中国医药
中國醫藥
중국의약
CHINA MEDICINE
2008年
4期
193-195
,共3页
蔡刁龙%夏金堂%朱光辉%翁杰锋
蔡刁龍%夏金堂%硃光輝%翁傑鋒
채조룡%하금당%주광휘%옹걸봉
肿瘤坏死因子相关细胞凋亡诱导配体%腺相关病毒%基因治疗
腫瘤壞死因子相關細胞凋亡誘導配體%腺相關病毒%基因治療
종류배사인자상관세포조망유도배체%선상관병독%기인치료
Tumor necrosis factor related apoptosis inducing ligand%Adeno-associated virus%Genetherapy
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.
目的 構建一種攜帶腫瘤壞死因子相關細胞凋亡誘導配體(TRAIL)基因的重組腺相關病毒(rAAV)載體.方法 首先構建攜帶可溶性腫瘤壞死因子相關細胞凋亡誘導配體(sTRAIL)基因的穿梭質粒腺相關病毒穿梭質粒-可溶性腫瘤壞死因子相關細胞凋亡誘導配體(pAAV-sTRAIL),將穿梭質粒轉染入HEK 293細胞(人胚腎細胞繫)中,採用細胞內質粒DNA同源重組法構建重組腺相關病毒載體rAAV-sTRAIL.以噬斑分析法篩選單剋隆rAAV-sTRAIL;PCR法鑒定暘性rAAV-sTRAIL;氯化銫密度梯度離心法純化rAAV-sTRAIL;紫外分光光度儀測定rAAV-sTRAIL顆粒數及純度,噬斑分析法測定rAAV-sTRAIL感染滴度;Western免疫印跡檢測rAAV-sTRAIL在HEK 293細胞中的錶達情況.結果 成功構建瞭rAAV-sTRAIL,製備的病毒純度好、滴度高,且在HEK 293轉導細胞中能有效錶達目的基因sTRAIL.結論 構建的重組腺相關病毒載體rAAV-sTRAIL,為研究TRAIL抗腫瘤細胞效應及臨床應用提供先進的載體繫統.
목적 구건일충휴대종류배사인자상관세포조망유도배체(TRAIL)기인적중조선상관병독(rAAV)재체.방법 수선구건휴대가용성종류배사인자상관세포조망유도배체(sTRAIL)기인적천사질립선상관병독천사질립-가용성종류배사인자상관세포조망유도배체(pAAV-sTRAIL),장천사질립전염입HEK 293세포(인배신세포계)중,채용세포내질립DNA동원중조법구건중조선상관병독재체rAAV-sTRAIL.이서반분석법사선단극륭rAAV-sTRAIL;PCR법감정양성rAAV-sTRAIL;록화색밀도제도리심법순화rAAV-sTRAIL;자외분광광도의측정rAAV-sTRAIL과립수급순도,서반분석법측정rAAV-sTRAIL감염적도;Western면역인적검측rAAV-sTRAIL재HEK 293세포중적표체정황.결과 성공구건료rAAV-sTRAIL,제비적병독순도호、적도고,차재HEK 293전도세포중능유효표체목적기인sTRAIL.결론 구건적중조선상관병독재체rAAV-sTRAIL,위연구TRAIL항종류세포효응급림상응용제공선진적재체계통.
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.
