TRAIL和HN基因融合表达重组腺病毒的构建及其对DT40细胞的抑瘤作用

Construction of recombinant adenovirus vector encoding TRAIL and HN fusion protein and its inhibition on avian leukosis tumor cell DT40 in vitro

分析携TRAIL和HN基因的重组腺病毒在DT40细胞中的表达规律及杀伤肿瘤细胞的生物学效应。通过RT-PCR分别从鸡外周血淋巴细胞和新城疫病毒(NDV)LaSota株中扩增出TRAIL和HN基因,通过定点突变和重叠延伸拼接法实现TRAIL和HN基因的2A连接,将获得的目的基因克隆到pShuttle-CMV穿梭载体中,与pAdeasy-1载体在BJ5183细菌内同源重组,构建了包含目的基因的重组腺病毒质粒pAd-TRAIL和pAd-TRAIL-2A-HN,将质粒线性化后转染AD293细胞,包装后的重组腺病毒,经RT-PCR检测、荧光显微镜观察和病毒滴度测定后,将获得的重组腺病毒体外转染禽淋巴白血病DT40细胞,通过RT-PCR和Western blotting检测重组腺病毒的感染性和外源基因的表达情况,采用流式细胞仪检测重组腺病毒对DT40细胞生长的影响以及Ad-TRAIL和Ad-TRAIL-2A-HN对DT40细胞的凋亡作用。PacⅠ酶切证实同源重组成功;RT-PCR、荧光显微镜检测证实重组腺病毒质粒成功导入AD293细胞中且具有良好的感染性,滴度为1.9×1010PFU/mL;Western blot检测结果显示,TRAIL-linker-HN基因能够在DT40细胞中稳定表达并对DT40细胞产生细胞毒作用,且诱导细胞的凋亡率为29.52%,表明TRAIL-linker-HN具有双基因抑瘤的协同效应。

Expression of reconstructed adenovirus with TRAIL and HN genes in avian leukemic lymphoid cell DT40 cells and its anti-tumor biological effects were analyzed. The TRAIL and HN genes were obtained by RT-PCR from the total RNA of chicken peripheral blood lym- phocyte and Newcastle disease virus （LaSota strain） , respectively. TRAIL and HN genes were linked using site mutation and splicing overlap extension by 2A peptide. And the fusion gene was cloned to pShuttle-CMV shuttling vector and co-transfected to BJ5183 competent cells by using pAd-Easy framework vector. Then the recombinant adenovirus plasmid with target genes pAd-TRAIL and pAd-TRAIL-2A-HN was constructed. The plasmid was digested and transfected to AD293 cells through ]iposome, thus being packed to be recombinant adenovirus. U- sing RT-PCR, immunofluorescence microscopy and viral titre assay, the exogenous genes were demonstrated to be expressed in the recombi- nant adenovirus correctly. The recombinant adenovirus was transfected to DT40 in vitro, and then its infectious ability and exogenous gene expression were detected by RT-PCR and western blotting. Its growth and influence on DT40 cell apoptosis were detected by flow cytometry, and anti-tumor cooperative-effect of the 2 genes was assessed too. Homologous recombination was proved by Pac I digestion. The reconstruc- ted adenovirus was successfully introduced into AD293 cells and viral titer was 1.9×10^10 PFU/ml. The western blot showed that TRAIL-lin- ker-HN gene could be expressed in DT40 cells, and cell apoptotic rate was 29. 52% , indicating that TRAlL-linker-HN have the synergistic effect of bi-gene anti-tumor.