Molecular characterization of Hor v 9. Conservation of a T-cell epitope among group IX pollen allergens and human VCAM and CD2.
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We have cloned, sequenced and expressed a recombinant group IX pollen allergen from barley (Hordeum vulgare). Hor v 9 is a polypeptide of 313 amino acids. The Hor v 9 cDNA clone was engineered into the E. coli protein expression vector pMAL and expressed as a fusion of maltose binding protein and truncated Hor v 9. Polyclonal antibodies to the fusion protein were raised in mice. Cross-reactive proteins, RNA and DNA homologues were found in many agricultural species including wheat, rye, triticale, oats, maize, sunflower and flax. The presence of group IX-like proteins in a variety of agricultural crops may represent a previously uncharacterized aeroallergenic occupational hazard. Sequence comparisons of the barley allergen, Hor v 9, with Poa p 9 and other cloned group IX pollen allergens revealed putative structural domains common to all. These include a signal peptide, two conserved immunoglobulin-like motifs, a 150 amino acid highly conserved carboxyterminal domain and a carboxyterminal transmembrane helix. This structural arrangement is also found in cell adhesion molecules. The highly conserved T-cell epitope previously characterized and mapped in group IX allergens (and present in Hor v 9) was found in several human cell adhesion molecule sequences (VCAM, NCAM and CD2). This T-cell epitope corresponded to the most highly conserved amino acid residues common to all group IX homologues sequenced to date. CD2 and VCAM are known to play a role in allergic inflammation: VCAM is involved in the recruitment of lymphocytes to sites of inflammation, and cross-linking CD2 leads to T-cell activation. We anticipate that the similar structural arrangement of group IX allergens and human cell adhesion molecules, as well as the presence of a T-cell epitope common to group IX pollen allergens and cell adhesion molecules, will have important consequences in the natural history of the atopic immune response.Keywords:
Polyclonal antibodies
Allergic Inflammation
Conserved sequence
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Objective:To prepare anti-NRBP antibody and study the titers and specificity of this antibody.Method:The full-length cDNA or cDNA encoding the first 99 Aa at N terminal of human NRBP was amplified from pEF-NRBP plasmid by PCR and then subcloned into prokaryotic expression vector pET-21a or pGEX-6P-1.The recombinant plasmids were then transformed into E.coli BL21 and induced.The fusion proteins purified were used to immune rabbit.The titers and specificity of the generated antibodies were analyzed by indirect ELISA and Western blot.Result:The human NRBP cDNA was obtained successfully.The recombinant NRBP or NRBP(1-99Aa) can be expressed by IPTG induction,the purified proteins were used to immune rabbit.The result of ELISA was positive,and the titers of antibody from four different immunized rabbits were from 1:5 120 to 1:40 000.Western blot analysis showed that the antibody can detect exogenous and endogenous NRBP protein(about 60 kDa) and had strong ability of immunoprecipitation.Conclusion:The anti-NRBP antibody was approved having good specificity and sensitivity.The polyclonal antibody of NRBP provided an important tool to study the function of NRBP.
Polyclonal antibodies
Antibody titer
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To obtain the expression products of goat IFN-γ,total RNA was extracted from activated goat PBMCs,and cDNA was synthesized and used as template for PCR.The recombinant cloning vector was constructed.The sequence encoding mature peptide region was cloned by PCR and inserted into the expression vector pET-32 a,and the recombinant plasmid was transformed into the competent cell BL21(Codon Plus),followed by sequencing and analysis.After induced by IPTG,the expression products were and analysized by SDS-PAGE and purified by Ni-NTA.The purified protein was immunize rabbits to prepare polyclonal antibodies.Sequence analysis suggested that sequence encoding the mature peptide was 432 bp.SDS-PAGE indicated that the fusion protein was about 34.9ku,and a large amount of soluble protein could be expressed when induced with a total concentration of 0.3mmol/L/L IPTG for 6hat 30℃.The titter of polyclonal antibodies was 1∶106 detected by indirect ELISA.
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Cloning (programming)
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In previous studies a subset of complement-component-C3 (C3) epitopes, C3(D), expressed in denatured and surface-bound C3 and C3 fragments, has been described. These epitopes were detected by antibodies raised against denatured C3. In the present study we used a cDNA expression strategy to localize epitopes recognized by monoclonal and polyclonal anti-C3(D) antibodies. First, DNAse I digestion of C3 cDNA was used to generate 200-300 bp fragments. These cDNA fragments were expressed as beta-galactosidase-C3 fusion proteins using the lambda gt11 vector. The fusion proteins were tested by Western-blot analysis for reactivity with monoclonal and polyclonal anti-C3 antibodies, and the location of the epitopes were determined by sequencing the cDNA fragments. Affinity-purified polyclonal anti-C3(D) antibodies specific for denatured C3 reacted strongly with the C3 fusion fragments corresponding to segments of the 40 kDa subunit of C3c (residues 1477-1510) and the C3d fragment (residues 1117-1155 and 1234-1294) of C3. Adsorption of the polyclonal antibodies with a mixture of EAC3b and EAC3bi (degradation fragments of C3 bound to sheep erythrocytes) abolished binding to fusion proteins spanning the C3d region, but not the 40 kDa fragment of C3c. No effect was seen with the corresponding soluble C3 fragments. The monoclonal anti-C3(D) antibodies (mAbs) 7D326.1 and 7D331.1, specific for EAC3b and EAC3bi, bound to a fusion protein corresponding to amino acid residues 1312-1404, whereas mAb 7D9.2, specific for EAC3d, reacted with a fusion protein spanning amino acid residues 1082-1118. mAbs 4SD11.1 and 4SD18.1, which did not bind to any physiological C3 fragment, detected a fusion protein covering residues 1477-1510. In summary, the segments of C3 represented by amino acid residues 1082-1118, 1117-1155, 1234-1294 and 1312-1404 accommodate C3(D) epitopes that are expressed by erythrocyte-bound C3 fragments, but not by the corresponding fluid-phase fragment, whereas the segments spanning residues 973-1026 and 1477-1510 contain C3(D) epitopes that are exposed exclusively in denatured C3 and therefore hidden in physiological fragments of the protein.
Polyclonal antibodies
Epitope mapping
Linear epitope
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Polyclonal antibodies
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Angioarrestin is one kind of potential antiangiogenesis factors.By DNA recombinant technique to construct E.coli BL21(DE3) expressed plasmid pMAL-C_2-hFD,and hFD cDNA fused to the 3'end of the gene encoding the MBP protein.The fusion protein was expressed in E.coli BL21(DE3)at 37℃ after 0.3 mmol/L IPTG induction for 4 hours.The fusion protein of high expression has a molecular weight 66 kD by SDS-PAGE.The yield of fusion protein was 20 percent in total proteins measured by SDS-PAGE.Supernatant of purified recombinant protein was used as immunogen to prepare the polyclonal antibody.The result of ELISA shows that antibody potency is 1∶10240.The fusion protein highly expressed in E.coli BL21(DE3)posses well antigen activity.And preparation of polyclonal antibody will provide material for further studies of angioarrestin.
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Polyclonal antibodies
Protein A/G
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To express and purify HIV—1 fusion multi-epitope gene in E.coli and to prepare polyclonal antibody.p24 gene was amplified by PCR, with the plasmid containing full-length of cDNA of HIV—1 standard strain as the template. The p24 gene and the gene coding cryptic epitopes by amino modified acid substitution were inserted into pET32a, obtained the recombinant plasmid pET32a/p24MEG. The recombinant vector was transformed into BL21(DE3). After induction, induced fusion protein was purified by purified with Ni2+-NTA affinity column. The purified protein was injected into a rabbit. And the titer of the rabbit’s anti-serum was measured by ELISA. The results stowed that p24 gene fragment and recombinant plasmid pET32a/p24MEG coding multi-CTL epitope gene and p24 gene were obtained successfully. The p24MEG fusion protein was successfully purified and the rabbit's anti-serum with high titer was obtained.The preparation of the purified p24MEG fusion protein and polyclonal antibody can be used for further investigation.
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Chimeric gene
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Objective: To obtain purified recombinant interleukin-28 of mouse(mIL-28) and prepare its polyclonal antibody.Methods: The cDNA fragment coding for mature mIL-28 protein was amplified by PCR and cloned into vector pET-30 to construct fusion expression vector pET-30a-mIL-28.After pET-30a-mIL-28 was transformed into E.coli BL21(DE3),the bacteria were induced by IPTG.The expressed mIL-28 fusion protein was purified by Ni-NTA affinity chromatography.Six to eight weeks old chickens were immunized with the purified protein for obtaining the antiserum.The titers of antibodies were measured by ELISA. Results: The DNA sequencing showed that the expression vector pET-30a-mIL-28 was constructed successfully.After induced by IPTG,the expressed mIL-28 fusion protein in E.coli cultured at 37℃ appeared a single band on SDS-PAGE.The result of ELISA indicated that the prepared polyclonal antibody had high titer and specificity. Conclusion: The purified recombinant interleukin-28 of mouse and polyclonal antibody have been acquired.
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To investigate the function of DJ-1 gene of chicken , a panel of polyclonal antibodies against DJ-1 was needed. In this paper , the CDS region of DJ-1 from cDNA of DF-1 cells were amplified by PCR and cloned it into pET-32a for prokaryotic expression. The BALB/c mice were immunized with the expressed recombinant protein. The results showed that the recombinant DJ-1 fusion protein was abundant and soluble under 3 h induced with 0.4mmol/L IPTG at 37 ℃. ELISA and Western blot results demonstrated that the anti-serum from mice specifically reacted with the recombinant DJ-1. The sera will support the further study of DJ-1 function in the avian leukosis virus subgroup J infection.
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Haven
Section (typography)
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According to the cDNA sequence (FJ608551)of the porcine Fc gamma receptor ⅡB's(swFcγ RⅡB's) RNA transcript variant in GenBank,a pair of primers was designed using Primer5.0 software.The gene was amplified from swFcγRⅡB abscised intracellular region and transmembrane domain.The PCR product was digested with KpnⅠ and EcoRⅠ.The truncated swFcγRⅡB gene was cloned into pET-32a vector to generate the recombinant plasmid pET-swFcγRⅡB for expression.The swFcγRⅡB-His fusion protein was successfully expressed with IPTG induction,and the protein predominantly existed as inclusion bodies in the cytoplasm.After expression in Escherichia coli and purification,the purified protein was used to immunize mice to prepare antisera.The results of ELISA and Western-blotting indicated that the prepared polyclonal antibodies had high titer and specificity.
Polyclonal antibodies
Inclusion bodies
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