Anti-mouse Serum Research Articles

Molecular characterization of an actin depolymerizing factor fromCryptocaryon irritans

Actin depolymerizing factors regulate actin dynamics involved in cellular processes such as morphogenesis, motility, development and infection. Here, a novel actin depolymerizing factor gene (CiADF 2 ) was cloned from the cDNA library of Cryptocaryon irritans, a parasitic ciliate causing cryptocaryonosis. The full-length cDNA of CiADF 2 was 531 bp. Its open reading frame (ORF) was 417 bp, encoding a polypeptide of 138 aa with typical features of the ADF/cofilin family. Reverse transcription-PCR suggested that CiADF 2 is expressed in all stages of the life cycle. After site-directed mutagenesis of a non-universal genetic code, the ORF was subcloned in Escherichia coli. The bacteria were induced with the addition of isopropylthio-β-D-galactoside to express a fusion protein of recombinant CiADF2 (rCiADF2) with glutathione S transferase. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blot confirmed the predicted molecular mass of rCiADF2 of 16·2 kDa. A mouse antibody against rCiADF2 recognized native CiADF2, and rCiADF2 reacted with mouse antisera against C. irritans trophonts. CiADF2 was abundant in the plasma around cytostomes, suggesting that CiADF2 is involved in ciliate movement. Moreover, rCiADF2 showed F-actin binding and depolymerizing activity. This study will help to clarify the pathogenic biology of the parasite and develop effective control measures for cryptocaryonosis.

Production of antisera against porcine haptoglobin: Potential for distinguishing haptoglobin subunits

Haptoglobin (Hp) is one of the acute phase proteins (APPs) that help to alleviate the immune oxidative damage. The present study expressed a truncated porcine Hp in Escherichia coli and produced rabbit and mouse antisera to the recombinant protein, in order to investigate Hp levels in sera from piglets infected with porcine reproduction and respiratory syndrome virus (PRRSV). Antisera prepared revealed both chains of porcine Hp in Western blot, and mouse antisera showed stronger binding activities than the rabbit antisera. With the combination of Hp monoclonal antibodies, this study confirmed that serum Hp was increased in piglets infected with PRRSV and offered a tool to know about subunit levels of Hp in porcine serum. But Hp itself could not be used as a specific biomarker for PRRSV infection, for elevated Hp levels were also obtained from pigs infected with other pathogens.

Characterization of immunologic and adhesive abilities of Mycoplasma pneumoniae P1 protein segment

Objective To determine the immunogenic and adhesive abilities of a segment (P1C protein) that located at the carboxy terminal region of P1 protein (1125 to 1395 amino acids).Methods A recombinant prokaryotic vector (pGEX6p-2/p1c) was constructed for P1C protein expression in E.coli BL21DE3.The expressed target recombinant protein (rP1C) was identified using SDS-PAGE and Western blot assay,and then extracted by GST-based affinity chromatography.The purified rP1C was used to immunize BALB/c mice to obtain rP1C-antiserum and titer of the antiserum was determined by ELISA.Immunoreactivity of the rP1C to the sera form M.pneumoniae-infected patients was detected using Western blot assay,while activity of the rP1C adhering to HeLa cells as well as adhesion blockage of the rP1C antiserum were detected using indirect immunofluorescence assays.Results The constructed prokaryotic expression system could efficiently express soluble rP1C with a relative molecular weight of 66×103.The antiserum from rP1Cimmunized mice showed an ELISA titer as high as 1:64 000.Both the M.pneumoniae-infected patients' sera and the mouse antiserum against rP1C could recognize as well as combine with the rP1C.rP1C could adhere to HeLa cells and the adhesion could be blocked by the mouse antiserum with an antiserum concentration-dependent manner.Conclusion P1C,a segment of M.pneumoniae P1 protein,possesses powerful immunogenicity and immunoreactivity and cell-adhered activity,indicating the protein segment can be used as an antigen candidate for developing vaccines and serological diagnostic methods of M.pneumoniae-induced diseases. Key words: Mycoplasma pneumoniae; P1 protein segment; Immunologic activity; Adhesion

Characterization and expression of cathepsin B2 in Fasciola gigantica

Fasciola gigantica cathepsin B belongs to a family of cysteine proteases which is involved in invasion of host tissues. In this study, the recombinant cathepsin B2 (rFgCatB2), synthesized in Pichia pastoris, showed enzymatic activity on a fluorometric substrate Z-Phe-Arg-AMC and gelatin. Furthermore, this recombinant enzyme could degrade IgG and type I collagen. Mouse antiserum against rFgCatB2 reacted with the native FgCatB2 in whole body (WB) extracts of metacercariae (MET), newly excysted juveniles (NEJ) and 2week-old juveniles, but not in 3, 4week-old juveniles and adult flukes. Immunolocalization showed the presence of cathepsin B2 only in the caecal epithelium of MET, NEJ and 2week-old juveniles. Co-localization of FgCatB2 and a prominent antigen of NEJ, FgCatB3, revealed that these proteins were expressed at the same regions in the caecal epithelium. Anti-rFgCatB2 showed no cross reaction with the other parasites’ antigens by Western blotting. These findings suggest that CatB2 is expressed only in early stages of the parasite and may be involved in digestion of host connective tissues and evasion of the host immune system during their penetration and migration. Thus, CatB2 could be considered as an immunodiagnostic and vaccine candidate for fasciolosis.

Identification and characterization of a novel Neospora caninum immune mapped protein 1

Immune mapped protein 1 (IMP1) is a newly discovered protein in Eimeria maxima. It is recognized as a potential vaccine candidate against E. maxima and a highly conserved protein in apicomplexan parasites. Although the Neospora caninum IMP1 (NcIMP1) orthologue of E. maxima IMP1 was predicted in the N. caninum genome, it was still not identified and characterized. In this study, cDNA sequence encoding NcIMP1 was cloned by RT-PCR from RNA isolated from Nc1 tachyzoites. NcIMP1 was encoded by an open reading frame of 1182 bp, which encoded a protein of 393 amino acids with a predicted molecular weight of 42.9 kDa. Sequence analysis showed that there was neither a signal peptide nor a transmembrane region present in the NcIMP1 amino acid sequence. However, several kinds of functional protein motifs, including an N-myristoylation site and a palmitoylation site were predicted. Recombinant NcIMP1 (rNcIMP1) was expressed in Escherichia coli and then purified rNcIMP1 was used to prepare specific antisera in mice. Mouse polyclonal antibodies raised against the rNcIMP1 recognized an approximate 43 kDa native IMP1 protein. Immunofluorescence analysis showed that NcIMP1 was localized on the membrane of N. caninum tachyzoites. The N-myristoylation site and the palmitoylation site were found to contribute to the localization of NcIMP1. Furthermore, the rNcIMP1-specific antibodies could inhibit cell invasion by N. caninum tachyzoites in vitro. All the results indicate that NcIMP1 is likely to be a membrane protein of N. caninum and may be involved in parasite invasion.

Predictors of long-term outcome from intraperitoneal radioimmunotherapy for ovarian cancer.

Data was analyzed from 92 patients > 5 years after intraperitoneal (IP) radionuclide therapy (RIT) with (90)Y- or (177)Lu-CC49 to determine prognostic factors. Patients had CC49 antibody-reactive ovarian cancer confined to the abdominal cavity after primary debulking and chemotherapy. The first 27 patients received IP (177)Lu-CC49 alone; the remainder received Interferon (IFN), to increase the expression of the tumor-associated glycoprotein-72 (TAG-72) antigen, +/- IP paclitaxel (25-100 mg/m(2)) 2 days before RIT. Factors assessed by univariate (and some multivariate) analysis included age, race, body size, interval between initial diagnosis and RIT, interval between 2nd look surgery and RIT, (90)Y versus (177)Lu, MBq dose, paclitaxel dose, grade of tumor, extent of initial surgery, size of disease deposits prior to RIT, intensity of TAG reactivity, the addition of unlabeled antibody, and the development of human anti-mouse antibody and/or serum sickness after murine antibody. A statistically significant improvement in progression-free survival (p ≤ 0.05) was noted for less bulky disease and younger age. Administration of paclitaxel plus IFN, an immune response, and use of (90)Y showed a favorable nonsignificant trend. Dose escalation of radionuclide did not change risk of progression; thus, this therapy may have therapeutic efficacy at modest dose levels.

Plasmodium vivax gametocyte protein Pvs230 is a transmission-blocking vaccine candidate

The malaria transmission-blocking vaccine (TBV) aims to interfere the development of malaria parasite in the mosquito and prevent further transmission in the community. So far only two TBV candidates have been identified in Plasmodium vivax; ookinete surface proteins Pvs25 and Pvs28. The pvs230 (PVX_003905) is reported as an ortholog of Pfs230, a gametocyte/gamete stage TBV candidate in Plasmodium falciparum, however its candidacy for TBV has never been tested. Therefore here, we have investigated whether Pvs230 can be a TBV candidate using P. vivax samples obtained from Thailand. The mouse antiserum raised against the plasmid expressing CRDs I-IV of Pvs230 detected Pvs230 protein in the lysate of P. vivax gametocyte in western blot analysis under non-reducing condition. From the localization of Pvs230 on the outer most regions of gametocyte in the immunofluorescence assay, it appears that Pvs230 is localized on the surface of gametes. Importantly, the anti-Pvs230 mouse serum significantly reduced the number of P. vivax oocysts developed in the mosquito midgut. Moreover, the polymorphism in Pvs230 CRDs I-IV is limited suggesting that it may not be an impediment for the utilization of Pvs230 as an effective TBV candidate. In conclusion, our results show that Pvs230 is a transmission-blocking vaccine candidate of P. vivax.

Preparation of a polyclonal antibody against immunogenic fragment of bovine intestinal peptide transporter I (<i>b</i>PepTI)

The goal of the current study is to prepare polyclonal antibody against bovine intestinal peptide transporter I (bPepTI) in order to develop assay for immunological assessment of protein levels. Antigenicity of the entire bPepTI was analyzed with DNAStar, and a fragment with high antigenicity (bPepTI ORF 1369 - 1695) was selected, cloned in pGEX-6p-1 vector, resulting in a recombinant plasmid GST-BP, which verified by double enzyme digestion and sequenced, the recombinant plasmid was introduced to BL21. Exogenous expression was induced by IPTG and validated by Western blot analysis. The recombinant protein was isolated, purified and used for production of antiserum in mice. The specificity of antiserum was evaluated with immunobloting and titer was estimated with ELISA. Results indicate that the antibody against bPepTI was produced. The optimal GST-BP antigen embedding concentration was 0.5 μg/ml. The optimal dilution was 1:400. An indirect ELISA assay indicates the effective dilution was 1:102400.

The sphingosine-1-phosphate receptor agonist FTY720 prevents the development of anti-glomerular basement membrane glomerulonephritis

The sphingosine-1-phosphate (S1P) agonist FTY720 prolongs the survival of organ allograft and attenuates autoimmune-mediated injury in experimental models. Most cases of glomerulonephritis (GN) in human appear to be immunologically initiated. In this study, we evaluated the potential therapeutic role of FTY720 in GN via a mouse anti-glomerular basement membrane (GBM) model. Mice were immunized with rabbit IgG in complete Freund's adjuvant (CFA) followed by an intravenous injection of a rabbit anti-mouse GBM serum. Disease and immune responses were assessed on day 14. Mice were treated with FTY720 (0.3 or 3mg/kg) and prednisone (10mg/kg) from days 0 to 14. The S1P modulator reduced proteinuria, serum creatinine, crescent formation and serum IgG level. The expressions of splenic S1P receptor and renal Th-1 cytokine were also inhibited at the transcription stage. Treatment with FTY720 increased splenocyte production of protective Th2 cytokine IL-4 and promoted the apoptosis of splenic CD4+ T cells in the animal models, which suggests that FTY720 played a protective role at the induction stage of GN by inhibiting mRNA expressions of splenic S1P receptor 1, S1P receptor 2, and S1P receptor 5.

The Mycoplasma gallisepticum α-enolase is cell surface-exposed and mediates adherence by binding to chicken plasminogen

The α-enolase protein is reported to be an adhesin in several pathogenic bacterial species, but its role in Mycoplasma gallisepticum is unknown. In this study, the M. gallisepticum α-enolase gene was adapted to heterologous expression in Escherichia coli by performing overlapping polymerase chain reaction with site-directed mutagenesis to introduce A960G and A1158G mutations in the nucleotide sequence. The full-length mutated gene was cloned into a pGEM-T Easy vector and subcloned into the expression vector pET32a(+) to construct the pET-rMGEno plasmid. The expression of rMGEno in E. coli strain DE3 was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis with Coomassie blue staining. Purified rMGEno exhibited α-enolase catalytic activity that it could reflect the conversion of NADH to NAD+. Mouse antiserum to α-enolase was generated by immunization with rMGEno. Immunoblotting and immunofluorescence assay with the antiserum identified α-enolase on the surface of M. gallisepticum cells. Enzyme-linked immunosorbent assay characterized rMGEno as a chicken plasminogen binding protein. An adherence inhibition assay on immortalized chicken fibroblasts (DF-1) demonstrated more than 77% inhibition of adhesion in the presence of mouse antiserum, suggesting that α-enolase of M. gallisepticum participates in bacterial adhesion to DF-1 cells.

Development of enzyme-linked immunosorbent assay for Δ 12-prostaglandin J 2 and its application to the measurement of the endogenous product generated by cultured adipocytes during the maturation phase

Peroxisome proliferator-activated receptor (PPAR)γ is a well-known master regulator for the differentiation and maturation of adipocytes. Prostaglandin (PG) D 2 can be produced in adipocytes and dehydrated to J 2 series of PGs including 15-deoxy-Δ 12,14-PGJ 2 (15d-PGJ 2) and Δ 12-PGJ 2, which serve as pro-adipogenic prostanoids through the activation of PPARγ. However, the quantitative determination of Δ 12-PGJ 2 has not been attempted during the life stage of adipocytes. In this study, we developed an enzyme-linked immunosorbent assay using mouse antiserum specific for Δ 12-PGJ 2. According to the standard curve, the amount of Δ 12-PGJ 2 can be measured from 0.5 pg to 14.4 ng in an assay. Our antiserum did not recognize most other prostanoids including 15d-PGJ 2, while it only showed the cross-reaction of 28% with unstable PGJ 2. This immunological assay was applied to the determination of the endogenous formation of Δ 12-PGJ 2 in cultured 3T3-L1 adipocytes during the maturation phase. The ability of cultured adipocytes to form endogenous Δ 12-PGJ 2 increased gradually at an earlier stage of the maturation phase and detectable at higher levels than 15d-PGJ 2. Treatment of cultured cells with either aspirin or indomethacin, a general cyclooxygenase inhibitor, significantly reduced the production of endogenous Δ 12-PGJ 2 in the maturation medium as expected. Furthermore, we evaluated individually the exogenous effects of PGJ 2 series at various doses on adipogenesis during the maturation phase. Although Δ 12-PGJ 2 was slightly less potent than 15d-PGJ 2, each of these PGJ 2 series rescued effectively both the accumulation of fats and the gene expression of typical adipocyte-markers that were attenuated in the presence of aspirin. Taken together, our findings indicate that endogenous Δ 12-PGJ 2 contributes substantially to the up-regulation of adipogenesis program through the activation of PPARγ together with 15d-PGJ 2 during the maturation phase of cultured adipocytes.

Protection of mice against enterotoxigenic E. coli by immunization with a polyvalent enterotoxin comprising a combination of LTB, STa, and STb

Currently available enterotoxigenic Escherichia coli (ETEC) vaccines are based on colonization factors and/or the heat-labile enterotoxin B subunit (LTB). However, the induction of antitoxic responses against heat-stable enterotoxin a (STa) and b (STb) has merit as these two poorly immunogenic toxins are frequently associated with ETEC strains. In this study, we genetically constructed a trivalent enterotoxin fusion protein (STa-LTB-STb, abbreviated to SLS) in an effort to develop a single toxoid containing these three enterotoxins for vaccination against ETEC. Mutagenesis at one disulfide-bridge-forming cysteine in STa led to a dramatic reduction in the STa toxicity of SLS; however, the fusion peptide retained the STb-associated toxicity. Immunization of mice with SLS protein elicited significant antibody responses to LTB, STa, and STb. Significantly, the mice antisera were able to neutralize the biological activity of both STa and STb. In the experiment to assess the protective effect of SLS immunization, the mortality of mice receiving SLS was significantly lower than their control cohorts (P < 0.01) after intraperitoneal challenge with ETEC. These results show that the trivalent fusion enterotoxin SLS has the potential to serve as a useful toxin-based vaccine against ETEC-induced diarrheal disease via a single immunogen.

Immunomodulative properties of conjugates composed of detoxified lipopolysaccharide and capsular polysaccharide of Vibrio cholerae O135 bound to BSA-protein carrier

O135 serotype Vibrio cholerae isolated from Slovak river was used as a source of surface polysaccharide antigens. Following detoxification procedure, fractions of polysaccharides were separated by size exclusion chromatography. Two resultant fractions were the capsular polysaccharide (Mw ∼ 197,000 Da) and the lipopolysaccharide fragment (Mw ∼ 13,300 Da). These materials were used for preparation of four novel glycoconjugates. Two of them containing detoxified lipopolysaccharide as antigen were prepared by original chemical method using the new biocompatible polymer as carrier of antigen. Additionally, other two conjugates were prepared by direct linking of capsular and detoxified lipopolysaccharide antigens to the protein carrier using adipic acid dihydrazide spacer. The immunogenicities (induced IgM, IgG, IgA antibodies) of all conjugates were determined by enzyme-linked immunosorbent assay. Polymer containing conjugates elicited higher levels of specific anti-lipopolysaccharide IgM and IgG antibodies in comparison with other conjugates without polymer carrier. Enhanced IgM vibriocidal activity of mice antisera was also evident here.

Knockout of the gene encoding the kinetoplast-associated protein 3 (KAP3) in Trypanosoma cruzi: Effect on kinetoplast organization, cell proliferation and differentiation

Kinetoplast DNA (kDNA) of trypanosomatid protozoa consists of an unusual arrangement of two types of circular molecules catenated into a single network: (1) a few maxicircles that encode various mitochondrial enzyme subunits and rRNA in a cryptic pattern and (2) thousands of minicircles that encode guide RNAs (gRNAs). kDNA is associated with proteins, known as kinetoplast-associated proteins (KAPs), which condense the kDNA network. However, little is known about the KAPs of Trypanosoma cruzi, a parasite that displays different kDNA condensation patterns during its complex morphogenetic development. We cloned the T. cruzi gene encoding TcKAP3 (kinetoplast-associated protein 3). TcKAP3 is a single-copy gene that is transcribed into a 1.8-kb mRNA molecule and expressed in all stages of the parasite. Mouse antiserum raised against recombinant TcKPA3 recognized a 21.8kDa protein, which was found, by indirect immunofluorescence and immunoelectron microscopy, to be associated with the T. cruzi kinetoplast. Several features of TcKAP3, such as its small size, basic nature and similarity with KAP3 from the insect trypanosomatid Crithidia fasciculata, are consistent with a role in DNA charge neutralization and condensation. This suggests that this protein is involved in organizing the kDNA network. Gene deletion was used to investigate TcKAP3 function. Here we investigated the T. cruzi KAP3 null mutant, analyzing its fitness during proliferation, differentiation and infectivity.

The cross-reactivity of the enterovirus 71 to human brain tissue and identification of the cross-reactivity related fragments

BackgroundEV71 occasionally cause a series of severe neurological symptoms, including aseptic meningitis, encephalitis, and poliomyelitis-like paralysis. However, the neurological destruction mechanism was remained to be clarified. This study described the cross reaction between EV71 induced IgG and human brain tissue.ResultsCross reaction of the IgG from 30 EV71 infected patients' sera to human tissues of cerebra was observed, which suggested that some EV71 antigens could induce IgG cross-reactivity to human cerebra. To identify the regions of EV71 virus that containing above antigens, the polypeptide of virus was divided into 19 peptides by expression in prokaryotes cell. Mouse anti-sera of these peptides was prepared and applied in immunohistochemical staining with human adult and fetus brain tissue, respectively. The result indicated the 19 peptides can be classified into three groups: strong cross-reactivity, weak cross-reactivity and no cross-reactivity with human brain tissue according the cross reaction activity. Then, the increased Blood Brain Barrier (BBB) permeability and permits IgG entry in neonatal mice after EV71 infection was determined.ConclusionEV71 induced IgG could enter BBB and cross-reacted with brain tissue in EV71 infected neonatal mice, and then the peptides of EV71 that could induce cross-reactivity with brain tissue were identified, which should be avoided in future vaccine designing.

Expression of EspA in Lactococcus lactis NZ9000 and the detection of its immune effect in vivo and vitro

Enterhemorrhagic Escherichia coli (EPEC), an important cause of severe infantile diarrheal disease in many parts of the developing world, produced several recently described virulence determinations. Several of its virulence factors are secreted by type III secretion including EspA, which forms filamentous structures on bacterial surface bridging to the host cell’s surface. These structures on bacterial surfaces may deliver other virulence factors directly into the host cell from EHEC. In this study, EspA was expressed in Lactococcus lactis NZ9000 (L. lactis NZ9000). BALB/C mice were immunized by recombinant EspA, and mice sera were assayed for the disruption of E. coli O157:H7 interaction with the host cell. BALB/C mice which were immunized with recombinant EspA produced specific antibody titers, and the difference between the control group and the immune group is marked (P<0.05). The polyclonal mice antisera blocked E. coli O157:H7-induced host cell actin rearrangement and could label E. coli O157:H7 EspA filaments in vitro. Our present findings suggest that L. lactis may be a good candidate to produce oral vaccine, and thus entertains the possibility of developing EspA-oral vaccine in the defense of E. coli O157:H7 infection.

Screening and identification of dominant functional fragments of human epididymal protease inhibitor

To lay a foundation for the further development of a novel contraceptive vaccine based on epididymal protease inhibitor (Eppin) protein, possible B-cell epitopes were predicted using bioinformatics. Overlapping KLH-coupled peptides corresponding to the 89–133 amino acid domain of Eppin protein were synthesized by the Fmoc method. The male mice were immunized with various peptides resulting in a gradual elevation of specific serum IgG antibodies until they peaked at the seventh week. Treatment with antiserum in immunized mice caused a significant decrease in the sperm–egg binding rate. Eventually, antifertility assays in vivo showed that, the pregnancy rate and the number of births per labor in experimental mice were significantly decreased. The decrease in the F5 and the F4 groups were more outstanding therefore these peptides might be the dominant functional fragments of Eppin protein and provide an experimental basis for the development of effective contraceptive vaccine based on Eppin protein.

HBsAg/HBsAb double positive hepatitis B virus infection model in vitro and in vivo

The pathogenesis of HBsAg (+)/HBsAb (+) double positive hepatitis B virus infection was investigated by simulating HBsAg/HBsAb coexistence in vitro and establishing HBsAg/HBsAb double positive model in vivo. Eukaryotic expression plasmids PCI-SY, PCI-adw, PCI-adr, PCI-ayw, which expressed S gene product of different serotypes, were constructed and transfected into HepG2 cells. Recombinant proteins were purified from the transfected cells. At the same time, HBsAg mouse antiserum was obtained by immunizing mice with PCI-SY plasmid. HBsAg/HBsAb coexistence was simulated using these antigens and antiserum. Furthermore, the expression plasmids expressing different serotypes of S gene product including PCI-adw, PCI-adr, and PCI-ayw were injected into mice via tail vein. HBsAg and HBsAb in mice sera were tested at the first and 7th day respectively after antigen plasmids injection. Both in vitro simulation and in vivo animal models demonstrated that HBsAg antigen and HBsAb of the same serotypes could not coexist, but HBsAg antigen and HBsAb of different serotype could coexist. HBsAg/HBsAb double positive hepatitis B virus infection could be due to infection of viruses of different serotypes.

EspA expressed in lettuce stimulated high immunisation and had a protective effect on HeLa cells

EspA proteins are virulence factors which are expressed by attaching and effacing Escherichia coli. In this study, BALB/C mice were immunised by a lettuce-derived EspA, and mice sera were assayed for the disruption of E. coli O157:H7 interaction with the host cell. BALB/C mice which were immunised with lettuce-derived EspA produced specific antibody titres with the highest immunoglobulin A titre of 3200 and IgG titre of 12,000. The polyclonal mice antisera blocked E. coli O157:H7-induced host cell actin re-arrangement and could label E. coli O157:H7 EspA filaments in vitro. Our present findings suggest that lettuce may be a good candidate to produce plant-derived proteins, and thus signifies the possibility of developing EspA–lettuce vaccine in defence of E. coli O157:H7 infection.

Cloning, expression and immunogenicity of ferric enterobactin binding protein Fep B from Escherichia coli O157:H7

The gene coding for ferric enterobactin binding protein from E. coli O157:H7 was amplifi ed. This gene was cloned and expressed as C-terminal His (6)-tagged protein. The SDS-PAGE analysis of the total protein revealed only two distinct bands, with molecular masses of 31kDa and 34kDa. The Ni-NTA chromatography purifi ed FepB and the osmotically shocked periplasmic fraction of IPTG induced cells showed only a single band of 31 kDa. Polyclonal mouse antibody was raised against the recombinant protein during 4 weeks after immunization. Western blot analysis of the recombinant FepB with mouse antiserum revealeda single band of 31 kDa. Identification and purification of FepB helped reveal its appropriate molecular mass. Polyclonal antibody raised against the recombinant protein reacted with bacterial FepB. The recombinant protein FepB could have a protective effect against E. coli O157:H7 and might be useful as an effective vaccine.