摘要
目的:构建F-actin结合结构域(F-actin binding domain,FABD)缺失腺病毒载体Ad-Bcr/Abl-ΔFABD和野生型Bcr/Abl腺病毒载体Ad-Bcr/Abl,探讨FABD结构域对Bcr/Abl蛋白定位及功能的影响。方法:PCR扩增bcr/abl和bcr/abl-ΔFABD片段,双酶切后克隆至穿梭质粒p Ad Track-CMV,经Ad Easy构建系统获得腺病毒Ad-Bcr/Abl-ΔFABD和Ad-Bcr/Abl,并在HEK293细胞中进行包装。将细胞分为3组,Ad-GFP组、Ad-Bcr/Abl组以及Ad-Bcr/Abl-ΔFABD组,分别加入对应的腺病毒。Western blot验证Bcr/Abl和Bcr/Abl-ΔFABD在293T细胞中的表达水平,间接免疫荧光法检测Bcr/Abl和Bcr/Abl-ΔFABD在293T细胞中的定位。流式细胞术、MTT法分别检测Bcr/Abl和Bcr/Abl-ΔFABD对293T细胞凋亡、增殖的影响。结果:双酶切以及测序验证穿梭质粒构建正确;PacⅠ酶切验证重组腺病毒质粒重组成功;绿色荧光显示重组腺病毒包装成功;Western blot验证Bcr/Abl和Bcr/Abl-ΔFABD可在293T细胞中表达;间接免疫荧光发现Bcr-Abl蛋白在细胞内的分布依赖于F-actin;FABD缺失后虽然不能使Bcr/Abl入核,但使其在细胞内的分布方式由均匀变为点状或块状聚集;FABD缺失后,Bcr/Abl在细胞内呈块状聚集的方式与IM处理后的结果相似。流式细胞术和MTT结果显示,与Bcr/Abl相比,Bcr/Abl-ΔFABD组293T细胞的凋亡比例增加(P<0.05),增殖能力降低(P<0.05)。结论:成功构建Ad-Bcr/Abl和Ad-Bcr/Abl-ΔFABD重组腺病毒,FABD缺失后使Bcr/Abl蛋白在细胞质中的分布方式发生改变,并能抑制Bcr/Abl的抗凋亡能力和促增殖能力。
Objective ：To construct the recombination adenoviruses Ad-Bcr/Ab1-△FABD and Ad-Bcr/Ab1,and to explore the effect of FABD on the cytoplasm localization and function of Bcr/Ab1 oncoprotein. Methods：The fragments of bcr/abl and bcr/abl-AFABD gene were amplified by PCR and cloned into shuttle vector pAdTrack-CMV. The adenoviruses Ad-Bcr/Ab1-△FABD and Ad-Bcr/Ab1 were constructed by Adeasy system and amplified in human embryonic kidney HEK293 cells. The expressions of Bcr/Ab1-△FABD and Bcr/Ab1 in 293T cells were detected by Western blot. The cytoplasm localization of Bcr/Ab1-AFABD and Bcr/Ab1 in 293T cells was detected by indirect immunofluorescence. The effect of Bcr/Ab1 and Bcr/Ab1-AFABD on apoptosis and proliferation in 293T cell was detected by flow cytometry and MTT. Results ： The shuttle plasmids of adenovirus were confirmed to be correctly constructed by double digestion and gene sequencing. The recombination adenovirns plasmids was verified by Pac I digestion. The expression of GFP indicated the adenoviruses were packed. The constructed adenoviruses were expressed in 293T ceils by Western blot. Bcr/Ab1-△FABD protein located in the cytoplasm not in nuclear as form of particles and did not depend on the distribution of the F-actin？ After treat- ing by IM, the distribution of Ber/Ab1 is similar with that of Bcr/Ab1-△FABD protein. As results of flow cytometry and MTT, effect of Bcr/Ab1-AFABD on apoptosis in 293T cell was promoted （P〈0.05） and effect on proliferation was reduced （P〈0.05） compared withthat of Bcr/Ab1. Conclusion：The adenoviruses Ad-Bcr/Ab1- △FABD and Ad-Bcr/Ab1 are constructed successfully. The dis- tribution of Bcr/Ab1-△FABD mutation protein has changed. The deletion of FABD inhibit the anti-apoptosis ability and prolif- eration of Bcr/Ab1.
出处
《重庆医科大学学报》
CSCD
北大核心
2017年第11期1477-1483,共7页
Journal of Chongqing Medical University
基金
国家自然科学基金资助项目(编号:81572060)
重庆市基础与前沿研究计划资助项目(编号:cstc2014jcyj A10081)
