关键词: 微小隐孢子虫, 微线体蛋白GP900, RAW264.7, NF-κB, MAPK

Abstract:

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.

Key words: Cryptosporidium parvum, Microneme protein GP900, RAW264.7, NF-κB, MAPK