Effect Of A Short CD59-ligand Peptide On The Blockade Of Complement-binding Sites Of CD59 Expressing On Prostate Cancer Cells And Construction Of Its Eukaryotic Expression System

Objective To investigate the effect of a short CD59-ligand peptide(SP22) on the blockade of complement-binding sites of normal or mutant CD59 molecules highly expressing on prostate cancer to evaluate the potential of SP22 to be used to improve the outcome of prostate neoplasms immnotherapy;to construct a eukaryotic expression system of SP22 to make preparation for furtherly optimizing the structure of SP22, studying the interaction mechanism between SP22 molecules and CD59 molecules in tumor cells and developing a novel strategy of hindering the expression CD59 molecules on the membrane of tumor cells.Methods plRES vectors carrying wild-type CD59 gene(wCD59) or mutant CD59 gene (mCD59) and empty vectors pIRES were transfected by liposomes into PC-3 cells respectively.Clones of PC-3 cells stably expressing transfected genes were screened by the addition of G418.The expression of CD59 was detected by FIH(fluorescence immunohistochemistry technology) and FACS(fluorescence activated cell sorter) while the level of CD59 mRNA was semiquantited by RT-PCR.The effect of SP22 on the blockade of activity sites of CD59 molecules was evaluated by complement lysis assays. According to the amino acid sequence of SP22 its coding gene(SP-dsDNA) was designed and synthesized to either of which termination a recognizing site of Nheâ… or EcoRâ… enzyme was added.SP-dsDNA was purified and subsequently linked with pMD18-T vector for the sake of being sequenced.After SP-dsDNA and the eukaryotic expression plasmid pIRES were double-digested respectively by Nheâ… and EcoRâ… ,their purified and recruited products were linked by T4 DNA ligase.The recombinant was transformed into E.coli.JM109 and subsequently identified by PCR,restrictiion endonuclease reaction and gene-sequencing methods.Results Successful transfection of wCD59 or mCD59 into PC-3 cells was testified by FIH,FACS and RT-PCR.pPC-3 cells into which empty vectors were transfected expressed lower level of CD59 than wPC-3 cells(wCD59 gene transfected) and mPC-3 cells(mCD59 gene transfected) while no obvious difference of the level of CD59 was found between wPC-3 and mPC-3 cells.There was significant difference among the lysis rate(LR) of the three kinds of transfected PC-3 cells before SP22 was administrated to complement lysis assays,i.e.LR of pPC-3 cells the highest while LR of wPC-3 cells the lowest.With the addition of SP22,LR of all kinds of transfected PC-3 cells rose significantly but in quite different modes.Successful recombination of SP22 gene and pIRES was proved by PCR,restriction endonuclease reaction and gene-sequencing methods.Conclusion SP22 can effectively block the complement-binding sites of normal CD59 molecules expressing on PC-3 cells and in return greatly enhance complement-mediated lysis to PC-3 cells although it fails to bind with mutant CD59 molecules.The eukaryotic expression system of SP22 gene has been successfully constructed,which makes it more facilitated to explore the possibility of SP22 to be used to prevent CD59 molecules excessively produced in tumor cells from expressing on the surface of these cells.SP22 may represent a very promising strategy to improve the outcome of prostate cancer immunotherapy.