表达分泌型脑源性神经营养因子的腺相关病毒载体的构建及其表达的检测

Construction and Expression of Recombinant Adeno-Associated Virus Expressing Brain-Derived Neurotrophic Factor

摘要:

采用PCR和体外连接的方法构建了带有信号肽的脑源性神经营养因子(BDNF)的融合基因, 将此基因插入到腺相关病毒载体穿梭质粒pSNAV, 然后用重组质粒pSNAV-Ig-BDNF转染包装细胞, 筛选出永久细胞株后, 用携带腺相关病毒rep及cap基因的单纯疱疹病毒超感染包装细胞, 成功包装出含有目的基因的一型血清型腺相关病毒。经PCR鉴定该病毒含有目的基因片段, 被该病毒感染的细胞裂解液经Western blotting 证实有BDNF表达, 其培养液经ELISA检测证实含有高水平的BDNF蛋白。该病毒与小量的非复制型腺病毒Ad-null联合应用时, 其BDNF蛋白的表达水平显著提高。可弥补腺相关病毒起效慢、对细胞转导效率低的弱点, 为今后应用AAV作基因治疗提供了实验证据。

Abstract:

A fusion gene called Ig-BDNF, in which brain-derived neurotrophic factor cDNA fused to the 3’ end of signal peptide of Ig coding sequence, was constructed by PCR, digested and subcloned into shuttle plasmid pSNAV to obtain a recombinant plasmid pSNAV-Ig-BDNF. Then the plasmid encoding fusion protein was transfected into 293 cell lines and the stably transfected clones were selected with neomycin. AAV1 containing Ig-BDNF fusion gene vectors were obtained by super-infection by Herpes virus. The resultant adeno-associated virus vectors AAV-Ig-BDNF were confirmed by PCR, Western blotting and a sandwich enzyme-linked immunosorbent assay (ELISA) after infection of 293 cell lines. The results indicated that AAV-Ig-BDNF contained the target gene, and infected cells and produced the fusion protein into the supernatant. The content of BDNF in medium per 5×104 cells over a 24 h incubation period reached 1000 pg/mL. With the help of non-replicative adenovirus during AAV-Ig-BDNF infection, the expression of BDNF increased 7~8 fold, and the enhancement of BDNF gene expression was observed in a concentration-dependent manner. These results suggested that a functional AAV-Ig-BDNF was successfully constructed and it offers basis for further study for gene therapy of neural degeneration diseases.