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Cloning, Expression And Activity Identification Of The Mouse Interleukin-17

Posted on:2011-05-29Degree:MasterType:Thesis
Country:ChinaCandidate:W ZhangFull Text:PDF
GTID:2120360308981787Subject:Microbiology
Abstract/Summary:PDF Full Text Request
Interleukin-17 is a powerful pro-inflammatory cytokine that is found in recent years, when it is combined with its receptor, it can induce the expression of many cytokines and adhesion molecules,which have different functions in hemopoiesis, inflammation and immune response. IL-17 is also a fine-tuning factor of the inflammatory response, so it can induce some diseases when IL-17 is over-expressed or insufficient. The current study find that IL-17 could participate in not only the immune regulation, but also other kinds of diseases (rheumatoid arthritis, gastrointestinal inflammation, systemic lupus and the immunoreaction after transplantantion, etc.), especially the rheumatoid arthritis. Although some of the drugs are available for treatment of the RA, but they are not entirely effective. So, better medicine is still needed. In view the IL-17 has an important role in RA, so studies of IL-17 have a profound significance for development of new drugs.This study is a part of the project to develop recombinant antibody therapeutics for treating RA. The aims of this study are to express the bioactive mIL-17, and prepare for animal pharmacology experiments.In this study, we used the cDNAs from the mouse thymus (immunited by hIL-1βprotein) as a template, cloned the mature mIL-17 gene fragment by RT-PCR. The result of DNA sequence indicated that the length of mIL-17 gene fragment was 402bp encoding 134aa, which had 100% homology with the published sequence in GenBank. The mature mIL-17 gene was subcloned into pHisSUMO Express expression vector and expressed in the host E. coli Rosetta. The inclusion body denatured by 8 mol/L urea was renatured by AKTA protein purification system. We could obtain the mature mIL-17 with the 95% purity. The molecular weight of the mature protein was 15.5kD in agreement with the published data. For evaluation of the bioactivity of the mature mIL-17, the pre-adipose cells 3T3-L1(a kind of mouse fibroblast) were treated with the recombinant mIL-17, the expression of IL-6 was detected by Realtime-PCR technology. The result showed that the transcription level of IL-6 was upregulated by the recombinant mIL-17 in a dose-dependent manner. In addition, this study also evaluated the bioactivity of the mature mIL-17 by injecting the protein into the RA model rats using peritoneal injection method. This result showed that the joint swelling of the RA model group injected with the mIL-17 was more severe than the control groups, and there was secondary pathological lesions with the RA group injected the mIL-17. The pathological sections also suggsted that the joint capsule of the RA group injected the mIL-17 is thicker than the control RA group, the infiltration of the inflammatory cells appered in the joint cavity, articular cartilage was destroyed severely and endostosis phenomenon was obvious. Above results proved the principle that the recombinant mIL-17 protein could improve the occurrence of the RA induced by typeⅡc ollagen, and aggravated the synovium inflammation and the joint damage.In this study, the Realtime-PCR was used to prove that the protein had bioactivity, which was a simple and effective method to evaluate the activity of the mIL-17. In this study, we used the recombinant protein that had been inactivated at 60℃water bath for 3h as negative control, it was more rigorous than using the endotoxin as negative control. Furthermore, this study used the peritoneal injection method to study the effect of mIL-17 on the the RA model induced by typeⅡc ollagen. Compared with the published method in which IL-17 protein was injected into the joint cavity, the peritoneal injection method eliminated the unnecessary damage of joint by local infection and reduced the stimulation factors, such as trauma and infection. So the design of this study was more rigorous and the result of this study was more believable.After cloning, expressing and purification, we obtain the bioactive recombinant mIL-17, and use more rigorous method (peritoneal injection) to prove that the mIL-17 can aggravate the pathological changes of RA, and it is an important factor for the occurrence and the development of RA. This study makes a foundation for the project to develop the new recombinant antibody therapeutics for treating RA.
Keywords/Search Tags:mouse interleukin-17, Reumatoid arthritis, prokaryotic expression, inclusion body purification, realtime-PCR, the rat animal model of the RA induced by by typeⅡcollagen
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