[Preparation and characterization of monoclonal antibodies against cytolethal distending toxin protein of Campylobacter jejuni].
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To express Campylobacter jejuni cytolethal distending toxin B protein (CdtB) in a prokaryote to prepare monoclonal antibodies (mAbs) against the protein, and to study their antitoxic effects.The C. jejuni cdtB gene was amplified and inserted into the expression plasmids pET-30a( + ) and pGEX-6p-1. The purified rGST-CdtB protein was used as the immunogen to screen hybridoma cells for mAbs against the protein. The mAb titers were determined with an indirect enzyme-linked immunosorbent assay (ELISA), and their specificity with a Dot-ELISA and western blotting analysis. We determined the antitoxic properties of the mAbs in CaCo-2 and HD-11 cells.Recombinant expression plasmids pET-30a (+)-cdtB and pGEX-6p-l-cdtB were successfully constructed, and fusion proteins rHis-CdtB and rGST-CdtB expressed, respectively. Five hybridoma cell lines, designated 1F3, IF5, 2E4, 2E11, and 2F2, were screened for the stable secretion of mAbs against CdtB. The immunoglobulin subclass of 2E11 was IgG2b and that of the other mAbs was IgG1. The mAb titers in the ascites fluids were 1:1 x 10(8) on indirect ELISA. Dot-ELISA demonstrated that the five mAbs reacted specifically with C. jejuni. Western blotting analysis confirmed that the five mAbs reacted well with the rGST-CdtB fusion protein. The mAbs significantly reduced the adhesion and invasion capacities of the bacterium in CaCo-2 cells (P < 0.01).The successful preparation of five mAbs specific for the CdtB protein will allow further study of the biological characteristics of CdtB and the pathogenesis of C. jejuni.Keywords:
Cytolethal distending toxin
Immunogen
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We aimed to prepare rabbit polyclonal and mouse monoclonal antibodies(mAbs) against human FHR4 and characterize these antibodies'properties for functional studies.Firstly we amplified FHR4 fragment from human liver cDNA library,and then constructed recombinant expression vectors pGEX-4T-1-FHR4 and PET-32a-FHR4.GST-FHR4 fusion protein was expressed in E.coli and then used as the immunogen.Properties of antiserum against human FHR4 were identified by ELISA and Western blot,while the mAbs against human FHR4 was characterized by Western blot,indirect immunofluorescent staining and immunohistochemistry staining.The GST-FHR4 fusion protein was highly expressed with molecular weight of 58 000;Western blot analysis proved the rabbit polyclonal antibody could specifically recognize proteins of 37 000 and 45 000 in expressed human liver total protein.All of the two established mAbs could recognize recombinant human FHR4 protein,and specifically bind to proteins in the cytoplasm of HepG2 cells or adult hepatocytes.In this study,polyclonal and monoclonal antibodies against human FHR4 are successfully prepared,which will provide efficient affinity reagents for functional studies of FHR4.
Polyclonal antibodies
Immunogen
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To prepare the immunological recombinant heat-stable enterotoxin(STp) and the monoclonal antibodies(MAb)against STp, a gene concatemer of estp5-his was constructed by five estp copies of tandem repeat, and inserted into p GEX-6p-1.The recombinant protein STp5-His(r STp5-His) was expressed. The r STp5-His was used as immunogen, the MBP-5STp as detection antigen of ELISA, and the positive hybridoma cells were prepared and screened. The specificity of the MAb obtained was examined by western blot assay and blocking ELISA. The results showed that the r STp5-His was expressed mainly in the form of inclusion bodies(about 38 ku) and it has immunogenicity. Using it as immunogen, four MAbs(C3, G1, F3 and G9) were prepared and all of them were able to react with natural STp, and with high specificity. This research conformed that the immunological recombinant protein of STp can be prepared by multicopy tandem repeat of estp, and the MAb prepared in this study there is the application value for detection of natural STp and recombinant protein of STp.
Immunogen
Hybridoma technology
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To express the human recombinant PCGF1 protein and prepare monoclonal antibody (mAb) against it.The recombinant expression plasmid pET32a-His-PCGF1-128/189 was made and transformed into E.coli (BL21), and then the recombinant fusion protein His-PCGF1-128/189 was expressed and purified. The BALB/c mice were immuned with purified protein His-PCGF1-128/189 as antigen. The mAbs against PCGF1 were prepared by using standard hybridoma technique. The hybridoma cell lines were obtained by ELISA and Western blot screening procedure, the isotype of the mAbs were further identified by immune-double diffusion. Ascites were prepared from one propagated hybridoma cell line and mAbs were purified by using the Kit from Millipore. The valence of mAb was detected by Western blot.The recombinant protein His-PCGF1-128/189 was expressed and purified. Two hybfidmas producing antibodies against PCGF1 were obtained, the isotypes of two mAbs were IgG1, Western blot showed that the antibodies were high sensitive(1:6 000) and high specific for PCGF1.The anti-PCGF1 mAb prepared by using recombinant His-PCGF1-128/189 protein as antigen can be used for detecting PCGF1 proteins which are either endogenous or exogenous.
Isotype
Myc-tag
Protein G
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The goose parvovirus(GPV) VP3 protein expressed in the recombinant E.coli Rosetta(DE3) was used as immunogen after purification by Ni-NTA.The 4 to 6 weeks-old BALB/c mice were intraperitoneally immunized with the VP3 protein for three times,then myeloma cells SP2/0 were fused with the spleen cells of the immunized BALB/c mice on day 3 post-last-immunization.Four hybridoma cell lines against the VP3 protein were obtained by screening with the indirect ELISA,named 1F1,2A9,3B11 and 4A2,respectively.The first monoclonal antibody(McAb) were identified to be IgG2b,and the others to be IgG1 with κ light chain.The ELISA titer of the 4 McAbs ascites were 1∶819 200,1∶1 638 400,1∶409 600 and 1∶819 200,respectively.Western-blot analysis showed that the four McAbs could react with the GPV recombinant VP3 protein specifically.IFA showed that the four McAbs could combine with the natural GPV.
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Aim To clone and express human alanine aminotransferase 2 (ALT2) in E.coli Rosetta (DE3), and to prepare monoclonal antibodies(mAb) against ALT2 for diagnostic purpose. Methods The gene encoding alanine aminotransferase 2 (ALT2) was cloned from hepatoma carcinoma cell by RT-PCR, and then inserted into pET28a vector. Recombination plasmids (pET28a-ALT2) were transformed into E.coli BL21. Human ALT2 was expressed as His-tagged fusion proteins and purified by immobilized Ni(2+);-affinity chromatography. The purified fusion ALT2 protein was used as an antigen to prepare mAb against it. Results The fusion ALT2 protein was expressed in recombinant E.coli Rosetta (DE3). The enzymatic activity of purified His-tag ALT2 is over 10 000 U/L. Mice were immunized with the purified fusion ALT2 protein, and 5 mAbs against ALT2 were generated. Conclusion Two mAbs with high specificity for ALT2 were selected for further quantitative diagnostic reagent development.
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To prepare the monoclonal antibody (mAb) against tissue inhibitor of metalloproteinases I (TIMP-I) fusion protein.TIMP-I gene was amplified from fibrotic human liver tissue by RT-PCR, then ligated with pQE31 to form recombinant plasmid pQE-TIMP-I and transformed into E. coli BL21. The protein induced by IPTG was purified by 6 x His-tag and used to immunize the BALB/c mice. The specific monoclonal antibodies (mAbs) were prepared by the cell fusion technique. Western Blot were used to detect specificity of mAbs.The prokaryotic plasmid expressing the recombinant protein was constructed, and the TIMP-I recombinant protein was expressed and purified. Four hybridoma cell lines that secreted anti-TIMP-I mAbs were obtained. 3 of 4 mAbs were the IgG1 subtype. Western Blot indicated the mAbs showed specific combination with TIMP-I protein.The TIMP-I recombinant protein is highly purified and has strong antigenicity. The anti- TIMP-I mAbs were prepared successfully.
Antigenicity
Myc-tag
Protein A/G
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Porcine interleukin-6 (PIL-6) protein without signal peptide was expressed as a glutathione S-transferase (GST) fusion protein in Escherichia coli. The fusion protein was expressed in an insoluble fraction, however, it was solubilized by refolding procedure using urea. From the solubilized protein, the recombinant PIL-6 (rPIL-6) was purified by a batch method using glutathione sepharose 4B and PreScissionTM protease cleavage. By the B3B1 hybridoma cell proliferation assay, biological activity of the purified rPIL-6 was confirmed. Three monoclonal antibodies (MAbs) named 2B-1, 5A-8 and 4C-3 were generated by using the rPIL-6 as an immunogen. Immunoglobulin isotypes of the MAbs were IgG2a (4C-3) and IgG2b (2B-1 and 5A-8). For the epitope analysis, additive enzyme-linked immunosorbent assay and immunoblot analysis using deletion mutants of PIL-6 were performed. These experiments revealed that the two MAbs (2B-1 and 5A-8) recognize an overlapped epitope and the other (4C-3) recognizes a distinct epitope, and all epitopes reside in the region of aa26-64 of PIL-6.
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Objective To prepare and identify the monoclonal antibody(MAb) against human Cu/Zn SOD.Methods BALB/C mice were immunized with immunogen human Cu/Zn SOD.The spleen cells of the immunized mice were isolated and fused with SP2/0 cells.After several rounds of detecting and cloning,a hybridoma cell line secreting anti-human Cu/Zn SOD MAb was obtained.Its specificity was evaluated with indirect-ELISA,spot-ELISA,Western blot and immunohistochemistry.The titer,immunoglobulin subtype and affinity of the MAb were measured.Results One cell line of hybridoma named 4H10H4B12 was obtained,which was identified that the heavy and light chains of anti-human Cu/Zn SOD MAb were IgG2a and κ,respectively,with high affinity of 1.101×109 L/mol and high titer of 1∶256,000.The spot-ELISA,Western blot revealed that the MAb was specifically against the immunogen and native proteins expressed in HL-7702 cells,human hematids and didn't react with rat Cu/Zn SOD or human Mn SOD.The cell immunohistochemistry proved that the MAb could recognize the human Cu/Zn SOD located in kytoplasm of HL-7702 cells.Conclusion The success in mouse anti-human Cu/Zn SOD MAb preparation provides the basis for further study of AAA.
Immunogen
Hybridoma technology
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To prepare and apply monoclonal antibody (mAb) against human LOC339524 protein.The non-glycosylated antigenic gene sequence of the LOC339524 protein was expressed in triplicate to enhance immunogenicity. Then this synthetic gene was connected to pET28a plasmid. Recombinant LOC339524 protein was obtained by E.coli expression system and was administered intraperitoneally as an immunogen to BALB/c mice to obtain mAb. The specificity and titer of the mAb were characterized by ELISA. Recombinant LOC339524 protein was identified through Western blotting. The expression of the LOC339524 protein in human myocardial tissues and H9C2 cells were detected by immunohistochemistry, and its level in the sera of patients with different heart diseases was detected with the antibody.We obtained two hybridoma cell lines, 5-D3 and 4-F8, secreting specific mAbs against LOC339524 protein. The titer of 5-D3 was up to 2×10(6), higher than the titer of 4-F8. Western blotting and immunohistochemistry demonstrated that 5-D3 could specifically recognize LOC339524 protein of Homo sapiens and Rattus norvegicus. With the antibody we obtained, we successfully detected the serum level of LOC339524 protein in patients with different heart diseases.The mAb against human LOC339524 protein with good specificity and high titer has been successfully prepared.
Immunogen
Protein A/G
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Objective To clone recombinant human IL-1B gene and express with a prokaryotic expression system and to prepare the monoclonal antibody against it.Methods The cDNA of HL-60 cell line was used as a template,the human IL-1β gene was detected by PCR.IL-1β expressed by a prokaryotic expression system was purified.Mice were immunized with purified fusion protein IL-1β for three times.The specificity and sensitivity of anti-human IL-1β monoclonal antibody were characterized by Western blot and indirect ELISA.Results Fusion protein IL-1β was highly expressed in E.coli with a molecular weight of about 51kd,its purity was 95%.Western blot and FACS results showed that the monoclonal antibodies could specifically recognize the target protein expressed in the E.coli expression system and HL-60 cell line.Conclusions The IL-1βsene is successfully cloned,the fusion protein of which and the mAb against recombinant human IL-1β are prepared,which provids a useful tool for laboratory and clinical research.
clone (Java method)
Cloning (programming)
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