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目的构建表达人Bcl2基因的重组腺病毒,并研究其在原代培养螺旋神经节细胞(spiralganglioncells,SGC)的表达特性。方法采用细菌内同源重组法,从pUCCAGGS/Bcl2质粒中获得人Bcl2cDNA,克隆到腺病毒穿梭质粒pAdTrackCMV上,后者和腺病毒骨架质粒pAdEasy1在细菌内同源重组,在HEK293细胞中包装成为重组Ad增强型绿色荧光蛋白/Bcl2腺病毒,电镜观察病毒颗粒,用病毒感染原代培养的大鼠螺旋神经节细胞,在荧光显微镜下观察感染效率,用逆转录聚合酶链反应方法检测Bcl2基因在螺旋神经节细胞的转录表达,用WesternBlot检测其蛋白的表达。结果经酶切鉴定证实重组腺病毒质粒构建成功,电镜显示包装细胞中有大量病毒颗粒存在,呈规则六边形。荧光显微镜下观测到感染病毒的SGC细胞发出明亮的绿色荧光。分别在mRNA水平及蛋白水平证实有外源性Bcl2基因的表达。结论细菌内同源重组法是一种简便、高效的制备方法。构建的重组Ad增强型绿色荧光蛋白/Bcl2腺病毒能有效的感染SGC,并可在SGC中正确地转录和翻译,为进一步研究腺病毒介导的Bcl2基因对螺旋神经节损伤的保护作用奠定基础。
Objective To construct a recombinant adenovirus expressing human Bcl2 gene and study its expression in primary cultured spiral ganglion cells (SGC). Methods Human Bcl2 cDNA was obtained from the pUCCAGGS / Bcl2 plasmid by homologous recombination in bacteria and cloned into the adenovirus shuttle plasmid pAdTrackCMV. The recombinant plasmid was homologously recombined with adenovirus backbone plasmid pAdEasy1 in bacteria and packaged in HEK293 cells for recombination Ad-enhanced green fluorescent protein / Bcl2 adenovirus was used to observe the virus particles by electron microscopy. Primary cultured rat spiral ganglion cells were infected with virus, and the infection efficiency was observed under a fluorescence microscope. The Bcl2 gene was detected by reverse transcription polymerase chain reaction Spiral ganglion cell transcriptional expression was detected by Western Blot protein expression. Results Restriction endonuclease confirmed the construction of recombinant adenovirus plasmid, electron microscopy showed a large number of packaging cells in the presence of virus particles, a regular hexagonal. The virus-infected SGC cells were observed to emit bright green fluorescence under a fluorescence microscope. The expression of exogenous Bcl2 gene was confirmed at mRNA level and protein level respectively. Conclusion Bacterial homologous recombination is a simple and efficient preparation method. The constructed recombinant adenovirus with enhanced green fluorescent protein / Bcl2 can effectively infect SGC and can be correctly transcribed and translated in SGC, which lays the foundation for further study on the protective effect of adenovirus mediated Bcl2 gene on spiral ganglion injury .