Basigin-CyP elevated porcine circovirus type2 replication.

Porcine circovirus type2 (PCV2) is a member of the circoviridae family. PCV2 was identified as the main pathogen of postweaning multisystemic wasting syndrome (PMWS) in weaned piglets and causes massive economic loss. Basigin, is a transmembrane glycoprotein belonging to the immunoglobulin superfamily; which is also a receptor for cyclophilins. CyP belongs to the immunophilin family that has peptidyl-prolyl cis-trans isomerase activity. Basigin-CyP interaction affects the replication stages of several viruses. In this study, we found that Basigin could elevate the replication of PCV2, and the Basigin only affected the replication stage rather than adsorption or endocytosis stages. In addition, the ligands of Basigin, CyPA and CyPB also elevated the replication of PCV2. Basigin-CyP interation was necessary for elevating PCV2 replication; At last, CyPs were proved to promote the replication of PCV2 by activating ERK signaling.

Molecular characterization of a fungal cyclophilin allergen Rhi o 2 and elucidation of antigenic determinants responsible for IgE-cross-reactivity.

Cyclophilins are structurally conserved pan-allergens showing extensive cross-reactivity. So far, no precise information on cross-reactive IgE-epitopes of cyclophilins is available. Here, an 18-kDa IgE-reactive cyclophilin (Rhi o 2) was purified from Rhizopus oryzae, an indoor mold causing allergic sensitization. Based on LC-MS/MS-derived sequences of natural Rhi o 2, the full-length cDNA was cloned, and expressed as recombinant (r) allergen. Purified rRhi o 2 displayed IgE-reactivity and basophil degranulation with sera from all cyclophilin-positive patients. The melting curve of properly folded rRhi o 2 showed partial refolding after heat denaturation. The allergen displayed monomeric functional peptidyl-prolyl cis-trans isomerase (PPIase) activity. In IgE-inhibition assays, rRhi o 2 exhibited extensive cross-reactivity with various other cyclophilins reported as allergens from diverse sources including its homologous human autoantigen. By generating a series of deletion mutants, a conserved 69-residue (Asn81-Asn149) fragment at C terminus of Rhi o 2 was identified as crucial for IgE-recognition and cross-reactivity. Grafting of the Asn81-Asn149 fragment within the primary structure of yeast cyclophilin CPR1 by replacing its homologous sequence resulted in a hybrid molecule with structural folds similar to Rhi o 2. The IgE-reactivity and allergenic activity of the hybrid cyclophilin were greater than that of CPR1. Therefore, the Asn81-Asn149 fragment can be considered as the site of IgE recognition of Rhi o 2. Hence, Rhi o 2 serves as a candidate antigen for the molecular diagnosis of mold allergy, and determination of a major cross-reactive IgE-epitope has clinical potential for the design of next-generation immunotherapeutics against cyclophilin-induced allergies.

Delineation of the Olive Pollen Proteome and Its Allergenome Unmasks Cyclophilin as a Relevant Cross-Reactive Allergen.

Olive pollen is a major allergenic source worldwide due to its extensive cultivation. We have combined available genomics data with a comprehensive proteomics approach to get the annotated olive tree (Olea europaea L.) pollen proteome and define its complex allergenome. A total of 1907 proteins were identified by LC-MS/MS using predicted protein sequences from its genome. Most proteins (60%) were predicted to possess catalytic activity and be involved in metabolic processes. In total, 203 proteins belonging to 47 allergen families were found in olive pollen. A peptidyl-prolyl cis-trans isomerase, cyclophilin, produced in Escherichia coli, was found as a new olive pollen allergen (Ole e 15). Most Ole e 15-sensitized patients were children (63%) and showed strong IgE recognition to the allergen. Ole e 15 shared high sequence identity with other plant, animal, and fungal cyclophilins and presented high IgE cross-reactivity with pollen, plant food, and animal extracts.

Critical role of TLR2 in triggering protective immunity with cyclophilin entrapped in oligomannose-coated liposomes against Neospora caninum infection in mice.

Neospora caninum is an intracellular protozoan parasite closely related to Toxoplasma gondii. N. caninum is thought to be a major cause of abortion in cattle worldwide. Given the current situation of drastic economic losses and a lack of efficient control strategies against such parasites, the challenge to develop potent vaccine candidates and technologies remains. We investigated the immune stimulating activity of N. caninum cyclophilin (NcCyp) with and without a formulation with oligomannose-coated-liposomes (OML) as the potential adjuvant. NcCyp-OML activated NF-κB in RAW 264.7 cells and triggered interleukin (IL)-12p40 production from murine peritoneal macrophages. In BALB/c mice, immunization with NcCyp-OML was associated with the production of specific antibodies (IgG1 and IgG2a). The specific antibody (IgG1) against naked NcCyp was also observed after the challenge infection, but it was significantly lower than those of NcCyp-OML. Moreover, significant cellular immune responses were induced, including spleen cell proliferation and interferon-gamma production. The immunization of mice with NcCyp-OML, and to lesser extent with naked NcCyp, induced significant protection against challenge with a lethal dose of N. caninum compared with the PBS control group. This protection was associated with a higher survival rate, slight changes in body weight, and lower clinical score of mice. In addition, the significant protective efficacy of NcCyp-OML was confirmed in another mouse strain, male C57BL/6 mice. The current study revealed the marked contribution of Toll-like receptor 2 (TLR2) to the protective immunity triggered by NcCyp-OML because higher numbers of TLR2-/- mice succumbed to a lethal dose of N. caninum compared with C57BL/6 mice. Moreover, prominent spleen cell proliferation and IFN-γ production was induced in NcCyp-OML-immunized mice by a TLR2-dependent mechanism.

Novel minor HLA DR associated antigens in type 1 diabetes.

Type 1 diabetes is an autoimmune disease leading to insulin deficiency. Autoantibodies to beta cell proteins are already present in the asymptomatic phase of type 1 diabetes. Recent findings have suggested a number of additional minor autoantigens in patients with type 1 diabetes. We have established luciferase immunoprecipitation systems (LIPS) for anti-MTIF3, anti-PPIL2, anti-NUP50 and anti-MLH1 and analyzed samples from 500 patients with type 1 diabetes at onset of clinical disease and 200 healthy individuals who had a family history of type 1 diabetes but no evidence of beta cell autoantibodies. We show significantly higher frequencies of anti-MTIF3, anti-PPIL2 and anti-MLH1 in recent onset type 1 diabetes patients in comparison to controls. In addition, antibodies to NUP50 were associated with HLA-DRB1*03 and antibodies to MLH1 were associated with HLA-DRB1*04 genotypes.

Mitochondrial cyclophilin D regulates T cell metabolic responses and disease tolerance to tuberculosis.

Mycobacterium tuberculosis (Mtb) is one of the most ancient human pathogens, yet the exact mechanism(s) of host defense against Mtb remains unclear. Although one-third of the world's population is chronically infected with Mtb, only 5 to 10% develop active disease. This indicates that, in addition to resistance mechanisms that control bacterial burden, the host has also evolved strategies to tolerate the presence of Mtb to limit disease severity. We identify mitochondrial cyclophilin D (CypD) as a critical checkpoint of T cell metabolism that controls the expansion of activated T cells. Although loss of CypD function in T cells led to enhanced Mtb antigen-specific T cell responses, this increased T cell response had no impact on bacterial burden. Rather, mice containing CypD-deficient T cells exhibited substantially compromised disease tolerance and succumbed to Mtb infection. This study establishes a mechanistic link between T cell-mediated immunity and disease tolerance during Mtb infection.

Peptidylprolyl Isomerases as In Vivo Carriers for Drugs That Target Various Intracellular Entities.

Analyses of sequences and structures of the cyclosporine A (CsA)-binding proteins (cyclophilins) and the immunosuppressive macrolide FK506-binding proteins (FKBPs) have revealed that they exhibit peculiar spatial distributions of charges, their overall hydrophobicity indexes vary within a considerable level whereas their points isoelectric (pIs) are contained from 4 to 11. These two families of peptidylprolyl cis/trans isomerases (PPIases) have several distinct functional attributes such as: (1) high affinity binding to some pharmacologically-useful hydrophobic macrocyclic drugs; (2) diversified binding epitopes to proteins that may induce transient manifolds with altered flexibility and functional fitness; and (3) electrostatic interactions between positively charged segments of PPIases and negatively charged intracellular entities that support their spatial integration. These three attributes enhance binding of PPIase/pharmacophore complexes to diverse intracellular entities, some of which perturb signalization pathways causing immunosuppression and other system-altering phenomena in humans.

Tracking the Antibody Immunome in Type 1 Diabetes Using Protein Arrays.

We performed an unbiased proteome-scale profiling of humoral autoimmunity in recent-onset type 1 diabetes (T1D) patients and nondiabetic controls against ∼10 000 human proteins using a Nucleic Acid Programmable Protein Array (NAPPA) platform, complemented by a knowledge-based selection of proteins from genes enriched in human pancreas. Although the global response was similar between cases and controls, we identified and then validated six specific novel T1D-associated autoantibodies (AAbs) with sensitivities that ranged from 16 to 27% at 95% specificity. These included AAbs against PTPRN2, MLH1, MTIF3, PPIL2, NUP50 (from NAPPA screening), and QRFPR (by targeted ELISA). Immunohistochemistry demonstrated that NUP50 protein behaved differently in islet cells, where it stained both nucleus and cytoplasm, compared with only nuclear staining in exocrine pancreas. Conversely, PPIL2 staining was absent in islet cells, despite its presence in exocrine cells. The combination of anti-PTPRN2, -MLH1, -PPIL2, and -QRFPR had an AUC of 0.74 and 37.5% sensitivity at 95% specificity. These data indicate that these markers behave independently and support the use of unbiased screening to find biomarkers because the majority was not predicted based on predicted abundance. Our study enriches the knowledge of the "autoantibody-ome" in unprecedented breadth and width.

Cyclophilin and enolase are the most prevalent conidial antigens of Lomentospora prolificans recognized by healthy human salivary IgA and cross-react with Aspergillus fumigatus.

PURPOSE: The study of the immunocompetent airways immune response may provide important information to improve the therapeutic efficacy against Lomentospora (Scedosporium) prolificans. So, this study aimed to identify the most prevalent conidial antigens of this multiresistant fungus recognized by healthy human salivary immunoglobulin A, and to study their expression and cross-reactivity with other fungal species. EXPERIMENTAL DESIGN: Twenty saliva from immunocompetent donors were used to detect and identify the immunoreactive proteins by 2D immunoblotting and LC-MS/MS. Moreover, anti-Aspergillus antibodies were purified to study their cross-reactivity. RESULTS: Ten proteins of L. prolificans conidia showed reactivity with more than 50% of the saliva samples. Among them, cyclophilin and enolase were the most prevalent antigens recognized by 85 and 80% of the samples, respectively. These enzymes were also identified on the cell wall surface of L. prolificans and on the immunomes of Scedosporium apiospermum and Scedosporium aurantiacum. Additionally, they showed cross-reactivity with the most common pathogenic filamentous fungus Aspergillus fumigatus. CONCLUSION AND CLINICAL RELEVANCE: These results show that the immunocompetent immune response might offer a pan-fungal recognition of conserved antigens such as enolase and cyclophilins, making them potential candidates for study as therapeutic targets.

The Activation of Phytophthora Effector Avr3b by Plant Cyclophilin is Required for the Nudix Hydrolase Activity of Avr3b.

Plant pathogens secrete an arsenal of effector proteins to impair host immunity. Some effectors possess enzymatic activities that can modify their host targets. Previously, we demonstrated that a Phytophthora sojae RXLR effector Avr3b acts as a Nudix hydrolase when expressed in planta; and this enzymatic activity is required for full virulence of P. sojae strain P6497 in soybean (Glycine max). Interestingly, recombinant Avr3b produced by E. coli does not have the hydrolase activity unless it was incubated with plant protein extracts. Here, we report the activation of Avr3b by a prolyl-peptidyl isomerase (PPIase), cyclophilin, in plant cells. Avr3b directly interacts with soybean cyclophilin GmCYP1, which activates the hydrolase activity of Avr3b in a PPIase activity-dependent manner. Avr3b contains a putative Glycine-Proline (GP) motif; which is known to confer cyclophilin-binding in other protein substrates. Substitution of the Proline (P132) in the putative GP motif impaired the interaction of Avr3b with GmCYP1; as a result, the mutant Avr3bP132A can no longer be activated by GmCYP1, and is also unable to promote Phytophthora infection. Avr3b elicits hypersensitive response (HR) in soybean cultivars producing the resistance protein Rps3b, but Avr3bP132A lost its ability to trigger HR. Furthermore, silencing of GmCYP1 rendered reduced cell death triggered by Avr3b, suggesting that GmCYP1-mediated Avr3b maturation is also required for Rps3b recognition. Finally, cyclophilins of Nicotiana benthamiana can also interact with Avr3b and activate its enzymatic activity. Overall, our results demonstrate that cyclophilin is a "helper" that activates the enzymatic activity of Avr3b after it is delivered into plant cells; as such, cyclophilin is required for the avirulence and virulence functions of Avr3b.

Primary identification, biochemical characterization, and immunologic properties of the allergenic pollen cyclophilin cat R 1.

Cyclophilin (Cyp) allergens are considered pan-allergens due to frequently reported cross-reactivity. In addition to well studied fungal Cyps, a number of plant Cyps were identified as allergens (e.g. Bet v 7 from birch pollen, Cat r 1 from periwinkle pollen). However, there are conflicting data regarding their antigenic/allergenic cross-reactivity, with no plant Cyp allergen structures available for comparison. Because amino acid residues are fairly conserved between plant and fungal Cyps, it is particularly interesting to check whether they can cross-react. Cat r 1 was identified by immunoblotting using allergic patients' sera followed by N-terminal sequencing. Cat r 1 (∼ 91% sequence identity to Bet v 7) was cloned from a cDNA library and expressed in Escherichia coli. Recombinant Cat r 1 was utilized to confirm peptidyl-prolyl cis-trans-isomerase (PPIase) activity by a PPIase assay and the allergenic property by an IgE-specific immunoblotting and rat basophil leukemia cell (RBL-SX38) mediator release assay. Inhibition-ELISA showed cross-reactive binding of serum IgE from Cat r 1-allergic individuals to fungal allergenic Cyps Asp f 11 and Mala s 6. The molecular structure of Cat r 1 was determined by NMR spectroscopy. The antigenic surface was examined in relation to its plant, animal, and fungal homologues. The structure revealed a typical cyclophilin fold consisting of a compact ß-barrel made up of seven anti-parallel ß-strands along with two surrounding α-helices. This is the first structure of an allergenic plant Cyp revealing high conservation of the antigenic surface particularly near the PPIase active site, which supports the pronounced cross-reactivity among Cyps from various sources.

Immunization with the Leishmania infantum recombinant cyclophilin protein 1 confers partial protection to subsequent parasite infection and generates specific memory T cells.

Control of zoonotic visceral leishmaniosis can be achieved using several available drugs. These drugs present high toxicity and require longer treatment regimens which complicate compliance to the treatment. Other control measures directed to the vector or the reservoirs are useful tools to restrain the spreading of this disease but the effects are transitory. A safe, affordable and efficient vaccine conferring long lasting immunity should be the most cost effective way of controlling zoonotic visceral leishmaniosis. The present study aims at characterizing a cyclophilin protein 1 of Leishmania infantum (LiCyP1) and investigating whether recombinant LiCyP1 (LirCyP1) is able to confer protection against infection by evaluating viable parasite load and the generation of specific CD4(+) and CD8(+) effector and central memory T cells in rodent model. LiCyP1 is present in the cytoplasm of L. infantum amastigotes and promastigotes. Immunization of BALB/c mice with LirCyP1 confers high protection to L. infantum infection, causing a marked reduction in parasite replication in the liver and spleen. Furthermore, helper and cytotoxic memory T cell subsets able to specifically recognize parasite antigens expanded in immunized and in challenged mice. CD4(+) T cell subpopulation of intermediate phenotype (CD62L(high)CD127(low)) of challenging mice also presented an accentuated expansion after the recall. This study demonstrated that LirCyP1 confers partial protection to L. infantum infection, promoting the generation of a desired long lasting immunity. LirCyP1 can be considered a potential candidate for the design of a vaccine against zoonotic visceral leishmaniosis.

Shockwave therapy differentially stimulates endothelial cells: implications on the control of inflammation via toll-Like receptor 3.

Shock wave therapy (SWT) reportedly improves ventricular function in ischemic heart failure. Angiogenesis and inflammation modulatory effects were described. However, the mechanism remains largely unknown. We hypothesized that SWT modulates inflammation via toll-like receptor 3 (TLR3) through the release of cytosolic RNA. SWT was applied to human umbilical vein endothelial cells (HUVECs) with 250 impulses, 0.08 mJ/mm(2) and 3 Hz. Gene expression of TLR3, inflammatory genes and signalling molecules was analysed at different time points by real-time polymerase chain reaction. SWT showed activation of HUVECs: enhanced expression of TLR3 and of the transporter protein for nucleic acids cyclophilin B, of pro-inflammatory cytokines cyclophilin A and interleukin-6 and of anti-inflammatory interleukin-10. No changes were found in the expression of vascular endothelial cell adhesion molecule. SWT modulates inflammation via the TLR3 pathway. The interaction between interleukin (IL)-6 and IL-10 in TLR3 stimulation can be schematically seen as a three-phase regulation over time.

Protective immunity induced by a recombinant BCG vaccine encoding the cyclophilin gene of Toxoplasma gondii.

The investigation of Toxoplasma gondii virulence factors can elucidate the immunopathology of T. gondii infection and identify potential candidates for effective human vaccines. The adjuvant is an important component of an effective vaccine. In this study, attenuated Mycobacterium bovis was used as a live vaccine vector with both antigen and adjuvant characteristics. Following amplification of the T. gondii cyclophilin gene, the shuttle expression plasmid pMV261-TgCyP and integrative expression plasmid pMV361-TgCyP were constructed, and their expression was stimulated after transfection into BCG. Both recombinant plasmids were highly immunogenic. Greater proliferation of CD4(+) and CD8(+) T cells was observed in the rBCG-vaccinated groups compared to the control groups. The levels of Th1-type IFN-γ, IL-2 and IL-12 were significantly increased following immunisation with the rBCG vaccines via the i.v. or oral route, which indicated that catalytic activity against T. gondii infection was generated in the mice. rBCGpMV361-TgCyP i.v. inoculation resulted in a higher protection efficiency, as demonstrated by the increased survival time and survival rate (17%) of BALB/c mice. The present study demonstrates that a BCG vector expressing a target antigen, TgCyP, represent an alternative system for the production of effective vaccines to prevent toxoplasmosis.

Análise da expressão imuo-histoquímica da ciclofilina A, EMMPRIN e MMP-7 na doença periodontal / Expression analysis imuo staining of cyclophilin A, EMMPRIN and MMP-7 in periodontal disease

A doença periodontal é uma entidade infecciosa que resulta da resposta imuno-inflamatória do hospedeiro aos microrganismos presentes no biofilme dentário, levando à destruição tecidual. O propósito do presente estudo foi avaliar a expressão imuno-histoquímica da ciclofilina A (CYPA), do indutor de metaloproteinases da matriz extracelular (EMMPRIN) e da metaloproteinase da matriz 7 (MMP-7) em espécimes humanos de gengiva clinicamente saudável (n=32), gengivite induzida por biofilme dentário (n=28) e periodontite crônica (n=30). Foram realizadas biópsias das três condições clínicas e feita a análise imuno-histoquímica através da contagem total do número de células positivas, correlacionando-a com parâmetros clínicos. A imunopositividade da CYPA, do EMMPRIN e da MMP-7 revelou diferença estatisticamente significativa entre os três grupos, com maiores percentuais de positividade nos espécimes de periodontite crônica, seguidos pelos espécimes de gengivite crônica e de gengiva saudável (p < 0,001). Foi evidenciada maior expressão de CYPA e MMP-7 nos grupos que tinham infiltrado inflamatório mais intenso. Foram observadas correlações das imunoexpressões de EMMPRIN, MMP-7 e CYPA, tanto entre si como com parâmetros clínicos (profundidade de sondagem e perda de inserção clínica). Foram verificadas correlações positivas entre a expressão de CYPA e as expressões da MMP-7 (r = 0,831; p < 0,001) e do EMMPRIN (r = 0,289; p = 0,006). Além disso, a profundidade de sondagem revelou correlação positiva, estatisticamente significativa, com as expressões de MMP-7 (r = 0,726; p < 0,001), EMMPRIN (r = 0,345; p = 0,001) e CYPA (r = 0,803; p < 0,001). Esses resultados evidenciam que a CYPA, o EMMPRIN e a MMP-7 podem estar associadas à patogênese e progressão da doença periodontal. (AU)

Periodontal disease is an infectious disease resulting from the immunoinflammatory response of the host to microorganisms present in the dental biofilm which causes tissue destruction. The objective of this study was to evaluate the immunohistochemical expression of cyclophilin A (CYPA), extracellular matrix metalloproteinase inducer (EMMPRIN) and matrix metalloproteinase 7 (MMP-7) in human specimens of clinically healthy gingiva (n=32), biofilm-induced gingivitis (n=28), and chronic periodontitis (n=30). Immunopositivity for CYPA, EMMPRIN and MMP-7 differed significantly between the three groups, with higher percentages of staining in chronic periodontitis specimens, followed by chronic gingivitis and healthy gingiva specimens (p < 0.001). Immunoexpression of CYPA and MMP-7 was higher in the intense inflammatory infiltrate observed mainly in cases of periodontitis. Analysis of possible correlations between the immunoexpression of EMMPRIN, MMP-7 and CYPA and between the expression of these proteins and clinical parameters (probing depth and clinical attachment loss) showed a positive correlation of CYPA expression with MMP-7 (r = 0.831; p < 0.001) and EMMPRIN (r = 0.289; p = 0.006). In addition, there was a significant positive correlation between probing depth and expression of MMP-7 (r = 0.726; p < 0.001), EMMPRIN (r = 0.345; p = 0.001), and CYPA (r = 0.803; p < 0.001). These results suggest that CYPA, EMMPRIN and MMP-7 are associated with the pathogenesis and progression of periodontal disease. (AU)

Cyclophilins in atherosclerosis: a new therapeutic target?

Atherosclerosis is a chronic disease with a significant inflammatory component. Recent studies indicate a role of extracellular cyclophilins as contributors to endothelial inflammation and pathogenesis of atherosclerosis. In this article, we review current literature on pro-inflammatory activities of extracellular cyclophilins and discuss possible approaches to selectively target this novel proinflammatory factor.

Is there a role for cyclophilin inhibitors in the management of primary biliary cirrhosis?

Autoimmune hepatitis (AIH) and primary biliary cirrhosis (PBC) are poorly understood autoimmune liver diseases. Immunosuppression is used to treat AIH and ursodeoxycholic acid is used to slow the progression of PBC. Nevertheless, a proportion of patients with both disorders progress to liver failure. Following liver transplantation, up to a third of patients with PBC experience recurrent disease. Moreover a syndrome referred to as "de novo AIH" occurs in a proportion of patients regardless of maintenance immunosuppression, who have been transplanted for disorders unrelated to AIH. Of note, the use of cyclosporine A appears to protect against the development of recurrent PBC and de novo AIH even though it is a less potent immunosuppressive compared to tacrolimus. The reason why cyclosporine A is protective has not been determined. However, a virus resembling mouse mammary tumor virus (MMTV) has been characterized in patients with PBC and AIH. Accordingly, we hypothesized that the protective effect of cyclosporine A in liver transplant recipients may be mediated by the antiviral activity of this cyclophilin inhibitor. Treatment of the MMTV producing MM5MT cells with different antivirals and immunosuppressive agents showed that both cyclosporine A and the analogue NIM811 inhibited MMTV production from the producer cells. Herein, we discuss the evidence supporting the role of MMTV-like human betaretrovirus in the development of PBC and de novo AIH and speculate on the possibility that the agent may be associated with disease following transplantation. We also review the mechanisms of how both cyclosporine A and NIM811 may inhibit betaretrovirus production in vitro.

The protective effect of a DNA vaccine encoding the Toxoplasma gondii cyclophilin gene in BALB/c mice.

Toxoplasmosis is a world-wide zoonosis that causes significant public health and veterinary problems. The study of vaccines remains the most promising method for the future prevention and control of toxoplasmosis. Recombinant Toxoplasma gondii cyclophilin has been shown to have potent PPIase and IL-12-inducing activities, thus promoting the stabilization of T. gondii's life cycle and maintaining the survival of its host during evolution. In this study, the T. gondii cyclophilin gene was used to construct a DNA vaccine (pVAX1-TgCyP). The immune response and protective efficacy of the vaccine against T. gondii infection in BALB/c mice were evaluated. All BALB/c mice that were vaccinated with pVAX1-TgCyP developed a high response with TgCyP-specific antibodies, and significant splenocyte proliferation (P < 0·05) compared with pVAX1 vector and PBS groups. pVAX1-TgCyP also induced a significant Th1 type immune response, indicated by the higher production of IL-2 and IFN-γ (P < 0·05). The survival rate of BALB/c mice increased significantly after vaccination with pVAX1-TgCyP (37·5%) (P < 0·05). These results indicate that TgCyP is a highly efficacious vaccine candidate that can generate protective immunity against T. gondii infection in BALB/c mice.

[Cloning, expression and bioinformatics analysis of cyclophilin of Echinococcus granulosus].

OBJECTIVE: To clone and express EgCyP gene of Echinococcus granulosas and analyze EgCyP using bioinformatics. METHODS: Total RNAS of adult E. granulosus was extracted and reversedly transcripted to cDNA. EgCyP gene was amplified from cDNA and inserted into vector pET28a. Recombinant plasmid pET28a-EgCyP was transformed into E. coli BL21 (DE3) for expression under the induction of IPTG. The expressed product was identified by SDS-PAGE and Western blotting. EgCyP was analyzed by the bioinformatics software. RESULTS: The EgCyP gene was successfully amplified from cDNA of adult E. granulosus and a fusion protein was expressed in E .coli BL21 (DE3). The molecular weight of the expressed protein was about 22 kDa. The Western blotting indicated that the antigenicity of the protein was specific. The bioinformatics analysis revealed that there were 7 antigen epitopes in EgCyP. CONCLUSION: EgCyP of E. granulosus is cloned and expressed in E. coli BL21 (DE3) successfully, which might be the foundation for the further study of its immunogenicity.