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Expression and Characterization of Canine Interferon-γ

Author: HanYang
Tutor: LiDeShan
School: Northeast Agricultural University
Course: Microbiology
Keywords: CaIFN-γ SUMO MTT P53
CLC: S858.292
Type: Master's thesis
Year: 2013
Downloads: 29
Quote: 0
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Abstract


Interferons comprise a family of cytokines with anti-virus, anti-tumor andimmmunomodulatory functions. There are two types ofIFNs,type I and type II. Type I consists of α,β, ω, type II consists of only IFN-gamma. IFN-gamma is mainly induced in lymphocytes cells,including NK cells, CD4+Th1cells and CD8+cells.At present, viral diseases of canine showing a worsening trend in the worldwide, the repeatedoccurrence of canine rabies and canine parvovirus disease brings harm to human and some virusesare the pathogens of zoonosis, which raises the attention of the world. Canine interferon hasbecome a hot pot for its significant therapy at variety of canine viral diseases, so research anddevelop it’s biological products is necessary.However, recombinant interferon-gamma usually expressed as inclusion bodies in E.coli. Theonly way to get mature protein is refolding. However, the inclusion body refolding efficiency isnot good enough to get mature protein and this has been a bottleneck for producing canineinterferon-gamma and applying in practice.So, this paper aim to solubly express CaIFN-gamma and get CaIFN-gamma protein withbiologically activity. Recent studies found that SUMO acts as a chaperone to increase the stabilityand solubility of heterologous proteins, and its mechanism may be SUMO acts as a highlyhydrophobic core, provide nucleation sites for the folding of the target protein, promotesinteraction and correct folding of the targetproteins and ultimately enhancesthe solubility of thefusion protein. In this study SUMO expression system was applied to express the CaIFN-gammaprotein.SDS-PAGE analysis showed that large quantity of the target protein was in supernatantafter sonication. Then, the expression conditions including induction time, inducer concentratinsand induction temperature were optimized. We found that the induction time have the most effecton CaIFN-gamma protein expression, the maximum expression was obtained after induction for4h. The inducer concentrations have little effect on CaIFN-gamma protein expression.In order tominumaze damage for the transformed bacteria we chose0.25mmol/L of IPTG in followingexperiments, temperature also affected the expression of the protein, the expression was lower andbacterial growth was slower at20°C compared to37°C,and therefore we chose37°C for theinduction temperature in the following experiments. Under the optimal expression condition: theinducer concentration was0.25mmol/L, induction temperature was37°C, induction time was4hours, SDS-PAGE analysis showed that the target protein in the supernatant accounted for50%oftotal bacterial proteins The results greatly improved expression of the soluble protein compared with the conventional expression system. In conclusion, we have overcome the bottlenecks todenature and renature the inclusion body and lay the foundation for later CaIFN-gamma researchand large-scale fermentation.Traditional method for assay vesicular stomatitis virus has to be used for detection ofbioactivity of the CaIFN-gamma protein.In addition it is time-consuming in culturing virus anddetecting TCID50, there is potential risk for VSV infection to operators.Therefore, search for a newsafe and convenient detection method is nessessary. In thisstudy a safe, fast and sensitive detectionmethod for evaluate the bioactivity of the CaIFN-gamma protein was established.First, MTTmethod was used to detect the inhibition of MDCK cell proliferation by canine interferon-gamma.The results showed that MDCK cells proliferation was significantly inhibited by canineinterferon-gamma. Second, apoptosis of MDCK cells induced by CaIFN-gamma was examinedusing Flow cytometry, the results showed that CaIFN-gamma significantly induced apoptosis ofMDCK cells. Third, mRNA level of p53was measured by Real-Time PCR in the MDCK cells afterstimulation by canine interferon-gamma,the results showed that p53mRNA level increased afterstimulation by canine interferon-gamma. All of the above methods demonstrate that the solubleCaIFN-gamma protein expressed in the SUMO expression system has biological activity.These studies provide methods for soluble expression of CaIFN-gamma and also establishsafe and convenient detection assays for evaluation of bioactivity of CaIFN-gamma. Thesemethods lay profound basis for further studies and manufactural production of CaIFN-gamma..

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CLC: > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Animal Medicine ( Veterinary Medicine) > Livestock, poultry, wildlife diseases > Livestock > Dog
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