| It is well known that tumor necrosis factor a(TNFa) can play very different character in defence mechnism of body and pathologic injury,in which it can inhibit or kill tumor cell,and induce inflammation against infection;on the other hand,TNFa can be as a very important mediator to cause some serious pathologic processes such as septic shock,GVHR and some autoimmune diseases. Therefore,scientists in many universities and pharmaceutic companies have afforded efforts on the improvement of TNFa and development of TNFa antagonists,especially in the latter. TNFa antagonists including mAb against TNFa and TNF receptor(TNFR),soluble TNFa receptor(sTNFR),mutant TNFa have been applied or tried to apply in the clinical with some positive therapy effects,but there were still some problems to limit the application of these agents. Recently,some new studies suggest that small cytokine peptide-mimics can function as the agonist or antagonist of cytokine receptor,and these small molecular peptides can be synthesized easily and oral adminstration with high concentration. Based on these work,we have tried to set up experiment on TNFa-binding peptides and TNFa mimotopes which may be as leads of TNFa antagonist. Our research can be divided into the following four parts:Part I Screening of TNFa-Binding Peptide from phage display peptide library:The TNFa binding-peptides were screened from c7c phage display peptide library by using rhTNFa as target protein and identified by sandwich ELISA,for exploring whether the binding-peptides can be used as antagonist of TNFa or not. After 3 rounds of screening,11 of 23 phage clones were identified as positive clones which can bind to rhTNFa. The amino acid sequence in two of these 11 clones is c-ALWHWWH-c,and inthe others is c-(T/S)WLHWWA-c.The results of competitive inhibition of binding between phage clone and TNFa by ELISA show that all of these phage clones can bind TNFa strongly.Part II Screening of TNFa mimotopes from phage display peptide library:Based on the results of screening TNFa binding-peptides,we have tried to use neutral TNFa McAb J1D9 as target to screen TNFa mimotopes from c7c phage display peptide library,which may be another form of antagonist for TNFa,and the mimotopes were identified by sandwich ELISA . After 3 rounds of screening,we got 9 phage clones identified as positive clones which can bind with McAb J1D9. We also identified the binding between mimotopes and TNF receptor by competitive ELISA,and the results showed strongly binding.The amino acid sequence results shown three different sequences:c-RRPAQSG- c-NKHNRKI-c and c-RGMSRKI-c. These phage display peptides may be the TNFa mimotopes.Part III Specific c7c TNFa mimotope phage clone as target for screening of TNFa specific Binding-peptides from 12mer phage display peptide library:By using the specific c7c TNFa mimotope as target,we developed a novel approach for screening phage peptide library targeting specific phage display peptides. We used c7c TNFa mimotope phage clone LCS-7(c-RRPAQSG-c)as target to screen 12mer phage peptide library and identify the positive clones by phage ELISA. After 3 rounds of screening,10 of 20 clones were identified as positive clones and all the 10 phage clones sharing the same amino acid sequence:EHMALTYPFRPP,and these positive 12mer phage clone peptide can bind with TNFa specifically. The results suggests that this method is very specific and simple for screening of binding epitope to small peptide molecule from phage display peptide library.Part IV Computer-Aided Molecule Modelling of TNFa mimotopes and TNFa-binding peptides:To investigate the interaction between TNFamimotopes and TNFa-binding peptides,the computational docking program AutoDock (with confirming calculations using Discover )was used to predict the binding modes of LLT-18 with TNFa firstly,then LCS-7 was docked to LLT-18 by manual. The interaction between LLT-18 and TNFa or LCS-7 showed electrostatic interaction and H-bond dominant. The Arg residues in TNFa or LCS-7 were important when TN...
|
PDF Full Text Request