摘要
目的：构建问号钩端螺旋体（简称钩体） tlyA基因原核表达系统，确定目的重组表达产物rTlyA溶解红细胞及诱导人THP-1和小鼠J774A．1巨噬细胞分泌致炎细胞因子的作用。方法采用PCR扩增问号钩体黄疸出血群赖型赖株tlyA基因，T-A克隆后测序。采用原核表达载体pET-42a及表达宿主菌E．coli BL21DE3构建tlyA基因原核表达系统。采用SDS-PAGE检测rTlyA表达情况， Ni-NTA亲和层析法提纯rTlyA。采用平板法和分光光度法测定rTlyA对绵羊红细胞的溶血活性。采用RT-PCR和Western blot分别检测问号钩体赖株感染THP-1和J774A．1细胞后tlyA基因mRNA水平变化及TlyA外分泌情况。采用ELISA检测rTlyA诱导THP-1和J774A．1细胞分泌致炎细胞因子IL-1β、IL-6和TNF-α的作用。结果所克隆的tlyA基因与GenBank中公布的相应基因核苷酸和氨基酸序列相似性分别为99．2％和99．8％。所构建的tlyA基因原核表达系统能有效表达rTlyA。10μg／ml rTlyA能显示较强的溶解绵羊红细胞活性。问号钩体赖株感染THP-1和J774A．1细胞1～8 h后，tlyA-mRNA水平显著升高（P＜0．05）且出现TlyA外分泌。0．1、1和10μg／ml rTlyA均能使THP-1和J774A．1细胞分泌致炎细胞因子IL-1β、IL-6和TNF-α的水平显著升高（P＜0．05）。结论问号钩体tlyA基因产物TlyA是溶血素并能诱导巨噬细胞分泌IL-1β、IL-6和TNF-α，可在钩体病炎症反应中发挥重要作用。
Objective To construct a prokaryotic expression system for tlyA gene of Leptospira in-terrogans （ L.interrogans） strain and to investigate the effects of the expressed rTlyA protein on the hemolysis of sheep erythrocytes and the secretion of pro-inflammatory cytokines by human THP-1 cells and murine J774A.1 macrophages.Methods The fragment of tlyA gene of L.inetrrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai was amplified by PCR.The PCR product was sequenced after T-A cloning.A prokary-otic expression system for the tlyA gene was constructed by using pET-42a as the expression vector and E.coli BL21DE3 strain as the host strain.The expression of rTlyA protein was detected by SDS-PAGE.Ni-NTA af-finity chromatography was performed for purification.The lytic activity of the rTlyA protein on sheep erythro-cytes was determined by plate hemolytic test and spectrophotometry.Real-time fluorescence quantitative PCR and Western blot assay were performed to respectively detect the expression of tlyA gene in THP-1 and J774A.1 cells at mRNA and protein levels after infection with L.interrogans strain Lai.ELISA was per-formed to evaluate the effects of the rTlyA protein on the secretion of several pro-inflammatory cytokines （ IL-β, IL-6 and TNF-α） by THP-1 and J774A.1 cells.Results The nucleotide and amino acid sequences of the cloned tlyA gene were 99.2% and 99.8% identical to those corresponding sequences in GenBank, re-spectively.The constructed prokaryotic expression system for tlyA gene successfully expressed the rTlyA pro-tein.The rTlyA protein at the concentration of 10μg/ml showed stronger lytic activity on sheep erythrocytes.nbsp;The transcription levels of tlyA gene （P〈0.05） and the secretion of TlyA protein in THP-1 and J774A.1 cells were significantly increased after infecting with L.interrogans strain Lai for 1 to 8 hours.The rTlyA pro-tein at concentrations of 0.1, 1 and 10 μg/ml significantly enhanced the secretion of IL-1β, IL-6 and TNF-αby THP-1 and J774A.1 cells （P〈0.05）.Conclusion The TlyA protein, encoded by the tlyA gene of L.interrogans strain, was confirmed to be a hemolysin with the ability to induce macrophages to secret IL-1β, IL-6 and TNF-α.This study suggested that the TlyA protein played an important role in inflammatory responses during leptospirosis.
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
2015年第5期341-347,共7页
Chinese Journal of Microbiology and Immunology
基金
国家自然科学基金（81301406）
浙江省自然科学基金（LQ13H190005
LQ12H16019）
