A20与EGFP共表达腺病毒载体的构建及转染THP-1细胞

Construction and identification of a recombinant adenoviral vector expressing A2 and EGFP

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摘要目的:构建A20和增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)共表达的腺病毒载体,观察其对人单核-巨噬细胞系THP-1的影响.方法:PCR扩增目的基因片段A20,将目的基因与载体GV314连接获得重组穿梭质粒pGV314-A20,在DH5a感受态细胞中扩增,同源重组得到含有目的基因的腺病毒载体,转染293T细胞,包装成具有感染能力的共表达A20和EGFP的腺病毒颗粒,转染目的细胞THP-1,通过Western blot检测A20蛋白在THP-1细胞的表达,EILSA检测脂多糖(lipopolysaccharide,L P S)刺激T H P-1后其上清肿瘤坏死因子-α(tumor necrosis factorα,TNF-α)和白介素-6(interleukin-6,IL-6)的表达.结果:限制性内切酶酶切鉴定与DNA测序证实重组载体构建成功,包装的腺病毒滴度达到1×1011 pfu/mL,感染了腺病毒后的293细胞出现了明显的气球样变,Western blot证实转染THP-1后A20蛋白表达显著增加,与空白组比较,THP-1上清中TNF-α和IL-6的表达下降,两者相比,有统计学差异(P<0.05).结论:成功构建A20与EGFP共表达腺病毒载体,能够在THP-1细胞表达,降低其炎性因子表达,为进一步研究其抗炎功能奠定基础. AIM： To construct an adenoviral vector expressing A20 and enhanced green fluorescent protein （EGFP） and evaluate its effects in human mononuclear cell line THP-1, METHODS： A20 gene fragment was amplified by PCR and inserted to GV314 vector to obtain a recombinant shuttle plasmid pGV314-A20. The recombinant vector was amplified in competent cells DH5α. A recombinant adenovirus co-expressing A20 and EGFP was generated by homologous recombination and packaged via 293T cells. The adenovirus was transfected into THP-1 cells, and then the expression of protein A20 was detected by Western blot. The levels of tumor necrosis factor alpha （TNF-α） and interleukin-6 （IL-6） in the culture supernatants of lipopolysaccharides （LPS）-stimulated THP-1 cells were assessed by ELISA. RESULTS： The recombinant vector was identi- fied and confirmed by restriction enzyme digestion and DNA sequencing. The titer of packaged adenoviruses was 1 × 10^11 pfu/L, and 293 cells infected with the adenovirus showed obvious ballooning. Western blot analysis indicated that A20 protein expression was increased in THP-1 cells transfected by the adenovirus. The levels of TNF-α and IL-6 in the supernatants declined significantly in the A20 group compared with the control group （P 〈 0.05）. CONCLUSION： A recombinant adenoviral vector which expresses A20 and EGFP has been successfully constructed, which lays a foundation for further study of anti-inflammatory function of A20.