人PBMCs内表达CCR5Delta32蛋白对HIV-1辅受体抑制作用的研究

The inhibitory effect of CCR5Delta32 protein on cell surface expression of the HIV-1 coreceptor CCR5 and CXCR4

目的:在人PBMCs内表达CCR5Delta32蛋白,研究其对细胞表面HIV-1辅受体CCR5和CXCR4的抑制作用。方法:构建pLenti-CCR5Delta32慢病毒载体,包装后产生重组慢病毒。将其转染PBMCs,Western blot检测目的蛋白的表达。继续培养靶细胞,FACS分析细胞表面CCR5和CXCR4分子的变化。结果:成功构建了pLenti-CCR5Delta32慢病毒载体,包装后产生重组慢病毒。将其转染PBMCs,Western blot检测到目的蛋白的表达。FACS分析表明,靶细胞内目的蛋白的表达对靶细胞表面辅受体CCR5和CXCR4的产生起抑制作用,抑制率在转染后第6天达到高峰(CCR5的抑制率为51.69%,CXCR4的抑制率为61.05%)。结论:靶细胞内目的蛋白的成功表达及其对靶细胞表面HIV-1辅受体CCR5和CXCR4产生的抑制作用,为后续的AIDS基因治疗研究奠定了基础。

Objective：To demonstrate that expression of the CCR5Delta32 protein in PBMCs able to down-regulate surface expression of the HIV-1 coreceptor CCR5 and CXCR4.Methods：CCR5Delta32 gene was amplified from human peripheral blood mononuclear cells（PBMCs） genomic DNA by using PCR,and then cloned into lentiviral vector pLenti6/V5-D-TOPO.Recombinant lentiviral particles were produced by packaging using 293T cells.Human PBMCs were transfected with the constructed recombinant lentiviral particles and the expression of CCR5Delta32 was detected by Western blot.The level of CCR5 and CXCR4 expression on transfected PBMCs was detected by FACS analysis.Results：The recombinant lentiviral vector pLentiCCR5Delta32 was constructed successfully,and the target protein was expressed in PBMCs.FACS analysis showed that CCR5Delta32 protein expressed in PBMCs was able to down-regulate cell surface expression of CCR5 and CXCR4.Conclusion：This study is expected to be used for the gene therapy on AIDS,which deserves further study.