Analysis of the T-cell receptor (TCR) repertoire of innate CD4+ T cells selected by major histocompatibility complex (MHC) class II-dependent thymocyte-thymocyte (T-T) interaction (T-T CD4+ T cells) is essential for predicting the characteristics of the antigens that bind to these T cells and for distinguishing T-T CD4+ T cells from other types of innate T cells. Using the TCRmini Tg mouse model, we show that the repertoire of TCRalpha chains in T-T CD4+ T cells was extremely diverse, in contrast to the repertoires previously described for other types of innate T cells. The TCRalpha chain sequences significantly overlapped between T-T CD4+ T cells and conventional CD4+ T cells in the thymus and spleen. However, the diversity of the TCRalpha repertoire of T-T CD4+ T cells seemed to be restricted compared with that of conventional CD4+ T cells. Interestingly, the frequency of the parental OT-II TCRalpha chains was significantly reduced in the process of T-T interaction. This diverse and shifted repertoire in T-T CD4+ T cells has biological relevance in terms of defense against diverse pathogens and a possible regulatory role during peripheral T-T interaction.
Amino Acid Sequence ; Animals ; Antigens, Surface/metabolism ; CD4-Positive T-Lymphocytes/cytology/*immunology/*metabolism ; Cell Communication ; Cell Differentiation/genetics/immunology ; Clonal Evolution ; Histocompatibility Antigens Class II/*immunology ; *Immunity, Innate ; Immunophenotyping ; Lymphocyte Count ; Mice ; Mice, Knockout ; Mice, Transgenic ; Peptide Fragments/chemistry ; Phenotype ; Receptors, Antigen, T-Cell/chemistry/*genetics/metabolism ; Receptors, Antigen, T-Cell, alpha-beta/chemistry/genetics ; Spleen/cytology ; Thymocytes/cytology/immunology/metabolism

The N-terminal fragment of prohormone brain natriuretic peptide (NT-proBNP) is a commonly used biomarker for the diagnosis of congestive heart failure, although its biological function is not well known. NT-proBNP exhibits heavy O-linked glycosylation, and it is quite difficult to develop an antibody that exhibits glycosylation-independent binding. We developed an antibody that binds to the recombinant NT-proBNP protein and its deglycosylated form with similar affinities in an enzyme immunoassay. The epitope was defined as Gly63-Lys68 based on mimetic peptide screening, site-directed mutagenesis and a competition assay with a peptide mimotope. The nearest O-glycosylation residues are Thr58 and Thr71; therefore, four amino acid residues intervene between the epitope and those residues in both directions. In conclusion, we report that an antibody reactive to Gly63-Lys68 of NT-proBNP exhibits O-glycosylation-independent binding.
Amino Acid Sequence ; Animals ; Antibodies/*immunology ; Antigen-Antibody Reactions ; Epitope Mapping ; Epitopes/chemistry/genetics/*immunology ; Glycosylation ; HEK293 Cells ; Heart Failure/immunology ; Humans ; Molecular Sequence Data ; Mutagenesis, Site-Directed ; Natriuretic Peptide, Brain/chemistry/genetics/*immunology ; Peptide Fragments/chemistry/genetics/*immunology ; Rabbits ; Recombinant Fusion Proteins/chemistry/genetics/immunology

BACKGROUNDWe previously reported that iodine-131((131)I)-labeled anti-pro-gastrin-releasing peptide (ProGRP(31-98)) monoclonal antibody D-D3 could selectively accumulate in the tumor sites of nude mice bearing small cell lung cancer (SCLC) xenografts. However, (131)I-D-D3 was cleared slowly from the body, and the best radioimmunoimaging time for SCLC was 72 - 96 hours after injection. The aims of this study were to radiolabel anti-ProGRP(31-98) D-D3 monoclonal antibody with technetium-99m ((99m)Tc) and to investigate the biodistribution of this antibody in healthy ICR mice.

METHODSD-D3 was labeled with (99m)Tc via the 2-mercaptoethanol reduction method. (99m)Tc-D-D3 was purified by the gel column separation method. The labeling efficiency and radiochemical purity were measured by thin-layer chromatography. The immunological activity of (99m)Tc-D-D3 was determined with cell conjugation assays. (99m)Tc-D-D3 was injected into healthy ICR mice via a tail vein, and all the healthy ICR mice were sacrificed by cervical dislocation at a designated time. Then, the blood and major organs were removed and weighed, and counted in a gamma scintillation counter to determine the percentage of the injected dose per gram (%ID/g).

RESULTSThe labeling rate and the radiochemical purity of (99m)Tc-D-D3 were (73.87 ± 2.89)% and (94.13 ± 4.49)%, respectively. The immunobinding rates of (99m)Tc-D-D3 to the human small cell lung cancer NCI-H446 cell line and lung adenocarcinoma A549 cell line were (81.2 ± 2.37)% and (24.3 ± 1.46)%, respectively. The distribution data of normal ICR mice demonstrated that (99m)Tc-D-D3 was mainly distributed in the liver, kidney and lung, and less in the brain tissue and muscle.

CONCLUSIONS(99m)Tc-D-D3 antibody not only had high radiochemical purity, but also had good stability both in vitro and in vivo, and maintained good immunological activity. (99m)Tc-D-D3 was metabolized mainly in the kidney and liver, and the blood radioactivity decreased rapidly. Thus, (99m)Tc-D-D3 is conducive to the radioimmunoimaging of SCLC.

Animals ; Antibodies, Monoclonal ; chemistry ; immunology ; metabolism ; Female ; Male ; Mice ; Mice, Inbred ICR ; Peptide Fragments ; immunology ; Recombinant Proteins ; immunology ; Technetium ; chemistry

Calreticulin (CRT) is a multifunctional molecule in both intracellular and extracellular environment. We have previously found that a recombinant CRT fragment (rCRT/39-272) could modulate T cell-mediated immunity in mice via activation and expansion of CD1d(hi)CD5⁺ B cells as well as induction of CRT-specific regulatory antibodies. Antibody secreting cells (ASCs) are terminally differentiated B cells responsible for producing antibodies to participate in positive immune response as well as immune regulation. In this study, we demonstrate that rCRT/39-272 differentiates murine CD1d(hi)CD5⁺ B cells into ASCs marked by increased expression of plasma cell-associated transcription factors and production of polyreactive antibodies against DNA and CRT in vitro. Intraperitoneal administration of rCRT/39-272 augmented differentiation of CD1d(hi)CD5⁺ B cells into ASCs in naïve mice or mice with experimental autoimmune encephalomyelitis. Thus, we propose that ASC differentiation and subsequent antibody production of CD1d(hi)CD5⁺ B cells are key steps in CRT-mediated immunoregulation on inflammatory T cell responses.
Animals ; Antigens, CD1d ; metabolism ; Autoantibodies ; biosynthesis ; B-Lymphocytes ; cytology ; drug effects ; immunology ; metabolism ; CD5 Antigens ; metabolism ; Calreticulin ; chemistry ; Cell Differentiation ; drug effects ; Encephalomyelitis, Autoimmune, Experimental ; immunology ; Humans ; Mice ; Peptide Fragments ; chemistry ; pharmacology ; Solubility

As the dominant antigens, early secreted antigenic target 6 (ESAT6, E6) and culture filtrate protein 10 (CFP10, C10) had once been the focus of tuberculosis (TB) vaccine due to their capability of inducing strong cell immune response in the host. They are also endowed with promising future of prevention against and diagnosis of TB. In this review, we systematically introduce recent research progress of E6 and C10, especially in structure-function, biological characteristics, protein expression and secretion, host immunity and vaccine development, and the prospects of their application are also discussed.
Antigens, Bacterial ; chemistry ; genetics ; immunology ; Bacterial Proteins ; chemistry ; genetics ; immunology ; Humans ; Immunodominant Epitopes ; immunology ; Molecular Biology ; Peptide Fragments ; chemistry ; genetics ; immunology ; Tuberculosis Vaccines ; genetics ; immunology ; Vaccines, DNA ; immunology

Prokaryotic expression plasmids carrying N-terminal(1-163aa) and C-terminal(141-306aa) gene of HCoV-NL63 nucleocapsid protein were constructed with pET-30a(+) vector. Consequently, we prepared two purified proteins, Np and Cp, respectively, and established a Western blotting-based line assay (WBLA) for detection of antibodies against HCoV-NL63 using three purified proteins: Np , Cp and Nf, a full-length HCoV-NL63 nucleocapsid protein as previously reported. We detected anti-HCoV-NL63 antibodies among 50 sera samples collected from adult for health-examination by WBLA. The results showed that: 25 (50%), 27 (54%), 36 (72%) of 50 sera were indentified as anti-HCoV-NL63 antibody positive when the antigen was from Nf, Np and Cp, respectively. Among these sera with positive anti-HCoV-NL63 antibody,Cp showed highest antibody positive rate in WBLA,and consistent rates of detection were 64% between Nf and Np, 54% between Nf and Cp, 54% between Np and Cp. Our study provides the foundation for development of HCoV-NL63 serological detection reagents and an experimental tool for immunological research of HCoV-NL63 infection.
Adult ; Antibodies, Viral ; blood ; Blotting, Western ; Coronavirus ; chemistry ; immunology ; Humans ; Nucleocapsid Proteins ; genetics ; immunology ; isolation & purification ; Peptide Fragments ; genetics ; isolation & purification ; Recombinant Proteins ; biosynthesis ; immunology ; isolation & purification ; Serologic Tests

OBJECTIVETo establish an effective method for purification of Helicobacter pylori UreB fragment and conduct functional analysis of the purified protein.

METHODSThe protein fragment expression was induced by IPTG and the expressed protein was purified through affinity chromatography and ion-exchange chromatography. The purity of the fragment was determined by high-performance liquid chromatography (HPLC), and the specific biological activity of the purified fragment was assayed by urease activity inhibition test.

RESULTSThe protein fragment was highly expressed in E. coli with a purity over 91%. The protein fragment showed highly specific biological activity and the specific antibody induced by this fragment in rabbits could inhibit the activity of urease in a dose-dependent manner.

CONCLUSIONThe UreB fragment with high purity and biological activity can be applied for further studies.

Amino Acid Sequence ; Animals ; Antibody Specificity ; Bacterial Proteins ; chemistry ; Bacterial Vaccines ; biosynthesis ; chemistry ; immunology ; isolation & purification ; Chromatography, High Pressure Liquid ; Electrophoresis ; Escherichia coli ; genetics ; Helicobacter pylori ; genetics ; immunology ; Molecular Sequence Data ; Peptide Fragments ; biosynthesis ; chemistry ; immunology ; isolation & purification ; Rabbits ; Urease ; antagonists & inhibitors

Cytokeratin 18 (CK18) protein was identified as an airway epithelial cell autoantigen associated with nonallergic asthma. Cleavage of CK18 protein by caspase-3 is a marker of early apoptosis in epithelial cells. It has been shown that the expression of active caspase-3 was increased in bronchial epithelial cells of asthmatic patients, when compared with healthy controls. To investigate the antigen-binding characteristics of IgG autoantibodies to CK18 protein in nonallergic asthma, the bindings of IgG autoantibodies to the fragments of CK18 protein cleaved by caspase-3 were analyzed by Western blot using serum samples from three patients with nonallergic asthma. Recombinant human CK18 protein was treated by caspase-3 and cleaved into N-terminal fragment (1-397 amino acids) and C-terminal fragment (398-430 amino acids). The binding capacity of IgG autoantibodies to N-terminal fragment of CK18 was maintained in one patient and reduced in other two patients. IgG autoantibodies from all three patients did not bind to C-terminal fragment of CK 18. In conclusion, IgG autoantibodies to CK18 protein from patients with nonallergic asthma seems to preferentially bind to the whole molecule of CK18 protein and their antigen-binding characteristics were heterogeneous among the patients with nonallergic asthma.
Protein Binding ; Peptide Fragments/immunology/metabolism ; Male ; Keratins/chemistry/*immunology/metabolism ; Immunoglobulin G/blood/*immunology/metabolism ; Hydrolysis ; Humans ; Female ; Epitopes/immunology/metabolism ; Caspases/metabolism ; Caspase 3 ; Blotting, Western ; Autoantibodies/blood/*immunology/metabolism ; Asthma/*blood/immunology ; Antigen-Antibody Reactions ; Antibodies, Monoclonal/immunology/metabolism ; Aged ; Adult

Human cytomegalovirus (HCMV) infection is an ubiquitous herpesvirus disease in human populations. It is rarely pathogenic to healthy adults, yet it may cause severe outcome to organ transplant recipients, the immunocompromised individuals and pregnant women. Using DNA from HCMV AD169 strain as template, the UL32 gene encoding pp150 protein fragment and the UL57 gene encoding MDBP protein fragment were amplified by PCR technique. After the construction of cloning vector pMD18-T-UL32, pMD18-T-UL57, pMD18-T-UL32/UL57 and expression vector pET-11a-UL32/UL57, the recombinant fusion proteins pp150/MDBP were induced with IPTG in BL21 host strain. The results showed that the relative molecular weight of recombinant fusion proteins pp150/MDBP is about 27 kD, the product of fusion proteins takes 17.45% in the total proteins in host bacteria, the analytical result was positive to the fusion proteins pp150/MDBP via Western blot technique, while the purified recombinant fusion proteins have strong antigenicity detected by ELISA and protein chip compared with whole virus antigens from HCMV. It was demonstrated that when used for the detection of serum from the clinical patients it has the same detection rate compared with the whole virus antigen. It needs further research for application.
Cytomegalovirus ; DNA-Binding Proteins ; chemistry ; Electrophoresis, Polyacrylamide Gel ; Enzyme-Linked Immunosorbent Assay ; Gene Expression ; Genetic Vectors ; genetics ; Humans ; Peptide Fragments ; blood ; genetics ; immunology ; isolation & purification ; Phosphoproteins ; chemistry ; Plasmids ; genetics ; Polymerase Chain Reaction ; Recombinant Fusion Proteins ; blood ; genetics ; immunology ; isolation & purification ; Transcription Factors ; chemistry ; Viral Matrix Proteins ; chemistry

AIMTo establish methods and requirements for the quality control of recombinant human neu epitope peptide 12.

METHODSBiological activity of recombinant human neu epitope peptide 12 was evaluated in FVB/N transgenic mice (TgN MMTV neu 202 Mul, Jackson Lab., USA) administered with samples. The percentage of antibody-positive mice detected by ELISA was used in the biological activity evaluation. The peptide map was performed by peptic digestion. The antigen content was determined by SEC-HPLC.

RESULTS AND CONCLUSIONThe quality control methods, such as biological activity, peptide map, antigen content, and the requirements for the quality control of recombinant human neu epitope peptide 12 were established. The established methods and requirements were already used for the quality control of recombinant human neu epitope peptide 12 products.

Animals ; Antibodies, Monoclonal ; analysis ; Biotechnology ; methods ; Epitopes ; analysis ; chemistry ; immunology ; Female ; Genes, erbB-2 ; Humans ; Mice ; Mice, Inbred BALB C ; Mice, Transgenic ; Peptide Fragments ; chemistry ; immunology ; Peptide Mapping ; Quality Control ; Receptor, ErbB-2 ; analysis ; chemistry ; immunology ; Recombinant Fusion Proteins ; analysis ; chemistry ; immunology ; Technology, Pharmaceutical ; standards