腺病毒载体介导的大鼠DNA多聚酶β重组体的构建及其在PC12细胞中的表达  

Construction of Recombinant Adenovirus Containing Rat DNA Polymerase β and Its Expression in PC12 Cells

作　　者：钟建辉[1] 银巍[2] 藏林泉[2] 江伟健[2] 邱鹏新[2] 颜光美[2] 

机构地区：[1]中山大学 基础医学院药理教研室附属第一医院肾内科,广东广州510080 [2]中山大学

出　　处：《中山大学学报（医学科学版）》2005年第6期622-625,共4页Journal of Sun Yat-Sen University:Medical Sciences

基　　金：国家自然科学基金(30472010) 广东省自然科学基金(039191)

摘　　要：【目的】为探讨DNA多聚酶β在神经细胞损伤中的作用与机制，构建其重组腺病毒载体，并在PC12细胞中表达。【方法】RT-PCR获取大鼠DNA多聚酶β(POLβ)的编码序列，TA克隆测序，酶切POLβ基因并连入穿梭质粒pAdTrack-CMV，电转至含腺病毒pAdeasy骨架质粒的大肠杆菌BJ5183。重组腺病毒质粒(pAd-polβ)和Lipofectamine2000转染293细胞，用重组腺病毒感染PC12细胞。【结果】10个TA克隆中有3个克隆的序列与GENE BANK中大鼠POLβ cDNA序列完全一致，同源重组检测时30个克隆中有8个含目标条带。DNA序列正确的POLβ重组腺病毒质粒转染293细胞后，感染Ad-polβ的293及PC12细胞荧光显微镜下可见绿色荧光，重组腺病毒Ad-polβPCR鉴定含目标条带，Ad-polβ感染PC12细胞Western Blot检测到POLβ蛋白表达。[Objective] The expression of rat DNA polymerase β was up-regulated after transient middle cerebral artery occlusion, using the technique of fluorescence differential display reverse transcription polymerase chain reaction （FDD RT-PCR）. To determine the role of DNA polymerase β （polβ） in brain injury. An adenoviral vector was constructed containing the coding sequence for rat DNA polymerase β.[Methods] The rat polβ coding sequence was cloned to pGEM-T Easy^TMvector by RT-PCR, and confirmed by DNA sequencing and subcloned into shuttle plasmid pAdTrack-CMV, then linearized and transferred to E. coli （BJ5183） contained adenoviral backbone vector pAdEasy-1. The recombinant plasmid was packaged and amplified in HEK 293 cells. PC12 cells were infected with the recombinant adenoviruses. [Results] Screening 10 pGEM-T Easy^TMclones, 3 clones have no base changes. Eight out of 30 homologous recombination plasmids have the target band. The Adpolβb recombinant plasmid was verified by DNA sequence analysis. After infected with Adpolβ adenovirus, strong green fluorescence were observed in HEK 293 and PC12 cells under fluorescence microscopy. Adpolβ DNA in 293 cells was identified by PCR. The expression of rat DNA polymerase β in Adpolβ infected PC12 cells are confirmed by immunoblot analysis. [Conclusion] Recombinant adenoviral vectors containing rat DNA polymerase β were constructed and efficiently expressed in PC 12 cells.

关 键 词：DNA多聚酶β 腺病毒载体 PC12细胞 

分 类 号：Q81[生物学—生物工程]