重组小鼠神经生长因子前导肽和人β-内啡肽融合基因非增殖型腺病毒的构建和鉴定  

Construction and identification of the incompetent-replication adenovirus carrying the fusion gene composed of prepropeptide of mouse nerve growth factor and human beta-endorphin gene.

作　　者：尤圣武 [1] 俞卫锋 [2] 于布为 [1] 钱其军 [3] 王星华 [3] 苏长青 [3] 曹云飞 [2] 杨立群 [2] 李晓青 [2] 

机构地区：[1]200025,上海市,上海第二医科大学瑞金医院麻醉科 [2]200025,上海市,上海第二医科大学东方肝胆外科医院麻醉科 [3]上海市东方肝胆外科医院肿瘤病毒基因治疗研究室

出　　处：《中华麻醉学杂志》2004年第12期896-900,共5页Chinese Journal of Anesthesiology

摘　　要：目的构建并鉴定携带小鼠神经生长因子前导肽(PN)和人β-内啡肽(β-EP)融合基因非增殖型腺病毒(Ad-NEP).方法取出生2周、雄性昆明小鼠的颌下腺组织,逆转录聚合酶链反应(RT-PCR)扩增得到PN上游部分序列,全基因合成PN下游部分和人β-EP序列并将其与PN上游部分连接成融合基因并测序.采用AdEasyTM System细菌内重组的方法构建携带小鼠PN和人β-EP融合基因非增殖型腺病毒,PCR鉴定后扩增不含野毒的阳性克隆并纯化,采用50%组织培养感染剂量(TCID50)法测定病毒滴度;体外转染人皮肤癌上皮(A431)细胞,3 d后RT-PCR观察融合基因的转录,免疫组化法检测细胞内β-EP的表达,放射免疫法检测感染后1、3、7 d时培养基内β-EP的浓度.结果融合基因序列正确,PCR可检测到475 bp的融合基因条带并排除野病毒株,腺病毒Ad-NEP滴度为1.5×1010pfu/ml.A431细胞转染病毒后3 d RT-PCR可以检测到475 bp大小的融合基因片段,免疫组化染色后细胞内出现橙黄色浓染颗粒,Ad-NEP组感染后1、3、7 d时细胞培养基内人β-EP浓度高于对照组(P＜0.01),感染后3、7 d Ad-NEP组β-EP浓度比感染后1 d高(P＜0.05或0.01).结论成功构建能在体外培养的非神经内分泌细胞内外高效转基因表达人β-EP的重组腺病毒.Objective To construct and identify the incompetent-replication adenovirus carrying the fusiongene composed of the encoding gene of prepropeptide of mouse nerve growth factor(PN)and human beta-endorphin(β-EP)geue.Methods The gene segments of PN obtained from total RNA of the submandibular glandof a 2-week old Kumning mouse were amplified by RT-PCR and joined with the segment of β-EP to form the fusiongene which was sequenced.The fusion gene contained in the incompetent-replication adenovirus was formed in theBJ-Ad Easy-1 susceptible cells and identified by PCR so as to choose the positive clone without wild vectors.Thecorrect clone was amplified and purified.The titers of adenovirus were determined using the specific 50% tissueculture infection dosage(TCID 50)method.Three days after the adenovirns was transferred into the cultured A431cells,RT-PCR was performed to showed the transcribed mRNA of this fusion gene and the intracellular β-EPexpression was quanlitatively detected by inummo-histological method.Finally the concentration of human β-EP inthe culture medium was determined by quantitative radio-immunoassay on 1st,3rd and 7th day afterinfeetion.Results The sequence of the fusion gene was correct.The titer of recombinant adenovirus Ad-NEP was1.5×10~10 pfu/ml.Three days after infection a 475 bp segment was amplified by RT-PCR and abundant orangegranules were shown in the infected cell.The β-EP concentration in the culture medium was significantly higher inAd-NEP group than in the control group on 1st,3rd and 7th day(P<0.01).Conclusion The recombinantadenoviruses which can make the cultured non-endocrine cell efficiently express human β-EP in vitro weresuccessfully constructed

关 键 词：融合基因 Β-EP 感染后 增殖型腺病毒 小鼠 Β-内啡肽 PN 阳性克隆 序列 基因合成 

分 类 号：R346[医药卫生—基础医学]