The characterization of the human T-cell receptor (TCR) repertoire has made remarkable progress, with most of the work focusing on the TCRβ chains. Here, we analyzed the diversity and complexity of both the TCRα and TCRβ repertoires of three healthy donors. We found that the diversity of the TCRα repertoire is higher than that of the TCRβ repertoire, whereas the usages of the V and J genes tended to be preferential with similar TRAV and TRAJ patterns in all three donors. The V-J pairings, like the V and J gene usages, were slightly preferential. We also found that the TRDV1 gene rearranges with the majority of TRAJ genes, suggesting that TRDV1 is a shared TRAV/DV gene (TRAV42/DV1). Moreover, we uncovered the presence of tandem TRBD (TRB D gene) usage in ~2% of the productive human TCRβ CDR3 sequences.
Complementarity Determining Regions ; genetics ; DNA Primers ; chemistry ; genetics ; Female ; Gene Rearrangement, beta-Chain T-Cell Antigen Receptor ; genetics ; Gene Rearrangement, delta-Chain T-Cell Antigen Receptor ; genetics ; Genes, T-Cell Receptor beta ; genetics ; Genetic Variation ; High-Throughput Nucleotide Sequencing ; Humans ; Immunoglobulin Joining Region ; genetics ; Immunoglobulin Variable Region ; genetics ; Male ; Receptors, Antigen, T-Cell, alpha-beta ; genetics

OBJECTIVETo develop a recombinant single domain antibody against hTERT, human telomerase catalytic subunit.

METHODSA previously prepared His-tagged hTERT fusion protein was used as the antigen, and the variable regions in heavy chain (VH) of immunized mice were RT-PCR amplified and cloned into the pCANTAB 5E, a phagemid vector. By transfection, the display library of mouse VH was developed. The candidate clones were selected by affinity panning, and soluble VH were obtained after expression in E. coli, HB2151. The resultant single VH antibodies were characterized on their binding potentials by western blotting.

RESULTSAn about 350 bp VH fragment was amplified from spleen cells of mice immunized by His-tagged hTERT and expressed by phage displayed as VH library. The size of the library was 8 x 10(4). After three rounds of affinity panning, 4 independent clones were chosen and consequently expressed as soluble single domain antibodies (Mr = 16 000). In Western blot analysis, the single domain antibody from 2 of 4 clones proved to react with the His-tagged hTERT fusion protein (Mr = 167 000) without dependence of His-tags and also detect the native hTERT (Mr = 127 000) extracted from the human HeLa cancer cell line. DNA sequencing showed both of the single domain antibodies were encoded by the heavy chain variable region of the mouse.

CONCLUSIONSThe single domain antibodies developed were hTERT recognizable and hTERT specific, thus providing a basis for application of recombinant single domain antibody in inhibition of telomerase activity and anticancer therapy.

Amino Acid Sequence ; Animals ; Antibodies, Monoclonal ; genetics ; immunology ; Base Sequence ; Cloning, Molecular ; Complementarity Determining Regions ; genetics ; DNA-Binding Proteins ; HeLa Cells ; Humans ; Immunoglobulin Heavy Chains ; genetics ; Immunoglobulin Variable Region ; genetics ; Mice ; Molecular Sequence Data ; Sequence Analysis, DNA ; Telomerase ; immunology

This study was purpused to analyze the characteristics of T cell receptor repertoire in target organs of murine graft-versus-host after haploidentical bone marrow transplantation (hiBMT) and the molecular characteristics of complementarity determining region3 (CDR3) repertoires of monoclonal T cell in liver, skin and ileum in murine after hiBMT. Murine haploidentical BMT model was established, CDR3-size spectratyping was used to study TCRBV repertoires in recipient liver, skin, ileum, spleen and a group of CDR3 molecules was obtained from GVHD-target tissues. The results showed that GVHD occurred as early as days 14 after transplantation and was proven by histology in liver, skin and ileum. A number of new monoclonal and oligoclonal T cells emerged in GVHD-target tissue. 45 CDR3 molecules had six C'-terminal motifs, which obtained from liver, skin, ileum in different times after hiBMT. It is concluded that target organs of murine graft-versus-host disease after hiBMT emerged a number of clonal or oligoclonal T cells, part of this T cell clones commonly uses some conserved CDR3 motifs and may recognize similar antigen.
Amino Acid Sequence ; Animals ; Bone Marrow Transplantation ; Complementarity Determining Regions ; genetics ; Graft vs Host Disease ; genetics ; immunology ; Immunoglobulin Variable Region ; genetics ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Molecular Sequence Data ; Receptors, Antigen, T-Cell ; genetics

Single chain antibody fragment (scFv) is a small molecule composed of a variable region of heavy chain (VH) and a variable region of light chain (VL) of an antibody, and these two chains are connected by a flexible short peptide. scFv is the smallest functional fragment with complete antigen-binding activity, which contains both the antibody-recognizing site and the antigen-binding site. Compared with other antibodies, scFv has the advantages of small molecular weight, strong penetration, low immunogenicity, and easy expression. Currently, the most commonly used display systems for scFv mainly include the phage display system, ribosome display system, mRNA display system, yeast cell surface display system and mammalian cell display system. In recent years, with the development of scFv in the field of medicine, biology, and food safety, they have also attracted much attention in the sectors of biosynthesis and applied research. This review summarizes the advances of scFv display systems in recent years in order to facilitate scFv screening and application.
Animals ; Immunoglobulin Variable Region/genetics* ; Immunoglobulin Fragments/metabolism* ; Single-Chain Antibodies/metabolism* ; Peptide Library ; Mammals/genetics*

OBJECTIVETo determine the expression of immunoglobulins in HT-29 cells (an established colon cancer cell line, and explore their effect on the biological activities of the cancer cells.)

METHODSThe transcripts of variable regions of immunoglobulin heavy chains in HT-29 cells were detected by RT-PCR. Antisense CDR3 (specific to HT-29)-pIRES 1 neo vector was constructed, then transfected into HT-29 cells by electroporation. Programmed cell death and growth inhibition of HT-29 cells were detected by FCM and MTT, respectively.

RESULTSThe transcripts of Ig heavy chain (V(H) CDR3 region) were expressed in HT-29 cells. Moreover, they showed a monoclonal characteristic after being sequenced. After transfection of the antisense vector of CDR3 (specific to HT-29)-pIRES 1 neo, expression level of Ig in HT-29 cells was significantly decreased, and growth inhibition (P < 0.05) and apoptosis (P < 0.01) were induced.

CONCLUSIONThese results suggest that tumor derived Ig could promote the survival and growth of tumor cells.

Apoptosis ; Cell Proliferation ; Complementarity Determining Regions ; biosynthesis ; genetics ; DNA, Antisense ; genetics ; Electroporation ; Genetic Vectors ; HT29 Cells ; HeLa Cells ; Humans ; Immunoglobulin G ; metabolism ; Immunoglobulin Heavy Chains ; biosynthesis ; genetics ; Immunoglobulin M ; metabolism ; Immunoglobulin Variable Region ; biosynthesis ; genetics ; Immunoglobulins ; metabolism ; RNA, Messenger ; biosynthesis ; genetics ; Recombinant Proteins ; genetics ; Transfection

To screen the specific anti-human intercellular adhesion molecule-1 (ICAM-1) single chain fragment variable (scFv) using phage display library technology and to identify its biological activity. P1 peptide was used as antigen, and the phage antibodies against human ICAM-1 antigen were panned by four binding-eluting-amplifying cycles using Tomlinson I+J phage display library. After four rounds of selective enrichment screening, the positive clones were determined by PCR, enzyme linked immunosorbent assay (ELISA)-based antigenic cross reaction and Dot blotting. Then the binding specificity and biological activity of purified scFv were identified by Western blotting, competitive ELISA and cell adhesion inhibition assay respectively. Furthermore, four positive clones were first panned through P1 peptide coated-ELISA assay, and then J-A1 was obtained and identified by PCR, ELISA-based antigenic cross reaction and Dot blotting, which could show a specific binding between P1 peptide and human ICAM-1 protein antigen. Subsequently, the purified scFv showed a satisfactory specificity and anti-adhesive activity in competitive ELISA and the cell adhesion inhibition assay. The specific anti-human ICAM-1 scFv was prepared successfully from Tomlinson I+J phage display library, which pave the way for further application of anti-human ICAM-1 scFv for inflammation diseases therapeutics.
Antibodies ; Enzyme-Linked Immunosorbent Assay ; Humans ; Immunoglobulin Variable Region ; Intercellular Adhesion Molecule-1 ; immunology ; Peptide Library ; Single-Chain Antibodies

OBJECTIVETo isolate single chain antibody fragments (scFv) against cell surface molecules by pathfinder selection from an anti-KG1a cell scFv phage library.

METHODSThe anti-KG1a scFv library was enriched by KGla cell panning for three rounds, or unenriched, then processed for pathfinder selection respectively using anti-CD34 monoclonal antibody as pathfinder molecule. ScFv phage clones were randomly picked and identified by binding KG1a cells using immunofluorescein and flow cytometry. The KG1a+ clones were further identified by KG1a, HL60, U937, and CEM cell lines and ELISA. Their antigenic molecules on cell surface were digested by chymopapain and analyzed by flow cytometry. DNAs from ten positive clones were sequenced. The scFv clones with different primary structure were used to analyze the molecular weight of their antigens by Western blot.

RESULTSOne hundred and two KG1a+ scFv phage clones were isolated from 144 enriched and 96 unenriched scFv phage library respectively, among which 47 bound KG1a, HL60, U937, and CEM cells, 55 bound KG1a cells exclusively. None of 28 KG1a+, HL60-, U937-, and CEM- scFv clones bound to the CD34 antigen, as confirmed by ELISA, although most of their antigens were sensitive to chymopapain digestion. DNA sequences from ten positive clones showed that they were from four different clones. They bound antigens with different molecular weight.

CONCLUSIONSOne hundred and two scFv phage clones specific for hematopoietic stem and progenitor cells have been isolated from an anti-KG1a cell scFv phage library. The pathfinder selection has showed advantages to improve the screening efficacy of scFv phage clones against antigens, which present at very low densities on the cell surface.

Antibodies, Anti-Idiotypic ; immunology ; Antibodies, Monoclonal ; genetics ; Antibody Specificity ; Bacteriophages ; genetics ; Cloning, Molecular ; Hematopoietic Stem Cells ; immunology ; Humans ; Immunoglobulin Fc Fragments ; biosynthesis ; genetics ; immunology ; Immunoglobulin Fragments ; genetics ; immunology ; Immunoglobulin Variable Region ; genetics ; immunology ; Peptide Library ; Single-Chain Antibodies

To prepare large naive phage antibody library, the host bacteria with high transformation efficiency is used in the Cre-LoxP recombination system. The variable regions of immunoglobulin light and heavy genes were amplified from lymphocytes collected from adult peripheral blood and newborn cord blood. The genes were spliced to form the single-chain variable fragments (scFv) by overlap PCR, cloned into pDAN5a vector and then transformed into XL2-blue MRF' with the Hte gene. Compared with XL1-blue strain, the size of the primary library was increased by 3.9 times. The primary library infected Cre recombinase-expressing bacteria, and the genes between phagemids created many new VH/VL combinations. The library was calculated to have a diversity of 1.7 x 10(11) and validated by the selection of antibodies against six different protein antigens. This library provides the basis for further selection of antibody-based drugs. It is the first time to report that XL2-blue MRF' can be used to improve the diversity of the library in the recombination system.
Adult ; Escherichia coli ; genetics ; immunology ; Genetic Vectors ; Humans ; Immunoglobulin Heavy Chains ; genetics ; Immunoglobulin Light Chains ; genetics ; Immunoglobulin Variable Region ; genetics ; Infant, Newborn ; Integrases ; metabolism ; Lymphocytes ; immunology ; Peptide Library ; Recombination, Genetic ; genetics ; Single-Chain Antibodies ; genetics ; metabolism ; Transformation, Genetic

The aim of this study is to construct a bifunctional fusion protein, which can conjugate both human red blood cells and antibodies against classical swine fever virus (CSFV). We respectively amplified 2E8ScFv and mE2 genes from different recombinant vectors, in which 2E8ScFv gene is the single chain Fv gene against H antigen of human red blood cells, whereas mE2 gene is the main antigen coding region gene of CSFV E2 protein. We used overlap extension PCR to obtain an artificial fusion gene segment 2E8mE2 containing genes of Both 2E8ScFv and mE2, then ligated into the expression vector pET-DsbA and expressed in Escherichia coli BL21(DE3) PlysS host cells, after induced with IPTG the target fusion protein was successfully expressed and identified in inclusion bodies by SDS-PAGE and Western blotting. We purified the fusion protein and renatured it from inclusion bodies to obtain a native state of well biological activity. The Erythrocyte agglutination test results indicated that the fusion protein can conjugate both human red blood cells and antibodies of CSFV.
Erythrocytes ; immunology ; Escherichia coli ; genetics ; metabolism ; Humans ; Immunoglobulin Fragments ; biosynthesis ; genetics ; immunology ; Immunoglobulin Variable Region ; biosynthesis ; genetics ; immunology ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Single-Chain Antibodies ; biosynthesis ; genetics ; Viral Envelope Proteins ; biosynthesis ; genetics

Activation-induced cytidine deaminase (AID) is required for the generation of antibody diversity through initiating both somatic hypermutation (SHM) and class switch recombination. A few research groups have successfully used the feature of AID for generating mutant libraries in directed evolution of target proteins in B cells in vitro. B cells, cultured in suspension, are not convenient for transfection and cloning. In this study, we established an AID-based mutant accumulation and sorting system in adherent human cells. Mouse AID gene was first transfected into the human non-small cell lung carcinoma H1299 cells, and a stable cell clone (H1299-AID) was selected. Afterwards, anti-hTNF-α scFv (ATscFv) was transfected into H1299-AID cells and ATscFv was displayed on the surface of H1299-AID cells. By 4-round amplification/flow cytometric sorting for cells with the highest affinities to hTNF-alpha, two ATscFv mutant gene clones were isolated. Compared with the wild type ATscFv, the two mutants were much more efficient in neutralizing cytotoxicity of hTNF-alpha. The results indicate that directed evolution by somatic hypermutation can be carried out in adherent non-B cells, which makes directed evolution in mammalian cells easier and more efficient.
Animals ; Antibody Affinity ; Cells, Cultured ; Cytidine Deaminase ; genetics ; metabolism ; HEK293 Cells ; Humans ; Immunoglobulin Variable Region ; genetics ; immunology ; Mice ; Mutation ; Single-Chain Antibodies ; chemistry ; genetics ; immunology ; Somatic Hypermutation, Immunoglobulin ; genetics ; immunology ; Tumor Necrosis Factor-alpha ; immunology