GPI Anchoring Cd46/cd55, Cd46/cd55/cd59 Chimeric Molecule Design And Construction Of Cloning Vectors

The Construction of GPI-anchored Form of Chimeric Human CD461CD55 and ChimericCD461CD551CD59 cDNAAbstractComplement activation is regulated by a number of plasma- and cellassociated proteins that function to inactivate specific steps of complement pathway. These proteins provide a mechanism for preventing the excessive generation of activated complement products, thereby protecting normal tissures from complement-mediated "bystand"lysis. Because of the integral involvement of the complement system in the inflammatory response, agents that block complement activation could have significant therapeutic potential for treatment of several human diseases. As means to specially bJ.ock complement activation, CD46, CD55 and CD59, which inhibit complement activity at two different key points either by blocking the assembly of the C3/C5 convatases or by preventing the formation of MAC. were reseached,We constructed two kinds of GPI-anchored form of chimeric CD461CD55 cDNA and two kinds of GPJ-anchored form of cbimeric CD46/CD551CD59 eDNA. First, Using CD46 eDNA and CD55 cDNA as templates, we amplified the cDNA which encode CD55 signal peptide, CD46 SCR(2-4) and CD55 SCR(2-4) as well as GPI-anchor, and linked them together by SOE (splicing by overlap extention) and cloned it into Bluescript-M13 vector. In order to avoid the change of the molecular structure and function, a polypeptide amino acid linker was added into one of recombinant molecules. DNA sequencing results showed that the sequence of two kinds of GPI-anchored form ofCD46/CD55 recombinant molecules in the reading frame and the ligation part are correct. Second, Using CD46 cDNA and mini-CD55-cd59 cDNA that was made by our Lab as templates, We amplified the cDNA that encode the functional domains of CD46,CD55 and the mature protein of CD59. and linked them together at the restriction sites and cloned it into Bluescript-M 13 vector. A polypeptide amino acid linker was also added into one of recombinant molecules. Then, we finished constructing two kinds of OPT-anchored form of CD46/CD55/CD59 chimeric cDNA from DNA sequencing results.In this experiment, two kinds of novel GPI-anchored form of recombinant molecules were constructed by two kinds of metheds. Our results paves a way for the study of the hyperacute rejection in the field of xenotransplant. Meanwhile, it places a base for the development of the novel complement inhibitor, which can apply as therapeutic agents in severe inflammation and autoimmune disease.