甲型副伤寒沙门氏菌cdtB亚基的原核表达及对巨噬细胞IL-6、IL-8、TNF-α分泌的影响  

Cloning and expression of recombinant Salmonella paratyphi A cytolethal distending toxin proteins and its effect on cytokine production by human monocyte-derived macrophages

作　　者：陈鸿鹄[1] 吴圆圆[2] 占利[1] 梅玲玲[1] 

机构地区：[1]浙江省疾病预防控制中心微生物所,杭州310051 [2]浙江大学医学院附属儿童医院实验检验中心,杭州310003

出　　处：《中国人兽共患病学报》2016年第6期535-538,546,共5页Chinese Journal of Zoonoses

基　　金：浙江省疾病预防控制中心青年科技项目(No.2013-11)资助~~

摘　　要：目的研究甲型副伤寒沙门氏菌感染过程中,cdtB对宿主巨噬细胞分泌促炎细胞因子的影响。NF-κB信号通路阻断剂对cdtB诱导的巨噬细胞分泌细胞因子的影响。方法对甲型副伤寒沙门氏菌cdtB亚基进行原核表达,制备并模型纯化重组蛋白,建立其刺激人THP-1巨噬细胞模型,ELISA检测THP-1分泌IL-6,IL-8和TNF-α等细胞因子。在共培养体系中加入NF-κB信号通路阻断剂,ELISA检测THP-1分泌IL-6,IL-8和TNF-α等细胞因子。结果成功构建甲型副伤寒沙门氏菌cdtB原核表达系统,表达并纯化重组cdtB蛋白,与空白对照相比,受到cdtB刺激的THP-1细胞上清中的IL-6,IL-8和TNF-α浓度显著上升,而在THP-1细胞培养基中加入NF-κB信号通路阻断剂SN50可以显著抑制重组cdtB诱导的IL-6、IL-8、TNF-α分泌。结论甲型副伤寒沙门氏菌cdtB能够通过NF-κB信号通路诱导巨噬细胞分泌IL-6、IL-8和TNF-α,在甲型副伤寒相关的炎症反应中发挥促进作用。To investigate the role of Salmonella paratyphi A cytolethal distending toxin（CDT）in pro-inflammatory cytokines induction in macrophages,we cloned and expressed cdtB,a subunit of CDT fromSalmonella paratyphi（S.paratyphi）A in Escherichia coli,and purified the recombinant cdtB proteins（SPA-rcdtB）to homogeneity by Ni-NTA affinity chromatography.By co-cultured SPA-rcdtB with human monocyte-derived macrophages（THP-1）and detected IL-6,IL-8and TNF-αin supernatant by ELISA,we determined if SPA-rcdtB were able to induce human monocyte-derived macrophages to produce cytokines.In addition,NF-κB inhibitor SN50 was used to research the effect of NF-κB signaling pathways in SPA-rcdtB induced macrophage IL-6,IL-8and TNF-αsecretion.In this study,we proved that the cdtb gene segment amplified fromS.paratyphi A CMCC 50001 strains showed high nucleotide and amino acid sequence identities（97.62% and 99.82%）compared with cdtb gene in the GenBank（GenBank accession No.NC_006511.1）.And the SPA-rcdtB were able to induce the synthesis by THP-1of interleukin-6,IL-8and TNF-α,and the IL-6,IL-8and TNF-αinduction by SPA-rCdtB was significantly suppressed by NF-κB inhibitor.This result indicated that SPA-rcdtB plays a promoting role inflammatory response associated with SPA infection.

关 键 词：甲型副伤寒沙门氏菌 cdtB 细胞因子 巨噬细胞 

分 类 号：R378.2[医药卫生—病原生物学]