中文摘要：

目的采用GatewayTM技术构建人Rb94基因重组腺病毒载体（Ad-hRb94）。方法从人类胚胎中提取总RNA,经反转录得到目的 cDNA,PCR扩增Rb94目的基因片段。设计含有attB侧翼序列的引物,用于重组片段的PCR扩增,在BP重组酶的作用下,将含attB位点的PCR产物与受体载体pDONRTM221发生重组反应,产生入门克隆。在LR重组酶作用下,将入门克隆与带attR1、at-tR2位点的目的载体Ad/CMV/V5-DEST体外重组形成表达克隆Ad-hRb94。经PCR和测序鉴定,将Ad-hRb94线性化后转入293A细胞进行病毒的包装、扩增及病毒滴度测定。结果经PCR和测序证实目的基因Rb94片段按正确方向重组入目的载体中,带Rb94基因的目的载体在293A细胞中包装成功,获得高滴度的病毒颗粒,滴度为9.41×10^10pfu/ml。结论本实验利用Gateway^TM技术成功构建了Ad-hRb94,为进一步进行肿瘤基因治疗研究奠定了实验基础。

英文摘要：

Objective To establish a recombinant adenovirus vector with hRb94 byλphage-site specific recombination systems.Methods Total RNA was extracted from human embryo and reversed transcript to get object cDNA,the hRb94 gene fragment was amplified by PCR.The attB-flanked PCR primers were designed and used to amplify hRb94 gene by PCR.An entry clone was performed by a BP recombination reaction with attB-PCR products and donor vector pDONRTM221.Then the entry clone and the target vector Ad/CMV/V5-DEST with attR1 and attR2 sites was recombined together in vivo to create the expression clone（Ad-hRb94）by an efficient LR recombination reaction.After the expression clone was confirmed by PCR and sequencing.Ad-hRb94 was digested with Pac I and transferred into 293A cells to be packaged into adenovirus stock.Ad-hRb94 was amplified by infection of 293A cells and the titer was measured.Results The target gene of hRb94 was transferred into Ad/CMV/V5-DEST vector correctly with the right ORF（open reading frame）by LR recombination reaction and it was confirmed by PCR and sequencing.The expression clone Ad-hRb94 was packaged into maturated adenovirus successfully.The titer of Ad-hRB94 was 9.41×10^10pfu/ml.Conclusion AdhRB94 was constructed with GatewayTMclone technology,which lays an experimental foundation for the further research on the genetic therapy of hRb94.