survivin基因RNAi逆转录病毒载体设计与构建方法实验研究

Report on design and construction of recombinant retrovirus vector expressing siRNA for survivin gene knockdown

目的 设计和构建survivin基因的表达si RNA逆转录病毒重组载体,探讨胶质瘤分子病因及用于基因治疗的可行性。方法 利用在线软件si Direct设计干扰survivin基因靶序列,合成回文DNA序列退火后克隆至线性化p SUPER质粒载体,重组质粒载体双酶切电泳鉴定和测序分析,再转染phoenix细胞产生病毒转染低分化SHG4 4 - 9胶质瘤细胞株。利用NIH3T3细胞测定病毒滴度。Western blot测定转染后SHG4 4 - 9细胞survivin表达量。结果 p SU PER表达si RNA重组质粒载体经双酶切电泳鉴定和DNA测序分析,证实插入6 0 bp序列与原序列一致,位置正确。测定p SUPER.retro- S1、p SU PER.retro- S2病毒滴度值分别为5 .5×10 5CFU/ m l和5 .75×10 5CFU/ ml,干扰效率分别为70 .5 %和接近10 0 .0 %。结论 survivin基因的表达si RNA逆转录病毒重组载体的构建成功,不但为研究胶质瘤分子病因和基因治疗提供了有用工具,而且也为研究高表达survivin的其它肿瘤构建了新的平台。

Objective To design and construct recom bi nant retrovirus vectors expressing siRNA for survivin gene knockdown, sequential ly as tools to explore the molecule pathogeny and new gene therapy of gliomas. Methods The target sequences of siRNAs for survivin knockdown w ere designed by siDirect,a on-line software. The DNA sequences containing a pal indrome structure were synthesized and annealed to form double-stands which wer e inserted into linear pSUPER plasmid vectors later. The recombinant plasmids w ere identifiedby agarose gel electrophoresis after cleaving of double enzymes a nd by sequencing, then were transfected into the phoenix cells, which produced r etrovirus to transfect a low-differentiated SHG44-9 glioma cell strain. The ti ters of the recombinant retroviruses were determined by NIH3T3 cell. The express ion level of survivin was detected by Western blot. Results The recombinant pSUPER plasmid vectors expressing siRNA were confirmed by agarose g el electrophoresis after cleaving of double enzymes and by sequence analysis. Th e inserted 60 bp sequences were identical to the original sequences and in corre sponding position. The titers of the pSUPER.retro-S1 and pSUPER.retro-S2 were 5.5×105 CFU/ml and 5.75×105 CFU/ml, respectively.The inhibitory effects o f pSUPER.retro-S1 and pSUPER.retro-S2 were 70.5% and almost 100.0%, respectiv ely. Conclusion Successful construction of pSUPER recombinant retro virus vectors expressing siRNA for survivin gene knockdown supplies useful tools for studying the molecule pathogeny and new gene therapy of gliomas sequentially, an d provides a new worktable for researching other tumors overexpressing survivin gene as well.