Development of a Synthetic Peptide ELISA Assay for the Detection of Foot-and-Mouth Disease Virus Nonstructural Protein Antibodies

In order to develop an ELISA assay with synthetic peptides for the detection of antibody to the nonstructural proteins of foot-and-mouth disease virus, specific peptides were synthesized by a solid-phase method according to FMDV NSPs B-cell epitopes, and were conjugated to carrier protein BSA. An ELISA system was developed to detect FMDV NSPs antibody with the conjugated proteins as the coating antigen. The optimal coating concentration of the antigen was determined as 2.5 μg mL-1. The comparative study of this assay with UBI NSP ELISA kit and national commercial 3ABC ELISA kit in the detection of 199 serum samples showed that they were very coincident, and the identity rates were 96.48 and 97.48%, respectively. The development of ELISA using the synthetic peptides as coating antigen is specific, reproducible, stable, and easy, and can be used to differentiate FMDV infected pigs from immunized pigs.

Rationally designed synthetic peptide as versatile calibrant to improve the accuracy of protein sequence analysis using MALDI mass spectrometry

Matrix-assisted laser desorption/ionization(MALDI)mass spectrometry(MS)plays an indispensable role in analyzing protein covalent structures.The reliable identification of amino acid residues and modifications relies on the mass accuracy,which is highly dependent on calibration.However,the accuracy provided by the currently available calibrants still needs further improvement in terms of compatibility with multiple tandem MS modes or ion polarity modes,calibratable range,and minimizing suppression of and interference with analyte signals.Here aiming at developing a versatile calibrant to solve these problem,we designed a synthetic peptide format of calibrant R_x(GDP_n)_m(referred to as“Gly-Asp-Pro,GDP”)according to the chemical natures of amino acids and polypeptide fragmentation rules in tandem MS.With four types of amino acid residues selected and arranged through rational designs,a GDP peptide produces highly regulated fragments that give rise to evenly spaced signals in each tandem MS mode and is compatible with both positive and negative ion modes.In internal calibration,its regulated fragmentation pattern minimizes interference with analyte signals,and using a single peptide as the input minimizes suppression of the analyte signals.As demonstrated by analyses of proteins including monoclonal antibody and Aβ-42,these features allowed significant increase of the mass accuracy and precision,which improved sequence coverage and sequence resolution in sequence analyses(including de novo sequencing).This rational design strategy may also inspire further development of synthetic calibrants that benefit structural analysis of biomolecules.

Immune Responses to Six Synthetic Peptides of Capsid Protein with Sera from HIV-1 Infected Individuals

Many B cell epitopes within p24 of human immunodeficiency virus type 1 （HIV-1） were identified, while most of them were determined by using murine monoclonal antibodies reacting with overlapping peptides of p24. Therefore these epitopes may not represent the actual epitopes recognized by the HIV-1 infected individuals. In the present study, immune responses of 67 HIV-1 positive sera from Yunnan Province, China to five peptides on p24 of HIV-1 and one of HIV-2 were analyzed. All of 67 sera did not recognize peptide GA-12 on HIV-1 and peptide AG-23 on HIV-2, which indicated that GA-12 was not human B cell epitope and AG-23 did not cross-react with HIV-1 positive serum. Except 13 sera （19.4%）, all remaining sera did not recognize peptides NI-15, DR-16, DC-22 and PS-18, which indicated that these four peptides represented B cell linear epitopes of HIV-1 p24 in some HIV-1 infected individuals but not the immuno-dominant epitopes in most individuals.

Selection and application of serotypical synthetic peptides derived from hepatitis C virus NS5A region

Background Numerous studies have reported a relationship between hepatitis C virus （HCV） genotype and the response to interferon therapy. Despite high sensitivity and specificity, genotyping methods can be performed only on HCV RNA positive samples. Serotyping might be a rapid and cost effective method for determining HCV genotypes, especially in patients with previously undetectable HCV RNA. In this study, an enzyme linked immunosorbent assay （ELISA） method for HCV serotyping with the genotype specific, synthetic peptides derived from HCV nonstructural 5a （NS5A） region was developed. Methods Based on 45 sequences, representing HCV genotypes 1-6 from Genebank, we synthesised 305 overlapping 30-mer peptides within NS5A region at positions 2182-2343 of HCV. All peptides for antigenic reactivity were tested by enzyme immunoassay with 69 human sera with antiHCV positive representing genotype 1-6. Forty hepatitis C patient sera were serotyped using serotype specific, synthetic peptides and genotyped by sequencing analysis. Results The correspondence of amino acids in HCV NS5A region with amino acids in positions 2182-2343 was very low among different genotype peptides. The highly conserved sequences were residues 2182-2211 （R1）, 2272-2301 （R7） and 2302-2331 （R9）： the highly variable 2212-2241 （R3） and 2257-2286 （R6）. Using 305 peptides, antigenic regions were located in R3, R7 and R9. Eighteen peptides from highly conserved region representing genotypes 1 to 6 showed broad immunoreactivity with sera containing antibody to all HCV genotypes. Immunoreactivity of the peptides from highly variable region was stronger with similar genotype sera. Twelve unique peptides showed highly, genotype specific, reactivity with types 1 and 3 sera. Type 2 genotype specific peptides had cross reaction with type 3 serum. No type 4, 5 or 6 specific peptides were selected. The serotyping results showed high agreement with sequencing analysis. Conclusions The major antigenic regions in HCV NS5A region were at 2212-2241（R3）, 2272-2301（R7） and 2302-2331（R9）. Eighteen peptides from highly conserved region show genotype independent, immunoreactivity, useful for antiHCV antibody test. Twelve peptides from highly variable region show genotype 1 and 3 dependent immunoreactivity, useful for determining HCV serotype, especially for patients with previously undetectable HCV RNA.

Synthetic Smac Peptide Enhances Chemo-sensitivity of Bladder Cancer Cells

The effects of synthetic Smac peptide （SmacN7） on chemotherapeutic sensitivity of bladder cancer cells were investigated. SmacN7 penetratin peptide was synthesized and delivered into T24 cells. MTT assay was used to evaluate the viability of T24 cells induced by low-dosage of MMC Flow cytometry was used to analyze the proportions of apoptosis. Western blot was used to detect the expression of XIAP and Caspase-3. The activity of Caspase-3 was measured and the effect of SmacN7 combined with MMC on T24 cell lines was also determined. The results showed that SmacN7 penetratin peptide could successfully interact with endogenous XIAP, increase the proportions of apoptosis of T24 cell lines induced by low-dosage of MMC in a dose-and time-dependent manner. An obvious down-regulation of XIAP expression and up-regulation of Caspase-3 was identi-fied by Western blot. The activity of Caspase-3 in experimental group was significantly increased as compared with that in the control group. As compared with MMC group, the viability of T24 cells in SmacN7 penetratin peptide＋MMC group was markedly decreased to 2.22 and 3.61 folds at 24 h and 48 h respectively. It was concluded that SmacN7 penetratin peptide could act as a cell-permeable IAP inhibitor, inhibit the proliferation, induce apoptosis and enhance the chemo-sensitivity of bladder cancer cells to MMC. These findings indicate that SmacN7 penetratin peptide may be a very promising ageut for bladder cancer treatment when used in combination with chemotherapy.

Identification and Characterization of Peptides Binding AgEG1 from a Phage Display Library

Endoglucanases are the main cellulolytic enzymes digestion as well as its good kinetic properties make it an attractive of Anoplophora glabripennis. Their high activities in cellulose target for development of cellulase inhibitors. In this study, random pepfide phage display technology was employed to identify peptides that bound the AgEG1, a member of endoglucanase isozymes. Phage clones with peptide LPPNPTK and XPP （X is residue T, L, A or H） motif frequently occurred in the selected phage population and showed a higher phage recovery than other clones. Peptide LPPNPTK was chemically synthesized and characterized tor its binding activities to AgEG1. The synthetic peptide exhibited high specificity for AgEG1. The peptide LPPNPTK has the potential to be developed into inhibitors of the endoglucanase of A. glabripennis.

DYNAMICAL OBSERVATION OF CARDIO-FUNCTIONAL IMPAIRMENT OF AUTOANTIBODIES AGAINST MITOCHONDRIA AND PEPTIDE IN RATS

27 rats were immunized with synthetic ANT peptide. Antibody titers in sera and heartfunctions were measured at day 14, 28 and 42 arter the latest immunization. The results showed thatIn the 27 rats immuuized with the ANT Peptide, 63% (17/27) showed marked increasing or specificanti-ANT-Peptide antibody titers in sera. The highest positive rate of antibodies in sera is on the 28th day after the latest immunization. Remarkabie impairment of cardiac functions was observed simultaneously. Correlaled analysis indicates that the degree of the declined cardiac function relatedclosely with the levels of antibody titers. It is indicated that anti-ANT-antibody is a specific,Pathogenic autoantibody to heart. The synthetic ANT peptide we select could replace the native ANTprotein in further studies of the pathogenesis of antiboies against ANT.

Insights into therapeutic peptides in the cancerimmunity cycle:Update and challenges

Immunotherapies hold immense potential for achieving durable potency and long-term survival opportunities in cancer therapy.As vital biological mediators,peptides with high tissue penetration and superior selectivity offer significant promise for enhancing cancer immunotherapies(CITs).However,physicochemical peptide features such as conformation and stability pose challenges to their on-target efficacy.This review provides a comprehensive overview of recent advancements in therapeutic peptides targeting key steps of the cancer-immunity cycle(CIC),including tumor antigen presentation,immune cell regulation,and immune checkpoint signaling.Particular attention is given to the opportunities and challenges associated with these peptides in boosting CIC within the context of clinical progress.Furthermore,possible future developments in this field are also discussed to provide insights into emerging CITs with robust efficacy and safety profiles.

Quick photofabrication of functional nanospheres from de novo designed peptides for NIR fluorescence and MR imaging

Combining the noncovalent and covalent interactions,a series of peptide amphiphiles were designed de novo and synthesized to architect functional assemblies by means of photochemistry.The strand of peptide sequence was structurally capped with photoactive tyrosine-tyrosine(YY)motifs at both termini,and the spacing was filled by alternating of hydrophilic D(L-aspartate)and hydrophobic X(ε-aminocaproic acid)structure.Upon visible-light irradiation,these de novo designed peptides underwent rapid photocrosslinking within merely 10 min.Interestingly,the modulation of alternating D-X pairs in occupying spacer would adjust molecular amphiphilicity,regulate charge distribution,and control particle size and loading capacity of peptide nanospheres(PNS)in aqueous media.With entirely peptide-based matrix,this PNS system could host cationic indicators of fluorescent rhodamine and magnetic GdIII for exemplar near infrared(NIR)fluorescence and magnetic resonance(MR)imaging,which paves a pathway to biomaterial and biomedical applications using de novo designed peptides.

Synthesis of an active peptide from Carapax Trionycis and its inhibitory effect on the proliferation of hepatic stellate cells

This study was aimed to synthesize an active peptide from Carapax Trionycis and to investigate its effect on the proliferation of hepatic stellate cells（HSCs）.An active peptide from Carapax Trionycis,which was shown to have significant anti-hepatic fibrosis activity,was synthesized by solid phase method and characterized by MALDI-TOF MS analysis.The HSCs in log growth phase was treated with the synthetic peptide at different concentrations.Viability and apoptosis of hepatic stellate cells were determined by MTS assay and Annexin V-FITC/PI staining,respectively.The active peptide showed strong inhibition of proliferation and induction of apoptosis of HSC-T6 in a concentration-dependent manner.The results suggest that the active peptide from Carapax Trionycis could be synthesized efficiently and has significant inhibitory effect on the proliferation of HSC-T6.

Metalloporphyrin receptors for histidine-containing peptides

Two new ditopic metalloporphyrin receptors constructed by combining metalloporphyrin with crown ethers have been prepared and characterized.1H NMR and MS spectra confirmed the complexation of receptor with peptide driven by coordination interaction and hydrogen bonding.UV/vis experiments revealed that the receptors exhibited high binding affinity to histidine-containing peptides.These receptors could differentiate short peptides of C-terminal histidine and N-terminal histidine and formed the most stable complexes with tripeptide.