人组织因子途径抑制物腺病毒表达系统的构建及体内表达

The construction of adenovirus vector carrying human tissue factor pathway inhibitor gene and its expression in vivo

目的构建携载人组织因子途径抑制物(tissuefactorpathwayinhibitor,TFPI)基因的重组腺病毒载体,为基因治疗提供实验基础。方法利用基因重组技术,将人TFPI基因连接到穿梭质粒pDC316中,然后将腺病毒骨架质粒pBHGlox△E1,3Cre以及重组穿梭质粒pDC316-TFPI共转染293细胞,并在其中发生Cre重组酶介导的位点,特异性重组及腺病毒包装,扩增后进行滴度测定。将包装成功的携带人TFPI基因的重组腺病毒(Ad-TFPI)转染兔颈动脉,并用携带LacZ报告基因的重组腺病毒(Ad-LacZ)作为对照,3d后RT-PCR、ELISA法检测人TFPImRNA、蛋白的表达。结果得到了携带人TFPI基因的重组腺病毒,包装的病毒蚀斑形成单位(plaqueformationunit,PFU)滴度为7.6×1012/L。在Ad-TFPI转染兔颈动脉后3d,RT-PCR法和ELISA法均检测出TFPI表达,Ad-LacZ转染后未测到人TFPI的表达。结论成功构建了人TFPI腺病毒表达载体,为下一步的基因治疗提供了基础。

Objective To construct adenoviral vector carrying human tissue factor pathway inhibitor gene （Ad-TFPI） for gene therapy. Methods First, the foreign TFPI cDNA was inserted into a small shuttle plasmid called pDC316. Then cotransfection was performed with plasmid pDC316-TFPI and pBHGlox△EI ,3Cre. Ad vector was generated as a result of Cre-mediated site-specific recombination between the two plasmids after their cotransfection into 293 cells. The titer was determined after virus amplication. The expression of human TFPI mRNA and protein would be detected by the techniques of RT-PCR and ELISA 3 days after Ad-TFPI or adenoviral vector carrying LacZ gene （Ad-LacZ） was transfered into the rabbit carotid arteries. Results The recombinant adenoviral vector carrying human TFPI was constructed successfully. The titer was 7.6 ×10^12 plaque formation unit （PFU）/L after amplification. The expression of human TFPI gene in rabbit carotid arteries was detected positively by both RT-Craned ELISA methods in group Ad-TFPI, but negtively in group Ad-LacZ. Conclusions The recombinant adenoviral vector carrying human TFPI has been successfully constructed and paved the way for further gene therapy.