携带CIP2A shRNA重组腺相关病毒载体的构建及其鉴定
Construction and appraisal of recombinant adeno-associated virus vector for expressing shRNA targeting CIP2A mRNA

摘要：目的 构建携带CIP2A shRNA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CIP2A shRNA，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-CIP2A shRNA，以鉴定正确的pDC316-EGFP-CIP2A shRNA克隆为模板PCR扩增EGFP-CIP2A shRNA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-CIP2A shRNA，建立载体细胞株BHK/CIP2A-shRNA，采用AAV MaxTM包装系统大规模制备rAAV2-EGFP-CIP2A shRNA并对其纯化及滴度测定。将rAAV2-EGFP-CIP2A shRNA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CIP2A shRNA基因沉默效果。结果 携带编码CIP2A shRNA腺相关病毒载体质粒pSNAV2.0-EGFP-CIP2A shRNA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-CIP2A shRNA，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CIP2A shRNA重组腺相关病毒载体rAAV2-CIP2A shRNA。
ABSTRACT: 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-CIP2A shRNA sequence was amplified by PCR, double digested with EcoRⅠ and SalⅠ and ligated to pSNAV2.0 plasmid digested with the same enzyme pair. pSNAV2.0-EGFP-CIP2A shRNA plasmid DNA was prepared, purified, identified and transfected into BHK-21 cells. BHK-21 cells expressing CIP2A shRNA (BHK-21/CIP2A-shRNA) were obtained and subsequently infected with VGTC’s proprietary AAV packaging system to package the rAAV2-CIP2A 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 CIP2A 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 CIP2A 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-CIP2A shRNA plasmid into BHK-21 cells, BHK-21/CIP2A shRNA cells were infected with helper virus HSV1-rc/ΔUL2 to package the rAAV2-CIP2A shRNA to obtain a functional and infectious virus. The titer of the recombinant virus was 0.25×1012 v.g./mL. The expression of CIP2A mRNA and protein was decreased significantly after infection for 24h and 48h as compared with that of the controls by screening value of 1×105 MOI effected HepG2