结膜吸吮线虫MIF蛋白原核表达及生物学效应鉴定  

Identification on prokaryotic expressions and biological effect of MIF proteins from Thelazia callipaeda

作　　者：黄琳[1,2] 莫兴泽 梁秋果[1] 党荣敏 王灵军[2] 贺莉芳[1] 刘晖[1] HUANG Lin;MO Xing-ze;LIANG Qiu-guo;DANG Rong-min;WANG Ling-jun;HE Li-fang;LIU Hui(Qiannan Medical College for Nationalities,Guizhou 558000,China;不详)

机构地区：[1]黔南民族医学高等专科学校,贵州558000 [2]遵义医科大学

出　　处：《医学动物防制》2022年第10期919-923,共5页Journal of Medical Pest Control

基　　金：国家自然科学基金项目(82060374);贵州省科技厅科技基金(黔科合基础[2020]1Y348);贵州省遵义市校联合基金(遵义市科合字[2017]08号);贵州省卫生健康委科学技术基金(黔卫健函[2022]101号,项目gzwkj2023-251)。

摘　　要：目的克隆、表达结膜吸吮线虫巨噬细胞迁移抑制因子(macrophage migration inhibitory factor,MIF),纯化重组Tcp-MIF蛋白,并验证其生物学效应。方法将筛选的MIF基因片段构建到pET-SUMO原核表达载体上,转化大肠杆菌BL21(DE3);异丙基硫代半乳糖苷(isopropyl thiogalactoside,IPTG)诱导表达;利用HisLink亲和层析柱进行蛋白纯化及质谱鉴定;将纯化的Tcp-MIF蛋白作用于THP-1巨噬细胞,运用CCK8、酶联免疫吸附试验(enzyme-linked immunosorbent assay,ELISA)、免疫印迹法(Western blotting,WB)检测MIF蛋白生物学效应。结果成功构建pET-SUMO-MIF重组原核表达质粒,Tcp-MIF重组蛋白主要以可溶形式表达,相对分子质量约为26.80 KD。纯化Tcp-MIF蛋白能够促进THP-1巨噬细胞的增殖,减少促炎因子(TNF-α),增加抑炎因子(IL-10)的产生;可通过与THP-1巨噬细胞表面TLR4受体结合,激活NF-κB通路。结论利用重组结膜吸吮线虫MIF蛋白初步证实了Tcp-MIF蛋白具有调控宿主免疫系统的作用,为进一步探讨MIF蛋白在寄生虫与宿主的相互作用研究奠定了基础。Objective To clone and express the macrophage migration inhibitory factor(MIF)of Thelazia callipoeda,purify the recombinant Tcp-MIF protein,and verify corresponding biological effect.Methods The gene fragment of MIF screened was constructed onto the prokaryotic expression vector of pET-SUMO and transformed into Escherichia coli(E coli)BL21(DE3).Isopropyl thiogalactoside(IPTG)was induced to express,MIF proteins were purified by HisLink affinity chromatography and identified by mass spectrometry.The purified MIF acted on THP-1 macrophages.CCK8,enzyme-linked immunosorbent assay(ELISA)and Western blotting(WB)were used to detect the biological effects of MIF protein.Results Recombinant prokaryotic expression plasmid pET-SUMO-MIF was successfully constructed,and Tcp-MIF recombinant protein was mainly expressed in soluble form,the molecular mass was about 26.80 KD.Purified Tcp-MIF protein could promote the proliferation of THP-1 macrophages,reduce the production of pro-inflammatory factor(TNF-α)and increase the production of anti-inflammatory factor(IL-10).NF-κB pathway was supported to be activated by binding to TLR4 receptors.Conclusion The recombinant MIF protein of Thelazia callipaeda preliminarily confirms that Thelazia callipaeda is effective in regulating the host immune system,laying a foundation for further studies on the interaction between parasites and hosts.

关 键 词：结膜吸吮线虫 巨噬细胞迁移抑制因子 原核表达 生物学效应 

分 类 号：R384.2[医药卫生—医学寄生虫学]