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Objective To study genetic polymorphism of surface adhesion protein 33 (AP33) gene on the seven isolates of Trichomonas vaginalis. Methods PCR technique was performed to amplify AP33 gene from the seven isolates, DNA sequences were obtained from the AP33 gene of the isolates and phylogenetic tree was built. Minimal lethal concentrations(MLC) of metronidazole on the isolates were measured in vitro. Results Percentage of the similarity between 7 isolates and U87098 in GenBank was 98.2%-100%,which indicated a high homology and belonged to isotype isolates. There were four branches between Beijing 1 isolate and Tangshan isolate, Jiujiang 1 isolate and Jiujiang 2 isolate, Beijing 2 isolate and Jiujiang 3 isolate, Chengde isolate and U87098 isolate in phylogenetic tree, which showed a close genetic relationship respectively. No relativity was detected between geographical origin and genetic relationship. Conclusion There is a close genetic relationship among the seven T. vaginalis isolates. MLC showed a difference between isolates which have close relationship.
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Objective To study the killing effect of polymorphonuclear neutrophils(PMNs) on Trichomonas vaginalis. Methods The vaginal secretion from a patient with vaginitis was incubated in the liver infusion liquid medium to get T. vaginalis. One ml serum was collected from the patient and heated for 30 min at 56 ℃ to inactivate complement in serum, and was absorbed three times with the parasites at 0 ℃ to make the serum free of antibodies. PMNs were separated from the patient’s blood and purified with density gradient centrifugation and polymer accelerating sedimentation. NBT and safranin O were used to stain the sample. The interaction between PMNs and the parasites was observed under microscope. 300 trichomonads and 3?104 PMNs were incubated for 10, 20, 30, 40, 50, 60 minutes under the conditions of aerobic or anaerobic, with superoxide dismutase (SOD) and catalase (CAT) or without SOD and CAT, and with complement or without complement. They were then inoculated in solid medium for another five days under the anaerobic condition, and surviving organisms were enumerated. Results PMNs were observed to surround and kill a single trichomonad. In the petri-dish containing PMNs, the surviving rate of the parasites in anaerobic condition was 85%, only 3% in aerobic condition (P
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Objective To observe the effect of dihydroartemisinin(DHA) on the ultrastructure of Trichomonas vaginalis cultured in vitro.Methods The trophozoites of T.vaginalis were cultivated with liver extract solution medium containing 1 mg/ml dihydroartemisinin,and were then observed by scanning and transmission electron microscopes after the treatment for 2.5-4.0 h.Results The cell membranes of the trophozoites treated with DHA were damaged considerably.The surface of T.vaginalis showed deepening folds,hollow,and cracks.The nuclear membrane appeared fractures.There were a few of crevices in the nucleus and cytoplasm.Disordered and dilated endoplasmic reticulum,injured and deformed hydrogenosomes were found.Cytoplasm of the damaged parasites spilled over from torn place.After the cell membrane was peeled off,the nuclei,hydrogenosomes and pelta were exposed,which finally resulted in the death of the denatured parasites.Conclusion Dihydroartemisinin can destroy membrane structure and organelles of Trichomonas vaginalis trophozoites,which leads to decomposition and necrosis of the parasites.
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Objective To Construct the prokaryotic expression vector of the fusion gene IFN-?1b/CSPⅡ. MethodsIFN-?1b was amplified from the human genomic DNA by PCR and cloned into prokaryotic expression vector pGEX-4T-1. The recombinant plasmid pGEX-4T-1/IFN-?1b was constructed. Circumsporozoite proteinⅡ(CSPⅡ) was amplified from the Plasmodium falciparum genomic DNA by PCR and was cloned into the prokaryotic expression vector pGEX-4T-1. The recombinant plasmid pGEX-4T-1/CSPⅡ was constructed. IFN-?1b was cut from the recombinant plasmid pGEX-4T-1/IFN-?1b digested with BamHⅠ and EcoRⅠ and ligated with the recombinant plasmid pGEX-4T-1/CSPⅡ also digested with BamHⅠ and EcoRⅠ . The recombinant prokaryotic plasmid pGEX-4T-1/IFN-?1b/CSPⅡ was constructed. The fusion gene IFN-?1b/CSPⅡ was expressed in E.coli by IPTG. Results The prokaryotic expression vector pGEX-4T-1/IFN-?1b, pGEX-4T-1/CSPⅡ and pGEX-4T-1/IFN-?1b/CSPⅡ were identified by PCR, enzyme digestion and gene sequencing. The expressed fusion protein/IFN-?1b/CSPⅡ in E.coli was identified by SDS-PAGE and Western blot. Conclusion The prokaryotic expression vector of the fusion gene IFN-?1b/CSPⅡ was successfully constructed, which was then expressed in E.coli .
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Objective To study genetic polymorphism of DNA on seven isolates of Trichomonas vaginalis. Methods The random amplified polymorphic DNA (RAPD) technique was performed to amplify genomic DNA of the seven T. vaginalis isolates, including Beijing 1, Beijing 2, Chengde, Tangshan, Jiujiang 1, Jiujiang 2 and Jiujiang 3. The DNA bands detected were analyzed by clustering analysis with SPSS software. Results The percentage of genetic similarity among the seven isolates was from 77.4% to (94.7%,) showing a close genetic relationship among them. The percentages between the isolates of Beijing 1 and Tangshan, Jiujiang 1 and Jiujiang 2, Beijing 2 and Jiujiang 3 were 89.2%, 92.1% and 94.7% respectively, while that of Jiujiang1 and Chengde was 77.4%, indicating a lower homology. Conclusion There are a close genetic relationship and certain gene polymorphism among the seven T. vaginalis isolates; geographical origin plays little role to the genetic characteristics.
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Objective To study the biological types on the seven isolates of Trichomonas vaginalis from Beijing, Hebei-Tangshan, Hebei-Chengde and Jiangxi-Jiujiang in the mainland of China. Methods The samples were analyzed by PAGE, isoenzyme stain and cluster analysis. Results The isoenzyme systems used in the study included MDH, LDH, G-6-PD, PGI and PGM. No difference in the isoenzyme patterns of G-6-PD and PGI was found among the seven isolates. The MDH and LDH patterns of Beijing 1, Beijing 2, Jiujiang 3 strains were identical, while they were distinguishable from those of Chengde, Tangshan, Jiujiang 1, Jiujiang 2 isolates. The PGM pattern of Beijing 1 and Beijing 2 isolates were same but was different from that of the remainders. Gene tree was constructed according to the isoenzyme profiles. The results showed that there are differences in the patterns of the five isoenzymes between the isolates of Beijing 1, Beijing 2, Jiujiang 3 and other four isolates, and Jiujiang 3 was different from Beijing 1, Beijing 2 slightly. Conclusion It seems reasonable to assume that there are at least three different biological types of Trichomanas vaginalis in China.
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After the merozoite entered the erythrocyte,the membrane debris in the parasitophorous vacuoles of early ring form was passed out through a narrow external aperture in erythro-cyte to the exterior.The trophozoite was oval or irregular in shape.Ingestion of host cell cytoplasm occurred cystostomally.The asexual parasite possessed acristate mitochondria and was surrounded by a single-membraned pellicle.The gametocyte possessed cristate mitochondria and was surrounded by two unit membranes.The cytoplasm of mature macrogametocytes contained many ribosomes,mitochondria and osmiophilic bodies and a small nucleus while microgametocytes contained fewer ribosomes,osmiophilic bodies and mitochondria and a large nucleus.Three characteristic morphological alterations were observed within the host cells,that is,small vesicles,cytoplasmic cleft and caveola-vesicle complex.The clefts within the cytoplasm of the host erythrocytes were present in all human malarial parasites.The small vesicles distributed all over the cytoplasm were surrounded by a unit membrane.The caveola-vesicle complex consisted of caveolae was surrounded by small vesicles and probably corresponds to a Schuffner's dot.(Figs.1-13)
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The ookinete formation and mature ookinete of Plasmodium yoelii yoelii were described. During the development from zygote to ookinete, at first a finger-shaped or a stick-shaped projection protruded from one side of zygote (Fig. l).The anterior end of the projection assumed a truncated cone shape (Figs. 1-5). Following enlargement of the projection, the body of zygote gradually wrinkled and shrank and became smaller (Figs. 2-5). An annular structure developed between the newly formed ookinete and the remaining body of zygote (Figs. 5-6), which ultimately separated from the ookinete.Tile ookinete was banana-shaped. The surface of mature ookinetes was smooth (Fig. 13). On the tip there was an apical complex showing truncated cone shape. Behind the apical end there were a few pellicular folds (Figs. 9-11).Through the course of development of the ookinete the parasite constantly showed obvious movement. As the parasites protruding forward, longitudinal and spiral wrinkles appeared on their body surface (Figs. 2-4, 10-12).
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100 kDa respectively. \ \{Conclusion\ Eight\} protein bands out of 26 soluble antigen bands of the parasite showed high immunogenicity. There were 9 main polypeptide spots in 43 polypeptide spots of the parasite.
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Hybridomas producing monoclonal antibodies (McAb) directed against Trichomonas vaginalis have been produced by fusing NSI myeloma cells with spleen cells of BALB/c mice immunized with Trichomonas vaginalis.IFA technique was used to test the binding activity of four McAbs produced.The McAb belonged to the IgG subtypes IgGl(2A2,2A4 McAb),IgG3 (2H9 McAb) and IgG 2b (2A12 McAb).Three McAbs,designated 2A2,2A4,2A12,reacted with a surface membrane component of live Trichomonas vaginalis.One (2A12) of them produced com-plement-dependent cytolysis of the parasites.Others (2A2.2A4) produced complement-independent cytotoxicity of the parasites.2H9 McAb which reacted with the nucleus of the organisms did not agglutinate the parasites.The four McAbs which did not have cross reaction with some protozoa of Zoomastigophorea species were specific antibodies against Trichomonas vaginalis.(Figs.1-3)