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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.
OBJECTIVE: To construct adenoviral vector coexpressing A20 and enhanced green fluorescent protein (EGFP), and to observe its effect on human monocyte-macrophage cell line THP-1.Methods: The target gene fragment A20, the target gene was ligated with the vector GV314 to obtain a recombinant shuttle plasmid pGV314-A20, amplified in DH5a competent cells, homologous recombination to obtain the adenoviral vector containing the gene of interest, transfected into 293T cells and packaged into a total infectious capacity The adenovirus particles expressing A20 and EGFP were transfected into THP-1 cells. The expression of A20 protein in THP-1 cells was detected by Western blot. The expression of A20 protein in THP-1 cells was detected by lipopolysaccharide (LPS) TNF-α and IL-6 were detected by Western blotting.Results: Restriction endonuclease digestion and DNA sequencing confirmed that the recombinant vector was successfully constructed and packaged Adenovirus titer of 1 × 1011 pfu / mL, 293 cells infected with adenovirus obvious balloon-like changes, Western blot confirmed that after transfection of THP-1 A20 protein expression was significantly increased compared with the blank group, THP- 1 supernatant TNF-α and IL-6 expression (P <0.05) .Conclusion: Adenovirus vector co-expressing A20 and EGFP can be successfully constructed, which can express in THP-1 cells and reduce the expression of inflammatory cytokines, so as to further study its anti-tumor effect Yan function to lay the foundation.