Table of Content

30 October 2004, Volume 22 Issue 5

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论著

Prokaryotic Expression of an Antigen Gene of Trichinella spiralis and Identification of the Recombinant Protein

LEILiping;ZHUXinping*;YANGJing;YANGYaping;DINGLi

2004, 22(5):  1-261. 

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　Objective To obtain the recombinant protein of an antigen gene Ts88 of Trichinella spiralis and identify the characteristics of the recombinant protein. Methods Ts88 cDNA obtained by immunoscreening the cDNA library of adult T. spiralis was subcloned into the pET 28c(+) expression vector and expressed in E.coli . Mice were immunized with the fusion protein incorporated into Freund’s adjuvant and the immune sera were collected. The titers of the Ts88 immune sera and the antigenicity of the recombinant protein were detected by ELISA and Western blotting. Immuno fluorescence test was performed in order to confirm the distribution of Ts88 protein in the worm. Results The fragment of Ts88 gene was expressed successfully in E.coli and a highly purified fusion protein was obtained. Immunization with the recombinant protein in mice produced high titers of antibodies, which recognized some components of native antigens of soluble proteins from adult worm of T. spiralis. Western blotting analysis showed that Ts88 recombinant antigen was recognized by all the positive sera, such as the sera from infected or immunized rabbits, from infected swine and from patients of trichinosis. Immuno fluorescence test confirmed that Ts88 protein mainly distributed in the cuticle surface of the worm. Conclusion The Ts88 antigen gene from T. spiralis was successfully expressed. The recombinant protein presented antigenicity.


Prediction of the Impact of Climate Warming on Transmission of Schistosomiasis in China

ZHOUXiaonong;YANGKun;HONGQingbiao;SUNLeping;YANGGuojing;LIANGYousheng;HUANGYixin

2004, 22(5):  2-265. 

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　Objective To predict the intensity and scale of impact on transmission of schistosomiasis japonica in China caused by the climate warming. Methods By using climate data from 193 weather stations in China from 1951 to 2000, the GIS database was created to analyze the tendency of average daily temperature. By using the results from the effective accumulated temperature models on Oncomelania snails and Schistosoma japonicum , the climate transmission model for schistosomiasis was established at country level, by which the spatio temporal analysis was performed to create the distribution maps of Oncomelania snails and Schistosoma japonicum , respectively, by means of GIS approaches based on the ratio of effective accumulated temperature to the snail or the parasite development temperature (ET/SDT) in all 193 stations. The potential distribution maps with the dispersal risk areas of schisotsomiasis japonica in 2030 and 2050 were created based on forecast data that the average temperature of the country will increase by 1.7 ℃ in 2030 and by 2.2 ℃ in 2050. Results The GIS database of climate schistosomiasis of the country was established. It was found that the average temperature in the last 5 decades inclined, especially after 1990 it increased significantly with its increasing regression formula T =0.0198X-28.476. The climate transmission model for schistosomiasis was established, and it was found that the geographical distribution of Schistosoma japonicum was much larger than that of Oncomelania snails based on the ratio of ET/SDT. The prediction maps for distribution of schistosomiasis in 2030 and 2050 were created, respectively, which showed that the sensitive areas were extended with the time, the risk of expansion northward for schistosomiasis will be increasing due to directly the climate warming. Conclusion It is predicted that a northward expansion of transmission area of schistosomiasis may occur due to the climate warming, the expanded potential area for schistosomiasis transmission will be important for future surveillance.


Expression of DNA Vaccine against Trichinella spiralis in Chinese Hamster Ovary Cells

CUIJing;WANGZhongquan*;HANHuamin;WEIHaiyan;ZHANGHongwei;LIYonglong

2004, 22(5):  3-270. 

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　Objective To observe the in vitro expression of DNA vaccine (recombinant eukaryotic expression plasmid pcDNA3 TspE1) encoding a Mr 31 000 antigen of Trichinella spiralis in Chinese hamster ovary (CHO) cells and analyze the antigenicity of the products expressed. Methods The recombinant plasmid pcDNA3 TspE1 was transfected into CHO cells by using cationic lipids (Lipofectamine 2000). The positive cell clones were screened by the selective antibiotic G418. The expressed products were identified by RT PCR, IFAT, SDS PAGE and Western blotting. Results The results of RT PCR amplification showed that there was one band with 876 bp in CHO cells transfected with pcDNA3 TspE1 and no any bands in CHO cells transfected with the empty plasmid pcDNA3. The IFAT demonstrated that the pcDNA3 TspE1 transfected CHO cells reacted with sera from mice immunized with the recombinant fusion protein and from mice infected with T. spiralis , the bright yellow green fluorescence staining appeared in the transfected CHO cells. The pcDNA3 transfected and un transfected CHO cells exhibited as orange color. The results of SDS PAGE showed that there was one band with Mr 31 000 in culture supernatant of CHO cells transfected with pcDNA3 TspE1. Western blotting confirmed that the band with Mr 31 000 could be recognized by sera from mice immunized with the recombinant fusion protein, from rabbits immunized with T. spiralis muscle larval soluble antigens, from mice infected with T. spiralis and from patients with trichinellosis. Conclusion The mammalian CHO cells were transfected by the recombinant plasmid pcDNA3 TspE1, and the TspE1 gene of T. spiralis was expressed in the transfected CHO cells. The proteins expressed are secreted into cell culture supernatants and show the antigenicity of T. spiralis.


The Clinical Significance of Interferon-γ and its Receptor in Patients with Schistosomiasis

SHAOPingyang;CAIWeimin;DINGRenye;WUWenlin;HUANGYuying

2004, 22(5):  4-273. 

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Objective To study the expression of interferonγ(IFNγ) and typeⅠinterferon γ receptor (IFN γR1) in the chronic and advanced patients of schistosomiasis japonica, and to discuss its clinical relationship with hepatic fibrosis. Methods IFN γR1 in the peripheral blood lymphocytes was detected by ELISA, and the hyaluronic acid (HA), collagen type Ⅳ(C Ⅳ), procollagen type Ⅲ (PCⅢ), laminin (LN) were detected by radio immunoassay (RIA) in schistosomiasis patients. The level of IFN γ, IFN γR1 and serum markers of hepatic fibrosis were observed, and the relationship with each other was analyzed by statistical method. Results There was no difference in the expression of IFN γ and IFN γR1 between the patients with chronic schistosomiasis and the normal group ( P>0.05), the IFN γR1 in advanced cases without splenectomy was low ( P <0.05), but IFN γ was high( P<0.01). The two indicators in the advanced schistosomiasis patients with splenectomy returned to normal. There was no corresponding relationship between the two indicators and HA, C Ⅳ,PCⅢ,LN with a r value of 0.19, 0.20, 0.14, and 0.21 respectively. Conclusion There is a corresponding relationship between IFN γ and IFN γR1; the expression of IFN γR1 is related to the course of schistosomiasis, and the relationship with hepatic fibrosis needs further study.


Analysis of Cytochrome c Oxidase I and Cytochrome b Genes of F1 in Laboratory Line Oncomelania hupensis hupensis

ZHANGYi;YamasakiHiroshi;LIUHexiang;FENGTing;FENGZheng

2004, 22(5):  5-276. 

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　Objective To analyze the diversity of F1 in laboratory line Oncomelania hupensis hupensis . Methods Genomic DNA was isolated, and cytochrome c oxidase I (COI) and cytochrome b (Cytb) genes were amplified by polymerase chain reaction (PCR). The nucleotide sequences of the PCR products were analyzed by GENETYX MAC software package (ver.9). Results Pairwise divergences among six F1 individuals were found in 12.2% nucleotides in COI gene fragments. 13.5% of genetic divergences between O.h.hupensis and O.h.robertsoni were identified. 94 amino acids were observed in difference. In Cyt b gene fragments, 6.4% of divergence was observed among four F1 samples. 25 amino acids were observed differently. The divergence of Cyt b genes from O.h.robertsoni and O.h.hupensis were 13.6%, included 6 amino acids. Conclusion Diversities were found in both COI and Cyt b gene sequences of F1 in laboratory line O.h.hupensis.


Analysis of the Sequences of the Ribosomal DNA ITS_2 Region for Differenciating Anopheline Mosquitoes within Hyrcanus Group

GAOQi;ZHOUHuayun;LIJulin;LIFenghua;ChoeTongGyu;YomSungChan;ZHUGuoding;CAOJun

2004, 22(5):  6-279. 

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　Objective To analyze the genetic characteristics of ribosomal DNA ITS2 region for differenciating anopheline mosquitoes within Hyrcanus group. Methods The ribosomal DNA ITS2 region of both laboratory line and filed collected An. anthropophagus and An. sinensis as well as the field collected An. yatsushiroensis and An. lester i were amplified and sequenced. The sequencing data were then analyzed for the restriction mapping using Omega Sequencing analysis program. Results and Conclusion The length of the sequences of An. sinensis, An. anthropophagus, An. yatsushiroensis and An. lesteri are 472, 452, 456 and 456 bp respectively. The restriction mapping showed that there were different restriction digesting sites among the ribosomal DNA ITS2 region sequences from An. sinensis, An. anthropophagus, An. yatsushiroensis and An. lesteri . On the basis of the sequence differences among the anopheline species within Hyrcanus group, it is possible to develop new technique for genetic identification of anopheline mosquitoes.


Change of Lipid,Esterase and Lipase in Mosquito Larvae Infected with Lagenidium giganteum

MOURong;BAOHuaien*;LIJianhua

2004, 22(5):  7-282. 

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　Objective To explore possible physiological and biochemical mechanisms of Lagenidium giganteum infection in killing mosquito larvae. Methods The content of lipid and the activities of esterase and lipase between the normal and infected mosquito larvae were observed with histochemical method. The results were photomicrographed and analysed by image analysis using computer. Results In 24 hrs after infection, the content of lipid in the infected Culex quinquefasciatus larvae was lower while the activities of esterase and lipase in the larvae were higher than the control. In 48 hrs and 78 hrs after infection, the content of lipid in the infected larvae of C. quinquefasciatus and Ae. albopictus were significantly lower while the activities of esterase and lipase were significantly higher than those of control. Conclusion The content of lipid in the mosquito larvae decreased while the activities of esterase and lipase increased after the infection of Lagenidium giganteum . The disorder of lipid metabolism might be part of the killing mechanisms to C. quinquefasciatus and Ae. albopictus larvae.


Biological Features of Mouse Macrophage Transfected with Toxoplasma gondii GRA-1 Gene

PENGBiwen;LINJianyin*;ZHANGTao;JIANGMingsen

2004, 22(5):  8-286. 

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　Objective To investigate the change of biological features of macrophages after transfected by Toxoplasma gondii GRA 1 genes (P24). Methods The transfected cells Cyto P24 RAW264.7, Nuc P24 RAW264.7, Mito P24 RAW264.7 and ER P24 RAW264.7 were studied by RT PCR to determine the P24 mRNA expression. Growth features of the cells were examined with microscopy and the cell growth curve was developed. Results In four cell lines, expression of ER P24 RAW 264.7 was found to be higher than the other three, and there was no P24 mRNA expression in either of the cells without P24 insert. The attachment and the proliferation of ER-P24-RAW264.7 were more rapid than normal RAW264.7. Conclusion Transfection of mouse macrophages ER RAW264.7 strain with T. gondii P24 gene leads to a prominent change of biological features in the studied cell line.


Prevalence of Anaphylaxis to Dust Mite in Human Population in Xuzhou

XINGDaorong;WENTinghuan

2004, 22(5):  9-289. 

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　Objective To study the prevalence of anaphylaxis to dust mite in normal individuals and patients with different allergic disorders in Xuzhou. Methods By skin prick test (SPT) with Dermatophagoides farinae allergen extract, the prevalence of the normal individuals and OPD patients visiting hospital with allergic symptoms were examined for hypersensitivity to mite. Histamine equivalent criteria were used to evaluate the reactivity rate and the strength of skin response. Results 34 (15.3%) out of 222 pupils and students of different age groups from different levels of schools regarded as the normal population showed positive reaction to the mite allergen with usually only very weak response(+). There were no significant differences between the different age groups (P>0.05), but it was different between 18.6% (22/118) in females and 11.5% (12/104) in males (P<0.05). Also the reactivity rate of younger female pupils was higher than that of older ones. Among 515 cases from the OPD patients with allergic symptoms from pediatrics, ENT, respiratory and dermatology departments, 424 (82.3%) were positive with different grades of skin reaction. The rate of strong reactivity(≧+++) was 68.6% in allergic children, 63.5% in patients with allergic rhinitis, 41.9 % in patients of respiratory department and 25.7% in patients with dermatitis. The allergic children of ≦10 years old showed highest rate of very strong reactivity (++++),the rates declined along with the growing age. Conclusion In Xuzhou area the SPT reactivity rate to dust mite for normal individuals was 15.3% with only weak response; while for allergic patients it was 82.3 %, 47.9% of them showed strong and very strong responses.


Molecular Cloning and Characterization of a Rac1 Homologue cDNA from Trichomonas vaginalis

FUYucai;ZHANGJiaxin;ZHENGXiaohong;LIUHong

2004, 22(5):  10-293. 

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　Objective To clone and characterize a Rac1 homologue from Trichomonas vaginalis for studying cell cycle of the organism. Methods A cDNA library derived from T. vaginalis mRNA was constructed into λ TriplEx2 phage vector. An expression sequence tag program was launched. Sequences of cDNA clones were analyzed using NCBI BLAST algorithms, and ClustalW and Treeview programs. Results A cDNA clone with a length of 714 base pairs was isolated. The sequence analysis showed that the cDNA clone has an open reading frame with 600 bp. The deduced amino acid sequence from the open reading frame contains 200 residuals and is most homologous to Rac1 subfamily of Rho GTPases with >60% identity. The conserved sequence elements of Rho GTPases, such as GTP binding sites, GTPase activating protein (GAP) interaction motifs, GTP dissociation inhibitors (GDI) interaction motifs, guanine nucleotide exchange factor (GEF) interaction elements, etc, were detected in the amino acid sequence. The phylogenetic analysis showed that the cDNA clone is grouped in the Rac subfamily and is more closely related to Rac1 proteins of protozoa. Conclusion The cDNA clone isolated belongs to Rac subfamily of Rho GTPases and is probably a Rac1 protein of T. vaginalis.


Cloning and Sequence Analysis of the Ribosomal DNA ITS Gene of Leishmania donovani Isolates from Hill Foci of China

TIANYu;CHENJianping*;HUXiaosu

2004, 22(5):  11-296. 

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　Objective To determine the nucleotide sequence of the ITS (internal transcribed spacer) gene of Leishmania donovani isolates from hill foci (L.d.SC10 and L.d.6), and to find out the difference of the gene sequences between the two isolates. Methods Specific ITS fragments from nuclear DNA of two Leishmania isolates were amplified by PCR, cloned into pMD18 T vector, and finally sequenced by the dideoxy chain termination method. Results Sequence analysis showed that the amplified DNA fragments of the two isolates were 1 027 bp (L.d.SC10) and 1 028 bp (L.d.6) respectively, showing a sequence difference. Conclusion Sequence difference exists between the Leishmania isolates L.d.SC10 and L.d.6 from hill foci in China.


REN Xiao yan,ZHANG Ling min,ZHU Pei xia,XIONG Zhong jin,WU Chun yun

RENXiaoyan;ZHANGLingmin*;ZHUPeixia;XIONGZhongjin;WUChunyun

2004, 22(5):  12-299. 

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　Objective To investigate the toxicity of alpha terthienyl to the larvae of Aedes albopictus , its influencing factors and effect on the larva deve lopment. Methods Under experimental ultraviolet A (UVA),the number of dead,pupal or eclosive mosquito larvae was determined on the condition of different doses of alpha terthienyl and different disposal time in the dark;the number of dead larvae was also determined under sunlight on the condition of different doses of alpha terthienyl and different disposal time to water. Results The LC 50 of alpha terthienyl to Aedes albopictus larvae was 2.37μg/L under UVA. The best effect was shown when the larvae were incubated with alpha terthienyl3 h in dark. Alpha terthienyl could significantly inhibit the larva deve lopment and the emergence of the pupae. Under strong sunlight, the larvae were quickly killed by high concentration alpha terthienyl. The 24 hours effect of alpha terthienyl was better when it was applied at 5AM than that of at 10AM and 1PM. Conclusion Alpha terthienyl is an effective, practicable larvicide which prohibits the growth and development of the larvae of Aedes albopictus .


实验报道

Studies on the Genetic Variation of Two Mitochondrial DNA Molecules of Schistosoma japonicum

GUOKaiwen;NIUAnou

2004, 22(5):  13-302. 

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　Objective To study the variation of Schistosoma japonicum through two mitochondrial DNA molecules. Methods Genomic DNA was isolated with kit, and the mitochondrial NADH dehydrogenase 1(ND1) and cytochrome c oxidase I(COI) gene fragments were amplified by polymerase chain reaction(PCR) and sequenced. The gene trees were constructed and the acquired data were analyzed with the help of bioinfotmatics. Results The gene trees showed that the Taiwan isolate and the mainland isolates can be divided in two groups: a group from the hilly region (Yunnan and Sichuan), another group from the lake region (Hunan, Jiangxi and Anhui); isolates from Hubei are at a different position on the gene trees. Conclusion There are variations among the geographic isolates of Schistosoma japonicum in China, nevertheless, they have close kinship.


Molecular Cloning of a Protein Antigen Gene of Cysticercus cellulosae

LIUDianwu;LIWenling;ZHANGLimei

2004, 22(5):  14-305. 

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　Objective To immunoscreen one protein antigen gene from a cDNA library of Cysticercus cellulosae . Methods A cDNA library of C. cellulosae was constructed after cDNA was synthesized, and immunoscreened using rabbit anti C. cellulosae polyclone antibody. The gene structure and its possible function were analyzed by comparing with the sequences available in the GenBank, after the insert of positive clone was subcloned to pBluescript SK plasmid and the nucleotide sequence of the cDNA was determined by dideoxynucleotide chain termination method using a Taq DyeDeoxy Terminator Cycle Sequencing Kit. The amino acid sequence was deduced from nucleotide sequence using GENETYX software. Homological search of the nucleotide sequences was done using BLAST in GenBank. Results and Conclusion A cDNA clone of 1 320bp was isolated. The clone contained one open reading frame composed of 1 260bp encoding 420 amino acids, in which two potential glycosylation sites were found. The partial nucleotide sequence of the gene fragment showed high homology with the essential spectrin gene of Caenorhabditis elegans and the erythrocyte surface antigen gene of Plasmodium falciparum , when the gene fragment was homologically analyzed in GenBank.


Amplification,Cloning and Expression of 3-Phosphoglycerate Kinase Gene from Clonorchis sinensis

WUDe;YUXinbing*;XUJin;WUZhongdao;CHENShouyi;HEDonggou

2004, 22(5):  15-308. 

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　Objective To construct a prokaryotic expression plasmid encoding 3 phosphoglycerate kinase(PGK) gene of Clonorchis sinensis and analyze its expression in E.coli JM109. Methods Total RNA was extracted with Trizol. The gene encoding PGK was amplified by RT PCR from the total RNA, and was cloned into the pGEX 4T1 vector. The recombinant plasmids pGEX4T1 PGK constructed were transferred into JM109, positive recombinants were screened and identified by tool enzyme digestion, PCR technique and sequencing. The recombinant was induced with IPTG to express the target protein. The expression product was characterized by SDS PAGE. Results There were three clear bands from extracted total RNA, the PGK gene (1 248bp) was amplified by RT PCR technique, the sequencing result was consistent with the known sequence. Ten clones were obtained by screening after transferring, six of which were positive recombinants. The positive recombinant was induced to express, and the SDS PAGE showed the expression products was about Mr 70 000. Conclusion The recombination prokaryotic expression vector pGEX 4T 1 PGK has been reconstructed and a fused protein was expressed which contained Sj26 GST.