Abstract: 【Abstract】〓Objective〓To generate lentiviral vector carrying HBEGF gene under the control of liver-specific albumin (Alb) promoter. Methods〓Firstly, the fragment of Alb-enhancer/promoter was amplified from pAlb-Cre-GH/BS, and subsequently subcloned into the Not I site in the polylinker of pTRECK6-GFP by In-Fusion cloning to generate pAlb-TRECK6..Secondly,.the fragment of Alb-TRECK6 was amplified from pAlb-TRECK6,.and then inserted into pCD823A-1 at the Xba I/BamH I sites by In-Fusion cloning, designated pLV-ATTG, as verified by restriction enzyme digestion and sequencing. According to the standard protocol from Invitrogen,.lentiviruses were produced and then infected BEL-7402 cells,.followed by copGFP assay under inverted fluorescence microscope, and detecting the expression of HBEGF transgene by qRT-PCR. Results〓the data from enzyme digestion and DNA sequencing demonstrated that pLV-ATTG was successfully constructed..Cells infected with LV-ATTG displayed the green fluorescence under inverted fluorescence microscope, and the increased expression of HBEGF detected by qRT-PCR. Conclusion〓The lentiviral vector harboring HBEGF gene under the control of liver-specific Alb promoter was successfully constructed,.which will lay a solid foundation for further research.

Key words: HBEGF, CopGFP, Lentivirus vector, Alb

摘要：

【摘要】 目的 构建肝特异性Alb(白蛋白)启动子调控HBEGF(Heparinbinding-epidermal growth factor)表达的慢病毒载体pLV-ATTG。方法 首先以pAlb-Cre-GH/BS为模板,PCR扩增目的基因Alb-enhancer/promoter,In-Fusion克隆至Not I 酶切的pTRECK6-GFP中,获得pAlb-TRECK6(TRECK: toxin receptor-mediated conditional cell knockout);接着以pAlb-TRECK6为模板,PCR扩增Alb-TRECK6,In-Fusion克隆至Xba I/BamH I酶切的pCD823A-1载体(该载体已卸去EF-1α启动子)中,获得最终的慢病毒载体pLV-ATTG,所构建的质粒均经测序和酶切鉴定。然后按Invitrogen公司推荐的标准程序进行慢病毒包装。包装成功的慢病毒LV-ATTG感染肝癌细胞株BEL-7402细胞,倒置荧光显微镜检测copGFP表达,并收集细胞提取总RNA,以检测HBEGF表达。结果 酶切鉴定和测序证实成功构建了慢病毒载体pLV-ATTG;LV-ATTG感染BEL-7402细胞后,倒置荧光显微镜下可见绿色荧光,同时qRT-PCR检测结果显示HBEGF转基因能够正常表达。结论 成功构建Alb启动子调控HBEGF表达的慢病毒表达载体,为相关后续研究奠定了坚实基础。

关键词: Alb, 慢病毒载体, HBEGF, copGFP