Construction And Immunogenicity Analysis Of Recombinant Each Peptide Constant Of Human IgMμ Chain Constant Region

Immunoglobulin M is one of immunoglobulin of the humanbeing, the others are lgA、lgG、IgD and lgE.5%-10%of serum immunoglobulin is IgM,it is the largest Ig,most ofIgM is in the blood,it can not cross the vessel wall. IgM is the the primary antibody inhumoral immune response.It is the “spearhead” of body’s resistance to infection. Ifdetect IgM in serum it suggest infection recently. So Detection of IgM in serum canused to earlier diagnosis of infection.During the infection IgM is the primaryantibody,but it dispappeared soon,so IgM antibody is the sign of recet refection.IgM include heavy chain(μ chain) and light chain(λchain).λchain is the common chainof variety Ig, μ chain is the specific chain of IgM. It is reported that human IgMμchainMonoclonal antibody can used to early diagnosis of some Infectious diseases andresearch for the immunology.But the nature IgMμchain which used to prepare humanIgMμchain Monoclonal antibody is shortage for provision.The method of preparat thenature IgMμchain is tedious,And the nature IgMμchain is expensive. Accordingly weused technique for Gene Engineering,we have constructed many prokaryotic expressionplasmids which were induced to express in E.coli to produce recombinant IgMμchain inorder to find the peptide whic the immunogenicity like natural one, It provided avaluable potential in further research on gene engineering obtained human IgMμmonoclonal antibodies.Part I: Construction and Prokaryotic Expression of each peptide constant ofhuman IgMμ chain constant regionAnalysis the constitution of IgMμ chain,the μ chain has a variable region（VH） and four constant regions（CHl、CH2、CH3and CH4）. A pair of light and a pair of heavychain composition the morphon of IgM.The heavy chain and light chain both havevariable region and constant region. IgM composited by heavy chain(μ chain) and lightchain(λchain), λchain is the common chain of variety Ig, μ chain is the specific chain ofIgM.The cDNA sequence of human IgM μ chain constant region（CH1-CH4） was obtainedfrom GenBank and necessary coden optimization was made due to the specialpreferance of E. coli, then synthesize the nucleotide sequence and its truncatedsequences CH1-CH2and CH3-CH4by OE-PCR. And clone the three DNA fragmentsonto vector pMD18-T. After being sequenced, the correct ones were cloned ontoexpression vector pET-32a to construct prokaryotic expression plasmids:PET32a-rMμCH1-CH4， PET32a-rMμCH1-CH2and PET32a-rMμCH3-CH4Then allthe plasmids were transformed into E.coli BL21(DE3) and induced to express by IPTG.Tree peptides recombinant were purified by affinity chromatography with themolecular weight69.8KD、43.8KD、46.9KD respectively.Part II: Immunogenicity Analysis of Recombinant each peptide of human IgMμchain constant regionThree truncated recombinant IgMμ chain each peptide PET32a-rMμCH1-CH4，PET32a-rMμCH1-CH2and PET32a-rMμCH3-CH4were used to immunize were usedto immunize BALB/c mice to compare their abilities of inducing antibodies to IgMμchain; Use native human IgM、IgMμchain and IgG immunize BALB/c mice,in orde tocompare abilities of inducing antibodies between recombinant protein and native nativehuman IgM、IgMμchain and IgG.And Analysis of cross-reactivity amonged humanIgM、IgMμchain and IgG. The results of ELISA showed that rabbit and mouse antisera raised by recombinantIgMμ chain described above and nateiv IgMμ chain reactivated specifically with IgMμchain protein, which demonstrated that the IgMμ chain constructed in this studypreserved its immunogenicity.①Immunogenicity Analysis IgG＞IgM＞IgMμchain＞rMμ1-4＞rMμ1-2＞rMμ3-4；②Specificity Analysis, intensity cross reaction betweenimmune sera of IgM and immune sera of IgG,it is no obviously cross reaction betweenimmune sera of IgMμchain Recombinant each peptide constant of human IgMμchainand immune sera of IgG;Summary:1. In this study, we successfully expressed recombinant proteins of by constructingIgMμ chain each peptiets prokaryotic expression plasmids which were transformed intoE.coli.2. All of three recombinant peptides (PET32a-rMμCH1-CH、PET32a-rMμCH1-CH2、PET32a-rMμCH3-CH) preserved its immunogenicity and antibodies as native IgMμchain. Three recombinant peptides can induct specific antibody to native IgMμ chain.This result is prompt that the recombinant peptides may replacement native IgMμ chainto be the material to prepare antibods. It provided a valuable potential in furtherresearch on gene engineering obtained human IgMμmonoclonal antibodies3. Preliminary study showed that IgMμmonoclonal antibodies is more specific thanIgMmonoclonal antibodies,is more fidelity in early diagnosis of Infectious Diseases andresearch of immunology. The Preliminary study providing clues for further study inanalyzing and screening the monoclonal antibodies for clinic and basis research.