CAJ | 학술논문

目的构建人肝细胞生长因子(hHGF)基因慢病毒表达载体并进行鉴定.方法采用聚合酶链反应(PCR)技术扩增IRES和hrGFP,拼接成IRES-hrGFP,并克隆到pLenti6.3载体;进一步采用PCR技术扩增hHGF,并克隆到plenti6.3-IRES-hrGFP载体,然后BamHI/AscI双酶切和测序鉴定构建的plenti6.3-hHGF-IRES-hrGFP重组载体.脂质体法转染293 T细胞,24 h、48 h、72 h后显微镜下观察细胞的绿色荧光蛋白表达情况和ElISA测定细胞上清液中HGF蛋白表达情况.最后利用ViraPowerTM慢病毒表达系统,将表达质粒与包装混合物(Packaging Mix)共转染293 T细胞产生包装病毒颗粒,以293 T细胞hrGFP表达水平(荧光显微镜下对hrGFP表达阳性细胞进行计数)测定病毒滴度.结果 IRES,hrGFP和hHGF基因片段重组到plenti6.3载体,电泳结果和双酶切鉴定均能得到与理论大小相符的片段,测序结果与Genebank序列完全一致.质粒转染293 T细胞后绿色荧光蛋白表达量和上清中HGF蛋白含量随时间增长而增多,72 h最高.24 h、48 h、72 h后HGF蛋白含量分别为(477±19),(1424±78),(4274±128)μg/L.测得病毒的滴度为1.9×108 TU/mL.结论携带hHGF基因的慢病毒载体构建成功,在293 T细胞中正确表达,并获得较高的病毒滴度,为其体内外研究提供基础.
목적구건인간세포생장인자(hHGF)기인만병독표체재체병진행감정.방법채용취합매련반응(PCR)기술확증IRES화hrGFP,병접성IRES-hrGFP,병극륭도pLenti6.3재체;진일보채용PCR기술확증hHGF,병극륭도plenti6.3-IRES-hrGFP재체,연후BamHI/AscI쌍매절화측서감정구건적plenti6.3-hHGF-IRES-hrGFP중조재체.지질체법전염293 T세포,24 h、48 h、72 h후현미경하관찰세포적록색형광단백표체정황화ElISA측정세포상청액중HGF단백표체정황.최후이용ViraPowerTM만병독표체계통,장표체질립여포장혼합물(Packaging Mix)공전염293 T세포산생포장병독과립,이293 T세포hrGFP표체수평(형광현미경하대hrGFP표체양성세포진행계수)측정병독적도.결과 IRES,hrGFP화hHGF기인편단중조도plenti6.3재체,전영결과화쌍매절감정균능득도여이론대소상부적편단,측서결과여Genebank서렬완전일치.질립전염293 T세포후록색형광단백표체량화상청중HGF단백함량수시간증장이증다,72 h최고.24 h、48 h、72 h후HGF단백함량분별위(477±19),(1424±78),(4274±128)μg/L.측득병독적적도위1.9×108 TU/mL.결론휴대hHGF기인적만병독재체구건성공,재293 T세포중정학표체,병획득교고적병독적도,위기체내외연구제공기출.
Objective To construct and identify a plenti6.3-hHGF-IRES-hrGFP lentivirus vector. Methods IRES and hrGFP gene cDNA were amplicated by polymerase chain reaction (PCR) and spliced into the IRES-hrGFP, which was cloned into the pLenti6.3 vector;hHGF gene cDNA was amplicated by PCR and cloned into the plenti6.3-IRES-hrGFP vector, then the plenti6.3-hHGF-IRES-hrGFP plasmid was identified by BamHI/AscI enzyme digestion and sequencing analysis. The hrGFP expression were observed under the microscope and the hHGF protein concentrations were detected by ELISA kit at 24 h, 48 h, 72 h after plenti6.3-hHGF-IRES-hrGFP plasmid transfected 293 T cells by liposome transfection. Results IRES, hrGFP, and hHGF gene fragments were successfully constructed into the plenti6.3 vector. The reconstructed plasmid was consistent with the theoretical fragment, and the sequence result was exactly the same with Genebank. The hrGFP expression and hHFP protein concentrantion increased with time after the plasmid transfected 293 T cells (72 h at maximum). The hHFP protein concentrantion at 24 h, 48 h and 72 h after transfection was (477±19), (1424±78), (4274 ±128)μg/L respectively. Conclusion Plenti6.3-hHGF-IRES-hrGFP lentivirus vector has been successfully constructed, correctly expressed in 293 T cells, and could be applied to further experiments in vivo and in vitro.