MUC1 mucin is a high molecular weight, type I transmembrane glycoprotein. High and aberrant expression of MUC1 is observed in various types of tumors, which make it an ideal target for tumor biotherapy as well as a biomarker for tumor diagnosis and prognosis. MUC1/Y is an isoform of MUC1 generated by alternative splicing. Specific expression of MUC1/Y in breast cancer as well as its involvement in tumor cell signal transduction have been reported. In order to purify peptides containing MUC1/Y-specific epitope in E. coli and prepare MUC1/Y-specific antibody, DNA fragment encoding the MUC1/Y-specific peptide was amplified by PCR using MUC1/Y full length cDNA as the template and cloned into fusion expression vector pGEX-2T, resulting pGEX-Y30. DNA sequencing was performed to confirm the correct amplification and orientation of the target sequence. Competent E. coli DH5alpha was transformed with pGEX-Y30 and the expression was induced for 4-5 hours in 0.2 mmol/L IPTG at 30 degrees C and 37 degrees C. Expressed proteins were released from the cells by ultrasonication or B-PER II reagent treatments. The fusion protein GST-Y30 were purified by affinity and anion exchange columns and identified by SDS-PAGE and Western-blotting. Polyclonal antibody was prepared by immunizing rabbits with the GST-Y30 protein for 4 times with intervals of 3 weeks and purified by GST column. Western blotting, ELISA and immunohistochemistry analysis were carried out using the purified antibody to confirm its MUC1/Y-binding capacity and specificity. The expressed fusion protein GST-Y30 is about 31 kD in size and represented about 20% of total cellular proteins. The majority of the GST-Y30 protein existed as soluble form when the induction was carried out at both 30 degrees C and 37 degrees C. After the two-step purification, the purity of GST-Y30 was about 94%. The titer of polyserum generated by GST-Y30 immunization was 1:320,000 by ELISA. The antiserum showed MUC1/Y specificity and can recognize MUC1/Y on MCF7 cell. The MUC1/Y-specific polyclonal antibody can be used for studying the role of MUC1/Y in carcinogenesis.
Animals ; Antibodies ; metabolism ; Antibody Specificity ; Cell Line, Tumor ; metabolism ; Epitopes ; chemistry ; genetics ; metabolism ; Escherichia coli ; genetics ; metabolism ; Humans ; Immunohistochemistry ; Models, Genetic ; Mucin-1 ; chemistry ; genetics ; metabolism ; Peptides ; chemistry ; genetics ; metabolism ; Rabbits

In recent years, the potential value of nonstructural protein (NSP) 2C was well documented for distinguishing foot-and-mouth disease virus (FMDV) in infected animals and vaccinated animals. In order to develop a more sensitive approach to detect natural infected FMDV while there is no interact with vaccinated FMDV, we incorporated a major epitope region of 2C with whole 3AB coding region within NSP and expressed in Escherichia coli. We got a 47.6 kD fusion protein named 2C'3AB. The product showed a specific reactivity with FMDV from serum of infected animal by using Western blotting analysis. This suggests that this protein could be applied to distinguish infected FMDV and vaccinated FMDV. We further compared 2C'3AB protein with 3ABC fusion protein, another available protein used for detecting infected FMDV, using indirect ELISA assay. The results showed that 2C'3AB-ELISA had higher sensitivity than that of 3ABC-ELISA for distinguishing infected FMDV and vaccinated FMDV of sera from epidemic region. Therefore, this recombinant protein 2C'3AB is a good candidate protein to develop more sensitive method to differentiate infected FMDV and vaccinated FMDV from vaccinated animals. This finding will increase our capability to check the infectious virus carrier and finally improve FMDV infection control.
Animals ; Antibody Specificity ; Carrier Proteins ; genetics ; immunology ; metabolism ; Epitopes ; immunology ; Escherichia coli ; genetics ; metabolism ; Foot-and-Mouth Disease Virus ; genetics ; Recombinant Fusion Proteins ; genetics ; immunology ; metabolism ; Viral Nonstructural Proteins ; genetics ; immunology ; metabolism

Untreated human immunodeficiency virus (HIV) infections usually lead to death from AIDS, although the rate of the disease progression varies widely among individuals. The cytotoxic T lymphocyte (CTL) response, which is restricted by highly polymorphic MHC class I alleles, plays a central role in controlling HIV replication. It is now recognized that the antiviral efficacy of CTLs at the single cell level is dependent on their antigen specificity and is important in determining the quality of host response to viruses so that the individual will remain asymptomatic. However, because of the extreme mutational plasticity of HIV, HIV-specific CTL responses are continuously and dynamically changing. In order to rationally design an effective vaccine, the questions as to what constitutes an effective antiviral CTL response and what characterizes a potent antigenic peptide to induce such responses are becoming highlighted as needing to be answered.
Animals ; Antigens, Viral ; immunology ; metabolism ; Epitopes, T-Lymphocyte ; Evolution, Molecular ; Genetic Variation ; HIV Infections ; immunology ; virology ; HIV-1 ; genetics ; pathogenicity ; physiology ; Host-Pathogen Interactions ; Humans ; Immunodominant Epitopes ; T-Lymphocytes, Cytotoxic ; immunology ; metabolism ; virology ; Virus Replication

Hepatitis B virus core (HBc) proteins have been used as carrier for foreign epitopes since the 1980s. They could self-assemble into icosahedral particles. Foreign epitopes could be inserted into HBc protein in various protein regions, including the N- or C-terminal and the major immunodominant region (MIR). The factors relevant in the design of HBc particles for vaccine purpose are summarized in this review.
Adjuvants, Immunologic ; pharmacology ; Drug Carriers ; Epitopes ; genetics ; immunology ; Genetic Vectors ; Hepatitis B Core Antigens ; genetics ; immunology ; Humans ; Recombinant Fusion Proteins ; genetics ; immunology ; metabolism ; Vaccines, Synthetic ; biosynthesis ; immunology

Rhesus macaque (Macaca mulatta) is the best model to study of human immunodeficiency virus (HIV) infection and to develop acquired immunodeficiency syndrome (AIDS) vaccine. The crystal structure of its major histocompatibility antigen complex (MHC) is helpful to understand the mechanism of HIV immune evasion. In this study, we cloned the light chain (beta2m) of MHC class I allele of rhesus macaques, Mamu-A*02, and inserted it into pET21a(+) vector. We transfected the recombinant plasmid pET21a(+)-Mamu-beta2m and pET21a(+)-Mamu-alpha into BL21(DE3). Mamu-A*02 and beta2m were expressed in the form of inclusion bodies in BL21 (DE3). We co-refolded the inclusion bodies of Mamu-alpha and Mamu-beta2m with SIV nonapeptide YY9 and obtained the correct refolded protein complex. Then we purified the protein complex by the gel filtration and anion-exchange column. With hanging-drop method, we screened and optimized for the protein crystal. We managed to collect a X-ray diffraction with the resolution to 2.8 angstroms in the condition of 0.1 mol/L BIS-TRIS (pH5.5), 2.0 mol/L(NH4)2SO4. This crystal belong to perpendicular space group P2(1)2(1)2(1), with unit-cell parameters a = 128.99 angstroms, b = 129.01 angstroms, c = 129.03 angstroms. This data is available for the structure determination.
Animals ; Crystallography, X-Ray ; Epitopes ; immunology ; Escherichia coli ; genetics ; metabolism ; Histocompatibility Antigens Class I ; biosynthesis ; genetics ; Macaca mulatta ; Oligopeptides ; biosynthesis ; genetics ; Simian Immunodeficiency Virus ; immunology

Receptor-like kinase involves self-incompatibility, male sterility, stress responses, and disease resistance. To better understand the physiological function and biological characteristics of rice receptor-like kinase, we cloned five predicted epitope fragments of rice receptor-like kinase. The purified fusion protein was used as antigen to immunize rabbit to get specific polyclonal antibodies. Western blotting analysis shows that the five receptor-like kinases were expressed in rice leaves.
Animals ; Antibodies, Monoclonal ; biosynthesis ; immunology ; Cloning, Molecular ; Epitopes ; immunology ; Immunization ; Oryza ; enzymology ; genetics ; Plant Proteins ; genetics ; immunology ; Plantibodies ; immunology ; metabolism ; Protein Kinases ; genetics ; immunology ; Rabbits

OBJECTIVETo construct the expression vectors of GPC-3 CTL epitope.

METHODSThe HBsAg gene with three different EYILSLEEL (EYI) sites was named EYI1, and another with one EYI replacing CTL epitope FLG or SIL of pcHBsAg were named EYI2 and EYI3, respectively. All the three DNAs were amplified by SEOing PCR from pcHBsAg plasmid and linked into pBSSK+ vector to construct Pbssk/EYI1, pBSSK/EYI2, and pBSSK/EYI3. The three plasmid were identified by PCR, double digestion and sequencing, and the fragments with EYI1-3 were obtained by double digestion and then inserted into pcDNA3.1+ vector.

RESULTS AND CONCLUSIONPCR, enzyme digestion and sequence analysis confirmed successful construction of the eukaryotic expression vectors pcDNA-EYI1/HBsAg, pcDNA-EYI2/HBsAg, pcDNA-EYI3/HBsAg, which facilitate further studies of the GPC3-HBsAg multiple peptides vaccine for HBV infection.

Amino Acid Sequence ; Epitopes ; chemistry ; genetics ; immunology ; Genetic Vectors ; genetics ; Glypicans ; genetics ; immunology ; metabolism ; Hepatitis B Surface Antigens ; genetics ; immunology ; Plasmids ; genetics ; metabolism ; Polymerase Chain Reaction ; T-Lymphocytes, Cytotoxic ; immunology ; Vaccines, Subunit ; chemistry ; genetics ; immunology

Flow cytometry was used to identify and characterize monoclonal antibodies (mAbs) that react with rabbit leukocyte differentiation molecules (LDM). Screening sets of mAbs, developed against LDM in other species, for reactivity with rabbit LDM yielded 11 mAbs that recognize conserved epitopes on rabbit LDM orthologues and multiple mAbs that recognize epitopes expressed on the major histocompatibility class I or class II molecules. Screening of mAbs submitted to the Animal Homologues Section of the Eighth Human Leukocyte Differentiation Workshop yielded 7 additional mAbs. Screening of mAbs generated from mice immunized with leukocytes from rabbit thymus or spleen or concanavalin A activated peripheral blood and/or spleen lymphocytes has yielded 42 mAbs that recognize species restricted epitopes expressed on one or more lineages of leukocytes. Screening of the anti-rabbit mAbs against leukocytes from other species yielded one additional mAb. The studies show that screening of existing sets of mAbs for reactivity with rabbit LDM will not be productive and that a direct approach will be needed to develop mAbs for research in rabbits. The flow cytometric approach we developed to screen for mAbs of interest offers a way for individual laboratories to identify and characterize mAbs to LDM in rabbits and other species. A web-based program we developed provides a source of information that will facilitate analysis. It contains a searchable data base on known CD molecules and a data base on mAbs, known to react with LDM in one or more species of artiodactyla, equidae, carnivora, and or lagomorpha.
Animals ; Antibodies, Monoclonal/*immunology ; Antigens, Differentiation/*metabolism ; B-Lymphocytes/cytology/metabolism ; Basophils/cytology/metabolism ; Epitopes/genetics/metabolism ; *Flow Cytometry ; Gene Expression Regulation ; Granulocytes/cytology/metabolism ; Leukocytes/immunology/*metabolism ; Mice ; Monocytes/cytology/metabolism ; Rabbits ; T-Lymphocytes/cytology/metabolism

To produce many epitopes of peptide mut, different tandem repeats containing HER-2 mimetic peptide mut were constructed and expressed. The oligonucleotide coding mut sequence was inserted into pET28a(+) to construct the prokaryotic recombinant plasmid pET28a-mutl. The mut coding sequence was repeatedly inserted into the vector in tandem to obtain a series of plasmids pET28a-mut2, pET28a-mut4, pET28a-mut8 and pET28a-mut16. The plasmids were transformed into E. coli. BL21 and induced by IPTG. The recombinant proteins were expressed in E. coli. The binding analysis verified that the interaction between the tandem peptides mut and Herceptin was increased. In conclusion, the tandem peptides consisted of many antigen epitopes, and laid the foundation for the further study of HER-2 peptide vaccine for biotherapy of cancer with HER-2 overexpression.
Breast Neoplasms ; metabolism ; Cancer Vaccines ; biosynthesis ; immunology ; Epitopes ; Escherichia coli ; genetics ; metabolism ; Female ; Gene Expression ; Humans ; Peptides ; genetics ; immunology ; metabolism ; Receptor, ErbB-2 ; biosynthesis ; chemistry ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; immunology

Binding sites of five monoclonal antibodies were obtained by reinforceable method of overlapping recombinant prion protein and synthetic peptide. Overlapping peptides of PrP core were expressed in Escherichia coli by insertion of serial PCR amplicons of ovine PrP gene fragments into pET32a. The expressed fusion peptides were then tested for the binding activity to PrP monoclonal antibodies in Western blotting. The binding sites of 5 monoclonal antibodies of ovine PrP were located respectively as follows: 2H3 in 199 aa-213 aa, 4C6, 5F11 and 7F11 in 139 aa-168 aa and 7F1 in 214 aa-227 aa. There oligo peptides were synthesized and used in ELISA test for more accurate localization of the binding sites. The binding sites of 4C6, 5F11 and 7F11 were further confirmed to be in 149 aa-158 aa. This conclusion may contribute to the research for pathogenesis and diagnostic method of scrapie and bovine transmissible spongiform encephalopathy.
Animals ; Antibodies, Monoclonal ; immunology ; metabolism ; Binding Sites, Antibody ; immunology ; Epitopes ; immunology ; Escherichia coli ; genetics ; metabolism ; Prion Diseases ; diagnosis ; Prions ; genetics ; immunology ; metabolism ; Recombinant Fusion Proteins ; genetics ; immunology ; metabolism ; Scrapie ; diagnosis ; Sheep