Sequence analysis and antigenic identification of mimic epitopes of peptidoglycan / 中国免疫学杂志

Objective:To analysis the Mimotopes of the peptide mimics to PGN using online softwares.Methods: Mimotopes of PGN were screened from 12-mers linear phage display peptide library by using anti-PGN McAb and the antigenicity of selected clones was identified by ELISA.The B cell epitopes,T cell epitopes of ′GRWxHxVxWAGL′ were estimated by DNAstar and online softwares.Results: 16 phage clones that bound with anti-PGN McAb were screened from 12-mers linear phage display peptide library.Among these positive clones,phage clones No.39 shared the conserved sequence:WxHx……AGL found in previous clone No.31(ATWxHxLxSAGL),which provoked an effective protective immunity against infection with S.aureus.To enhance the stability of the conformation as well as adding biotin on the N-ter-minal as a tag,the sequences ′39′ were redesigned and synthesized by adding S(serine)A(alanine) and GG(glycine)on the C-terminal of origin sequence(named SP39).Next,we estimated or predicted antigenic epitopes,T cell epitopes and scores binding to MHC of these peptides by using DNASTAR and online softwares(http://bio.dfci.harvard.edu/Tools/antigenic.pl,www.syfpeithi.de,http://www.darrenflower.info/mhcpred),indicating that SP39 contains sites bound both mice and human MHC.The sequences ′WxHxVxW-′ may be antigenic epitope as SP39,which contains a T cell epitope.Our results showed that both SP39 could bind to both anti-PGN McAb and a polyclonal antibody against S.aureus.Moreover PGN could inhibit the binding of SP39 to the anti-PGN McAb.These data indicated that SP39 mimic to epitopes on PGN.Conclusion: SP39(GRWxHxVxWAGLAGGS) probably display the mimotopes of PGN.

Screening and identification of peptide mimics to lipoteichoic acid by phage displayed random peptide library / 中国免疫学杂志

Objective:To screen epitope mimics to lipoteichoic acid from a random 12-mer phage display peptide library and i-dentify the specificity of the mimotopes of LTA.Methods:The monoclonal antibody against LTA was used as a target to screen the 12-mer phage display peptide library and the specificity of phage clones were identified by sandwich ELISA.The amino acid sequences of positive phage clones were deduced from DNA sequencing.The specificity of synthetic peptide were identified by sandwich ELISA.Results:4 clones were obtained after 3 rounds of screening.Amino acid sequence analysis revealed four different types of mimotope sequence.A linear peptide (GHxDFRQxxQPS),named L2,which derived from positive sequence was synthesized.ELISA result indicates that L2 can bind to anti-LTA mAb specifically in a dose-dependent manner.Conclusion:The mimotopes of LTA were obtained by using the phage display technology.

Preparation and application of antibodies against advanced oxidation protein products / 中国免疫学杂志

Objective:To prepare the polyclonal antibodies against advanced oxidation protein products (AOPP),and to provide an effective agent for research on the pathogenesis of AOPP and assess exactly the relationship between AOPP and relative diseases.Methods:AOPP-rabbit serum albumin (AOPP-RSA) was prepared by treating RSA with hypochloric acid.The rabbit anti-AOPP-RSA polyclonal antibodies were generated and purified by affinity chromatography. The titers and the specificity of the antibodies were measured by ELISA.The plasma AOPP and the localization of AOPP in nephridial tissues of some patients with chronic kidney disease (CKD) were determined using rabbit anti-AOPP-RSA.Results:Titers of the antibodies were 10-6.Purified antibodies reacted specifically with oxidized albumin from different genus,but could not react with normal albumin and glycosylated albumin.The high level of AOPP in plasma from CKD patients was confirmed by Western blot.The antibodies could be used to immunostain AOPP deposition in different regions of kidney tissues from both CKD patients and CKD rat models.Conclusion:We successfully generate rabbit anti-AOPP polyclonal antibodies with high titers and striking specificity.The presence of plasma AOPP and localizations of AOPP in kidney tissues of CKD patients can be demonstrated using the antibodies.The development of anti-AOPP polyclonal antibodies may provide a new tool to explore the pathogensis of AOPP and assess exactly the relationship between AOPP and relative diseases.

Screening and identification of HIV-1 gp41 core structure epitope from random phage display peptide library / 中国免疫学杂志

Objective:To identify and characterize the epitopes on core structure of HIV 1 gp41.Methods:A random phage displayed dodecapeptide library was screened with a conformation specific monoclonal antibody NC 1 specifically against the core structure of HIV 1 gp41.The positive clones were identified by sandwich ELISA,soluble NC 1 blocking assay and competitive inhibition assay.Results:After three rounds of screening,10 of 24 phage clones were identified as positive clones which can bind to NC 1.Amino acid sequences deduced from DNA sequences showed five different sequences:HDVHHRWVYLLS?ITVNEWLYTSEQ?HGRSHGMFKPKR?MGPIARPHWHLN?DMYRSPRPKPDT.The binding between phage clones(displayed HDVHHRWVYLLS,VNEWLYTSEQ and MGPIARPHWHLN, respectively)and NC 1 could be inhibited by N36 C34 complex.Soluble NC 1 could block the binding between phage clones and NC 1.Conclusion:The results indicate that HDVHHRWVYLLS,VNEWLYTSEQ and MGPIARPHWHLN are the mimotopes which could mimic the core structrue epitopes of six helix bundle of HIV 1 gp41.

Screening and identification of HIV-1 gp41 C-helix mimotopes displayed on phages / 中国免疫学杂志

Objective:To find small molecular leads for inhibition on early stage of HIV infection by identification and characterization of the HIV-1 gp41 C-helix mimotopes.Methods:For identification of the gp41 C-helix mimotopes,C7C phage display peptide library was biopanning by using a synthetic peptide N36 which was derived from the gp41 N-helix as target.After three rounds of screening,positive phage clones were identified by ELISA and sequenced.Results:16 of 26 phage clones were identified to bind with peptide N36,and 10 of them were sequenced.Every clone of ten clones contains at least two hydrophobic residues,which may dock into the hydrophobic pocket in the gp41 N-helix domain.9 of the 10 clones have a conservative sequence WW,which may mimic the W628 and W631 in C-helix to interact with the hydrophobic residues in the gp41 pocket.One clone expressing the conservative sequence named clone No.8(CYWWHRLHC) was selected for characterization.The binding between the clone No.8 and N36 was blocked by free peptide N36.And the binding between clone No.8 and peptide N36 was inhibited by peptide C34(IC 50=12.5 ?g/ml).Conclusion:The short circular peptides displayed on phages containing WW residues may mimic the conformational epitope of the HIV-1 gp41 C-helix to interact with the N-helix.This information may be useful for design of HIV-1 fusion inhibitors.

Studies on a high-throughput screening method for identification of HIV fusion inhibitors targeting gp41 / 中国药理学通报

AIM To modify and improve a screening assay so that it becomes more convenient, economic and adaptable in China for high throughput screening of HIV fusion inhibitors targeting gp41. METHODS The original screening method reported by Jiang et al (J Virol. Methods 1999;80:85 96) was modified by: ① using a conformation specific monoclonal antibody to replace a polyclonal antibody for coating plates; ②simplifying the procedures; ③using parts of the reagents produced in China. RESULTS The modified screening assay is simpler, more convenient, and more economic than the original assay, but its sensitivity is comparable to and specificity is a little better than the original method. CONCLUSIONS The modified screening assay is more convenient and economic and can be used in China for high throughput screening of HIV fusion inhibitors from complex sample, such as phage display peptide libraries, microorganism fermentation liquids, herbs and other natural products.

Characterization of the epitopes recognized by monoclonal antibody 3A8 specific to broad-spectrum Gram-negative bacteria / 中国免疫学杂志

Objective:To screen and characterize the common epitopes recognized by monoclonal antibody 3A8,which binds several kinds of Gram negative bacteria,which were recognized by monoclonol antibody 3A8 agaisnt different strains of LPS.Methods:Phage clones binding to 3A8 were screened from C7C phage displayed peptide library,the attached phage clones were eluted by LPS2630(O111:B4).After 3 rounds of panning,the positive phage clones were identified by ELISA,and the amino acid sequences of these positive clones were deduced from DNA sequences.In order to predict how these peptide mimics interaction with 3A8,peptides(A2 and A5) based on conserved sequences of positive clones were synthesized and conjugated with KLH for further study.The binding of A2-KLH and A5-KLH to 3A8 were identified by ELISA.Results:15 of 33 phage clones were identified as positive clones and shown specific binding with 3A8.LPS2630 potently inhibited the binding of phage clone to 3A8.In analysis of the amino acid sequence,there were seven kinds of sequences containing highly hydrophilic residues,and Ser Pro Pro/Pro X Pro was the conserved sequence.The peptides A2 and A5 could bind to 3A8 specially.Conclusion:The conserved sequences containing Ser Pro Pro/Pro X Pro are obtained,which are recognized by mAb 3A8 against broad spectrum Gram negative bacteria and several strain LPS.The synthetic peptides based on the conserved sequence can bind to 3A8,indicating that the peptides can mimic a common epitope of Gram negative bacteria,which be expected as candidate epitope for vaccinization.