携带肿瘤坏死因子相关细胞凋亡诱导配体重组腺相关病毒的构建及鉴定

Construction and identification of recombined adeno-associated virus with tumor necrosis factor related apoptosis inducing ligand

目的:构建一种携带肿瘤坏死因子相关细胞凋亡诱导配体(tumor necrosis factor related apoptosis inducing ligand,TRAIL)基因的重组腺相关病毒(recombined adeo-associated virus,rAAV)载体。方法:实验于2005-09/2006-04在中山大学附属第一医院普通外科实验室完成。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基因的穿梭质粒pAAV-sTRAIL,利用磷酸钙共沉淀法将穿梭质粒共转染入HEK293细胞中,采用细胞内质粒DNA同源重组法构建重组腺相关病毒rAAV-sTRAIL。以噬斑分析法筛选单克隆重组腺相关病毒;聚合酶链反应法鉴定阳性重组腺相关病毒;氯化铯密度梯度离心法纯化病毒;紫外分光光度仪测定病毒颗粒数及纯度,噬斑分析法测定病毒感染滴度;Western blot检测rAAV-sTRAIL在293细胞中的表达情况。结果:成功构建了重组腺相关病毒载体rAAV-sTRAIL,制备的病毒纯度好、滴度高,且在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,为组织工程相关细胞转基因构建及临床应用提供先进的载体系统。

AIM： To construct a kind of recombined adeno-associated virus （rAAV） encoding soluble tumor necrosis factor related apoptosis inducing ligand （sTRAIL） gene cDNA. METHODS： The shuttle plesmid pAAV-sTRAIL was first constructed with sTRAIL, end then transfected into HEK 293 cells by calcium phospheteprecipitation method. Recombinant edeno-essociated virus rAAV-sTRAIL was constructed by homologous recombination in 293 calls. Monoclonel rAAV-sTRAIL was screened by plaque assay; positive virus was identified by PCR; purified virus were prepared by CsCl density gradient centrifugation; virus particle number and purity were determined by recording spectrophotometer measurements end functional titers in plaque-forming units were determined by end-point dilution infection on 293 cells. Western blot was employed to detect the expression of rAAV-sTRAIL in 293 calls. RESULTS： rAAV-sTRAIL was constructed successfully and verified with high particles titers and good purification. The western blot showed that rAAV-s TRAIL expressed sTRAIL proteins in 293 calls. CONCLUSION： rAAV-sTRAIL provides an advanced gene expression vector tool for the study of the related transgenic construction in tissue engineering end the clinical application of TRAIL.