Prokaryotic Expression、Purification Of IgE Cε3-Cε4, Analysis Of The Function IgE Cε3-Cε4 Interact With FcεR Ⅰ And The Identification Of Polyclonal Antibody

Allergic disease is a kind of very common disease including allergic shock,allergic purpura,allergic rhinitis and so on.It is caused by typeⅠhypersensitivity reaction and the most important two parts are immunoglobulin E and high affinity IgE receptorⅠ（FcεRⅠ）.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 FcεRⅠexpresses 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.Ⅰ-type allergic diseases include asthma,allergic rhinitis,atopic dermatitis and food allergy,etc.In the past 20 years the incidence ofⅠ-type allergic diseases increased year by year.All over the world there are about 1.0～150 million people suffering from asthma which could lead to about 18 million deaths Each year.It is estimated that the global economic cost of prevention and treatment of asthma is much more than the use for tuberculosis and AIDS combined.Some mature anti-allergic therapy and desensitization therapy only reach the goal of anti-inflammatory and alleviate the symptoms and have a lot of serious side effects,however the treatment effect is still not satisfied.Therefore,looking for newer and more specific therapeutic approaches are imperative.Allergy can cause typeⅠhypersensitivity 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.IgE and its high affinity receptor are the key steps of triggering anti-parasitic immunity and allergic reactions。Recent structural studies of the receptor,the IgE-Fc and the IgE-Fc:FcεRI complex have revealed how these two proteins interact to prime mast cell responses to antigen.The structures have revealed a novel arrangement for the FcεRI ectodomains that is also observed in homologous members of this antibody receptor family.The crystal structure of the IgE-Fc:FcεRI complex clarified how a 1:1 complex between the antibody and receptor is formed,with the receptor binding each chain of the antibody Fc dimer.The IgE-Fc structure in the absence of the receptor revealed the potential for large conformational rearrangements within the IgE that may affect receptor binding.These studies provide the basis for further investigation of the specificity of antibody:receptor binding and for the development of new treatments for allergic hypersensitivities.Twelve amino acids from the IgE and eight amino acids from the receptor form site 1.The IgE residues are from four distinct regions of the IgE-Fc sequence, including the Cε2-Cε3 linker（residues 334-336）,the BC loop（362-365）,the DE loop （393-396） and the FG loop（residue 424）.The receptor residues derive from two regions of the D2 domain,involving the C strand（residues 117,119 and 121） and the C’-E region（residues 126 and 129-132）.Two potential salt bridges（aK117-Cε3D362 and aE132-Cε3R334） and four potential hydrogen bonds（aK117-Cε3G335,aY129-Cε3D362,aY131-Cε3D364 and aY131-Cε3H424） are formed across the site 1 interface.Y131 from the receptor projects into a pocket on the IgE-Fc formed by the BC and FG loops of Cε3 and the Cε2-Cε3 linkerInteractions at site 2 includ ten amino acids from IgE and eight from FceRIa.The IgE residues are localized to two distinct segments of Cε3:the Cε2-Cε3 linker region （residues 332-337）;and the FG loop（424-427）.The FceRIa contributes residues from three regions:the D1-D2 linker region（85-87）;the D2 BC loop（residues 110 and 113）;and the D2 FG loop（156-158）.Residues from the D1 domain of thereceptor do not form direct interactions with the IgE-Fc.Site 2 primarily contains hydrophobic amino acids with three potential hydrogen bonds across the interface （aW156-Cε3G335,aQ157-Cε3N332 and aQ157-Cε3R334）.P426 from the IgE-Fc intercalates between receptor residues W87 and W110,forming a hydrophobic proline sandwichIgE/FcεRⅠcomplex crystal structure shows that many interaction sites are located in Cε3 opened N-terminal region and Loop which directly contact with the receptor.IgE Fc Cε2 domain bend towards the Cε3 domain Districtly and Violently that Cε3 have a accessible site and a hidden site.The crystal structure of IgE Fc which contains a complete Cε2 domain show that monomer IgE Fc has only one accessible site between the two sub-sites.This requires that conformation changes should happen so that the second locus can combine with IgE Fc.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.Since there is more basophils and IgE in allergic patients than in health adult,After getting the IgE Cε3-Cε4 gene from peripheral blood lymphocytes of an allergic disease patient by RT-PCT,it was ligated into the vector pET28a（+） and constructed an expression vector IgE Cε3-Cε4-pET28a（+）.The recombinant plasmid was transformed into E.coli BL21（DE3）.The target protein was expressed in BL21（DE3）.The fusion protein was purified by Ni Sepharose 6 Fast Flow affinity chromatography.The interreaction beteewn IgE Cε3-Cε4 and FcεRⅠwas identified by cell immunofluorescence.The fusion protein was quantitated using automatic analysis detecting instrument UniCAP 100.BALB/c mice were immunized with recombinant IgE Cε3-Cε4,anti-mouse polyclonal antibody was prepared and identified by western blot.Sequencing showed that the success of access to the IgE Cε3-Cε4 gene sequence and has been reported in line.The fusion protein was analysed by SDS-PAGE and the relative molecular mass was correct as predicted.Cell immunofluorescence experiments confirmed that IgE Cε3-Cε4 could specifically bind to FcεR I obtained from human basophils.Through the analysis of automatic detecting equipment UniCAP 100,the recombinant protein could be detected to precision of the international unit.Western blot confirmed the specific combination of anti-mouse serum polyclonal antibody and human IgE.The recombinant protein was expressed in E coli with recombinant plasmid IgE Cε3-Cε4-pET28a（+）.After the optimization of expression, products were presented mainly as inclusion body.The molecular weight of recombinant protein is about 30kDa,consistant with the theoretical value. The expression quantity is about 60%of the total protein of E coli.The purity quotient of the recombinant proteins is about 80%.Cell immunofluorescence experiments confirmed that IgE Cε3-Cε4 could specifically bind to FcεRⅠobtained from human basophils.Through the analysis of automatic detecting equipment UniCAP 100,the recombinant protein could be detected to precision of the international unit.The recombinant protein could be detected to precision of the international unit.Western blot confirmed the specific combination of anti-mouse serum polyclonal antibody and human IgE.The above results showed that recombinant expression plasmid IgE Cε3-Cε4-pET28a（+） has been constructed successfully,recombinant protein was expressed in E coli and then polyclonal antibody was identified by human IgE which makes foundations for researching the protein’s function, preparation of McAbs,effection in signal transmition and treatment of allergic disease.