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1.
Article in English | WPRIM | ID: wpr-194442

ABSTRACT

Phage display technology provides a powerful tool to screen a library for a binding molecule via an enrichment process. It has been adopted as a critical technology in the development of therapeutic antibodies. However, a major drawback of phage display technology is that because the degree of the enrichment cannot be controlled during the bio-panning process, it frequently results in a limited number of clones. In this study, we applied next-generation sequencing (NGS) to screen clones from a library and determine whether a greater number of clones can be identified using NGS than using conventional methods. Three chicken immune single-chain variable fragment (scFv) libraries were subjected to bio-panning on prostate-specific antigen (PSA). Phagemid DNA prepared from the original libraries as well as from the Escherichia coli pool after each round of bio-panning was analyzed using NGS, and the heavy chain complementarity-determining region 3 (HCDR3) sequences of the scFv clones were determined. Subsequently, through two-step linker PCR and cloning, the entire scFv gene was retrieved and analyzed for its reactivity to PSA in a phage enzyme immunoassay. After four rounds of bio-panning, the conventional colony screening method was performed for comparison. The scFv clones retrieved from NGS analysis included all clones identified by the conventional colony screening method as well as many additional clones. The enrichment of the HCDR3 sequence throughout the bio-panning process was a positive predictive factor for the selection of PSA-reactive scFv clones.


Subject(s)
Antibodies , Bacteriophages , Chickens , Clone Cells , Cloning, Organism , Complementarity Determining Regions , DNA , Escherichia coli , Immunoenzyme Techniques , Mass Screening , Methods , Polymerase Chain Reaction , Prostate-Specific Antigen , Single-Chain Antibodies
2.
Blood Research ; : 55-61, 2017.
Article in English | WPRIM | ID: wpr-226881

ABSTRACT

BACKGROUND: This study characterized clonal IG heavy V-D-J (IGH) gene rearrangements in South Indian patients with precursor B-cell acute lymphoblastic leukemia (precursor B-ALL) and identified age-related predominance in VDJ rearrangements. METHODS: IGH rearrangements were studied in 50 precursor B-ALL cases (common ALL=37, pre-B ALL=10, pro-B ALL=3) by polymerase chain reaction (PCR) heteroduplex analysis. Twenty randomly selected clonal IGH rearrangement sequences were analyzed using the IMGT/V-QUEST tool. RESULTS: Clonal IGH rearrangements were detected in 41 (82%) precursor B-ALL cases. Among the IGHV1-IGHV7 subgroups, IGHV3 was used in 25 (50%) cases. Among the IGHD1-IGHD7 genes, IGHD2 and IGHD3 were used in 8 (40%) and 5 (25%) clones, respectively. Among the IGHJ1-IGHJ6 genes, IGHJ6 and IGHJ4 were used in 9 (45%) and 6 (30%) clones, respectively. In 6 out of 20 (30%) IGH rearranged sequences, CDR3 was in frame whereas 14 (70%) had rearranged sequences and CDR3 was out of frame. A somatic mutation in Vmut/Dmut/Jmut was detected in 14 of 20 IGH sequences. On average, Vmut/Dmut/Jmut were detected in 0.1 nt, 1.1 nt, and 0.2 nt, respectively. CONCLUSION: The IGHV3 gene was frequently used whereas lower frequencies of IGHV5 and IGHV6 and a higher frequency of IGHV4 were detected in children compared with young adults. The IGHD2 and IGHD3 genes were over-represented, and the IGHJ6 gene was predominantly used in precursor-B-ALL. However, the IGH gene rearrangements in precursor-B-ALL did not show any significant age-associated genotype pattern attributed to our population.


Subject(s)
Child , Clone Cells , Complementarity Determining Regions , Gene Rearrangement , Genotype , Heteroduplex Analysis , Humans , Immunoglobulins , Polymerase Chain Reaction , Precursor Cell Lymphoblastic Leukemia-Lymphoma , Precursor Cells, B-Lymphoid , Young Adult
3.
Article in Chinese | WPRIM | ID: wpr-328232

ABSTRACT

<p><b>OBJECTIVE</b>To observe anti-cancer effects of Jianpi Jiedu Recipe (JJR) on liver cancer (LC) rats with Pi deficiency syndrome (PDS) and its relation with the third complementary-determining region gene spectratyping of TCRVβ-chain (TCRVβCDR3).</p><p><b>METHODS</b>Rats were divided into 8 groups according to random digit table, i.e., the blank control group (normal), the PDS group, the LC model group, the LC-PDS group, high, middle, and low dose JJR groups (75.00, 37.50, 18.75 g/kg, respectively by gastrogavage, once per day), the thymus pentapeptide group (5 mg/kg, intramuscular injection, twice per week), 8 in each group. Rats in the normal group were administered with physiological saline by gastrogavage once per day. PDS rat model was prepared by bitter-cold purgation. LC model was prepared by orthotopic transplantation method. Twenty gene subfamilies of TCRβCDR3 in the thymus, liver, and LC tissues were detected by Gene Scan.</p><p><b>RESULTS</b>High and middle dose JJR could postpone the growth of LC volume (P < 0.05), with equivalent liver index and thymus index to those of the normal group (P > 0.05). In thymus and liver tissue of the normal group, the number of clones (20 and 19), gene fragment number (220 and 113), Quasi-Gaussian distribution ratio of TCRVβCDR3 gene repertoire (100.0% and 42.1%), and fragment fluorescence peak area (6,539 ± 2,325 and 1,238 ± 439) were at the highest level among the 8 groups. TCRVβCDR3 expressions in thymus and liver tissue of high and middle dose JJR groups were approximate to those of the normal group. They were in the middle of the thymus pentapeptide group, the PDS group, the LC model group, and poorest in the LC-PDS group. TCRVβCDR3 in liver tissue expressed the best in the thymus pentapeptide group.</p><p><b>CONCLUSION</b>JJR might inhibit the growth of LC cells, and its mechanism might be related to enhancing TCRVβCDR3 spectratype expression.</p>


Subject(s)
Animals , Complementarity Determining Regions , Genetics , Drugs, Chinese Herbal , Pharmacology , Gene Expression Regulation, Neoplastic , Genes, T-Cell Receptor beta , Liver Neoplasms , Drug Therapy , Genetics , Random Allocation , Rats
4.
Article in Chinese | WPRIM | ID: wpr-239556

ABSTRACT

To explore the association between T-cell receptor beta variable (TCR BV) complementarity determining region 3 (CDR3) spectratyping and CMV activation in the recipients of allogeneic hematopoietic stem cell transplantation (HSCT).Fluorescence quantitative PCR melting curve analysis was used to sequence 24 TCR BV families in 7 HSCT recipients and 3 healthy controls. CMV-pp65 antigenemia was measured by immunohistochemical staining. Plasma IgM specific for CMV was identified using ELISA. Relationship between TCR BV families and CMV activation was statistically analyzed.Twenty-four TCR BV families were expressed in 3 healthy controls, while TCR BV CDR3 sequencing results in 7 recipients turned out to be BV9, BV11, BV17, BV20 and so on. Amino acid sequence features were as follows:TCR BV9 contained "QVRGGTDTQ", TCR BV11 contained "VATDEQ" and "LGDEQ", TCR BV17 contained "IGQGNTEA", and TCR BV20 contained "VGLAANEQ". Five recipients suffered from pp65 antigenemia in 3 month after transplantation, and pp65-positive cells ranged from 2 to 15 per 5×10white blood cells. Three recipients were CMV-IgM positive. No significant differences were found in TCR BV families between pp65-positive recipients and pp65-negative recipients (all>0.05). But there was statistically significant difference in frequency of TCR BV11 between CMV-IgM negative recipients and CMV-IgM positive recipients (<0.05).T cell immune response was characterized by special TCR BV CDR3 spectratyping in HSCT recipients, and TCR BV11 expression may be associated with CMV activation.


Subject(s)
Amino Acid Sequence , Complementarity Determining Regions , Genetics , Allergy and Immunology , Cytomegalovirus , Genetics , Allergy and Immunology , Cytomegalovirus Infections , Genetics , Genotype , Hematopoietic Stem Cell Transplantation , Humans , Lymphocyte Activation , Genetics , Phosphoproteins , Polymerase Chain Reaction , Polymorphism, Genetic , Allergy and Immunology , Receptors, Antigen, T-Cell, alpha-beta , Genetics , Allergy and Immunology , Spleen , T-Lymphocytes , Allergy and Immunology , Virology , Viral Matrix Proteins
5.
Article in English | WPRIM | ID: wpr-210166

ABSTRACT

The C-terminal domain of RNA polymerase II is an unusual series of repeated residues appended to the C-terminus of the largest subunit and serves as a flexible binding scaffold for numerous nuclear factors. The binding of these factors is determined by the phosphorylation patterns on the repeats in the domain. In this study, we generated a synthetic antibody library by replacing the third heavy chain complementarity-determining region of an anti-HER2 (human epidermal growth factor receptor 2) antibody (trastuzumab) with artificial sequences of 7–18 amino-acid residues. From this library, antibodies were selected that were specific to serine phosphopeptides that represent typical phosphorylation patterns on the functional unit (YSPTSPS)₂ of the RNA polymerase II C-terminal domain (CTD). Antibody clones pCTD-1stS2 and pCTD-2ndS2 showed specificity for peptides with phosphoserine at the second residues of the first or second heptamer repeat, respectively. Additional clones specifically reacted to peptides with phosphoserine at the fifth serine of the first repeat (pCTD-1stS5), the seventh residue of the first repeat and fifth residue of the second repeat (pCTD-S7S5) or the seventh residue of either the first or second repeat (pCTD-S7). All of these antibody clones successfully reacted to RNA polymerase II in immunoblot analysis. Interestingly, pCTD-2ndS2 precipitated predominately RNA polymerase II from the exonic regions of genes in genome-wide chromatin immunoprecipitation sequencing analysis, which suggests that the phosphoserine at the second residue of the second repeat of the functional unit (YSPTSPS)2 is a mediator of exon definition.


Subject(s)
Antibodies , Chromatin Immunoprecipitation , Clone Cells , Complementarity Determining Regions , DNA-Directed RNA Polymerases , Exons , Peptides , Phosphopeptides , Phosphorylation , Phosphoserine , ErbB Receptors , RNA Polymerase II , RNA , Sensitivity and Specificity , Serine
6.
Protein & Cell ; (12): 603-615, 2014.
Article in English | WPRIM | ID: wpr-757472

ABSTRACT

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.


Subject(s)
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
7.
Acta Pharmaceutica Sinica ; (12): 1651-1656, 2013.
Article in Chinese | WPRIM | ID: wpr-298030

ABSTRACT

To rapidly select potent anti-VSTM1-v2 scFv (single-chain antibody fragment) by construction and screening of a humanized scFv library in which a murine VH-CDR3 library was grafted onto a human scFv framework. A murine VH-CDR3 library was amplified from anti-VSTM1-v2 murine cDNA and grafted on human scFv (VH3-VK1) framework. Anti-VSTM1-v2 scFv templates were selected and enriched through ribosome display, TA-cloned into expression vector, and transformed into BL21 (DE3) for soluble expression of target scFv. A total of 1000 clones were randomly picked. Positive ones were first identified using colony PCR, indirect ELISA, Western blotting and then verified with sequencing and dose response ELISA. At last an anti-VSTM1-v2 humanized scFv with good binding affinity (EC50 = 21.35 nmol x L(-1)) was selected from the humanized library of 10(12) members generated in this study. This scFv antibody might have potential applications. This study provides a new approach for rapid screening of humanized antibodies.


Subject(s)
Animals , Complementarity Determining Regions , Genetics , Allergy and Immunology , Cytokines , Allergy and Immunology , Humans , Immunoglobulin Fragments , Genetics , Allergy and Immunology , Immunoglobulin Heavy Chains , Genetics , Allergy and Immunology , Mice , Peptide Library , Protein Binding , Receptors, Immunologic , Allergy and Immunology , Single-Chain Antibodies , Genetics , Allergy and Immunology
8.
Article in Chinese | WPRIM | ID: wpr-318054

ABSTRACT

<p><b>OBJECTIVE</b>To study the expression of TCR BV CDR3 family in fulminant hepatitis B (FHB) patients.</p><p><b>METHODS</b>Totally 28 patients with fulminant hepatitis B (FHB) (FHB group), who were treated in our hospital from Oct. 2010. to Mar. 2012, and 20 healthy controls( HC group) were included in the study. PBMCs were isolated from anticogulated blood, and RT-PCR was used to detect the levels of TCR BV CDR3 family in the 2 groups.</p><p><b>RESULTS</b>The levels of DeltaCt1, DeltaCt12 and DeltaCt20 in FHB group were higher than those in HC group (P < 0.05); The levels of DeltaCt5, DeltaCt7, DeltaCt13, DeltaCt14, DeltaCt15, DeltaCt22, DeltaCt23 in FHB group were lower than those in HC group (P < 0.05).</p><p><b>CONCLUSIONS</b>The result indicates that cellular immunology is involved in the pathogenesis of the liver inflammation process of FHB.</p>


Subject(s)
Adolescent , Adult , Aged , Case-Control Studies , Complementarity Determining Regions , Chemistry , Genetics , Allergy and Immunology , Female , Hepatitis B , Genetics , Allergy and Immunology , Virology , Humans , Male , Middle Aged , Receptors, Antigen, T-Cell , Chemistry , Genetics , Allergy and Immunology , Young Adult
9.
Acta Pharmaceutica Sinica ; (12): 322-331, 2012.
Article in Chinese | WPRIM | ID: wpr-323040

ABSTRACT

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.


Subject(s)
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
10.
Article in Chinese | WPRIM | ID: wpr-268965

ABSTRACT

<p><b>OBJECTIVE</b>To analyze the drift of T cell receptor (TCR) Vα and Vβ gene family CDR3 spectratype in response to ovarian carcinoma cells mediated by an anti-human ovarian carcinoma/CD3 bispecific single-chain antibody (BHL-1), and explore the mechanism of the bispecific single-chain antibody-mediated T cell immune response.</p><p><b>METHODS</b>Immunoscopic spectratyping technique was used to analyze the TCR repertoire diversity (CDR3 spectratype distribution) of the T cells from 6 healthy donors before and after stimulation of the cells with human ovarian carcinoma in the presence of BHL-1. The predominant usage of TCR α and Vβ chain CDR3 was analyzed after the stimulation, and sequence analysis was performed for the CDR3 region of the monoclonal T cells.</p><p><b>RESULTS</b>The spectratypes of Vα and Vβ gene family TCR CDR3 region showed a Gaussian distribution before stimulation of the T cells from the 6 donors. After stimulation of the T cells, CDR3 spectratype drift occurred in the T cells, and some TCR Vα and Vβ families showed an anomalous and oligoclonal expansion. Different CDR3 sequences of the Vα and Vβ gene family TCR were found in the monoclonal T cells stimulated with BHL-1.</p><p><b>CONCLUSION</b>CDR3 spectratype drift occurs in TCR α and Vβ chains of T cells after stimulation with human ovarian carcinoma cells and BHL-1, indicating that the predominant usage of TCR Vα and Vβ families is associated with the specific T cell immune response mediated by BHL-1.</p>


Subject(s)
Antibodies, Bispecific , Allergy and Immunology , Cell Line , Cell Line, Tumor , Complementarity Determining Regions , Allergy and Immunology , Female , Humans , Monocytes , Allergy and Immunology , Ovarian Neoplasms , Allergy and Immunology , Receptors, Antigen, T-Cell, alpha-beta , Allergy and Immunology , Single-Chain Antibodies , Allergy and Immunology
11.
Article in English | IMSEAR | ID: sea-135768

ABSTRACT

Background & objectives: The unique immunological functions of γδ T lymphocytes to contribute immunity against Mycobacterium tuberculosis attracted interest of researchers. However, little is known about the specificity of γδ Τ cell in tuberculosis patients and the lack of exact tuberculosis antigen recognized by γδ T cells limited its application. The analysis of complementary determinant region (CDR)3 sequence characteristic in γδ T cells of tuberculosis patients would contribute to understand the distribution specificity of γδ T cell. In present study, we investigated the diversity of the γ9/δ2 T cell immunorepertoire and analysed the specificity of the expressed CDR3 in pulmonary tuberculosis patients. Methods: The total RNA in peripheral blood mononuclear cell of 50 pulmonary tuberculosis patients and 10 healthy controls was extracted. The polymerase chain reaction was used to specifically amplify the CDR3 region of γ9 and δ2 chain. The PCR products were ligated into the pGEM-T easy vector. The plasmid DNA was sequenced using the ABI3700 and the T7 primer. Results: Our findings showed that predominant CDR3 sequence of δ2 chain in pulmonary tuberculosis patients was CACDTLVSTDKLIFGKG. The sequence specifically exists in almost all pulmonary tuberculosis patients. The conserved hydrophobic acid residue in 97 positions is present in the γδ T cell reactive to M. tuberculosis. The length of δ2 CDR3 in pulmonary tuberculosis patients has no relation with the disease progress. Interpretation & conclusions: Our results suggest that γδ T cells appear to use CDR3 sequence to recognise M. tuberculosis antigen. γδ T cells reactive to M. tuberculosis were diverse and polyclonal.


Subject(s)
Amino Acid Motifs/genetics , Complementarity Determining Regions/metabolism , DNA Primers/genetics , Female , Genetic Vectors , Humans , Male , Molecular Sequence Data , Mycobacterium tuberculosis/immunology , Polymerase Chain Reaction , Receptors, Antigen, T-Cell, gamma-delta/immunology , Receptors, Antigen, T-Cell, gamma-delta/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Sequence Analysis, DNA , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Tuberculosis, Pulmonary/immunology
12.
Rev. bras. cir. plást ; 25(2): 297-303, abr.-jun. 2010. ilus
Article in Portuguese | LILACS | ID: lil-579333

ABSTRACT

Método: Foram avaliadas as rugas das regiões frontal e glabelar da face em pacientes submetidas a aplicações seriadas da toxina botulínica tipo A com finalidade estética, analisando-se os intervalos entre as aplicações e as doses utilizadas. Vinte e quatro pacientes do sexo feminino receberam de 5 a 7 aplicações da toxina botulínica tipo A no terço superior da face. O tempo médio de acompanhamento foi de 42,2 ± 4 meses e o intervalo médio entre as aplicações de 8 ± 0,51 meses. As rugas frontais e glabelares foram avaliadas segundo escala padronizada, comparando-se fotografias previas à primeira e à ultima aplicação, em repouso e em movimentos da mímica facial. Resultados: No longo prazo, as rugas estáticas da região frontal foram amenizadas em 62,5% das pacientes e inalteradas em 37,5%, enquanto as rugas estáticas da região glabelar foram amenizadas em 47,2% das pacientes, inalteradas em 50% e pouco acentuadas em2,8%. As dinâmicas da região frontal foram amenizadas em 84,7% das pacientes e inalteradas em 15,3%, enquanto as dinâmicas verticais da região glabelar (corrugadores) foram amenizadasem 63,9% das pacientes, inalteradas em 26,4% e pouco acentuadas em 9,7%. As rugas dinâmicas horizontais da região glabelar (prócero) foram amenizadas em 57% das pacientes, inalteradas em 32% e pouco acentuadas em 11%. A média da dose total de toxina botulínica utilizada por sessão foi de 43,83 ± 1,25 U, e a dose total cumulativa foi de 272,08 ± 20,42 U.


Methods: Wrinkles in the frontal and glabellar regions of the face were evaluated in patients submitted to a series of cosmetic botulinum toxin applications, with analysis of the intervals between applications and doses used. Twenty-four female patients who received 5 to 7 botulinum toxin applications to the upper third of the face were evaluated. Mean follow-up timewas 42.2 ± 4 months and the mean interval between applications was 8 ± 0.51 months. The forehead and glabellar wrinkles were evaluated by a standardized scale comparing photos taken prior to the first application, at rest and during facial expressions. Results: Over time, static wrinkles in the frontal region were reduced in 62.5% of the patients and remained the same in 37.5%, while static wrinkles in the glabellar region were reduced in 47.2% of the patients, remained the same in 50% and were slightly accentuated in 2.8%. Dynamic wrinkles of the frontal region were reduced in 84.7% of the patients and remained the same in 15.3%, while the vertical dynamic wrinkles in the corrugator region were reduced in 63.9% of the patients, remained the same in 26.4% and were slightly accentuated in 9.7%. Horizontal dynamic wrinkles in the procerus region were reduced in 57% of the patients, remained the same 32% and were slightly accentuated in 11%. The mean total dose of toxin used per session was 43.83 ± 1.25 U, and the total cumulated dose was 272.08 ± 20.42 U.


Subject(s)
Humans , Female , Adult , Middle Aged , Botulinum Toxins, Type A , Complementarity Determining Regions , Esthetics , Face/surgery , Postoperative Complications , Rejuvenation , Diagnostic Techniques and Procedures , Methods , Patients , Surgery, Plastic
13.
Chinese Journal of Hepatology ; (12): 184-188, 2010.
Article in Chinese | WPRIM | ID: wpr-247561

ABSTRACT

<p><b>OBJECTIVE</b>To analyze the characteristics of CDR3 of TCRbeta on CD8+ T cells in chronic hepatitis B patients.</p><p><b>METHODS</b>Eight patients with chronic hepatitis B (ALT more than 2 ULN) were enrolled in this study. CD8+ T cells were isolated from peripheral blood. RT-PCR was proformed to amplify the CDR3 of TCRbeta, and the PCR products were sequenced and analyzed.</p><p><b>RESULTS</b>The chronic hepatitis B patients showed obvious clonal expansion of T cell, and three perturbation patterns of T cell expansion were showed in the CDR3 of TCRbeta, including monoclonicity, oligoclonicity and skewed peak patterns. The number of perturbation families of CD8+ subpopulation was significantly higher than that of CD8- subpopulation (10.6+/-4.7 vs. 4.1+/-3.1, t = 6.619, P less than 0.01). In 3 out of 8 patients, the number of perturbation families of CD8+ subpopulation was also higher than that of PBMCs without depleting CD8+ subpopulation.</p><p><b>CONCLUSIONS</b>The characteristics of CDR3 of TCRbeta may help to understand the inflammatory response in CHB patients.</p>


Subject(s)
Adult , CD8-Positive T-Lymphocytes , Allergy and Immunology , Complementarity Determining Regions , Genetics , Genes, T-Cell Receptor beta , Hepatitis B, Chronic , Blood , Allergy and Immunology , Humans , Male , Receptors, Antigen, T-Cell , Allergy and Immunology , Young Adult
14.
Article in Chinese | WPRIM | ID: wpr-343362

ABSTRACT

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.


Subject(s)
Amino Acid Sequence , Animals , Bone Marrow Transplantation , Complementarity Determining Regions , Genetics , Graft vs Host Disease , Genetics , Allergy and Immunology , Immunoglobulin Variable Region , Genetics , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Molecular Sequence Data , Receptors, Antigen, T-Cell , Genetics
15.
Article in Chinese | WPRIM | ID: wpr-349078

ABSTRACT

<p><b>OBJECTIVE</b>To 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.</p><p><b>METHODS</b>mtDNAs obtained from 119 unrelated individuals were amplified and directly sequenced.</p><p><b>RESULTS</b>One 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.</p><p><b>CONCLUSION</b>There 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.</p>


Subject(s)
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
16.
Article in Chinese | WPRIM | ID: wpr-302117

ABSTRACT

This study was purposed to investigate the expression and clonal proliferation of receptor (TCR) Vbeta subfamilies of the T-cells in acute leukemic patients at different disease status (onset, complete remission or relapse) and to analyze the influence of the leukemic cell load on anti-leukemic effect of peripheral T-lymphocytes of the patients. Gene sequences of peripheral TCR Vbeta 24 families from 11 leukemic patients and 3 normal donors were expanded by RT-PCR. Genescan technique was applied to evaluate clonal expression of the TCRVbeta subfamilies, clonal characteristics of the CDR3 from peripheral blood of AML patients at different disease status. The application, clonal proliferation, cellular complexity of T-cells, and the variation of immunotypes of T-cells were compared. The results indicated that the lower and partial distribution of TCR Vbeta subfamily was found in all 11 patients when firstly diagnosed; the expression of TCR Vbeta subfamilies after induction in vitro increased; obvious elevation of TCR Vbeta subfamilies was observed in patients at complete remission although expression level was still lower than normal, whereas the significant descent of TCR Vbeta subfamilies was detected in 4 relapsed patients. Only 1 - 2 clonal proliferation of TCR Vbeta subfamilies existed in 9 out of 11 patients at initial diagnosis which increased at remission. The status of clonal proliferation of Vbeta subfamily T-cells continued regardless of any different disease status in most patients. There was an obvious decrease of CDR3 complexity at initial diagnosis or relapse, while CDR3 complexity would be partially improved at remission. It is concluded that the restrict distribution and expression of TCR Vbeta subfamilies were found in AML patients. Clonal proliferation of T-cells Vbeta subfamily continuously exists regardless of any different disease status in most patients. Some Vbeta subfamilies sustain clonal proliferation at different disease status. Some clonal proliferations of Vbeta subfamilies are associated with the effects of leukemic cells, CDR3 complexity obviously decreases under disease status which can be partially improved at remission.


Subject(s)
Adult , Aged , Clone Cells , Complementarity Determining Regions , Genetics , Female , Humans , Leukemia, Myeloid, Acute , Genetics , Male , Middle Aged , Receptors, Antigen, T-Cell , Genetics , Reverse Transcriptase Polymerase Chain Reaction , Young Adult
17.
Article in Chinese | WPRIM | ID: wpr-287394

ABSTRACT

<p><b>OBJECTIVE</b>To investigate the association of the mitochondrial DNA region np16181-16193 variations with type 2 diabetes mellitus (T2DM).</p><p><b>METHODS</b>Blood samples of 199 unrelated T2DM patients and 205 normal controls were collected to detect the mitochondrial DNA region np16181-16193 variations by PCR and sequencing, and to analyze the association of the variations with the major clinical symptoms.</p><p><b>RESULTS</b>The mitochondrial DNA np16181-16193 region is a hypervariable area, with several polymorphisms. Four types of np16181-16193 region variations were found only in T2DM. The 1-hour postprandial blood glucose (P1BG) in the T2DM individuals with np16181-16193 region variations was significantly higher than those without variations (P<0.05), while there was no significant difference in other biochemical parameters (P>0.05).</p><p><b>CONCLUSION</b>The mitochondrial DNA np16181-16193 variations could not be regarded as a risk factor for T2DM.</p>


Subject(s)
Adult , Complementarity Determining Regions , Genetics , DNA Mutational Analysis , DNA, Mitochondrial , Diabetes Mellitus, Type 2 , Genetics , Female , Genetic Predisposition to Disease , Genome, Mitochondrial , Genetics , Humans , Male , Sequence Analysis, DNA
18.
Chinese Journal of Virology ; (6): 249-254, 2008.
Article in Chinese | WPRIM | ID: wpr-334815

ABSTRACT

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.


Subject(s)
Amino Acid Sequence , Animals , Complementarity Determining Regions , Allergy and Immunology , Cross Reactions , Enzyme-Linked Immunosorbent Assay , Epitopes , Hepacivirus , Allergy and Immunology , Humans , Mice , Mice, Inbred BALB C , Molecular Sequence Data
19.
Article in Chinese | WPRIM | ID: wpr-280157

ABSTRACT

<p><b>OBJECTIVE</b>To study the CDR3 spectratyping and clonal expansion of T cells in the peripheral blood mononuclear cells (PBMCs) of patients with beta-mediterranean anemia patient undergoing allogeneic peripheral blood stem cell (PBSC) transplantation.</p><p><b>METHODS</b>The total RNA was isolated from the PBMCs of a nine-year-old boy with beta-mediterranean anemia before and after PBSC transplantation, and at the time of occurrence of graft-versus-host disease (GVHD). The CDR3 length was analyzed using immunoscope technique, and the characteristics of the T cell receptor (TCR) on the T cells with clonal expansion were examined by gene sequencing.</p><p><b>RESULTS</b>The 24 TCR BV CDR3 repertoire showed Gaussian distribution in the PBMCs isolated before the transplantation, and some of the TCR BV family CDR3 showed skewing in the PBMCs isolated 23 days after transplantation and at the onset of GVHD (28 days after transplantation), suggesting the clonal expansion of the donor PBSCs.</p><p><b>CONCLUSION</b>The host PBMCs show muti-clonal expansion 23 days after PBSC transplantation, and in the stage of GVHD, some of the TCR BV family T cells show significant monoclonal expansion. Analysis of TCR CDR3 spectratyping and the molecular characteristics of specific TCR may help evaluate the immune reconstitution following the transplantation and indicate specific treatment for potential GVHD.</p>


Subject(s)
Child , Complementarity Determining Regions , Genetics , Allergy and Immunology , Graft vs Host Disease , Allergy and Immunology , Humans , Male , Peripheral Blood Stem Cell Transplantation , Methods , Receptors, Antigen, T-Cell, alpha-beta , Genetics , Allergy and Immunology , beta-Thalassemia , Allergy and Immunology , General Surgery
20.
Article in Chinese | WPRIM | ID: wpr-303962

ABSTRACT

<p><b>OBJECTIVE</b>To observe the TCR V gamma gene repertoire diversity in the peripheral blood mononuclear cells of the patients with the chronic benzene poisoning.</p><p><b>METHODS</b>Complementarity determining region 3 (CDR3) of TCR V gamma subfamily genes were amplified in 10 patients with the chronic benzene poisoning using RT-PCR. The PCR products were further analyzed by genescan to evaluate clonality of T cells. 8 healthy persons served as control.</p><p><b>RESULTS</b>All V gamma subfamilies were detected in the 8 healthy donors. (1.30 +/- 0.48) V gamma subfamilies were detected in 10 patients. The number of detectable V gamma subfamilies present in the patients with the chronic benzene poisoning was significantly lower than in the control group (P < 0.01). The most frequently used V gamma genes in clonally expanded T-cells were V gamma II.</p><p><b>CONCLUSION</b>Skewed distribution and clonal expansion of TCR V gamma subfamily T cells could be found in the patients with the chronic benzene poisoning. This is the first report of clonal expansion TCR V gamma T cells in patients with chronic benzene poisoning. The bias pattern of TCR V alpha T cells may be due to the immune cytotoxicity from benzene.</p>


Subject(s)
Adult , Benzene , Poisoning , Chronic Disease , Complementarity Determining Regions , Genetics , Female , Humans , Leukocytes, Mononuclear , Allergy and Immunology , Male , Middle Aged , Occupational Diseases , Genetics , Allergy and Immunology , Receptors, Antigen, T-Cell, gamma-delta , Genetics , Allergy and Immunology , Young Adult
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