Prokaryotic expression of mouse interleukin 17A and its ability of stimulating RAW264.7 cell inflammatory factors expression
10.3760/cma.j.issn.0254-5101.2010.07.012
- VernacularTitle:小鼠白细胞介素17A的原核表达及对RAW264.7细胞分泌炎症因子的影响
- Author:
Sheng GUO
;
Xiaoyong FAN
;
Chunli HAO
;
Hui MA
;
Ling CHEN
;
Jianhua ZHANG
- Publication Type:Journal Article
- Keywords:
Recombinant interleukin 17A;
Macrophage inflammatory protein;
Anti-infection immunity;
Beta-defensin
- From:
Chinese Journal of Microbiology and Immunology
2010;30(7):635-640
- CountryChina
- Language:Chinese
-
Abstract:
Objective To express and purify mouse interleukin 17A(mIL-17A) in E. coli and to analyze its ability of stimulating macrophage inflammatory factors expression. Methods The coding gene of mouse mIL-17A mature protein was amplified from mouse spleen cells by RT-PCR. PCR product was cloned into the prokaryotic expressing vector pET28a, and the resulting recombinant plasmid pET28a/mIL-17a was then transformed into the host E. coli strain BL21(DE3) for expression. The mIL-17A protein was identified by SDS-PAGE and Western blot. The recombinant protein was purified by the Ni-NTA affinity chromatography, and was further tested on the stimulation of cytokine and chemokine of RAW264.7 cells by ELISA and real-time quantity PCR in vitro. Results The mIL-17A with bioactivity was over-expressed and purified successfully, and the results of real-time PCR and ELISA showed that recombinant mIL-17A stimulated macrophage mRNA upregulation of IL-6, defensin β2 and Cxcl3 and secretion of defensin β2, Ccl3, Cxcl3,IFN-γ, IL-6 and IL-4. Interestingly, these effects could be blocked by the addition of anti-IL-17A neutralizing antibody partly. After treatment with mIL-17, 74. 87-fold of defensin β2 mRNA expression was increased comparing with that of untreated cells( P ＜0.01 ), while blocking with anti-IL-17A antibody the increase was only 5.4-fold(P ＜ 0.01 ). Conclusion The recombinant mIL-17A has a strong stimulation on secretion of cytokine and chemokine of macrophage, that maybe result to the enhancement of anti-infection ability of macrophage.
