摘要
目的构建携带CIP2AshRNA重组腺相关病毒载体,制备靶向性沉默CIP2A表达的高浓度重组腺相关病毒。方法设计合成CIP2AshRNA,退火形成双链与pDC316-EGFP-U6质粒BamHⅠ和HindⅢ双酶切产物相连接构建形成质粒pDC316-EGFP-CIP2AshRNA,以鉴定正确的pDC316-EGFP-CIP2AshRNA克隆为模板PCR扩增EGFPCIP2AshRNA片段,EcoRⅠ和SalⅠ双酶切PCR扩增产物及pSNAV2.0质粒后进行连接形成重组质粒pSNAV2.0-EGFP-CIP2AshRNA,建立载体细胞株BHK/CIP2A-shRNA,采用AAV MaxTM包装系统大规模制备rAAV2-EGFPCIP2AshRNA并对其纯化及滴度测定。将rAAV2-EGFP-CIP2AshRNA感染肝癌细胞HepG2,利用空载病毒载体rAAV2-EGFP作对照,采用Real-time PCR及Western blot方法检测CIP2AshRNA基因沉默效果。结果携带编码CIP2AshRNA腺相关病毒载体质粒pSNAV2.0-EGFP-CIP2AshRNA经双酶切及测序鉴定,质粒构建正确;将重组质粒与辅助病毒HSV1-rc/ΔUL2共转染包装细胞BHK-21,成功制备重组腺相关病毒rAAV2-CIP2AshRNA,经测定纯化所得rAVV2病毒滴度为0.25×1012v.g./mL。筛选MOI值为1×105感染肝癌细胞HepG2,CIP2A mRNA及蛋白表达水平分别于感染后24h和48h与对照细胞相比出现明显下降。结论成功制备高滴度携带CIP2AshRNA重组腺相关病毒载体rAAV2-CIP2AshRNA。
Objective To construct the recombinant adeno-associated virus vector carrying cancerous inhibitor of PP2A（CIP2A）short hairpin RNA（shRNA）for preparation of high-titer viruses.Methods The small hairpin RNA of CIP2A（CIP2A shRNA）was designed,synthesized and cloned into pDC316-EGFP-U6 plasmid which was double digested by BamHⅠ and HindⅢ.The resultant plasmid pDC316-EGFP-shRNA was confirmed and served as template to appraise primers.EGFP-CIP2 A shRNA sequence was amplified by PCR,double digested with EcoRⅠand SalⅠ and ligated to pSNAV2.0plasmid digested with the same enzyme pair.pSNAV2.0-EGFP-CIP2 A shRNA plasmid DNA was prepared,purified,identified and transfected into BHK-21 cells.BHK-21 cells expressing CIP2 A shRNA（BHK-21/CIP2A-shRNA） were obtained and subsequently infected with VGTC＇s proprietary AAV packaging system to package the rAAV2-CIP2 A shRNA.After purification,the functional and infectious virus was obtained and the titer of virus was detected.Real-time PCR and Western blot methods were used to detect the expression of CIP2 A after infection with HepG2 cells,and the empty viral vector rAAV2-EGFP was used as control.Results A recombinant adeno-associated virus-2 vector carrying CIP2 A shRNA was constructed successfully.The presence of the target sequence in the vector was confirmed by double enzyme digestion and sequencing.By transfecting the pSNAV2.0-EGFP-CIP2 A shRNA plasmid into BHK-21 cells,BHK-21/CIP2 A shRNA cells were infected with helper virus HSV1-rc/ΔUL2 to package the rAAV2-CIP2 A shRNA to obtain a functional and infectious virus.The titer of the recombinant virus was 0.25×1012 v.g./mL.The expression of CIP2 A mRNA and protein was decreased significantly after infection for 24 h and 48 h as compared with that of the controls byscreening value of 1×105 MOI effected HepG2 cells.Conclusion A high-titer recombinant adeno-associated virus-2 vector carrying CIP2 A shRNA has been constructed successfully.
出处
《西安交通大学学报（医学版）》
CAS
CSCD
北大核心
2015年第6期743-748,共6页
Journal of Xi’an Jiaotong University（Medical Sciences）
关键词
CIP2A
RNA干扰
重组腺相关病毒
短发卡RNA
cancerous inhibitor of PP2A（CIP2A）
RNA interference
recombinant adeno-associated virus（rAAV）
short hairpin RNA（shRNA）
