三重表达肺结核DNA疫苗在细胞水平的检测  

The Detection of Triple Expression of Tuberculosis DNA Vaccines on the Cell Level

作　　者：马爱丽[1] 曹以诚[1] 张珍武[1] 

机构地区：[1]华南理工大学生物科学与工程学院,广州510006

出　　处：《中国生物工程杂志》2008年第7期78-86,共9页China Biotechnology

基　　金：国家自然科学基金重大研究项目(90412015);教育部中国网格计划生物信息网格平台子项目(B1-137040130);国家科技基础条件平台项目(2005DKA64001);科技部基础学科平台建设生物计算网络平台项目(2005DKA64001);广州市科技攻关计划(2005Z1-E4023)资助项目

摘　　要：目的:评估整合siRNA、融合抗原基因、hIL-12的新型肺结核DNA疫苗在人胚肾细胞293中三个表达单元独立互不干扰表达。方法在已构建含有靶向抗调亡基因Mcl-1L的siRNA、结核分枝杆菌抗原基因Ag85B-ESAT6(PVAE)、hIL-12三个独立表达单位的新型肺结核DNA疫苗pVAX-siRNA-PVAE-IL-12基础之上,将抗原基因与增强型绿色荧光蛋白(EGFP)基因融合,得到真核表达质粒pVAX1-siRNA-PVAE[EGFP]-hIL12。并将DNA疫苗中的靶向抗凋亡基因Mcl-1L的序列置换为靶向EGFP基因的siRNA序列[siEGFP],得到pVAX1-[siEGFP]-PVAE[EGFP]-hIL12表达质粒。用质粒pVAX1-siRNA-PVAE[EGFP]-hIL12、pVAX1-[siEGFP]-PVAE[EGFP]-hIL12转染人胚肾细胞293,以EGFP为报告基因,分别证实融合抗原基因与siRNA编码序列的表达。以ELISA测定培养细胞上清中hIL-12的表达。结果经酶切鉴定和测序证实肺结核DNA疫苗改造成功。转染细胞中检测到绿色荧光,证实了融合抗原基因的表达。对照组证实了siRNA编码序列表达的抑制效果。转染48h后细胞培养液中hIL-12的检出量为1571.63pg/ml;72h后细胞培养液中hIL-12的检出量为2392.25pg/ml。结论已构建的肺结核DNA疫苗能在真核细胞中有效表达siRNA、抗原基因与hIL-12。为进一步研究该DNA疫苗抗肺结核的免疫保护率和基因治疗效果打下基础。Objective： A novel tuberculosis DNA vaccine integrating siRNA, fusional antigen and hIL-12 triple expression units was constructed in our laboratory. Methods： To evaluate the independent expression of the three expression units, two eukaryotic expression plasmid pVAX1-siRNA-PVAE[ EGFP] -hiLl2 with TB fusional gene Ag85B -ESAT6 （PVAE） and enhanced green fluorescent protein （ EGFP）, and pVAX1 -siEGFP-PVAE [ EGFP]-hiLl2 with siRNA coding sequence targeting EGFP instead of Mcl-1L were constructed. Then two plasmids were used to transfer human embryonic kidney 293 cells. Based on EGFP report gene, it was demonstrated that siRNA expression unit and fusional antigen gene were independently expressed. Results： The hiLl2 expression at 48h and 72h post transfection were also detected by ELISA analysis up to 1571.63pg/ml and 2392.25pg/ml respectively in the cell culture fluid. Conclusion： The results demonstrated that the novel DNA vaccine with siRNA, TB fusional antigen and hiLl2 three expression units in the same plasmid frame is successfully constructed and independently expressed in eukaryotic cells. It laid a foundation for further animal model study on anti-tuberculosis effects of this novel DNA vaccine.

关 键 词：肺结核 DNA疫苗 SIRNA 融合抗原 hIL-12 

分 类 号：Q786[生物学—分子生物学]