒(HIV)调节蛋白Rev与Rev反应区(RRE)的高亲和性建立引导HIV感染细胞凋亡的结构。方法:用分子克隆技术合成含RRE和肿瘤坏死因子受体-1(TNF-R1)的Rev依赖性凋亡引导质粒,流式细胞仪检测质粒表达。结果:新质粒pDM128-TNF-R1(pT128)HindIII内切有3.1、2.7、1.0和0.87kb片段;聚合酶链反应(PCR)法检测示TNF-R1的1360bp片段。DNA测序法确认其准确性。单纯Hup60TNF-R1在pDC302(pT60)转染Hela,TNF-R1表达可明显杀伤Hela(P<0.01),Rev存在时,pT128也能表达TNF-R1杀伤Hela(P<0.01),但不及pT60的作用(P<0.01);无Rev时,pT128不表达TNF-R1,不杀伤Hela(P>0.01)。单纯Rev不杀伤Hela(P>0.01)。pT128与pRev共转,TNF-R1表达较pT60慢(P<0.01),40h后才接近单纯pT60。结论:新质粒具有Rev依赖性表达作用,进而引导Rev表达细胞凋亡。

Objective: To design a construct that can specifically and directly induce HIV infected cells into apoptosis based on the high affinity of regulatory protein Rev and Rev response element (RRE). Methods Molecular cloning technique was used to synthesis Rev selected killing plasmid that contains RRE and TNF-R1. The expression of the new plasmid was further tested by flow-cytometry. The new plasmid pDM128-TNF-R1 (pT128) is composed of 7744 base pairs and contains Amp R SV40 promoter TNF-R1 gene and segment of HIV-1-RRE. The construct can be cut with Hind III into the segments of 3.1 kb 2.7 kb 1 kb and 0.87 kb. Results It was shown that the 1360 bp segment of TNF-R1 was inserted in and completely substituted the CAT gene in pDM128. DNA sequencing showed that inserted TNF-R1 sequence had no mutation. TNRG1 expression from simply transfected Hu p60TNF-R1 in pDC302 (pT60) significantly killed the Hela cells (P < 0.01). The pT128 also expressed TNF-R1 and killed the Hela cells only when pRev existence (P < 0.01). But without pRev co-transfection it did not express TNF-R1 and also did not kill the Hela cells (P > 0.01). Also simply pRev expression could not kill the Hela cells (P > 0.01). The expression of TNF-R1 in the cells of pT128 and pRev co-transfection was slower than that of pT60 (P < 0.01). The expression of TNF-R1 in pT128 infected cells gradually rose to that of pT60 after 40 hrs culture. Conclusions The new plasmid can express TNF-R1 with the manner of Rev dependence and induce apoptosis of the HIV infected or Rev expressed cells.