人类免疫缺陷病毒-1vpr基因干扰RNA载体构建及筛选的体外研究

Construction and screening of human immunodeficiency virus-1 vpr gene RNA interference vector in vitro

目的筛选鉴定针对HIV-1vpr基因的小干扰RNA（siRNA）干扰片段，探讨siRNA对HIV-1vpr基因的干扰效率。方法根据siRNA设计要求合成针对HIV-1vpr靶点的2段寡核苷酸片段，并构建相应载体，转染含HIV-1vpr质粒的HEK293T细胞。设2个实验组（siRNA56、siRNA160组），1个阴性对照组（NC组），空白HEK293T细胞为对照组（Con组）。各组分别进行总RNA、蛋白提取，实时PCR和蛋白质印迹分别从核酸和蛋白水平验证有效的靶向HIV-1vpr的siRNA片段。ELISA检测各组培养细胞上清液中IL-17、丫干扰素水平。结果DNA测序结果表明成功构建了哺乳动物细胞pRNAT—U6．1／Neo—Vpr-56／160（siRNA56和s．RNA160）表达载体。siRNA56和siRNAl60干扰使HIV1vpr基因在mRNA水平的表达分别下降69．0％和76．1％；在蛋白表达水平分别下降76．3％和86．5％。Con组、NC组、siRNA56组和siRNA160组细胞培养上清液中IL-17分别为（1．936±0．415）、（1．815±0．393）、（1．935±0．356）和（2．03±0．421）pg／mL；了干扰素分别为（1．673±0．234）、（1．648±0．332）、（2．169±0．362）和（2．301±0．4125）pg／mL。结论表达pRNAT—U6．1／Neo—vpr-56／160（sirNA56和siRNA160）的质粒构建成功，针对不同基因片段的siRNA均可以下调HIV-1vpr的表达水平。

Objective To screen the small interfering RNA （siRNA） fragment targeted on human immunodeficiency virus-1 （HIV-1） vpr gene and to investigate the efficacy of siRNA interference. Methods Two oligonucleotide fragments were designed and synthesized targeted on HIV-1 vpr gene, and the expression vector was constructed. Plasmids containing HIV1 vpr gene were transfected into the human embryonic kidney 293 T （HEK293T） cells. Set up two experimental groups （siRNA56, siRNA160）, and a negative control group. Blank HEK293T cells were set as control group. The total RNA and protein were extracted from cells. Real-time polymerase chain reaction and Western blot were used to confirm the efficacy of siRNA targeted on HIV-1 vpr gene at nucleic acids level and protein level. The concentrations of interleukin-17 （IL-17） and interferon-＇/ （IFN-7） were detected by enzyme-linked immunosorbent assay. Data were analyzed by one-way ANOVA. Results The pRNAT U6. 1/Neo-vpr-56/160 expression vectors were successfully constructed and confirmed by DNA sequencing in mammalian cells, siRNA56 and siRNA160 could down-regulate the expression of HIV-1 vpr gene by 69.0 % and 76.1 % at mRNA level, respectively and 76.3 % and 86.5 %in protein level, respectively. The concentrations of IL-17 in control group, negative control group, siRNA56 and siRNA160 group were （1. 936±0. 415）, （1. 815±0. 393）, （1. 935±0. 356）, and （2. 034±0. 421） pg/mL, respectively. And the concentrations of IFN7 in four groups were （1. 673±0. 234）, （1. 648±0. 332）, （2. 169±0. 362）, and （2. 301±0. 412） pg/mL, respectively. Conclusions The plasmid expressing pRNAT U6. 1/Neo-vpr-56/160 is successfully constructed, siRNA targeted on different gene fragment can down-regulate the expression of HIV-1 vpr gene.