The nfa1 gene was cloned from a cDNA library of pathogenic Naegleria fowleri by immunoscreening; it consisted of 360 bp and produced a 13.1 kDa recombinant protein (rNfa1) that showed the pseudopodia-specific localization by immunocytochemistry in the previous study. Based on the idea that the pseudopodia-specific Nfa1 protein mentioned above seems to be involved in the pathogenicity of N. fowleri, we observed the effect of an anti-Nfa1 antibody on the proliferation of N. fowleri trophozoites and the cytotoxicity of N. fowleri trophozoites on the target cells. The proliferation of N. fowleri trophozoites was inhibited after being treated with an anti-Nfa1 polyclonal antibody in a dose-dependent manner for 48 hrs. By a light microscope, CHO cells co-cultured with N. fowleri trophozoites (group I) for 48 hrs showed severe morphological destruction. On the contrary, CHO cells co-cultured with N. fowleri trophozoites and anti-Nfa1 polyclonal antibody (1: 100 dilution) (group II) showed less destruction. In the LDH release assay results, group I showed 50.6% cytotoxicity, and group II showed 39.3%. Consequently, addition of an anti-Nfa1 polyclonal antibody produced a decreasing effect of in vitro cytotoxicity of N. fowleri in a dosedependent manner.
Animals ; Antibodies, Protozoan/*immunology ; Antigens, Protozoan/genetics/*immunology ; CHO Cells ; Dose-Response Relationship, Immunologic ; Female ; Hamsters ; Mice ; Mice, Inbred BALB C ; Naegleria fowleri/growth & development/immunology/*pathogenicity ; Protozoan Proteins/genetics/*immunology ; Recombinant Proteins/immunology ; Support, Non-U.S. Gov't

Trichomoniasis is a sexually transmitted disease due to infection with Trichomonas vaginalis, and it can cause serious consequences for women's health. To study the virulence factors of this pathogen, T. vaginalis surface proteins were investigated using polyclonal antibodies specific to the membrane fractions of T. vaginalis. The T. vaginalis expression library was constructed by cloning the cDNA derived from mRNA of T. vaginalis into a phage lambda Uni-ZAP XR vector, and then used for immunoscreening with the anti-membrane proteins of T. vaginalis antibodies. The immunoreactive proteins identified included adhesion protein AP65-1, alpha-actinin, kinesin-associated protein, teneurin, and 2 independent hypothetical proteins. Immunofluorescence assays showed that AP65-1, one of the identified immunogenic clones, is prevalent in the whole body of T. vaginalis. This study led us to identify T. vaginalis proteins which may stimulate immune responses by human cells.
Animals ; Antigens, Protozoan/genetics/*immunology ; Female ; Humans ; Molecular Sequence Data ; Protozoan Proteins/genetics/*immunology ; Rats ; Trichomonas Infections/parasitology ; Trichomonas vaginalis/genetics/*immunology

OBJECTIVETo characterize the immune response induced by SAG1 encoding plasmid combined with IL-2 gene adjuvant in mice and to assess the protective effect of this vaccination against toxoplasmosis.

METHODSMice were co-injected intramuscularly with plasmid encoding Toxoplasma gondii SAG1 plus murine IL-2 expression vector at a dose of 100 microg. Booster immunizations were employed 2 more times at 3-week interval. As controls, mice were inoculated with PBS or empty plasmid pcDNA3. Humoral and cellular responses were assayed using ELISA for the determination of Ab, Ab isotype and IFN-gamma, as well as IL-4. To detect the integration and dissemination of DNA in the injected mice, PCR and in situ hybridization were performed. All mice were then infected with highly virulent RH tachyzoites of Toxoplasma gondii intraperitoneally.

RESULTSSignificant increases in specific IgG levels were observed in mice after immunization three times with SAG1 expression plasmid. With respect to the IgG isotype, co-inoculation of IL-2 expression plasmid enhanced the level of IgG2a and the production of IFN-gamma. Challenging mice by vaccinating with combined plasmids with RH tachyzoites resulted in prolonged survival.

CONCLUSIONHumoral and cytokine responses elicited by SAG1 DNA immunization can be modulated by co-inoculation with IL-2 expression plasmid. The use of DNA vaccine in combination with an appropriate cytokine gene to prevent T. gondii infection warrants further investigation.

Animals ; Antibodies, Protozoan ; blood ; Antigens, Protozoan ; Cytokines ; biosynthesis ; Female ; Immunization ; Immunoglobulin G ; blood ; classification ; Interleukin-2 ; genetics ; Mice ; Protozoan Proteins ; genetics ; Protozoan Vaccines ; immunology ; Toxoplasma ; immunology ; Toxoplasmosis, Animal ; prevention & control ; Vaccines, DNA ; immunology

A Cryptosporidium parvum sporozoite and oocyst lambda gt11 cDNA library was screened with a hyperimmune rabbit serum that was developed against insoluble fragments of ultrasonicated oocysts. A clone named Cp22.4.1 encoding a protein of 231 amino acids with 4 zinc-finger domains characterized by a Cys-X2-Cys-X4-His-X4-Cys motif was isolated and characterized. There was a complete match between the sequencing data of the coding region of Cp22.4.1 and the corresponding gene at chromosomal level. Cloning in a pBAD-TOPO-TA expression vector permitted to evaluate the antigenicity of the recombinant His-tagged antigen. This antigen was recognized by 2 out of 5 sera from Cryptosporidium immune calves and not by sera from parasite naive animals.
Amino Acid Sequence ; Animals ; Antibodies, Protozoan/*blood ; Antigens, Protozoan/chemistry/*immunology ; Base Sequence ; Cattle/*immunology ; Cryptosporidium parvum/*immunology ; Molecular Sequence Data ; Protozoan Proteins/chemistry/genetics ; Rabbits ; Recombinant Proteins ; Zinc Fingers/genetics/*immunology

OBJECTIVETo identify and clone a new full ORF gene of PyDyn (Plasmodium yoelii dynamin-like protein), and examine the protection of their expression products.

METHODUsing the P. yoelii Genome technology and RT-PCR.

RESULTSThe full ORF gene of PyDyn was amplified from mRNA of the erythrocytic stage of P. yoelii., three domains of PyDyn were expressed in E. coli., and the fairly positive immunogenicity of them was showed by IFA. The full ORF gene of PyDyn was 2,433 bp and encode 811 amino acids. Its Gene Bank access number is AF458071. PyDyn belongs to the dynamin-like protein family according to its property.

CONCLUSIONThe new full ORF gene of PyDyn is obtained and identified; their expressed domains are probably new candidates for malaria vaccine.

Amino Acid Sequence ; Animals ; Base Sequence ; Cloning, Molecular ; Dynamins ; genetics ; immunology ; Escherichia coli ; genetics ; Genes, Protozoan ; genetics ; immunology ; Malaria Vaccines ; immunology ; Molecular Sequence Data ; Plasmodium yoelii ; chemistry ; genetics ; Protozoan Proteins ; genetics ; immunology ; Vaccines, Synthetic ; immunology

OBJECTIVETo develop a technology for production of recombinant SAG1 of Toxoplasma gondii (T.g) in batches.

METHODSThe rSAG1 of T.g was expressed in E.coli by high-density fermentation and purified by Sephadex G-75 column chromatography after Ni-NTA agarose at native condition. The activity of rSAG1 and its efficacy in T.g diagnosis were identified by Western blotting and ELISA, respectively.

RESULTSThe optical density (OD) of the bacteria reached 20.21 after induction, and 300 g bacteria were harvested from 11.5 L broth. The rSAG1 was highly expressed in E.coli as a fusion protein, accounting for about 25.82% of the total bacterial protein. The purity of rSAG1 reached 98.54% after purification by Ni-NTA combined with Sephadex G-75 column chromatography. Western blotting revealed a distinct band reacting with the sera of rabbits vaccinated by T.g. Twenty-four of the 25 sera of mice infected with T.g and 36 of the 38 sera of human subjects with IgG antibody against T.g were detected by rSAG1-ELISA.

CONCLUSIONA large-scale production of immunoreactive SAG1 of T.g is developed by high-density fermentation and purification with Ni-NTA combined with Sephadex G-75 column chromatography.

Animals ; Antigens, Protozoan ; biosynthesis ; genetics ; immunology ; Antigens, Surface ; immunology ; Blotting, Western ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli ; genetics ; metabolism ; Fermentation ; Protozoan Proteins ; biosynthesis ; genetics ; immunology ; Recombinant Fusion Proteins ; biosynthesis ; immunology ; isolation & purification ; Toxoplasma ; immunology

Serologic tests are widely accepted for diagnosing Toxoplasma gondii but purification and standardization of antigen needs to be improved. Recently, surface tachyzoite and bradyzoite antigens have become more attractive for this purpose. In this study, diagnostic usefulness of 3 recombinant antigens (SAG1, SAG2, and SAG3) were evaluated, and their efficacy was compared with the available commercial ELISA. The recombinant plasmids were transformed to JM109 strain of Escherichia coli, and the recombinants were expressed and purified. Recombinant SAG1, SAG2, and SAG3 antigens were evaluated using different groups of sera in an ELISA system, and the results were compared to those of a commercial IgG and IgM ELISA kit. The sensitivity and specificity of recombinant surface antigens for detection of anti-Toxoplasma IgG in comparison with commercially available ELISA were as follows: SAG1 (93.6% and 92.9%), SAG2 (100.0% and 89.4%), and SAG3 (95.4% and 91.2%), respectively. A high degree of agreement (96.9%) was observed between recombinant SAG2 and commercial ELISA in terms of detecting IgG anti-Toxoplasma antibodies. P22 had the best performance in detecting anti-Toxoplasma IgM in comparison with the other 2 recombinant antigens. Recombinant SAG1, SAG2, and SAG3 could all be used for diagnosis of IgG-specific antibodies against T. gondii.
Antibodies, Protozoan/*blood ; Antigens, Protozoan/diagnostic use/*genetics ; Enzyme-Linked Immunosorbent Assay ; Humans ; Immunoglobulin G/blood ; Immunoglobulin M/blood ; Membrane Glycoproteins/*genetics ; Protozoan Proteins/*genetics ; Recombinant Proteins/diagnostic use/immunology ; Sensitivity and Specificity ; Toxoplasma/immunology ; Toxoplasmosis/blood/*diagnosis

Major surface protein (p30) and Dense Granule Antigen GRA6 of Toxoplasma gondii have good antigenicity, and could be used for detection of IgM against Toxoplasma gondii. GRA6 may complement P30 to reach more high sensitivity for detection of antibodies to Toxoplasma gondii, so, we try to express the chimeric protein of GRA6 and P30 by genetic engineering, identify its antignenicity and use for developing diagnosis reagent. Antigenic domains of p30 and GRA6 of Toxoplasma gondii were screened by analyzing their sequences using the software ANTHEWIN. Two DNA fragments encoding respectively antigenic domains of p30 and GRA6 were cloned, they were inserted into the same expression vector pET28a( + ) and expressed as a chimeric protein in Escherichia coli. BL21(DE3), the expressed chimeric protein of p30 with GRA6 in a form of inclusion body was about 25% of total proteins of E. coli. BL21(DE3). The inclusion body was washed once with 0.5% Triton X-100 and dissolved with 0.5% SKL, after renaturation by gradient dialysis, the recombinant protein was purified by DEAE-Sepharose FF cation column and then detected with 12% SDS-PAGE, it exists mainly in the eluted peak with 300 mmol/L NaCl and has high purity. By using enzyme-linked immunosorbent assay (ELISA), the recombinant protein was examined for reactivity with immunoglobulin M (IgM) antibodies in 6 sera from patients infected with Toxoplasma gondii ., it was reactive with all the 6 sera but not with sera from normal people, these results showed that the recombinant chimeric antigen has good antigenicity and specificity and could be used for detection of IgM against Toxoplasma gondii. The expressed chimeric protein could be used for epidemic investigation of Toxoplasma gondii, blood donor screening, especially for detection of pregnant women, and is of great significance in prevention of Toxoplasma gondii infection.
Animals ; Antigens, Protozoan ; genetics ; immunology ; metabolism ; Electrophoresis, Polyacrylamide Gel ; Enzyme-Linked Immunosorbent Assay ; Female ; Humans ; Immunoglobulin M ; immunology ; Models, Genetic ; Polymerase Chain Reaction ; Pregnancy ; Protozoan Proteins ; genetics ; metabolism ; Recombinant Fusion Proteins ; genetics ; immunology ; metabolism ; Toxoplasma ; genetics ; immunology

Relatively little has been studied on the AMA-1 vaccine against Plasmodium vivax and on the plasmid DNA vaccine encoding P. vivax AMA-1 (PvAMA-1). In the present study, a plasmid DNA vaccine encoding AMA-1 of the reemerging Korean P. vivax has been constructed and a preliminary study was done on its cellular immunogenicity to recipient BALB/c mice. The PvAMA-1 gene was cloned and expressed in the plasmid vector UBpcAMA-1, and a protein band of approximately 56.8 kDa was obtained from the transfected COS7 cells. BALB/c mice were immunized intramuscularly or using a gene gun 4 times with the vaccine, and the proportions of splenic T-cell subsets were examined by fluorocytometry at week 2 after the last injection. The spleen cells from intramuscularly injected mice revealed no significant changes in the proportions of CD8+ T-cells and CD4+ T-cells. However, in mice immunized using a gene gun, significantly higher (P<0.05) proportions of CD8+ cells were observed compared to UB vector-injected control mice. The results indicated that cellular immunogenicity of the plasmid DNA vaccine encoding AMA-1 of the reemerging Korean P. vivax was weak when it was injected intramuscularly; however, a promising effect was observed using the gene gun injection technique.
Animals ; Antigens, Protozoan/administration & dosage/genetics/*immunology ; CD8-Positive T-Lymphocytes/*immunology ; COS Cells ; Cercopithecus aethiops ; Humans ; Lymphocyte Activation ; Malaria, Vivax/*immunology/parasitology ; Membrane Proteins/administration & dosage/genetics/*immunology ; Mice ; Mice, Inbred BALB C ; Plasmodium vivax/genetics/*immunology ; Protozoan Proteins/administration & dosage/genetics/*immunology ; Protozoan Vaccines/administration & dosage/genetics/*immunology ; Vaccines, DNA/administration & dosage/genetics/*immunology

Recombinant antigenic proteins of Toxoplasma gondii are alternative source of antigens which are easily obtainable for serodiagnosis of toxoplasmosis. In this study, highly antigenic secretory organellar proteins, dense granular GRA2 and GRA3, rhoptrial ROP2, and micronemal MIC2, were analyzed by bioinformatics approach to express as water-soluble forms of antigenic domains. The transmembrane region and disorder tendency of 4 secretory proteins were predicted to clone the genes into pGEX-4T-1 vector. Recombinant plasmids were transformed into BL21 (DE3) pLysS E. coli, and GST fusion proteins were expressed with IPTG. As a result, GST fusion proteins with GRA225-105, GRA339-138, ROP2324-561, and MIC21-284 domains had respectively higher value of IgG avidity. The rGST-GRA225-105 and rGST-GRA339-138 were soluble, while rGST-ROP2324-561 and rGST-MIC21-284 were not. GRA231-71, intrinsically unstructured domain (IUD) of GRA2, was used as a linker to enhance the solubility. The rGST-GRA231-71-ROP2324-561, a chimeric protein, appeared to be soluble. Moreover, rGST-GRA231-71-MIC21-284 was also soluble and had higher IgG avidity comparing to rGST-MIC21-284. These 4 highly expressed and water-soluble recombinant antigenic proteins may be promising candidates to improve the serodiagnosis of toxoplasmosis in addition to the major surface antigen of SAG1.
Animals ; Antibodies, Protozoan/immunology ; Antibody Affinity ; Antigens, Protozoan/chemistry/*diagnostic use/genetics/immunology ; *Gene Expression ; Immunoglobulin G/blood/immunology ; Mice, Inbred BALB C ; Recombinant Proteins/chemistry/*diagnostic use/genetics/immunology ; Serologic Tests/methods ; Solubility ; Toxoplasma/genetics/immunology/*metabolism ; Toxoplasmosis/diagnosis