人CUIAA重组腺病毒载体的构建及其在PC-12细胞中的表达特点

目的构建并鉴定带有绿色荧光蛋白（green fluorescence protein，GFP）报告基因的人源CUL4A（hCUIAA）基因腺病毒表达载体Ad—hCUIAA—GFP，探求bCUIAA在PC-12细胞中的表达特点。方法扩增hCUIAA基因，并通过In—FusionPCR克隆技术构建穿梭质粒pDC315-EGFP—hCUIAA，利用AdMaxTM腺病毒包装系统将该穿梭质粒与腺病毒表达载体骨架质粒pBHGloxAEl，E3Cre共转染HEK293细胞，经GFP荧光检测和Western印迹检测确认hCUIAA的表达后，进一步通过病毒扩增及纯化得到hCUL4A重组腺病毒载体Ad—hCUIAA—GFP。将该载体转染Pc—12细胞，观察hCUIAA—GFP融合蛋白在Pc-12细胞中的表达情况。结果成功获得了较高滴度的腺病毒载体Ad—hCUIAA—GFP（1．6×10^12pfu／m1）。荧光检测表明，Ad—hCUIAA—GFP转染Pc-12细胞后72h内，病毒转染率随着时间和病毒转染滴度的增加而增加。DAPI细胞核荧光染色结果表明，hCUIAA—GFP的表达主要集中在细胞质部分。GFP荧光检测及Western印迹检测结果显示，hCUIAA—GFP在Pc-12细胞中的表达量随时间和病毒转染滴度的增加而增加。结论带GFP的hCUIAA重组腺病毒载体Ad—hCUIAA—GFP构建成功，掌握了其转染Pc-12细胞的最佳滴度及其在Pc-12细胞中的时空表达特点，为今后对hCUIAA在PC-12细胞中的功能性研究奠定了基础。

Construction of Human CUL4A Recombinant Adenovirus Vector and Expression Features of CUL4A in PC-12 Cells

Objective To construct and human CUIAA （hCUIAA） adenovirus expression vector carrying green fluorescence protein （GFP） report gene and identify the expression features of hCUL4A in PC-12 cells. Methods Human CUIAA gene was amplified and cloned into shuttle vector （pDC315-EGFP-hCUIAA） by In-Fusion PCR cloning technique, pBHG lox AE1, E3 Cre, the skeleton plasmid of adenovirus expressioin vector, was then eo-transfected with pDC315-EGFPhCUIAA into HEK293 cells using AdMaxTM vector system. Following GFP-hCUIAA fluorescence detection and Western blot analysis, hCUL4A adenovirus expression vector Ad-CUIAA-GFP was obtained through further viral amplification and purification. The expression patterns of hCUL4A in PC- 12 cells were studied after transfeetion of Ad-hCUL4A-GFP into PC-12 cells. Results High titer hCUL4A adenovirus expression vector Ad-hCUL4A-GFP （ 1.6 × 10^12 pfu/mL） was obtained. The Ad-hCUL4A-GFP vector was successfully tranfeeted into PC-12 cells. Fluorescence results showed that in the oeriod of 72 hours after transfeetion, transfection efficiency of Ad-hCUIAA-GFP changed in a time and titer dependent manner. DAPI staining showed that hCUIAA-GFP was mainly expressed in the cytoplasm of PC-12 cells. GFP fluorescence detection and western blot showed that the expression level of hCUL4A-GFP was time-course and titer dependent. Conclusion Human CUIAA adenovirus expression vector Ad-hCUIAA-GFP was successfully constructed and its optimal transfection titer was determined. The expression patterns of hCUL4A were revealed. These results may help further functional study of hCUL4A in PC-12 cells.