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目的:构建RGD修饰的ING4和PTEN双基因共表达重组腺病毒载体(Ad.RGD-ING4-PTEN),研究其对人白血病细胞MEG01的抑制作用。方法:采用AdEasyTM腺病毒重组系统,在本科室已成功构建的pAdTrack-CMV-ING4-polyA-promoter、pAdTrack-CMV-polyA-promoter腺病毒转移质粒的基础上,在多克隆酶切位点的Not I、Xho I间插入PTEN片段,得到pAdTrack-CMVING4-polyA-promoter-PTEN重组转移载体,与RGD-4C修饰的腺病毒骨架质粒pAdEasy-1(RGD)同源重组后,经包装和扩增获得ING4和PTEN双基因共表达重组腺病毒Ad.RGD-ING4-PTEN。将Ad.RGD-ING4-PTEN体外感染人白血病细胞MEG01,检测腺病毒对MEG01细胞的感染效率,Western blot法检测外源基因ING4和PTEN在MEG01细胞中的表达,CCK8法检测外源基因的导入对MEG01细胞的生长抑制作用,流式细胞仪检测外源基因导入对MEG01细胞的凋亡和周期的影响。结果:鉴定结果显示,成功构建了Ad.RGD-ING4-PTEN双基因共表达腺病毒,其能有效地感染MEG01细胞。Western blot检测到ING4和PTEN在MEG01细胞中的表达;ING4和PTEN基因能明显抑制MEG01细胞的生长,诱导其凋亡并产生G2/M期阻滞作用,且双基因联合作用较单基因作用更明显。结论:成功构建了RGD-4C修饰的ING4和PTEN双基因共表达腺病毒载体Ad.RGD-ING4-PTEN。收获的腺病毒能有效感染人白血病MEG01细胞。Ad.RGD-ING4-PTEN具有抑制MEG01细胞的生长、诱导其凋亡及G2/M期阻滞的作用。
OBJECTIVE: To construct a recombinant adenovirus vector (RGD-ING4-PTEN) co-expressing ING4 and PTEN modified by RGD to study its inhibitory effect on human leukemia cells MEG01. Methods: Based on the AdEasyTM adenovirus recombination system and the adenovirus transfer plasmid pAdTrack-CMV-ING4-polyA-promoter and pAdTrack-CMV-polyA-promoter successfully constructed in our department, PTEN fragment was inserted between I and Xho I to get pAdTrack-CMVING4-polyA-promoter-PTEN recombination transfer vector. After homologous recombination with RGD-4C adenovirus backbone plasmid pAdEasy-1 (RGD) ING4 and PTEN double gene co-expression of recombinant adenovirus Ad.RGD-ING4-PTEN. Ad.RGD-ING4-PTEN was transfected into human leukemia cells MEG01 in vitro to detect the infection efficiency of adenovirus against MEG01 cells. The expression of foreign genes ING4 and PTEN in MEG01 cells was detected by Western blot and the expression of foreign genes was detected by CCK8 On the growth inhibition of MEG01 cells, flow cytometry detection of exogenous gene transfection on MEG01 cells apoptosis and cycle. Results: The results of identification showed that Ad.RGD-ING4-PTEN double gene co-expression adenovirus was successfully constructed and could effectively infect MEG01 cells. The expression of ING4 and PTEN in MEG01 cells was detected by Western blot. ING4 and PTEN genes could significantly inhibit the growth of MEG01 cells, induce their apoptosis and induce G2 / M arrest, and the combined effect of two genes was more than that of single gene obvious. CONCLUSION: Recombinant adenoviral vector, Ad.RGD-ING4-PTEN, co-expressing ING4 and PTEN gene modified by RGD-4C was successfully constructed. Harvested adenovirus can effectively infect human leukemia MEG01 cells. Ad.RGD-ING4-PTEN can inhibit the growth of MEG01 cells and induce apoptosis and G2 / M arrest.