携带抑癌基因的重组腺相关病毒载体的构建及病毒包装滴定方法探讨

Construction, packaging and titration of recombinant adeno-associated virus vectors contaning antitumor genes

目的 构建携带多肿瘤抑制基因p16 (mts 1)及抑癌基因p2 1WAF1的腺相关病毒 (AAV)载体 ,通过病毒包装、纯化及滴定 ,获得高纯度高感染性的病毒液 ,为进行肝癌基因治疗的实验研究奠定基础。方法 以p16cDNA及p2 1cDNA全长为目的基因 ,构建AAV载体prAAV AFP p16 ,prAAV AFP p2 1,prAAV CMV p16及prAAV CMV p2 1。其中前两种载体能在人肝癌细胞中特异表达目的基因。应用脂质体将重组载体与辅助质粒PspRC72、腺病毒粘粒共转染包装细胞 ,2d后感染野生型腺病毒 ,至细胞裂解病变明显时收获。纯化后用聚合酶链反应法扩增倍比稀释后病毒液内的甲胎球蛋白或巨细胞病毒启动子DNA ,进行病毒DNA颗粒滴度测定。然后用重组腺相关病毒与腺病毒 (MOI均为 10 )共感染C12细胞 ,至细胞裂解病变明显时再收获。结果 重组载体经酶切鉴定测序证实。 2 93细胞、C12细胞包装病毒效果良好 ,病毒颗粒滴度介于 10 13 ～ 10 14 个 /ml之间。携带GFP的感染性AAV滴度为 5× 10 10 个 /ml。电镜下病毒呈小圆形空斑状 ,直径 2 5～ 30nm。MOI值 10 0时 ,rAAV AFP GFP病毒感染肝癌细胞的阳性率达 70 % ,为携带目的基因的AAV病毒用于肝癌细胞的促凋亡研究提供了定量指标。结论 成功构建了AAV载体 ,探讨了病毒包装方法 ,即第一次转染成功后 ,

Objective To construct, package, and titrate vectors that express antitumor genes using adeno associated virus (AAV) containing human alpha fetoprotein (AFP) promoter. Methods Recombinant AAV vectors were constructed by blunted ligation of plasmids rAAV AFP GFP and pTR UF5 and p16 cDNA and p21 cDNA. Packaging cell lines, 293 and C12 cells, were transfected with rAAV vectors, plasmid PspRC72, and adonovirus cosmid pAdc with the help of LipofectAMINE and then infected by wild adenovirus 48 hours later. The total viral particle titre was measured by PCR. Electron microscopy was used to observe the morphology of rAAV. C12 cells were infected with rAAV and wild adenovirus (MOI 10) and the titre of infectious virus was measured. by RCA method. Hepatocellular carcinoma cells were cultured and infected with rAAV contaiing green fluorescent protein (GFP), the infectious rate was observed 48 hours later. Results Recombinant vectors, rAAV AFP p16, rAAV AFP p21, rAAV CMV p16, and rAAV CMV p21, were constructed and verified by DNA sequencing and enzyme digestion. The total viral titre was 10 13 to 10 14 /ml. The titre of infectious rAAV containing GFP was 5×10 10 /ml. Electron microscopy showed that the rAVV was round and plaqued with a diameter between 20 and 30 nm. When the MOI was 100, the infectious rate of hepatocellular carcinoma cells was at least 70%. Four rAAV vectors carrying p16 or p21 genes have been constructed. rAAV packaging and titration methods were developed to produce highly purified AAV stocks. These approaches help in research of gene therapy in liver cancer.