人神经营养素-3基因重组腺病毒载体的构建、表达和生物活性检测  

CONSTRUCTION OF A RECOMBINANT ADENOVIRUSVECTOR, EXPRESSION OF HUMAN NEUROTROPHIN-3 WITH BIOLOGICAL ACTIVITY

作　　者：王俊梅[1] 曾园山[1] 刘然义 吴立志 薛刚 黄文林 熊轶[1] 王延华[3] 

机构地区：[1]中山大学中山医学院组织胚胎学教研室神经科学研究室 [2]肿瘤防治中心,广州510080 [3]昆明医学院神经研究所,昆明650031

出　　处：《解剖学报》2005年第5期460-464,共5页Acta Anatomica Sinica

基　　金：国家自然科学基金(30270700);广东省社会发展攻关项目(2003C33808);高等学校博士学科点专项科研基金(2004558068)资助

摘　　要：目的构建具有生物活性的人神经营养素_3(NT_3)基因重组腺病毒表达载体。方法从人脑组织mRNA中扩增NT_3基因全长cDNA,定向克隆于穿梭质粒pShuttle中,获得一个带有CMV启动子的质粒。再与腺病毒骨架DNA(Adeno-X viral DNA)体外连接,形成重组腺病毒质粒(pAd_NT_3)。用pAd_NT_3转染人胚肾293细胞后包装成有感染能力的重组腺病毒颗粒(Adeno_NT_3)。结果NT_3基因RT-PCR扩增产物约801 bp。Adeno_NT_3 PCR鉴定为正确重组子。RT_PCR、免疫细胞化学、ELISA及Western blot检测显示,Adeno_NT_3能在其转染的293细胞表达和分泌NT_3。这种NT_3能使体外培养的神经干细胞更多地分化为神经元样细胞。结论应用体外连接法成功构建了人NT_3基因重组腺病毒表达载体,其表达产物具有促进神经干细胞分化为神经元样细胞的活性作用。Objective To construct a recombinant adenovirus vector with biological activity expressing human neurotrophin-3 （NT-3）. Methods Using the mRNA extracted from human brain tissue, the full-length eDNA of human NT-3 gene was amplified by RT-PCR, First, a expression cassette with CMV promoter was made by cloning NT-3 into pShuttle vector. Next, the expression cassette was transferred to Adeno-X viral DNA to form a recombinant adenoviral plasmid （pAd-NT-3） by in vitro ligation reaction, pAd-NT-3 was packaged into infectious recombinant adenoviral particles （Adenc-NT-3） by transfeeting human embryonic kidney 293 cells. Results The amplified NT-3 gene was about 801bp in length by RT-PCR, Adeno-NT-3 was proved correct recombinant with PCR, The expression of NT-3 in 293 eells was confirmed by RT-PCR, immonucytochemistry, ELISA and Western blot. This kind of NT-3 was able to make more neural stem cells differentiated into neuron-like cells in vitro. Conclusion A recombinant adenovirus carrying human NT-3 has be generated successfully by in vitro ligation reaction. NT-3 expressed in Adeno-NT-3 transfected 293 cells is able to make neural stem cells differentiated into neuron-like cells. Present study may provide the basis on gene therapy of central nervous injury using further NT-3.

关 键 词：神经营养素-3 基因克隆 重组腺病毒 基因表达 神经干细胞 免疫细胞化学 小鼠 

分 类 号：R322.85[医药卫生—人体解剖和组织胚胎学]