作　　者： ; ;

机构地区： 长春农牧大学

出　　处： 《中国兽医学报》 1995年第3期228-234,共7页

摘　　要： 以构建的重组肿瘤坏死因子逆转录病毒载体ｐＺＩＰＴＮＦＤＮＡ转染Ｃｏｓ７细胞，获得了ＴＮＦα暂时性表达。采用ＤＮＡ－磷酸钙共沉淀法，将ｐＺＩＰＴＮＦＤＮＡ导入了单向性逆转录病毒包装细胞ψ２，在Ｇ４１８选择下，克隆出了ψ／ｐＺＩＰＴＮＦ细胞系。该细胞可产生重组ＴＮＦ逆转录病毒。本研究利用ψ／ｐＺＩＰＴＮＦ细胞分泌的重组逆转录病毒连续传代感染ψ２或ＰＡ３１７细胞以及病毒交替感染ψ２和ＰＡ３１７细胞，制备了高滴度单向和双向性重组ＴＮＦ逆转录病毒，其滴度可高达１．４×１０６，并发现获得的病毒滴度在传代感染的第３代达到高峰，随之趋于平衡。利用重组ＴＮＦ逆转录病毒感染ＮＩＨ３Ｔ３细胞，建立了３Ｔ３／ｐＺＩＰＴＮＦ细胞，该细胞的４８ｈ培养上清中可产生３２Ｕ／ｍＬ的ＴＮＦα。利用重组ＴＮＦ逆转录病毒直接感染ＮＩＨ３Ｔ３细胞后的４８ｈ培养上清中，ＴＮＦα活性也可达到８Ｕ／ｍＬ。实验还观察到３Ｔ３／ｐｚＩＰＴＮＦ细胞有自身生长抑制现象。本研究结果成功地实现了重组逆转录病毒介导的ＴＮＦα基因在哺乳动物细胞中的转移与表达，为进一步实施肿瘤基因治疗奠定了基础。 The temporary expression of pZIPTNF vector was confirmed in COS7 cells. Two kinds of retrovirus packaging cell lines/pZIPTNF and/pZIPTNeo were developed by transfecting 2 cells with the recombinant and primal vector DNAs under G418 resistance selection, respectively. In order to prepare the ectropic or amphotropic recombinant TNF retroviruses with high titers,we developed two methods of serially virus-generated infecting 2 or PA317 cells and virus-alternately infecting 2 and PA317 cells. The titers of recombinant TNFα retrovirus prepared by these methods reached up to 1.2 × 106～1.4 × 106.It was found that the titers of third passage of recombinant retroviruses could reach at the peak,and then tended to maintain evenly in titers during the course of latter passages.The studies successfully carried out retrovirus-mediated transfer and expression of TNFα gene in mammalian cells. The cloned 3T3/pZIPTNF cells could be able to produce TNFα with 32 U/mL in the culture supernatants after 48 h incubation, and the 3T3 cells infected directly with recombinant retrovirus could se- crete TNFα of 8 U/mL in culture supernatants too. On the other hand, the experiment showed the phenomena of self-growth-inhibition of 3T3/pZIPTNF cells. These results provided a fundamental for researching the gene therapy for cancers using recombinant retrovirus-mediated TNFα gene tranfer.

关 键 词： 重组逆转录病毒 肿瘤坏死因子 基因转移 动物

领　　域： [农业科学] [农业科学] [农业科学]