Table of Content

30 October 2003, Volume 21 Issue 5

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论著

Protective Immunity Induced by Recombinant Signaling Protein 14-3-3 Vaccine of Schistosoma japonicum

LIUQing-zhong*;SHENJi-long**

2003, 21(5):  1-260. 

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　Objective To evaluate the immunoprotective effect of Schistosoma japonicum (Chinese strain) recombinant signaling protein 14-3-3(rSj14-3-3), and to observe the synergism of rSj14-3-3 and rSjGST proteins as candidate vaccine and the effect of γδ-T cells activated by Mtb against Schistosoma japonicum. Methods BALB/c mice immunized with rSj14-3-3 and rSjGST purified through SDS-PAGE, electroelution and dialysis were challenged by cercaria infection. Six weeks after challenging infection, the mice were killed and the worm and egg reduction rates were calculated. Results Worm reduction rate was found to be 32.20% in rSj14-3-3+Freund adjuvant group, 31.10% in rSj14-3-3+rSjGST+Freund adjuvant group, 27.96% in rSj14-3-3+Mtb group, 26.00% in rSj14-3-3+rSjGST+Mtb group, and 27.10 % in rSjGST+Mtb group, respectively, number of eggs in liver tissue was reduced by 50.40%, 53.30%, 51.10%, 58.60% and 51.30%, respectively. Conclusion rSj14-3-3 could induce partial immunity against Schistosoma japonicum in BALB/c mice, and might serve as a candidate vaccine; γδ-T cell activated by Mtb played a role in anti-Schistosoma japonicum similar to the immune reactions induced by Freund adjuvant, but no synergistic effect combined with rSjGST was observed.


Screening and Identification of Phage Antibodies from Phage Display Library Against Schistosoma japonicum

CHENDai-xiong;YUMu-hua;LEIZhi-gang;ZHENGBin;HEAi;ZHANGRui-lin;ZHANXi-mei

2003, 21(5):  2-263. 

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　Objective To obtain single chain variable fragment(ScFv) against the circulating antigen(CAg) from Schistosoma japonicum(Sj). Methods Metabolic antigen of adult worm of Sj (Sj-MAg) was used in the panning of phage library against Schistosoma japonicum. The activity of Sj-MAg-binding phage clones was assayed by ELISA. The specificity of expression products of the positive clones was analyzed by ELISA, SDS-PAGE and Western blotting. Results Seventy-two randomly selected clones were tested for the presence of anti-Sj-MAg ScFvs, 6 clones showed positive. The specificity of these 6 clones was confirmed by binding them to antigens of other four trematodes. Two clones (B04,C24) were found to bind to Sj-MAg but not to any of the antigens of other four trematodes and their expression products were about 31kDa in size. Conclusion ScFv antibodies against the circulating antigens from Schistosoma japonicum Sj-MAg can be selected and manufactured from the antibody library.


The Internal Control Role of Ribosomal Protein S7 in the Defense of Anopheles dirus Against Plasmodium Infection

XUWen-yue;HUANGFu-sheng;DUANJian-hua

2003, 21(5):  3-267. 

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　Objective To investigate the role of ribosomal protein S7(rpS7) in the defense of Anopheles dirus against infection. Methods rpS7 was amplified from Anopheles dirus hemocytes with degenerated primers designed according to the conservative region of S7, rpS7 was then cloned using T/A cloning kit and the inserted fragment was sequenced. The difference of the transcript abundance of rpS7 from Anopheles dirus hemocyte among non-blood-fed (N), normal-blood-fed (B) and Plasmodium yoelii infected groups (I) was also analyzed by RT-PCR and gel scanning system at d1, d2, d3, d4, d7 and d11 after blood feeding. Results There is no significant difference of rpS7 signal between the three groups. Conclusion Anopheles dirus S7 can be used as an internal control to study the role of Anopheles dirus related immune factors in Plasmodium infection.


Studies on Specific Diagnostic Antigens in Excretory-secretory Products from Trichinella spiralis Muscle Larvae

CUIJing;WANGZhong-quan*;ZHANGDeng

2003, 21(5):  4-271. 

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　Objective To find out the specific diagnostic antigens in excretory-secretory (ES) products from muscle larvae of Trichinella spiralis. Methods The ES antigens (ESA) of Trichinella spiralis muscle larvae cultured in vitro at 18 h and 30 h were analyzed by SDS-PAGE and Western blotting. Results At different times after cultivation, the protein components of ESA of T. spiralis muscle larvae were similar. SDS-PAGE revealed that the molecular weight(MW) of the major bands of 2 ES antigens were 112, 110, 108, 97, 53, 49, 45, 42, 35, 23 and 16 kDa. Western blotting showed that the protein bands with 102, 97, 95 and 53 kDa in 18 h ESA and the protein bands with 53, 49, 45 and 43 kDa in 30 h ESA cross-reacted with sera from the patients with paragonimiasis, clonorchiasis, schistosomiasis, and cysticercosis, respectively. The protein component with 23 kDa in ESA only reacted with sera from the rats and mice infected with T. spiralis and the patients with trichinellosis, but not reacted with sera from animals and patients infected with other parasites, and sera from normal rats, mice and persons. Conclusion The protein component with 23 kDa in T. spiralis ESA is the specific antigen of T. spiralis muscle larvae and it could be applied to the serodiagnosis and seroepidemiological survey of trichinellosis.


Studies on the Antigens of Invasive Stages of Plasmodium yoelii and Plasmodium berghei

JINLi-qun;LUOJian-min;FUYu-cai;XUShi-e;GUOYan;XIELin-chong;T.Tsuboi;M.Torii

2003, 21(5):  5-274. 

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　Objective To detect the rhoptry and surface proteins of invasive stages of Plasmodium yoelii and P. berghei with monoclonal antibodies. Methods Subcellular localization of antigens was detected by IFA. The antigens of different stages of the two species malaria parasites were analyzed by Western blotting. Results The antigens of rhoptry are very complicated. There are similar epitopes of the rhoptry proteins detected between the two species of Plasmodium. The similar epitopes were also detected between ookinetes and merozoites of P. yoelii, and ookinete antigens between the two species. But there are different antigens detected between merozoites and ookinetes in P. yoelii. The sporozoite surface antigen of P. yoelii was not detected in the ookinetes and merozoites in the same species. Conclusion There are similar epitopes in the rhoptry and surface antigens of different stages and different species of rodent malaria parasites. There are also distinct antigens among them.


Screening of Schistosomiasis japonica Diagnostic Antigen with Phage 12-mer Peptide Library

ZHUXiao-hua;JIANGChang-fu;WEILan-ying;LEIJia-hui;SHIHong-bo;PANHong

2003, 21(5):  6-278. 

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　Objective To obtain the mimic epitope of specific and sensitive diagnostic antigen in schistosomiasis japonica from phage 12-mer peptide library. Methods Specific Ig was purified from sera of patients with acute schistosomiasis and used to immunoscreen the phage peptide library (PH.D.-12). After 3 rounds of panning, 10 positive plaques were selected and amplified. The immunoactivity of each clone was examined by ELISA. The sensitivity and specificity of immunoactive clones were confirmed by detecting the sera of patients with different parasitosis. Results Six clones could bind to the specific Ig purified from sera of patients with acute schistosomiasis. One clone with the highest A 492 value showed a high sensitivity and specificity. Conclusion The clone (SjA1)identified by the specific Ig from the library played a better part in the immunodiagnosis of schistosomiasis.


Cloning and Expression of the Signaling Protein 14-3-3 of Toxoplasma gondii

DUJian*;SHENJi-long**;WANGXue-long;WANGWei

2003, 21(5):  7-281. 

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　Objective To clone and express the cell signaling protein 14-3-3 gene from Toxoplasma gondii RH strain. Methods Toxoplasma RH strain tachyzoites, which maintained by mouse passage, were harvested from ascites of mice and genomic DNA was prepared. A pair of primers were designed and synthesized based on the sequence of Toxo 14-3-3 cDNA. A specific fragment of Toxo 14-3-3 gene was obtained by RT-PCR amplification from Toxoplasma genomic DNA. The PCR products were ligated to pGEM-T. The EcoRI / Xho I restricted fragments, confirmed by PCR and EcoRI / XhoI digestion, were cloned into expression vector pET28a and the recombinants were transformd into E.coli BL21. Fusion expression was induced by isopropyl-beta-D-thiogalactoside (IPTG) and confirmed by Western blotting with rabbit anti-Toxoplasma sera. Results The molecular size of Toxo 14-3-3 was 803 bp, which is highly homologous to the previous report cloned from the parasites of intestinal epithelial stage in cat. High expression was obtained in pET28a/ Toxo 14-3-3/E.coli BL21 when confirmed by Western blotting. Conclusion The recombinant construction of Toxo 14-3-3 was generated and expression was induced.


Cloning and Sequencing of Cathepsin L1 FheCL1 Gene cDNA of Fasciola hepatica

ZHANGRen;LIHai-yun*

2003, 21(5):  8-285. 

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　Objective To search for a candidate DNA vaccine of Fasciola hepatica. Methods Using RT-PCR and digestion with Hind III and BamHI, Fasciola hepatica secreted cathepsin L1(FheCL1) cDNA was cloned into the expression vector pcDNA3.1. Results The cloning was successful, the cDNA sequence and its deduced amino acid sequence were analyzed. There was much difference between the cloned FheCL1 and the published one. But the first 20 residues of their amino acid sequences were the same. Conclusion The recombinant plasmid pcDNA3.1-FheCL1 may be a new type of candidate DNA vaccine candidate for Fasciola hepatica. It is possible that Fasciola hepatica presents different sub-species but their amino acid residues (1 to 20) encoded by FheCL1 might build up membrane spanning helix.


Schistosoma japonicum: Screening of cDNA Library with Sera from Self-cured Water Buffaloes(Bos buffelus)

WANGWen-qin;LIUShu-xian;SONGGuang-cheng;XUYu-xin;CAOJian-ping;CHENJia-xu

2003, 21(5):  9-288. 

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　Objective To understand and identify the molecules related to the self-cure of Schistosoma japonicum infection in water buffaloes. Methods The S. japonicum adult worm cDNA library was immunologically screened with the sera of self-cured water buffaloes. The positive clones were identified, cloned, sequenced and analysed with software. Results Three genes encoding antigens relevant to sera antibodies in water buffaloes were cloned and sequenced. These antigens included paramyosin (Sj97), GST, carbonyl reductase-like 20-β-hydroxysteroid dehydrogenase(CR/20-β-HSD). Conclusion Sera from self-cured S. japonicum infected water buffaloes can be used to screen adult worm cDNA library for vaccine development.


Immunoscreening of Schistosoma japonicum Adult Worm cDNA Library with Sera Vaccinated with Cercaria Antigen and Analysis of Novel Genes

YUANShi-shan;YIXin-yuan*;ZENGXian-fang;OUYANGLi;WANGMin;TANGLian-fei;LarryMcReynolds

2003, 21(5):  10-292. 

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　Objective To explore antigens possessing common immunogenicity with Schistosoma japonicum (Sj) cercariae antigens, and to find out new candidate antigens for schistosomiasis diagnosis and vaccine. Methods Sj adult cDNA library was screened using sera from rabbits vaccinated with Sj cercariae antigen, the inserts of positive clones were amplified by PCR, all positive clones were sequenced and the data were analysed using Nucleotide BLAST software of NCBI and Expert Protein Analysis System of Swiss Institute of Bioinformatics. Results Thirteen positive clones were obtained after three rounds of immunoscreening, and all amplified by PCR.Among four novel genes, SjCAI, SjCA, SjCAI-2 and SjCAI-3(GenBank accession number:AF495883, AF515834, AY118086 and AY129303,respectively)encoded proteins with 353, 161, 137 and 72 animo acids respectively. Sj CAI protein contained six DNA-binding zinc fingers and showed some homology to gastrula zinc finger protein XLCGF48.2; proteins encoded by SjCA, SjCAI-2 and SjCAI-3 respectively contained N-glycosylation sites and phosphorylation sites. Conclusion Novel genes were obtained by immunoscreening Sj adult cDNA library.


Assay of Haemolymph Protein Concentration in Anopheles stephensi

YANGSong;HUANGFu-sheng;KUANGMing-shu;DUANJian-hua;ZHANGJing-ru

2003, 21(5):  11-295. 

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　Objective To ascertain the changes of haemolymph protein concentration in adult Anopheles stephensi mosquitoes under different feeding conditions. Methods Haemolymph samples from four groups of adult An.stephensi, fed with sucrose solution, normal blood, plasmodium-infected blood and nitroquine, respectively, were collected by expulsion method. The concentration of haemolymph protein was examined by Bradford method. The results were analyzed automatically by Excel program. Results The level of protein concentration in the infected blood-fed group was higher than the sucrose solution group and normal blood group at day 8 after Plasmodium yoelii infection,the average concentration was 4.436,3.080 and 3.092 μg/μl,respectively. The haemolymph protein concentration (2.264 μg/μl) in the nitroquine-administered mosquitoes was lower than the infected blood-fed mosquitoes. Conclusion The haemolymph protein concentration of the adult An. stephensi decreases after the nitroquine administration, indicating that the haemolymph proteins may be involved in the melantotic encapsulation reaction of plasmodial oocysts.


实验研究

Signaling Role of Exogenous Arachidonic Acid in the Invasion of Macrophages by Toxoplasma gondii

PENGBi-wen;HUANGQing-ling;LINJian-yin*;JIANGMing-sen

2003, 21(5):  12-299. 

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　Objective To investigate the signaling role of arachidonic acid in the invasion of RAW264.7 macrophage by Toxoplasma gondii . Methods Rate of infection was calculated by both light microscope and flow cytometer. Fluorescent emission spectra were recorded using a microspectrofluometer for the concentration of cytoplasmic free calcium. Results Calcium concentration in macrophages and rate of infection increased with a higher concentration of exogenous arachidonic acid in a dose-dependent manner. The invasion was dependent on the mobilization of calcium from the extracellular medium and from intracellular stores and followed the influx of calcium into the parasitized cell. Conclusion Arachidonic acid may enhance the rate of infection via calcium transduction pathway.


A Rapid Procedure to Purify Serum IgG from Microtus fotis

JIANGShou-fu;PANCai-e;HEYan-yan;ZHUMin;LIHao;SHIYao-jun;WEIMei-xiong

2003, 21(5):  13-302. 

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　Objective To evaluate the procedure to purify IgG antibodies from Microtus fotis serum. Methods IgG antibodies from sera of three groups of Microtus fotis were purified by protein G or protein A affinity chromatography, their purity and binding capacity were compared. Results The protein G affinity chromatography was more efficient than protein A affinity chromatography. The antibodies isolated from protein G affinity chromatography showed a higher purity and better activity than that from protein A affinity chromatography monitored by SDS-PAGE and ELISA. The ability of the purified IgG to bind the second antibodies were 8.5 times and 3.1 times that of non-IgG proteins and unpurified sera, respectively. Conclusion The protein G affinity chromatography is a rapid, convenient and reliable procedure for Microtus fotis serum IgG purification.


Initial Temperature for the Development of Schistosoma japonicum Larvae in Oncomelania hupensis

SUNLe-ping;ZHOUXiao-nong;HONGQing-biao;HUANGYi-xin;YANGGuo-jing;XIWei-ping;JIANGYu-ji

2003, 21(5):  14-306. 

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　Objective To study the impact of environmental temperature on the development of Schistosoma japonicum larvae within the Oncomelania hupensis. Methods Oncomelania snails, collected from the field and free of S. japonicum infection, were exposed to miracidiae of S. japonicum in a ratio of 1∶20 and raised at 30 ℃, 27 ℃, 24 ℃, 21 ℃ and 18 ℃, respectively. The prepatent period of larvae within the Oncomelania hupensis and the developmental velocity were determined, of which the relationship with the temperature was analysed. Results The average prepatent period of cercariae in snail was (128.89±16.05) d,(95.00±21.03) d,(71.93±12.74) d and (62.74±14.19) d at 21 ℃, 24 ℃, 27 ℃, 30 ℃, respectively. The regression formulation between prepatent period and temperature was y =730.68x -0.8918 (r=0.9976, P<0.01). And the regression formulation between developmental velocity of S. japonicum larvae in snail and temperature was y =0.0235ln(x)- 0.0639(r=0.9973, P<0.01). It was derived that the unitial temperature for the development of S. japonicum within the snails was 15.17 ℃±0.43 ℃. Conclusion The development of S. japonicum larvae within the Oncomelania snails declines with the decrease of temperature.


防治经验

Evaluation of Intervention Strategy and Measures on the Control of Intestinal Parasitic Infections

TUXing-guo;YAOLi-nong

2003, 21(5):  15-310. 

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　Objective To evaluate the effect of intervention strategy and measures for intestinal parasite control in Zhejiang Province. Methods The protective rate (PR) and the index of effectiveness (IE) on the overall prevalence of parasites after and before interventions for intestinal parasitic infections were compared in 30 villages of 10 counties randomly selected as investigation spots. Results After the implementation of the interventions in the past decade, the total parasite prevalence declined significantly from 77.0% in 1989 to 22.84% in 1998 in the Province, the PR was 70.34%, the IE was 3.37. In each of the 10 counties, the PR was above 45%, the IE was between 1.85 and 14.47. Lavatory improvement, socioeconomic development and health education were among the first three factors that affected the effectiveness of the intervention. Conclusion The comprehensive intervention combining the socioeconomic development, health education, environmental improvement with mass chemotherapy has been proved an effective strategy.


调查报告

Epidemiological Investigation of Taenia saginata asiatica in Duyun, Guizhou and Detection of Amino Acids and Elements of Adult Worms

CHENYan;BAOHuai-en;LIJin-fu;LANGShu-yuan;QIUXue-li;HUANGJiang;WUYuan-ming;ZHANGChao-yun

2003, 21(5):  16-313. 

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　Objective To study epidemiological factors of taeniasis and to detect amino acid and element components of adult worms in Duyun of Guizhou Province. Methods ① Traditional methods were used for epidemiological investigation. ② Automatic amino acid analyzer and bioassay were applied for the detection. Results Among 70 persons with clinical symptoms, 25 patients (24 men and 1 woman) were found to have adult taenia worms in their faeces after taking Areca catechu L. and other drugs. Sixteen amino acids and 12 elements were determined in adult worms. Conclusion Duyun area in Guizhou is a highly endemic area of taeniasis. The pathogenic parasite is identified as Taenia saginata asiatica. Its clinical symptoms are similar to that of Taenia saginata saginata.