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应用DNA重组技术把微小隐孢子虫(Cryptosporidium parvum)子孢子抗原p23基因克隆入表达载体pMG36e当中,成功构建了可在干酪乳杆菌(Lactobacillus casei Zhang)胞浆中表达的重组载体pMG-p23。并以SDS-PAGE、Western blotting以及IFAT试验方法,分别在破碎菌体组分和活菌体当中鉴定p23基因表达产物并确定其在细胞中表达的位置。本试验为进一步构建分泌型或锚定型干酪乳杆菌(L.casei Zhang)活菌载体奠定了基础。 相似文献
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为将微小隐孢子虫(Cryptosporidium parvum)P23基因在巴斯德毕赤酵母(Pichia pastoris)系统中进行表达,利用表达蛋白初步建立隐孢子虫病间接ELISA诊断技术,设计引物从微小隐孢子虫基因组DNA中扩增P23基因序列,构建pPIC9K-P23重组质粒,在毕赤酵母中进行表达,用阴离子交换层析柱进行纯化。以重组P23纯化蛋白为抗原建立间接ELISA检测方法,对现场采集的猪血清样品进行检测。SDS-PAGE显示所表达的蛋白大小约为23 kDa。Western blot检测表明该蛋白能与兔抗P23蛋白血清特异性结合。用建立的间接ELISA技术对186份猪血清样品进行检测,阳性率为83.3%。本研究获得了真核表达的P23重组蛋白,初步建立了微小隐孢子虫病间接ELISA诊断技术,为隐孢子虫病的诊断和流行病学调查打下了基础。 相似文献
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以CP23重组蛋白为抗原建立检测微小隐孢子虫抗体间接ELISA方法的研究 总被引：3，自引：0，他引：3
摘要：以在E.coli高效表达的微小隐孢子虫子孢子表面抗原CP23为抗原，以辣根过氧化物酶(HRP)标记的山羊抗鼠IgG为二抗，建立了检测微小隐孢子虫抗体的间接ELISA方法。经检测筛选出最佳反应条件为1μg／孔纯化的E.coli表达的CP23抗原包被酶标板，用10％免血清进行封闭，以正常E.coli裂解上清液稀释待检血清。实验表明应用CP23重组蛋白作为诊断C.parvum抗原具有特异性高、抗原易纯化和成本低等特点。 相似文献
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利用重组的微小隐孢子虫（Cryptosporidium parvum）P23蛋白作为ELISA诊断抗原,对来自青海地区的部分放牧犬和宠物藏獒犬进行隐孢子虫特异性抗体的检测。结果在497份血清中检出150份阳性血清,阳性率为30.18%。其中放牧犬阳性率为32.77%,藏獒的阳性率为37.95%,宠物犬的阳性率为10.42%。 相似文献
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应用间接ELISA检测微小隐孢子虫抗体的研究 总被引：5，自引：0，他引：5
以在大肠埃希氏菌中表达的微小隐孢子虫(Cryptosporidum parvum)子孢子表面抗原CPl5为抗原，以辣根过氧化物酶(HRP)标记的山羊抗鼠IgG为二抗，建立了检测微小隐孢子虫抗体的间接ELISA。试验筛选出的最佳反应条件为：大肠埃希氏菌CPl5抗原包被量为1μg/孔，用100mL／L的兔血清进行封闭，以正常大肠埃希氏菌裂解上清液稀释待检血清。试验结果表明，应用CPl5重组蛋白作为诊断微小隐孢子虫抗体的抗原具有特异性高、抗原易纯化和成本低等特点。 相似文献
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微小隐孢子虫病毒衣壳蛋白抗体检测ELISA方法的建立 总被引：1，自引：0，他引：1
为建立快捷的含病毒隐孢子虫的检测方法,本研究利用已构建的微小隐孢子虫病毒衣壳蛋白重组表达质粒表达并纯化重组蛋白,建立以该重组衣壳蛋白为包被抗原检测抗体的间接ELISA检测方法.经优化后间接ELISA的最佳条件为:抗原包被浓度为0.25 μg/孔,被检血清稀释度为1:200,酶标二抗稀释度为1:2000,封闭液为含5％脱脂奶粉的PBST溶液.该方法检测安氏隐孢子虫阳性血清,柔嫩艾美尔球虫阳性血清,蓝氏贾第虫阳性血清,弓形虫阳性血清与微小隐孢子虫阳性结果均为阴性.该方法灵敏度为1:1600,特异性较强且具可重复性,可用于含病毒隐孢子虫的抗体检测及流行病学调查. 相似文献
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采用PCR技术扩增了微小隐孢子虫Cp23基因,将Cp23基因克隆至原核表达载体pET-28a(+)中,构建重组表达载体pET28a-Cp23。将其转化至宿主菌E.coli BL21(DE3)中,用IPTG诱导表达后进行SDS-PAGE和Western blot分析。SDS-PAGE结果表明,蛋白相对分子质量为25 000,目的蛋白高效表达,且为包涵体蛋白。Western blot结果表明,表达产物可被微小隐孢子虫阳性血清识别,具有良好的免疫反应原性。本研究为抗微小隐孢子虫疫苗和免疫学诊断方法的研制提供候选抗原。 相似文献
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应用从鼠粪便中分离的小球隐孢子虫(Cryptosporidium parvum)卵囊,经阿拉伯胶梯度离心后,超声粉碎制成可溶性抗原.按常规方法免疫BALB/c小鼠,经融合、克隆筛选建立了5个单克隆抗体株(1A_8、2B_2、3C_4、1A_3和2C_3).通过免疫荧光和免疫组化检测,发现1A_8、2B_2和3C_4克隆株针对子孢子,而lA_3和2C_3针对卵囊壁.1A_8、2B_2和3C_4株单抗腹水分别以10%、20%和30%的量与小球隐孢子虫子孢子(约4×10~4个)共同孵育1h后,接种到人胎肺细胞上培养,结果观察到30_8株腹水、10+_2株腹水和20<_4株腹水对子孢子有明显细胞毒作用.进而观察了2B_2株单抗腹水对培养虫体的反应性,发现其对裂殖体期虫体亦有作用;进行了2B_2株腹水对BALB/c 小鼠的被动保护性试验,发现以1mL/只剂量分别于攻虫前后1d接种,其对5×10~5个卵囊攻击的BALB/c鼠有明显的保护作用.Western印迹结果表明,2B_2株单抗是针对小球隐孢子虫抗原的28000蛋白质分子. 相似文献
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Different isoenzyme activities have been assayed in three strains of Cryptosporidium parvum, C1 (C. parvum from infected calves, UK), C2 (C. parvum from infected calves, Egypt) and C3 (C. parvum from infected goats, Egypt). The electrophoretic variations of five enzymes; lactate dehydrogenase (LDH), glucose phosphate isomerase (GPI), hexokinase (HK), malate dehydrogenase (MDH) and glutamate dehydrogenase (GLDH) were compared among the three different isolates using native polyacrylamide gel-electrophoresis. LDH showed an identical pattern in the three isolates. GPI showed two different bands in C3 and C1, with both bands present in C2. HK activity showed a weak band in C1 but no reaction was detected with C2 and C3. Malate dehydrogenase (MDH) showed no reaction in C1, but similar bands in C2 and C3. Glutamate dehydrogenase (GLDH) showed two different patterns, C2 and C3 had one pattern and C1 showed additional zones of reaction. Rat liver homogenate was run at the same time as the parasite extracts as a positive control. This investigation suggests that GPI, HK and GLDH could be used to characterise different Cryptosporidium isolates. 相似文献
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为了探讨我国微小隐孢子虫(C.parvum)的端粒序列,试验根据国外已报道的端粒重复序列5’-CCTAAA-3’设计寡聚核苷酸探针(CCTAAA)5,并用地高辛标记,C.parvum基因组DNA经Bal31-HindⅢ酶切后进行Southern印迹杂交鉴定;并在此基础上,采用改进的端粒重复序列扩增(TRAP)法首次对子孢子和卵囊时期C.parvum的端粒酶活性进行检测。结果表明:C.parvum基因组DNA与探针(CCTAAA)5杂交,获得了较好的杂交条带,随着Bal31-HindⅢ酶切时间的延长,杂交信号逐渐减弱,说明C.parvum端粒序列定位在染色体的末端,与国外报道的C.parvum端粒序列一致,为5’-CCTAAA-3’;检测C.parvum端粒酶活性时发现,子孢子时期端粒酶具有活性,卵囊中未检测到端粒酶活性。 相似文献
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Summary Four groups of calves (A, B, C and D) each consisting of five calves were used for the present study. Group A calves were givenCorynebacterium parvum alone. Group B calves were inoculated with inactivated ground-up-tick supernate (GUTS) prepared fromHyalomma anatolicum anatolicum infected withTheileria annulata plusC. parvum. Group C received only inactivated GUTS. All the surviving calves of groups A to C were exposed on day 45 post-inoculation to a lethal tick challenge along with susceptible control calves of group D. All the calves of groups A and B withstood the challenge whereas all the calves of groups C and D died of theileriosis. Complement fixing antibodies were detected in calves of groups B and C. A significant decrease in neutrophils and a significant increase in monocytes was observed in calves of groups A and B. No significant changes were seen in other cell types. The results of this study demonstrated thatC. parvum alone may be used as an immunostimulant for producing non-specific resistance againstT. annulata.
Proteccion Del Ganado Contra La Infeccion DeTheileria annulata UtilizandoCorynebacterium parvum
Resumen Se utilizaron para el presente estudio, cuatro grupos de terneros (A, B, C y D), cada uno conformado por cinco animales. Los terneros del grupo A fueron inoculados conCorynebacterium parvum únicamente. El grupo B, se inoculó con un sobrenadante inactivado de garrapatas molidas preparado deHyalomma anatolicum anatolicum infectadas conTheileria annulata másC. parvum; el grupo C recibió molido de garrapatas inactivado. Todos los terneros sobrevivientes de los grupos A y C fueron expuestos, a los 45 días de la inoculación, a una descarga letal de garrapatas infectadas, como tambien los terneros del grupo D. Todos los terneros de los grupos A y B sobrevivieron la descarga, mientras que los animales de los grupos C y D murieron de theileriosis. Se detectaron anticuerpos fijadores de complemento, en terneros de los grupos B y C. Los resultados demuerstran que elC. parvum solo, podría utilizarce como inmunoestimulante para crear resistencia no específica contraTheileria annulata.
Protection Des Bovins Contre L'infection ATheileria annulata Par L'utilisation DeCorynebacterium parvum
Résumé Quatre groupes de veaux (A, B, C et D), chacun composé de cinq veaux, ont été utilises dans la présente étude. On a administré desCorynebacterium parvum uniquement aux veaux du groupe A. Les veaux du groupe B ont été inoculé avec le surnageant de broyat de tique (SBT préparé à partir deHyalomma anatolicum anatolicum infecté parTheileria annulata et avec duC. parvum. Le groupe C a reçu seulement du SBT inactivé. Tous les veaux des groupes A à C qui avaient survécu ont été exposés le 45e jour après inoculation à une épreuve léthale de tiques avec les veaux du groupe D. Tous les veaux des groupes A et B ont résisté à l'épreuve tandis que les veaux des groupes C et D sont morts de theileriose. Des anticorps fixant le complément ont été décelés chez des veaux des groupes B et C. Les résultats de cette étude ont démontré queC. parvum seule peut être utilisée comme immunostimulant pour créer une résistance non spécifique àT. annulata.相似文献
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通过PCR扩增出小球隐孢子虫(Cryptosporidium parvum)P15基因片段。将该片段连接到克隆质粒裁体pUC19的BamH Ⅰ酶切位点上，并对其序列进行了测定。结果表明，该基因长度为479bp，编码的表面蛋白由148个氨基酸残基组成。再将P15蛋白基因片段分别在大肠杆菌和哺乳动物细胞中表达，经SDS-PAGE分析和Western blot检测，结果表明，小球隐孢子虫P15蛋白基因在大肠杆菌内获得表达的融合蛋白相对分子质量为50000；在哺乳动物细胞中，重组痘病毒表达的蛋白相对分子质量为18000-22000，与直接从自然感染牛小球隐孢子虫分离的P15蛋白的相对分子质量相似。 相似文献
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A.M.A.M.Z. Siddiki Jonathan M. Wastling 《Journal of veterinary science (Suw?n-si, Korea)》2009,10(3):203-210
Cryptosporidium (C.) spp. are important zoonotic parasites causing widespread diarrhoeal disease in man and animals. The recent release of the complete genome sequences for C. parvum and C. hominis has facilitated the comprehensive global proteome analysis of these opportunistic pathogens. The well-known approach for mass spectrometry (MS) based data analysis using the BLAST tool (MS BLAST) is a database search protocol for identifying unknown proteins by sequence similarity to homologous proteins using peptide sequences produced by mass spectrometry. We have used several complementary approaches to explore the global sporozoite proteome of C. parvum with available proteomic tools. To optimize the output of the MS data, a sequence similarity-based MS BLAST strategy was employed for bioinformatic analysis. Most significantly, almost all the constituents of glycolysis and several mitochondrion-related proteins were identified. In addition, many hypothetical Cryptosporidium proteins were validated by the identification of their constituent peptides. The MS BLAST approach was found to be useful during the study and could provide valuable information towards a complete understanding of the unique biology of Cryptosporidium. 相似文献
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Cryptosporidium DNA was extracted from 134 faecal specimens from pre-weaned calves from different German Federal States (age range, 3–15 days old), which tested positive for oocysts by microscopic analysis. The 18S rDNA gene and the oocyst wall protein gene (COWP) were used as targets for PCR and RFLP techniques. Cryptosporidium species were identified by using SspI, MboII and RsaI endonucleases for the digestion of 18S rDNA and COWP amplified fragments, respectively. In all samples, restriction patterns corresponding to Cryptosporidium parvum were identified, which is in agreement with abundant literature data indicating C. parvum as the most common species in pre-weaned calves. In order to estimate the genetic heterogeneity among C. parvum calf isolates, 53 samples chosen to represent different German Federal States were successfully subtyped by sequence analysis of the highly polymorphic 60-kDa glycoprotein gene. All isolates belonged to the allele IIa (with seven subtypes), with the exception of one isolate that belonged to the allele IId. Moreover, three novel subtypes of the allele family IIa have been found. This study confirms the utility of genotyping and subtyping tools in characterizing the transmission of Cryptosporidium spp. This is the first molecular epidemiological report about subtyping of Cryptosporidium bovine isolates in Germany. 相似文献