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

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 HIV-1 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-γ (IFN-γ ) 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 IFN-γ 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. Key words: Human immunodeficiency virus-1; Genes, vpr; RNA interference; Interleukin-17; Interferon type Ⅱ