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Find video protocols related to scientific articles indexed in Pubmed.
Construction, characterization and evaluation of the protective efficacy of the Streptococcus suis double mutant strain ?SsPep/?SsPspC as a live vaccine candidate in mice.
Microbiol. Res.
PUBLISHED: 07-08-2014
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Streptococcus suis serotype 2 (S. suis 2) causes sepsis and meningitis in piglets and humans, and results in one of the most serious bacterial diseases affecting the production of commercial pigs around the world. Due to the failure of the current inactivated vaccine to protect against the disease, development of a new attenuated live vaccine against S. suis 2 by deleting essential virulence factors is urgently needed. We have previously reported the construction and characterization of an SsPep single gene deletion mutant strain ?SsPep based on S. suis 2. Our previous results have shown that SsPep plays a critical role in the pathogenesis of S. suis 2. In this study, a precisely defined double-deletion mutant ?SsPep/?SsPspC of S. suis 2 without antibiotic-resistance markers was constructed based on ?SsPep, and the levels of virulence of the wild-type (WT) and ?SsPep/?SsPspC were compared in a mouse experimental infection model. We demonstrated that the double mutant ?SsPep/?SsPspC was less virulent than the WT, and could induce a noticeable antibody response. Analysis of IgG subclasses (IgG1 and IgG2a) indicated that both Th1 and Th2 responses were induced by ?SsPep/?SsPspC, although the IgG2a (Th1) response predominated over the IgG1 (Th2) response. Moreover, ?SsPep/?SsPspC could confer 90% protective efficacy against challenge with a lethal dose of fully virulent S. suis 2. Taken together, these data demonstrate that ?SsPep/?SsPspC can be used as an effective live vaccine and provide a novel strategy against infection of S. suis 2.
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Lithium chloride suppresses colorectal cancer cell survival and proliferation through ROS/GSK-3?/NF-?B signaling pathway.
Oxid Med Cell Longev
PUBLISHED: 02-09-2014
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Glycogen synthase kinase-3? (GSK-3?), a serine/threonine protein kinase, has been regarded as a potential therapeutic target for multiple human cancers. In addition, oxidative stress is closely related to all aspects of cancer. We sought to determine the biological function of lithium, one kind of GSK-3? inhibitors, in the process of reactive oxygen species (ROS) production in colorectal cancer. In this study, we analyzed the cell apoptosis and proliferation by cell viability, EdU, and flow cytometry assays through administration of LiCl. We used polymerase chain reaction and Western blotting to establish the effect of GSK-3? inhibition on the nuclear factor-?B (NF-?B) pathway. Results showed administration of LiCl increased apoptosis and the level of ROS in colorectal cancer cells. Furthermore, the underlying mechanisms could be mediated by the reduction of NF-?B expression and NF-?B-mediated transcription. Taken together, our results demonstrated that therapeutic targeting of ROS/GSK-3?/NF-?B pathways may be an effective way for colorectal cancer intervention, although further preclinical and clinical testing are desirable.
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Autocrine interleukin-6 drives skin-derived mesenchymal stem cell trafficking via regulating voltage-gated Ca(2+) channels.
Stem Cells
PUBLISHED: 01-25-2014
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Mesenchymal stem cells (MSCs) have demonstrated promising therapeutic potential for a variety of diseases including autoimmune disorders. A fundamental requirement for MSC-mediated in vivo immunosuppression is their effective trafficking. However the mechanism underlying MSC trafficking remains elusive. Here we report that skin-derived MSCs (S-MSCs) secrete high levels of interleukin-6 (IL-6) in inflammatory conditions. Disruption of the il6 or its signaling transducer gp130 blocks voltage-gated calcium (Ca(2+) ) channels (VGCC) critically required for cell contraction involved in the sequential adhesion and de-adhesion events during S-MSC migration. Deletion of il6 gene leads to a severe defect in S-MSC's trafficking and immunosuppressive function in vivo. Thus, this unexpected requirement of autocrine IL-6 for activating Ca(2+) channels uncovers a previously unrecognized link between the IL-6 signaling and the VGCC and provides novel mechanistic insights for the trafficking and immunomodulatory activities of S-MSCs.
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CCR6 is a prognostic marker for overall survival in patients with colorectal cancer, and its overexpression enhances metastasis in vivo.
PLoS ONE
PUBLISHED: 01-01-2014
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The chemokine receptor CCR6 has been recently shown to be associated with colorectal cancer (CRC) progression. However, the direct evidence for whether CCR6 in tumors is a prognostic marker for the survival of patients with CRC and whether it plays a critical role in CRC metastasis in vivo is lacking. Here we show that the levels of CCR6 were upregulated in CRC cell lines and primary CRC clinical samples. CCR6 upregulation was closely correlated with disease stages and the survival time of CRC patients. Knockdown of CCR6 inhibited the migration of CRC cells in vitro. Overexpression of CCR6 in CRC cells increased their proliferation, migration, and colony formation in vitro and promoted their metastatic potential in vivo. CCR6 activated Akt signaling, upregulated metastasis genes and downregulated metastasis suppressor genes. Selective targeting of CCR6 in tumors dramatically inhibited the growth of CRC in mice. Thus, the tumor expression of CCR6 plays a critical role in CRC metastasis, upregulated CCR6 predicts poor survival in CRC patients, and targeting CCR6 expression in tumors may be a potential therapeutic strategy for CRC.
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Expression of T-cell immunoglobulin- and mucin-domain-containing molecules-1 and -3 (Tim-1 and Tim-3) in Helicobacter pylori infection.
Helicobacter
PUBLISHED: 09-20-2011
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Th immune response plays an important role in Helicobacter pylori (H. pylori) infection. Tim-1 and Tim-3 are expressed on terminally differentiated Th2 and Th1 cells, respectively, and participate in the regulation of Th immune response. Until now, the role of Tim in H. pylori infection remains unclear.
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Influences of ORF1 on the virulence and immunogenicity of Actinobacillus pleuropneumoniae.
Curr. Microbiol.
PUBLISHED: 08-01-2011
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Actinobacillus pleuropneumoniae is a Gram-negative pathogen that causes porcine pleuropneumonia. The pathogenicity of A. pleuropneumoniae is strongly correlated with the production of active repeat-in-toxin (RTX) proteins such as ApxIVA. We evaluated the contribution of a potential ApxIVA activator, ORF1, to the virulence and immunogenicity of A. pleuropneumoniae in pigs. The orf1 gene in A. pleuropneumoniae SLW03 (serovar 1, ?apxIC?apxIIC) was deleted, producing strain SLW05 (?apxIC?apxIIC?orf1). The virulence of strains SLW03 and SLW05 was compared in pigs. Clinical signs and pulmonary lesions induced by strain SLW05 were slighter than that of strain SLW03 (P < 0.05). The immunogenicity and protective efficacy of strains SLW03 and SLW05 were similar. All pigs immunized with strain SLW03 or SLW05 developed high antibody titers against ApxIA, ApxIIA, and ApxIVA before challenge. Two weeks after a second immunization, pigs were challenged intratracheally with either a fully virulent A. pleuropneumoniae serovar 1 or serovar 3 strain. Vaccination with strains SLW03 or SLW05 provided significantly greater protection compared to the negative control (P < 0.01). Immunized pigs displayed significantly fewer clinical signs and lower lung lesion scores than non-immunized pigs. These results suggested that ORF1 plays an important role in the development of ApxIVA toxicity. Furthermore, strain SLW05 is a highly attenuated strain able to induce protective immunity against A. pleuropneumoniae infection.
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Cloning, expression, and characterization of TonB2 from Actinobacillus pleuropneumoniae and potential use as an antigenic vaccine candidate and diagnostic marker.
Can. J. Vet. Res.
PUBLISHED: 06-09-2011
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In this study the tonB2 gene was cloned from Actinobacillus pleuropneumoniae JL01 (serovar 1) and expressed as a glutathione-S-transferase (GST) fusion protein in Escherichia coli BL21(DE3). The GST fusion protein was recognized by antibodies in serum positive for A. pleuropneumoniae by Western blot analysis. Purified soluble GST-TonB2 was assessed for its ability to protect BALB/c mice against A. pleuropneumoniae infection. Mice were vaccinated with GST-TonB2 subcutaneously and challenged intraperitoneally with either ~4.0 × 10(5) colony-forming units (CFU) or ~1.0 × 10(6) CFU of A. pleuropneumoniae 4074. They were examined daily for 7 d after challenge. The survival rate of the TonB2-vaccinated mice was significant higher than that of the mice given recombinant GST or adjuvant alone. These results demonstrate that A. pleuropneumoniae TonB2 is immunogenic in mice and should be further assessed as a potential candidate for a vaccine against A. pleuropneumoniae infection. In addition, an indirect enzyme-linked immunosorbent assay (ELISA) based on the GST-TonB2 recombinant protein was developed. Compared with the ApxIVA ELISA, the TonB2 ELISA provided earlier detection of antibodies in pigs at various times after vaccination with A. pleuropneumoniae live attenuated vaccine. When compared with an indirect hemagglutination test, the sensitivity and specificity of the TonB2 ELISA were 95% and 88%, respectively. The TonB2 ELISA provides an alternative method for rapid serologic diagnosis of A. pleuropneumoniae infection through antibody screening, which would be especially useful when the infection status or serovar is unknown.
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Tumor-associated macrophages recruit CCR6+ regulatory T cells and promote the development of colorectal cancer via enhancing CCL20 production in mice.
PLoS ONE
PUBLISHED: 04-07-2011
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Tumor-associated macrophages (TAMs) remodel the colorectal cancer (CRC) microenvironment. Yet, findings on the role of TAMs in CRC seem to be contradictory compared with other cancers. FoxP3(+) regulatory T (Treg)-cells dominantly infiltrate CRC. However, the underlying molecular mechanism in which TAMs may contribute to the trafficking of Treg-cells to the tumor mass remains unknown.
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Generation and immunogenicity of transgenic potato expressing the GP5 protein of porcine reproductive and respiratory syndrome virus.
J. Virol. Methods
PUBLISHED: 01-26-2011
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Porcine reproductive and respiratory syndrome virus (PRRSV) is an important pathogen that has caused huge economic losses in the global swine industry. The advent of molecular farming has provided a cost-effective strategy for the development of transgenic plants as bioreactors to produce recombinant proteins. In this study, transgenic potato expressing GP5 protein of PRRSV was produced by Agrobacterium-mediated transformation, and confirmed using Southern blot and RT-PCR analyses. Recombinant GP5 protein was detected by ELISA and Western blot analyses. Mice immunized with transgenic potato extracts generated both serum and gut mucosal-specific antibodies, although low levels of neutralizing antibodies were elicited. This study provides a new approach for the production of vaccines against PRRSV.
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[Construction and characterization of Actinobacillus pleuropneumoniae serovar 7 live attenuated vaccine strain co-expressing ApxIA].
Sheng Wu Gong Cheng Xue Bao
PUBLISHED: 06-04-2010
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Actinobacillus pleuropneumoniae (A. pleuropneumoniae), the causative agent of porcine contagious pleuropneumonia (PCP), is a significant pathogen of the world pig industry, vaccination is potentially an effective tool for the prevention of PCP. The purpose of present study was to enhance the immunogenicity of A. pleuropneumoniae live vaccine strain HB04C- (serovar 7), which was unable to express ApxIA, and to develop effective multivalent vaccines for the respiratory pathogens based on the attenuated A. pleuropneumoniae. We introduced a shuttle vector containing intact apxIA gene into HB04C-, generating HB04C2, an A. pleuropneumoniae serovar 7 live attenuated vaccine strain co-expressing ApxIA. Then we investigated the biological characteristics of HB04C2. We found that the shuttle vector expressing ApxIA was stable in HB04C2, and the growth ability of HB04C2 was not affected by the shuttle vector. We observed that HB04C2 elicited detectable antibodies against ApxIA and ApxIIA when it was administrated intratracheally as a live vaccine in pigs, and all immunized pigs were protected from heterologous virulent A. pleuropneumoniae (serovar 1) challenge. In conclusion, we demonstrated that A. pleuropneumoniae live vaccine could be used as a vector for expression of heterologous antigens.
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Expressions of connexin and par-3 in the distal margin of rectal cancer after ultra-low anterior resection.
J. Huazhong Univ. Sci. Technol. Med. Sci.
PUBLISHED: 06-10-2009
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This study examined the expression of connexin and protease-activated receptor 3 (par-3) in the distal resection margin of rectal cancer and the correlation of the expression of the two proteins with tumor relapse. A total of 40 patients with rectal cancer underwent ultra-low anterior resection with curved cutter stapler. The pathological specimens were divided into 3 groups in terms of sampling sites: tumor group, 2.0-cm group (in which the tissues were harvested 2.0 cm distal to the tumor tissues), 3.0-cm group (in which the tissues were taken 3.0 cm away from the tumor tissues). All the samples were pathologically observed and then measured for the expression of connexin and par-3 by employing immunohistochemistry and Western blotting. The operations in this series went uneventfully. No anastomotic stoma bleeding, stenosis and death occurred postoperatively. Histopathologically, in the tumor group, epithelial cells lost normal pattern of arrangement and polarity, and were loosely connected and even detached. In the 3.0-cm group, the epithelia had normal appearance, obvious cell polarity and essentially intact cell junction. Immunohistochemistry and Western blotting indicated that the 3.0-cm group had the strongest expression of connexin and par-3, and the expression in the 2.0-cm group and the tumor group was relatively weak. There existed significant difference in the expression of the two proteins among the three groups (P<0.05 for all). It was concluded that the down-regulated connexin and par-3 in the distal margin of rectal cancer tissues may indicate the progression of the disease and high likelihood of recurrence and metastasis. Although no tumor cells were found in the sections of the 2.0-cm group, the decreased expression of connexin and par-3 may suggest the development of anaplasia and the increased odds of tumor relapse. Therefore, we are led to speculate that tumor resection only including 2.0 cm of unaffected rectum could not completely avoid the distant metastasis and local relapse.
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Vaccination with Streptococcus suis serotype 2 recombinant 6PGD protein provides protection against S. suis infection in swine.
FEMS Microbiol. Lett.
PUBLISHED: 05-05-2009
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Streptococcus suis serotype 2 (S. suis 2 or SS2) is the causative agent of several diseases in both pigs and humans. 6-Phosphogluconate dehydrogenase (6PGD) is a cell surface protein in SS2. In this study, the immunogenicity and protective efficacy of recombinant 6PGD (r6PGD) from SS2 was evaluated in piglets. Immunization with an r6PGD-containing adjuvant induced a vigorous immunoglobulin G (IgG) response, with the titer of IgG2 being higher than that of IgG1. Immunization of piglets with r6PGD yielded 50% survival upon an intravenous challenge with a lethal dose of SS2. Piglets immunized with the r6PDG vaccine were better protected than those immunized with the adjuvant control. The clinical signs and histopathological changes in the piglets were recorded. Collectively, these results suggest that r6PGD can confer partial protection against SS2 infection and could be useful for the development of subunit vaccines against SS2.
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In vivo induced RTX toxin ApxIVA is essential for the full virulence of Actinobacillus pleuropneumoniae.
Vet. Microbiol.
PUBLISHED: 01-02-2009
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Actinobacillus pleuropneumoniae is a Gram-negative pathogen. It is the aetiological agent of porcine contagious pleuropneumonia (PCP), a severe and highly contagious and severe respiratory disease of swine. Four sets of RTX (repeats in toxin) exotoxins have been described in A. pleuropnuemoniae and three of them have been characterized as important virulence determinants. The aim of this study was to determine the pathogenicity of the invivo induced RTX toxin ApxIVA during infection of piglets with A. pleuropnuemoniae. An A. pleuropnuemoniae apxIVA mutant was obtained based on an A. pleuropnuemoniae apxIIC-deleted mutant strain. An experimental infection assay was performed to evaluate the virulence of ApxIVA in piglets. Clinical signs, lung lesion scores, blood biochemical parameters and histopathologic changes in the piglets were recorded. The results indicated that the pathogenicity of A. pleuropnuemoniae was greater when ApxIVA was present, suggesting that ApxIVA is essential for expression of the full virulence of A. pleuropnuemoniae.
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Protective effects of saponin on a hypertension target organ in spontaneously hypertensive rats.
Exp Ther Med
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The present study was undertaken to investigate the protective effects of saponin on a hypertensive target organ (the kidney) in spontaneously hypertensive rats (SHRs) and also to explore the effect of saponin on the renin-angiotensin-aldosterone system (RAAS). A total of 24, 14-week-old SHRs were randomly divided into three groups; the first was administered low-dose saponin, the second with high-dose saponin and the third with a placebo as the control group. An additional eight healthy male Wistar rats were used as the normal group. The blood pressures (BPs) of the rats were determined using an animal BP-6 non-invasive blood pressure tester. Furthermore, the gene expression of TGFB1, collagen I and prorenin receptor (PRR) was determined by quantitative real time (qRT)-PCR. The histopathological and morphological features of the tissue samples were assessed semi-quantitatively. The content of saponin in the renal samples was lower in SHRs than in the normal healthy rats, but the plasma levels of saponin were similar. Mean arterial pressure (MAP) was reduced 5 days subsequent to saponin treatment by 36±3 and 51±4 mmHg in the low- and high-dose saponin groups, respectively. The anti-hypertensive effect of saponin was dose-related during the first 4 weeks of treatment. The gene expression of TGFB1 and collagen I in the renal samples was significantly suppressed in the low- and high-dose saponin groups compared with that in the control group. The gene expression of PRR was significantly and dose-dependently increased in the saponin-treated groups. These findings suggested that saponin reduced systemic BP and blocked the circulating and tissue RAAS.
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The live attenuated Actinobacillus pleuropneumoniae triple-deletion mutant ?apxIC ?apxIIC ?apxIV-ORF1 strain, SLW05, Immunizes pigs against lethal challenge with Haemophilus parasuis.
Clin. Vaccine Immunol.
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Haemophilus parasuis and Actinobacillus pleuropneumoniae both belong to the family Pasteurellaceae and are major respiratory pathogens that cause large economic losses in the pig industry worldwide. We previously constructed an attenuated A. pleuropneumoniae serovar 1 live vaccine prototype, SLW05 (?apxIC ?apxIIC ?apxIV-ORF1), which is able to produce nontoxic but immunogenic ApxIA, ApxIIA, and ApxIVA. This triple-deletion mutant strain was shown to elicit protective immunity against virulent A. pleuropneumoniae. In the present study, we investigated whether immunization with SLW05 could also protect against lethal challenge with virulent H. parasuis SH0165 (serovar 5) or MD0322 (serovar 4). The SLW05 strain was found to elicit a strong humoral antibody response in pigs and to confer significant protection against challenge with a lethal dose of H. parasuis SH0165 or MD0322. IgG subtype analysis revealed that SLW05 induces a bias toward a Th1-type immune response and stimulates interleukin 2 (IL-2) and gamma interferon (IFN-?) production. Moreover, antisera from SLW05-vaccinated pigs efficiently inhibited both A. pleuropneumoniae and H. parasuis growth in a whole-blood assay. This is the first report that a live attenuated A. pleuropneumoniae vaccine with SLW05 can protect against lethal H. parasuis infection, which provides a novel approach for developing an attenuated H. parasuis vaccine.
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Immunogenicity and protective efficacy of recombinant Haemophilus parasuis SH0165 putative outer membrane proteins.
Vaccine
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Haemophilus parasuis (H. parasuis), the causative agent of swine polyserositis, polyarthritis, and meningitis, is one of the most important bacterial diseases of pigs worldwide. Little vaccines currently exist that have a significant effect on infections with all pathogenic serovars of H. parasuis. H. parasuis putative outer membrane proteins (OMPs) are potentially essential components of more effective vaccines. Recently, the genomic sequence of H. parasuis serovar 5 strain SH0165 was completed in our laboratory, which allow us to target OMPs for the development of recombinant vaccines. In this study, we focused on 10 putative OMPs and all the putative OMPs were cloned, expressed and purified as HIS fusion proteins. Primary screening for immunoprotective potential was performed in mice challenged with an LD50 challenge. Out of these 10 OMPs three fusion proteins rGAPDH, rOapA, and rHPS-0675 were found to be protective in a mouse model of H. parasuis infection. We further evaluated the immune responses and protective efficacy of rGAPDH, rOapA, and rHPS-0675 in pig models. All three proteins elicited humoral antibody responses and conferred different levels of protection against challenge with a lethal dose of H. parasuis SH0165 in pig models. In addition, the antisera against the three individual proteins and the synergistic protein efficiently inhibited bacterial growth in a whole blood assay. The data demonstrated that the three proteins showed high value individually and the combination of rGAPDH, rOapA, and rHPS-0675 offered the best protection. Our results indicate that rGAPDH, rOapA, and rHPS-0675 induced protection against H. parasuis SH0165 infection, which may facilitate the development of a multi-component vaccine.
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Monoclonal antibodies against Actinobacillus pleuropneumoniae TonB2 protein expressed in Escherichia coli.
Hybridoma (Larchmt)
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TonB is known to be a bacterial periplasmic protein that transduces proton from the inner membrane to the outer membrane receptor in complex with the ExbB and ExbD proteins. Actinobacillus pleuropneumoniae TonB2 protein is the second TonB protein that is important for iron acquisition and virulence. The TonB2 protein was verified to be immunogenic and could afford partial protection for animals from lethal infection. In the present study, the recombinant TonB2 (rTonB2) was overexpressed in Escherichia coli BL21(DE3) and purified. The rTonB2 was then used as antigen to immunize BALB/c mice for the production of monoclonal antibodies (MAb). Four clones of TonB2-specific MAb secretion hybridomas--2F2, 2G8, 3D2, and 6F10--were selected. The MAbs 2F2, 3D2, and 6F10 were classified as IgG1 isotype and 2G8 was of IgG2a isotype. Western blot and ELISA results indicated that MAbs had specific binding activity to rTonB2. The MAbs generated here will be used for further functional analyses of the TonB2 protein.
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Construction and immune effect of Haemophilus parasuis DNA vaccine encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in mice.
Vaccine
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Haemophilus parasuis, the causative agent of swine polyserositis, polyarthritis, and meningitis, is one of the most important bacterial diseases of pigs worldwide. The development of a vaccine against H. parasuis has been impeded due to the lack of induction of reliable cross-serotype protection. In this study the gapA gene that encodes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was shown to be present and highly conserved in various serotypes of H. parasuis and we constructed a novel DNA vaccine encoding GAPDH (pCgap) to evaluate the immune response and protective efficacy against infection with H. parasuis MD0322 serovar 4 or SH0165 serovar 5 in mice. A significant antibody response against GAPDH was generated following pCgap intramuscular immunization; moreover, antibodies to the pCgap DNA vaccine were bactericidal, suggesting that it was expressed in vivo. The gapA transcript was detected in muscle, liver, spleen, and kidney of the mice seven days post-vaccination. The IgG subclass (IgG1 and IgG2a) analysis indicated that the DNA vaccine induced both Th1 and Th2 immune responses, but the IgG1 response was greater than the IgG2a response. Moreover, the groups vaccinated with the pCgap vaccine exhibited 83.3% and 50% protective efficacy against the H. parasuis MD0322 serovar 4 or SH0165 serovar 5 challenges, respectively. The pCgap DNA vaccine provided significantly greater protective efficacy compared to the negative control groups or blank control groups (P<0.05 for both). Taken together, these findings indicate that the pCgap DNA vaccine provides a novel strategy against infection of H. parasuis and offer insight concerning the underlying immune mechanisms of a bacterial DNA vaccine.
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