携带人livin α基因腺病毒载体的构建及其在树突状细胞的表达

Construction of a recombinant adenoviral vector encoding functional human livin α gene and its expression in dendritic cells

目的:本研究旨在利用AdMax系统构建携带人抗凋亡基因livinα的重组腺病毒载体(rAd-livinα),并感染树突状细胞(Dendritic cells,DCs),制成DCs疫苗,为下一步用此疫苗抗肿瘤实验奠定基础。方法:以质粒pIRES2-EGFP-livinα为模板,通过PCR扩增出人livinα基因cDNA序列,再将livinα基因cDNA亚克隆于穿梭质粒pDC316-EGFP-cmv,获得重组质粒pDC316-EGFP-cmv-livinα。将鉴定后的重组穿梭质粒pDC316-EGFP-cmv-livinα与腺病毒辅助质粒pBHGlox(delta)E1,3Cre共转染HEK293细胞,同源重组获得重组腺病毒rAd-livinα。用重组腺病毒rAd-livinα感染人DCs后,Western blot方法分析livinα蛋白表达,流式细胞仪检测DCs的表型变化。结果:在HEK293细胞内能观察到重组腺病毒rAd-livinα绿色荧光蛋白的表达。rAd-livinα经PCR鉴定特异性地扩增出符合预期大小的片段。rAd-livinα感染的DCs中可检测到livinα蛋白的表达,并且感染的DCs与未感染DCs比较,可明显提高了细胞表面分子CD83、CD86和HLA-DR的表达(P<0.05)。结论:通过AdMax系统成功构建了重组腺病毒rAd-livinα,使livinα蛋白有效表达于DCs中,并且重组腺病毒感染的DCs的成熟度得到提高。

Objective：To construct the recombinant adenovirus vector carrying human antiapoptosis gene livin α（rAd-livin α） by AdMax system and make vaccine using the rAd-livin α to infect dendritic cells（DCs）.This will provide experimental basis for next antitumor researches using the vaccine.Methods：The full-length cDNA of human livin α was amplificated from the plasmid pIRES2-EGFP-livin α and was subcloned into the adenovirus shuttle plasmid pDC316-EGFP-cmv.The newly constructed plasmid pDC316-EGFP-cmv-livin α transfected into HEK293,together with the helper plasmid pBHGlox（delta）E1,3Cre.Based on the homologous recombination of the two plasmids within HEK293 cells,rAd-livin α was generated.Then human DCs were transduced with rAd-livin α.The expression of human livin α protein was assessed by Western blot in lysate of rAd-livin α DCs.Phenotype of cells was analysed by flow cytometry.Results：The bands of recombinant plasmid pDC316-EGFP-cmv-livin α were in the right range corresponding with expectation by enzyme digestion.Sequence of inserted livin α was correspond to the report in GeneBank.EGFP expression could be observed in rAd-livin α-infected HEK293 cells and PCR amplification of the rAd-livin α specifically produced the bands with expected sizes.By Western blot,a band corresponding to the expected full-length human livin α protein was detected in rAd-livin α DCs.Compared with the non-infected DCs,the CD83,CD86,and HLA-DR expression levels on cell surface were upregulated after DCs were infected by rAd livin α.Conclusion：The rAd-livin α has been successfully constructed using AdMax system,and effective expressions of livin α protein also have been verified in rAd-livin α DCs.Infection of recombinant adenovirus enhances maturation of DCs.