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1.
为对犬弓首蛔虫(T.canis)免疫抑制卵巢信息蛋白(Tc-iom)、卵黄原蛋白(Tc-vit)、酪氨酸蛋白激酶(Tctpk)和丝/苏氨酸蛋白磷酸酶(Tc-stp)四个性别相关基因进行差异表达分析。本研究采用SYBR Green I qRT-PCR方法检测Tc-vit、Tc-iom、Tc-tpk、Tc-stp在T.canis雌虫卵巢、输卵管、子宫和雄虫精巢、贮精囊、输精管的转录情况。结果显示,Tc-vit在雌虫卵巢、输卵管和子宫中均有转录,在雄虫精巢和贮精囊中也有转录,但在输精管中无转录;Tc-iom在雌虫卵巢、输卵管和子宫中高水平转录,在雄虫精巢和贮精囊中转录量较低,输精管中无转录;Tc-tpk在雄虫精巢和贮精囊中的转录明显高于其他生殖组织;Tc-stp在雄虫精巢和输精管中转录量较高,在雌虫卵巢、输卵管和子宫中转录量较低。本试验为T.canis性别相关基因的进一步研究奠定了基础。  相似文献   

2.
为探讨犬弓首蛔虫(Toxocara canis,T.canis)软骨素蛋白多糖TcCPG1的表达特征及其与几丁质的结合方式,为犬弓首蛔虫软骨素蛋白多糖的功能研究提供依据,笔者克隆T.canis软骨素蛋白多糖1基因(Tc-cpg-1)并原核表达重组蛋白TcCPG1,采用镍柱亲和层析技术对重组蛋白进行纯化,并与几丁质进行结合试验;采用实时荧光定量PCR (quantitative real-time polymerase chain reaction, qRT-PCR)进行Tc-cpg-1基因的转录水平的性别和组织差异表达分析。结果显示:Tc-cpg-1基因编码区(coding sequence, CDS)的长度为1 251 bp,共编码417个氨基酸;SDS-PAGE电泳结果显示重组蛋白TcCPG1以包涵体形式存在,相对分子质量大小约为70 ku;对表达条件进行优化后,以0.8 mmol·L~(-1) IPTG于16℃诱导14 h时可获得大量目的蛋白;利用镍柱亲和层析纯化可以获得具有较高纯度的目的蛋白,与几丁质进行结合试验,验证了重组蛋白TcCPG1能够与几丁质结合;通过qRT-PCR对Tc-cpg-1基因转录水平的组织差异性进行分析,发现Tc-cpg-1在雌虫中高量表达,尤其在雌虫的生殖组织中表达量最高,表明Tc-cpg-1可能参与了T.canis雌虫的生殖过程,推测TcCPG1可能通过与卵壳中几丁质的相互作用从而影响T.canis的胚胎和生殖发育过程。  相似文献   

3.
为了探明猪鞭虫(Trichuris suis)雌、雄虫的转录组差异,丰富T.suis转录组数据信息,利用Illumina Hiseq2000对临床采集的T.suis雌、雄虫进行高通量测序,将组装得到的Unigenes在NR、GO、COG和KEGG数据库中比对注释,并进行差异表达基因分析。结果,T.suis雌、雄虫分别获得59 657 854条和48 414 184条高质量测序数据,分别组装获得21 026个和28 886个Unigenes。注释到NR、GO、COG和KEGG数据库的雌虫Unigenes数量分别为11 700、2 287、1 650和9 690个,雄虫Unigenes数量分别为12 902、2 414、1 791和10 377个,在NR数据库比对显示84.90%以上信息均来源于猪鞭虫。T.suis雌、雄虫共有4 320个差异表达基因。以雌虫为对照,雄虫中共有3 496个基因表达上调,有824个基因表达下调。共有906个差异表达基因获得160个KEGG通路富集,其中显著富集通路7个。在T.suis转录组中发现了许多重要基因的表达,包括与T.suis繁殖相关的主要精子蛋白、组蛋白H2A/H2B及卵黄蛋白原,与T.suis致病性、免疫及炎症调节相关的蛋白酶、丝氨酸蛋白酶抑制分子和半胱氨酸蛋白酶抑制分子,以及众多未知功能的基因。本研究揭示了T.suis雌、雄虫差异表达基因的数量,获得了这些差异表达基因的功能、分类和代谢通路注释,为丰富T.suis转录组信息,揭示鞭虫与宿主互作特点及抗鞭虫靶点药物研发奠定基础。  相似文献   

4.
为克隆、表达犬链球菌(S.canis)保护性抗原基因,并对其表达产物进行免疫原性分析.本研究以Lambda ZAP Ⅱ载体构建了S.canis的3 kb～8 kb随机基因组文库,通过S.canis阳性血清筛选和质粒拯救,获得一株阳性克隆重组质粒并进行了序列测定.通过BLAST分析并与GenBank中登录的相近基因序列比较发现,该基因为S.canis的1个保护性抗原(SPASc)基因,该基因与兽疫链球菌和产脓链球菌的保护性抗原及马链球菌M蛋白基因具有一定的同源性.根据该序列设计特异性引物,经PCR扩增、克隆并进行了SPASc基因的原核表达.用纯化SPASc重组蛋白免疫兔制备的血清对小鼠具有保护性作用.  相似文献   

5.
日本血吸虫成熟雌虫所产虫卵是造成宿主病理损害及疾病再传播的根源,因此从控制雌虫生殖发育入手对控制血吸虫病的发生具有重要意义。本研究通过5′-RACE和3′-RACE获得了在发育阻遏雌虫中高表达的SjELAV-like 2基因的全长序列,并对其进行克隆和表达,制备了相应的多克隆抗体,Western blotting分析结果表明该蛋白质具有良好的反应原性;实时定量分析发现该基因在血吸虫童虫时期高表达,成熟后趋于稳定,且在雌虫体内的表达量显著高于同时期合抱雄虫;该基因在雄虫体内的表达量基本恒定;免疫组化研究发现,在血吸虫成虫雌虫该蛋白主要定位于虫体体被。利用RNA干扰技术对该基因的生物学功能进行了初步研究,RT-qPCR结果显示,RNAi可明显抑制SjELAV-like 2基因的表达;对该基因表达的长期干扰导致受干扰虫体产卵能力及卵胚孵化能力的下降,统计分析发现,干扰组宿主每克肝荷卵减少达39.67%(P0.01),虫卵的卵胚孵化减少达75.56%(P0.01)。扫描电镜观察发现,与NC组相比,siRNA干扰组虫体体被上泡状突出物明显增多,这些结果表明SjELAV-like 2基因对日本血吸虫生长发育具有重要影响。  相似文献   

6.
旨在探讨犬弓首蛔虫(Toxocara canis,T.canis)C型凝集素4(C-type lectin 4,C-TL-4)的基因特征，并对其组织分布进行研究。根据Tc-ctl-4的基因组数据(GenBank:AF126830.1),以T.canis为模板对Tc-ctl-4全长基因进行扩增，并进行序列分析和多重序列比对；构建Tc-ctl-4/pCold TF原核表达载体，对重组蛋白进行纯化并制备多克隆抗体；同时采用实时荧光定量PCR(quantitative real-time PCR,qRT-PCR)和间接免疫荧光组化检测Tc-ctl-4基因的转录水平和Tc-CTL-4的组织分布。结果显示，Tc-ctl-4基因的全长序列为842 bp,共编码280个氨基酸，多重序列比对显示，犬弓首蛔虫和猫弓首蛔虫的C型凝集素均含有高度保守的CTLD结构域；SDS-PAGE检测发现重组蛋白Tc-CTL-4的大小约为86 ku,以可溶性形式表达；利用Ni-NTA亲和层析柱纯化蛋白，以500 mmol·L-1咪唑进行洗脱时可获得高纯度的目的蛋白；将纯化后的重组蛋白免疫新西兰大白兔制...  相似文献   

7.
为检测犬布氏杆菌(Brucella canis)重组Cu/Zn-SOD蛋白的抗原性,本研究通过PCR方法扩增B.canis的Cu/Zn-SOD基因,将其克隆于表达载体pET-28a(+)中,构建重组表达质粒pET-SOD.将该质粒转化受体菌E.coli BL21 (DE3),经IPTG诱导后,SDS-PAGE电泳分析,...  相似文献   

8.
本研究根据曼血吸虫抱雌沟蛋白（Gyncophoral canal protein)基因SmGCP保安区片设计－对引物,以日本血吸虫中国大陆株成虫mRNA为模板,用TR－PCR法扩增出一大小为868bp的基因片段。测序后分析序列推断该片段与SgGCP相应片段碱基同源性86．6％。说明所克隆的基因为编码日本血吸虫中国大陆株抱雌沟蛋白基因,推断的氨基酸序列同源性为84．1％,将其克隆到表达载体pET28（a)中,在大肠杆菌BL21中获得了较高量的融合表达,表达产物分子量为29KD。利用因吸虫成虫粗抗原免疫大白兔所得抗血清对该表表达产物进行Westernblto检测,在预测位置出现了明显的识别条带,显示了该表达产物良好的抗原性。将该表达产物用佐剂处理免疫昆明系小鼠攻击血吸虫尾蚴,7周后剖杀小鼠冲虫,进行成虫和虫卵计数,其数虫率为35．2％,减卵率为37．8％,与对照组比较差异显著,初步证明血吸虫抱雌沟蛋白具有一定的免疫保护功能。  相似文献   

9.
为探讨日本血吸虫雌虫生长发育、性成熟及产卵的调控机制,本研究采用RNA-seq高通量技术对25日龄两性感染而来的正常发育雌虫(MF)和单性感染而来的发育阻遏雌虫(SF)的转录组进行测序、定量及差异基因的筛选。从MF和SF中分别获得3 696 376条和3 662 109条clean reads,将clean reads与日本血吸虫基因组数据库进行匹配发现2组样本的匹配率皆达79%以上。以|Log_2 Ratio|≥1, p-Value0.001为筛选条件,得到775个在MF和SF间差异表达的基因,其中有401个基因在MF中高表达,374个基因在SF中高表达。利用荧光实时定量RT-PCR对差异基因进行验证,结果与RNA-seq相符。生物信息学分析表明,在MF中高表达的基因多与代谢和生物合成过程相关,这些过程对于促进和维持雌性性成熟和产卵至关重要;而在SF中高表达的基因多与运动和细胞通讯等功能相关。这些发现可为深入研究日本血吸虫雌虫生殖发育以及产卵的生物学机理提供一定的理论依据。  相似文献   

10.
山羊PTHrP基因的多克隆抗体制备和组织表达图谱分析   总被引：1，自引：0，他引：1  
旨在获得山羊PTHrP多克隆抗体,检测其在山羊各组织中的表达图谱,以便进一步研究山羊PTHrP的功能。本研究构建山羊PTHrP基因的原核表达载体,转化E.coliBL21(DE3)菌株,IPTG诱导表达获得融合蛋白,SDS-PAGE电泳检测融合蛋白,然后将融合蛋白纯化后多点免疫大耳白兔,制备多克隆抗体,最后使用West-ern blot检测PTHrP在山羊各组织中的表达。结果,酶切和测序显示原核表达载体构建成功;SDS-PAGE电泳结果说明融合蛋白(约36.5 ku)被成功纯化;间接ELISA检测所制备的抗体效价达1∶51 200;表达谱显示PTHrP在山羊乳腺、肾脏、垂体、脑、心脏、肝脏、骨、肌肉、脾脏等组织中均有表达。本研究成功制备了山羊PTHrP的多克隆抗体,发现PTHrP在山羊各组织中广泛表达。  相似文献   

11.
An analysis of the protein profiles of intact worms and isolated tissues of adult male and female Toxocara canis worms was conducted. Soluble proteins recovered from homogenized whole specimens and dissected tissues (body wall, reproductive tract, esophagus and intestine) of T. canis adults from several different canine hosts were separated by size using gradient sodium dodecyl sulfate electrophoresis (SDS-PAGE) and visualized with silver staining. SDS-PAGE profiles of worms from different hosts were found to be virtually identical irrespective of sex or tissue type. Recovered proteins ranged in size from 3.4 to 325 kDa. As expected, variations existed between the protein profiles of different body tissues, with only slight variations between the sexes. The largest number of recovered proteins was present in the female reproductive tract extracts.  相似文献   

12.
菌壳技术是一种新型的灭活疫苗制备方法,通过非变性的灭活方式保存细菌表面多个抗原表位,所制备的菌壳可作为预防细菌病的理想疫苗。本试验从噬菌体PhiX174 DNA钓取裂解E基因,连接至温控原核表达载体pBV220,通过PCR在所构建的pBV220+E上扩增出蛋白裂解部件（protein lysis component,PLC）,该部件包含阻遏蛋白cI857、溶菌E基因及终止序列rrnbT1T2,然后将其克隆至广宿主表达载体pBBR1MCS-2中,最终将构建的广宿主裂解质粒pBBR+PLC电转入犬布鲁氏菌RM6/66中。试验结果表明,经42 ℃诱导后,广宿主裂解质粒对犬布鲁氏菌RM6/66的裂解率达100%,成功制备了犬布鲁氏菌RM6/66菌壳疫苗。本试验通过菌壳技术制备的犬布鲁氏菌疫苗对预防宠物犬布鲁氏菌病起到重要作用,同时也对人兽布鲁氏菌疫苗的研制提供新策略。  相似文献   

13.
The efficacy of selamectin against adult ascarids was evaluated in eight controlled and masked studies in dogs. Three laboratory studies evaluated selamectin against experimentally induced infections of Toxocara canis; three laboratory studies evaluated selamectin against naturally acquired infections of T. canis; one laboratory study evaluated selamectin against naturally acquired infections of both T. canis and Toxascaris leonina; one field study evaluated selamectin against naturally acquired infections of ascarids (T. canis and/or T. leonina) in dogs presented as veterinary patients. Selamectin was administered topically to the skin of dogs in unit doses designed to deliver a minimum of 6mgkg(-1) (range, 6-12mgkg(-1)). In all studies, dogs were allocated randomly to treatment assignments (selamectin or vehicle control in laboratory studies: selamectin or reference product in the field study) on the basis of pretreatment fecal egg counts. For induced infections, there were significant reductions in geometric mean numbers of adult T. canis after a single application of selamectin (93.9-98.1%, P=0.0001), after two monthly applications (> or =88.3%, P< or =0.0001), and after three monthly applications (100%, P< or =0.0002). In the natural infection laboratory studies, when selamectin was administered twice at an interval of 30 days, the percentage reductions in geometric mean numbers of adult T. canis at necropsy were 84.6, 91.3, and 97.9%, and when selamectin was administered on days 0, 14, and 30, the percentage reductions were 91.1 and 97.6%. Geometric mean fecal T. canis egg counts were reduced by > or =92.9% (P< or =0.0067) at the end of the studies. In the field study, geometric mean fecal ascarid egg counts were reduced by 89.5 and 95. 5% (P=0.0001) for 14 and 30 days, respectively, after a single treatment with selamectin, and by 94.0% (P=0.0001) 30 days after the second treatment with selamectin. These reductions compared favorably with the egg count reductions from dogs treated with a reference product containing praziquantel, pyrantel embonate, and febantel. There were no adverse drug experiences or treatment-related mortalities during any of the studies. Selamectin, when administered topically in a unit dose providing a minimum dosage of 6mgkg(-1), was safe and effective against adult T. canis and T. leonina and in reducing the fecal excretion of T. canis eggs in dogs.  相似文献   

14.
In this study, we investigated the morphological identification of Toxocara canis and T. cati eggs on the basis of light and scanning electron microscopic (SEM) observations. T. canis and T. cati eggs used in this study were recovered from the uteri of respective gravid female worms. Measurement of egg size was not helpful in the differentiation of these species, because approximately 90% of eggs measured were of similar size. Using SEM, we were able to differentiate T. canis eggs from T. cati eggs based on their respective characteristic surface structures. Both species have subspherical eggs with markedly pitted surfaces like those of a golf ball, but the surface pitting in T. canis is more coarse than that in T. cati. In this study, however, these differences were not absolute, as 16% of T. canis and 29% of T. cati eggs showed surface pitting that was uncharacteristic of their species. Of the 16% of T. canis eggs that could not be differentiated by surface structure, 3% had pitting resembling T. cati, and the remaining 13% showed intermediate type surface pitting. Similarly, 5% of T. cati eggs resembled T. canis, and 25% of these were of intermediate type. Light microscopic observation yielded results similar to those of SEM, indicating that light microscopy is also a useful tool for the identification of Toxocara eggs.  相似文献   

15.
Aim of the investigation was to examine whether two administrations of moxidectin to pregnant dogs could prevent pre-natal and lactogenic infections of puppies with reactivated Toxocara canis larvae. Four pregnant beagles, infected experimentally with 20 000 embryonated eggs of T. canis, were treated subcutaneously with 1 mg moxidectin per kg body weight on days 40 and 55 of pregnancy (5-13 days before parturition). One further dam and its puppies served as untreated control. Two applications of moxidectin completely prevented pre-natal and lactogenic infections in the puppies. Neither intestinal stages nor somatic larvae were found in the dams or their corresponding puppies. All puppies and dams of the treatment group remained coproscopically negative until 42 days after parturition. The administration of moxidectin did not show any side effects in the dams. None of the puppies of the treated dams showed any pathological abnormalities. In the untreated dam one adult and 26 somatic larvae of T. canis were detected at necropsy. All puppies of the untreated dam showed a patent T. canis infection from day 28 post-natum (p.n.); 296 pre-adult and adult stages of T. canis were spontaneously eliminated and 51 intestinal stages and five somatic larvae of T. canis were recovered at necropsy. In contrast to the puppies of the treated dams all negative control puppies showed blood eosinophilia after parturition and elevated liver enzyme levels.  相似文献   

16.
To determine the efficacy of a formulation of milbemycins in treating patent infection with Toxocara canis, 8 male and 7 female, 10-week-old, ascarid-free Beagles each were given 125 embryonated eggs of T canis. All dogs developed patent infection within 56 days. On post-infection day 70, the dogs were assigned to 1 of 3 groups of 5 dogs each; members of 1 group were given a placebo, while dogs of the other 2 groups were given either 5.68 or 34.08 mg of the milbemycin formulation, respectively. In both groups of dogs given the drug, the number of eggs passed per gram of feces decreased precipitously. However, a few eggs still were found in the feces of several dogs of each group on the day of necropsy (postinfection day 75). Worms or fragments of worms were passed by the treated dogs from the day of treatment until the day on which necropsy was performed; however, most worms were passed during the first 2 days after treatment. At necropsy, only dogs of the control group were found to harbor adult T canis.  相似文献   

17.
丙酮酸脱氢酶激酶(PDK)在生物体的新陈代谢中具有重要的生理功能。为了探讨家蚕(Bombyx mori)PDK基因的表达特性,用蛾区半分法将二化性家蚕品种的活化越年卵(丙2期)分别以常温(25℃)光照和低温(15℃)黑暗催青,通过实时荧光定量PCR技术检测分析2种温度催青处理后家蚕不同发育阶段和不同组织的BmPDK表达水平。常温光照催青区BmP-DK的表达水平表现出发育时期和组织差异:胚胎发育阶段BmPDK在己4期的表达水平最高,其次是戊2、己3和己5期;幼虫发育阶段BmPDK的表达水平在蚁蚕期极显著高于其它龄期,5龄期在卵巢和精巢的表达水平高于其它组织;蛹期BmPDK的表达水平比其它发育时期低;成虫期的表达水平与胚胎发育末期相当,其中雌蛾的脂肪体和卵巢中BmPDK的表达水平较高。推测BmPDK在家蚕胚胎发育末期及产卵过程中有重要作用。催青温度影响BmPDK在家蚕各发育时期及卵巢组织中的表达水平:胚胎发育阶段戊2期BmPDK的表达水平为常温催青区显著高于低温催青区,己3～己5期常温催青区BmPDK的表达呈低-高-低的趋势,而低温催青区则呈上升趋势;蚁蚕期BmPDK表达水平为低温催青区极显著低于常温催青区;蛹期和成虫期卵巢中,常温催青区BmPDK的表达水平极显著高于低温催青区。催青温度对二化性家蚕BmPDK的表达的影响主要出现在胚胎戊2期、胚胎己3期～蚁蚕期、蛹期、成虫期。  相似文献   

18.
Six techniques for recovering unembryonated Toxocara canis eggs from sand samples were tested for efficiency and suitability for routine use. The tests were done under standardised conditions on 50g of sand samples contaminated experimentally with 10, 100 and 500 eggs of T. canis. Best result was achieved by the method of Dunsmore et al. [Dunsmore, J.D., Thompson, R.C.A., Bates, I.A., 1984. Vet. Parasitol. 16, 303-311]. The results were expressed as the number of T. canis eggs recorded and percentage rates of recovery in sand samples.  相似文献   

19.
Two strains of Rhipicephalus sanguineus acquired Ehrlichia canis by feeding as either larvae or nymphs on acutely infected dogs and, in subsequent instars, transmitted the agent to normal dogs. Three strains of R sanguineus transmitted E canis as adults after their larval and nymphal stages fed on infected dogs. More than 400 adult female ticks were fed on infected dogs as larvae or nymphs or both, but none transmitted E canis transovarially.  相似文献   

20.
克隆了犬布氏杆菌外膜蛋白Omp31基因并构建原核表达系统,并对表达产物进行了初步的血清学鉴定。利用PCR技术扩增犬布氏杆菌RM6/66参考株Omp31基因,然后将其克隆到pGEMT-easy载体上进行测序。测序正确后,将该基因插入到pET-32a载体中构建原核表达载体,转化大肠杆菌BL21感受态细胞,诱导表达融合蛋白,Western blot分析融合蛋白的免疫反应性。结果构建了犬布氏杆菌Omp31基因的原核表达载体pET-Omp31,并且在大肠杆菌中成功表达融合蛋白,经Western Blot鉴定该蛋白能被犬布氏杆菌阳性血清所识别。犬布氏杆菌外膜蛋白Omp31的表达成功,为犬布氏杆菌病血清学诊断方法的建立提供了基础资料。  相似文献   

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