重组腺病毒载体Ad5-CCL20的构建及其表达

Construction and expression of recombinant adenovirus vector Ad5-CCL20

目的:构建表达趋化因子CCL20的重组腺病毒载体Ad5-CCL20,检测体外转染小鼠结肠癌细胞CT-26后的产物表达。方法:通过EcoR I/Sal I双酶切CCL20质粒和pDC316质粒,将获取编码CCL20的基因片段连接到pDC316重组穿梭质粒。将测序正确的穿梭质粒pDC316-CCL20与骨架质粒pBHGlox_E1,3Cre共同转染293T细胞,构建重组腺病毒载体Ad5-CCL20。予Ad5-CCL20体外转染结肠癌细胞CT-26,Western blotting和Elisa检测转染后不同时间段CCL20的表达情况,并以含CCL20的上清分别对mDC、iDC进行趋化实验。结果:成功构建表达趋化因子CCL20的重组腺病毒载体Ad5-CCL20;Western blotting和Elisa均检测到CCL20表达,且CCL20表达量随病毒转染CT-26细胞的时间延长而逐渐增加;趋化实验表明,趋化因子CCL20对iDC、mDC都有趋化作用,但对iDC的趋化作用更加明显(P<0.05)。结论:重组腺病毒载体Ad5-CCL20的构建及获取为后续开展肿瘤的免疫治疗提供实验基础。

Objective： To construct recombinant adenovirus vector Ad5 - CCL20, and detect the expression of CCL20 after Ad5-CCL20 transfected colon cancer cells CT-26. Methods： Genes encoding CCL20 was obtained from original plasmid double-digested with EcoR I/Sal I enzymes.The CCL20 DNA segments were linked into pDC316 to recombine shuttle plasmid pDC316-CCL20. After genome sequencing,we take shuttle plasmid pDC316-CCL20 and plasmid backbone pBHGIox_E1, 3Cre co-transfecting 293T cells in mediation of liposome. The constructed recombinant adenovirus vector was named Ad5 - CCL20.Lastly,after Ad5 - CCL20 transfected CT-26 cells in vitro,the expression of CCL20 at different time points （12h,24h,36h and48h）was detected by Western blot and Elisa.Then,Culture supernatant was added into iDC and mDC to evaluate the chemotactic activity of CCL20. Results： The recombinant adenovirus AdS-CCL20 were successfully constructed.The expression of CCL20 was detected by Western blot and Elisa.The level of CCL20 expression was increased with prolonged incubation of the infected CT-26 cells. Chemotaxis experiments show that the chemokine CCL20 had chemotactic activity to the iDC and mDC, but more obviouly for iDC （P〈0. 05）, Conclusion： The construction and obtain of recombinant adenovirus vector Ad5 - CCL20 pro vide a new method for developing tumor immunotherapy.