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Optimization Of Expression Condition Of Canine Interferon Alpha By Pichia Pastoris And Study On Its Purification And Activity

Posted on:2017-06-17Degree:MasterType:Thesis
Country:ChinaCandidate:Y H FuFull Text:PDF
GTID:2310330503466357Subject:Biochemistry and Molecular Biology
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Canine interferon alpha is a cytokine from canine leukocyte by external stimulation. The antiviral effect of canine interferon alpha was mediated by a series receptor(IFN-R1 and IFN-R2) for signal transduction, which also resulted in the generation of ISGs(interferon stimulated gene product).ISGs(interferon stimulated gene production) have antiviral effect by inhibiting of viral nucleic acid transcription and of protein synthesis. A series of features of ISGs are determined with broad-spectrum antiviral, enhanced immune response and weak antigenicity, and have good prospects for clinical application.Pichia pastoris expression system is an eukaryotic protein expression system which is widely used in vitro. Compared with other expression system, the advantage of this expression system are outstanding including easy purification, high density fermentation, and the good foundation of industrialization. It is one of the important ways to realize the commercialization of exogenous protein.On the basis of mating factor MF- alpha factor leading signal peptide, we construct Ca IFN- alpha recombinant strains GS115-p PIC9K-MF-Ca IFN-?. The following studies were carried out.Research content 1:Ca IFN- alpha and MF- alpha gene sequences were verified by the means of PCR and enzyme digestion. The primers were designed according to the Ca IFN- alpha gene sequence. By using single factor experiment and orthogonal experiment, the growth and expression conditions of the engineering bacteria were optimized. The results showed that the optimum growth condition is: 30?initial temperature, initial p H6.0, 2%(m/v) initial glycerol, 20%(v/v)loading volume of the shake flask, and 10%(v/v)inoculation amount. The optimized engineering strain induced conditions included: 1%(v/v)inducer methanol concentration, the initial p H6.0, 72 h induction time, 28? induction temperature, and addition of 1% casein every 12 h. The result of engineering bacteria cultured by optimizing the condition showed that: the expression of target protein was increased by 76%.Research content 2:on the basis of the optimization of shake flask culture, the fermentation culture of GS115 yeast engineering bacteria was carried out with 50 L fermentation tank produced by Shanghai Wanmuchun Company. The growth curve of dissolved and oxygen curve were determined and rendered in the fermentation process. The test method of methanol content in fermentation process was determined. And the content of methanol in the induction process was detected. The result shows that methanol content in the fermented liquid was close to the optimization of shake flask by adding methanol feed by the way associated with the dissolved oxygen. At the same time we make sure that the optimum p H was 7.0, and the best induction time is 62 h, when the GS115 engineering bacteria induced in fermentation tank. Finally we obtained the target protein which was about 80% of the total protein. And its concentration was about 0.74mg/m L.Research content 3:the supernatant of shake flask culture and fermentation inducted was purified through G-25 desalination, hydrophobic chromatography, and ion exchange chromatography. The purity of the target protein was 91.4% and 95.7%. The concentration of the purified target protein was 0.24mg/ml and the recovery rate was about 58% after the shaking culture. The purified target protein concentration was 0.56mg/ml and the recovery rate was 43% after fermentation. The target protein was identified by Western-blot.Research content 4: the activity of the target protein(flask culture and fermentation inducted) was examined by the MDCK-VSV detection system. The results of the activity were 1.08 × 10~8 U/mg and 1.92 × 10~8 U/mg. The activity of anti-CDV on MDCK cells was 3.15×10~6 U/mg and 5.74×10~6 U/mg.
Keywords/Search Tags:CaIFN?, Pichia pastoris, Expression optimization, fermentation, protein purification, activity detection
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