猪带绦虫丝氨酸蛋白酶抑制剂Ts-serpin-1对人巨噬细胞THP-1的免疫调节功能研究

Study on immunomodulatory function of Taenia solium serine protease inhibitorTs-serpin-1 in THP-1 cells

主要研究猪带绦虫丝氨酸蛋白酶抑制剂Ts-serpin-1(WormBase:TsM_000065700)对宿主THP-1细胞的免疫调节作用。通过设计特异性引物和RT-PCR扩增技术,获得Ts-serpin-1编码序列,用qRT-PCR分析Ts-serpin-1基因在猪带绦虫成虫和中绦期幼虫的表达情况;构建pCold-Ts-serpin-1原核表达载体,诱导表达纯化重组蛋白Ts-serpin-1;用重组蛋白Ts-serpin-1处理THP-1细胞,采用qRT-PCR和ELISA方法检测Ts-serpin-1处理THP-1细胞后,各炎性细胞因子的变化情况。结果显示:获得的Ts-serpin-1目的基因长度为1149 bp,编码382个氨基酸,含有Serpin家族特有的反应中心环。Ts-serpin-1基因在猪带绦虫成虫和中绦期幼虫均表达,且成虫表达量显著高于幼虫。重组蛋白Ts-serpin-1的分子质量约为43 ku,可抑制THP-1细胞促炎性细胞因子IL-6、IL-1β、IL-12、TNF-α、IFN-γ和iNOS2的表达,促进抗炎性细胞因子IL-10和TGF-β的分泌表达。以上结果提示,Ts-serpin-1在寄生虫-宿主互作过程中可能发挥重要作用。

A serine protease inhibitor Ts-serpin-1(WormBase:TSM000065700)of Taenia solium was investigated for the immunomodulatory effects on THP-1 cells.The coding DNA sequence(CDS)of Ts-serpin-1 was amplified by RT-PCR with specific primers.qRT-PCR was used to analyze the expression of Ts-serpin-1 gene in adult and larval stages.The recombinant protein Ts-serpin-1 was expressed and purified based on prokaryotic expression vector pColdⅠ.After treatment with recombinant protein Ts-serpin-1,the changes of cytokines in THP-1 cells were detected by qRT-PCR and ELISA.The sequence analysis showed that Ts-serpin-1 CDS was 1149 bp in length,encoding a protein with 382 amino acids,which contains a specific reactive centre loop motif.The results of qRT-PCR revealed that expression of Ts-serpin-1 significantly higher in adult stage than that in larval stage.The recombinant protein Ts-ser pin-1 was expressed as a fusion with a molecular weight of 43 ku.The recombinant protein Ts-serpin-1 decreased the expression of pro-inflammatory cytokines IL-6,IL-1β,IL-12,TNF-α,IFN-γand iNOS2 in the THP-1 cells that were stimulated by the recombinant protein and LPS and promoted the secretion and expression of anti-inflammatory cytokines IL-10 and TGF-βin THP-1 cells stimulated by IL-4.These results suggest that Ts-serpin-1 may play an important role in parasite-host interactions.