| Bronchial Asthma (Bronchial Asthma), referred to as Asthma, is a chronicinflammatory disorder characterized by reversible airflow limitation, often associatedwith symptoms as airway hyperresponsiveness(AHR)，mucus hypersecretion，bronchialsmooth muscle contraction，reversible airway obstruction[1]..At present, there are about300million people suffered from asthma, at the same time, there are30million patientswith asthma in China. Asthma has become one of the important respiratory systemdisease which effect deeply on people’s physical and mental health[2].Generally, asthma is due to various cytokines released from Th2cells, like CD4T-lymphocytes. These cells can cause local inflammatory reaction in the lung.Interleukin-5is one of the most important cytokines. IL-5seems to be the primarycytokine involved in vivo in the production, differentiation, maturation and activation ofthe eosinophils（Eos）[4]. IL-5receptor complex includes alpha(IL-5Rα) and beta (IL-5Rβ)two subunits. IL-5Rαcombined with IL-5to activate the downstream channeland play a biological function. Splicing of the IL-5Rα gene can generates two differenttranscripts: one encoding a membrane-anchored protein through alternative splicing, anda second one encoding a soluble form of this receptor, by normal splicing events. Thesoluble isoform competes with the membrane anchored receptor for IL-5binding, andtherefore this variant is considered to be a potential natural negative regulator of IL-5function in vivo[5].At present, inhibiting Eos in bronchial by IL-5antagonist to alleviateasthma has become hot spot of bronchial asthma research. IL-5monoclonal antibody caninhibit asthma allergic inflammation to some extent, but its application is restrictedbecause of its foreign immunogenicity[6]. So finding and researching a kind of drugwhich can alleviate overall asthma symptoms and cure various kinds of asthma isimminent.Objective：Expression and purification mouse soluble IL-5receptor α protein withBac to Bac baculovirus expression system to investigate the effects of IL-5receptor αprotein on murine model of allergic asthma to provide a new approach for the treatmentfor asthma.Methods: Application Vector NTI Advance II software to optimize the solublemurine IL-5α extracellular region of codon and then the sequence was synthesised. UsingBamHI and HindIII cloned sIL-5α into the baculovirus transfer vector pFastBacI/HTB.Then the pFastBacI/HTB was transformed into competent DH10BacTME.coil cells．Thetransposition occurred between pFastBacI/HTB and Bacmid and a recombinant Bacmidwas obtained．The positive clones were picked out and the recombinant Bacmid wasisolated，and then complete transfected into sf9cells for producing complete recombinantbaculovirus. Determination reconstruct virus’ TCID50(Tissue culture infective dose) andthen calculation the MOI (Motiplicity of infection)to amplification the recombinantbaculovirus. The sIL-5Rα was expressed when the baculovirus stock was amplified. Therecombinant protein was Purificated with Ni+affinity chromatography and wasidentificated with Western Blot. Concentration of purified recombinant protein is0.22mg/ml. Application the recombinant protein to asthma model mice and evaluationeosinophils, mast cells, neutrophils and lymphocytes cell percentage, IL-5, IL-4, Extoxincontent of BALF and the pathological changes of lung tissue.Results：The recombinant baculovirus which contains sIL-5α gene was obtained and the recombinant protein was expressed and purificated successfully. It was identifiedby Western Blot. Treatment with sIL-5α protein significantly reduced the percentage ofeosinophils and concentrations of IL-5and Extoxin in bronchoalveolar lavage fluid andlung tissue pathology compared with administration of saline. This shows that solubleIL-5receptor α protein plays a certain treatment function in murine model of asthma. |
PDF Full Text Request