人CCR5Delta32基因重组慢病毒载体的构建及表达

目的:构建含人CCR5Delta32基因的重组慢病毒载体并鉴定其表达性能。方法:从CCR5Delta32突变个体外周血单个核细胞(PBMCs)内提取人基因组DNA,利用PCR技术扩增CCR5Delta32全长基因,经EcoRI单酶切后与pUCm-T载体连接,随后转化感受态E.coliDH5α,提取质粒进行酶切鉴定及DNA测序。再将鉴定正确的CCR5Delta32基因亚克隆至慢病毒载体pLenti6/V5-D-TOPO并进行酶切鉴定及DNA序列分析。最后用pLP1、pLP2、pLP/VSVG及pLenti-CCR5Delta32四种质粒共转染293T细胞,产生重组慢病毒并通过Western blot鉴定目的基因在靶细胞内的表达。结果:经PCR扩增获得约650bp的DNA片段,测序结果与发表于GenBank上的序列完全一致。经酶切鉴定,克隆的目的基因已经正确插入到慢病毒载体pLenti6/V5-D-TOPO中。四种质粒共转染293T细胞,产生出5×105TU/ml高滴度的重组慢病毒。用其感染靶细胞,Western blot结果显示有目的蛋白表达。结论:成功构建了含人CCR5Delta32基因的重组慢病毒载体并将其在293T细胞内表达,为进一步AIDS基因治疗研究奠定基础。

Construction and expression of the recombinant lentiviral vector containing human CCR5Delta32 gene

Objective: To construct the recombinant lentiviral vector containing human CCR5Delta32 gene and observe its expression in 293T cells. Methods: CCR5Delta32 gene was amplified from human peripheral blood mononuclear cells (PBMCs) genomic DNA by using PCR,and then cloned into pUCm-T. After sequence analysis,the CCR5Delta32 gene was subcloned into Lentiviral vector pLenti6/V5-D-TOPO. The 293T cells were cotransfected with the four plasmids (pLP1, pLP2, pLP/VSVG, and pLenti-CCR5Delta32) and the expression of CCR5Delta32 was detected by Western blot. Results: A DNA fragment of 650bp was obtained by PCR and its sequence was in conformity with the sequence deposited in Gen Bank. After the constructed lentiviral vector pLenti-CCR5Delta32 was transfected into 293T cells, the titer of the acquired recombinant lentivirus reached 5×105TU/ml, and CCR5Delta32 was expressed in target cells. Conclusion: The recombinant lentiviral vector pLenti-CCR5Delta32 was constructed successfully, and the target protein was expressed in 293T cells. All these lay a foundation for further studying the gene therapy for AIDS.