Table of Content

30 August 2005, Volume 23 Issue 4

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论著

Screening of a Hepatic Stellate Cells Subtracted cDNA Library of Differentially Expressed Genes in Mice with Schistosomiasis japonica

ZHENGMin;CHENFeng;CAIWei-min;LIURong-hua

2005, 23(4):  1-197. 

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Objective To construct and screen a hepatic stellate cells(HSCs) subtracted cDNA library, in order to seek differentially expressed genes in mice infected with Schistosoma japonicum. Methods HSCs were isolated from mouse as targets, and cDNA fragments of normal mouse were compared to those of S.japonicum infected mice to find differentially expressed genes by technique of suppression subtractive hybridization(SSH). Differentially expressed cDNA fragments were then directly inserted into T/A cloning vector to set up the subtractive library. Amplification of the library was carried out with transformation of DH5α. A subtracted cDNA library was constructed and then hybridized with forward and backward subtracted probes for differential screening. One hundred positive bacterial clones were randomly selected for sequencing and BLAST analysis. Finally, virtual Northern blot confirmed such differential expression. Results The amplified library contained more than 400 positive bacterial clones. Random analysis of 100 clones with restriction enzyme digestion showed that all clones contained 200-600 bp inserts. 76 ESTs were obtained with 70 related to fibrosis caused by schistosomiasis or other etiological factors. Other 6 ESTs were not found in PubMed. Conclusion The subtracted cDNA library of differentially expressed genes of HSC in normal mice and schistosome-infected mice was constructed successfully with SSH and T/A cloning techniques.


Study on a Grey Model for Evaluation of Anopheles minimus Density

YUGuo-wei;TANGLin-hua

2005, 23(4):  2-201. 

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Objective To establish Anopheles minimus density evaluation model based on climate, environmental and remote sensing data. Methods 27 townships in 10 counties of Yunnan Province were chosen as field spots. Data on climate, environment, remote sensing and An. minimus density were collected from 1984 to 1993. Grey correlation analysis was carried out to study the relationship of 18 indices with An. minimus density. E variable was developed and its relation to the An. minimus density was analyzed to establish an evaluation model. Results Eight indices were selected based on a grey threshold 0.70: Dry season average temperature>dry season temperature_min>wet season temperature_min>wet season NDVI>wet season average temperature>the ratio of paddy field in total arable land>dry season temperature_max>wet season temperature_max. The An. minimus density evaluation model was derived as follows: y=0.0578e0·0780(8X10'+7X12'+6X11'+5X15'+4X9'+3X4'+2X8'+1X7') The correct rate of evaluation by the model was 92.0%. e0.5=18%, with an average relative error of 21%. Conclusion A quasi-evaluation on the An. minimus density can be made by applying the grey model.


论文

Transformation of Schistosomulae by Electroporation and Transient Expression of the Enhanced Green Fluorescent Protein (EGFP) Gene

YUANXiao-song;SHENJi-long;WANGXue-long;HUYuan-sheng;LUOQing-li

2005, 23(4):  3-205. 

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Objective To explore the possibility of heterogenous gene to express in juvenile Schistosoma japonicum and the application of electroporation in transformation of schistosomulae. Methods The plasmids of pEGFP-C1 were introduced into mechanically transformed schsitosomula with electroporation. The presence, transcription and translation of the transgene in electroporated schistosomula were confirmed by PCR, RT-PCR and Western blotting analysis respectively using the genomic DNA, total RNA and protein extracted and isolated from schistosomula cultured in vitro for 48 hours. Meanwhile, localization of EGFP within electroporated schistosomula was performed with confocal laser scanning microscope. Results 760 bp and 276 bp amplified products by PCR and RT-PCR were found coincident with the expected size and expression of EGFP gene in elctroporated schistosomula was confirmed by Western blotting. Fluorescence of EGFP was localized in tegument and subtegument of the electroporated schisto?鄄somula with confocal microscopy, especially in the anterior part of the worm. Conclusion The heterogenous gene of EGFP has been successfully introduced into juvenile S. japonicum by electroporation and the expression of transgene was confirmed with molecular and microscopical methods.


论著

Purification of the Effective Component from Solanum xanthocarpum and its Effect against Oncomelania Snails

LIZhou;CHENGXi;WANGChun-jing;LIGui-ling;XIAShu-zhen;WEIFeng-hua

2005, 23(4):  4-208. 

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Objective To extract and purify the active component from Solanum xanthocarpum for Oncomelania snail control, evaluate its effect against the snails and determine its chemical structure. Methods Pedicel and seed were moved and the remaining part of the Solanum xanthocarpum fruit was torrefied and ground into powder. The powder was soaked into 95% ethanol and extracted. The residue was treated by ethyl acetate. 7 components were separated by methods of thin layer chromatography (TLC) and column chromatography (CCL). 4 main components were observed for the effect of snail-killing by immersion method. An alkaloid with Rf=0.58, named as component A, was confirmed as one of the effective components for Oncomelania snail control. The component A was further purified and received A1 and A2 by proper elusion, and their effect was re-tested. The molecular weight and chemical structure of A2 were determined by MS, NMR and IR respectively. Results The death rate of Oncomelania snails was 94.2% when the concentration of component A was 2.50 mg/L(28 ℃). The flow liquid system (ethyl acetate: chloroform: methanol=11∶11∶35) applied was the best reagent for separating component A. The death rate of Oncomelania snails in solution of A2 (0.2 mg/L) was 100%(28 ℃).The molecular weight of A2 was 867 with an mp 298-305 ℃. Conclusion The effective agent (A2),one of the active components from the fruit of Solanum xanthocarpum, is α-solamarrgine which shows an excellent effect in killing Oncomela snails.


Preparation and Preliminary Application of Monoclonal Antibodies Against Adult Worm of Angiostrongylus cantonensis

TANFeng;PANChang-wang;LIANGShao-hui;HUANGHui-cong

2005, 23(4):  5-212. 

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　Objective To develop and identify monoclonal antibodies (McAbs) against adult worm of Angiostrongylus cantonensis and observe its applicability. Methods BALB/c mice were immunized with soluble antigen of adult worms of A.cantonensis. The spleen cells of immunized mice were fused with myeloma cell, and the hybridoma secreting high titer of McAbs with high specificity was screened. By using the McAbs, serum of angiostrongyliasis patient and sera of the rats infected with A.cantonensis were detected by Western blotting and double antibody sandwich ELISA respectively. Results Three McAbs were established (2A2,3F1,4H2), which all showed no cross reaction with antigens of Schistosoma japonicum, Paragonimus westermani, Cysticercus cellulosae and Trichinella spiralis. Western blotting analysis demonstrated that the three McAbs recognized a Mr 15 000 soluble antigen of adult worm of A.cantonensis and recognized the Mr 24 000 and Mr 15 000 circulating antigens from the serum of angiostrongyliasis patient. The double antibody sandwich ELISA detection showed a positive rate of 76.5%. Conclusion Three hybridoma cell lines against adult worm of A.cantonensis have been established which secret high titer of McAbs with high specificity and seem promising in detecting the circulating antigen of the angiostrongyliasis patient.

Preparation of Monoclonal Antibodies Specific to Lactate Dehydrogenase of Plasmodiun falciparum

WANGJun-yun;BAOYi-fang;YANGYue-tao;TANGLin-hua

2005, 23(4):  6-216. 

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Objective To prepare monoclonal antibodies specific to lactate dehydrogenase of Plasmodiun falciparum. Methods The Plasmodium falciparum lactate dehydrogenase (pLDH) gene was amplified from whole blood of malaria patients by PCR and cloned into expression vector pGEX-3X. Recombinant pLDH protein was expressed and purified, and used for immunizing mice to prepare monoclonal antibodies (McAbs). The McAbs were characterized by Western blotting analysis. Results The Plasmodium falciparum lactate dehydrogenase gene was amplified and cloned into expression vector pGEX-3X. The recombinant pLDH plasmid was expressed in E.coli) BL-21 cells. 15 cell lines of McAbs with high titer against pLDH were obtained using the recombinant pLDH as immunogen. Western blotting analysis showed that these McAbs recognized a Mr 33 000 of native Plasmodiun falciparum protein without cross-reaction with constituents of red blood cell of febrile patients from endemic area of malaria. Conclusion Fifteen hybridoma cell lines secreting high titer of McAb specific to Plasmodium falciparum LDH were established based on the recombinant pLDH.


Consistency Analysis in the Use of Abdominal Ultrasonography for Diagnosing Schistosomiasis japonica-Related Morbidity

ZHOUYi-biao;ZHAOGen-ming;OUYANGShan-wen;JIANGQin-wu

2005, 23(4):  7-220. 

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Objective To explore the consistency among different indices of abdominal ultrasonography for the diagnosis of schistosomiasis japonica-related morbidity and the best combination of the indices. Methods Six indices of abdominal ultrasonography were selected to investigate schistosomiasis-related morbidity in residents in a village of Hunan Province, and the Kappa coefficients of diagnostic consistency among different indices and Cronbach's alpha coefficients of different combinations of indices were computed. Results The Kappa coefficients of 'liver parenchyma≥gradeⅡ' with 'right midclavicular subcostal' and with 'portal vein diameter' were 0.4131 and 0.4655 respectively, higher than normal level. The degree of their consistency was fair, and others showed poor or almost no consistency. Among the combinations made up of different indices, the Cronbach's alpha coefficient of the combination made up of 'liver parenchyma≥gradeⅡ', 'right midclavicular subcostal' and 'portal vein diameter' was 0.6566 which was the highest, showing the strongest internal consistency. Conclusion The six indices can not be replaced with each other in assessing schistosomiasis-related morbidity. Before abdominal ultrasonography is used extensively to assess the morbidity, it is necessary to study the diagnostic consistency of these indices and the best combination of the indices.

Experimental and Clinical Studies on the Effect of Sodium Dodecylbenzene Sulfonate in in vitro Killing Demodex and in Treating Demodicidosis

ZANGYun-shu;WUDa-jun;SONGJian-bo

2005, 23(4):  8-224. 

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Objective To observe the effect of sodium dodecylbenzene sulfonate (SDBS) in killing Demodex in vitro and in the treatment of demodicidosis. Methods ① The experiment of in vitro killing Demodex with 1% and 2% SDBS was conducted. ② A clinical trial was carried out to evaluate the therapeutic effect in the treatment of demodicidosis with 2% SDBS and 2% metronidazole emulsion. Patients with demodicidosis were randomly divided into trial and control groups (31 cases each). They were treated with 2% SDBS ointment and 2% metronidazole ointment twice a day in the early morning and evening respectively for eight weeks consecutively. Inflammatory lesions of face, Demodex infestation and scores of erythema were measured to evaluate the effect before and after treatment. ③ Follow-up was carried out for two months to evaluate the effect and side-effects after 8 weeks' treatment. Results ① 2% SDBS killed all Demodex in vitro after 5h, there was significant difference between the 2% SDBS and 2% metronidazole (69.4%), or between SDBS and peanut oil (9^1%)(P<0.05). There was no significant difference in killing Demodex folliculorum and Demodex brevis with 2% SDBS. ② Clinical observation showed that there was a significant difference in facial inflammatory lesions, Demodex infestation and scores of erythema before and after treatment with 2% SDBS(P<0.05). The effective rate was 87^1%, 65^5% in 2% SDBS group and 2% metronidazole group respectively, with a significant difference between the two groups (P<0.05). Follow-up showed same results. ③ A slight burning sensation was the only side-effect in 3 cases with 2% SDBS ointment. Conclusion Sodium dodecylbenzene sulfonate has the effect of in vitro killing Demodex and is highly effective in the treatment of demodicidosis.


Preliminary Survey on Concentration of Indoor Allergens Derived from Dust Mites in Residential Homes in Shanghai

MAOZuo-hua;LIUShi-wei;ZHANGFeng-xia;ZHOUShu-jun;HEYu-liang

2005, 23(4):  9-227. 

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Objective To investigate the level of indoor dust mite allergens in residential homes in Shanghai. Methods The allergenic proteins, Dff F3 and Der fⅢ (well known allergen) from the whole culture extract of Dermatophagoides farinae were purified by chromatography and sedimentation techniques. Allergic activities of both proteins were identified by skin prick test and RAST. After preparation of specific IgG anti-Dff F3, anti-Der fⅢ, and extraction of indoor allergens from 200 indoor dust samples collected from residential homes, the allergen concentration was measured by sandwich ELISA. Allergen level was expressed in geometric mean and range, the analysis of variance (ANOV) was used to determine the level of significance between groups. Results Dff F3 and Der fⅢ were demonstrated strongly allergic activities, which can be highly recognized with IgE from sera of the mite-allergic patients with asthma. In the sampled 200 homes, the proportion of homes with Dff F3 level of >10 μg/g was 57.0%, 29.5% for group of 2-10 μg/g, and 13.5% for group of <2 μg/g. The proportion of homes with Der fⅢ level of >10 μg/g was 53.5%, 32.0% for group of 2-10 μg/g, 14.5% for group of <2 μg/g. Conclusion Selected residential homes in Shanghai were found more likely to have high level of dust mite allergens. Dff F3 was identified as the stronger allergic fraction.

论文

Preparation of DNA from Cryptosporidium parvum Oocysts for PCR Detection

SHENYu-juan;CAOJian-ping;LUWei-yuan;LIXiao-hong;LIUHai-peng;XUYu-xin;ZHOUXiao-nong;TANGLin-hua;LIUShu-xian

2005, 23(4):  10-230. 

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Objective To establish three methods of DNA extraction from Cryptosporidium parvum oocysts and test by PCR. Methods After three freeze-thaw cycles, three kinds of templates were extracted from the oocysts by Chelex-100, phenol/chloroform or genomic DNA purification system kit, and used for PCR detection. According to the sequence of a C.parvum gene (L16996), a pair of primers was designed and synthesized, and used for PCR. The sensitivity of the template by Chelex-100 method was also tested by PCR. Results One 446 bp PCR product was observed by agarose gel electrophoresis for all three kinds of templates. The PCR sensitivity by Chelex-100 extracted DNA reached for detection of a specimen containing only 1/2 oocyst. Conclusion The three kinds of extraction can all be served as templates for PCR detection of C.parvum oocysts, while Chelex-100 method is simpler, quicker and more reliable for DNA extraction of the parasite.


实验报道

Purification, Enzyme Activity and Immunology Study of Recombinant Protein Glyceraldehyde-3-phosphate Dehydrogenase of Clonorchis sinensis

ZHANGYong-li;WUDe;YUXin-bing

2005, 23(4):  11-235. 

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Objective To produce prokaryotic recombinant protein glyceraldehyde-3-phosphate dehydrogenase of Clonorchis sinensis (CsGAPDH), analyze its enzyme activity and immunological function. Methods The recombinant CsGAPDH was purified according to the protocol of GST·BindTM kit and was digested with thrombin proteinase and eluted with wash buffer. The BALB/c mice were inoculated with the purified protein. The antisera collected from the mice were used to detect the titres of IgG antibodies by ELISA, and Western blotting was used to identify the specificity of the antisera with the purified CsGAPDH. S-P immunohistochemistry method was used to confirm the expression and distribution of CsGAPDH in adult Clonorchis sinensis with the polyclonal antibodies from immunized BALB/c mice. The CsGAPDH catalytic activity was evaluated employing the conventional substrate glyceraldehydes-3-phosphate (3-GAP). Results SDS-PAGE showed a single purified protein band. Gel scanning analysis revealed that the protein purity of CsGAPDH was 90%. ELISA analysis showed an increased IgG value. S-P immunohistochemistry analysis demonstrated that the recombinant plasmid pGEX-4T-1-GAPDH expressed and distributed in muscle cell membrane of immune mice. Western blotting result suggested that CsGAPDH protein contained essential epitopes with high antigenic activities. This protein CsGAPDH could catalyzed 3-GAP with enzymatic active unit of 2 872 U min^-1ml^-1. Conclusion The recombinant protein CsGAPDH shows a proper enzymatic activity and immunogenicity.

Construction and Expression of the Ts87 Gene of Trichinella spiralis in Pichia pastoris

WANGShao-hua;ZHUXin-ping*;GUYuan;YANGYa-ping;YANGJing

2005, 23(4):  12-239. 

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Objective To construct the recombinant plasmid P^PICZαA-Ts87 and obtain secretive protein in Pichia pastoris. Methods Ts87 was amplified by PCR and cloned into the expression vector P^PICZαA of P. pastoris to form P~PICZαA-Ts87. After the recombinant was isolated and linearized, it was transformed into P. pastoris GS115 by EasyComp^TM kit and screened for zeocin resistant with different conditions and Mut phenotype. The high resistant Mut~+ clones were cloned and the product induced by methanol was tested by dot-hybridization. The expression product was further identified by SDS-PAGE and Western blotting. Results and Conclusion The recombinant plasmid P^PICZαA-Ts87 has been constructed. Ts87 gene has been expressed and secreted in Pichia pastoris GS115 strain.

Gimenez Staining: A Rapid Method for Initial Identification of Legionella pneumophila in Amoeba Trophozoite

SHENJie;JIANGQing-wu;LIQing-xue;CHENHong-you;LIZi-hua

2005, 23(4):  13-242. 

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Objective To establish a rapid staining method for facilitating initial identification of Legionella pneumophila in amoebal trophozoite. Methods Acanthamoeba polyphaga and Legionella pneumophila were co-cultured under laboratory condition. At consecutive time points during the culture, smears of the cultured products were made on glass slides for staining purposes. Different types of stainings including Gram′s staining, Gimenez staining, Giemsa staining and immunofluorescence were used to determine the best method for the identification of amoebal pathogens. Results Gimenez staining technique is simpler and yields better results as compared with the other three stainings. Gimenez stain gives the best color and contrast for amoeba and amoebal Legionella Amoeba trophozoites and/or cysts showed a distinct purplish blue with amoebal Legionella in red. Amoebal Legionella can be distinguished clearly at an earlier time of co-culture, providing a proper sensitivity. It takes only 10 minutes to finish the operation. The other techniques require the use of expensive reagents, are relatively time-consuming, and involve complex staining procedures. Conclusion Gimenez staining is of value for the initial identification of amoebal pathogens, and it is suitable for laboratory diagnosis.

病例报告

2005, 23(4):  22-201. 

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