人肝细胞生长因子基因腺病毒重组体的构建及转染血管平滑肌细胞的表达

为探讨构建能表达肝细胞生长因子基因的腺病毒重组体的方法，以及这种腺病毒重组体能否在血管平滑肌细胞表达。将含有肝细胞生长因子基因片段的质粒用限制内切酶酶切，以琼脂糖凝胶电泳回收得到目的基因片段：将它用DNA连接酶插入至腺病毒穿梭质粒中，并用限制内切酶鉴别插入方向，得到插入方向正确的重组腺病毒穿梭质粒。用限制内切酶酶切重组腺病毒穿梭质粒和表达载体，使它们线性化；以电转化方法使它们共转化至BJ5183细胞中进行同源重组，构成肝细胞生长因子基因腺病毒重组体。以脂质体为转染介质，将肝细胞生长因子基因腺病毒重组体转染到人胚肾细胞中进行包装，使病毒复性具有感染能力。用包装后的肝细胞生长因子基因腺病毒重组体感染体外培养的大鼠主动脉平滑肌细胞；经逆转录一聚合酶链反应和蛋白印迹检测发现肝细胞生长因子呈现过表达。此外，酶切及测序发现重组腺病毒穿梭质粒和肝细胞生长因子基因腺病毒重组体是正确的；绿色荧光蛋白标记对重组体在人胚肾细胞中的包装及包装后感染大鼠主动脉平滑肌细胞进行了荧光示踪。结果提示，肝细胞生长因子基因腺病毒重组体构建成功，为血管疾病转基因治疗奠定了基础，具有一定临床价值，

Construction of Hepatocyte Growth Factor Gene Recombinant Adenovirus Vector and its Expression in Vascular Smooth Muscle Cells

Aim To construct an adenovirus expression vector which can express hepatocyte growth factor (HGF) in vascular smooth muscle cells (SMC). Methods The plasmid containing HGF fragment was cleaved by restriction enzyme digestion, and the resultant fragment was inserted directionally into adenoviral shuttle plasmid. The linearized recombinant adeno viral shuttle plasmid and adenovirus expression vector were cotransformed into Escherichia coli BJ5183 cells for homologous recombination . The resultant recombinant plasmid, pAd-HGF, then was transfected into HEK293 cells with liposome for packaging. The recombinant adenoviral shuttle plasmid and pAd-HGF were identified by enzyme digestion and sequencing. The package of pAd-HGF in HEK293 cells was tracked by fluorescent microscope, and was observed by electronic microscope. The expression of packaged pAd-HGF in abdominal aortic SMCs of rat was identified by RT-PCR and Western blotting. Results HGF fragment was inserted correctly into the adenoviral shuttle plasmid and adenovirus expression vector. High-liter packaged adenovirus vector was produced and expressed in abdominal aortic SMCs of rat. Conclusions A recombinant adenovirus expression vector of HGF was constructed successfully. This study suggested that HGF may be a potential target for the gene therapy of vascular diseases and established a foundation for further study.