核糖体蛋白RPS2对刚地弓形虫体外侵入小鼠巨噬细胞Ana-1功能的影响  

Effect of ribosomal protein RPS2 on the function of Toxoplasma gondii invading mouse macrophage Ana-1 in vitro

作　　者：杨晨晨 齐玮瑜 王晓娟[1] 姜宇宸 李相辰 张莉[1] YANG Chen-chen;QI Wei-yu;WANG Xiao-juan;JIANG Yu-chen;LI Xiang-chen;ZHANG Li(College of Animal Science and Technology,Ningxia University,Yinchuan 750021,China)

机构地区：[1]宁夏大学动物科技学院,宁夏银川750021

出　　处：《中国预防兽医学报》2024年第11期1174-1182,共9页Chinese Journal of Preventive Veterinary Medicine

基　　金：宁夏引才专项(2021BEB04010)。

摘　　要：为研究刚地弓形虫核糖体蛋白RPS2(TgRPS2)对弓形虫侵入小鼠巨噬细胞Ana-1功能的影响,本研究采用系列分子生物学软件分析TgRPS2的生物信息学,通过PCR从弓形虫中扩增TgRPS2基因构建重组表达质粒pET32a-TgRPS2,经双酶切和测序鉴定正确后与pET-32a分别转化大肠杆菌BL21,经IPTG诱导后采用镍柱亲和层析法纯化表达的重组TgRPS2蛋白(r TgRPS2)和空载体蛋白,采用SDS-PAGE检测两种蛋白的表达及纯化效果并经BCA法测定两种纯化蛋白的浓度。将r TgRPS2以300μg的剂量免疫小鼠6次,制备TgRPS2多克隆抗体(PAb),采用western blot鉴定r TgRPS2与弓形虫抗体、弓形虫可溶性蛋白与rTgRPS2 PAb的反应性。结果显示,分别在47.3 ku及18 ku处出现目的蛋白和空载体蛋白条带,且目的蛋白主要以包涵体的形式表达,空载体蛋白以上清形式表达,纯化后二者在相应位置均出现单一目的条带,测得rTgRPS2及空载体蛋白的浓度分别为1 949.54μg/mL和2 827.15μg/mL。Western blot结果显示,rTgRPS2能够与兔源弓形虫PAb反应,rTgRPS2 PAb也可以与弓形虫可溶性蛋白反应。将20μg/mL及不同浓度的rTgRPS2分别与小鼠巨噬细胞Ana-1共培养后,采用间接免疫荧光试验(IFA)检测rTgRPS2与小鼠巨噬细胞Ana-1的结合;利用CCK-8试剂盒及流式细胞术分别检测不同浓度rTgRPS2对巨噬细胞增殖能力、吞噬能力及凋亡的影响,利用ELISA检测各组巨噬细胞中肿瘤坏死因子α(TNF-α)、IL-6、IL-10和转化生长因子β1(TGF-β1)的分泌水平,利用试剂盒检测各组巨噬细胞NO的含量。结果显示,rTgRPS2能够与小鼠巨噬细胞紧密结合,在细胞质中出现红色荧光,而阴性对照及Mock组细胞均无红色荧光。不同浓度的rTgRPS2对巨噬细胞的增殖均无影响。与空载蛋白对照组相比,不同浓度rTgRPS2作用后巨噬细胞Ana-1的吞噬能力均极显著增强(P<0.001);10μg/mL~40μg/mL的rTgRPS2作用后,巨噬细胞Ana-1的凋亡率均极显著升�In order to investigate the effect of Toxoplasma gondii ribosomal protein RPS2(TgRPS2)on the invasion of Toxoplasma gondii into mouse macrophages Ana-1,the TgRPS2 gene was amplified from Toxoplasma gondii by PCR,and the recombinant expression plasmid pET32a-TgRPS2 was constructed.After confirmed by double digestion and sequencing,the plasmid was transformed into E.coli BL21 and then induced by IPTG.The empty vector protein and the expressed recombinant TgRPS2 protein(rTgRPS2)were purified by nickel column affinity chromatography,respectively,and the expression and purification effects of the two proteins were detected by SDS-PAGE and the concentration of purified proterins were was determined by BCA.Polyclonal antibody(PAb)against TgRPS2 was prepared by immunizing mice with rTgRPS2 six times at a dose of 300μg,and the reactivity of rTgRPS2 with Toxoplasma gondii antibodies and Toxoplasma gondii soluble proteins with rTgRPS2 PAb was identified by western blot.The results showed that the target protein band appeared at 47.3ku and the empty vector protein at 18ku,and the target protein was mainly expressed in the form of inclusion bodies,and the empty vector protein was expressed in the form of supernatant.After purification,both proteins showed a single band at the corresponding position.The concentrations of the two proteins were determined to be 1949.54μg/mL and 2827.15μg/mL,respectively.Western blot results showed that rTgRPS2 protein could react with rabbit-derived PAb against Toxoplasma gondii,and rTgRPS2 PAb could also react with Toxoplasma gondii soluble protein.After rTgRPS2 of 20μg/mL and other different concentrations was co-cultured with mouse macrophage Ana-1,the binding of rTgRPS2 to mouse macrophage Ana-1 was detected by indirect immunofluorescence assay(IFA);CCK-8 kit and flow cytometry were used to detect the effects of different concentrations of rTgRPS2 on the proliferation,phagocytic ability and apoptosis of macrophages;The secretion levels of tumor necrosis factor-α(TNF-α),IL-6,IL-10 and t

关 键 词：刚地弓形虫 核糖体RPS2蛋白 原核表达 小鼠巨噬细胞 免疫功能 

分 类 号：S852.6[农业科学—基础兽医学]