Table of Content

30 June 2004, Volume 22 Issue 3

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论著

Mucosal Immunization of Recombinant Schistosoma japonicum Ferritin

CHENLi-yu;YIXin-yuanZ;ENGXian-fang;ZHANGShun-ke;LarryMcReynolds

2004, 22(3):  1-132. 

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　Objective To subclone and express the gene encoding Schistosoma japonicum ferritin(SjFer)and study its immune protection against challenge infection in mice vaccinated intranasally. Methods The SjFer gene was amplified by PCR, and subcloned into the N-terminal of intein 2 of the pTWIN1 vector. The positive recombinant was screened by PCR, restriction enzyme digestion and sequence analysis. The positive recombinant was transformed into E. coli ER2566. The soluble recombinant fusion protein (rSjFer-intein 2) was expressed in E. coli by induction of low IPTG concentration under low temperature, and analyzed by SDS-PAGE and Western blotting. Mice were immunized intranasally with rSjFer, using chitosan as adjuvant. Two weeks after the third vaccination, challenge infection with S. japonicum cercariae was carried out. Worms and eggs collected from the livers of mice were counted at 42 days after the challenge. Levels of specific antibodies were detected by ELISA before infection. Results SjFer was successfully subcloned into pTWIN1 vector and expressed in E. coli. In mice vaccinated intranasally with rSjFer and adjuvant chitosan, the worm reduction rate was 35.51% and the reduction rate of eggs per gram liver tissue (LEPG) was52.17%. As compared with the control groups, levels of IgG, IgA in sera and SIgA in saliva increased significantly. Conclusion The expressed rSjFer can induce partial protective immunity against S. japonicum infection in mice when they were vaccinated intranasally, with chitosan as adjuvant.


Cloning and Expression of Aldolase Encoding Gene of Plasmodium falciparum FCC1/HN Strain

ZHANGRui-juan;ZHUHuai-min;CAOYi;ZHOUAi-guo;ZHENGZhi-qiang;ZHANGQing-feng;ZHENGHui

2004, 22(3):  2-135. 

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　Objective To clone, sequence, and express the aldolase(ALD)encoding gene of Plasmodium falciparum FCC1/HN strain. Methods The ALD encoding gene was amplified by PCR from genomic DNA of FCC1/HN strain. The positive clones were screened and identified by agarose gel electrophoresis and endonuclease. The recombinant plasmid was transformed into E. coli M15. The fusion protein was expressed by IPTG induction and purified by Ni-NTA affinity chromatography and anion exchange column. Results The ALD gene of P. falciparum was amplified. Analysis of sequencing showed that the ALD gene of P. falciparum was identical with the sequence of other reported isolates. A Mr 41 000 fusion protein was induced by IPTG and was purified by chromatography. Conclusion The ALD gene of P. falciparum FCC1/HN strain was identical to the other reported isolates. ALD fusion protein of P. falciparum was expressed and purified.


Modulation of Immune Response to Plasmodium falciparum Apical Membrane Antigen 1 DNA Vaccine by Cytokine Plasmids

LIXun;MIAOJun;LEIJun-chuan;XUECai-fang;WANGXian-feng;LIUZhong-xiang;LIShu-mei

2004, 22(3):  3-143. 

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　Objective To explore the effect of cytokine encoding plasmids on DNA immunization in mice. Methods Prototype DNA vaccine VR1020/E which contain Plasmodium falciparum apical membrane antigen 1 (AMA1) ectodomain was constructed, and eukaryotic expression vectors pcDNA3/GM-CSF, pcDNA3.1(-)/IL-4, pIL-12 and pGM-CSF/pTPA-E were also built. BALB/c mice were immunized with VR1020/E alone or with VR1020/E plus different cytokine plasmids. Serum IgG and its subtype were determined by ELISA and in vitro splenocyte proliferation assay was done. Results GM-CSF, IL-4 and IL-12 encoding plasmids all promoted mice immune response to VR1020/E, the antibody level increased 7 to 10 times and splenocyte proliferation was enhanced too. Plasmid pcDNA3/GM-CSF induced much more IgG1 whereas plasmid pIL-12 induced much more IgG2a. Conclusion Cytokine encoding plasmids might be used as adjuvant in AMA1 DNA immunization.


Construction of the Subtracted cDNA Libraries Related to Artemisinin-resistance of Plasmodium berghei

BEIZhu-chun;WANGJing-yan

2004, 22(3):  4-143. 

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　Objective To construct the subtracted cDNA libraries related to artemisinin-resistance of Plasmodium berghei using suppression subtractive hybridization PCR(SSH PCR). Methods Total RNA was extracted from the artemisinin-sensitive(NS)and artemisinin-resistant(AR)strains of Plasmodium berghei K173. The cDNA synthesis followed the protocol of super SMART cDNA synthesis kit. Taking the NS as driver, AR as tester and reverse,two subtractions were performed by SSH PCR. Enriched different expressed cDNA was cloned into pMD18-T vector to construct subtractive libraries. Results The subtracted cDNA libraries of NS-AR and AR-NS contained 395 and 506 positive clones respectively. The PCR results of 108 clones picked randomly from each library showed 100 and 104 positive inserts contained in the plasmids respectively, and distributing in 250-2 000 bp. Conclusion The successful construction of the subtracted cDNA libraries related to artemisinin-resistance of P. berghei enable us to identify the different expressed genes involved in the resistance mechanism.


The Application of Remote Sensing Data in Epidemic Situation Analysis of Malaria and Encephalitis B

YUGuo-wei;TANGLin-hua;ZENGGuang;TANGYin;MEIJia-mo

2004, 22(3):  5-147. 

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　Objective To explore the epidemic regularity of malaria and encephalitis B by using the data of remote sensing (Rs) in flood area. Methods The demarcation standards in counties with flood disaster were formed depending on the descriptive analysis results of remote sensing data and combined with that of field survey. Three indicators were used to analyze the epidemic situation of malaria and encephalitis B in Jiangxi Province during a heavy flooding in 1998: the increasing percentage of incidence in 1998 comparing with the median of past five years (1993-1997), the increasing percentage of incidence in 1998 comparing with that of 1997, the increasing percentage of incidence in 1999 comparing with that of 1998. Results The demarcation standards of flooding counties were defined as follows: by Rs, a county with a flood area of over 100 thousand mu was classified into group one, a county with a flood area under 100 thousand mu was classified into group two, a county with reported flood but not identified by RS was classified into group three, the other counties in the province were classified into group four. The malaria incidence in the province in 1998 was at an average historical level. Compared with 1997, malaria incidence in each group increased in 1998 by 111.61% in group one, 97.50% in group two, 43.63% in group three. So there is an evident correlation between the flood area by Rs and the increasing of malaria incidence(Rs=0.893,P<0.05). Malaria incidence in 1999 in non-flood area increased by 83.39% in comparison with that of 1998. The encephalitis B incidence increased by 252.03% in 1998 in group four compared with that of 1997; while the incidence increased in all the four groups in 1999 than that of in 1998. Conclusion The remote sensing data on flood can help fully analyze the epidemic situation of malaria and encephalitis B.


Effect of Artemether on Schistosoma mansoni: Dose-Efficacy Relationship, and Changes in Worm Morphology and Histopathology

XIAOShu-hua*;GUOJian;JacquesChollet;WUJia-tong;MarcelTanner;JurgUtzinger;

2004, 22(3):  6-153. 

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　Objective To investigate the effects of artemether on Schistosoma mansoni harboured in mice, with particular consideration on single dose-efficacy relationship, hepatic shift and artemether-induced alterations in worm morphology and histopathology. Methods Groups of mice, infected with 21d old S.mansoni, were treated with artemether at single oral doses of 12.5mg/kg to 600mg/kg. Worm burden reduction was assessed 28d post-treatment. The hepatic shift was investigated in mice infected with 46d old S. mansoni and treated with artemether at a single oral dose of 400 mg/kg within a period of 14d post-treatment. Morphological and histopathological observations were made in adult worms in mice, subject to single oral dose of artemether at 400mg/kg. Results The minimum effective dose of oral artemether against juvenile worms in mice was 200 mg/kg, resulting in a worm burden reduction of 81%. The hepatic shift commenced 8 h post-treatment, and all worms shifted to the liver 3-7d post-treatment. Fourteen days post-treatment, 31% of the worms returned to the mesenteric veins. Treatment with artemether resulted in decreased worm body size, expansion of the pharynx and dilation of the gut with marked reduction in pigment. Focal tegumental damage was observed among female worms with adherence of host leukocytes and degeneration of ovary and vitelline glands, as well as atrophy of testis in male worms. Artemether-damaged worms were surrounded and infiltrated by eosinophils. Conclusion The minimum effective dosage of artemether against 21d old S.mansoni in mice is 200mg/kg. Artemether also exhibits effect against adult schistosomes, including shrinkage and degeneration, and can lead to worm death. The predominant inflammatory cell surrounded and infiltrated into the artemether-damaged worm is eosinophil.


Studies on Smads at Transcription Level in Liver Fibrosis of Mice with Schistosomiasis japonica

ZHANGBin-bin;JIAOYang-wen;CAIWei-min;TAOJun;ZHENGMin;DONGFeng-qin;LIURong-hua

2004, 22(3):  7-156. 

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　Objective To study Smads involved in TGF-β signal transduction at the transcription level during the development of liver fibrosis in BALB/c mice infected by Schistosoma japonicum. Methods BALB/c mice infected with cercariae of S. japonicum were used as liver fibrosis models. Liver specimens were harvested at 8,12,16 and 24 weeks after infection and normal control were sacrificed at the 24th week. A part of the liver specimens were preserved for pathologic examination and the other part was frozen for the detection of mRNA level of Smad 2,Smad 3,Smad 4 and Smad 7. Results The level of Smad 3 mRNA was significantly higher than that of control at the later stage, while the mRNA level of Smad 2 decreased at 12 and at 24 weeks,respectively. No significant difference in the mRNA level of Smad 4 and Smad 7 was observed between the infection group and the control. Conclusion Smad 3 may induce the development of liver fibrosis in mice infected by S. japonicum while Smad 2 may induce the development of liver fibrosis at early stage and inhibit it at later stage.


Expression of Inducible Nitric Oxide Synthase in the Livers of Mice Infected with Schistosoma japonicum

LONGXiao-chun;LIYong-long*;AndreasRuppel

2004, 22(3):  8-159. 

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　Objective To study the expression of inducible nitric oxide synthase (iNOS) in livers of mice infected with Schistosoma japonicum. Methods The livers of NMRI mice infected with S. japonicum were collected on day 21,28,38,45 post infection(p.i.), total RNA of livers were extracted and kinetics of the mRNA expression of iNOS were detected by RT-PCR, the protein expression of iNOS was then confirmed by Western blotting and the distribution of iNOS in the infected liver was determined by immunohistochemical methods. Results The mRNA expression of iNOS was not detectable in the uninfected liver, iNOS mRNA expression was detected on day 21 p.i, the expression increased on day 28 p.i and peaked on day 38 p.i, then decreased slightly on day 45 p.i. Western blotting showed an iNOS expression in the livers only on day 38, 45 p.i. IFA test showed that the expression of iNOS was maily distributed in the granuloma of the livers. Conclusion S. japonicum infection can induce the expression of iNOS in a time-dependent manner in the liver of the host,and eggs may be the main factor in inducing the expression.


Screening of cDNA Clone for Putative RNA Polymerase Subunit of Cysticercus cellulosae

LUOXue-nong;ZHENGYa-dongDOUYong-xi;HOUJun-lin;JINGZhi-zhong.CAIXue-peng

2004, 22(3):  9-163. 

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　Objective To obtain related genes of Cysticercus cellulosae from spliced leader (SL) cDNA library. Methods Spliced leader library of Cysticercus cellulosae was constructed using SL specific primer and oligo (dT)15 with M13M4 primer, and positive clones were then screened randomly, identified with enzyme restriction, followed by sequencing and homologous analysis. Results The amino acid sequence, encoded by the positive clone with a poly (A) 22 tail and a complete open reading frame (ORF), was with homology of RNA polymerase subunit genes of human, B. napus, fission yeast, A. thaliana, C. elegans and fruit fly up to 71.6%. Conclusion The protein, RNA polymerase subunit encoded putatively by the clone, is high conservative in different species.


Additive Therapeutic Effect of a Combination of Artemether and Daphnetin against Plasmodium berghei in Mice

GUOJian;NIYi-chang;WUJia-tong;WANGQin-mei

2004, 22(3):  10-166. 

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　Objective To investigate the therapeutic effect of a combination of artemether and daphnetin against Plasmodium berghei ANKA strain in mice. Methods Groups of P. berghei infected mice were treated with various oral doses of artemether and daphnetin according to “4-day suppress assay”. Thin blood smears were made on the fifth day after inoculation of parasites and the parasitemia reduction rate was calculated. The ED 50 values obtained were plotted on isobolograms. A combined action of artemether and daphnetin was assessed. Results Artemether 0.4mg/(kg·d)×4d exhibited no detectable antimalarial effect, while artemether 0.4mg/(kg·d)×4d combined with daphnetin 7.7 mg/kg.d×4d showed potent antiparasile efficacy. The ED 50s of artemether in combination with daphnetin were lower than that of single artemether or daphnetin. The R-values were higher than 0.4, but lower than 2.7. Conclusion The combination of artemether with daphnetin showed an additive antiparasile effect.


Amplification and Sequencing of the Gene Coding for Mucin-like Protein from Schistosoma japonicum

LIUYan;XIAOJian-hua;LIAOLi;ZENGGu-qing;ZHANGYu-kuai;YANGSheng;LIANGYu

2004, 22(3):  11-169. 

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　Objective To amplify and sequence the partial gene coding for mucin-like protein from Chinese isolates of Schistosoma japonicum (SjMLP). Methods The antigenic determinants of SmMLP were predicted by PCGENE software and specific oligonucleotide primers were designed and synthesized. Total RNA was isolated from adult worms of S.japonicum using Trizol reagent and the coding region gene of SjMLP was amplified by RT-PCR technique. Results The coding region of SjMLP gene was specifically amplified by RT-PCR and the size of amplified fragment was 756 base pairs. The DNA sequence analysis result indicated that the coding sequence of the MLP was highly homologous between S. mansoni and S.japonicum. Conclusion The amplified fragment is consistent to the predicted one, providing a basis for cloning and further study on DNA immunization.


Prediction of the Spatial Distribution of Oncomelania Snails in Marshland of Jiangning County using the Ordinary Kriging

ZHANGZhi-ying;XUDe-zhong;PENGHua;ZHOUYun;ZHANGBo;LIUShi-jun;ZHOUXiao-nong;GONGZi-li

2004, 22(3):  12-172. 

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　Objective To explore the methods to predict the distribution of Oncomelania hupensis in the marshland of Jiangning County. Methods Semi-variogram was used to analyze the spatial autocorrelation of snails’distribution in the marshland of Jiangning using the Arcview8.1. A prediction map for the snails’distribution was established using the Ordinary Kriging and evaluated using the cross-validation. Results Analysis showed that the distribution of alive snails in the marshland of Jiangning in the year 2000 was auto-correlated in spatial. The semi-variogram model which was spherical demonstrated that the variation of alive snails in spatial were related with distance apart when the distance was less than 0.0301. The prediction map of the snail distribution in the marshland of Jiangning was established based on the semi-variogram using the Ordinary Kriging. The cross-validation showed that the prediction map could estimate the distribution of snails in the marshland of Jiangning correctly. And the determinant coefficient for the prediction model was 0.973. Conclusion It is feasible to predict the snail distribution in the marshland of Jiangning County by using Ordinary Kringing and data from the surveillance spot.


实验报道

Cloning, Sequencing and Subcloning of cDNA Coding for GroupⅠAllergen of Dermatophagoides farinae

YANGQing-gui;LIChao-pin

2004, 22(3):  13-175. 

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　Objective To clone,sequence and subclone the cDNA coding for group 1 allergen of Dermatophagoides farinae(Derf1). Methods The cDNA of Derf1 was amplified by RT-PCR and PCR. After purified, the gene fragment was cloned into a vector pMD-18T. The recombinant plasmid pMD-18T-Derf1 was transformed into E.coli JM109. Positive clones were screened and identified by PCR and digestion with restriction enzyme. The sequence of inserted Def1 gene fragment was also detected. Derf1 was then subcloned into the vector of pET-32a(+). Results The Derf1 gene fragment of Dermatophagoides farinae was specifically amplified from RNA by RT-PCR and PCR. The recombinant plasmid pMD-18T-Derf1 and pET-32a(+)-Derf1 was constructed and digested by Bam HⅠand SacⅠ, the size of gene fragment was 646 bp and in accordance with the expected one. Conclusion The pET-32a(+)-Def1 subcloning has been constructed successfully.