The Cloning And Expression Of The Extracellular Part Of FcεR Ⅰα Chain And Preparation And Identification Of McAbs

Allergic disease is a kind of very common disease including allergic shock,allergic purpura,allergic rhinitis and so on.It is caused by typeâ… hypersensitivity reaction and the most important two parts are immunoglobulin E and high affinity IgE receptorâ… (FcεRâ… ).When the allergen ingresses the human body for the first time,it can derivation the B lymphcell,and the specific IgE is produced subsequently.Then the specific IgE binds to the FcεRâ… expresses on the surface of basophil and mast cell. When the same allergen ingresses the human body for the second time,there will be a series of signal transmit in the two kinds of target cells.Target cells release bioactive compounds like histamine and leukotriene which can induce allergic disease.According to this,FcεRⅠαchain is the most important part of allergic disease.Allergy can cause typeâ… hypersensitivity reaction.Food,fur,insect, fungus,drug and so on are all allergen around us.When they ingress in our body,they can make us allergized,and specific IgE is released by plasma cell.FcεRâ… is a kind of glycosidoprotein composed of three parts,αchain, βchain andγchain.The a chain can be separated into three parts,the intracellular part,transmembrane part and extracellular part.The extracellular part is the most important in allergic disease for its combination with IgE.It is constructed with two immunoglobulin-like parts named D1 and D2 and the part near to the membrane can be combound to IgE. Extracellular part of the FcεRⅠαchain can bind to the two CH3 of IgE in the shape of a dissymmetry arch and a chain is just sit in the middle of the two Fc parts of IgE.When the specific antigen binds with more than two IgE on the surface of target cell,the conformation of FcεRⅠαchain is changed. Two protein kinase,Lyn and Syk in cytoplasm is actived by the change.Then Ca²⁺ is released,which can cause the release of bioactive substance. FcεRâ… is mainly expressed on the surface of basophil and mast cell,and it also can be found on the surface of eosinophil,monocyte and thrombocyte.The expression of FcεRⅠαchain and its C terminal is regulated by several transcription factors and the level of IgE in peripheral blood.J.Kochan found seven glycosylation site,which play an important role in the regulation of the secretion,but it is not necessary to the correct fold ofαchain.Some researchers find that the single extracellular part ofαchain can bind with IgE and the mouse without FcεRⅠαchain can escape from typeâ… hypersensitivity.According to this,our design about the receptor is feasible. Recently,the reseach of FcεRⅠαchain and its antibody is more and more popular.Professor Renshan Sun has successfully interrupt the typeâ… hypersensitivity in mice with recombinant extracellular part of FcεRⅠαchain.Since there is more basophils and FcεRâ… in allergic patients than in health adult,we extrat the gene of FcεRⅠαchain from allergic patients, amplified the gene with RT-PCR.Then cloned the product into the vector pET-28a(+),a great quantity of insoluble product was undertaken in E coli. Then the recombinant protein was purified by affinity chromatograph and molecular sieve.After the recombinant protein is renatured by dialysis, BALB/c mice were immunized to prepare monoclonal antibody.Monoclonal antibody was detected and identified by ELISA and immunofluorescence of cell from peripheral blood of allergic patient.Results showed that the FcεRⅠαgene which is 696bp was cloned by RT-PCR successfully,completely consistent with sequence in GenBank.The FcεRⅠαgene was ligated into the vector pET-28a(+),the product was identified by PCR.Restrictive digestion and sequencing proved that prokaryotic expression plasmid FcεRⅠα-pET28a(+) was constructed correctly.The recombinant protein was expressed in E coli with recombinant plasmid FcεRⅠα- pET28a(+).After the optimization of expression,products were presented mainly as inclusion body.The molecular weight of recombinant protein is about 23kDa,consistant with the theoretical value. The expression quantity is about 30%of the total protein of E coli.The purity quotient of the recombinant proteins is about 15%.BALB/c mice were immunized with purified recombinant protein to prepare immuned serum. ELISA proved that recombinant proteins could react with prepared immuned serum,no cross reactivity to normal mice serum,that is to say prepared fusion proteins have antigen activity.Twelve hybridoma strains of monoclonal antibodies were prepared by immuned mice with purified recombinant protein.The titer of monoclonal cell strains cultivation supernatant is 1.628～2.512 to recombinant protein as antigen.Immunofluorescence of cell from peripheral blood of allergic patient reveals that four monoclonal antibodies of anti-FcεRⅠαcan react to native protein,while control groups are both negative.The above results showed that recombinant expression plasmid FcεRⅠα- pET28a(+) has been constructed successfully,recombinant protein was expressed in E coli;constructing four hybridoma cell lines of anti-FcεRⅠα,makeing foundations for researching the protein's function, effection in signal transmition and treatment of allergic disease.