复制缺陷型重组腺病毒Adeno-X-脑源性神经营养因子的构建与鉴定

目的制备能高水平表达大鼠脑源性神经营养因子（BDNF）的复制缺陷型重组腺病毒。方法首先TRIzol法提取大鼠脊髓组织总RNA。逆转录-聚合酶链反应（RT—PCR）方法扩增BDNF基因并将其克隆入pMD18-T载体测序，测序后将其插入pShuttle2中。然后利用体外连接法将BDNF基因克隆入Adeno-X^TM腺病毒骨架中。脂质体法转染HEK 293细胞包装携带BDNF基因的重组腺病毒。少量提取重组腺病毒DNA利用PCR法检测为复制缺陷型BDNF基因重组腺病毒后大量扩增，蛋白电泳及Western blot法检测BDNF基因在HEK 293细胞中的表达。结果成功构建了能高水平表达大鼠BDNF的复制缺陷型重组腺病毒。结论利用Adeno-X^TM系统体外连接法可方便、快捷的构建BDNF基因复制缺陷型重组腺病毒，并可在体外高水平表达其所携带的目的基因。

The construction and identification of replication-deficient recombinant Adeno-X-BDNF

Objective To construct and identify a strain of replication-deficient recombinant adenovirus which can express the SD rat BDNF gene in a high level. Methods Extract the total RNA of a SD rat spinal cord, BDNF gene is synthesized and amplified using the method of RT-PCR, then BDNF gene is cloned into the pMDI8-T vector using a method of A-T cloning, and then clone the BDNF gene which sequences correctly into pShuttle2 vector. Subsequently clone it into Adeno-X^TM backbone by ligation in vitro, transfect HEK 293 cells using Lipofectamine^TM 2000 and package for the recombinant adenovirus particles. Amplify it largely after make sure it is the correct replication-deficient recombinant adenovirus which carry the BDNF gene. Finally, determine the levels of secreted recombinant adenovirus-derived BDNF via protein electrophoresis and Weston blot. Results Construct successfully the replication-deficient recombinant adenovirus which can express the BDNF gene in a high level in vitro. Conclusion Ligation in vitro is a short-cut method for construction of replication-deficient recombinant adenovirus cloned with a SD rat BDNF gene and the gene of interest can be expressed in a high level in cultured HEK 293 cells.