• Biomolecules & Therapeutics
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• 제2권4호
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• pp.303-309
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• 1994

The analysis of membrane receptors for hormones and neurotransmitters has progressed considerably by pharmacological and biochemical means and more recently through the use of specific antibodies. Two kinds of antibodies could be produced, one is from synthetic peptides and the other from proteins such as purified receptor. Anti-peptide antibodies gave some advantages; epitope is evident and also receptor purification in quantity is not prerequisite. It can be also applied to the study of receptor structure-activity relationship. The purpose of the present study was 1) to produce and characterize a polyclonal antibody against a synthetic $\beta$2-adrenergic receptor peptide(Phe-Gly-Asn-Phe-Trp-Cys-Phe-Trp-Thr-Ser-Ile-Asp-Val-Leu) and 2) to determine the effects of this antibody on the $\beta$-adrenergic receptor ligand interaction. The peptide sequence contains an amino acid residue such as Asp-113 which was identified as one of important component for receptor-ligand interaction in site-directed mutagenesis studies. Production of antibody was performed by immunization of rabbits through popliteal lymph node with the peptide coupled with Keyhole Limpet Hemocyanin (KLH). The titer of antibody against this peptide was 1 : 1000. The anti-peptide antibody was able to detect a 67 kDa protein band in western blot corresponding to the molecular weight of the $\beta$-adrenergic receptor in partially purified receptor fraction derived from guinea pig lung. The antisera inhibited the specific binding of [$^3$H]dihydroalprenolol to $\beta$-adrenergic receptor in a concentration-dependent manner. The results from this study suggest that the peptide sequence selected in the present study is important for the receptor ligand interaction.

• Bulletin of the Korean Chemical Society
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• 제25권12호
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• pp.1845-1849
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• 2004

Apolipoprotein B-100 (Apo-B100) is a major protein component for low density lipoproteins (LDL). A number of mimetic peptides of Apo-B100 were screened from the phase-displayed random peptide library by utilizing monoclonal antibody (B9). Mimetic peptide for B9 epitope against apo B-100 was CRNVPPIFNDVYWIAF (pB1). From the BLAST search, the mimetic peptide pB1 had 40% homology with apo B-100. As a result of the structural determination of this mimotope using homo/hetero nuclear 2D-NMR techniques and NMR-based distance geometry (DG)/molecular dynamic (MD) computations, DG structure had low penalty value of 0.3-0.6 ${\AA}^2$ and the total RMSD was 0.5-1.5 ${\AA}. Moreover, pB1 structure included a weak $3_{10}$-helix from $Ile^7$,/TEX> to $Trp^{13}$.

• IMMUNE NETWORK
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• 제14권4호
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• pp.207-218
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• 2014

Chronic virus infection leads to the functional impairment of dendritic cells (DCs) as well as T cells, limiting the clinical usefulness of DC-based therapeutic vaccine against chronic virus infection. Meanwhile, B cells have been known to maintain the ability to differentiate plasma cells producing antibodies even during chronic virus infection. Previously, ${\alpha}$-galactosylceramide (${\alpha}GC$) and cognate peptide-loaded B cells were comparable to DCs in priming peptide-specific $CD8^+$ T cells as antigen presenting cells (APCs). Here, we investigated whether B cells activated by ${\alpha}GC$ can improve virus-specific T cell immune responses instead of DCs during chronic virus infection. We found that comparable to B cells isolated from naïve mice, chronic B cells isolated from chronically infected mice with lymphocytic choriomeningitis virus (LCMV) clone 13 (CL13) after ${\alpha}GC$-loading could activate CD1d-restricted invariant natural killer T (iNKT) cells to produce effector cytokines and upregulate co-stimulatory molecules in both naïve and chronically infected mice. Similar to naïve B cells, chronic B cells efficiently primed LCMV glycoprotein (GP) 33-41-specific P14 $CD8^+$ T cells in vivo, thereby allowing the proliferation of functional $CD8^+$ T cells. Importantly, when ${\alpha}GC$ and cognate epitope-loaded chronic B cells were transferred into chronically infected mice, the mice showed a significant increase in the population of epitope-specific $CD8^+$ T cells and the accelerated control of viremia. Therefore, our studies demonstrate that reciprocal activation between ${\alpha}GC$-loaded chronic B cells and iNKT cells can strengthen virus-specific T cell immune responses, providing an effective regimen of autologous B cell-based therapeutic vaccine to treat chronic virus infection.

• Asian Pacific Journal of Cancer Prevention
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• 제17권sup3호
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• pp.293-297
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• 2016

TRAIL, tumor necrosis factor (TNF)-related apoptosis-inducing ligand belongs to one of important cytokine superfamilIES, tumor necrosis factor ($TNF{\alpha}$). TRAIL-2 receptor agonists activate several cell signaling pathways in cells in different manners and could lead to apoptosis or necrosis. Agonistic egg yolk antibodies like IgY which have been developed in a selective manner could activate TRAIL death receptors such as TRAIL-2 (DR5) and thus apoptosis signaling. We here investigated induction of apoptosis in human breast cancer cells (MCF7 cell line) by an IgY produced against an 21 aminoacid epitope of the human TRAIL-2 receptor. As the first step a small peptide of 21 aminoacids choosen from the extracellular domain of DR5 protein was produced with a peptide synthesizer. After control assays and confirmation of the correct amino acid sequence, it was injected to hens immunized to achieve high affinity IgYs. At the next step, the produced IgYs were extracted and examined for specificity against DR5 protein by ELISA assay. Subsequently, the anticancer effect of such IgYs was determined by MTT assay in the MCF7 human breast cancer cell line. The produced peptides successfully immunized hens and the produced antibodies which accumulated in egg yolk specifically recognized the DR5 protein. IgYs exerted significant toxicity and killed MCF7 cells as shown by MTT assay.

• Journal of Veterinary Science
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• 제23권3호
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• pp.50.1-50.12
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• 2022

Background: There is an urgent need to find reliable and rapid bovine tuberculosis (bTB) diagnostics in response to the rising prevalence of bTB worldwide. Toll-like receptor 2 (TLR2) recognizes components of bTB and initiates antigen-presenting cells to mediate humoral immunity. Evaluating the affinity of antigens with TLR2 can form the basis of a new method for the diagnosis of bTB based on humoral immunity. Objectives: To develop a reliable and rapid strategy to improve diagnostic tools for bTB. Methods: In this study, we expressed and purified the sixteen bTB-specific recombinant proteins in Escherichia coli. The two antigenic proteins, MPT70 and MPT83, which were most valuable for serological diagnosis of bTB were screened. Molecular docking technology was used to analyze the affinity of MPT70, MPT83, dominant epitope peptide of MPT70 (M1), and dominant epitope peptide MPT83 (M2) with TLR2, combined with the detection results of enzyme-linked immunosorbent assay to evaluate the molecular docking effect. Results: The results showed that interaction surface Cα-atom root mean square deviation of proteins (M1, M2, MPT70, MPT83)-TLR2 protein are less than 2.5 A, showing a high affinity. It is verified by clinical serum samples that MPT70, MPT83, MPT70-MPT83 showed good diagnostic potential for the detection of anti-bTB IgG and M1, M2 can replace the whole protein as the detection antigen. Conclusions: Molecular docking to evaluate the affinity of bTB protein and TLR2 combined with ELISA provides new insights for the diagnosis of bTB.

• 약학회지
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• 제50권1호
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• pp.33-39
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• 2006

We have previously reported the minimum criteria that can be applied to evaluate efficacy and safety of a DNA vaccine with use of Streptococcus pneumoniae DNA vaccine (SPDNA). The SPDNA was formulated by inserting the DNA sequences that are codons specific for the carbohydrate epitope in the capsule of S. penumoniae by phage display peptide library. Administration of the SPDNA into mice induced both humoral and cell-mediated immunities. The induction was protective even in the absence of CD4+ T lymphocyte in mice. Profiles of cytokine and isotyping of antibody displayed tendency of the Th1. In continuation of these studies, we examined if the efficacy of the SPNDA was provoked by the peptide recognized by codons specific for the capsule. Results showed that the peptide vaccine formulae (SPP) induced protective antibody in mice as did the SPDNA. Involvement of the cell-mediated immunity was also determined. Possible side effects of autoimmune diseases such as myositis and C3a production and tumor-formation were undetectable in mice given 7 times of SPDNA vaccination during entire of 92 days. Even after the frequent immunization, immunogenicity of the SPDNA was observed as determined for antibody production, suggesting that there was no immunotolerance provoked. All together, these examining factors would be applied to measurement of a DNA vaccine safety regarding the immunopathological aspect.

• Journal of Pharmaceutical Investigation
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• 제38권3호
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• pp.191-197
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• 2008

The purpose of the present study was to examine the pharmacokinetics and lymphatic delivery of the oligopeptide, a model peptide of X antigen epitope peptides, after the intramuscular administration of the peptide-bearing liposomes in rats. $^{14}C$-labelled peptide was used as a tracer to analyze the peptide levels in plasma, bile, urine, tissue homogenates, and lymph nodes (superior cervical nodes, brachial nodes and superior mesenteric nodes). Model peptide rapidly disappeared from the plasma by 30 min (${\alpha}$ phase) after i.v. administration, which was followed by the late disappearance. The apparent plasma half-lives ($t_{1/2({\alpha}),app}$) of the peptide at the ${\alpha}$ phase when administered at a dose of 0.2-1.0 mg/kg were about 5 min. The maximum plasma concentration ($C_{max}$) was $1.52\;{\mu}g/mL$, after the i.m. administration of the peptide at a dose of 1.0 mg/kg. The bioavailability, which was calculated from the time zero to last quantitative time, of the i.m. administered peptide was over 60%. Of the various tissues tested, the peptide was mainly distributed in the kidney after the i.m. administration. The peptide levels in the kidney 3 hr after the i.m. administration were higher than those of maximum plasma concentration ($C_{max}$). The cumulative amounts of the peptide found in the urine 72 hr after the administration of 1.0 mg/kg were 2-folder higher than those in the bile, suggesting that the peptide is mostly excreted in the urine. Moreover, the concentrations of the peptide in the lymph nodes were as high as that of the plasma and the tissues. In conclusion, the peptide concentration in the lymph nodes was maintained by 24 hr after the i.m. administration of the peptide-bearing liposomes.

• Parasites, Hosts and Diseases
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• 제54권4호
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• pp.431-437
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• 2016

The study of antigenic epitopes from Toxoplasma gondii has not only enhanced our understanding of the structure and function of antigens, the reactions between antigens and antibodies, and many other aspects of immunology, but it also plays a significant role in the development of new diagnostic reagents and vaccines. In the present study, T. gondii GRA6 epitopes were identified using bioinformatics tools and a synthetic peptide technique. The potential B cell epitopes of GRA6 predicted by bioinformatics tools concentrated upon 3 regions of GRA6, 1-20 aa, 44-103 aa, and 172-221 aa. Ten shorter peptides from the 3 regions were synthesized and assessed by ELISA using pig sera from different time points after infection. Three of the 10 peptides (amino acids 44-63, 172-191, and 192-211) tested were recognized by all sera and determined to be immunodominant B-cell epitopes of GRA6. The results indicated that we precisely and accurately located the T. gondii GRA6 epitopes using pig sera collected at different time points after infection. The identified epitopes may be very useful for further studies of epitope-based vaccines and diagnostic reagents.

• Asian Pacific Journal of Cancer Prevention
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• 제14권7호
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• pp.4167-4175
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• 2013

A najor current challenge and constraint in cervical cancer research is the development of vaccines against human papilloma virus (HPV) epitopes. Although many studies are done on epitope identification on HPVs, no computational work has been carried out for high risk forms which are considered to cause cervical cancer. Of all the high risk HPVs, HPV 16, HPV 18 and HPV 45 are responsible for 94% of cervical cancers in women worldwide. In this work, we computationally predicted the promiscuous epitopes among the E6 proteins of high risk HPVs. We identified the conserved residues, HLA class I, HLA class II and B-cell epitopes along with their corresponding secondary structure conformations. We used extremely precise bioinformatics tools like ClustalW2, MAPPP, NetMHC, Epi,Jen, EpiTop 1.0, ABCpred, BCpred and PSIPred for achieving this task. Our study identified specific regions 'FAFR(K)DL' followed by 'KLPD(Q)LCTEL' fragments which proved to be promiscuous epitopes present in both human leukocyte antigen (HLA) class I, class II molecules and B cells as well. These fragments also follow every suitable character to be considered as promiscuous epitopes with supporting evidences of previously reported experimental results. Thus, we conclude that these regions should be considered as the important for design of specific therapeutic vaccines for cervical cancer.

• 융합정보논문지
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• 제10권6호
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• pp.209-216
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• 2020

단백질 항원에 존재하는 에피토프는 에피토프를 기반으로 한 백신 개발의 표적이 되고 있다. 인간의 주조 직적합 복합체 (MHC-1)에 결합하는 펩타이드를 확인할 수 있는 여러 서버들이 보고되고 있으나 인간의 MHC-I 분자의 수가 매우 많고 각 서버 검색 방법의 표준화 부재 등의 문제로 인해 펩타이드 예측에 적절한 서버를 선정하는 것이 쉽지 않다. 본 논문에서는 MHC-I 결합 펩타이드를 예측하는 서버 30 종을 비교하였으며, 다발성 골수종에 적용하기 위해 survivin 단백질로부터 사람의 HLA-A2 제한 펩타이드를 예측하였다. 본 연구의 결과는 MHC-I 결합 예측의 표준화된 방법을 제시하고 펩타이드 에피토프를 예측하는데 도움을 줄 것이다.