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Polymorphisms of the oxidant enzymes glutathione S-transferase and glutathione reductase and their association with resistance of Plasmodium falciparum isolates to antimalarial drugs 被引量:3
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作者 Raewadee Wisedpanichkij Wanna Chaicharoenkul +2 位作者 Poonuch Mahamad Prapichaya Prompradit Kesara Na-Bangchang 《Asian Pacific Journal of Tropical Medicine》 SCIE CAS 2010年第9期673-677,共5页
Objective:To investigate the association between amplification of the two regulatory genes controlling glutathione(GSH) levels,glutathione reductase(PfGR) and glutathione S-transferase (PfGST) genes and sensitivity of... Objective:To investigate the association between amplification of the two regulatory genes controlling glutathione(GSH) levels,glutathione reductase(PfGR) and glutathione S-transferase (PfGST) genes and sensitivity of Plasmodium falciparum(P.falciparum) isolates collected from different malaria endemic areas of Thailand to standard antimalarial drugs.Methods:A total of 70 P.falciparum isolates were collected from endemic areas of multi-drug resistance (Tak,Chantaburi and Ranong Provinces) during the year 2008-2009.The in vitro assessment of antimalarial activity of P.falciparum clones(K1- and Dd2 chloroquine resistant and 3D7- chloroquine sensitive) and isolates to chloroquine,quinine,mefloquine and arteusnate was performed based on SYBR Green modified assay.Results:68(97.14%),11(15.71%) and 28(40%) isolates respectively were classified as chloroquine-,quinine- and mefloquine-resistant isolates. With this limited number of P.falciparum isolates included in the analysis,no significant association between amplification of PfGST gene and sensitivity of the parasite to chloroquine, quinine,mefloquine and quinine was found.Based on PCR analysis,Dd2,Kl and 3D7 clones all contained only one copy of the PfGST gene.All isolates(70) also carried only one copy number of PfGST gene.There appears to be an association between amplification of PfGR gene and chloroquine resistance.The 3D7 and Dd2 clones were found to carry only one PfGR gene copy, whereas the K1 clone carried two gene copies.Conclusions:Chloroquine resistance is likely to be a consequence of multi-factors and enzymes in the GSH system may be partly involved. Larger number of parasite isolates are required to increase power of the hypothesis testing in order to confirm the involvement of both genes as well as other genes implicated in glutathione metabolism in conferring chloroquine resistance. 展开更多
关键词 plasmodium falciparum Drug RESISTANCE GLUTATHIONE reductase(PfGR) GLUTATHIONE S-transferase(P/GST)
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Bioinformatics analysis for structure and function of CPR of Plasmodium falciparum 被引量:3
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作者 Zhigang Fan Lingmin Zhang +4 位作者 Guogang Yan Qiang Wu Xiufeng Gan Saifeng Zhong Guifen Lin 《Asian Pacific Journal of Tropical Medicine》 SCIE CAS 2011年第2期85-87,共3页
Objective:To analyse the structure and function of NADPH-cytochrome p450 reductase(CYPOR or CPR) from Plasmodium falciparum(Pf),and to predict its’ drug target and vaccine target. Methods:The structure,function,drug ... Objective:To analyse the structure and function of NADPH-cytochrome p450 reductase(CYPOR or CPR) from Plasmodium falciparum(Pf),and to predict its’ drug target and vaccine target. Methods:The structure,function,drug target and vaccine target of CPR from Plasmodium falciparum were analyzed and predicted by bioinformatics methods.Results:PfCPR,which was older CPR,had close relationship with the CPR from other Plasmodium species,but it was distant from its hosts,such as Homo sapiens and Anopheles.PfCPR was located in the cellular nucleus of Plasmodium falciparum.335aa-352aa and 591aa - 608aa were inserted the interior side of the nuclear membrane,while 151aa-265aa was located in the nucleolus organizer regions.PfCPR had 40 function sites and 44 protein-protein binding sites in amino acid sequence.The teriary structure of laa-700aa was forcep-shaped with wings.15 segments of PfCPR had no homology with Homo sapien CPR and most were exposed on the surface of the protein.These segments had 25 protein-protein binding sites.While 13 other segments all possessed function sites. Conclusions:The evolution or genesis of Plasmodium falciparum is earlier than those of Homo sapiens.PfCPR is a possible resistance site of antimalarial drug and may involve immune evasion, which is associated with parasite of sporozoite in hepatocytes.PfCPR is unsuitable as vaccine target,but it has at least 13 ideal drug targets. 展开更多
关键词 plasmodium falciparum NADPH-cytochrome p450 reductase Origin Immune EVASION Drug TARGET Vaccine TARGET
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Subcloning and expression of two conserved regions (Ⅰ,Ⅴ) onP190 antigen of Plasmodium falciparum in E.coli
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作者 潘卫庆 杨树桐 +1 位作者 邓海琳 陆德如 《Journal of Medical Colleges of PLA(China)》 CAS 1994年第1期36-39,共4页
Two DNA fragments, designated as P190CRI (AA1-55) and P190CRV (AA1597-1667)respectively, which encode amino acid residues of conserved region Ⅰ and Ⅴ on the P190 antigen were amplified by polymerase chain reaction f... Two DNA fragments, designated as P190CRI (AA1-55) and P190CRV (AA1597-1667)respectively, which encode amino acid residues of conserved region Ⅰ and Ⅴ on the P190 antigen were amplified by polymerase chain reaction from genomic DNA in FCCl/HN strain of Plasmodium falciparum isolated from Hainan Province, China. It was found that there were five base substitutions in the P190CR V, in comparison with the nucleotide sequences of MAD20 strain. These two fragments sequenced were inserted into pGEX-2T plasmid. E. coli JM109 (DE3) were transformed with the recombinant plasmids and the parental plasmid. The results showed that the two fragments were expressed as high-level C-terminal fusions with glutathione S-transferase (GST). The fusion proteins were easily purified from bacterial lysates by affinity chromatography using glutathione Sepharose 4B. 展开更多
关键词 P190 ANTIGEN plasmodium falciparum malarial VACCINE genetic engineering
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中缅边境恶性疟原虫Pfcrt基因76位点突变研究 被引量:2
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作者 邓艳 王珊珊 +1 位作者 董莹 王剑 《中国人兽共患病学报》 CAS CSCD 北大核心 2014年第11期1137-1140,共4页
目的检测分析中缅边境恶性疟原虫氯喹抗性基因(pfcrt)及其76位点氨基酸的突变情况。方法采用巢式PCR方法扩增含76位点的pfcrt基因,并对扩增产物进行限制性内切酶及测序分析。结果对恶性疟现症病人血样作pfcrt基因的巢式PCR扩增,目的基... 目的检测分析中缅边境恶性疟原虫氯喹抗性基因(pfcrt)及其76位点氨基酸的突变情况。方法采用巢式PCR方法扩增含76位点的pfcrt基因,并对扩增产物进行限制性内切酶及测序分析。结果对恶性疟现症病人血样作pfcrt基因的巢式PCR扩增,目的基因片段(145bp)检出率为74.05%(117/158),对PCR扩增阳性的产物进行RELP分析,检查突变型酶切片段,突变率为95.73%(112/117)。结论恶性疟原虫pfcrt基因可以作为一个分子标记用于监测中缅边境地区恶性疟原虫的氯喹抗性。 展开更多
关键词 恶性疟原虫 氯喹抗性转运蛋白基因(pfcrt)突变型 抗性
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艾康恶性疟/间日疟免疫层析检测试剂盒效果评价 被引量:8
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作者 孙晓东 邓艳 +8 位作者 张再兴 冯舜 罗存富 苏红梅 陈忠明 汪红蕾 刘慧 汪丽波 徐万红 《中国热带医学》 CAS 2007年第1期28-29,共2页
目的评价艾康恶性疟/间日疟(P.f/P.v)胶体金免疫层析检测试剂盒诊断疟疾效果。方法以镜检法为金标准,采用盲法实验室测试疟疾阳性和阴性血样。结果共测试331例血样,其中疟疾血样121例。对疟疾的灵敏性和特异性分别为92.56%和98.57... 目的评价艾康恶性疟/间日疟(P.f/P.v)胶体金免疫层析检测试剂盒诊断疟疾效果。方法以镜检法为金标准,采用盲法实验室测试疟疾阳性和阴性血样。结果共测试331例血样,其中疟疾血样121例。对疟疾的灵敏性和特异性分别为92.56%和98.57%,与镜检符合率为96.38%;其中恶性疟的灵敏性和特异性分别为100.00%和98.57%,重复性为100.00%;对间日疟的灵敏性和特异性分别为86.96%和100.00%,恶性疟和间日疟之间无交叉反应。结论艾康P.f/P.v胶体金免疫层析检测试剂盒诊断疟疾具有快速、简便、准确、直观以及不需要特殊仪器优点,能同时诊断单纯恶性疟和单纯间日疟,但不能鉴别诊断混合感染。 展开更多
关键词 艾康(p.f/P.v)免疫层析检测试剂盒 诊断 疟痰 恶性疟 间日疟
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万孚疟疾快速诊断试剂盒效果评价 被引量:6
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作者 刘慧 康可人 +7 位作者 李春富 熊新灿 聂仁华 孙院红 李贵深 沈加员 孙晓东 王剑 《寄生虫病与感染性疾病》 CAS 2008年第4期224-225,共2页
目的评价万孚恶性疟快速检测试剂盒的检测效果。方法以镜检法为金标准,采用单盲法检测临床"四热"患者血样。结果使用万孚恶性疟快速检测试剂盒检测"四热"患者547例,检出疟疾阳性323例。敏感性和特异性分别为95.00%... 目的评价万孚恶性疟快速检测试剂盒的检测效果。方法以镜检法为金标准,采用单盲法检测临床"四热"患者血样。结果使用万孚恶性疟快速检测试剂盒检测"四热"患者547例,检出疟疾阳性323例。敏感性和特异性分别为95.00%和100.00%,与镜检符合率为96.89%。结论万孚恶性疟快速检测试剂盒敏感性和特异性高,质量稳定,适合于恶性疟流行区使用。 展开更多
关键词 恶性疟 万孚恶性疟快速检测试剂盒 诊断
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抗恶性疟SERA噬菌体抗体库的构建及单链抗体的筛选与鉴定(英文)
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作者 徐伟文 李文全 +2 位作者 李明 毕惠祥 Toshihiro Horii 《细胞与分子免疫学杂志》 CAS CSCD 北大核心 2002年第1期58-62,共5页
目的 研制用于疟疾简便、快速诊断试剂盒的试剂。方法 利用噬菌体抗体库技术,构建抗恶性疟原虫丝氨酸重复抗原(SERA)的噬菌体抗体库。经3轮“吸附-洗脱-扩增”的富集反应后,从中筛选抗SERA的阳性克隆株,并进行可溶性诱... 目的 研制用于疟疾简便、快速诊断试剂盒的试剂。方法 利用噬菌体抗体库技术,构建抗恶性疟原虫丝氨酸重复抗原(SERA)的噬菌体抗体库。经3轮“吸附-洗脱-扩增”的富集反应后,从中筛选抗SERA的阳性克隆株,并进行可溶性诱导表达,最后用ELISA和Western blot等进行鉴定。结果 成功地构建了中等库容的噬菌体抗体库,并从中筛选到9株抗SERA的阳性克隆株,表达的单链抗体的相对分子质量大小约为31kDa,能与 SE47'抗原起特异性结合反应。结论 成功地构建了抗SE47'的噬菌体抗体库,从中筛选制备的单链抗体为研制疟疾快速诊断试剂盒奠定了基础。 展开更多
关键词 噬菌体抗体库 单链抗体 丝氨酸重复抗原 SERA 恶性疟
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恶性疟原虫子孢子CS抗原融合基因的克隆
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作者 李杰之 马清钧 +2 位作者 曹诚 石成华 张京生 《生物化学杂志》 CSCD 1993年第4期429-433,共5页
利用381-A型DNA合成仪,参照恶性疟原虫子孢子CS抗原决定簇相应氨基酸的核苷酸序列,设计了CS-Ⅰ和CS-Ⅱ两个片段。以噬菌体M13mp18质粒作为载体,将两片段进行磷酸化、退火、连接和克隆,经过原位杂交,序列分析,获得了(NANP)_(10)重复串联... 利用381-A型DNA合成仪,参照恶性疟原虫子孢子CS抗原决定簇相应氨基酸的核苷酸序列,设计了CS-Ⅰ和CS-Ⅱ两个片段。以噬菌体M13mp18质粒作为载体,将两片段进行磷酸化、退火、连接和克隆,经过原位杂交,序列分析,获得了(NANP)_(10)重复串联基因的重组质粒M13mp18-CS。再以酶切,从重组质粒中回收(NANP)_(10)次的片段,将其片段基因与CT-B基因融合,最后将融合基因CT-B-CS插入载体PMC055中,成功地得到含有(NANP)_(10)与CT-B基因融合的重组质粒pMC055-CS。 展开更多
关键词 恶性疟原虫 子孢子 抗原 生物工程
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应用(Dig)-DNA探针进行恶性疟流行病学调查
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作者 张兆松 王荣芝 陈淑贞 《南京医学院学报》 CSCD 1993年第1期40-41,共2页
重组的恶性疟原虫DNA片段用洋地黄毒苷配基标记后作为探针(pPF_(14)-F-Dig),以斑点杂交试验对海南省恶性疟流行区人群的274份血样进行检测,并同时作厚薄血片镜检。结果显示,274例样本中血片镜检阳性1例,带虫率为0.36%;pPF_(14)-F-Dig ... 重组的恶性疟原虫DNA片段用洋地黄毒苷配基标记后作为探针(pPF_(14)-F-Dig),以斑点杂交试验对海南省恶性疟流行区人群的274份血样进行检测,并同时作厚薄血片镜检。结果显示,274例样本中血片镜检阳性1例,带虫率为0.36%;pPF_(14)-F-Dig 探针检出阳性15例(其中1例为镜检阳性者),阳性率为5.47%;pPF_(14)-F-Dig 与镜检的阳性符合率为1/1,阴性符合率为94.87%。每张硝酸纤维素膜可查96份样本,故该探针具有用于大规模流行病学调查的价值。 展开更多
关键词 DNA探针 斑点杂交试验 疟疾
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利用Lifeact法检测恶性疟原虫F-actin的研究
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作者 王卫峰 张青锋 潘卫庆 《同济大学学报(医学版)》 CAS 2014年第6期44-48,共5页
目的通过构建游离表达绿色荧光蛋白融合Lifeact多肽(Lifeact-GFP)的转基因虫株,在不影响恶性疟原虫actin动态平衡的前提下特异标识纤维状肌动蛋白(F-actin),为研究F-actin在恶性疟原虫中的特性及其对毒力基因的转录调控作用打下基础。... 目的通过构建游离表达绿色荧光蛋白融合Lifeact多肽(Lifeact-GFP)的转基因虫株,在不影响恶性疟原虫actin动态平衡的前提下特异标识纤维状肌动蛋白(F-actin),为研究F-actin在恶性疟原虫中的特性及其对毒力基因的转录调控作用打下基础。方法构建重组转染质粒Lifeact-GFP/p LN,经电转化方法将质粒转入恶性疟原虫3D7标准虫株中,BSD药物筛选获得携带游离Lifeact-GFP/p LN质粒的转基因虫株,并经免疫印迹(Western Blot)和活细胞荧光检测,证实Lifeact-GFP融合基因的细胞内表达,以期通过免疫共沉淀实验(Co-IP)检测Lifeact与恶性疟原虫F-actin的相互作用。结果成功获得了恶性疟原虫Lifeact-GFP转基因虫株,并通过W estern Blot和荧光检测证实Lifeact-GFP成功获得表达;但是,免疫共沉淀实验结果初步显示,Lifeact不能直接结合恶性疟原虫F-actin。结论虽然Lifeact多肽能在酵母等细胞中特异结合F-actin,但是不能直接识别恶性疟原虫F-actin。 展开更多
关键词 恶性疟原虫 纤维状肌动蛋白 免疫共沉淀
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Malaria:role of antibodies in protection and pathogenesis:an overview
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作者 Tyagi P Biswas S 《Asian Pacific Journal of Tropical Medicine》 SCIE CAS 2008年第1期69-82,共14页
The research scenario for malaria has improved in the last three decades to understand the epidemiology and host immune responses to plasmodial infection.Due to the augmented episodes of resistance development against... The research scenario for malaria has improved in the last three decades to understand the epidemiology and host immune responses to plasmodial infection.Due to the augmented episodes of resistance development against the commonly used antimalarials in plasmodium parasites,especially in Plasmodium falciparum,neutralization of infection through effective vaccine(s) remains the feasible alternative in malaria control.In this direction,lot of attention was paid towards the identification of stage specific malaria antigens targeted by host ’s immune system.Preparation of synthetic or recombinant peptides and evaluation of their immunogenecity in naturally occurring antibody response were also given much importance,as these studies could help in finding potential candidates for future malaria vaccine(s).Attention was also paid.on the pathogenic consequences of antibody formation in malaria infection as polyclonal activation of B cells,which is a very prominent feature in malaria infection.Formation of circulating immune complexes in chronic malaria infection was also viewed as pathogenic parameter of severe malaria.The present survey focuses mainly on protective and pathogenic aspects of malaria antibodies(eliciting against various,stage specific antigens),and future research plan in antibodymediated immune response. 展开更多
关键词 MALARIA plasmodium falciparum P.vivax antigen antibobody immune complex
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Observation on the specific binding site of p195 protein on human erythrocytes using colloidal gold labelling
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作者 方军 何斌 管惟滨 《Journal of Medical Colleges of PLA(China)》 CAS 1997年第2期116-120,共5页
Objective: p195, the major protein on the surface of Plasmodium falciparum merozoites, has been found to have ability to bind sialic acid residues on the surface of human erythrocytes, and this binding is thought to b... Objective: p195, the major protein on the surface of Plasmodium falciparum merozoites, has been found to have ability to bind sialic acid residues on the surface of human erythrocytes, and this binding is thought to be a prerequisite for recognition of human erythrocyte by merozoite- This study attempted to map out the binding site of p195, thus providing a theoretical clue to developing an antimalaria vaccine which blockades invasion of merozoites into human erythrocytes. Methods: Eight proteins derived from pl95 were expressed in E. coli, and purified by Ni-chelate affinity chromatography. The re folded proteins were labelled with colloidal gold. The labelled protein complexes were co-incubated with human erythrocytes separately and simultaneously, and the proteins were put into the culture supernatant of P- falciParum to observe their effect on invasion of merozoites into erythrocytes. Results: A fragment of p195, M6 (amino acid sequence: 384 - 595), was found to have the ability to bind human erythrocytes. M6 gold complexes showed no ability to bind erythrocytes treated with trypsin or neuraminidase. M6 was also found to have the ability to inhibit invasion of merozoites of P. falciparym into human erythrocytes. Conclusiou: A fragment of p195, M6, has the ability to bind human erythrocytes. The binding is dependent on sialic acid residues, and may be a prerequisite to recognition of erythrocytes by merozoites. 展开更多
关键词 plasmodium falciparum p195 COLLOIDAL GOLD erythrocytes
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输入性恶性疟原虫Pfcrt和pfmdr1基因突变关联性分析 被引量:1
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作者 周水茂 李联军 +3 位作者 贾西帅 杨燕 徐明星 陈芳 《中国媒介生物学及控制杂志》 CAS 2018年第3期242-245,共4页
目的了解输入性恶性疟原虫氯喹抗性转运蛋白基因(Pfcrt)和恶性疟原虫多药抗性基因1(pfmdr1)相关位点突变情况及其关联性。方法 2009-2016年,采集武汉市从非洲和东南亚等回国人员恶性疟患者血样,利用巢式PCR扩增恶性疟原虫Pfcrt和pfmdr1... 目的了解输入性恶性疟原虫氯喹抗性转运蛋白基因(Pfcrt)和恶性疟原虫多药抗性基因1(pfmdr1)相关位点突变情况及其关联性。方法 2009-2016年,采集武汉市从非洲和东南亚等回国人员恶性疟患者血样,利用巢式PCR扩增恶性疟原虫Pfcrt和pfmdr1基因,分别用限制性内切酶ApoⅠ、AseⅠ和EcoRv对产物进行酶切,分析相关位点的突变性。结果 232例输入性恶性疟患者Pfcrt76和pfmdr186、1042、1246位点的突变率分别为55.2%、17.2%、5.2%和8.6%,pfmdr1总突变率为26.3%;东南亚患者血样未检出pfmdr186和pfmdr11246位点突变;非洲输入性恶性疟患者Pfcrt76突变和未突变中pfmdr186、1042、1246位点及pfmdr1总突变率分别为28.6%、3.8%、12.4%、36.2%和10.4%、2.1%、7.3%、17.7%。患者症状与Pfcrt76、pfmdr186、1042、1246位点突变连锁不平衡分析,D′和r^2分别为0.230和0.018、0.290和0.004、0.996和0.012、0.150和0.035。结论输入地为非洲和东南亚缅甸的恶性疟原虫pfmdr1基因突变位点存在差异,Pfcrt76突变与pfmdr1突变和pfmdr186点突变呈正相关。 展开更多
关键词 输入病例 恶性疟原虫 恶性疟原虫氯喹抗性转运蛋白基因 恶性疟原虫多药抗性基因1 基因突变
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艾博混合疟/恶性疟快速诊断试剂效果评价
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作者 沈旭 刘慧 李春富 《寄生虫病与感染性疾病》 CAS 2011年第4期235-237,共3页
目的评价艾博混合疟/恶性疟(Malaria Pan./p.f..)快速诊断试剂的检测效果。方法采集疟疾、疑似疟疾、不明原因发热及感冒"四热"患者血样,以显微镜镜检方法为金标准,比较艾博混合疟/恶性疟试剂检测效果结果共采集584份血样,艾... 目的评价艾博混合疟/恶性疟(Malaria Pan./p.f..)快速诊断试剂的检测效果。方法采集疟疾、疑似疟疾、不明原因发热及感冒"四热"患者血样,以显微镜镜检方法为金标准,比较艾博混合疟/恶性疟试剂检测效果结果共采集584份血样,艾博试剂检测恶性疟原虫277份,灵敏度为98.9%,特异性为97.7%,与显微镜符合率为98.3%。艾博试剂检测混合疟377份的灵敏度为99.5%,特异性为99.0%,与显微镜符合率为99.3%,总符合率为98.8%,上述2方面的检测结果经Kappa一致性检验,Kappa值均大于0.97。结论艾博混合疟/恶性疟试剂盒实验室检测敏感性和特异性高,能检测混合疟并区分恶性疟,结果与血涂片吉氏染色镜检结果高度一致,适用于疟疾流行的基层使用。 展开更多
关键词 艾博混合疟/恶性疟检测试剂 富组氨酸蛋白Ⅱ 醛缩酶 混合疟 恶性疟
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