SH2-ODC/AZ1腺病毒的构建及对BCR-ABL蛋白的降解效应

Construction of adenovirus SH2-ODC/AZ1 and its degradation effect on BCR-ABL

目的构建重组腺病毒Ad-SH2-ODC和Ad-AZ1,检测共表达SH2-ODC和AZ1蛋白对BCR-ABL癌蛋白的降解效应。方法PCR扩增src同源结构域2(src homology 2,SH2)、鸟氨酸脱羧酶(ornithine decarboxylase,ODC)及抗酶蛋白1(antizyme 1,AZ1)基因,构建携带SH2-ODC(SO)或AZ1基因的p Ad Track-CMV腺病毒穿梭质粒,经双酶切和测序鉴定后与腺病毒骨架质粒p Ad Easy-1同源重组,获得重组腺病毒质粒。后者经PacⅠ酶切鉴定后,在人胚肾细胞系AD293中包装、扩增,获得重组腺病毒;腺病毒Ad-SO和Ad-AZ1共感染人白血病细胞系K562,western blot检测目的蛋白的表达。结果双酶切和测序证实,腺病毒穿梭质粒构建正确;PacⅠ酶切结果显示,重组腺病毒质粒构建成功;western blot证实目的蛋白在K562细胞中表达正确,共表达SO和AZ1可以降低BCR-ABL蛋白水平。结论成功构建了Ad-SO和Ad-AZ1重组腺病毒,共表达SO和AZ1可以降解K562细胞中的BCR-ABL蛋白。

Objective To construct the recombination adenoviruses Ad-SH2-ODC and Ad-AZ1, and explore the degradation effect of SH2-ODC and AZl co-expression system on BCR-ABL oncoprotein. Methods The fragments of SH2 （src homology 2） , ODC（ orni- thine decarboxylase） and AZ1 （antizyme 1 ） gene were amplified by PCR and cloned into shuttle vector pAdTrack-CMV. The shuttle plasmids were digested by Pme I and homologously recombined with backbone plasmid AdEasy-1. After identification by Pac I diges- tion, adenoviruses were packaged and amplified in human embryonic kidney AD293 cells. The expressions of fusion protein and BCR- ABL in human leukocyte K562 cells co-infected with adenoviruses Ad-SO and Ad-AZl were detected by western blot. Results The shuttle plasmids of adenovirus were confirmed to be correctly contructed by double digestion and gene sequencing. The fusion proteins expressed by recombination adenovirus plasmids were verified by Pac I digestion. The constructed adenoviruses were expressed in K562 cells. BCR-ABL protein level decreased in SO/AZ1 co-expressed K562 cells. Conclusion The adenoviruses Ad-SO and Ad- AZ1 were constructed successfully. The SO/AZl co-express system may degrade BCR-ABL protein in K562 cells.