Expression of Cryptosporidium parvum GP900829-1099 protein and its immunomodulatory effects on mouse macrophages cells
Objective To probe the immunomodulatory effects of Cryptosporidium parvum micronemal glycopro-tein 900(GP900)829-1 099aa fragments(GP900829-1099)on mouse macrophages(RAW264.7 cells)through the nuclear factor kappa-B(NF-KB)/mitogen-activated protein kinase(MAPK)signalling pathways.Methods The amino acid se-quence of GP900829-1099 was obtained from the NCBI database for bioinformatics analysis.Sequence fregment of gp900829-1099 was amplified with PCR using cDNA from C.parvum oocysts as a template.After gel extraction,gp900829-1099 were inserted into the pET-32a-gp900829-1099 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 endo-toxin.The GP900829-1099 recombinant protein at concentration of 0.16,0.80,4.00,20.00,100.00 and 500.00 μg/ml was ap-plied 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-αmRNA in RAW264.7 cells.ELISA was used to detect the expression levels of IL-6 and TNF-α 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 GP900829-1070 proteins β comers 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 GP900829-1099 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 tran-scription 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-α 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 lev-els 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-α 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 GP900829-1070 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-αand IL-6 mRNA,thereby,participating in macrophage immunomodulation.
Cryptosporidium parvumMicroneme protein GP900RAW264.7NF-κBMAPK
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