Whole cell screening of phage-display peptide library for mimicry peptides of glioma SWO-38

AIM and METHODS:The Ph.D.-7 phage display library was used to isolate peptides specific for glioma SWO-38 cell by whole cell screening.Moreover,binding efficiency analysis was carried out to test the binding specificity of the clones obtained.RESULTS:After three rounds of biopanning,a high concentration of phage clones was obtained and two of them were found to be highly specific to glioma SWO-38.CONCLUSION:Highly specific clones against neurtral glioma cel s can be obtained from a phage display library by simple procedures.     

茉莉酸甲酯诱导葡萄悬浮细胞防卫反应机制的研究

为明确茉莉酸甲酯(methyl jasmonate,Me JA)诱导葡萄果实抗病反应的机制,以‘巨峰’葡萄悬浮细胞为试材,将其继代培养一次后分别在含10μmol·L-1 Me JA的B5培养基和含100 nmol·L-1隐地蛋白的B5培养基中(模拟病原菌接种)振荡培养15 d,每3 d测定1次细胞质量及抗病相关指标。结果显示:单一Me JA处理未激活葡萄悬浮细胞内的防卫反应;而单一隐地蛋白处理则可立即显著诱导葡萄悬浮细胞内源H2O2迸发,提升vv NPR1.1、PR1和PR2表达水平和植保素含量;经Me JA处理的葡萄悬浮细胞在添加隐地蛋白后,其细胞内出现较单一隐地蛋白处理更为显著的H2O2迸发、PR基因表达和植保素合成现象,说明经Me JA处理的悬浮细胞只在病原激发子胁迫时才表达出较强的系统抗性。因此,Me JA诱导的葡萄悬浮细胞抗病反应可归因于Priming(植物敏化过程或防御准备过程)机制。此外,经Me JA诱导的Priming反应对葡萄悬浮细胞生长无显著影响,暗示其未抑制葡萄细胞生长和胞内物质积累。     

In vivo screening and characterization of peptides specifically binding to vasculature endothelial cells of liver in mice with septic shock

AIM: To screen peptides specifically binding to vasculature of liver in mice with septic shock. METHODS: Peptide display libraries were injected into septic shock mice by tail vein injection. After circulating for 10 min, the phages from mouse livers were recovered, amplified and purified following a routine protocol, and then re-injected for the next round screening. After screening for 4 rounds in vivo, the phage libraries were injected into normal mice by tail vein injection and the phages were recovered by serum collection to subtract the peptides binding with endothelial cells of normal mice. Then the phage clones were sequenced and further analyzed by bioinformatics. The interested phage clones were reinfused to the mice with septic shock, and appraised by titering the phage distribution in vivo and immunohistochemical staining of the mouse liver. RESULTS: After 4 rounds of biopanning, the phage recovery rates increased step by step, indicating that the phage library was successfully enriched in the livers of mice with septic shock. Fifty percent (5/10) of the analyzed sequences were identical, and with a sequence of LTTWAPA. In vivo titering and immunohistochemical staining displayed that this sequence selectively targets liver of mice with septic shock. CONCLUSION: The peptide of LTTWAPA specifically binds to vascular endothelial cells of liver in septic shock mice, which may have important significance for the therapy of septic shock.     

Screening of lung cancer specific peptide ZS-9 from a phage display peptide library and NCI-H1299 cells

〓AIM: To obtain the polypeptides specifically bound to NCI-H1299 cell line from peptide libraries and to identify these polypeptides affinity and specific to lung cancer. METHODS: The lung cancer NCI-H1299 cell line was used as the antigen and MRC-5 was used as control for subtraction biopanning from a phage display peptide library. The positive and specific binding clones were identified by cell enzyme-linked immunosorbent assay (ELISA) and immunochemistry staining. Those DNA sequences of identified clones were sequenced, and the amino acid sequence was deduced and analyzed with bioinformatics. RESULTS: After 3 rounds of panning, 9 phage clones were identified by ELISA, one of them specially bound to the NCI-H1299 and A549. The DNA sequence result of ZS-9 was CAT AAT AAG CAT CTT CCG TCT ACG CAG CCT CTT GCG. Hence, the polypeptide sequence was HNKHLPSTQPLA deduced from its DNA sequence. The amino acid sequence was analyzed in NCBI and Swiss-Prot, the results showed that it has no similarity with the known proteins sequence in the database. All these results showed that we have discovered a novel lung cancer surface associated antigen ligand. CONCLUSION: A peptide which is specific binding to lung cancer cell line NCI-H1299 and A549 has been selected from phage display peptide libraries. Therefore, it provides a potential tool for early diagnosis of lung cancer or targeted drug delivery in chemotherapy.     

EGCG depresses migration and invasion of human glioma cell line by downregulating COX-2 and MMP-2 expression

AIM: To investigate the mechanism that epigallocatechin-3-gallate (EGCG) depresses the migration and invasion in human glioma cell line SWO-38 by downregulation of cyclocxygenase-2(COX-2) and matrix metalloproteinase-2(MMP-2). METHODS: The effect of EGCG on the apoptosis of SWO-38 cell line was examined by the method of MTT. The migration and invasion of the SWO-38 cells were determined by Transwell assay. The expression of COX-2 and MMP-2 was measured by Western blotting. Meanwhile, TNF-α was used to stimulate the expression of COX-2 for determining if the mechanism of COX-2 pathway is involved in the inhibitory effect of EGCG on the migration and invasion of the tumor cells. RESULTS: After treated with EGCG for 24 h, the migration and invasion abilities of SWO-38 cells were lower than that of the cells before treatment. The results of Western blotting revealed that the 24 h treatment of EGCG on SWO-38 cell line inhibited the expression levels of COX-2 and MMP-2, indicating that the degradation of the extracellular matrix in SWO-38 cells was related to the COX-2 signaling pathway. CONCLUSION: EGCG inhibits the migration and invasion of SWO-38 cell line. The correlation between COX-2 expression and enzymatic degradation in the extracellular matrix determines the abilities of migration and invasion of tumor cells.     

Screening of tissue factor targeting peptides alternately from phage display peptide library

AIM: To screen tissue factor (TF) targeting peptides by establishment of a new and effective phage display method to acquire the peptides specifically bound to the transmembrane receptor. METHODS: Five rounds of panning were alternately conducted by targeting TF and HT-29 cell line which showed the detectable TF expression (screen for targeting receptor and cell alternately with phage display, STRCA). The 30 phage clones were assessed by enzyme-linked immunosorbent assay (ELISA). DNA sequencing was performed for the phage clones. The affinity of synthetic peptides was verified with competitive inhibition ELISA. The repeated experiment was conducted to verify the reliability of the results. RESULTS: The phages were effectively enriched after 5 rounds of panning with the improvement of the recovery rate from (2.25×10^-4)% to (1.32×10^-2)%. In 30 individual phages, ELISA positive rate was 76.7%, and the repetition of A, B, C and D peptides showed 23.3% (7/30), 23.3% (7/30), 26.7% (8/30) and 10.0% (3/30),respectively. E peptide constructively consisted of A and B. The five synthetic peptides were verified by ELISA, and the IC₅₀ of each peptide showed 3.25 nmol/L, 6.72 mol/L, 3.24×10³ mol/L, 2.08×10² mol/L and 45.77 mol/L,respectively. The positive phages were selected again in the second experiment to compare the results of the first experiment and the repeatability was 33.3%. CONCLUSION: STRCA can select TF targeting peptides with high affinity, which has the potential to become a therapeutic screening of transmembrane receptor-binding peptides.     

Screening of binding proteins of HMGB1 promoter by phage display technique

AIM: To screen the binding proteins to HMGB1 promoter by phage display technique. METHODS: HMGB1 promoter was incubated with phage display library. Unbound phages were eluted and phages bound to HMGB1 promoter were amplified. Twenty individual clones were randomly selected and identified by enzyme-linked immunosorbent assay (ELISA). Positive clones were characterized by DNA sequencing and the sequences were subjected for computer analysis. RESULTS: Positive phages binding to HMGB1 promoter were enriched after 4 rounds of biopanning. Twenty phage clones were selected and eleven clones of which were identified to bind specifically to HMGB1 promoter. The sequences in full length were obtained and searched for homologous sequences from GenBank. Altogether eight coding sequences were obtained, six of which were known proteins including activator protein-1(AP-1) and two of which were uncharacterized ones. CONCLUSION: Several proteins were obtained that bind specifically with HMGB1 promoter. The results will be useful for further studying the expression and regulation mechanism of HMGB1.     

Advances in research of phage peptide library

Phage peptide libraries are collections of the specific length of short peptides, and they are based on phages as their carriers. They include mimotope libraries and pept ide antibody libraries. Phagedisplayed pept ide libraries have been used to isolate specific ligands for numerous protein targets, and they have been proven useful in defining antigen momitopes, rapid determination of binding energetics at protein-protein interfaces, designing of vaccine and tumor research aspects. This review summarized the research progression of phage peptide library.     

Change of nestin expression in differentiation of human glioma cells induced by CDA-2

AIM: To investigate the change and significance of nestin expression in differentiation of human glioma cell line SWO-38 induced by CDA-2 (uroacitide, a healthy human urine extract). METHODS: Cellular differentiation of SWO-38 cells induced by CDA-2 was determined by light microscopy. The change of nestin expression in SWO-38 cells induced by CDA-2 was detected by munofluorescence, RT-PCR and Western blotting. RESULTS: Light microscopic observation revealed that CDA-2 induced SWO-38 cells to differentiate into astrocytes with increased cytoplasm and cytoplasmic processes and decreased nucleus/cytoplasm ratio. Nestin was expressed in cytoplasm and stained like filament by immunofluorescence staining. Nestin expression was downregulated in differentiated SWO-38 cells induced by CDA-2. CONCLUSION: Nestin expression is downregulated in differentiation of SWO-38 cells induced by CDA-2, which verifies the relationship between nestin expression and cell differentiation. Nestin may be a new differentiation marker of glioma.     

Inhibitory effect of different target-side antisense oligonucleotides on bFGF expression in SWO-38 cells

AIM:Three different antisense oligonucleotides complementary to basic fibroblast growth factor (bFGF) mRNA were compared in inhibitory effect on gene targeted expression.METHODS:After transfecting bFGF antisense oligonucleotides (asODN) into SWO-38 cells by lipofectin, the proliferation of cells was identified by MTT method, apoptosis was examined by flow cytometric cell cycle analysis and the expression levers of bFGF were detected by Western-blotting.RESULTS:There were 49%, 33%, 51% inhibition of cell growth and 35%, 27%, 18% cell apoptosis after asODN1, asODN2 and asODN3 treatment.In addition, the decrease in bFGF protein was 63%, 42%, 11%, respectively.CONCLUSION:The data suggeste that asODN1 is a potent target to bFGF mRNA, which inhibits cell growth and induces apoptosis in SWO-38 cells.     

Investigation of amino acid sequence of specific ouabain conjugated peptides

AIM: The purpose of the study was to investigate the gene and amino acid sequence of specific ouabain conjugated peptides (OCP) in order to get an experimental bases to block or antagonist the actions between endogenous ouabain(EO) and sodium pump in hypertension. METHODS: Screening the phage displayed 12-peptide library by biopaning for OCP. The sequence of each selected peptide was determined and the sequence was analyzed through internet. The bioactivity was determined by erythrocyte [⁸⁶Rb] uptaking. RESULTS: Three kinds of peptides were screened out. Peptide A (12 peptide) was occupied in 66.7%(8/12), peptide B (8 peptide) 16.7% (2/12) and peptide C (12 peptide) 8.3% (1/12). There was only one case without insertron. The analysis of protein showed that there were no homogenous between peptide A, B, C and sodium pump. The amino acid sequence of specific OCP was Leu-Leu-Ala-Asp-Thr-Thr-His-His-Arg -Pro-Trp-Thr. CONCLUSIONS: Determination of the sequence of OCP supplies an important experimental foundation for ouabain research. The results also show that phage display peptide library is an effective, simple and efficient method to select specific steroid receptors.     

CDA-2 induces differentiation and inhibits proliferation of human SWO-38 glioma cells

AIM: To investigate the differentiation-inducing effect of cell differentiation agent-2 (CDA-2) in human SWO-38 glioma cell line in vitro.METHODS: The inhibitory effect of CDA-2 on cell proliferation was assessed by MTT assay and colony formation assay.Cell morphology was determinded by light microscopy observation,and the expression of GFAP (glial fibrillary acidic protein) was detected by immunohistochemistry and Western blotting.Western blotting was also applied to explore the expression of PPARγ and COX-2.RESULTS: The data showed that CDA-2 inhibited proliferation and induced differentiation of SWO-38 cells.The inhibition efficiency was time-dependent and dose-dependent .The IC₅₀ of CDA-2 was (2.33±0.37) g/L and (0.51±0.01) g/L,respectively when cells were treated for 72 h and 10 days.CDA-2 caused differentiation of human glioma cells as indicated by outgrowth of long processes and expression of astrocyte marker GFAP.Simultaneously,the expression of PPARγ increased after 3 h of CDA-2 treatment，while the expression of COX-2 decreased after 48 h of CDA-2 treatment.CONCLUSION: CDA-2 inhibits proliferation and induces differentiation of SWO-38 cells.These effects may be through increasing cellular GFAP,PPARγ level and decreasing COX-2 expression induced by CDA-2.     

Screening of phage antibodies against HBsAg from the constructed phage display library and sequencing of the positive clones

AIM:Screening of phage antibodies against HBsAg from the constructed phage display library and sequencing of the positive clones.METHODS:RNAs were extracted from blood lymphocytes of a HBsAg-immunized volunteer. Then cDNAs were synthesized by RT-PCR. To construct the phage antibody display library, cDNAs of Fd fragments and κ chains of the antibodies were amplified through PCR and were inserted into vector pComb3H. Three rounds panning against coated HBsAg showed the specific enrichment of phage antibody. Positive clones were selected and sequenced.RESULTS:A phage antibody display library was constructed after amplifing of Fd fragments and κ chains of IgG1. Three Fab antibodies from the library were selected and sequenced. The result of sequencing showed that two of the three antibodies had the same Fd fragment and all of the three had the same light chain. CONCLUSION:The phage antibody display library was constructed successfully and three specific antibodies (Fab fragments) have been obtained from it.     

Identification of tumor antigen epitopes by MHC-I/peptide binding prediction and immunological experiments

AIM:To establish the methods to identify immune epitopes of tumor antigens restricted by human leukocyte antigen (HLA) alleles frequent in the Chinese population for the isolation and identification of tumor-specific T cells and the cloning of tumor antigen-specific T-cell receptor gene. METHODS:Three cancer/testis antigens NY-ESO,MAGE-A1 and KK-LC-1 were selected as the model antigens to predict the MHC-I binding peptides by major histocompatibility complex (MHC)-peptide binding prediction software. The HLA alleles of healthy volunteers were determined by PCR. The predicted epitopes restricted by HLA-A*11:01 and HLA-B*46:01 of the volunteers were selected for the long peptide design. ELISPOT and flow cytometry were used to analyze the interferon-γ (IFN-γ) spots of the activated T-cells and the up-regulation of CD137 to verify the effectiveness of peptides for T-cell-specific stimulation. We selected long peptides with the best response to verify short peptides. RESULTS:Three cancer/testis antigens containing many strong binding epitopes restricted by 35 HLA alleles common in Chinese population were found. It was found that the predicted epitopes were not equally distributed in the protein sequences. KK-LC-1 antigen was selected for further study. The 2 long peptides stimulated T-cell activation:the release of IFN-γ and up-regulation of CD137 molecules by T cells of the volunteers with the matched HLA alleles. The short peptides worked better than the long peptides.CONCLUSION:Tumor antigen epitopes are quickly identified by MHC/peptide binding prediction and T-cell stimulation analysis. According to the reaction of the long peptide, the short peptide is synthesized to verify the precise epitope. This study provides a new way to determine tumor antigen epitopes quickly.     

Identification of HLA-A3 restricted cytotoxic T-lymphocyte epitopes from cancer-testis antigen MAGEC2

AIM:To predict and identify an HLA-A3 supertype-restricted cytotoxic T-lymphocyte (CTL) epitope derived from MAGEC2, which is utility in epitope design for the development of HLA-based vaccines and immunotherapeutics. METHODS:HLA-A3 epitopes from MAGEC2 protein were predicted by BIMAS, SYFPEITHI and IEDB. The binding affinity of the peptides to HLA-A*03 molecule was evaluated by T2A3 cell binding assay. ELISPOT assay was used to investigate the ability of the peptides inducing specific restricted CTLs to release interferon-γ (IFN-γ). The ability of the peptides to induce T-cell response was investigated by cytotoxicity assay in vitro. RESULTS:The candidate peptides P147, P167, P196, P229 and P251 showed moderate affinity toward HLA-A3 molecule. ELISPOT assay showed that P167, P196 and P251 were able to induce specific CTLs and higher levels of IFN-γ were released. The CTLs induced by P196 and P251 were able to lyse target cells. CONCLUSION:The peptides P196 and P251 have higher binding affinity with HLA-A3 and retain immunogenicity. They are excellent HLA-A3-restricted CTL epitopes from tumor antigen MAGEC2, which could serve as new candidates towards antitumor peptide vaccines.     

Identification of HLA-A2 restricted cytotoxic T lymphocyte epitopes from tumor antigen PIWIL2

AIM: To identify the human leucocyte antigen A2 (HLA-A2) restricted cytotoxic T lymphocyte (CTL) epitopes from tumor antigen PIWIL2. METHODS: RT-PCR and Western blot was used to determine the expression of PIWIL2 in cancer cell lines MCF-7, SW480 and HT-29. HLA-A2 epitopes from PIWIL2 protein were predicted by the software of BIMAS, RankPep, NetMHC, NetCTL1.2 and IEDB. The peptides were synthesized by standard solid-phase methods. The binding affinity of the peptides to HLA-A2 molecules was evaluated by T2 cells binding assay. ELISPOT assay was used to investigate the levels of IFN-γ. The cytotoxicity assay in vitro was also used to determine the ability of inducing T cell response by the peptides. RESULTS: The expression of PIWIL2 was observed in MCF-7, SW480 and HT-29. The candidate peptide P485, P493 and P965 showed moderate affinity toward HLA-A2 molecule. ELISPOT assay showed P485 and P965 induced CTLs of IFN-γ release form CTLs. The CTLs induced by P485 and P965 lysed the MCF-7 cells. CONCLUSION: The peptides P485 and P965 are excellent HLA-A2 restricted cytotoxic T lymphocyte epitopes from the tumor antigen PIWIL2, which could serve as new candidates towards antitumor peptide vaccines.     

In vivo immunogenic characteristics of cytotoxic T lymphocyte epitopes from pancreatic tumor antigen MUC4

AIM: Peptide vaccines have been conceived as promising therapies for tumor-inflicted patients due to their easy production and capability of inducing specific immune response required for defending the tumor. During our previous research, 4 HLA-A2-restricted peptides had been identified as immunogenic in vivo. In this study, we aimed to testify the in vivo immunogenicity of the 4 peptides. METHODS: BALB/c mice were vaccinated with HLA-A2 restricted peptides emulsified in incomplete Freund's adjuvant (IFA) subcutaneously in combination with the epitope at the adjacent location. After the 3rd peptide vaccination for 10 d, the peptide-specific immune response was evaluated by ELISPOT and ELISA. The ability to induce T cell response was investigated by using cytotoxicity assay in vivo and the presence of peptide-specific CD8⁺ T cells capable of recognizing the MHC-peptide was detected by flow cytometry. RESULTS: Among the 4 candidate HLA-A2 restricted peptides, the immune response elicited by P2004-1Y9V was superior to that of the other 3 peptides. The CTLs induced by P2004 and P2004-1Y9V lysed CAPAN-2 cells. P2004-1Y9V peptide-specific CTLs showed higher cytotoxicity against pancreatic tumor cell lines of CAPAN-2 than the native peptide-specific CTLs. Intracellular cytokine staining assay indicated the presence of P2004-1Y9V specific CD8⁺T cells in the P2004-1Y9V vaccinated mice. CONCLUSION: P2004-1Y9V is the most immunogenic peptide in vivo, and can be explored as potential tumor peptide vaccine in the future clinical study.     

Construction of phage random eight-peptide library

AIM: To construct random eight-peptide library for the study on atherosclerosis and restenosis. METHODS and RESULTS: Random oligodeoxynucleotides encoded eight peptides were synthesized and amplified by polymerise chain reaction( PCR).The product was cloned into phage surface display vector fUSE5 in Sfi I site and electroporated into competent MC1061. The library was identified through PCR, hybridization, DNA sequencing and affinity biopanning of streptavidin. Because the upstream primer is complementary to part vector clone site sequences and part exogenous gene sequences, and the other one complementary to pⅢ gene of vector, thus only clones inserted exogenous gene could be amplified easily. Additionally we used the probe oligodeoxynucleotide complementary to vec for clone site sequences to identify clones which were not inserted exogeneous genes. Furthermore, two hybridizing positive clones were sequenced. Their sequences are consistent with two oligodeoxynucleotide probe sequences. As a result, 2.1×10⁸ special clones were obtained. Affinity biopanning proved that the libraries could be amplified steadily.CONCLUSION: The eight-peptide library is reliable.     

Identification and molecular modification of cytotoxic T-lymphocyte epitopes from osteosarcoma high-expressing antigen PBF

AIM: To observe whether modified epitopes from osteosarcoma high-expressing antigen papillomavirus-binding factor (PBF) have HLA-A2 restricted antitumor ability, and to develop peptide-based immunotherapy for osteosarcoma. METHODS: RT-PCR and Western blot were used to determine the expression of PBF in the osteosarcoma cell lines U2OS and Saos-2. HLA-A2 epitopes from PBF protein were predicted by NetCTL 1.2, SYFPEITHI and IEDB. The modified peptides from PBF containing HLA-A2 binding anchor motifs were designed by replacing the anchor residues. The peptides were synthesized by standard solid-phase methods, and the binding affinity of the peptides to HLA-A*0201 was evaluated by T2A2 cell binding assay. ELISPOT assay was used to investigate the seretion of interferon-γ (IFN-γ) from the peptide-induced specific cytotoxic T-lymphocytes (CTLs). The ability of inducing T-cell response was analyzed by lactate dehydrogenase (LDH) release assay and carboxyfluorescein succinimidyl ester (CFSE) cytotoxicity assay in vitro. RESULTS: The expression of PBF was observed in the U2OS and Saos-2 cells. The candidate peptides P75-1Y2L, P412-1Y, P416-1Y2L9V, P107-1Y and P435-1Y2L showed moderate affinity toward HLA-A2 molecule. The modified peptides showed significantly higher affinity with HLA-A2 than the native peptide. ELISPOT assay showed that P412, P412-1Y, P416, P416-1Y2L9V and P435-1Y2L induced specific CTLs to secrete IFN-γ, and P412-1Y and P416-1Y2L9V induced more secretion of IFN-γ than the native peptide. The CTLs induced by P412, P412-1Y, P416 and P416-1Y2L9V lysed U2OS cells. P412-1Y and P416-1Y2L9V peptide-specific CTLs showed higher cytotoxicity against U2OS cells than the native peptide-specific CTLs. CONCLUSION: Compared with the native peptide, modified epitopes P412-1Y and P416-1Y2L9V have higher binding affinity with HLA-A*0201 and retain immunogenecity. In addition, the anti-tumor immunity effects of modified epitopes P412-1Y and P416-1Y2L9V are stronger than the native peptide. The peptides P412-1Y and P416-1Y2L9V is excellent HLA-A*0201 restricted CTL epitopes from tumor antigen PBF, which could serve as new candidates towards antitumor peptide vaccines.     

Construction of human immunoglobulin combinatorial library containing D-Dimer on the surface of phage

AIM: To construct a human recombinant immunoglobulin library containing D-Dimer by using phage surface display technology. METHODS: Human immunoglobulin heavy chain and light chain genes were amplified respectively by RT-PCR from different human lymphocytes using family specific primers and signal sequences of immunoglobulin as half-nested PCR primers. The heavy chain and light chain PCR products were cloned into phagemid vector pComb3H and the human immunoglobulin recombination library was generated with helper phage VCSM13. RESULTS: A human combinatorial antibody library consisting of 2.8×108 in dependent clones was generated with a titer of 4.1×1017PFU/L. The recombinant frequency of Fab genes was 46%. CONCLUSION: A human combinatorial antibody library was generated. It will be beneficial for selecting Fab antibodies of D dimer from the library.