中文摘要：

目的构建pGC-FU-survivin慢病毒质粒,旨在下一步实验中利用survivin蛋白的表达影响原代肝细胞的增殖。方法从高表达survivin的小鼠淋巴瘤细胞株YAC-I中获取存活素（survivin）基因,连接到pcDNA3.1（＋）质粒上,双酶切及双向测序鉴定。构建pGC-FU-survivin慢病毒载体,从基因水平和蛋白质水平鉴定含有survivin的目的片段表达。经包装、纯化,获得效价较高的病毒颗粒进行下一步生物学功能研究。结果双酶切及测序证实从YAC-I中获得的survivin基因连接到pcDNA3.1（＋）质粒上,除第317位碱基由腺苷酸突变为胸腺嘧啶外,其余均与NCBI基因文库中survivin基因切合,该突变不影响蛋白质空间结构及蛋白质生理功能。构建pGC-FU-survivin表达质粒的阳性克隆菌株转染293T细胞后,荧光显微镜下观察可见绿色荧光,Western blot结果见一与标准品大小相同的条带。在293T细胞中进行病毒包装,最终纯化出pGC-FU-survivin慢病毒颗粒。结论借助T载体及pcDNA3.1（＋）质粒,成功地在pGC-FU慢病毒表达质粒上建立了pGC-FU-survivin慢病毒表达质粒。

英文摘要：

Objective To construct a pGC-FU-survivin lentivirus plasmid and to study its further biological functions.Methods Mouse survivin gene was obtained from YAC-I cell line,a mouse lymphoma cell line,which highly expresses survivin protein,then the gene was linked to pcDNA3.1（＋ ） plasmid.The accuracy of the procedure was determined by restriction endonuclease digestion and sequencing.Survivin gene was acquired from pcDNA3.1（＋ ）-survivin plasmid and then connected with linearized pGC-FU plasmid,a lentivirus vector,then the accuracy was determined by sequencing and Western blot.After package and purifying,high titer of virus particles were established for further study.Results Restriction endonuclease digestion and sequencing confirmed that survivin obtained from YAC-I cell line was connected to pcDNA3.1（＋ ） plasmid correctly.Compared with survivin gene nucleotides in Gene Bank,all the sequences coincided with the Bank sequences except an A to T mutation at 317th gene position,which attributed an aminoglutaminic acid to aminoisovaleric acid at 68th protein position,but didn t affect spacial configuration and biological functions of survivin protein,according to NCBI protein library.After transfecting pGC-FU-survivin plasmid to 293T cells,obvious green fluorescence was observed.Protein was obtained from Western Blotting.Packaging virus particles in 293T cells,after purifying,high titer of pGC-FU-survivin plasmid was received eventually.Conclusion PGC-FU-survivin plasmid was constructed successfully with the assistance of pMD18-T vector and pcDNA3.1（＋ ） plasmid.