人前列腺特异性膜抗原基因重组腺病毒的构建及其表达

Construction and expression of recombinant adenovirus vector containing human prostate specific membrane antigen gene

目的体外构建含有人前列腺特异性膜抗原(PSMA)基因的重组腺病毒载体,并检测其在HEK293细胞中的表达。方法设计一对含有SfiI酶切位点的PSMA基因上下游引物,以质粒pCMV-SPORT6/PSMA为模板,通过PCR扩增获得PSMA基因全序列。片段回收后经酶切处理,连接到穿梭质粒pShuttle-CMV-EGFP上,获得重组穿梭质粒pShuttle-EGFP-PSMA。经SfiI酶切、PCR及插入片段测序鉴定正确后,将其用I-CeuI和I-SceI双酶切处理,转移到pAdxsi载体上,得到pAdxsi-GFP-PSMA病毒质粒,线性化后经HEK293细胞包装成复制缺陷型腺病毒Ad-PSMA,通过观察绿色荧光蛋白(GFP)的表达,RT-PCR,Western印迹检测目的基因人PSMA的表达。结果成功构建了含有人PSMA基因的重组腺病毒,病毒滴度为2×1010 pfu/ml。Western印迹检测可见PSMA蛋白的正确表达。结论该重组腺病毒载体的成功构建及表达,为下一步以人PSMA作为靶抗原构建DC疫苗和基因免疫治疗奠定基础。

Objective To construct the recombinant adenovirus vector which containing human prostate specific membrane antigen（PSMA） gene and check the target gene expression in HEK293 cells.Methods Primers containing SfiⅠ was designed and full length PSMA cDNA was obtained from the pCMV-SPORT6/PSMA plasmid by PCR,the PCR product was digested with restrictive endonucleases SfiI,then being inserted directionally into pShuttle-CMV-EGFP.The plasmid of pShuttle-EGFP-PSMA was lined with SfiⅠ,the fragment containing PSMA was reclaimed and then transfected into pAdxsi vector after being digested with I-CeuI and I-SceI,the recombinant adenovirus plasmid was transfected into HEK293 and packed,and the expression of PSMA was proved by observing the green fluorescence protein（GFP） and detected by RT-PCR and Western blot method.Results The recombined adenovirus PSMA was constructed successfully and the titer was about 2×1010 pfu/ml.PSMA was expressed efficiently in HEK293 cells after infection.Conclusions The successful construction of recombinant adenovirus Ad-PSMA lays a good foundation for the further research on producing DC vaccines for the prostate cancer therapy.