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目的:在人外周血单个核细胞(PBMC)内表达CCR5Delta32蛋白,研究该蛋白与CCR5和CXCR4分子间是否存在相互作用。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PBMC,荧光显微镜观察转染情况,Western blot鉴定目的蛋白表达,免疫共沉淀检测目的蛋白与CCR5和CXCR4分子间的相互作用。结果:成功构建了pLenti-CCR5Delta32慢病毒载体,经包装后产生重组慢病毒,滴度约为5×108TU/L。将其转染PBMC,观察到约半数PBMC胞质内有绿色荧光蛋白表达,经Western blot鉴定有目的蛋白表达。免疫共沉淀结果显示,CCR5Delta32与CCR5、CCR5Delta32与CXCR4以异源二聚体形式结合,之间确实存在着相互作用。结论:成功地构建重组慢病毒载体并将其转染PBMC靶细胞,观察到目的蛋白与HIV-1两类辅受体(CCR5和CXCR4)间确实存在相互作用。这些工作为后续的AIDS基因治疗研究奠定了基础。
AIM: To express CCR5Delta32 protein in human peripheral blood mononuclear cells (PBMCs) and investigate whether there is interaction between CCR5 and CXCR4. Methods: The pLenti-CCR5Delta32 lentiviral vector was constructed and packaged to produce recombinant lentivirus. The transfected PBMCs were transfected into PBMCs. The transfected cells were observed under a fluorescence microscope. The expression of the target protein was identified by Western blot. The interaction between the target protein and the CCR5 and CXCR4 molecules was detected by co-immunoprecipitation. Results: The pLenti-CCR5Delta32 lentiviral vector was successfully constructed and packaged to produce a recombinant lentivirus with a titer of about 5 × 10 8 TU / L. Transfection of PBMCs revealed the expression of green fluorescent protein in about half of PBMCs, and the expression of target protein was confirmed by Western blot. Co-immunoprecipitation showed that there was indeed an interaction between CCR5Delta32 and CCR5, CCR5Delta32 and CXCR4 as heterodimers. CONCLUSION: The recombinant lentiviral vector is successfully constructed and transfected into PBMC target cells. It is observed that there is a real interaction between the target protein and two co-receptors of HIV-1 (CCR5 and CXCR4). These work laid the foundation for the subsequent AIDS gene therapy research.