微小隐孢子虫GP900^(829~1099)蛋白的表达及其对小鼠巨噬细胞的免疫调节作用

Expression of Cryptosporidium parvum GP^(900829-1099) protein and its immunomodulatory effects on mouse macrophages cells

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(GP^(900^(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GP^(900^(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、500.00μg/ml的GP^(900^(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、4.00μg/ml的GP^(900^(829~1099))重组蛋白刺激RAW264.7细胞,以PBS为对照组,脂多糖(1μg/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-α 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 GP^(900^(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GP^(900^(829~1099))重组蛋白的相对分子质量约为49 000。CCK-8结果显示,GP^(900^(829~1099))对细胞没有毒性作用,后续实验采用0.16、0.80和4.00μ/g/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4.00μg/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-α 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4.00μg/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-α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4.00μg/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GP^(900^(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)829-1099aa fragments(GP^(900829-1099))on mouse macrophages(RAW264.7 cells)through the nuclear factor kappaB(NFκB)/mitogenactivated protein kinase(MAPK)signalling pathways.Methods The amino acid sequence of GP^(900829-1099) was obtained from the NCBI database for bioinformatics analysis.Sequence fregment of gp^(900829-1099) was amplified with PCR using cDNA from C.parvum oocysts as a template.After gel extraction,gp^(900829-1099) were inserted into the pET32agp^(900829-1099) 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 GP^(900829-1099) recombinant protein at concentration of 0.16,0.80,4.00,20.00,100.00 and 500.00μg/ml was applied to stimulate RAW264.7 for 24 h,subsequently,its regulatory effect on the cell viability was detected by CCK8 assay.Using PBS as the control group and lipopolysaccharide(1μg/ml)as the positive control group,the recombinant protein at the concentration of 0.16,0.80 and 4.00μg/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 IL6 and TNFɑmRNA in RAW264.7 cells.ELISA was used to detect the expression levels of IL6 and TNFɑin the supernatant of RAW264.7 cell culture.The phosphorylation levels of P65 protein and extracellular regulated protein kinase(ERK)in NFκB and MAPK signaling pathways were detected by Western blotting assay.Results In the secondary structure of GP900829-1070 proteinsβcorners 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 GP^(900829-1099) 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 4.00μg/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 IL6 mRNA in macrophages in 0.16,0.80 and 4.00μg/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 TNFɑmRNA 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 IL6 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 4.00μg/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 TNFɑcytokine 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 pP65 protein and pERK protein in macrophages in 0.16,0.80 and 4.00μg/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 GP900829-1070 recombinant protein stimulated macrophages at different concentrations,which activates NFκB and MAPK signalling pathways to induce activation of macroopages and promote the transcription of TNFαand IL6 mRNA,thereby,participating in macrophage immunomodulation.