| Asthma is a chronic allergic airway inflammation mediated by various kinds of cells including Eosinophils (Eos) and mast cells. During the pathological process,the accumulation and activation of Eos piay critical role,while interleukin-5 (IL-5) selectively acts on Eos and thus is the most important Eos inducer. SIL-5Rαis an important negative regulator,which can prevent Eos mediated by IL-5 and Alkalophilic cell's activation in vitro. It has potential anti-inflammatory function . This experiment will apply the sIL-5Rαto asthma mouse model .the purpose is to explore the sIL-5Rα's role in the course of the asthma treatment. It has been reported that Chinese hamster ovary cells (CHO) expression system was used to express mice IL-5R protein and that E.coli expression system was used to express pig's IL-5Rαextracellular protein. But the protein's activity and quantity from both expression system are unsatisfactory.At present, Pichia pastoris has become an important expression system to produce heterologous protein. Compared with other eukaryotic expression systems,Pichia pastoris has many advantages,such as absence of bacterial endotoxin, absence of protein contamination in the process of animal cell's culture when producing proteins by viral . Currently,that the mouse sIL-5Rαis secretorily expressed in Pichia pastoris expression system has not been reported . Pichia pastoris expression system is used to secretorily express target gene in this experiment. The aim is to obtain the murine recombinant sIL-5Rαprotein. We believe that this work will do some good for the further study of sIL-5Rα's role in the treatment of asthma and that sIL-5Rαhas the potential to develop new drug for asthma.Objective : To clone mouse soluble IL-5 receptorαgene into Pichia pastoris secretion expression plasmid pPICZαA and to obtain a recombinant yeast strain that expresses IL-5 receptorαprotein. Methods : gene segment of soluble IL-5 receptorα(sIL-5Rα) was amplified and obtained by RT-PCR from spleen tissue of BALB/c mouse. After being verified by restriction enzyme,the segment was inserted into eukaryotic expression vector pMD18-T to construct the recombinant plasmid pMD18-T-sIL-5Rα. After restriction enzyme verification,the recombinant plasmid was used for the construction of recombinant plasmid pPICZαA-sIL-5Rα,with Escherichia coli (E. coli) DH5αas the vector. Thereby,pPICZαA-sIL-5Rαwas linearized and was inserted into Pichia pastoris secretion expression vector pPICZαA , which was then electro-transformed into Pichia pastoris GS115. Highly-resistant transformant was screened using gradient concentrations of anti-biotic Zeocin. PCR was used to verify positive recombinant yeast strains. Recombinant yeast strain with high-copy number was selected and cultivated in BMGY/BMMY medium added with methanol as expression inducer. SDS-PAGE and western blotting assays were used to detect the expression of target protein. Results : experimental methods including PCR,double restriction enzyme verification and DNA sequencing demonstrated that target gene sIL-5Rαwas cloned and obtained and that target gene was successfully inserted into Pichia pastoris secretion expression vector pPICZαA; moreover,the vector was transformed into Pichia pastoris GS115. As demonstrated by SDS-PAGE and western blotting assays , the recombinant Pichia pastoris cells showed secretion expression of sIL-5Rαprotein when induced by methanol. Conclusion : a Pichia pastoris secretion expression vector carrying sIL-5Rαgene was constructed and a recombinant yeast strain expressing IL-5 receptorαwas obtained.
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