Screening and identification of the polypeptides specifically binding to the adhesion protein of Myco-plasma genitalium / 中华微生物学和免疫学杂志

Objective To screen and identify the polypeptides specifically binding to the adhesion protein of Mycoplasma genitalium(MgPa) by using the Ph. D.-12TM phage display peptide library for further understanding the biological function and the possible pathogenic mechanism of the MgPa. Methods The Ph. D.-12TM phage display peptide library was used for 3 rounds of biopanning with the purified recombinant MgPa ( rMgPa) as the given target. The phages were collected for amplification after biopanning. The single strand DNA of phage clones were extracted and purified by using the sodium iodide method for further se-quencing. ELISA, competitive binding assay and dot immunobinding assay were performed to analyze the specific binding of positive phages to rMgPa. Results A significant enrichment of phages was achieved after 3 rounds of biopanning. Eleven different phage exogenous sequences (P1-P11) were detected among the 38 phages randomly selected from the agar. Two core sequences were deduced according to the repeating times of amino acids among the 11 polypeptide sequences, which were V-H-W-D-F-R-Q-W-W-Q-P-S and D-W-S-S-W-V-H/Y-R-D-P-Q-T/S. Ten out of the 11 representative phages ( P1-P10 ) specifically combined with the rMgPa. Conclusion Two polypeptides specifically binding to rMgPa were successfully screened out, which provided the tool for further investigation on the biological function of MgPa and the pathogenic mecha-nism of Mycoplasma genitalium.

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.

The screening of liver cancer specific binding peptide from serum / 中华肝胆外科杂志

Objective To find the liver cancer specific peptide for serological screen of liver cancer patients via screening phage-display peptide library. Methods Fifteen sera from liver cancer patients and physical examinates were collected for the four-round screening with Ph. D. 12TM phage display peptide kit. Highly specific phage monoclones were selected based on the ELISA results of the serological assay. The peptide labeled with FITC was synthesized according to the DNA sequencing of the optimal monoclone and tested with serum via fluorescent imagery. Results Nine highly specific monoclones were found among 50 selected ones after 4 rounds of screenings. The positive rate of the optimal monoclone,ZH-3, reached 46.7 %. The peptide sequence of ZH-3 was concluded by DNA sequencing as SAHGTSTGVPWP. Desirable specificity and affinity were also shown in the serum of liver cancer patients. Conclusion The peptide ZH-3 can be used as a diagnostic reagent for liver cancer.

Panning and identification of humanized antinuclear antibody Fab fragment / 第二军医大学学报

Objective: To prepare humanized antinuclear antibody Fab fragment. Methods: The reconstructed humanized antinuclear antibody (ANA) Fab phage display library was enriched by 4 rounds of panning and was identified by indirect ELISA method. Phasmid DNA isolated from positive clones was deprived of g III gene. After self-ligation the recombinant plasmid was used to transform E. coli. XL1-Blue, then XL1-Blue was induced by IPTG to product soluble human antinuclear antibody Fab fragment. Finally, soluble human antinuclear antibody Fab in the supernatant was identified by indirect ELISA method and immunofluorescence. Results: The eluted phages were enriched by more than 200 folds after 4 rounds of panning. Two positive clones were isolated from the ANA Fab library. Electrophoresis after Xho I digestion proved that the self-ligation was successful after deletion of g III gene. The results of indirect ELISA indicated that the 2 positive clones of Fab had specific anti-dsDNA activity. Indirect immunofluorescence showed homogeneous fluorescence within nuclei of Hep2 and monkey hepatic cells and in the Crithidia kinetoplast. Conclusion: We have successfully prepared soluble, specific human antinuclear antibody Fab fragment, which pares a way for preparation of high affinity antinuclear antibody Fab fragment.

Selection of peptides bind to PreS1 antigen from a phage-displayed peptide library / 第二军医大学学报

Objective: To screen for peptides that specifically bind to PreS1 antigen from a phage-displayed peptide library. Methods: The PreS1 antigen was used as the target molecule to screen the binding peptide from the Ph. D. -7 peptide library with phage display technique, and the positive clones were identified by ELISA. Results: After three rounds of biopanning, the binding peptides were screened from the peptide library by ELISA and competitive inhibiting ELISA. Sequencing result showed that the binding peptides had high affinity and specificity. Conclusion: A peptide binding PreS1 antigen has been successfully obtained by screening the phage display library, which paves a way for the diagnosis and treatment of hepatitis B infection.

Protective effects of ouabain conjugated peptide from Ph. D.-7 Library on vascular endothelial cell / 细胞与分子免疫学杂志

AIM: To find one kind of peptide that will conjugate with ouabain and inhibit its biological function, and provide a new treatment strategy for primary hypertension. METHODS: In this study, ouabain was used as a target to screen ouabain conjugated peptide (OCP) from Ph. D. -7 phage display peptide library. After 3 rounds of bio-panning, the products were identified by ELISA and DNA electrophoresis analysis and sequencing. Peptide was synthesized and analyzed the activity by radioligand binding assay. The inhibitory ratio of cell proliferation was measured by MTT and the cell morphology changing was measured by Hoechst 33342/PI staining. The expression of Na~+-K~+-ATPase α1-subunit and β1-subunit were detected by RT-PCR and immunocytochemistry. The levels of the free intracellular Na~+ in EAhy926 cells were measured by laser confocal microscope. RESULTS: The ouabain conjugated peptide was found out, and it was occupied in 0.64(9/14). The analysis of protein showed that the obtained peptides had no homology with Na~+-K~+-ATPase. The amino acid sequence of synthesized peptide was Arg-Cys-Met-Thr-Ser-Arg-Ser. There was binding activity between OCP and ~3H-ouabain. The MTT assay showed that OCP could reverse the inhibition action of ouabain on vascular endothelial EAhy926 cells in a dose and time-dependent manner. The number of apoptotic cells had significantly decreased detected by Hoechst 33342/PI staining. The results of RT-PCR and immunocytochemistry showed that OCP could inhibit the up-regulated expression of Na~+-K~+-ATPase α1-subunit and down-regulated expression of Na~+-K~+-ATPase β1-subunit induced by ouabain in EAhy926 cells. CONCLUSION: The OCP could reverse the growth inhibition and death induction of ouabain in EAhy926 cells, which would provide the basis for studying the interaction between ouabain and Na~+-K~+-ATPase and explore novel anti-ouabain agents.

Selection of HFRSV-specific epitopes by screening random peptide libraries with HFRSV-positive sera / 医学研究生学报

Objective: To obtain the 12-mer phage clones displaying the Hantaan virus mimic epitopes.Methods: HFRSV-positive sera were used as selective molecules for biopanning.A 12-mer phage peptide library was bio-panned for 5 rounds,and the result was confirmed by sandwich ELISA,competition ELISA and DNA sequencing.Results: After 5 rounds of effective screening,the results detected by sandwich ELISA and competition ELISA showed that the majority of the selected clones could react to positive sera in a dose-dependent manner,but could not bind to BSA and the control sera.Sequencing and alignment analyses indicated that amino acid sequences of 45 positive clones fell into 8 groups,and 7 of them exhibited putative motifs: LVXKR,LTXR,IXKP,LXPA,VGA,KXIR and EKXP.Four of the putative motifs had a homologous region within the structural proteins of HFRSV.Conclusion: The peptides displayed by the phage can mimic the epitopes of HFRSV antigens,which provides the potential for preparing more effective epitope-based vaccines and specific diagnostic reagents.

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.

Selection of peptides bind to PreS1 antigen from a phage-displayed peptide library / 第二军医大学学报

Objective:To screen for peptides that specifically bind to PreS1 antigen from a phage-displayed peptide library.Methods: The PreS1 antigen was used as the target molecule to screen the binding peptide from the Ph.D.-7 peptide library with phage display technique,and the positive clones were identified by ELISA.Results: After three rounds of biopanning,the binding peptides were screened from the peptide library by ELISA and competitive inhibiting ELISA.Sequencing result showed that the binding peptides had high affinity and specificity.Conclusion: A peptide binding PreS1 antigen has been successfully obtained by screening the phage display library,which paves a way for the diagnosis and treatment of hepatitis B infection.

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.