Table of Content

28 February 2001, Volume 19 Issue 1

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论著

Genotypes of Merozoite Surface Protein 2 of Plasmodium falciparum in Different Malaria Endemic Areas *

ZHUXinping;ZHOULei;ZHANGXinmei;YANGJing;YANGYaping

2001, 19(1):  1-3. 

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　Objective To determine the genotypes and distribution of MSP2 of Plasmodium falciparum isolates in Yunnan and Hainan provinces, China.Methods The central polymorphic region of MSP2 allele was amplified by the nested PCR for genotyping of P.falciparum. Results The higher degree of polymorphism of MSP2 of P.falciparum was observed in Yunnan and Hainan. Distribution and allele frequencies differed in both provinces, indicating considerable geographical heterogeneity of parasite populations. The mixed infection of different allele type and multiplicity of infection was more frequent in Hainan than in Yunnan.Conclusion There were obvious differences in the distribution and frequencies of MSP2 alleles between Yunnan and Hainan endemic areas. MSP2 is suitable to be used as a marker gene for the genotyping of P.falciparum infection.


Gene Typing of Merozoite Surface Protein 1 of Plasmodium falciparum isolates from Hainan Province *

JIANGGangfeng;HONGJiadong;CHENPeiquan;WANGShanqing;MENGFeng

2001, 19(1):  2-6. 

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　Objective To identify the genotype of merozoite surface protein 1 (MSP1) of Plasmodium falciparum isolates from Hainan Province. Methods Nested PCR was applied to amplify the MSP1 of Blocks 2 and 3 Plasmodium falciparum isolates from Hainan Province. Two allelic family representitive gene fragments were sequenced.Results From 36 out of 39 blood samples from Plasmodium falciparum patients, 44 gene fragments of blocks 2 and 3 of the MSP1 were amplified, of which the MAD20 type allele was dominant(75%). followed by K1 type allele. No RO33 type allele was found. The mixed infection rate of the two different allelic type was 19 4%. Sequence analysis showed that the sequences of MAD20 and K1 type isolates from Hainan Province were highly homologous to that of the MAD20 and K1 allelic prototypes.Conclusion Two principal allelic types of MSP1 gene, MAD20 and K1 type, exist in malaria endemic areas in Hainan Province, the MAD20 type being the dominant.


Comparison of Immune Responses Elicited by Recombinant Protein and Eukaryotic Expression Plasmid Based on Histidine Rich Protein 2 of Plasmodium falciparum *

LIXun;MIAOJun;XUECaifang;ZHENRongfen;LIUZhongxiang;WANGXianfeng;MUShijie

2001, 19(1):  3-10. 

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　Objective To identify the immune characteristics of different vaccine prototypes based on HRP2 and to provide experimental evidence for developing P f. blood stage vaccines.Methods BALB/c mice were immunized with recombinant protein TP HRP2 or eukaryotic expression plasmid pcDNA3 1(-)/HRP2. The kinetics and specificities of antibody responses were analyzed. The proliferation tests of spleen cells were done, and P f. growth inhibition assays were done with immune sera.Results The mice immunized with TP HRP2 in Freund′s adjuvant produced high level and high specificity antibody response. The antibodies appeared rapidly and lasted for a longer time. Cellular responses were induced simultaneously, and the immune sera could inhibit the development of parasite in IRBCs. The mice immunized with pcDNA3 1(-)/HRP2 produced middle level antibody response which had some specificity,however,the induction of antibodies required repeated inoculation and a longer duration. Immune cells were well primed and the memorial immune response was obvious but the immune sera had no effect on the growth of P f. in vitro .Conclusion Both the recombinant protein and plasmid DNA based on HRP2 have different immune characteristics in mice. HRP2 recombinant protein has the potential in practical application.


Screening the Mimic Antigen Epitopes of Triosephosphate Isomerase of Schistosoma japonicum Chinese Strain (Sjc Tpi) with Random Phage Peptide Library

YUChuanxin;ZHUYinchang;YINXuren;HEWei;XUYongliang;GUANXiaohong

2001, 19(1):  4-14. 

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　Objective To screen the mimic antigen epitopes of the triose phosphate isomerase of Schistosoma japonicum Chinese strain (SjC TPI) and investigate their immunogenicity. Methods The random phage peptide library (PH.D. 12) was screened with the purified antibody(IgG) against SjC TPI to get the positive phage which contained the mimic antigen epitopes of SjC TPI, and the immuno characterization of the mimic antigen epitopes were investigated. Results Two mimic antigen epitopes (M1, M2) of SjC TPI were obtained. The immuno sera of mice (Kunming strain) against the positive phages could recognize both the SjC TPI and the protein of the positive phages. The DNA sequencing data showed no homology between the sequences of the deduced amino acid of the two mimic antigen peptides and the amino acid of SjC TPI. Conclusion The two mimic antigen epitopes of SjC TPI obtained are imitative epitopes of the configuration antigen of SjC TPI.


Cloning and Expressing of the Three Repeat Fragments of Plasmodium falciparum 11 1 gene *

HUWei;TasakiYamasaki;FENGZheng;YANGBailin;XUXuenian;WANGJujun

2001, 19(1):  5-18. 

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　HT5"H]Objective To clone and express the 3R, 6R and 9R repeat fragments of Plasmodium falciparum (Pf11 1) gene. Methods Three repeat fragments from the genomic DNA of Plasmodium falciparum 3D7 strain cultivated were amplified by using the designed primers.The PCR products were cloned into the pT7 vector for bi direction sequencing.The sequencing results were analysised by GENETYX MAC. And then the amplified fragments were subcloned into pET32a(+) or pET32b(+) in order to express the recombinant proteins under the induction of IPTG in E coli BL21. Results 3R,6R and 9R fragments with sizes of 552 bp, 630 bp and 444 bp respectively were successfully amplified by PCR. The sequence analysis showed that there were 4 more 3AA units and one more 6AA unit in Pf11.1 gene of 3D7 strain as compared with Palo Alto strain. The homologies of the nucleotide sequence between the 3R fragment and the 6R fragment of the two strains were 92.8% and 95 1%,respectively. The amplified 9R fragment contains 13 9AA repeat units. The three recombinant proteins were expressed in BL21 strain with molecular weights of 45,60 and 42 kDa. Conclusion We got the 3R, 6R and 9R fragments separately by PCR and expressed them in E.coli successfully. The Pf11.1 gene of 3D7 strain is highly homologous to that of the Palo Alto strain.


Analysis of the Mitochondria Related Protein of Schistosoma japonicum and its Antigen Epitopes

HuXuemei;WuHaiwei;ZhangZhaosong;SuChuan;ZhaoWei;MaLei;ZhouJili;WuGuanling

2001, 19(1):  6-21. 

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　Objective To sequence the cloned gene Sj338 and to identify the encoded protein and its antigen epitopes.Methods The Sj338 gene fragment obtained from adult S. japonicum cDNA library amplified by PCR method was subcloned into pGEM T vector for sequencing. The sequence of nucleotides and the characteristics of the encoded protein were analyzed by DNASIS Program and Goldkey DNA and Protein Analytical Program, and then the homology of the amino acid sequence was searched on the BLAST net.Results The cloned rSj338 gene was demonstrated to be 487 bp containing one 459bp ORF, encoding a protein consisted of 153 amino acids with a molecular weight of 17 6 kDa. The amino acid sequence of the recombinant protein rSj338 shared 46% identity with that of the corresponding part of human mitochondrial import receptor and 44% identity with that of the Rattus sp. mitochondrial precursor receptor. The possible antigen epitopes were predicted within the peptide fragments of 26-32 aa, 37-46 aa and 147-151 aa.Conclusion The protein encoded by rSj338 gene fragment might be the mitochondria related protein of Schistosoma japonicum .


Cloning and Expression of the Gene Encoding Schistosoma japonicum Tropomyosin *

CAOJianping;LIUShuxian

2001, 19(1):  7-25. 

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　Objective To clone and express the cDNA encoding Schistosoma japonicum tropomyosin. Methods The cDNA was amplified by reverse transcription polymerase chain reaction (RT PCR). The PCR products were ligated with pGEM T vectors and then for transformations. After characterization of white clones by agarose gel electrophoresis, endonucleases digestion and PCR, some recombinant plasmids with inserts were used for sequencing. Then the gene was subcloned into prokaryotic expression vector pQE30 and expression was induced by IPTG. Results The PCR products was 823 bp judged by agarose gel electrophoresis and sequencing. A cDNA encoding S japonicum tropomyosin, except for 14 amino acids at the amino terminus and 2 at the carboxyl terminus, has been constructed and cloned successfully. The colony, designated pGSjcTM12, was sequenced and shown to be 91 1% identical at the nuclei acid level and 98.1% identical in deduced amino acid sequence to that of S mansoni tropomyosin. The gene was subcloned into pQE30 and an expressed protein of about 32 kDa was obtained.Conclusion The cloning and expression of the gene encoding S japonicum tropomyosin had been successfully made.


Dynamic Changes in Collagen Type Ⅰ and Collagen Type Ⅲ in Rabbits Infected with Schistosoma japonicum and the Effect of Gamma Interferon *

WENGHonglei;CAIWeimin;YANGYanhong

2001, 19(1):  8-29. 

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　Objective To observe the dynamic changes in collagen type Ⅰ and collagen type Ⅲ in rabbits with schistosomiasis japonica and the treatment effect of gamma interferon on the degradation of collagens in schistosomal hepatic fibrosis.Methods Each rabbit was infected with 80±1 S japonicum cercariae. Liver operations were done at different time points after infection and the liver specimens were embedded with paraffin and stained with α SMA, HE and picric acid Sirius red. The stained slides were observed under polarizing microscope and different collagen areas calculated by computer imagine analysis system. At the 16th week after infection, the infected rabbits received a single dose of praziquantel and gamma interferon for 8 weeks.Results The area percent of collagen type Ⅰ at the 28th week after infection (40 14±17 00) increased about seven fold compared with the 8th week group (5 73±3 40). The area percent of collagen type Ⅲ at the 28th week after infection (6 80±5 19) increased about six fold compared with the 8th week group (1 15±1 34). The α SMA positive cells also increased significantly. After gamma interferon treatment, the area percent of collagen type Ⅰ and type Ⅲ decreased significantly, from 18 51±7 52 and 4 63±3 64 (before treatment) to 3 09±1 54 and 0 40±0 37 (0 and 4 weeks after treatment) ( P <0 01). However, after the withdrawal of gamma interferon treatment,the collagen degradation was reversible.Conclusion Gamma interferon is effective in the treatment of hepatic fibrosis in rabbits infected with S japonicum ,the effect being reversible.


Effect of Daphnetin on the Exo erythrocytic Stage of Rodent Malaria *

LIUYunguang;WANGQinmei;XUYueqin;NIQizhen;NIYichang**

2001, 19(1):  9-32. 

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　Objective To investigate the activity of daphnetin(DPNT) against the exo erythrocytic stage of rodent malaria. Methods Groups of ten male ICR mice were infected by intraperitoneal injection with sporozoites of P yoelii .Mice were administered daphnetin 0 5 hr postinfection on d 0 and once per day for three additional consecutive days(d 1-d 3) by the i g route. The effects of daphnetin at various dosages and those of the combination of daphnetin with primaquine were assessed by the number of mice with negative Giemsa stained slides from tail blood on the seventh day after infection and by the average number of red blood cells (RBC) infected in 1 000 RBC observed on the eleventh or twelfth day after infection. We also observed the effect of daphnetin on the concentration of Hb in ICR mice.Results Daphnetin exhibited no detectable antimalarial effect on the exo erythrocytic stage of P yoelii ,while the antimalarial efficacy of DPNT 50 mg/(kg·d) combined with 5 mg/(kg·d) PQ, was comparable to PQ 10 mg/(kg·d)×4 d i g. in mice infected with sporozoites of P yoelii . The concentration of Hb in ICR mice administered with DPNT 50 mg/(kg·d) ×4 d decreased on the eighth day after administration.Conclusion Daphnetin alone showed no anti exoerythrocytic activity in vivo . The combination of DPNT 50 mg/(kg·d) with PQ 5 mg/(kg·d) showed promising antimalarial efficacy comparable to that of PQ 10 mg/(kg·d).Administration of DPNT caused anemia in ICR mice.


Effects of Anti Idiotypic Antibody NP30 on Modulation of Egg Granuloma Formation and Hepatic Fibrosis of Schistosomiasis *

FENGZhenqing;ZHURong;LIYuhua;QIUZhenning;LIYunqian;WANGZhuming;XUEWanfen;GUANXiaohong

2001, 19(1):  10-36. 

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　Objective To study the effects of the monoclonal anti idiotypic antibody NP30 active immunization on egg granuloma formation and hepatic fibrosis in Schistosoma japonicum infection. Methods ICR mice were actively immunized with NP30 100 μg ×3 ip. every 10 days while the mice in control group were injected with SP2/0 ascites ip. simultaneously. After cercariae challenging,the mice were killed at the 4th, 8th,12th, 16th, 20th and 24th week, respectively.Mouse livers were removed and stained histochemically with VG and subjected to immunohistochemical assay of collagen type Ⅰ,Ⅲ and fibronectin(FN).The volume of egg granulomas and the content of collagen type Ⅰ,Ⅲ and FN were determined quantitatively by NYD 1000 Image Analysis System. Results The volume of egg granulomas in NP30 immunized group was much smaller than that of control group from the 12th week after cercariae challenge. The cellular components of egg granulomas in NP30 immunized group were significantly different from those of the control group,exhibiting two types of atypical egg granulomas were found.VG stain revealed that the average optical density of collagen in hepatic granulomas of experimental group was lower than that of control group.Immunohistochemical assay revealed that the contents of collagen type Ⅰ,Ⅲ and fibronectin in egg granulomas of experimental group were lower than those of control group. Conclusion NP30 vaccination may induce both cellular and humoral protective immunity to modulate egg granulomas and suppress liver fibrosis of schistosomiasis japonica.


Cytotoxic Effect of Acanthamoeba Trophozoite on Hela Cells

QIANMin;ZHANGXiuhua;MEIBing;ZHANGPing;YANZheng;YUMeng

2001, 19(1):  11-40. 

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　Objective To investigate the cytotoxic effect(CTE) on human cervix cancer HeLa cells induced by five strains of pathogenic free living Acanthamoeba .Methods The cytotoxic effect of five isolates of Acanthamoeba on HeLa cells was investigated by light microscopy and MTT method.Results The photomicrographs of HeLa cells showed a sequence of cardinal morphological features of apoptosis when HeLa cells were exposed to Acanthamoeba in a time dependent manner at a ratio of 1∶1 for 12 h. MTT method showed more than 50% of tumor cells underwent cytolysis following exposure to A lugdunensis trophozoites,and only 18% of cells treated with A polyphaga underwent CTE. The CTE produced by A lugdunensis and A quina trophozoites was more rapid than the others, beginning as early as 6 h after coincubation and resulting in cytolysis by 72 h.Conclusion These five strains of Acanthamoeba exhibit cytotoxic effects of varying degrees on HeLa cells,inducing apoptosis.


Preliminary Study on Cytochrome C Oxidase 1 Gene of Oncomelania hupensis from Miao River Area in Hubei Province *

SHIChaohui;QIUChiping;XIAMingyi;FENGZheng;GeorgeM.Davis

2001, 19(1):  12-44. 

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　Objective To study the mitochondrial cytochrome C oxidase 1(CO1) gene of Oncomelania snails from Miao River area in Hubei Province.Methods Oncomelania snails were collected from Miao River area, including upstream and downstream. Genomic DNA was extracted from the tissue of the snail. PCR was used to amplify a fragment of the CO1 gene. Sequences of the CO1 fragment were determined directly from the purified PCR products by an automated sequencer. Sequences for each individual were assembled and edited using ESEE 3 0 s. A distance matrix was computed using program DNADIST of PHYLIP(3 57). Unrooted maximum likelihood trees were calculated from program FITCH.Results The amplified CO1 gene of the snail was a fragment of 638 bp in length. Sequence analysis showed that the accumulated variable sites were significant different between upstream and downstream populations, being 29 and 46, respectively. From the number of variable sites in the gene,snails in this area were roughly separated into two groups. Each of them was a mixture of both upstream and downstream snails.Same haplotypes were confirmed to be present among the collected sites along the river. From the distance matrix of sequence divergence, the population upstream vs downstream differed by 0 0221±0 0105.Conclusion There were more variation in downstream population than that in upstream.Gene flow was identified in these populations. The phylogenetic trees suggest the existence of two groups,but all of them belong to O h hupensis .


实验报道

Dynamic Changes in Amino Acid and Glucose in Culture Medium with Adult Schistosoma japonicum *

FANHong**;DongHuifen;JIANGMingsen;ZHONGQinping;MINGZhenping

2001, 19(1):  13-47. 

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　Objective To observe the dynamic changes in the contents of amino acid(AA),glucose(Gluc) and triglyceride(TG) in the culture medium containing adult Schistosoma japonicum .Methods The contents of AA, Gluc and TG in the culture medium during the incubation period from d 0 to 6 d were detected by amino acid automatic analyzer and automatic biochemical analyzer.Results The contents of Arg,Thr,Met,and Lys and Gluc were reduced, Asp and Ala increased apparently.Concluasion Increasing the levels of Arg,Thr,Met Lys and Gluc,reducing the levels of Asp and Ala,and changing the culture medium in time might be in favor of the in vitro cultivation of S japonicum .