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ABSTRACT: The combination of IL-2 (interleukin-2) and GM-CSF (granulocyte/macrophage colony-stimulating factor) has been broadly studied in antitumour immune therapy, but its efficacy is uncertain. To better exert the activities of the two cytokines and study them in a mouse model, we have constructed a bifunctional protein, hIL-2–mGM-CSF (human IL-2–mouse GM-CSF), fused to a C-terminal tag of six histidine residues (His6). The fusion protein was expressed in Escherichia coli as IBs (inclusion bodies). After extracting and clarifying the IBs, four methods of protein purification and refolding were compared in order to optimize the preparation technique. Of these methods, the best result was obtained with a four-step process consisting of (1) purification with denaturing affinity chromatography, (2) followed by fully denaturing the protein with system conversion, (3) then refolding by isovolumetric ultrafiltration and (4) finally, purification by anion-exchange chromatography. The purity of the hIL-2–mGM-CSF was approx. 95%, yielding approx. 20 mg of protein/l of culture. The fusion protein retained the natural activities of IL-2 and GM-CSF, with specific activities of 8.7×106 and 1.1×107 i.u./mg respectively. Flow-cytometric analysis indicated that hIL-2–mGM-CSF could specifically bind to the corresponding receptor-positive cells. The present study provides important preliminary information for studying the antitumour activity of hIL-2–mGM-CSF in vivo, which will facilitate future clinical research into the use of hIL-2/hGM-CSF in immune therapy.
Biotechnology and Applied Biochemistry 12/2010; 50(1):41 - 48. · 1.53 Impact Factor
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Wei Luo,
Li Ma,
Qian Wen,
Xin-Sheng Yao,
Na Wang,
Hong-Yun Zou,
Ming-Qian Zhou,
Ying Lin,
Zhen-Qiang Wu, Xiao-Wei He,
Ju-Fang Wang,
Xiao-Ning Wang
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ABSTRACT: ABSTRACT: OBJECTIVE: To find conserved motifs in specific T cell receptor (TCR) alpha and beta chains, and to analyze the association between Complementarity Determining Region 3 (CDR3) spectratype and Systemic Lupus Erythematosus (SLE) activity. METHODS: TCR alpha and beta chain CDR3 spectratype were analyzed in 20 SLE patients. The CDR3 spectratypes of three patients were monitored over time, and the CDR3 regions of clonally expanded T cells were sequenced. A possible specific TCR structure was modeled. RESULTS: CDR3 spectratype analysis showed prominent usage of TCR AV8, AV14, AV23, AV30, AV31, BV2, BV8, BV11, BV14, BV16, BV19 and BV24 families in SLE patients. The CDR3 spectratype showed dynamic change correlating with SLE activity. The sequence of the CDR3 region in clonally expanded T cells suggested a conserved GGX amino acid motif in both alpha and beta chains. The Jalpha34Jbeta2s1 and Jbeta2s7 region genes were found in high frequency. CONCLUSION: Both TCR Valpha and Vbeta gene usage is highly restricted in SLE, suggesting that the TCRs recognize a limited number of antigenic epitopes. The conserved motifs and limited use of joining region genes may indicate the recognition of similar antigenic epitopes in multiple individuals.
Journal of Autoimmune Diseases 08/2008; 5:4.
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ABSTRACT: The combination of IL-2 (interleukin-2) and GM-CSF (granulocyte/macrophage colony-stimulating factor) has been broadly studied in antitumour immune therapy, but its efficacy is uncertain. To better exert the activities of the two cytokines and study them in a mouse model, we have constructed a bifunctional protein, hIL-2-mGM-CSF (human IL-2-mouse GM-CSF), fused to a C-terminal tag of six histidine residues (His(6)). The fusion protein was expressed in Escherichia coli as IBs (inclusion bodies). After extracting and clarifying the IBs, four methods of protein purification and refolding were compared in order to optimize the preparation technique. Of these methods, the best result was obtained with a four-step process consisting of (1) purification with denaturing affinity chromatography, (2) followed by fully denaturing the protein with system conversion, (3) then refolding by isovolumetric ultrafiltration and (4) finally, purification by anion-exchange chromatography. The purity of the hIL-2-mGM-CSF was approx. 95%, yielding approx. 20 mg of protein/l of culture. The fusion protein retained the natural activities of IL-2 and GM-CSF, with specific activities of 8.7 x 10(6) and 1.1 x 10(7) i.u./mg respectively. Flow-cytometric analysis indicated that hIL-2-mGM-CSF could specifically bind to the corresponding receptor-positive cells. The present study provides important preliminary information for studying the antitumour activity of hIL-2-mGM-CSF in vivo, which will facilitate future clinical research into the use of hIL-2/hGM-CSF in immune therapy.
Biotechnology and Applied Biochemistry 06/2008; 50(Pt 1):41-8. · 1.53 Impact Factor
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Wei Luo,
Li Ma,
Qian Wen,
Xin-Sheng Yao,
Na Wang,
Hong-Yun Zou,
Ming-Qian Zhou,
Ying Lin,
Zhen-Qiang Wu, Xiao-Wei He,
Ju-Fang Wang,
Xiao-Ning Wang
Journal of Autoimmune Diseases 02/2008; 5:5.
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ABSTRACT: Recent 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.
TCR 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.
RAG1, 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.
RAG1 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.
Chinese medical journal 04/2007; 120(5):410-5. · 0.86 Impact Factor
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ABSTRACT: HLA-A*0201 alpha chain and beta2m were expressed from a prokaryotic system, and after refolding and purification, the alpha chain and beta2m were used to immunize eight laying hens. The titer of egg yolk antibody against alpha chain increased from 10(2) to 10(5.3) The titer of egg yolk antibody against beta2m increased from 10(1) to 10(4.7). The extent of titer increase is similar between the two antigens. An average of 135 mg purified polyclonal antibody (IgY) can be easily obtained from one egg yolk. The use of egg collection rather than serum collection is compatible with modern animal protection regulations. An average of 28 eggs were obtained from a laying hen every month, with a total amount of 3780 mg immunoglobulin extracted from one immunized hen every month, which would be equivalent to 630 mL of serum or 1260 mL of blood per month. Chickens are an optimal host for the production of polyclonal antibodies with high titer and high yield. Purified IgY was labeled with horseradish peroxidase and reacted with PBMC on nitrocellulose membranes indicating that the antibody can bind to the native conformation of class I HLA molecule on PBMC.
Journal of Immunoassay and Immunochemistry 02/2007; 28(1):35-45. · 0.69 Impact Factor
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Xin-Sheng Yao,
Guang-Wen Zhang,
Li Ma,
Qian Wen,
Jin-Lin Hou,
Min-Jie Meng,
Shi-Wu Ma,
Min-Feng Liang,
Ying Lin,
Zhen-Qiang Wu, Xiao-Wei He,
Ju-Fang Wang,
Xiao-Ning Wang
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ABSTRACT: To clarify the characteristics of TCR alphabeta T cells in the peripheral blood of patients with chronic hepatitis B (CHB), we investigated the patterns of Complementarity Determining Region3 (CDR3) length distribution for all 24 TCR BV gene families and 32 TCR AV gene family in the peripheral blood lymphocytes of two patients with CHB and two healthy controls by immunoscope spectratyping technique. We found that the profiles of CDR3 length distribution for all TCR AV and TCR BV family showed Gaussian distribution (the polyclonal TCR alphabeta T cells) in healthy controls, however, the restricted usage of TCR BV and TCR AV family (the oligoclonal TCR alphabeta T cells) has been monitored in two CHB patients, furthermore, the oligoclonal TCR alphabeta T cells showed the different CDR3 sequences and length, it might be correlated to the different epitope of hepatitis B virus (HBV) or the different HLA type of the patients. Detailed analysis of the CDR3 length of TCR alphabeta T cells might be interesting to light on the further study of the individualized immunotherapy of CHB and the further research of the new T lymphocyte epitope vaccine of HBV.
Hepatology Research 06/2006; 35(1):10-8. · 2.20 Impact Factor
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ABSTRACT: Different methods were used to prepare HLA tetramers and the yields of each method were compared. Our results indicate that preliminary refolding of the heavy chain (Hc) and light chain (beta 2m) yields more monomer than the typical conventional method with urea-solubilized Hc and beta 2m. We then used the corresponding tetramers to detect cytomegalovirus (CMV)-specific cytotoxic T lymphocytes (CTL). Increasing data suggest that the adoptive transfer of CMV-specific CTL constitutes an effective strategy against CMV infections. We designed a method that efficiently induces CMV-specific CTL to a higher frequency in vitro than is currently achieved. This method increased the percentage of CMV-specific CTL from below 1% to 20% of PBL, accounting for more than 40% of CD8+ T cells. Successful HLA tetramer preparation provides the basis for the subsequent detection of CMV-specific CTL in clinical applications.
Journal of Immunological Methods 06/2006; 312(1-2):148-56. · 2.20 Impact Factor
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ABSTRACT: The cynomolgus monkey (Macaca fascicularis) is one of the most widely used surrogate animal models for an increasing number of human diseases and vaccines, especially immune-system-related ones. Towards a better understanding of the gene expression background upon its immunogenetics, we constructed a cDNA library from Epstein-Barr virus (EBV)-transformed B lymphocytes of a cynomolgus monkey and sequenced 10,000 randomly picked clones.
After processing, 8,312 high-quality expressed sequence tags (ESTs) were generated and assembled into 3,728 unigenes. Annotations of these uniquely expressed transcripts demonstrated that out of the 2,524 open reading frame (ORF) positive unigenes (mitochondrial and ribosomal sequences were not included), 98.8% shared significant similarities (E-value less than 1e-10) with the NCBI nucleotide (nt) database, while only 67.7% (E-value less than 1e-5) did so with the NCBI non-redundant protein (nr) database. Further analysis revealed that 90.0% of the unigenes that shared no similarities to the nr database could be assigned to human chromosomes, in which 75 did not match significantly to any cynomolgus monkey and human ESTs. The mapping regions to known human genes on the human genome were described in detail. The protein family and domain analysis revealed that the first, second and fourth of the most abundantly expressed protein families were all assigned to immunoglobulin and major histocompatibility complex (MHC)-related proteins. The expression profiles of these genes were compared with that of homologous genes in human blood, lymph nodes and a RAMOS cell line, which demonstrated expression changes after transformation with EBV. The degree of sequence similarity of the MHC class I and II genes to the human reference sequences was evaluated. The results indicated that class I molecules showed weak amino acid identities (<90%), while class II showed slightly higher ones.
These results indicated that the genes expressed in the cynomolgus monkey could be used to identify novel protein-coding genes and revise those incomplete or incorrect annotations in the human genome by comparative methods, since the old world monkeys and humans share high similarities at the molecular level, especially within coding regions. The identification of multiple genes involved in the immune response, their sequence variations to the human homologues, and their responses to EBV infection could provide useful information to improve our understanding of the cynomolgus monkey immune system.
BMC Genomics 02/2006; 7:82. · 4.07 Impact Factor
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ABSTRACT: The importance of eggs as a source of specific antibodies is well recognized. Egg yolk contains 8--20mg immunoglobulins (IgY) per milliliter. However, the major problem in separating IgY is to remove the high concentrations of lipids in egg yolk. We first used water dilution method to get the supernatant containing IgY, then purified the antibody by caprylic acid-ammonium sulfate method, and obtained specific antibody with satisfactory purity and activity. By comparison of these several methods, each has its advantages, one can be chosen to purify IgY according to practical need. The purified IgY produced by the immunized chickens can stain the human peripheral blood mononuclear cell effectively when labeled with fluorescent FITC.
Protein Expression and Purification 12/2005; 44(1):45-51. · 1.59 Impact Factor
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ABSTRACT: To describe the emperipolesis of lymphocytes and investigate the factors affecting lymphocyte emperipolesis in the KB cells and the relation of the emperipolesis to the biological behaviors of the lymphocytes.
After pre-stimulation with phytohemagglutinin (PHA), interleukin (IL)-2, a-CD3, and PHA+IL-2, respectively, the peripheral blood lymphocytes (PBLs) isolated from healthy donors were mixed with KB cells to observe the emperipolesis of the PBLs under inverted microscope every other hour, and calculate the tumor-adhesion/emperipolesis indices of the PBLs. The cell mixture was then fixed with cool acetone and stained by Giemasa to observe emperipolesis between PBLs and KB cells. The occurrence and development of emperipolesis between PBLs and KB cells was also observed under scanning electron microscope (SEM) at 1, 2, 4, and 6 hours after fixation.
The tumor-adhesion/emperipolesis indices of pre-stimulated PBLs were significantly higher than those of non-stimulated PBLs, and the indices of PHA+IL-2 group were the highest. The PBLs underwent morphological changes in the course of PBL emperipolesis mixed with KB cells as observed under SEM.
Pre-stimulation can enhance the tumor-adhesion/emperipolesis indices of the PBLs. The emperipolesis might be a means of cytotoxicity of the PBLs.
Di 1 jun yi da xue xue bao = Academic journal of the first medical college of PLA 10/2005; 25(9):1124-7.
