bcl-2重组腺病毒载体的构建

本研究目的在于构建 bcl-2的正义和反义重组腺病毒载体 ,以研究 bcl-2过度表达对周围神经损伤后脊髓前角运动神经元抗凋亡能力和对轴突再生的影响。为此 ,将正义和反义 bcl-2 c DNA克隆到腺病毒穿梭质粒 p Ad CMV,利用体内同源重组原理 ,用该重组质粒与 p JM17质粒共转染 2 93细胞 ,获得复制缺陷型重组腺病毒 Ad CMV/sense-bcl-2和 Ad CMV/antisense-bcl-2 ,其中 bcl-2 c DNA插入腺病毒基因组的 E1区 ,并由 CMV启动子控制。利用原位杂交、免疫组化反应鉴定重组腺病毒。结果显示 :从感染 Ad CMV/sense-bcl-2重组腺病毒的 2 93细胞中检测到 bcl-2的表达 ;重组腺病毒可感染 2 93细胞并在 2 93细胞内进行有效复制。从而表明本研究成功地构建了 bcl-2重组腺病毒 ,为进一步研究 bcl-2重组腺病毒的功能提供了条件

CONSTRUCTION OF bcl-2 RECOMBINANT ADENOVIRUS VECTOR

To construct bcl-2 recombinant adenovirus vector and study the effect of bcl-2 over-expression on anti-apoptosis of anterior horn motoneurons of spinal cord and regeneration of neurite after peripheral nerve injury, sense and anti-sense bcl-2 cDNA were cloned into adenovirus shuttle vector pAdCMV-linker by standard procedures.bcl-2 cDNA was rescued into adenovirus by cationic lipid based cotransfection of pAdCMV/bcl-2 and pJM17 plasmids into 293 cells. bcl-2 positive recombinant adenovirus was screened by in situ hybridization and immunohistochemical staining. The results showed that bcl-2 positive recombinant adenovirus could express bcl-2. bcl-2 recombinant adenovirus infected 293 cells and was duplicated in 293 cells. bcl-2 recombinant adenovirus has been constructed and can be used for further research.