Table of Content

30 June 2005, Volume 23 Issue 3

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论著

Expression of Truncated Toxoplasma gondii GRA8in the Prokaryotic Expression Plasmids

YUANShi-shan*;WUShao-ting;ZHANGRen-li;GAOShi-tong;HUANGDa-na;YUXin-bing

2005, 23(3):  1-134. 

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Objective To construct the prokaryotic recombinant expression plasmids of Toxoplasma gondii GRA8 and analyze their expression in E. coli containing the prokaryotic recombinant plasmids. Methods The full gene and its truncated fragment of GRA8 were amplified by PCR from T. gondii genomic DNA, and cloned into pMD18-T vector. The right genes in positive clones sequenced with ABI PRISMTM 377XL DNA Sequencer were digested with restrictive endonucleases and subcloned into pGEX-4T-2. The recombinant plasmids were transformed into E. coli JM109. The recombinant clones were characterized by PCR and digested with restriction endonucleases. Positive clones were induced with IPTG to express target protein and characterized by SDS-PAGE. The recombinant protein was purified from E. coli lysate by affinity chromatography and SDS-PAGE. The immunological activity of this protein was analyzed by Western blotting. Results The gene fragments encoding GRA8 were amplified by PCR from T. gondii genomic DNA. The inserts of GRA8 in positive clones were coincident with the original sequence of GRA8 gene from GenBank. The recombinant expression plasmids were contructed through subcloning the right inserts of GRA8 into pGEX-4T-2. The expression level of GRA8 from the recombinant expression plasmids in E. coli was low, and there was almost no full-length GRA8 expressed in E. coli. The purified protein reacted with the sera from rabbits infected with T. gondii RH strain. Conclusion The expression level of GRA8 from the recombinant expression plasmids in E. coli is low and the purified truncated GRA8 shows certain antigenicity.

The Point Mutations in Pfcrt and Pfmdr1 Genes in Plasmodium falciparum Isolated from Hainan Province

GUANYa-yi;TANGLin-hua;HULing;FENGXiao-ping;LIUDe-quan

2005, 23(3):  2-139. 

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Objective To evaluate the point mutations in Pfcrt and Pfmdr1 genes in Plasmodium falciparum isolated from Hainan Province. Methods Nested polymerase chain reaction and restriction fragment length polymorphism were used to detect the point mutations at codon 76 of Pfcrt and at codon 86, 1246 of Pfmdr1 in P. falciparum isolates. Chloroquine resistance was measured by the in vitro microtest recommended by WHO. Results In 36 samples tested， 28 were successfully amplified for Pfcrt, 64.3% of them carried mutant allele at codon 76, 21.4% with wild allele K76 and 14.3% with mixed allele mutation. While for Pfmdr1, 3.4% isolates displayed the 86Y mutation, 89.7% with wild allele N86 and 6.9% with the mixed alleles in 29 isolates which were amplified successfully for N86Y. No point mutation in Pfmdr1 at codon 1246 was found in 13 isolates from the total 36 samples. By the in vitro test, 72.2% (26/36) showed resistance to chloroquine. The 76T and 86Y mutant alleles were present in both in vitro susceptible and resistant isolates. There was a significant difference between susceptible and resistant isolates carrying 76T mutant codon（P<0.05）， but no significant difference was found in Pfmdr1（P＞0.05）. Conclusion There is a significant difference of the 76T prevalence in Pfcrt gene between the susceptible isolate and resistant one of P. falciparum to chloroquine in vitro. The Pfcrt 76T may be used as a predictive marker for chloroquine resistance surveillance.

Tag Primer-Nested / Multiplex PCR for Detectionof Plasmodium falciparum and Plasmodium vivax

HUANGTian-yi;WANGShi-hai;LIXue-ming;GUOChuan-kun;XUJian-jun;TANGLi-na;LULi-dan

2005, 23(3):  3-142. 

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Objective To establish a sensitive, simple to use and low noise nested/multiplex PCR for simultaneously detection of Plasmodium falciparum （P.f） and Plasmodium vivax （P.v）. Methods The tag primer amplification technique, software Primer Premier 5.0, NCBI-BLAST web resources and the matrix test were used to optimize the nested / multiplex PCR for detection of P.f and P.v with filter paper blood samples taken from malaria patients diagnosed by microscopy, and the results of the optimized nested/multiplex PCR and microscopy were evaluated. Results The sensitivity of the optimized PCR, determined by the examination of imitative filter paper blood samples, was about 1-2 parasites / μl for P.f and 5-10 parasites / μl for P.v. Primer-dimer and other PCR noise were removed. When 71 field filter paper blood samples taken from microscopically diagnosed patients (24 P.f, 47 P.v) were examined, the concordance between the optimized PCR and microscopy was 87.5% for P.f and 100% for P.v. Conclusion The nested/multiplex PCR optimized by tag primer amplification technique is simple, with low noise and being able to detect P.f and P.v simultaneously. It is more sensitive in detecting cases with low parasitaemia and more accurate in identifying Plasmodium species than microscopy.

Detection of Anti-Trichinella spiralis Antibody by Indirect ELISA Using Recombinant Protein as Antigen

NIUTing-xian;PANFei-Fei;LIUMing-yuan;LUQiang;FUBao-quan;PascalBoireau

2005, 23(3):  4-145. 

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Objective To study the specificity, sensitivity of diagnostic antigen for Trichinella spiralis（T. s）. Methods T668 recombinant protein from highly efficient expression of newborn larvae stage-specific gene of T. s in E. coli as diagnostic antigen. By using negative and positive sera from rabbit, pig, human as the first antibody, and goat-anti-rabbit IgG, goat-anti-pig IgG, goat-anti-human IgG labeled with HRP as the secondary antibody, indirect ELISA method was established for detecting anti-T.s antibody, while excretory-secretory (ES) antigen of T.s muscle larvae was used as control. Results Sera from rabbit, pig and human were detected by T668 recombinant protein as antigen, which showed a positive rate of 100% with 0.016 μg/well. There was no difference on the results between the T668 recombinant antigen and the ES antigen for diagnosing T. s-infection. Conclusion The T668 recombinant antigen is promising in substituting ES antigen in the detection of anti-T. s antibody.

Comparative Study on the Resting Habit of Anopheles minimus Aand Anopheles minimus C in Yunnan Province

ZHENGBin;TANGLin-hua;MAYa-jun;SHIWen-qi;ZHOUShui-sen;WANGXue-zhong

2005, 23(3):  5-149. 

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Objective To compare the difference of resting habitat between Anopheles minimus A and An. minimus C in Yunnan Province. Methods A village was selected in Mengla and Yuanjiang County respectively to collect mosquitoes by hanging ultraviolet lamps inside the cattle pen and human dwellings. The samples were identified by multiplex-PCR after morphological examination. Suspicious An. minimus A/C hybrid samples were identified by PCR-ASA, PCR-RFLP and D3 region sequence. Results In human dwellings, the proportion of An. minimus A and C was 21.35% and 78.65%, and in cattle pen, 28.54% and 71.46% respectively. As for the An. minimus A/C hybrid, the result of PCR-ASA and PCR-RFLP displayed the pattern of both An. minimus A and An. minimus C (PCR-ASA: 376、 294 and 112 bp, PCR-RFLP:108、 268 and 376 bp), the D3 sequence displayed heterozygosis in the variation place of An. minimus A and An. minimus C, which means that the hybrid possesses the signal of both An. minimus A and An. minimus C. Conclusion There is no resting habit difference between An. minimus A and An. minimus C in Yunnan Province. In Mengla County, 5 An. minimus A/C hybrids have been found for the first time.

The Adjuvant Effect of IL-12 on Protective Immunity of Schistosoma japonicum Fatty Acid Binding Protein (Sj14FABP)

ZHUXiao-hua;SHIYou-en;NINGChang-xiu;ZHUHong-gang

2005, 23(3):  6-154. 

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Objective To study the immune protection of Schistosoma japonicum fatty acid binding protein （Sj14FABP） DNA vaccine enhanced by IL-12. Methods The recombinant plasmids pVIVO2-Sj14FABP and pVIVO2-IL12-Sj14FABP were constructed respectively, and were prepared on a large scale after identification. 48 male BALB/c mice were divided into 4 groups randomly. In group A, B, C, and D, each mouse was injected intramuscularly with 100　μl 0.9% NaCl, 100μg pVIVO2, 100μg pVIVO2-Sj14FABP and 100　μg pVIVO2-IL12-Sj14FABP respectively. 30 days after immunization each mouse was challenged with 40±2 cercariae of S. japonicum. On day 45 after challenge, all mice were sacrificed to count the number of recovered adult worms and the hepatic eggs. Sera from mice were used to detect IgG antibody. The production of IL-2, IL-4 and IFN-γ in the supernatant of spleen cells was observed by means of sandwich ABC-ELISA. Results The recombinant plasmids pVIVO2-Sj14FABP and pVIVO2-IL12-Sj14FABP were constructed. The worm reduction rate in group C and D was 24.11% and 39.4%, as well as liver egg reduction rate of 27.2% and 32.8% respectively. The level of IL-2 and IFN-γ in group D increased significantly, while IL-4 secretion decreased(P<0.01). 30 days after immunization, no higher titer of IgG antibody was shown in all groups. Furthermore, no significant difference on the level of IgG was found among the groups (P>0.05). Conclusion Sj14FABP DNA vaccine induces partial protective immunity in BALB/c mice. IL-12 drives the immune response toward a Th1 direction, and enhances the protective immune effect of the vaccine.

Fusion Expression and Antigenicity Analysis of MiracidialAntigen from Eggs of Schistosoma japonicum

LIUJian-fa;YUChuan-xin;ZHUYin-chang;YINXu-ren;XUYong-liang

2005, 23(3):  7-158. 

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Objectives To express the miracidial antigen from eggs of Schistosoma japonicum (Chinese mainland strain) (SjMP10)， and investigate the role of the miracidial antigen during the hepatic granuloma formation of schistosomiasis. Methods A pair of specific primers was designed and synthesized according to the nucleotide sequence of the open reading frame of the miracidial antigen gene. The open reading frame of the miracidial antigen gene was amplified, digested by restrictive enzyme(BamHI, SalI)， and cloned directly into the expression plasmid pGEX-4T-3 to construct the recombinant plasmid. The recombinant plasmids were transformed into E. coli BL21(DE3), and induced by IPTG to express the fusion protein of GST-SjMP10. The expressed fusion protein was purified by electric elution method, and its antigenicity was examined by Western blotting and lymphocyte proliferation test. Results The gene of miracidial antigen was cloned into the expression plasmid pGEX-4T-3. After induced by IPTG, the recombinant expressed a fusion protein of GST-SjMP10, with a molecular weight of 39 000 approximately. The purified fusion protein showed proper antigenicity that could be recognized by the sera of rabbits heavily infected by Schistosoma japonicum and could stimulate the proliferation of splenic lymphocytes of infected BALB/c mice. Conclusion The miracidial antigen from eggs of Schistosoma japonicum was expressed successfully.

Oral Mixed DNA Vaccine Protects Mice from Infection ofToxoplasma gondii

CONGHua;GUQin-min;ZHOUHuai-yu;GUOLan;YANGTing-ting;HEShen-yi;LIYing;ZHAOQun-li

2005, 23(3):  8-162. 

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Objective To examine the immune response in BALB/c mice induced by oral mixed Toxoplasma gondii DNA vaccine delivered by live attenuated Salmonella typhimurium. Methods Gene fragments SAG1 and SAG2 were amplified from the genomic DNA of T. gondii RH strain by PCR and were subcloned into pcDNA3.1(±) eukaryotic expression vector. The positive recombinant plasmid was transformed into aroA- and aroD-attenuated Salmonella typhimurium BRD509 (BRD509/pSAG1/SAG2). After screened by PCR, restrictive enzyme digestion and sequencing, recombinant Salmonella strain was used to immunize BALB/c mice by i.g. route, three times at 2-week interval. Humoral and cellular responses were assayed using ELISA for determining antibody, IFN-γ and IL-4. MTT assay was used to detect the proliferation activity of T lymphocytes and the activity of NK killers. FCM was also used to sort the lymphocyte subsets of spleen cells. All mice were then infected with highly virulent RH tachyzoites of T. gondii intraperitoneally. Results Significant increase of specific IgG level was observed in immunized mice with a titer of 1∶100. Proliferation activity of specific NK cells and T lymphocytes was highly enhanced in BRD509/ pSAG1/SAG2 vaccinated mice： the killing activity of NK cells was 85%±7%， the proliferation SI of T lymphocytes was 2.83， which resulted in a 5 days longer survival time than mice in control group after challenge infection. Conclusion The oral mixed DNA vaccine delivered by attenuated Salmonella typhimurium shows an immunoprotection against T. gondii in mice.

Impact of Chronic Schistosomiasis japonica on the Protective Immunity Induced by Vaccine Against Hepatitis B Virus

SONGWen-jian;CHENGYu-li;LIULa-zhen;KONGZheng;HUSong;LIUKai;LINLi;LIUCun-xi

2005, 23(3):  9-165. 

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Objective To study the impact of chronic schistosomiasis on the protective immunity induced by vaccine against hepatitis B virus. Methods 24 patients with chronic or advanced schistosomiasis (experimental group) and 26 healthy volunteers (control group) all without hepatitis B virus infection were selected for the study. Sera of the subjects in the two groups were collected before inoculation and on the 35th day after inoculation with yeast-derived recombinant hepatitis B vaccine. The level of anti-Hbs, IL-2 and TNF-α in sera was examined by ELISA respectively. Results Anti-Hbs in both groups were negative before inoculation, with an average absorbance （A value） of 0.134 and 0.150 respectively. After inoculation, positive rate of anti-Hbs was 17% (4/24, average A value 0.145 ) in experimental group and 92% (24/26, average A value was 1.210) in control group. The vaccine against hepatitis B induced significantly higher level of anti-Hbs in healthy volunteers compared with that in schistosomiasis patients (P < 0.01). The level of IL-2 and TNF-α increased in both groups after inoculation without significant difference compared with the level before inoculation. Conclusion The results suggest that the protective immunity of patients with chronic schistosomiasis is deficient to the stimulation of hepatitis B virus and it may involve in a higher incidence of hepatitis B among schistosomiasis patients.

Study on the Relationship of Immune Status with Severity ofSchistosomiasis japonica in Fishermen in the Dongting Lake

HOUJian-wei;ZENGQing-ren;HEYong-kang;LUOXin-song;SIMAYan-xiang;HuangZhi-hui;ZHANGShun-ke

2005, 23(3):  10-170. 

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Objective To investigate the relationship of the immune status and the intensity of infection or the severity of the hepatosplenic pathology among fishermen with schistosomiasis japonica in the Dongting Lake region. Methods Inquiring and physical examination (IPE), stool examination, B-ultrasonography of the liver and spleen, flow cytometry, turbidimetry and ELISA were undertaken to acquire or determine the intensity of infection (EPG in stool), pathological change in the liver and spleen and the level of cellular and humoral immunity. Data were analyzed with SPSS 10.0 statistics software. Results Compared with subjects from non-endemic area, the CD4+ T cells and the CD4+/CD8+ ratio in fishing population in the endemic area significantly decreased. The decrease of the CD4+/CD8+ ratio was more significant among population with positive stool exam and with the increase of EPG and/or severity of pathological change in the liver and spleen. Contrarily, the level of the total IgM and the anti-SEA IgG in serum from fishing population in the endemic area was significantly higher than those from non-endemic area. High level serum antibodies in those stool positives were remarkable with the increase of EPG and/or the severity of hepatosplenic pathological change. The total IgA increased considerably in the subjects with significant pathological change of the liver and spleen. A high total IgG was only detected in those stool positives. Conclusion The immune status in fishermen with schistosomiasis in the Dongting Lake showed a suppressed cellular immunity and a hyper functioning humoral immune response. The imbalance of the immunity was related to the increase of the intensity of infection and the progress of the hepatosplenic pathology．

实验报道

Expression of Taenia solium Oncosphere 45WB2 Gene in Pichia pastoris System

WANGJin;LUOXue-nong;YUECheng;HUZhi-min;CAIXue-peng

2005, 23(3):  11-174. 

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Objective To amplify and express the gene from Taenia solium oncosphere in Pichia pastoris system. Methods Total RNA was extracted from hatched and activated oncospheres. A 459bp specific fragment was amplified by RT-PCR. It was subcloned into the secreted expression vector pPIC9K, and identified by sequencing and then transformed into P. pastoris SMD1168 by electroporation. PCR analysis showed that 45WB2 was integrated into the genomic DNA of yeast. The recombinants were induced by 1% methanol and the culture supernatant was collected and tested by SDS-PAGE and Western blotting. Results 45WB₂ gene was expressed successfully in P. pastoris and the product was recognized by human positive serum against cysticercosis. Conclusion The recombinant 45WB2 expressed in P. pastoris may be used to prevent pigs from the infection of Taenia solium.

Measurement of Morphological Parameters of Cryptosporidium murisOocysts by Digital Image Processing Technology

BINGYu-yan;LIYan;ANChun-li

2005, 23(3):  12-177. 

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Objective To establish and analyze the morphological parameters of the oocysts of Cryptosporidium muris for defining their morphological change. Methods Oocysts were collected from KM mice（immunodepressed by dexamethasone for 10 days） and examined with modified acid-fast staining. Images of 1 190 oocysts were acquired by photograph system. The length, width, perimeter, area and equivalent diameter of the oocysts were obtained by computer digital image processing system and analyzed by SPSS software (Version 11.0). Result The average length of the oocysts was 5.93 μm， ranging from 3.36 μm to 8.51 μm in 95% confidence interval of them. The average width was 4.96 μm, ranging from 3.26 μm to 6.66 μm in 95% confidence interval of them. The average perimeter was 18.03 μm and the average area was 16.08 μm2. Conclusion Data obtained from the computer system are objective and precise, offering scientific foundation for measuring the oocysts and for identifying Cryptosporidium spp.

简报

张鸣青;王爱民;张荔群;杨青平

2005, 23(3):  17-158. 

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