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

OBJECTIVETo analyze the drift of the complementarity-determining region 3 (CDR3) of T cell receptor (TCR) beta chain variable region (TCR BV) in peripheral blood mononuclear cells (PBMCs) of patients with systemic lupus erythematosus.

METHODSImmunoscope spectratyping techniques was used to analyze the distribution of TCRbeta chain CDR3 in 5 normal blood donors and the dominant CDR3 in the PBMCs in 5 SLE patients. Sequence analysis of the CDR3 region in monoclonal or oligoclonal T cells was performed.

RESULTSThe spectratypes of TCR BV gene CDR3 region showed Gaussian distribution in the 5 normal blood donors. The 5 SLE patients, however, displayed anomalous proliferation and oligoclonal expansion of the T cells was observed in different TCR BV families with different CDR3 sequences.

CONCLUSIONNoticeable drift of TCRbeta chain CDR3 can be seen in active SLE, indicating possible association of selective expression of TCR with immune pathogenesis in SLE. Determination of specific TCR CDR3 sequence provides a new means for studying the pathogenesis and personalized treatment of SLE.

Amino Acid Sequence ; Base Sequence ; Cloning, Molecular ; Complementarity Determining Regions ; genetics ; Female ; Genes, T-Cell Receptor beta ; genetics ; Humans ; Leukocytes, Mononuclear ; cytology ; metabolism ; Lupus Erythematosus, Systemic ; genetics ; immunology ; Molecular Sequence Data ; Sequence Analysis, DNA

OBJECTIVESTo understand the role cellular immunology plays in the pathogenesis of chronic hepatitis B (CHB) through analysis of T cell receptor (TCR) beta chain variable region gene (BV) family dominant usage and beta chain complementarity determining region3 (CDR3) sequences of peripheral blood mononuclear cells of the patients.

METHODSTCR BV families were amplified by inverse polymerase chain reaction (RT-PCR), and the dominant usage of BV families and CDR3 repertoire were analyzed by immunoscope technology for 8 CHB patients during their acute exacerbations and for 4 healthy blood donors who served as controls. The clonality of the T cells suspected by immunoscope was further confirmed by CDR3 sequencing.

RESULTSThe TCR BV CDR3 repertoire of the 4 healthy blood donors showed a Gaussian distribution. In the 8 CHB patients, however, the clonal expansion of T cells showed different TCR BV families with each patient. The T cells of the clonal expansion shared different CDR3 sequences.

CONCLUSIONThe peripheral blood T cells of CHB patients during their acute exacerbation showed significantly a clonal expansion and their T cell clonal expansion may be stimulated by several HBV epitopes. These results indicate that cellular immunology is involved in the pathogenesis of the liver inflammation process of CHB.

Adult ; Amino Acid Sequence ; Base Sequence ; Cloning, Molecular ; Complementarity Determining Regions ; genetics ; Female ; Gene Rearrangement, beta-Chain T-Cell Antigen Receptor ; genetics ; Hepatitis B, Chronic ; genetics ; Humans ; Male ; Middle Aged ; Molecular Sequence Data ; Receptors, Antigen, T-Cell ; genetics

OBJECTIVETo analyze the sequence polymorphisms of the mitochondrial DNA hypervariable regions I (HVR I) and HVR II in the Deng population in Linzhi area of Tibet.

METHODSmtDNAs obtained from 119 unrelated individuals were amplified and directly sequenced.

RESULTSOne hundred and ten variable sites were identified, including nucleotide transitions, transversions, and insertions. In the HVR I region (nt16024-nt16365), 68 polymorphic sites and 119 haplotypes were observed, the genetic diversity was 0.9916. In the HVR II (nt73-nt340) region, 42 polymorphic sites and 113 haplotypes were observed, and the genetic diversity was 0.9907. The random match probability of the HVR I and HVR II regions were 0.0084 and 0.0093, respectively. When combining the HVR I and HVR II regions, 119 different haplotypes were found. The combined match probability of two unrelated persons having the same sequence was 0.0084.

CONCLUSIONThere are some unique polymorphic loci in the Deng population. There are different genetic structures between Chinese and other Asian populations in the mitochondrial DNA D-loop region. Sequence polymorphism of mitochondrial DNA HVR I and HVR II can be used as a genetic marker for forensic individual identification and genetic analysis.

Asian Continental Ancestry Group ; ethnology ; genetics ; Base Sequence ; China ; Complementarity Determining Regions ; DNA, Mitochondrial ; genetics ; Female ; Humans ; Male ; Molecular Sequence Data ; Pedigree ; Polymorphism, Genetic ; Tibet

OBJECTIVETo investigate the anti-human CEM lymphoma cell activities induced by TCR idiotypic DNA vaccine containing different antigen determinants in BALB/c mice.

METHODSThe specific rearranged gene fragment encoding TCRVbeta region of CEM cell line was obtained by RT-PCR technique. The PCR product was cloned into eukaryocytic expression vector pcDNA3, which was used as DNA vaccine and template for PCR amplifying different antigen determinant. Gene fragments encoding different antigen determinant were amplified and cloned into pcDNA3, separately. The experimental mice were immunized by intramuscular injection of the DNA vaccines. The specific anti-idiotype antibodies were detected by indirect immunofluorescence assay.

RESULTSTCRbetaV of CEM cell line contains five antigen determinants. Specific anti-idiotype antibody was detected in all of the six mice immunized with DNA vaccine containing all the five determinants (the highest titer was 1:480). Although the antibody could also be detected in four of the six mice immunized with DNA vaccine containing four of the five antigen determinants, the antibody titer was lower (the highest titer was 1:80). DNA vaccine containing two of the five determinants could not induce the specific antibody.

CONCLUSIONThe idiotypic DNA vaccine containing the whole TCRbetaV five antigen determinants could induce the specific anti-lymphoma idiotypic antibody in BALB/c mice.

Amino Acid Sequence ; Animals ; Antibodies, Anti-Idiotypic ; blood ; immunology ; Base Sequence ; Complementarity Determining Regions ; genetics ; immunology ; Epitopes ; genetics ; immunology ; HL-60 Cells ; Humans ; Lymphoma ; immunology ; Mice ; Mice, Inbred BALB C ; Molecular Sequence Data ; Receptors, Antigen, T-Cell, alpha-beta ; genetics ; immunology ; Sequence Analysis, DNA ; Tumor Cells, Cultured ; Vaccines, DNA ; genetics ; immunology

The cross reactivity of mimotopes of hepatitis C virus (HCV) hypervariable region 1 (HVR1) was investigated to obtain epitopes that have high cross reactivity. Five expression vectors encoding B cell mimotopes fused with Trx were constructed, and the mimotope proteins were purified. The cross reactivity of mimotope proteins with HCV positive sera was determined by ELISA. HCV pseudotype particles (HCVpp) were generated and applied to evaluate neutralization effects of the sera of BALB/c mice immuned with the mimotope proteins on infection of Huh7. 5 cells. Our data showed that the mimotope proteins (P1, P2, P5, P6, P8) could react to the HCV positive sera. The HCVpp infection inhibition of the sera of BALB/c mice immuned with P6 or P8 was detectable. These results suggest that the mimotopes may be valuable in the studies of anti-HCV infection and development of HCV vaccines.
Amino Acid Sequence ; Animals ; Complementarity Determining Regions ; immunology ; Cross Reactions ; Enzyme-Linked Immunosorbent Assay ; Epitopes ; Hepacivirus ; immunology ; Humans ; Mice ; Mice, Inbred BALB C ; Molecular Sequence Data

OBJECTIVETo investigate the effects of T cell receptor (TCR) BD2-BJ2 gene rearrangement on the complementary-determining region (CDR) 3 of TCR beta chain (TCR BV CDR3) in Jurkat cells.

METHODSTCR BV gene subfamilies were detected by RT-PCR in Jurkat cells during proliferation and after induction with non-specific T cell activators and SEA, respectively. To determine the clonality of TCR BV subfamilies and the lengths of CDR3, the PCR products were analyzed by TCR GeneScan technique, and the sequences of CDR3 were further analyzed by DNA sequencer.

RESULTSNo new TCR BV subfamilies were found in Jurkat cells, a monoclonal BV8(+)cell line, either during cell proliferation or after stimulation with different treatments, nor were any differences found in CDR3 size or sequences.

CONCLUSIONTCR BD2-BJ2 rearrangement in Jurkat cells may not play a role in modification of TCR BV CDR3 domains or the consequent antigen immunorecognition of BV CDR3, but the possibility of TCR modification can not be excluded.

Amino Acid Sequence ; Base Sequence ; Complementarity Determining Regions ; genetics ; Gene Rearrangement, beta-Chain T-Cell Antigen Receptor ; genetics ; Humans ; Immunologic Factors ; genetics ; Jurkat Cells ; Leukemia, T-Cell ; genetics ; immunology ; pathology ; Molecular Sequence Data ; Receptors, Antigen, T-Cell ; genetics ; T-Lymphocytes ; immunology ; metabolism

BACKGROUNDRecent studies have suggested that mature T cells can change their specificity through reexpression of recombination-activating genes (RAG) and RAG-mediated V(D)J recombination. This process is named receptor revision and has been observed in mature peripheral T cells from transgenic mice and human donors. However, whether thebreceptor revision in mature T cells is a random or orientated process remains poorly understood. Here we used the Jurkathuman T cell line, which represents a mature stage of T cell development, as a model to investigate the regulation of Tcell receptor (TCR) gene recombination.

METHODSTCR Dbeta-Jbeta signal joint T cell receptor excision DNA circles (sjTRECs) were determined by nested and seminested PCR. Double-strand DNA breaks at recombination signal sequences (RSSs) in the TCRVbeta chain locus were detected by ligation-mediated-PCR. Further analysis of the complementarity-determining region 3 (CDR3) size of the TCRVbeta chain was examined by the TCR GeneScan technique.

RESULTSRAG1, RAG2, and three crucial components of the nonhomologous DNA end-joining (NHEJ) pathway were readily detected in Jurkat. Characteristics of junctional diversity of Dbeta2-Jbeta2 signal joints and ds RSS breaks associated with the Dbeta2 5' and Dbeta 2 3' sites were detected in DNA from Jurkat cells. CDR3 size and the gene sequences of the TCRVbeta chain did not change during cell proliferation.

CONCLUSIONSRAG1 and RAG2 and ongoing TCR gene recombination are coexpressed in Jurkat cells, but the ongoing recombination process may not play a role in modification of the TCR repertoire.However, the results suggest that Jurkat could be used as a model for studying the regulation of RAGs and V(D)J recombination and as a "special" model of the coexistence of TCR gene rearrangements and "negative" receptor revision.

Antigens, Nuclear ; genetics ; Base Sequence ; Complementarity Determining Regions ; DNA Breaks ; DNA-Binding Proteins ; genetics ; Genes, RAG-1 ; Genes, T-Cell Receptor ; Humans ; Jurkat Cells ; Ku Autoantigen ; Leukemia, T-Cell ; genetics ; Molecular Sequence Data ; Nuclear Proteins ; genetics ; Recombination, Genetic

Since the publication of a high-throughput DNA sequencing technology based on PCR reaction was carried out in oil emulsions in 2005, high-throughput DNA sequencing platforms have been evolved to a robust technology in sequencing genomes and diverse DNA libraries. Antibody libraries with vast numbers of members currently serve as a foundation of discovering novel antibody drugs, and high-throughput DNA sequencing technology makes it possible to rapidly identify functional antibody variants with desired properties. Herein we present a review of current applications of high-throughput DNA sequencing technology in the analysis of antibody library diversity, sequencing of CDR3 regions, identification of potent antibodies based on sequence frequency, discovery of functional genes, and combination with various display technologies, so as to provide an alternative approach of discovery and development of antibody drugs.
Animals ; Antibody Diversity ; genetics ; Base Sequence ; Complementarity Determining Regions ; chemistry ; genetics ; DNA ; genetics ; DNA, Complementary ; genetics ; Drug Discovery ; methods ; Gene Library ; High-Throughput Nucleotide Sequencing ; methods ; Humans ; Polymerase Chain Reaction ; Sequence Analysis, DNA ; methods

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