针对人免疫缺陷病毒1型辅助受体CCR5 RNA干扰表达载体的构建

Construction of human immunodeficiency virus type I co-receptor CCR5 target RNAi expression vector

目的:抑制细胞表面人免疫缺陷病毒1型(HIV-1)的辅助受体CCR5表达,阻止HIV-1进入靶细胞。方法:设计CCR5靶向的发夹状siRNA,合成2条互补的寡核苷酸链,退火后重组入pRNAT-U6.2载体,转化扩增后进行序列测定。用脂质体包裹pRNAT-U6.2-siCCR5后转染U937细胞,采用RT-PCR和流式细胞术分别检测CCR5基因mRNA和蛋白表达的变化。结果:重组子pRNAT-U6.2-siCCR5,经过测序鉴定与设计一致。RT-PCR检测显示,转染pRNAT-U6.2-siCCR5细胞的CCR5基因表达水平显著降低于未转染对照组和转染空载体细胞(P<0.05)。未转染对照组U937细胞表面CCR5的表达率为(83.10±6.52)%,转染空载体组为(81.44±4.97)%,转染pRNAT-U6.2-siCCR5组为(1.94±0.06)%,3组间差异有统计学意义(P<0.05)。结论:成功构建出沉默细胞CCR5基因表达的siRNA载体。

Aim：To inhibit the expression of human immunodeficiency virus type I（ HIV-1 ） co-receptor CCR5 and prevent HIV-I entrying into the target cells. Methods：Specific short chain oligonucleotide was designed by using the siRNA software according to the mRNA sequence, the double chain DNA sequene was gained through annealing after chemosynthesis and was inserted to pRNAT-U6.2. The recombinant expression vector was evaluated by sequencing and transfected into U937 cell line by Liposome. The mRNA and protein expressions of the CCR5 gene in cells were determined by RT-PCR and flow cytometry. Results ： Sequencing showed that the expression vector was constructed successfully. The result of RT- PCR and flow cytometry showed that CCR5 gene expression level transfected cells. Conclusion： The constructed siRNA expression HIV-1 on U937. was dramatically suppressed on pRNAT-U6.2-siCCR5 vector can decrease the expression of eo-receptors for HIV -1 on U937.