大鼠PEDF基因的克隆及腺病毒表达载体的构建

Cloning of rat PEDF gene and construction of its adenovirus expression vector

目的构建PEDF基因腺病毒表达载体,为基因治疗年龄相关性黄斑变性(AMD)奠定基础。方法从大鼠视网膜组织中提取总RNA,RT-PCR扩增PEDF基因cDNA,并将回收的PEDF基因克隆入质粒pGEM-Teasy载体中,亚克隆入腺病毒表达载体质粒pDC316中,DNA序列分析加以证实。利用腺病毒Admax系统,通过含有目的基因片段PEDF的穿梭载体pDC316/PEDF和病毒骨架质粒共转染293细胞的方法包装出重组腺病毒AdPEDF。Western Blot对重组腺病毒进行鉴定。结果基因测序表明PEDF基因包含了大鼠PEDF基因阅读框内的全部序列,与NCBI Sequence Viewer中公布的大鼠PEDF mRNA序列(NM-177927)完全一致。获得了表达PEDF蛋白的重组腺病毒。结论成功构建了PEDF基因腺病毒表达载体,为进一步研究基因治疗脉络膜新生血管奠定了基础。

Objective Inhibition of choroidal neovescularization（CNV） is an effective approach to the treatment of wet age-related macular degeneration. Researches showed that pigment epithelium-derived factor（PEDF） can inhibit CNV. Our study was to construct the adenovirus expressing vector of PEDF for gene therapy in experimental CNV. Methods 0. 2 g of retina tissue was collected from rat for the extract of total RNA. The PEDF cDNA were amplified by RT-PCR using the template mRNA isolated from the rat retinal tissue. The cDNA fragments were cloned into the vector pGEM-Teasy. The PEDF coding sequence was subcloned into an adenovirus expression plasmid pDC316 and then sequenced. The Ad-max adenoviral vector system was used in this experiment. PEDF gene was linked to pDC316, and adenovirus back-bone plasmid were transferred into 293 cells and the recombinant Ad-PEDF （Adeno-vector Pigment Epithelium-Derived Factor） was packaged in the cell. The insertion of the PEDF into the adenovirus was identified by Western blot. Results The RNA was to have a high purity with an absorbance 1.86. Compared with the sequences of the rat PEDF mRNA published in NCBI Sequence Viewer （NM-177927） , the PEDF cDNA fragment contained all the sequences of open reading frame of the rattus PEDF gene. The results of identification showed a dominant amplifying band in 1 300 bp, and the gene sequencing of recombination plasmid evidenced that the insertion was consistent with objective gene and the recombinant adenovirus contained the PEDF gene. Conclusion The recombination AdPEDF provides a feasible tool for further research on the gene therapy of CNV.