人B7-1基因的cDNA克隆及其重组腺病毒载体制备

目的：构建并制备含人B7-1基因的重组腺病毒（Ad-B7-1)载体，为后续的肿瘤基因治疗研究提供实验基础。方法：应用逆转录-套式PCR方法从人骨髓细胞中克隆B7-1的cDNA后，将其克隆至小片段腺病毒载体pCI′中，后与pHBG10在293细胞内重组包装产生腺病毒，感染SGC7901细胞。结果：成功克隆了人B7-1的cDNA并构建Ad-B7-1载体，且经过酶切鉴定和测序验证，感染Ad-B7-1的细胞经PCR鉴定，表明B7-1基因已整合入细胞基因组。结论：本实验构建的腺病毒载体可有效地转B7-1基因进入肿瘤细胞，该结果为下一步应用于体内肿瘤基因治疗研究提供了实验基础。

Cloning of human B7-1 gene cDNA and preparation of adenovirus vector carrying B7-1

Objective: To construct adenovirus vector carrying human B7 1 gene for tumor gene therapy. Methods and Results: RT nest PCR was used to clone human B7 1 gene cDNA from mixed human bone marrow cell culture. Then a recombinant adenovirus vector was constructed, which carrying B7 1 or LacZ. Human B7 1 gene cDNA and adenovirus vector carrying B7 1 were identified by enzyme digestion and DNA sequencing. The infectious adenovirous carrying B7 1 gene were produced by 293 packaging cells co transfected by the recombinant plasmid and plasmid BHG10. The adenovirus were used to infect stamoch cancer cells SGC7901 in vitro . Cells infected by Ad LacZ were dyed blue by X gal while B7 1 gene was detected by PCR in cells infected by Ad B7 1. Conclusion: The recombinant adenovirus vector can efficiently transfer LacZ and B7 1 gene into tumor cells. This result suggests that it can be used in the future for tumor gene therapy. [