人CCR5Delta32基因的克隆及其重组慢病毒载体的构建

Cloning of human CCR5Delta32 gene and construction of the recombinant lentiviral vector pLenti-CCR5Delta32

目的:克隆人CCR5Delta32基因并构建含目的基因的重组慢病毒载体pLenti-CCR5Delta32,用于获得性免疫缺陷综合征(AIDS)的基因治疗研究.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.coli DH5α,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.结果:经PCR扩增获得约650bp的DNA片段,测序结果与GenBank上的序列完全一致,克隆的目的基因已经正确插入到慢病毒载体pLenti6/V5-D-TOPO中.结论:成功克隆了人CCR5Delta32基因并构建了含目的基因的重组慢病毒载体pLenti-CCR5Delta32,为进一步AIDS基因治疗研究奠定基础.

AIM： To clone human CCR5Delta32 gene and construct the recombinant lentiviral vector pLenti-CCR5Delta32 for the further gene therapy of AIDS. METHODS： Full-length CCRSDeha32 gene was amplified by using PCR from human peripheral blood mononuclear cells （PBMCs） genomic DNA, and then cloned into pUCm-T. After sequence analysis, the CCR5 Deha32 gene was subcloned into lentiviral vector pLenti6/V5-D- TOPO. RESULTS： A DNA fragment of 650 bp was obtained by PCR and its sequence was in conformity with the sequence reported in GenBank. The cloned gene was inserted into the lentiviral vector exactly. CONCLUSION： Human CCRSDelta32 gene is cloned correctly and the recombinant lentiviral vector pLenti- CCR5Deha32 is constructed successfully, and all these lay a foundation for further studying the gene therapy of AIDS.