AKT3基因重组腺病毒表达载体的构建及其对BT474乳腺导管癌细胞增殖的影响  

Construction of AKT3 Gene Recombinant Adenovirus Expression Vector and Study Its Effect on the Proliferation of Breast Ductal Cancer Cells

作　　者：韩俊永[1] 林炎鸿[1] 黄俏佳[1] 

机构地区：[1]南京军区福州总医院分子医学研究中心,福州市350025

出　　处：《医学分子生物学杂志》2014年第1期12-16,共5页Journal of Medical Molecular Biology

基　　金：国家自然科学基金（No.81372788）,南京军区医药卫生科研基金重点项目（No.112032）

摘　　要：目的构建人AKT3基因的重组腺病毒表达载体，探究其对人乳腺导管癌细胞增殖的影响。方法应用RT-PCR方法从流产胎儿脑组织总RNA中扩增出AKT3eDNA，以XhoI及最g21I酶切位点克隆入腺病毒pShuttle-IRES-hrGFP-1穿梭载体，测序鉴定后与骨架载体PAdeasy-1在BJ5183细菌中进行同源重组，获得重组腺病毒表达载体pAd．AKT3后，转染293A细胞，进行病毒的包装。荧光显微镜下观察细胞内绿色荧光，测定病毒效价，Western印迹方法分析细胞内AKT3蛋白质的表达。通过MTr实验，研究AKT3过表达前后BT474乳腺导管癌细胞增殖的变化。结果重组载体经酶切鉴定和测序证实目的基因正确无误。重组腺病毒效价为2．6×10^8pfu／ml，重组腺病毒转导后，BT474乳腺导管癌细胞中可检测到AKT3的过表达：Western印迹检测结果显示AKT3融合蛋白在BT474细胞中表达良好。而转导空载体腺病毒及未转导细胞对照中未见有此融合蛋白质条带；MTr结果显示AKT3表达上调的重组腺病毒组，其增殖活性显著高于转导空载体腺病毒组及未转导细胞组，差异具有统计学意义（P〈0．01），而后两者差异无统计学意义（P〉0．05）。结论成功地构建了AKT3重组腺病毒表达载体，AKT3过表达可增强BT474乳腺导管癌的增殖，为进一步研究AKT3的功能奠定了基础。Objective To construct human AKT3 gene recombinant adenovirus expression vector and to study its effect on the the proliferation of breast ductal cancer cells. Methods AKT3 cDNA was amplified by RT-PCR with the RNA extracted from brain tissue of aborted fetus as template, and was then cloned into adenovirus shuttle vector pShuttle-IRES-hrGFP-1 by XhoI and BglII restriction sites. The recombinant shuttle vector was confirmed by sequencing, and then transformed into Escherichia coli BJ5183 carrying backbone plasmid pAdEasy-1 to obtain adenovirus plasmid through homologous recombination. The recombinant adenovirus vector pAd-AKT3 was transfected into 293A cells to form adenovirus particles. Intracellular green fluorescence was observed by fluorescent microscopy, and the virus titer was determined. The adenovirus particles were then transduced into BT474 breast ductal cancer cells. Western blotting was carried out to analyze the expression of AKT3. The effects of AKT3 on the proliferation of BT474 cells were detected by MTT assay. Results DNA sequencing confirmed that the AKT3 adenovirus expression vector was successfully constructed with the virus titer of 2. 6 × 10^8 PFU/mL. Western blotting revealed that AKT3-GFP fusion protein was only detected in BT474 cells infected with AKT3 adenovirus particles but not in those transduced with the adenovirus particles containing empty vector or untransduced cells. MTI＂ assay showed that the proliferation of BT474 cells transduced with AKT3 adenovirus particles was significantly greater than in BT474 cells with empty vector or untransduced cells （P 〈 0. 01 ） . There was no significant difference between the latter two groups （ P 〉 0. 05 ） . Conclusion AKT3 can increase the proliferation of BT474 breast ductal cancer cells. Overexpression

关 键 词：Akt3 重组腺病毒表达载体 基因表达 BT474细胞 四唑盐比色测定 

分 类 号：Q78[生物学—分子生物学]