携带表皮生长因子受体反义cDNA的重组腺病毒的构建

Construction of the Recombinant Adenovirus Vector with EGFR Antisense cDNA of Controlling Cell Cycle

目的构建携带参与细胞周期调控的表皮生长因子受体(EGFR)反义cDNA的重组腺病毒载体。方法将1032 bp EGFR反义cDNA片段正向及反向插入腺病毒载体质粒pA dT rack-CM V上,与骨架质粒在大肠杆菌B J5183胞内进行同源重组,经293细胞包装、扩增后得到携带EGFR反义cDNA的重组腺病毒A dE asy-GFP-EGFR-sense/an tisense。结果成功地构建了A dE asy-GFP-EGFR-sense/an tisense的重组腺病毒载体系统,经测定腺病毒载体滴度分别2.2×109efu/mL和2.5×109efu/mL。结论构建的重组腺病毒A dE asy-GFP-EGFR-an t-isense可望有效地将EGFR-an tisense cDNA基因导入喉癌细胞株/组织内,为进一步研究喉癌细胞信号转导干预机制和治疗研究提供实验基础。

Objective To recombine the adenovirus vector carrying EGFR sence/antisense cDNA which takes part in control of cell cycle. Methods The 1032 bp EGFR sence/antisense cDNA fragment was cloned into the shuttle plasmid pAdTrack-CMV. The resultant plasmid and the backbone plasmid pAdEasy-1 were transferred into E.coli BJ5183 for homologous recombination, and the recombinant adenoviruses were generated in cells. The recombinant adenoviruses were packaged and amplified in the 293 cells. Then the viral titer was detected by GFP. Results The recombinant adenovirus vector carrying EGFR sence/antisense cDNA to control the cell cycle was constructed successfully. The viral titers were 2.2×10^9 efu/mL and 2.5×10^9 efu/mL respectively. Conclusion The recombinant adenovirus vector constructed by us could introduce EGFR antisense cDNA into the laryngeal squamous cell carcinoma line or tumor tissue, which would provide an experiment basis to study further the interfered mechanism of signal transdution and the therapies of laryngeal squamous cell carcinoma.