1.Identification of the binding site on glycophorin A for Plasmodium falciparum EBA-175.
Xiao-min SUN ; Wen-bo HAO ; Ming LI ; Ren LUO ; Yu-hua JIA
Journal of Southern Medical University 2007;27(11):1696-1698
OBJECTIVETo identify the binding site on glycophorin A (GPA) for EBA-175 to provide clue for developing short peptide vaccine and therapeutic agents against Plasmodium falciparum.
METHODSWith the recombinant protein of EBA-175 as the target molecule, the mimetic peptides of GPA were screened from a 12-mer random peptide library. Three rounds of biopanning were carried out, and enzyme-linked immunosorbent assay (ELISA), competitive ELISA, Dot-ELISA and Western blotting used to evaluate the binding between the phage-borne peptides and EBA-175. The insert DNA sequences of positive clones were determined and their amino acid sequences deduced.
RESULTSThirty clones from the third round were randomly selected, of which 27 were found positive by sandwich ELISA. Competitive ELISA proved that most of the phage-borne peptides could competitively inhibit the binding of antibody (EBA-175 Ab) with EBA-175. Analysis of DNA and amino acid sequences indicated that 24 positive phage clones contained the conservative sequence of IRR, which was highly homologous with the 114-116 amino acids of GPA.
CONCLUSIONThese phage-displayed peptides can bind with EBA-175, and the amino acid sequence IRR might play an important role in the binding between EBA-175 and GPA.
Antigens, Protozoan ; metabolism ; Binding Sites ; Enzyme-Linked Immunosorbent Assay ; Glycophorin ; chemistry ; Humans ; Peptide Library ; Plasmodium falciparum ; Protozoan Proteins ; metabolism ; Sequence Analysis, DNA ; Sequence Analysis, Protein
2.Study of the relations between toxoplamosis and bronchial asthma.
Hong LIAO ; Long XU ; Yi-ming GUO ; Yi ZHAO ; Zhen-ying DING ; You-yuan ZENG ; Hong TANG ; Wen-yi ZHOU ; Song ZHANG ; Li-min ZHANG ; Wei WU ; Li ZHANG
Chinese Journal of Pediatrics 2003;41(6):470-470
Animals
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Antibodies, Protozoan
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analysis
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Antigens, Protozoan
;
analysis
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Asthma
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blood
;
parasitology
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Child
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Child, Preschool
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Female
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Humans
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Infant
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Male
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Toxoplasma
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immunology
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Toxoplasmosis
;
blood
3.Detection and characterization of excretory/secretory proteins from Toxoplasma gondii by monoclonal antibodies.
The Korean Journal of Parasitology 2001;39(1):49-56
Excretory/secretory proteins (ESP) from Toxoplasma gondii were analyzed to define the function in the penetration process into host cells. Whole ESP obtained at 37 degrees C were composed of 15 bands with molecular mass of 110, 97, 86, 80, 70, 60, 54, 42, 40, 36, 30, 28, 26, 22, and 19 kDa. Five ESP of 86, 80, 42, 36, and 28 kDa were reacted with monoclonal antibodies (mAb), named as Tg386 (microneme), Tg485 (surface membrane), Tg786 (rhoptry), Tg378, and Tg556 (both dense granules), respectively. The ESP was released by a temperature-dependent/-independent manner and all at once whenever ready to pour out except Tg786. Each ESP was not exhausted within the parasite but the amount was limited. Tg786 was released continuously with increment, whereas Tg378 and Tg556 were ceased to release after 3 and 4 hr. Dense granular Tg378 and Tg556 were released spontaneously and constitutively before the entry into host cells also. The entry of T. gondii was inhibited by all the mAbs differentially. And the parasite deprived of ESP was inhibited to enter exponentially up to 90.1%. It is suggested that ESP play an essential function to provide appropriate environment for the entry of the parasite into host cells.
Animals
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*Antibodies, Monoclonal
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*Antibodies, Protozoan
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Antigens, Protozoan/*analysis/physiology
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Mice
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Mice, Inbred BALB C
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Support, Non-U.S. Gov't
;
Temperature
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Toxoplasma/*chemistry/pathogenicity
4.Antigenic diversity of Theileria major piroplasm surface protein gene in Jeju black cattle.
Myung Soon KO ; Kyoung Kap LEE ; Kyu Kye HWANG ; Byung Sun KIM ; Gui Cheol CHOI ; Young Min YUN
Journal of Veterinary Science 2008;9(2):155-160
Piroplasms are tick-transmitted, intracellular, hemoprotozoan parasites that cause anorexia, fever, anemia, and icterus. Theileriosis is caused by Theileria sergenti and causes major economic losses in grazing cattle in Japan and Korea. In May 2003, we examined the antigenic diversity of the major piroplasm surface protein (MPSP) gene in 35 healthy Jeju black cattle that were born and raised at the National Institute of Subtropical Agriculture. On microscopic examination of Giemsa-stained blood smears, 9 of 35 cattle had intra-erythrocytic piroplasms. Hematological data were within normal range for all 35 cattle. Amplification of DNA from all blood samples using universal MPSP gene primers showed mixed infections with C, I, and B type Theileria spp. Type C was identified in 20 of 35 blood samples, and type B was identified in 17 samples. Allelic variation was seen in type B.
Animals
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Antigens, Protozoan/*genetics
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Base Sequence
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Cattle
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DNA Primers/genetics
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*Genetic Variation
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Korea
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Molecular Sequence Data
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*Phylogeny
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Protozoan Proteins/*genetics
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Sequence Analysis, DNA
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Theileria/*genetics
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Theileriasis/*parasitology
5.Two-dimensional gel electrophoresis and immunoblot analysis of Neospora caninum tachyzoites.
Eung Goo LEE ; Jae Hoon KIM ; Yong Seung SHIN ; Gee Wook SHIN ; Yong Hwan KIM ; Gon Sup KIM ; Dae Yong KIM ; Tae Sung JUNG ; Myung Deuk SUH
Journal of Veterinary Science 2004;5(2):139-145
Identification of expressed protein profiles and antigenic determination are some of the most challenging aspects of proteomics. Two-dimensional gel electrophoresis (2-DE) combined with immunoblot analysis were employed to study the N. caninum proteome. Protein sample preparation was carried out by first conducting sonication, followed by adding lysis buffer containing 7M urea plus 2M thiourea to the purified tachyzoites in order to complete disruption. A total of 335 differentially expressed protein spots were detected using pH 4-7 IPG strip (7 cm) that were run in a 56 kVh isoelectric focusing (IEF) system. Of the spots analyzed, 64 were identified as antigenic spots on immunoblot profile. Major antigenic spots appeared at 65 kDa (pI 5.2-5.3), 51 kDa (pI 5.5), 38 kDa (pI 5.1), 33 kDa (pI 4.4), 29 kDa (pI 5.6) and 15.5 kDa (pI 5.0) were observed to be significantly distinct compared to the rest of the antigenic spots. The results indicate that combination of 2-DE and immunoblotting methods were thought as very useful tools in defining both proteins and antigens of N. caninum tachyzoites. Additionally, present 2-DE profiles may be valuable in further proteomic approaches and study of the pathogen.
Animals
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Antibody Formation
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Antigens, Protozoan/*analysis/isolation&purification
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Electrophoresis, Gel, Two-Dimensional/methods
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Electrophoresis, Polyacrylamide Gel
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Epitopes/analysis
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Image Processing, Computer-Assisted
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Immunoblotting/methods
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Isoelectric Focusing
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Neospora/*chemistry/immunology
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Proteome/analysis
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Proteomics
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Protozoan Proteins/*analysis/isolation&purification
6.Toxoplasma gondii: ultrastructural localization of specific antigens and inhibition of intracellular multiplication by monoclonal antibodies.
Boo Young LEE ; Myoung Hee AHN ; Hyun Chul KIM ; Duk Young MIN
The Korean Journal of Parasitology 2001;39(1):67-75
This experiment was focused on the characterization of anti-Toxoplasma monoclonal antibodies (mAbs) and the effect of mAbs on the parasite invasion of mouse peritoneal macrophages. Twenty eight mAbs including M110, M556, R7A6 and M621 were characterized by Ab titer, immunoglobulin isotyping and western blot pattern. Antibody titer (optical density) of 4 mAbs, M110, M556, R7A6 and M621, were 0.53, 0.67, 0.45 and 0.39 (normal mouse serum; 0.19) with the same IgG1 isotypes shown by Enzyme-linked immunosorbent assay (ELISA). Western blot analysis showed that M110, M556, R7A6 and M621 reacted with the 33 kDa (p30), 31 kDa (p28), 43 kDa and 36 kDa protein. Immunogold labelling of mAbs M110, M556, R7A6 and M621 reacted with the surface membrane, dense granules and parasitophorous vacuolar membrane (PVM), rhoptries and cytoplasm of tachyzoite, respectively. For in vitro assay, preincubation of tachyzoites with four mAbs, M110, M556, R7A6 and M621 resulted in the decrease of the number of infected macrophages (P < 0.05) and the suppression of parasite multiplication at 18 h post-infection. Four monoclonal antibodies including M110 (SAG1) were found to have an important role in the inhibition of macrophage invasion and T. gondii multiplication in vitro, and these mAbs may be suitable for vaccine candidates, diagnostic kit and for chemotherapy.
Animals
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*Antibodies, Monoclonal/pharmacology
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*Antibodies, Protozoan/pharmacology
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Antigens, Protozoan/*analysis/immunology
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Cells, Cultured
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Depression, Chemical
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Macrophages, Peritoneal/parasitology
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Mice
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Mice, Inbred ICR
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Support, Non-U.S. Gov't
;
Toxoplasma/growth & development/*immunology
7.Ultrastructural Localization of Cryptosporidium parvum Antigen Using Human Patients Sera.
Jong Gyu LEE ; Eun Taek HAN ; Woo Yoon PARK ; Jae Ran YU
The Korean Journal of Parasitology 2009;47(2):171-174
The antigen location of Cryptosporidium parvum, which stimulates antibody formation in humans and animals, was investigated using infected human sera. Immuno-electron microscopy revealed that antigenicity-inducing humoral immunity was located at various developmental stages of parasites, including asexual, sexual stages, and oocysts. The amount of antigen-stimulating IgG antibodies was particularly high on the oocyst wall. The sporozoite surface was shown to give stimulation on IgG and IgM antibody formation. Trophozoites implicated the lowest antigenicity to humoral immunity, both IgG and IgM, by showing the least amount of gold labeling. Immunogold labeling also provided clues that antigens were presented to the host-cell cytoplasm via feeder organelles and host-parasite junctions.
Animals
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Antibodies, Protozoan/*immunology
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Antigens, Protozoan/*analysis
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Cryptosporidium parvum/*chemistry/*immunology/ultrastructure
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Female
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Humans
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Immunoglobulin G/immunology
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Immunoglobulin M/immunology
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Mice
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Microscopy, Immunoelectron
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Sporozoites/chemistry/immunology/ultrastructure
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Staining and Labeling/methods
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Trophozoites/chemistry/immunology/ultrastructure
8.Analysis of polymorphic regions of Plasmodium vivax Duffy binding protein of Korean isolates.
Weon Gyu KHO ; Joon Yong CHUNG ; Eun Jeong SIM ; Dong Wook KIM ; Woo Chul CHUNG
The Korean Journal of Parasitology 2001;39(2):143-150
The present study was designed to investigate polymorphism in Duffy binding protein (DBP) gene of Plasmodium vivax isolates of Korea. Thirty samples were obtained from P. vivax patients in Yonchon-gun, Kyonggi-do in 1998. The PCR products of the samples were subjected to sequencing and hybridization analyses of the regions II and IV of P. vivax DBP gene. Two genotypes, SK-1 and SK-2, were identified on the basis of amino acid substitution and deletion. The genotype of 10 isolates was SK-1 and that of 20 isolates was SK-2. Most of the predicted amino acids in the region II of DBP gene were conserved between the Korean isolates and Belem strain except for 4-5 amino acid substitutions. In the region IV of DBP, a 6-bp insert that was shown in the Sal-1 allele type was found in SK-1, and a 27-bp insert that was shown in the Papua New Guinea allele type was found in SK-2. In conclusion, the present findings suggest that two genotypes of P. vivax coexist in the endemic area of Korea.
Amino Acid Sequence
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Animals
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*Antigens, Protozoan
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Base Sequence
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Carrier Proteins/*analysis/chemistry/*genetics
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DNA, Protozoan/genetics
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Genotype
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Human
;
Korea
;
Malaria, Vivax/parasitology
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Molecular Sequence Data
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Plasmodium vivax/*genetics/isolation & purification
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Polymerase Chain Reaction
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*Polymorphism (Genetics)
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*Protozoan Proteins
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Receptors, Cell Surface/*analysis/chemistry/*genetics
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Support, Non-U.S. Gov't
9.Genotype of Toxoplasma gondii from Blood of Stray Cats in Gyeonggi-do, Korea.
Hye Youn KIM ; Yun Ah KIM ; Ho Sa LEE ; Ho Gun RHIE ; Shin Hyeong CHO ; Jae Ran YU ; Sang Eun LEE
The Korean Journal of Parasitology 2009;47(4):413-415
Genotyping of Toxoplasma gondii has been performed in 23 PCR positive blood samples from stray cats in Korea. We used 2 separate PCR-restriction fragment length polymorphism (RFLP) patterns of SAG2 gene, amplifying the 5'and 3'ends of the locus. The results revealed that all samples belonged to the type I clonal lineage. Although T. gondii organisms were not isolated from the samples, the results of the present study represent that stray cats with T. gondii infection should be seriously concerned in our environment. Adequate and continuous control programs of stray cats are needed to reduce the risk of transmission of T. gondii as a zoonotic infection threatening the public health.
Animals
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Antigens, Protozoan/genetics
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Blood/*parasitology
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Cat Diseases/*parasitology
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Cats
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Cluster Analysis
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DNA Fingerprinting/methods
;
DNA, Protozoan/genetics/isolation & purification
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Genotype
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Korea
;
Polymerase Chain Reaction/methods
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Polymorphism, Restriction Fragment Length
;
Protozoan Proteins/genetics
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Toxoplasma/*classification/*genetics/isolation & purification
;
Toxoplasmosis, Animal/*parasitology
10.Primary structure of mature SAG1 gene of an Indonesian Toxoplasma gondii and comparison with other strains.
Sri HARTATI ; Asmarani KUSUMAWATI ; Hastari WURYASTUTI ; J Sri WIDADA
Journal of Veterinary Science 2006;7(3):263-270
Toxoplasma gondii is a persistent protozoan parasite capable of infecting almost any warm-blooded vertebrates. SAG1 (p30) is the prototypic member of a superfamily of surface antigens called SRS (SAG1-related sequence). It constitutes the most abundant and predominant antigen. In this paper the primary structure of mature SAG1 gene of an Indonesian T. gondii isolate is described and sequence comparison is made with published sequence data of 7 other strains or isolates. Sequence comparison indicated that SAG1 is highly conserved through evolution and despite parasite spreading world-wide. Sequences may be divided into two major families, independent of the strain/isolate geographic origin. Variations were mainly localized at the C-terminal half or domain 2 and some clustered in restricted areas. Sequence comparison allowed us to define the Indonesian isolate as genuine virulent RH strain. A phylogenetic tree of Toxoplasma strains/isolates was constructed based on SAG1.
Amino Acid Sequence
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Animals
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Antigens, Protozoan/chemistry/*genetics
;
Base Sequence
;
Cloning, Molecular
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DNA, Protozoan/chemistry/genetics
;
Goat Diseases/parasitology
;
Goats
;
Indonesia
;
Molecular Sequence Data
;
Phylogeny
;
Polymerase Chain Reaction
;
Protozoan Proteins/chemistry/*genetics
;
Sequence Alignment
;
Sequence Analysis, DNA
;
Toxoplasma/*genetics/*immunology/isolation&purification
;
Toxoplasmosis/parasitology
;
Zoonoses/parasitology