目的 初步探究微小隐孢子虫微线体蛋白（MIC）糖蛋白900（GP900）829~1 099aa片段（GP900^829~1099）通过核因子-κB（NF-κB）/丝裂原活化蛋白激酶（MAPK）信号通路对小鼠巨噬细胞（RAW264.7细胞）的免疫调节作用。方法 从NCBI数据库中获取微小隐孢子虫GP900^829~1099氨基酸序列进行生物信息学分析。以微小隐孢子虫卵囊cDNA为模板，PCR扩增gp900^829~1099基因片段，构建pET-32a-gp900^829~1099重组质粒并转化至BL21感受态细胞中诱导表达。纯化、超滤浓缩重组蛋白并去除内毒素。采用0.16、0.80、4.00、20.00、100.00、500.00 μg/ml的GP900^829~1099重组蛋白刺激RAW264.7细胞24 h后，CCK-8法检测其对RAW264.7细胞活力的调节作用。以0.16、0.80、4.00 μg/ml的GP900^829~1099重组蛋白刺激RAW264.7细胞，以PBS为对照组，脂多糖（1 μg/ml）为阳性对照，24 h后流式细胞术检测CD86表达量，qPCR检测RAW264.7细胞中IL-6和TNF-ɑ mRNA的相对转录水平，ELISA检测RAW264.7细胞培养上清中IL-6、TNF-α的表达水平。采用蛋白质免疫印迹分析NF-κB和MAPK信号通路中P65蛋白和细胞外调节蛋白激酶（ERK）蛋白磷酸化水平。结果 GP900^829~1099蛋白二级结构中β转角占比9.23%，无规则卷曲占比64.58%，且含有较丰富的T、B细胞抗原表位。表达的GP900^829~1099重组蛋白的相对分子质量约为49 000。CCK-8结果显示，GP900^829~1099对细胞没有毒性作用，后续实验采用0.16、0.80和4.00 μg/ml作为作用浓度。流式细胞术结果显示，CD86⁺的阳性表达率分别为（13.500 ± 0.815）%、（18.670 ± 0.657）%、（20.470 ± 1.271）%，后两者均高于对照组的（14.500 ± 0.872）%（t = 3.818、3.872，P < 0.05）。qPCR结果显示，0.16、0.80和4.00 μg/ml组RAW264.7细胞的IL-6 mRNA相对转录水平分别为1.409 ± 0.050、2.052 ± 0.098和3.284 ± 0.097，均高于对照组的1.010 ± 0.096（t = 3.700、7.595、16.700，均P < 0.05）；TNF-ɑ mRNA相对转录水平分别为1.077 ± 0.034、1.440 ± 0.021和2.378 ± 0.037，后两者均高于对照组1.000 ± 0.025（t = 13.380、30.850，均P < 0.05）。ELISA检测结果显示，RAW264.7细胞培养上清中，0.16、0.80和4.00 μg/ml组IL-6的表达水平分别为（535.400 ± 17.230）、（572.800 ± 8.286）、（555.600 ± 23.940）mg/L，均高于对照组的（454.400 ± 18.630）mg/L（t = 3.193、5.809、3.339，P < 0.05）；TNF-ɑ细胞因子的表达水平分别为（351.800 ± 12.270）、（386.400 ± 10.250）、（489.800 ± 10.540）mg/L，后两者均高于对照组的（324.200 ± 11.070）mg/L（t = 4.125、10.830，P < 0.05）。蛋白质免疫印迹结果显示，0.16、0.80和4.00 μg/ml组RAW264.7细胞p-P65、p-ERK蛋白的相对表达水平分别为2.294 ± 0.254、1.714 ± 0.205、1.877 ± 0.309，1.522 ± 0.054、1.760 ± 0.066、1.582 ± 0.027，均高于对照组的1.0 ± 0.0（t = 5.100、3.489、2.836，9.737、11.450、21.900，均P < 0.05）。结论 不同浓度GP900^829-~1099重组蛋白刺激巨噬细胞后，通过激活NF-κB和MAPK信号通路诱导巨噬细胞活化, 通过促进TNF-α和IL-6 mRNA的转录参与巨噬细胞的免疫调节。

Objective To probe the immunomodulatory effects of Cryptosporidium parvum micronemal glycoprotein 900 (GP900) 829-1 099aa fragments (GP900^829-1099) on mouse macrophages (RAW264.7 cells) through the nuclear factor kappa-B (NF-κB)/mitogen-activated protein kinase (MAPK) signalling pathways. Methods The amino acid sequence of GP900^829-1099 was obtained from the NCBI database for bioinformatics analysis. Sequence fregment of gp900^829-1099 was amplified with PCR using cDNA from C. parvum oocysts as a template. After gel extraction, gp900^829-1099 were inserted into the pET-32a-gp900^829-1099 vector and transformed into BL21 competent cells for induced expression. The recombinant proteins were purified and filtered, concentrated by ultrafiltration and further cleaned by removing endotoxin. The GP900^829-1099 recombinant protein at concentration of 0.16, 0.80, 4.00, 20.00, 100.00 and 500.00 μg/ml was applied to stimulate RAW264.7 for 24 h, subsequently, its regulatory effect on the cell viability was detected by CCK-8 assay. Using PBS as the control group and lipopolysaccharide（1 μg/ml）as the positive control group, the recombinant protein at the concentration of 0.16, 0.80 and 4.00 μg/ml was applied to stimulate the RAW264.7 cells for 24 h to detect CD86 expression by flow cytometry. qPCR was used to detect the relative transcription levels of IL-6 and TNF-ɑ mRNA in RAW264.7 cells. ELISA was used to detect the expression levels of IL-6 and TNF-ɑ in the supernatant of RAW264.7 cell culture. The phosphorylation levels of P65 protein and extracellular regulated protein kinase (ERK) in NF-κB and MAPK signaling pathways were detected by Western blotting assay. Results In the secondary structure of GP900^829-1070 proteins β corners account for 9.23%, irregular curls account for 64.58%, and contain abundant T and B cell antigenic epitopes. The molecular mass of the expressed recombinant protein GP900^829-1099 was consistent with the theoretical value. The CCK8 results showed no toxic effect on the cells, and the subsequent working concentrations were selected as 0.16, 0.80 and 4.00 μg/ml. Flow cytometry results showed that the positive expression rate of CD86⁺ was (13.500 ± 0.815)%, (18.670 ± 0.657)% and (20.470 ± 1.271)%, respectively, and the latter two were higher than that of the blank control group (14.500 ± 0.872)% (t = 3.818, 3.872; both P < 0.05). qPCR results showed that the relative transcription levels of IL-6 mRNA in macrophages in 0.16, 0.80 and 4.00 μg/ml groups were 1.409 ± 0.050, 2.052 ± 0.098 and 3.284 ± 0.097, respectively, which were higher than those in the blank control group (1.010 ± 0.097) (t = 3.700, 7.595, 16.700; all P < 0.05). The TNF-ɑ mRNA relative transcription levels in macrophages of the three concentration gradient groups were 1.077 ± 0.034, 1.440 ± 0.021 and 2.378 ± 0.037, respectively, with the latter two being higher than that of the blank control group (1.000 ± 0.025) (t = 13.380, 30.850; both P < 0.01). ELISA results showed that the expression levels of IL-6 cytokines in macrophage culture supernatants were (535.400 ± 17.230), (572.800 ± 8.286) and (555.600 ± 23.940) mg/L in the 0.16, 0.80 and 4.00 μg/ml groups, respectively, which was higher than that in the blank control group [(454.400 ± 18.630) mg/L] (t = 3.193, 5.809, 3.339; all P < 0.05); the expression levels of TNF-ɑ cytokine were (351.800 ± 12.270), (386.400 ± 10.250) and (489.800 ± 10.540) mg/L, respectively, and the latter two were higher than that of the blank control group [(324.200 ± 11.070) mg/L] (t = 4.125, 10.830; both P < 0.01). Western blotting results showed that the relative expression levels of p-P65 protein and p-ERK protein in macrophages in 0.16, 0.80 and 4.00 μg/ml groups were 2.294 ± 0.254, 1.714 ± 0.205, 1.877 ± 0.309 and 1.522 ± 0.054, 1.760 ± 0.066, 1.582 ± 0.027, all were higher than that of the blank control group (1.0 ± 0.0) (t = 5.100, 3.489, 2.836 and 9.737, 11.450, 21.900; all P < 0.05). Conclusion GP900^829-1070 recombinant protein stimulated macrophages at different concentrations, which activates NF-κB and MAPK signalling pathways to induce activation of macroopages and promote the transcription of TNF-α and IL-6 mRNA, thereby, participating in macrophage immunomodulation.