plenti6.3-hHGF-IRES-hrGFP慢病毒表达载体的构建和鉴定

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293T细胞,24h、48h、72h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(PackagingMix)共转染293T细胞产生包装病毒颗粒,以293T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293T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,72h最高。24h、48h、72h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1.9×108TU/mL。结论携带hHGF基因的慢病毒载体构建成功,在293T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。

Objective To construct and identify a plenti6.3-hHGF-IRES-hrGFP lentivirus vector. Methods IRES and hrGFP gene eDNA were amplicated by polymerase chain reaction （PCR） and spliced into the IRES-hrGFP, which was cloned into the pLenti6.3 vector; hHGF gene eDNA was amplicated by PCR and cloned into the plenti6.3-IRES-hrGFP vector, then the plenti6.3-hHGF-IRES-hrGFP plasrnid 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 pIenti6.3-h/-/GF-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 eoncentrantion 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 transfeetion 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.