人鸟氨酸脱羧酶和S-腺苷甲硫氨酸脱羧酶双反义腺病毒载体的构建

Construction of an antisense bicistronic recombinant adenovirus vector of ornithine decarboxylase and S-adenosylmethionine decarboxylase

目的:构建人鸟氨酸脱羧酶(ODC)第三外显子和S-腺苷甲硫氨酸脱羧酶(SAMDC)翻译起始位点双反义RNA腺病毒载体。方法:应用RT-PCR方法扩增出SAMDCmRNA翻译起始位点区205bp的基因片断。插入PMD-18T载体中,经XbaI、XhoI酶切回收,反向插入穿梭质粒pAdTrack-CMV-ODCr,形成重组质粒pAdTrack-ODC-SAMDCr。经PmeI酶切线性化后,转入Adeasy-1细菌与pAdeasy-1质粒发生同源重组。挑选阳性重组质粒pAdeasy-ODC-SAMDCr,经PacI酶切后,转染293细胞,包装出腺病毒颗粒,经荧光显微镜和PCR方法对重组腺病毒进行鉴定。结果:利用RT-PCR方法成功地从大肠癌细胞扩增出205bp的cDNA片断,经测序证实为SAMDC翻译起始位点序列。测序证实,重组质粒pAdTrack-ODC-SAMDCr两个基因插入方向和序列均正确,转入Adeasy-1细菌获得多个阳性重组克隆。pAdeasy-ODC-SAMDCr转染293细胞进行包装扩增,可见明显病毒空斑形成,荧光显微镜下可见绿色荧光蛋白在293细胞中表达,PCR法证实含有目的基因。结论:成功构建并扩增出ODC和SAMDC双反义RNA腺病毒载体,为以后肿瘤的基因治疗和预防研究提供了必要工具。

Objective: To construct an antisense bicistronic recombinant andenovirus vector of the third extron in ornithine decarboxylase (ODC) gene and the region around start codon in S-adenosylmethionine decarboxylase (SAMDC) mRNA. Methods: A 205 bp cDNA around start codon in SAMDC mRNA was amplified by RT-PCR method and subcloned to shuttle vector pAdtrack-ODCr. The resulted vector pAdTrack-ODC-SAMDCr was linearized by PmeI and then transformed into adeasy-1 cells. The positive recombinant vectors pAdeasy-ODC-SAMDCr were digested by PacI and then transfected to HEK293 cells to pack and amplify recombinant adenovirus particles. Transfection and viral production were monitored and proved by fluorescent microscope and PCR technique. Results: A 205 bp cDNA was successfully amplified by RT-PCR from colorectoral cancer cells and confirmed by sequence. Several positive recombinant bacterial clones were identified after transformation of Adeasy-1 cells with pAdTrack-ODC-SAMDCr. During the viral package and amplification in 293 cells, lots of obvious viral plagues and GFPs were detected under the microscope and fluorescent microscope. The exist of target gene in adenovirus genome was confirmed by PCR technique. Conclusion: The antisense bicistronic recombinant adenoviruses of ODC and SAMDC are successfully constructed and amplified.