Construction and Expression of a Recombinant Adenovirus Vector Containing the Human Prostate-Specific Membrane Antigen Gene on Dendritic Cells
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摘要:
目的 构建含有人前列腺特异性膜抗原基因（prostate specific membrane antigen，PSMA）的重组腺病毒，并将其感染树突状细胞（dendritic cell，DC）后检测其在DC上的表达。 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基因序列。片段回收、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，转移至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-GFP；感染从健康志愿者外周血来源的DC，荧光倒置显微镜下观察绿色荧光蛋白（GFP）的表达，Western blot检测PSMA基因在DC上的表达。 结果 成功构建含有人PSMA基因的重组腺病毒，病毒滴度为2×1010 pfu/mL。构建好的Ad-PSMA-GFP可以在DC上高效和正确地表达。 结论 人PSMA重组腺病毒载体的成功构建及其在DC上的表达，为下一步研究奠定了基础。 -
关键词:
- 腺病毒 /
- 前列腺特异性膜抗原（PSMA） /
- 树突状细胞 /
- DC疫苗
Abstract:Objective To construct the recombinant adenovirus vector containing the human prostate-specific membrane antigen (PSMA) gene; and to check the target gene expression on dendritic cells (DCs). Methods Primers containing the restrictive endonuclease Sfi I were designed, and a full-length PSMA cDNA was obtained from the pCMV-SPORT6 /PSMA plasmid via polymerase chain reaction (PCR). The PCR product was digested with Sfi I and then inserted directionally into pShuttle-CMV-EGFP. The pShuttle-EGFP-PSMA plasmid was then lined with Sfi I. The fragment containing PSMA was reclaimed and transfected into a pAdxsi vector after digestion with I-CeuI and I-SceI. The recombinant adenovirus plasmid was then transfected into human embryonic kidney 293 (HEK293) cells and packed, and Ad -PSMA-green fluorescence protein (GFP) was infected into the DCs from the peripheral blood of healthy volunteers. The PSMA protein expression in the DCs was confirmed via GFP observation and detected using Western blot analysis. Results The recombined adenovirus PSMA was successfully constructed at a titer of approximately 2× 1010 pfu/mL. PSMA can be expressed efficiently and correctly in DCs after infection. Conclusion The DC vaccine was successfully infected using the recombinant adenovirus Ad-PSMA. The current results can serve as a good foundation for further research on prostate cancer therapy. -
Key words:
- Adenovirus /
- Prostate-specific membrane antigen /
- Dendritic cell /
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图 1 pShuttle-GFP-FOLH1鉴定结果
M：Marker（1kb DNA ladder）；1，2：阳性克隆：2.2kb/5.1kb（选择测序验证正确的1#进行下游实验）；3：阴性对照
Figure 1. Identification of pShuttle-GFP-FOLH1
图 2 pAdxsi-GFP-FOLH1鉴定结果
M：Marker（1kb DNA ladder)；1~3：阳性克隆14kb/11.8kb/4.8kb/2.66kb/2.47kb/1.45kb/0.6kb；4：阴性克隆14kb/11.8kb/4.0kb/2.47kb/1.45kb/0.6kb
Figure 2. Identification of pAdxsi-GFP-FOLH1
图 3 重组纯化腺病毒PCR电泳
M：Marker；1，2：质粒pShuttle-EGFP-PSMA/pAdxsi-GFP-PSMA（引物PSMA-SfiⅠ-F和PSMA-SfiⅠ-R扩增结果）；3：Ad-GFP病毒（引物PSMA-SfiⅠ-F和PSMA-SfiI-R扩增结果）；4，5：Ad-GFP-PSMA（引物PSMA-SfiⅠ-F和PSMA-SfiⅠ-R扩增结果）
Figure 3. Amplification of adenovirus fragment by PCR
图 4 Ad-GFP-PSMA转染DC细胞48h后状态（×100）
A：荧光显微镜下观察Ad-GFP-PSMA转染；B：同一视野下普通光镜观察；C：同一视野叠加后观察转染效率
Figure 4. Ad-GFP-PSMA transfected DC cells at 48 hours
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