肿瘤坏死因子相关细胞凋亡诱导配体表达载体的制备

摘要

Objective To construct and identify recombined adeno associated virus encoding soluble tumornecrosis factor related apoptosis inducing ligand(sTRAIL)gene cDNA.Methods The shuttle plasmid pAAV-sTRAIL was first constructed.then transfected into HEK 293 cells by calcium phosphateprecipitation method.Recombinant adeno-associated viruS rAAV-sTRAIL Was produced by homologous recombination in 293 cells.Monoclonal rAAV-sTRAIL Was screened by plaque assay;virus identity was confirmed by PCR;purified virus stockswere prepared by CsCl gradients banding;virus particle titers were determined by OD260 measurements andfunctional titers in plaque forming units were determined by endpoint dilution infection on 293 cells. Western blotwas employed to detect the expression of the rAAV-sTRAIL in 293 cells.Results rAAV-sTRAIL was constructedand verified with hish particles titers and good purification.Furthermore,western blot showed that rAAV-sTRAILexpressed sTRAIL proteins in 293 cells.Conclusion The development of rAAV-sTRAIL provided an effective geneexpression vector tool for studv of the anti-tumor effect and for the clinical application of TRAIL cell.%目的 构建一种携带肿瘤坏死因子相关细胞凋亡诱导配体(TRAIL)基因的重组腺相关病毒(rAAV)载体.方法 首先构建携带可溶性肿瘤坏死因子相关细胞凋亡诱导配体(sTRAIL)基因的穿梭质粒腺相关病毒穿梭质粒-可溶性肿瘤坏死因子相关细胞凋亡诱导配体(pAAV-sTRAIL),将穿梭质粒转染入HEK 293细胞(人胚肾细胞系)中,采用细胞内质粒DNA同源重组法构建重组腺相关病毒载体rAAV-sTRAIL.以噬斑分析法筛选单克隆rAAV-sTRAIL;PCR法鉴定阳性rAAV-sTRAIL;氯化铯密度梯度离心法纯化rAAV-sTRAIL;紫外分光光度仪测定rAAV-sTRAIL颗粒数及纯度,噬斑分析法测定rAAV-sTRAIL感染滴度;Western免疫印迹检测rAAV-sTRAIL在HEK 293细胞中的表达情况.结果 成功构建了rAAV-sTRAIL,制备的病毒纯度好、滴度高,且在HEK 293转导细胞中能有效表达目的基因sTRAIL.结论 构建的重组腺相关病毒载体rAAV-sTRAIL,为研究TRAIL抗肿瘤细胞效应及临床应用提供先进的载体系统.