人前列腺特异性膜抗原胞外区基因重组腺病毒的构建

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。

Construction of Human Prostate Specific Membrane Antigen Extracellular Domain Gene Recombinant Adenovirus

Objective:To construct a recombinant adenovirus containing human prostate specific membrane antigen (hPSMA) extracellular domain gene. Methods: Primers containing BglⅡ and HindⅢ were designed, and extracellular domain gene tPSMA was amplified from plasmid of pCR3.1-Uni-hPSMA by PCR and subcloned into an adenovirus shuttle plasmid pAdTrflck-CMV. Subsequently, the recombinant shuttle plasmid pAdTrack-CMV-tPSMA was co-transformed into BJ5183 cells with the adenovirus backbone plasmid pAdeasy-1 to obtain the homologous recombination. The adenovirus was generated in HEK293 cells. The expression of extracellular domain gene of PSMA was detected by RT-PCR and Western blot.Results: Recombinant adenovirus Ad-tPSMA was constructed successfully and the titer of virus was 2.0×1011 pfu/L. Conclusion: The successful construction of recombinant adenovirus Ad-tPSMA laid a good foundation for the further research on producing dendritic cell vaccines for the therapy of prostate cancer.