携带凋亡素、内皮抑素双基因腺病毒载体的构建

Construction of adenovirus vector carrying apoptin gene and endostatin gene

目的构建携带有凋亡素(Apoptin、VP3)、内皮抑素(Endostatin)双基因的重组腺病毒载体,为其在肿瘤治疗的应用研究打下基础。方法将VP3基因和Endostatin基因克隆入腺病毒穿梭质粒pDC316中,将该穿梭质粒与腺病毒骨架质粒pBHGloxE1,3Cre共转染HEK293细胞,包装出重组腺病毒颗粒Ad-vp3-IRES-sEndo-his。其后挑选病毒空斑获取克隆进行小剂量扩增并提取病毒DNA进行PCR、RT-PCR检查以筛选和鉴定毒种,之后大剂量扩增所选得的病毒克隆并进行纯化、测定病毒的滴度。结果所得腺病毒经PCR、RT-PCR检测表明重组腺病毒Ad-vp3-IRES-sEndo-his包装成功。测定病毒50%组织培养感染剂量(TCID50)为5.7×109/ml,病毒颗粒滴度(VP)为1.9×1011/ml。结论成功构建携带凋亡素、内皮抑素双基因腺病毒载体并对其进行了大剂量扩增及提纯,达到了细胞及动物实验使用的标准。

Objective To construct recombinant adenovirus vector carrying apoptin （VP3） gene and end- ostatin gene. Methods vp3 gene and endostatin gene were cloned into adenovirus shuttle plasmid pDC316. Then the recombinant shuttle plasmid was cotransformed into HEK293 cells with adenovirus backbone plasmid pBH- GloxE1 to obtain recombinant adenovirus particles （Ad-vp3-IRES-sEndo-his）. The adenovirus particles was screened and confirmed by PCR and RT-PCR, and the correct adenovirus clones screened were amplified in large scale and puff- fled. The viral particle titration such as tissue culture infectious dose 50 （TCID50） and virus particles （VP） was detected. Results PCR and RT-PCR test indicated that apoptin gene and endostatin gene were integrated into the adenoviral genome correctly. Conclusion The recombinant adenovirus vector carrying VP3 gene and endostatin gene was suecessfully constructed, which qualifies for use in cell and animal experiments.