The Construction And Eukaryotic Expression Of A Novel Chimeric Complement Inhibitor HCI-3

The complement system is a native protective system . It consists of a set of over 30 soluble glycoproteins and has a complex reaction system in human. In vivo,the major functions of the complement system are (a) initiation of acute inflammation by direct activation of mast cells.(b) Attraction,by C5a,of neutrophils to the site of microbial attack(chemotaxis). (c) Enhancement of the attachment of the microbe to the phagocyte (opsonization) and killing of the microbe activating the mebrane attack complex (lysis). Complement can be activated directly by microbes,or by antibodies bound to a microbe or any other antigen through three activated pathways,but excessive activation of the complement system can cause many kinds of diseases such as infection, myocardium ischemia injury reperfusion (MI/R), glomerulonephritis, arthrositis and so on. It should be effective to therapy these diseases through inhibiting the activation of complement.Regulation proteins in complement system is promising to inhibit complement activation.And the production of C3/C5 invertase and the formation of MAC is crucial in complement activation.In order to find a more powerful and useful complement inhibitor interfering both the two key ways.We selected three membrane regulatory proteins:CD46(member cofactor protein.MCP), CD55(decay-accelerating factor,DAF) and CD59 constructed two forms of chimeric CD46/CD55/CD59 molecular(one is GPI-anchored and the other is soluble) with the function region of the three proteins and named the two new complement inhibitors as mHCI-3 and sHCI-3 respectivly.Our work provide a new method to prevent and therapy SIRS,ARDS,MOF and MI/R in clinic.To accomplish this.we first use CD46 cDNA ,CD55 cDNA and CD59 cDNA as templats, select the sequences encoding the protecting region of complement-mediated lysis: CD46 (SCR2-4), CD55(SCR2-4) and CD59 correlated region.Link them withGPI-anchor by SOE(splicing by overlap extension).The PCR product is mHCI-3 cDNA.To avoid the change of the molecular structure and function ,a polypeptide amino acid linker was added into one of recombinant molecules CD46/CD55,as well as CD55/CD59.Then insert it into Bluescript ?M13 vector ,named the new vector mHCI-3/Bluescript-M13 .DNA sequencing results showed the open reading frame and the ligation part of the GPI-anchored form CD46/CD55/CD59 recombinant molecule are correct.Second,using GPI-anchored form of mHCI-3 cDNA as templet, cut off the signal peptide and transmembrane region of CD59.we designed new primers, amplied the cDNA and add suitable restriction sites in the ends with PCR,and then insert it into the eukaryotic expressing vector pSecTagA.The DNA sequencing results showed that we acquired the correct sHCI-3/pSecTagA vector.Third ,we transfect sHCI-3/pSecTagA plasmid into COS-7 cells with liposome transient expression system.After several weeks cell culture, we use SDS-PAGE and Western-Blotting to testified the condensed supernatantThe results showed that the molecular weight is correct, and the immune activity is moderate.So we sucessfully expressed sHCI-3 recombinant molecule in souble form in eukaryotic cells.