大鼠BDNF基因复制缺陷型重组腺病毒的构建与鉴定  

Construction and Identification of Replication-deficient Recombinant Adenovirus Cloned with a SD Rat BDNF Gene

作　　者：王鹏[1] 吴燕峰[2] 黄文林[3] 唐勇[1] 黄霖[1] 杨睿[1] 沈慧勇[1] 

机构地区：[1]中山大学附属第二医院矫形外科学,广东广州510120 [2]中山大学附属第二医院脐血库,广东广州510120 [3]中山大学肿瘤防治中心实验研究部,广东广州510060

出　　处：《中山大学学报（医学科学版）》2006年第2期135-139,共5页Journal of Sun Yat-Sen University:Medical Sciences

基　　金：国家自然科学基金资助项目(30271311);广东省自然科学基金资助项目(36642);广东省科技厅重大项目(2004A30201002)

摘　　要：【目的】构建携带大鼠脑源性神经营养因子(brainderivedneurotrophicfactor,BDNF)目的基因的复制缺陷型重组腺病毒,并初步检测其体外表达目的基因的能力。【方法】提取大鼠脑组织总RNA,使用RT-PCR方法扩增BDNF目的基因并将其克隆入pMD18-TSimple载体测序,将测序完全正确的BDNF目的基因克隆入pShuttle2中,然后利用体外连接法将BDNF目的基因克隆入Adeno-XTM腺病毒骨架中,LipofectamineTM2000法转染HEK293细胞包装携带BDNF目的基因的重组腺病毒,少量提取重组腺病毒DNA检测证实为复制缺陷型BDNF基因重组腺病毒后大量扩增,蛋白电泳及Westernblot法初步检测BDNF目的基因在HEK293细胞中的表达。【结果】成功构建了携带大鼠BDNF目的基因的复制缺陷型重组腺病毒,并证实其在HEK293细胞中可高水平表达BDNF目的基因。【结论】利用体外连接法可方便、快捷地构建携带BDNF目的基因的复制缺陷型重组腺病毒,并可在体外高水平表达表达其所携带的目的基因。[Objective] To construct a strain of replication-deficient recombinant adenovirns which were cloned with a SD rat brain derived neurotrophic factor （BDNF） gene and examine its ability to express the BDNF gene in vitro. [Methods] The total RNA of a SD rat brain were extracted, BDNF gene was synthesized and amplified by using the method of RT-PCR, then BDNF gene was cloned into the pMD18-T simple vector by using a method of A-T cloning, and then the BDNF gene, which sequences were correct, was cloned into pShuttle2 vector. Subsequently it was cloned into Adeno-XTM backbone by ligation in vitro, HEK 293 cells were transfected by using LipofectsmineTM 2000 and packaged for the recombinant adenovirns particles. Replication-deficient recombinant adenovirus which carried the BDNF gene were identified and amplified. Finally, the levels of secreted recombinant adenovirns-derived BDNF were determined by protein electrophoresis and Western blot analysis. [Results] The replication-deficient recombinant adenovirns which carried the BDNF gene were constructed successfully and could express the BDNF gene in a high level when transfected HEK 293 cells in vitro. [Conclusion] The method of ligation in vitro is a short cut for construction of replication-deficient recombinant adenovirus cloned with a SD rat BDNF gene and the gene of interest can be expressed in high level in cultured HEK 293 cells .

关 键 词：脑源性神经营养因子 基因重组 腺病毒载体 

分 类 号：R342.3[医药卫生—基础医学]