Table of Content

30 August 2006, Volume 24 Issue 4

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论著

Cloning， Expression， Purification and Identification of Der f6 Gene andits Immunological Characteristics from the Dust House Mite

ZHUYong-feng;LIUZhi-gang;GAOBo

2006, 24(4):  1-246. 

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Objective To construct, purify and characterize a recombinant expression plasmid containing Der f6 gene of Dermatophagoides farinae. Methods A pair of primers was designed according to the known sequence of Der f6 gene. The live mites identified and cultured locally were picked and the total RNA was extracted. The Der f6 gene fragment was amplified by RT-PCR，and cloned into pMD18-T vector， and then transferred into E.coli Top10. The target gene obtained from the recombinant plasmid by digestion with EcoRⅠ and XhoⅠ was connected to the prokaryotic expression vector pET-24a. The expressed recombinant plasmid containing Der f6 gene was constructed by cloning target gene into pET-24a and first transferred into E.coli Top10，then into E.coli Bl21（DE3）. The expressed recombinant protein was analyzed by SDS-PAGE and Western blotting，and was purified by immobilized metal ion affinity chromatography（IMAC）. Results The two recombinant plasmids，pMD18-T-Der f6 and pET24a-Der f6，were constructed. SDS-PAGE showed a correct molecular weight of the recombinant Der f6 protein. After purification by affinity chromatography，the protein showed only one strip on SDS-PAGE gel and appropriate combination ability with IgE in sera of allergic patients. Conclusion The Der f6 gene has been cloned into plasmid pMD18-T vector and sub-cloned into the expression vector pET-24a，the recombinant plasmid pET24a-Der f6 has been expressed in E.coli BL21（DE3），purified by IMAC，and showed approprriate IgE-combined ability.

病例报告

Preparation and Characterization of Monoclonal Antibody Specific to PfCP-2.9 Chimeric Protein of Plasmodium falciparum

WANGRui;QIANFeng;QULi;PANWei-qing

2006, 24(4):  2-250. 

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Objective To prepare and characterize monoclonal antibody against a malaria vaccine candidate， PfCP-2.9 chimeric protein of Plasmodium falciparum. Methods BALB/c mice were immunized with PfCP-2.9，and the spleen cells were used for fusion with SP2/0 cells. The monoclonal antibodies were analyzed by ELISA，Western blotting as well as growth inhibition assay. Result A monoclonal antibody was obtained. It interacted with the PfCP-2.9 recombinant protein by ELISA and Western blotting. The interaction of the monoclonal antibody with the protein was reduction-sensitive，indicating that the antibody recognized a conformational epitope. Moreover，the antibody also recognized the cultured parasites of P.falciparum by indirect immunofluorescent antibody test（IFA）. When tested by growth inhibition assay，the antibody significantly inhibited parasite growth in vitro of 56% inhibition rate at the antibody concentration of 0.3 mg/ml. Conclusion A monoclonal antibody against PfCP-2.9 malaria vaccine candidate has been obtained，which recognizes a conformational epitope of the protein and natural protein.

论著

Change of Cytoskeleton and Variance of Ca2+ in Cultured Cells During the Invasion of Toxoplasma gondii

LILi-wei;SHAOZhe-xin;YANJie

2006, 24(4):  3-256. 

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Objective To explore the change of cytoskeleton and the variance of Ca²⁺ in cultured cells during the invasion of Toxoplasma gondii. Methods Tachyzoites suspensions were gathered by routine method and used to infect phagocytic cells（J774A.1）and non-phagocytic cells （HUVEC）. The ability of T. gondii invading into the cells and the influence of cytoskeleton inhibitor，colchicine and cytochalasin D，were observed by microscopy. The rearrangement of cytoskeleton of cells was observed by fluoromicroscopy. By using laser scanning confocal microscope，the variance of Ca²⁺ in J774A.1 and HUVEC was detected. Results Ca²⁺ increased greatly in J774A.1 during the invasion of T.gondii（P<0.01）and PLC inhibitor，U73122，could block the increase of Ca²⁺（P>0.05）. The microfilaments of J774A.1 were agglomerated during the invasion of T. gondii. Cytoskeleton inhibitor，cytochalasin D（P<0.01）and colchicine（P<0.05）significantly reduced the infection rate of J774A.1 cells. No considerable change of Ca²⁺ in HUVEC was found （P>0.05）during the invasion and cytoskeleton was not changed. Cytochalasin D and colchicine showed little effect on the infection rate of HUVEC. Conclusion The concentration of Ca²⁺ increases greatly and cytoskeleton（mainly the microfilament）has been rearranged in phagocytic cell during the invasion of T. gondii，while both of them show no significant change in non-phagocytic cell.

Analysis on the Anatomic Features of 47 Cases of Hepatic Hydatidosis Complicated with Biliary Fistula

YANGHong-qiang;PENGXin-yu;NIUJian-hua;ZHANGＳhi-jie;SUNHong;PANHui-zhong;Mulati;WUXiang-wei

2006, 24(4):  4-260. 

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Objective To propose a criterion and its significance of clinical classification of hepatic hydatidosis complicated with biliary fistula. Methods 47 hepatic hydatidosis with biliary fistula cases who were given a sub-adventitial pericystectomy were observed from 2000 to 2005 in a retrospective study. The methods included observation of the different anatomic features of hepatic hydatidosis complicated with biliary fistula during the surgical operation and
evaluation of the curative effect. Results All the 47 patients recuperated successfully and had no complication. Based on the anatomic features of hepatic hydatidosis complicated with biliary fistula，a criterion on clinical classification was proposed as three types: tangential，transfixional and terminal types. Conclusions Hepatic hydatidosis complicat-ed with biliary fistula can be classified as three types according to its anatomic features.

Establishment of In vitro Cultivation of Giardia canis Trophozoites Infected with Giardia canis Virus

CHENLi-feng;LIJian-hua;ZHANGXi-chen-LIUQuan-ZHAOYue-ping;CAOLi-li

2006, 24(4):  5-265. 

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Objective To cultivate a Giardia canis isolate with G.canis virus（GCV）. Methods Five-day-old Meriones unguiculatus was infected with the cysts of G. canis isolated from dogs in Changchun and purified by sucrose density gradient centrifugation-G1 acid funnel filtration method. Trophozoites were isolated aseptically from the duodenum of the infected rodent after 8 days，then transferred to modified TYI-S-33 medium and cultivated at 37 ℃. The trophozoites were centrifuged with 3 000 × g，15 min after liquid nitrogen freeze-thawing three times and the supernatant stained negatively by phosphotungstic acid was observed with transmission electron microscope. Results G. canis trophozoites which adapted gradually to the environment and grew a cellular monolayer after 14 days were examined by freezing and thawing experiment，purity quotient，stability，biology characteristics and microbial contamination detection. The results demonstrated that a stable G.canis trophozoite cell isolate was established. G. canis virus with icosahedron spherical shape and 36 nm in diameter was observed by electron microscope. Conclusion In vitro cultivation of G.canis trophozoites with GCV is established.

Detection of P-4 and GP-46 Expression in Leishmania amazonensis Amastigotes and Promastigotes by RT-PCR

WANGHua-min;SoongLynn

2006, 24(4):  6-268. 

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Objective To detect the expression level of stage-specific genes in Leishmania promastigotes and amastigotes. Methods Total RNAs were isolated from Leishmania amazonensis stationary promastigotes and three sources of amastigotes: freshly obtained from mouse skin lesions，infected J774.G8 macrophages，and transformed from the cultured promastigotes. mRNAs were conversely transcribed into cDNA with SuperScripII reverse transcriptase and oligo dT primers. The polymerase chain reaction（PCR）was used to amplify the specific fragments of amastigote-specific nuclease（P-4）and promastigote-specific membrane glycoprotein（GP-46）. PCR products were analyzed in 1.5% agarose gel. Results A P-4-specific band（273 bp）was observed in all three types of amastigotes with similar density，but it was almost undetectable in promastigotes. In contract，a GP-46-specific band（325 bp）was expressed at a higher level in promastigotes than in all three types of amastigotes. Conclusion Promastigote-derived amastigotes express high level of P-4-specific gene and can be used as a source of amastigotes for biochemical and immunological studies.

Studies on the Growth-Development and Infectivity of Angiostrongylus cantonensis in Dormant Pomacea canaliculata

LIUHe-xiang;ZHANGYi;ZHOUXiao-nong;LVShan;ZHUDan;LINJin;Xiang;LILi-sha;LIYou-song

2006, 24(4):  7-272. 

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Objective To study the impact of dormancy of Pomacea canaliculata on the growth-development and infectivity of Angiostrongylus cantonensis. Methods The intermediate host snails（P. canaliculata）were infected with the first stage larvae of A. cantonensis from the laboratory. One day after infection the snails were kept dormant under 25.0-25.5 ℃ ，and a sample of the snails was selected and dissected to examine the larval growth-development at various interval. Twenty days after infection，they were placed in room with natural winter conditions. Every 10 days a sample of the snails was dissected for larval activity. The third-stage larval infectivity from each group was identified by infecting SD rats. Meanwhile the survival and weight change of snails in the two groups were recorded，and were compared with those snails cultured in water under the corresponding temperature conditions mentioned above. Results The time for first-stage larvae of A. cantonensis in dormant snails to develop to third stage was shorter than that in the snails in aquarium. All the third stage larvae at various degree of activity recovered from snails in winter room conditions，including dormant snails and active snails in water，infected rats successfully. The dormant snails in winter room conditions stopped growing with decreased weight，but the survival rate was significantly lower than that of the snails in aquarium with the same condition along with an extending time of dormancy. Conclusion The development of A. cantonensis larvae has not been affected when snails are kept dormant under 25.0-25.5 ℃. The third stage larvae from snails at natural winter room temperature or in aquarium were all infective. As of the overwintering ways，it is better to keep the infected snails dormant than in the aquarium.

Molecular Identification of Naturally Acquired Plasmodium knowlesi Infection in a Human Case

ZHENGHui;ZHUHuai-min;NINGBei-fang;LIXiang-yu

2006, 24(4):  8-276. 

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Objective To confirm the diagnosis of a human case with atypical vivax-malaria from Yunnan Province by molecular technique. Methods DNA was extracted from blood films of unidentified sample， and of four known Plasmodium species （P. vivax， P. falciparum， P. knowlesi， and P. cynomolgi）. A DNA-based diagnosis with the poly-merase chain reaction （PCR） method targeting the small subunit ribosomal RNA （SSU rRNA） genes of genus- and species-specific （two human malaria species and P. knowlesi） was introduced. Results The PCR amplification with primer pair specific for P. knowlesi produced a single fragment of 150 bp. Sequence analysis showed that the amplified fragment was identical to the sequence of P. knowlesi. Conclusion The patient was naturally infected with P. knowlesi.

Experimental Study on Compatibility of Three Species of Freshwater Snails with Angiostrongylus cantonensis

LVShan;ZHANGYi;WANGXian-hong;LIUHe-xiang;ZHUDan;YINWei-gang;ZHOUXiao-nong

2006, 24(4):  9-280. 

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Objective To compare the compatibility of three species of freshwater snails，Pomacea canaliculata，Cipangopaludina chinensis，Bellamya aeruginosa，with Angiostrongylus cantonensis. Methods The snails were infected by the first-stage larvae of A. cantonensis under the same conditions. Twenty snails of each species were randomly sampled after exposed to the larvae for 1，3，6，12，24 hours，respectively. Each group was placed into an aquarium. Each species with same number was established as control. All the aquaria were equipped with a filter，the water temperature was kept at（24±1）℃. In the first two weeks，the number of death was recorded. Later，the snails were successively examined to record the weight and worm burden of each snail. Results Some snails were dead which mainly happened in the first week postinfection. The death rate and infection rate were not associated with exposure time and snail species. Worm burden of P. canaliculata was significantly higher than the other two species，however，the worm density of P. canaliculata and B. aeruginosa was not significantly different but higher than that of C. chinensis. The worm burden and worm density of P. canaliculata and B. aeruginosa，respectively，were significantly different among five exposure-time groups， but that of C. chinensis was not. Conclusion All the three snail species show a high compatibility with A. cantonensis. In general，the compatibility of P. canaliculata is superior to the other two species.

Construction of Suppression Subtracted cDNA Library of Deltamethrin-resistant Aedes albopictus

WUJia-hong;ZHAOTong-yan;DONGYan-de

2006, 24(4):  10-284. 

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Objective To construct the suppression subtracted cDNA library of deltamethrin-resistant Aedes albopictus. Methods Total RNA was extracted from the deltamethrin-resistant （R-lab） and -sensitive （S-lab） isolates， mRNA was obtained after purification. Double stranded cDNAs were synthesized after reverse transcription. Two subtractions were performed by suppression subtractive hybridization with S-lab as tester and R-lab as driver or S-lab as driver and R-lab as tester. Enriched different expressed cDNA was cloned into pMD18-T vector to construct subtractive libraries. Results The subtracted cDNA libraries contained 580 and 477 positive clones respectively. The PCR results of 150 clones picked randomly from each library showed that the positive ratio of constructed cDNA libraries was 93%， with a length of cDNA fragments ranged from 150 bp to 750 bp. Conclusion The suppression subtracted cDNA library of deltamethrin-resistant Ae. albopictus is constructed.

实验报道

Combined Expression of TSO45W-4BX from Taenia solium and Porcine CD58 in Escherichia coli

LUOXue-nong;ZHENGYa-dong;DOUYong-xi;HOUJun-lin;JINGZhi-zhong;CAIXue-peng

2006, 24(4):  11-289. 

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Objective To express the TSO45W-4BX of Taenia solium in combination with CD58 as a molecular adjuvant for improving the protective efficacy of the TSO45W-4BX recombinant vaccine. Methods TSO45W-4BX and porcine CD58 genes were amplified by PCR， using recombinant plasmids pGEM-4B and pGEM-CD58 as template respectively. The CD58 fragment was inserted into the recombinant plasmid pGEX-4T-1 with directly ligated TSO45W-4BX. The transformant was induced with IPTG and followed by identifying the integrity of the recombinant containing TSO45W-4BX and porcine CD58 with PCR and sequencing. The products were analyzed by SDS-PAGE and Western blotting. Results The expression products of Mr 69 000 GST-4BX/CD58 and Mr 41 000 GST-4BX were present mainly in the form of inclusion bodies and soluble substance respectively， and both were recognized by sera of cysticercosis patients. Conclusions The TSO45W-4BX co-expressed with porcine CD58 conserves its immune reactivity.

DNA Amplification of Plasmodium vivax Parasites from Giemsa-stained Blood Smears

IAOFang-zhen;ZHANGShan-ying;XULong-shan;HUANGJiang-hong;XIEHan-guo;OUYang-rong

2006, 24(4):  12-292. 

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Objective To develop methods of extracting DNA from malaria parasites on Giemsa-stained blood smears. Methods Improved Na₂HPO₄ method and Chelex-100 ion-exchange technique were used to extract DNA from Giemsa-stained or unstained blood smears. Nested PCR was employed for amplification and identification of allelotypes in the Plasmodium vivax merozoite surface protein-1（PvMSP-1）. Results Target DNA bands appeared in all samples of unstained thick blood smears， while no DNA bands were visible in the fixed and stained thin smears. Both methods identified PvMSP-1 alleles from smears with parasitemia of ≥0.01%. Conclusion It is feasible to identify PvMSP-1 alleles from Giemsa-stained blood smear.

综述

Research Progress on the Resting Habits of Major Sandflies and Control Strategy

XIONGGuang-hua;JINChang-fa

2006, 24(4):  13-298. 

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Based on the comprehensive studies in the past years， the authors reviewed important findings on biology， especially the resting habits， of the four major species of sandflies transmitting visceral leishmaniasis. The effective ways for sandfly control were also discussed.

An Update on the Research of Human Thelaziosis

WangZeng-xian;ShenJi-long;WangHong-yan;DomenicoOtranto

2006, 24(4):  14-303. 

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Thelaziosis is one of the parasitic zoonoses which affects the eyes of humans and domestic animals. This review covers its distribution，morphology and life cycle of the parasite，pathogenesis，clinical diagnosis，treatment，and prevention of the disease.

Application of Filamentous Phage Display Technology in Parasitology

GUOAi-jiang;CAIXue-peng

2006, 24(4):  15-308. 

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This review focuses on the application of phage display technology in the prevention，diagnosis and treatment of parasitic diseases. It covers: ① an introduction of phage display technology，filamentous phage and the advantage of carrier. ② construction of phage antibody libraries of parasites，epitope mapping and mimotope，and their potential application in the development of novel diagnostic reagents and vaccines.

信息报道

Analysis on Theses，Funding Ratio and Impact Factor ofthe 《Chinese Journal of Parasitology and ParasiticDiseases》 in 2000-2004

SHENGHui-feng;FUXiu-lan;BOWei;HUYa-qing;DAIJing

2006, 24(4):  16-312. 

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Number of theses published and the major evaluation indicators of the 《Chinese Journal of Parasitology and Parasitic Diseases》 in 2000-2004 were analyzed. Together 833 papers and communications were published in the 5 years and the original articles, reviews and experiment reports occupied 39.4%, 9.1% and 4.4% respectively. The key authors were from universities (53.4%) and disease control institutions (29.4%). 20 universities/institutions published over 8 papers in the period occupying 38.1%. The average rate of papers supported by various funds and by international a-gencies was 0.50 and 0.09 respectively, and higher in 2004, 0.52 and 0.07 respectively. The ratio of national, ministry (provincial) and international projects was 29.9%、43.9% and 20.4% respectively. The overall citation and impact factor of this journal increased from 325 and 0.377 in 2000 to 437 and 0.462 in 2004 respectively, being among the best of the periodicals and expressing its high academic level and its domestic and abroad importance in the field of parasitology.

研究简报

Study on the Relationship Between the Degree of Periportal Fibrosis， Hepatic Parenchymatous Fibrosis and Diameter of Portal Vein in Schistosoma japoncium Infection

FUXiao;LUOXin-song;HOUXun-ya;HEHong-bin;ZHOUJie;WANGYuan-yuan;HEYong-kang;AlainDessein;LIYue-sheng

2006, 24(4):  17-265. 

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The degree of periportal fibrosis，hepatic parenchymatous fibrosis and the diameter of portal vein in fishermen from highly endemic area of schistosomiasis japonica in Dongting Lake region were measured. The results showed a significant correlation between the degree of periportal fibrosis and parenchymatous fibrosis and the portal venous diameter with a correlation coefficient of 0.375 and 0.332 respectively. The authors consider that the diameter of the portal vein can be used to assess the hepatic morbidity of patients.

The Current Prevalence of Intestinal Parasites in Beijing

JIALei;WANGXiao-mei;MAXiao-yan;TANGYao-wu;WUJiang;HEXiong;GAOGui-hua;HANQing-ying;PENGTao;YUHai-zhu;WANGHua-yong

2006, 24(4):  18-314. 

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Following the requirements of the “National survey on the current status of the major human parasitic diseases”，the investigation was conducted in June-October 2002 in 5 districts （counties） of Beijing with a sample of 7912 people. The overall prevalence rate of intestinal parasites was 2.9%，significantly lower than the result from the first survey in 1988-1989 （34.8%）（x^２=3 227.45，P<0.05），revealing that intestinal parasitic infections are not an important risk for people in Beijing Municipality in general.

A Searching System of Parasite Pictures and Relevant Information

CHENHai-ning;HAOZhI-ming;ZHUXian-yin

2006, 24(4):  19-316. 

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Over 3 000 pictures on five major parasites（schistosome，filaria，hookworm，leishmania and plas-modium）collected between 1950 and 1990 were edited and a searching system was established. The data can be used with a network-based version through a LAN system in the Institute. The adoption of Digital Computerized Management makes it possible for sharing resources in human parasitology.

CTL Response to Pre-erythrocytic Stage Vaccine Candidate of Plasmodium falciparum in HLA-A*0201 Transgenic Mice Detected by ELISPOT Assay

QIANFeng;ZHANGQing-feng;SHENLu-hui;PANWei-qing

2006, 24(4):  20-320. 

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The importance of cytotoxic T-lymphocyte（CTL）against malaria parasite in pre-erythrocytic stage has been presented in relevant researches. In order to investigate whether one CTL epitope（YLNKIQNSL）involved in a chimeric pre-erythrocytic stage vaccine candidate of Plasmodium falciparum which was expressed and purified in the laboratory can stimulate in vivo CTL response，HLA-A*0201 transgenic mice were immunized with this vaccine candidate. Enzyme-linked immunosorbent spot（ELISPOT）assay was performed on the splenocytes from the immunized transgenic mice. Positive result indicated that this CTL epitope can be in vivo processed and correctly presented.