B区缺失人凝血因子Ⅷ基因在293T细胞表达.docVIP

B区缺失人凝血因子Ⅷ基因在293T细胞表达 　 作者：程海，徐开林， 孙海英， 杜冰， 曾令宇， 鹿群先， 何徐彭， 潘秀英 【摘要】 本研究目的是构建含有人凝血因子Ⅷ(FⅧ)基因的慢病毒载体，观察其在293T细胞中的表达情况。 用限制性内切酶法获得B区缺失的人凝血因子Ⅷ基因(BDDhFⅧcDNA)片段,将其克隆至慢病毒载体pXZ208，构建了慢病毒表达载体pXZ208BDDhFⅧ；用限制性内切酶法鉴定载体的连接方向，用磷酸钙共沉淀法将重组质粒pXZ208BDDhFⅧ分别与包装质粒ΔNRF、包膜蛋白质粒VSVG共转染293T包装细胞，包装后感染293T细胞，并以pXZ171作为对照。在感染后用逆转录聚合酶链反应(RTPCR)检测BDDhFⅧ基因的转录，一期法检测细胞培养上清FⅧ的活性，流式细胞仪(FCM)检测载体的感染效率，PCR检测BDDhFⅧ基因的整合。结果表明： 成功构建了慢病毒表达载体pXZ208BDDhFⅧ，其基因转导染效率达到了59.57%。RTPCR法能够检测到BDDhFⅧ转录的mRNA。感染后24、48、72小时检测到细胞上清中FⅧ活性(FⅧ∶C)分别为12%、43%、87%。PCR法扩增出了534 bp的特异性片段。结论： 成功构建的慢病毒表达载体pXZ208BDDhFⅧ，在体外可以有效感染293T细胞并表达有活性的FⅧ，提示基因治疗可应用于血友病A。 【关键词】 慢病毒 Expression of B DomainDeleted Human Coagulant Factor Ⅷ Gene in 293T Cells Mediated by Lentiviral Vector In Vitro Abstract This study was aimed to construct a lentiviral vector carrying human coagulant factor Ⅷ (FⅧ) and to investigate its expression in 293T cells. Bdomaindeleted factor Ⅷ gene fragment (BDDhFⅧcDNA) was obtained by enzyme digestion and cloned into lentiviral vector pXZ208 to establish the expression vector pXZ208BDDhFⅧ. Recombinant viral particles were prepared by cotransfection with packaging plasmid ΔNRF and envelope plasmid VSVG using calcium phosphate precipitation method. 293T cells were transfected by viral supernatant. Coagulant activity of FⅧ， BDDhFⅧmRNA and genome integration were assayed by onestep method, RTPCR and PCR after transfection. The results showed that 293T cells could be transfected by recombinant virus. The transfection rate of 293T was 59.57%. After transfection, the cells expressed FⅧ efficiently. Detection confirmed that the activity of FⅧ was 12%,43% and 87% respectively at 24,48 and 72 hours after infection. BDDhFⅧ transcription was detected by RTPCR from the infected cells. The gene integration in the targeted cells was also observed. It is concluded that the successfuly constructed lentiviral vector is able to generate high level expression of human FⅧ in 293T cells, which may provide a potential application of