pGC-FU-存活素慢病毒表达质粒的构建

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,...

Construction of pGC-FU-survivin lentivirus expression plasmid vector

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.