摘要
目的研究创伤弧菌溶细胞素融合蛋白(rVvhA)对人肠上皮细胞(human intestinal epithelial cell,Caco-2)IL-8基因表达的影响。方法IPTG诱导、表达、纯化、复性及Western blot鉴定rVvhA表达和纯化情况;CCK-8法检测rVvhA对Caco-2的细胞毒性作用;RT—PCR检测rVvhA诱导Caco-2细胞IL-8基因的表达情况;ELISA检测Caco-2细胞培养上清IL-8的分泌情况。结果Ni^2+-NTA亲和层析柱对rVvhA进行纯化后纯度可达95%以上;CCK-8结果显示rVvhA活性蛋白显著降低了Caco-2细胞的存活率,有效浓度为1.5HU/ml(P〈0.05);RT—PCR结果显示,0.6HU/ml rVvhA 30 min即可诱导Caco-2细胞IL-8mRNA基因表达上调;ELISA结果显示,Caco-2细胞经rVvhA作用后,培养液上清中IL-8多肽的表达时相在4h。RT—PCR与ELISA结果皆显示,IL-8基因的转录及IL-8表达皆具有时间-剂量依赖性。结论rVvhA在转录水平上能诱导人Caco-2细胞IL-8 mRNA表达,促进IL-8的合成,在创伤弧菌引起的过度炎症反应和败血症的发生、发展中可能具有重要意义。
Objective To investigate the effect of VvhA recombinant protein to the expression of IL-8 in human intestinal epithelial Caco-2 cells. Methods The VvhA recombinant protein(rVvhA) was expressed by prokaryotic expression vector pET-28a ( + )-vvhA in E. coli BL21 (DE3) , purified by Ni^2+- NTA affinity chromatography refolded by stepwise deliquation together with dialysis methods and identified by Western blot. The cytotoxic of rVvhA to human Caco-2 cells was measured by CCK-8. The transcription of IL-8 mRNA in human Caco-2 cells induced by rVvhA was determined by RT-PCR, and the expression of IL- 8 in human Caco-2 cells induced by rVvhA was determined by ELISA assay. Results rVvhA was purified with high purity up to 95%. The viability of human Caco-2 ceils treated with 1.5 HU/ml rVvhA was inhibited significantly (P 〈 0.05). The rVvhA can induce human Caco-2 cells to increase the transcription and expression of IL-8 in dose- and time-dependent manner. The transcription of IL-8 gene in human Caco-2 cells treated with 0.6 HU/ml rVvhA in 30 rain can be up-regulated significantly, and the expression of IL-8 in human Caco-2 cells treated with rVvhA in 4 h can be increased significantly. Conclusion rVvhA has cytotoxic to human Caco-2 and can increase the expression of IL-8, it might play a major role in the inflammatory reaction of rVvhA-exposed cells.
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
2010年第1期60-65,共6页
Chinese Journal of Microbiology and Immunology
基金
浙江省自然科学基金(X205004)
浙江省科技计划项目(2008C-33059)