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Objective To study the serum levels of low molecule weight IgM (LMW IgM) in ankylosing spondylitis (AS) patients and rheumatoid arthritis (RA) patients and to evaluate the relationship of LMW IgM levels with the disease activities. Methods The levels of LMW IgM and pentameric IgM in AS patients, RA patients and healthy controls were measured with ELISA after separated using ultrafiltration assay. Differences in the percentage of LMW IgM between subject groups were analysed using Mann-Whitney U test. Results The percentages of LMW IgM increased dramatically in AS patients and RA patients compared with healthy controls (0.194 ± 0123, 0.061 ±0.026, 0.028 ±0.165 separately). The LMW IgM percentages were not correlated with the disease activities. Conclusion The increase of LMW IgM indicates humoral immune function abnormality in AS patients and RA. However, the mechanism needs further study.
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Objective To explore the role of sialic acid-binding immunoglobulin-like lectin-one (Siglec-1) in the process of atherosclerotic inflammation induced by oxidized low-density hpepmtein (ox-LDL). Methods Ox-LDL was synthesized by oxidization of native LDL and different concentration of ox-LDL was added to the culture medium of RAW264.7. Forty-eight hours later, cells and supernatants were collected separately. The expression of Siglec-1 protein and mRNA were measured by flow cytometry(FCM) and real-time quantitative RT-PCR, respectively. The levels of monocyte chemotactic protein-1 (MCP-1), macrophage inflammatory protein-1α(MIP-1α) and IL-8 in supernatants were determined by ELISA. Re-sults By the stimulation of ox-LDL, Siglec-1 protein and mRNA on RAW264.7 cells were significantly in-creased, meanwhile, the cytokines levels in culture supematants were significantly higher than that in the control group. And both Siglec-1 expression and cytokine secretion were ox-LDL dose-dependent. Conclu-sion Ox-LDL can increase Siglec-1 protein and mRNA expression and some inflammatory cytokines secre-tion on RAW264.7 cells in a dose-dependent manner. Manifested by enhanced Siglec-1 expression, the acti-vated macmphages may take a part in the development and progression of atherosclerosis.
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Objective To examine the suscepribility of the single nucleotide polymorphisms(SNPs)in IL-1 gene in Chinese Han population to ankylosing spondylitis (AS). Methods An correlation analysis was performed in a case-control cohort of 162 AS cases, 58 patients with other autoimmune diseases and 162 controls. Four SNPs located in the IL-1 gene (rs16944, rs3811058, rs419598, rs315952) were examined by polymerase chain reaction restriction fragment length polymorphism(PCR-RFLP). Results The frequencies of allele C at position rs16944, rs3811058, rs419598 significantly increased in AS cases VS controls, so did their genotype frequencies (50% vs 36.3%, 57.9% vs 52.8%, 45.7% vs 12.3%, P＜0.05). The SNPs of these sites significantly influenced the prevalence of AS. Conclusion We discover that SNPs of IL-1 gene Rs16944, Rs419598, Rs3811058 is closely related to the occurrence of AS.
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Objective To construct a phage antibody Fab library of human antinuclear antibody. Methods Total RNA was extracted from peripheral blood lymphocytes of 4 patients suffering from autoallergic disease, with antinuclear IgG antibody titers in the serum higher than 1∶10 000 as estimated by indirect immunofluorescence technique. Taking oligo-dT as the primers, human immunoglobulin light chains ?/? genes as well as heavy chains Fd genes were reversely transcribed to cDNA by PCR. The ?/? light chains were initially cloned into pComb3Hss vector to construct a human recombinant light chain library, and the heavy chain genes were subsequently inserted into the corresponding sites of ?/?-pComb3Hss plasmid to generate a recombinant ?/?-Fd-pComb3Hss plasmid. The plasmid was then transformed into E. coli XL1-Blue, which was subsequently infected by the helper phage M13KO7. A random recombinant antibody library was expressed on the surface of filamentous phage. Results Human immunoglobulin ?/? light chain and heavy chain Fd genes (660bp) were amplified successfully. The light chain library with the capacity size of 2?104, and the human recombinant Fab antibody library with the capacity size of 4?104 were also successfully obtained. Finally, the phage antibody library of the Fab fragment of human antinuclear antibody, containing 2?109 CFU/ml phage, was constructed. Conclusion A phage antibody library of Fab fragment of human antinuclear antibody is constructed successfully. This research lays a foundation for the preparation for phage library of Fab fragment of human antinuclear antibody, and also paves the way for the advanced assessment for its effect on the immuno-targeting therapy of tumors.
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Objective:To investigate the possible role of chemokines monocyte chemoattractant protein-1(MCP-1) and macrophage inhibitory protein-1(MIP-1?) in pathogenesis of rheumatoid arthritis(RA).Methods: Enzyme linked immunosorbent assay(ELISA) was used to determine the serum expression of MCP-1 and MIP-1? in 17 patients with early active RA,18 with advanced active RA,and 15 healthy controls(all aged 18-79 years).Clinical activity indices such as the strength of grip,joint pain index,and joint swelling index were assessed in all subjects;and the serological indices such as erythrocyte sedimentation rate(ESR),C-reactive protein(CRP),and rheumatoid factors were also determined.The relationships between serum levels of MCP-1, MIP-1? with the clinical activity indices and serological indices were analyzed.Results: The serum levels of MCP-1 in patients with early and advanced RA were higher than that in the controls(P
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Objective Construct an expression vector for anti-HBs Fab fragment and IFN-? fusion protein, then transform it into E.coli Top10 in order to produce a targeting drug for hepatitis B. Methods Using PCR and molecular clone techniques, we amplify the gene fragment of IFN-? with corresponding endonuclease sites and artificial linker at 5′,3′ termini,then recombine it within the vector of anti-HBs Fab gene in correct endonuclease sites at 3′ termini of the Lc gene, choosing the positive clone with PCR and transform it into E.coli Top10,after the expression and purification of the fusion protein, we use ELISA and Dot-blot to verify the antigenicity and binding activity to HBsAg. Results We got fusion protein consisting Lc and IFN-?. It has the IFN-? activity and HBsAg affinity which is similar to anti-HBs Fab fragment. Conclusions The successful expression of the fusion protein with both HBsAg affinity and IFN-? activity make it possible for immunobiotherapy drug targeted to HBV.
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Objective:To show whether the Fas Ligand gene induces mast cells apoptosis.Methods:RT-PCR was used to amplify the gene of rat Fas ligand extracelluar domain and transmembrane domain and cloned it into eukaryotic expression plasmid pcDNA3.1.Transcent transfect RBL-2H3,the expression of Fas ligand RBL-2H3 was detected by RT-PCR、Western blot.The Annexin V FCM was used to detect the RBL-2H3 apoptosis after the transfection of Fas Ligand.Results:It is successful to obtain the gene of rat Fas Ligand extracellular domain and transmembrane segment,cloning it into pcDNA3.1,FasL was expressed on the surface of RBL-2H3 and it's supernatant after the transfection of pcDNA3.1/FasL.The cell start to be apoptosis.Conclusion:Our study reveals that Fas Ligand gene transfection in RBL-2H3 can effectively induced apoptosis.It is a promising strategy for Fas Ligand to be used in the therapy of allergic disease. [
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The purpose of this study was to compare the efficiency of 3 different methods in purifying engineered anti-HBsFab. Anti-HBsFab Supernatants containing anti-HBsFab were prepared by ultrasound lysis of host cells of anti-HBsFab positive clone. Anti-Fab-sepharose gel, streptococcal protein G-sepharose gel (Prot G gel) and Ni-NTA-agarose gel were used to purify Fabs according to the reagents protocols. Then the concentrations of purified proteins were determined. SDS-PAGE was used for the measurement of purified Fabs'purity. HBsAg based ELISA was chosen to determine the Fabs' bio-activity. The results indicated that Fab recovery of different gels in equal volume were different. The recovery of Ni-NTA GEL was greater than that of Prot G gel, and the recovery of Prot G gel was greater than that of anti-Fab gel. Purity of the Fab isolated by 3 different gels was confirmed by SDS-PAGE. The binding capacity of anti-HBs Fab to HBsAg of these three gels had no signifticant difference. Our data suggested that each method had its advantage and usage limitation, Ni-NTA method demonstrated much better performance in economy, management, and efficiency over the other two methods.