N-甲基-D-天冬氨酸受体2B亚基基因重组腺病毒镇痛疫苗的构建  

Construction of analgesic vaccine with recombinant adenovirus encoding NMDA receptor 2B gene

作　　者：王公明[1] 陈建平[1] 杨少兵[1] 田学愎[1] 高峰[1] 杨辉[1] 安珂[1] 田玉科[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院麻醉学教研室,武汉市430030

出　　处：《中华麻醉学杂志》2007年第8期699-702,共4页Chinese Journal of Anesthesiology

基　　金：国家自然科学基金资助项目（30471660）

摘　　要：目的构建携带N-甲基-D-天冬氨酸受体2B亚基（NR2B）基因的重组腺病毒镇痛疫苗。方法以携带NR2B基因的腺病毒穿梭质粒pDC515-NR2B和腺病毒骨架质粒pBHGfrt（del）E1，3FLP共转染腺病毒包装细胞293细胞，连续观察细胞的形态学。取形成病毒空斑的293细胞，分别采用PCR、RT-PCR及Western blotting的方法从基因水平、转录水平和蛋白水平对病毒空斑筛选和鉴定，对鉴定正确的病毒空斑进行扩增和纯化。结果转染后12d可观察到细胞病变效应，15d后形成病毒空斑。NR2B基因不仅正确插入重组腺病毒载体，而且可在293细胞正确表达NR2B蛋白。鉴定正确的病毒空斑命名为NR2B基因重组腺病毒5型镇痛疫苗，滴度为5×10^11VP／ml，纯度100％。结论成功地构建了NR2B基因重组腺病毒镇痛疫苗，可用于疫苗镇痛的研究。Objective To construct the recombinant adenovirus analgesic vaccine encoding NR2B gene. Methods The shuttle plasmid pDC515-NR2B was cotransfected with an Adenovirus genomic plasmid pBHGfrt （del） E1,3FLP into E1 complementing cell line 293 cells. The transfected 293 cells were observed continuously for the detection of cytopathic effect and the viral plaques. As the formation of the viral plaques, some viral plaques were picked and were identified using PCR, RT-PCR and Western blotting. The amplification and purification of the identified viral plaques were done and the titer and purity of the virus were assayed. Results Cytopathic effect were observed on posttransfectional day 12 and well-defined plaques were observed on posttransfectional day 15. The presence and expression of transgene NR2B in the recombinant adenovirus vector were confirmed using PCR, RT-PCR and Western blotting. The identified viral plaques were designated as the recombinant adenovirus serotype 5 analgesic vaccine encoding NR2B （rAd5/NR2B）. The titer of rAd5/NR2B was 5 × 10^11 VP/ml and the purity was 100%. Conclusion The recombinant adenovirus analgesic vaccine encoding NR2B are constructed successfully and can be put into researching about vaccine analgesia.

关 键 词：受体 N-甲基-D天冬氨酸 腺病毒科 疫苗 镇痛 

分 类 号：R3[医药卫生—基础医学]