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目的:体外构建和表达人前列腺特异性膜抗原胞外区(tPSMA)基因重组腺病毒。方法:首先设计一对含BglⅡ和HindⅢ酶切位点的tPSMA引物,以质粒pCR3.1-Uni-hPSMA为模板,通过PCR扩增tPSMA并克隆到腺病毒穿梭质粒中,获得pAdTrack-CMV-tPSMA质粒,将其与含有pAdeasy-1的BJ5183菌电转化,筛选阳性克隆,重组子经线性化后转染HEK293细胞,通过观察绿色荧光蛋白(GFP)的表达、RT-PCR、Western blot法检测目的基因tPSMA的表达。结果:成功构建了含tPSMA的重组腺病毒载体Ad-tPSMA,病毒滴度为2.0×1011pfu/L。结论:该重组腺病毒的构建为下一步研究用其制备树突状细胞疫苗基因治疗提供基础。
Objective: To construct and express the recombinant adenovirus of extracellular domain of human prostate specific membrane antigen (tPSMA) in vitro. Methods: A pair of tPSMA primer containing Bgl Ⅱ and Hind Ⅲ restriction sites was designed. Plasmid pCR3.1-Uni-hPSMA was used as a template to amplify tPSMA by PCR and cloned into adenovirus shuttle plasmid to obtain pAdTrack-CMV-tPSMA The recombinant plasmid was transformed into HEK293 cells by linearization and then transfected into HEK293 cells. The expression of green fluorescent protein (GFP) was observed by RT-PCR and Western blot. Gene tPSMA expression. Results: The recombinant adenovirus Ad-tPSMA containing tPSMA was successfully constructed and its titer was 2.0 × 10 11 pfu / L. Conclusion: The construction of the recombinant adenovirus provides the foundation for the further study of gene therapy of dendritic cell vaccine.