摘要
目的 构建含有大鼠 β NGF基因的重组腺病毒载体并观察其在星形胶质细胞的表达 ,为下一步在动物体内进行中枢损伤的基因治疗提供基础。方法 设计一对含有HindⅢ及SalⅠ酶切位点的 β NGF基因上下游引物 ,PCR法扩增含有大鼠 β NGFcDNA的质粒pUC19 β NGF ,产物经HindⅢ及SalⅠ双酶切 ,插入腺病毒穿梭质粒 pAdTrack CMV ,获得重组质粒pAdTrack β NGF ,经PmeⅠ酶线性化后 ,与腺病毒骨架质粒 pAdEasy 1共同转入BJ5 183中进行同源重组 ,重组子经卡那霉素抗性筛选及酶切分析 ,阳性克隆经 2 93细胞包装 ,扩增 ,空斑法测定病毒滴度 ,转化体外培养的星形胶质细胞。通过RT PCR、ELISA、Westernblot法检测其在星形胶质细胞中的表达。结果 PCR及酶切鉴定确认获得重组腺病毒载体 pAd β NGF ,重组腺病毒在 2 93细胞中包装成功 ,病毒滴度达 2× 10 11PFU/ml。病毒上清液经ELISA检测 ,β NGF表达的量为 (94 5 0± 4 5 8)ng/L。 结论 该重组腺病毒载体的构建为下一步在动物体内的表达及中枢神经系统损伤的转基因治疗提供了一定的基础。
Objective To construct a recombinant adenovirus vector containing β-NGF gene and detect the expression of β-NGF in astrocyte infected by this recombinant adenovirus. Methods Rat β-NGF cDNA was amplified by PCR from plasmid pUC19-β-NGF and inserted into shutter plasmid pAdTrack-CMV by enzyme digestion with HindⅢ and Sac Ⅰ. The resultant plasmid was linearized by enzyme Pme Ⅰ and subsequently contransformed into BJ5183 with the adenoviral backbone plasmid pAdEasy-1. So the homologous recombinantion took place in the bacteria. Recombinants were selected for kanamymic resistance and confirmed by restriction endonuclease analyses. The adenoviruses were packaged and amplified in 293 cells. Finally the cells of astrocyte were transfected with the resultant adenoviruses. Recombinant adenovirus production was confirmed by PCR. ELISA and Western blot methods were employed to identify the expression of NGF in astrocyte. Results PCR and enzyme digestion demonstrated that the recombinant adenovirus vector pAd-β-NGF was obtained.The recombinant adenovirus was packaged in 293 cells and the viral titer was 2×10 11 PFU/ml. ELISA methods revealed that β-NGF was expressed in astrocyte.In the supernatant of infected astrocyte β-NGF obtained a high concentration to (94.50±4.58) ng/L. Conclusion The β-NGF recombinant adenovirus has been successfully built,which makes the basis for further research and transgene therapy for central nervous system injury.
出处
《安徽医科大学学报》
CAS
2004年第2期122-126,共5页
Acta Universitatis Medicinalis Anhui
基金
江苏省科委应用基础研究计划资助项目 (编号 :BJ990 62)
