Table of Content

30 June 2002, Volume 20 Issue 3

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论著

Identification of the Binding Site on ICAM-1 for Red Blood Cells Infected by Plasmodium falciparum

HAOWenbo;XUWeiwen;LIMing;CHENBaihong;WANGPing;LIZhongqi

2002, 20(3):  1-132. 

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　Objective To identify the binding site on ICAM 1 to PRBCs in order to explore anti adhesive agent against cerebral malaria. Methods Monoclonal antibody 15.2 against ICAM 1 domain 1 was chosen as target molecule to screen mimetic peptides of ICAM 1 from a 12 mer random peptide library. Three rounds of biopanning were carried out and then ELISA, competitive ELISA, dot ELISA and Western blotting were used to evaluate the binding character between phage borne peptides and McAb 15.2. The insert DNA sequences of positive clones were determined and their amino acid sequences were deduced. Results Thirty clones from the third round were randomly selected, and 26 of them were found positive by sandwich ELISA. The competitive ELISA test proved that most phage borne peptides could competitively inhibit the binding of antibody (15.2 McAb) with ICAM 1. Analysis of DNA and amino acid sequences indicated that over a half positive phage clones expressed 12 mer peptide KLYLIAEGSVAA. Comparison of peptide K(XX)L(XXX)GSV with the 64-73 aa of primary sequence of ICAM 1 showed a 50% homogeneity. Conclusion These peptides displayed by phage may be analogs of ICAM 1, K··L···GSV probably plays a significant role on the binding reaction of ICAM 1 and PRBCs.


Preliminary Observation on Specific Antibody Level and Reduction of Ovulation Induced by Recombinant Schistosoma japonicum 26 kDa GST Antigen in Water Buffaloes

HEYongkang;LIUShuxian;YUXinling;SONGGuangcheng;XUYuxin;CAOJianping;YANGRuiqing;HOUXunya;ZHANGXinyue

2002, 20(3):  2-135. 

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　Objective To observe the specific antibody level and reduction of egg laying induced by a recombinant Schistosoma japonicum 26 kDa GST antigen (rSjc26 GST) in water buffaloes. Methods 20 water buffaloes were randomly divided into two groups, the vaccination group and control group, with 10 buffaloes each. The subjects in vaccination group were immunized with rSjc26 GST antigen while the control received adjuvant only. After challenged with S. japonicum cercariae, the anti rSjc26 GST antibody level and the numbers of eggs and miracidia in stool were detected. Results The anti rSjc26 GST antibody appeared 1 month after immunization with rSjc26 GST antigen and maintained a high level for 12 months. Numbers of eggs (EPG) and miracidia (MPG) in vaccination group were significantly lower than those in control group during the period of day 50 to day 90 post challenge. However, EPG and MPG tended to decrease starting from day 100 post challenge in both groups. The difference of EPG and MPG between the two groups diminished progressively, and both groups showed zero egg count from day 330 on post challenge. Conclusion The specific anti rSjc26 GST antibody was detected in vaccinated water buffaloes and maintained a high level for 12 months. The vaccination showed a significant effect to the reduction of ovulation in the first three months after S. japonicum cercariae challenge.


Proliferation and Cytokine Production of Lymphocytes from Clonorchis sinensis-infected Rats in Response to Stimulators in vitro

FuShiQuan;SungWeonCho;KyoungHwanJoo*

2002, 20(3):  3-140. 

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　Objective To study the rat lymphocyte proliferation, differentiation and cytokine production in response to Clonorchis sinensis infection. Methods The lymphocyte proliferation and cytokine production (IFN γ, IL 2, IL 4, IL 10) in response to mitogen phytohaemagglutinin (PHA), C.sinensis excretory secretory antigen (ES Ag), C.sinensis crude antigen (crude Ag) and Anisakis larvae antigen were detected in vitro from splenic lymphocytes (SLC) and mesenteric lymph node cells (MLNC) of rats infected with C.sinensis . Statistical analysis was performed by Sigma Plot System. Results Lymphocyte proliferations in MLNC were higher than that in SLC. At concentrations of 3×10 6 or 9×10 6 cells/well, lymphocyte proliferations were significantly higher in both SLS and MLNC than in the control with cell alone ( P <0.01). At the lymphocyte concentration of 5×10 6 cells/well and stimulator concentration of 5 or 10 μg/ml, significant lymphocyte activation was observed. Under the same culture condition (5×10 6 cells/well with 10 μg/ml stimulator ), cytokine IFN γ and IL 10 production in vitro increased significantly in MLNC. Conclusion Concentrations of 5×10 6 lymphocytes/well and 10 μg/ml stimulator were selected as the optimal culture condition for activation of lymphocyte proliferation and cytokine production. Since the production of Th1 type cytokine IFN γ and Th2 type cytokine IL 10 was much enhanced from MLNC of C.sinensis infected rats, it is considered that C.sinensis ES Ag may stimulate lymphocyte proliferation and cytokine production in C.sinensis infected rats in vivo .


Purification and Immunogenicity Identification of the Recombinant Protein Related with Specific IgE against Schistosoma japonicum

WANGYong;SUChuan;ZHANGZhaosong;HUXuemei;LIUFeng;LIChunlin;JIMinjun;ZHUXiang;WUGuanling

2002, 20(3):  4-144. 

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Objective To purify the specific IgE antibody related recombinant protein of Schistosoma japonicum and to identify its immunogenicity. Methods The recombinant plasmid Sj43B/pGEX 6p 1 was expressed in E. coli BL 21. The inclusion body of the fusion protein was washed by TNMFX buffer and separated by FPLC. After renaturation, the fusion protein was used to vaccinate the mice. The specific IgG and IgE antibodies were detected by dot ELISA and Western blotting analysis, respectively. Results Most of the proteins mixed with the inclusion body of the recombinant protein could be eliminated by washing with TNMFX buffer. The purified recombinant fusion protein could be obtained by FPLC separation. The experiment on mice immunized with the fusion protein showed that the specific IgE antibody was generated against the target part of the fusion protein,but not the specific IgG antibody. Conclusion The fusion protein expressed by the recombinant plasmid Sj43B/pGEX 6p 1 could induce specific IgE response of the immunized mice.


Application of Multifactor Spatial Composite Model to Predict Transmission Tendency of Malaria at National Level

YANGGuojing;ZHOUXiaonong;J.B.Malone;J.C.McCarroll;WANGTianping;LIUJianxiang

2002, 20(3):  5-147. 

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　Objectives To predict the transmission tendency of malaria at national level by application of geographic information system(GIS) technique. Methods With the assistance of ArcView 3.0a software and its spatial analyst extension, the surface spatial analysis on three natural factors, namely, total growing degree days(TGDD), precipitation and relative humidity, were conducted individually. The map calculation was preformed based on the three factors′ ratio of 5∶3∶2 resulted from the Delphi investigation. Results The individual maps and composition map of TGDD, precipitation and relative humidity were created, respectively, based on the spatial composite model, which were used to predict the transmission tendency of malaria at national level. Conclusion The high risk areas for malaria transmission, predicted by the spatial composite model based on the multilayers of environmental factors, are correlated with the previous reports. This will, therefore, provide information for predicting malaria transmission by multiple factors in a larger area.


Reexamination of Specific Antibodies in Sera of Cystic Echinococccosis Patients with IgG Negative Seroresponse

XUMingqian;ZHUBing;XUEHaichou;LIXiong;GUOXiangrong;ZHANGYonghong;LIHao

2002, 20(3):  6-151. 

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　Objective To explore the factors of false negative antibody response in patients with echinococcosis granulosus (Eg) for improving immunodiagnosis. Methods Indirect ELISA and sandwich ELISA were used to detect the specific antibody of IgG subclass (IgG1, IgG2, IgG3, IgG4) and IgA, IgM, IgE, as well as circulating antigen (CAg) and immunocomplex (CIC) in sera of Eg patients with negative response of total IgG. Results Among 42 sera with IgG negative seroresponse, 32 were positive with IgG subclass, IgA, IgM or IgE antibody, 10 were negative in all 7 kinds of antibody response. The detection rate of specific IgG1, IgG4, IgA, IgM and IgE was 42.9%, 11.9%, 28.6%,26.2%and 21.4% respectively, being significantly higher than in sera of the control. IgM level in children was higher than that in adults. IgG subclass in patients with liver Eg was higher than that of pulmonary hydatidosis, when testing with IgG1 combined one or two of the other six Ig antibodies. The highest positive rate (64.3%) was seen in IgG1+IgA+IgM antibody system. The positive rate of CAg and CIC in IgG negative patients was 28.57% and 30.95%, respectively. Conclusion The factors involved with seronegative response of total IgG in Eg patients might be low specific IgG, variant Ig antibody expression and formation of CIC. Combined detection of IgG1+IgA+IgM could enhance the sensitivity of serological tests in Eg patients.


Construction and Expression of Single Chain Fv Gene of Anti- idiotypic Monoclonal Antibody NP30 of Schistosoma japonicum

SONGXiaotong;FENGZhenqing;WANGZhuming;QIUZhenning;LIYunqian;GUANXiaohong;HUANGHualiang

2002, 20(3):  7-154. 

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　Objective To construct single chain Fv (scFv) gene of anti idiotypic monoclonal antibody NP30 of Schistosoma japonicum . Methods The heavy and light chain variable region genes of anti idiotypic monoclonal antibody NP30 of Schistosoma japonicum were inserted into two corresponding sites of expression vector pTHA90,and a scFv gene was constructed with a short peptide (Gly 4Ser) 3 linker gene. The recombinants were determined by digesting with Xho I/ Spe I, Xba I/ Eco RI and Xho I/ Eco RI,and then were introduced into E.coli Top10. The antigen binding activity of expressed product was detected with ELISA. Results The recombinants were determined by digesting with endonucleases and expected bands were identified. The value of expressed scFv was 3 times higher than negative control by ELISA(OD 492 =1.06). Conclusion The scFv gene of anti idiotypic monoclonal antibody NP30 of Schistosoma japonicum was successfully cloned, and the expressed scFv fragment could interact specifically with antigen NP48.


Improvement in the Preparation of Paragonimus westermani Chromosome for Karyotype Analysis

CHENShaohong;CHANGZhengshan;CHENMinggang;ZHANGYongnian;FENGZheng

2002, 20(3):  8-157. 

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　Objective To improve Paragonimus westermani chromosome preparation technique. Methods After being exposed to colchicine, the gonadal cells of P.westermani were treated by the following procedures: hypotonicity, fixation, dropping onto a slide and staining. Results The chromosome number of P. westermani is 22,and the karyotype is 2m+6Sm+14t chromosome. Conclusion The improved technique of chromosome preparation is feasible to operate and the chromosome is clear enough for karyotype analysis.


Expression and Characterization of 21.7 kDa Membrane Protein of Schistosoma japonicum

LIUJianfa

2002, 20(3):  9-160. 

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　Objective To express, purify and characterize the 21.7 kDa membrane protein of Chinese strain S. japonicum (SjC21.7). Methods The gene of SjC21.7 was subcloned into the expression vector pGEX 4T 3 to form recombinant plasmid. The recombinant plasmids were transformed into E. coli BL21 and the GST SjC21.7 fusion protein was expressed by IPTG induction. The recombinant SjC21.7 molecule was prepared by affinity chromatography and digested by thrombin. The Kunming strain mice were immunized with the recombinant SjC21.7 molecule to produce anti SjC21.7 antibody. The purified SjC21.7 was recognized by the immunized mouse serum and the sera of rabbits infected by S. japonicum . Results The SjC21.7 gene was subcloned into expression vector pGEX 4T 3, then transformed into E.coli BL21 to express the GST SjC21.7 fusion protein. The recombinant SjC21.7 molecule obtained from the fusion protein could stimulate the mice to produce a high titer of specific antibody and could be recognized by sera of both the immunized and infected rabbits. The sera of immunized mice could also recognize the 21.7 kDa protein molecule of the adult worm antigen (AWA). Conclusion The recombinant and purified SjC21.7 was prepared and showed similar immunological characteristics to the natural SjC21.7 molecule, providing a basis for further investigation of the immunological protection of the recombinant SjC21.7.


Cloning and Sequencing of the gp23 Gene Encoding A Surface Antigen on Sporozoites of Cryptosporidium parvum

HEHongxuan;ZHANGXichen;OUYANGHongsheng;XUWeidongCHENJianbao;YINJigang;LIJianhua;YANGJu

2002, 20(3):  10-163. 

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　Objective To clone and sequence the gp23 gene encoding a surface antigen on the sporozoites of Cryptosporidium parvum . Methods Genomic DNA was isolated from oocysts of Cryptosporidium parvum . The gp23 gene was amplified by polymerase chain reaction (PCR) and cloned into pMD18 T vector and sequenced by the methods of dideoxy mediated chain termination. Results and Conclusion The gp23 gene was 345 bp in length and included an open reading frame encoding a protein of 114 amino acid residues. The homology of the nucleotide and amino acid sequences of the gp23 gene was 97.3% and 98.2% to that in the GenBank, respectively. The gp23 gene cloned contained 6 nucleotides more than that in the GenBank.


Identification of Epitope of 22.6 kDa Antigen of Schistosoma japonicum with Phage-displayed Random Peptide Library

HUXuemei;ZHANGZhaosong;WUHaiwei;SUChuan;JIMinjun;WANGYong;CHENShuzhen;WUGuanling

2002, 20(3):  11-167. 

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　Objective To identify the epitopes of 22.6 kDa antigen of Schistosoma japonicum (Sj22.6). Methods A 12 mer library displayed on pIII of fd phage was used to screen the epitopes of Sj22.6 with the purified multiple clone IgG antibody against the Sj22.6 antigen. Five rounds of biopanning were carried out and fourteen clones from the fifth round biopanning were randomly selected and identified by Western blotting. The mice were immunized with the obtained positive clones and the antibody titers of sera from the mice were detected. The clones which could stimulate the mice to produce anti Sj22.6 antibodies were sequenced and their amino acid sequences were compared with that of Sj22.6. Results Six clones selected from the fourteen ones could stimulate the mice to produce anti Sj22.6 antibodies analysed by Western blotting. The amino acid sequence of one epitope showed high homology with Sj22.6. Conclusion Four epitopes of Sj22.6 were obtained. One may be a structure epitope and the other three were mimic epitopes.


Molecular Variation Based on mtDNA-COII Gene of Members of the Anopheles minimus Group

ZHOUShuisen;TANGLinhua;ZHENGXiang

2002, 20(3):  12-170. 

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　Objective To investigate intra and inter species molecular variations and phylogeny of the five members of the Minimus Group of Anopheles subgenus Cellia in China: An. aconitus, An.varuna, An.jeyporiensis, An.minimus A and An.minimus C, and to report the DNA sequence divergence among these species at a mitochondrial locus (cytochrome oxidase II). Methods Single mosquito′s legs were digested to extract DNA. COII gene was amplified, sequenced and analysed; and the phylogenetic tree of members of An.minimus group was reconstructed by maximum likelihood method (DNAML). Results and Conclusion The data confirmed the presence of two cryptic species, A and C, within An.minimus complex in the research localities; and two species differed by 2.3% in the COII sequences owing to 16 nucleotide substitutions. There was less variation between the two species than other members of the Minimus Group.


Changes in Element Content in Aedes albopictus Infected by Dengue Virus Type II

CHENHong;CHENHanbin

2002, 20(3):  13-173. 

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　Objective To study the changes in the content of elements in Aedes albopictus after dengue virus typeⅡ(DV2) infection. Methods DV2 were injected into the thorax of Ae.albopictus . The content of elements in Ae. albopictus was determined on days 5, 10 and 20 after infection respectively and compared with those in the same instar non infected mosquitoes. Results and Conclusion For the group of day 5 after DV2 infection, the content of elements Ca, S and Cr increased remarkably compared to those in the control group; while the content of elements Zn, Mo, Pb, Ce and Cl reduced remarkably. For the group of day 10 after infection, the content of Na, Mg, Zn, Se, Ce and S increased remarkably compared to those in the control; only that of Fe was lower remarkably than the control. For the group of day 20 after infection, the content of elements Ti and Pb increased remarkably than that of the control; while the content of Na, Zn, Cr and Ce reduced remarkably.


防治经验

Target Chemotherapy of Intestinal Nematode Infection in Area with Low Endemicity

ZHANGTao*;SHENYiping;LIUYing**;YANGWeiping;SHAOJingou;JUShaoyou;DONGKai;XUJuling;JIANGJimin

2002, 20(3):  14-176. 

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　Objective To evaluate the control measures for intestinal nematodiasis in endemic area with low prevalence and intensity of infection. Methods Target chemotherapy was carried out in high risk population based on the epidemiological characteristics such as age and clinical findings. Albendazole and mebendazole were administered each 200 mg once daily every year for 3 or 5 years. Saturated brine floatation and Kato Katz thick smear techniques were used for stool examination to evaluate the efficacy of treatment. Results Two hundred residents from each of the three investigation villages were selected for target chemotherapy once a year for three years. The prevalence of intestinal nematodes decreased from 6.2% in 1995 to 5.4% in 1996 and 3.2% in 1997, and remained at 2.3% after three years in 2000. One control village where only primary school students were treated once a year for 5 years, the prevalence of Ascaris and Trichuris infection also decreased from 1.4% and 4.2% in 1995 to 0.9% and 1.4% in 2000, respectively. The target chemotherapy on the predisposed population to hookworm infection showed that the prevalence in the population above 41 years old was declined from 19.4% to 10.9%. Conclusion The target chemotherapy is an economical and effective approach for the control of intestinal nematode infection in endemic area with low prevalence and intensity of infection.


临床研究

Investigation of Inpatient Cases of Food-borne Parasitic Encephalopathy

YINChunyu;SHIYaozhong

2002, 20(3):  15-179. 

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　Objective To investigate the clinical features of the patients with encephalopathy caused by food borne parasites. Methods Questionnairing was carried out to collect and analyze clinical data of cerebral form of food borne parasitic diseases in the hospital during the past five years. Results Among 190 discharged medical histories, 115 cases were valid for investigation, the number of males was 73, females 42, with a ratio of 1.74∶1. Among these patients, 20.9% (24/115) had a history of eating raw meat. For discharge diagnosis, neurocysticercosis accounted for 92.2% (106/115),cerebral paragonimiasis 3.5% (4/115), sparganosis 2.6% (3/115), and angiostrongyliasis cantonensis and gnathostomiasis 0.9%(1/115) each. 13.9% (16/115) of the patients were hospitalized for three times or more. Conclusion More attention should be paid to food borne parasitic encephalopathy.


Therapeutic Effect of Dihydroartemisinin Combined with Naphthoquine Phosphate in Patients with Falciparum Malaria

WANGShanqing;MENGFeng;SHENHeng;WENYan;ZHUOKairen;ZHUQixian;PANGXuejian;LINShigan;ZENGLinhai

2002, 20(3):  16-182. 

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　Objective To observe the therapeutic efficacy of dihydroartemisinin combined with naphthoquine phosphate in patients with falciparum malaria. Methods Patients with Plasmodium falciparum were selected as the subjects, treated with a single dose of dihydroarteminisinin 160 mg combined with naphthoquine phosphate 400 mg (for adults) and followed up in preselective time by blood and temperature examination for 28 days after drug administration. Results 37 patients with falciparum malaria were treated and followed up. One patient had recrudescence and the cure rate in 28 days was 97.3%(36/37). The mean fever clearance time was (15.8±8.7) hours; the mean parasite clearance time was (27.6±13.2) hours; the mean reduction parasite rate in 24 hours was 96.7%±26.5%. No apparent side effect was observed. Conclusion A combination of dihydroartemisinin and naphthoquine is effective for the treatment of patients with falciparum malaria.