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1.
为研究马链球菌马亚种FNEB蛋白的免疫保护性,本研究采用PCR方法从马链球菌马亚种新疆分离株中扩增FNEB基因部分片段,克隆于原核表达载体p ET-30a中,转化大肠杆菌BL21(DE3)感受态细胞,以IPTG诱导表达,经SDS-PAGE和western blot分析显示,重组蛋白大小约60 ku,具有良好的抗原性。将纯化的重组蛋白免疫小鼠后,以马链球菌马亚种新疆分离株进行攻毒,结果显示该重组蛋白对免疫组小鼠具有保护力,保护率为65%。本研究克隆了马链球菌马亚种的FNEB截短基因并表达了相应重组蛋白,免疫小鼠后能够提供较好的保护,为重组FNEB蛋白亚单位疫苗的研制奠定基础。  相似文献   

2.
目的将小反刍兽疫病毒M蛋白基因截短表达后用于特异性单抗制备及临床抗体水平检测。方法:用在线分析软件BepiPred分析小反刍兽疫病毒M蛋白潜在的B细胞线性表位,以本实验室构建的pCR2.1T-PPRV M质粒为模板,扩增三段截短的M基因,纯化后的PCR产物分别与克隆载体pCR2.1T连接,筛选出的阳性重组质粒经双酶切后,分别与表达载体pET-32a(+)及pGEX-6p-1连接,再将鉴定为阳性的重组质粒转化入E.coli BL21(DE3)菌株诱导表达,并用SDS-PAGE及Western blot验证。结果 PCR产物电泳,得到与预期大小相符的特异性片段。对连接克隆载体及表达载体的重组质粒双酶切后,均出现与预期一致的片段,DNA测序表明,插入片段序列与小反刍兽疫Nigeria75/1株M蛋白基因完全一致。重组蛋白经SDS-PAGE及Western blot鉴定,证明所构建的重组蛋白均获得高效表达,并均具有良好的反应原性。结论成功表达了小反刍兽疫截短M基因的蛋白,为制备特异性单抗及小反刍兽疫抗体检测奠定了一定基础。  相似文献   

3.
根据GenBank登录的非洲猪瘟病毒(ASFV) VP73基因序列,人工合成VP73主要抗原表位区基因片段,克隆至pUC57载体中.设计1对引物,PCR扩增获得429 bp的VP73基因片段;将VP73基因片段使用EcoRI和SalI酶切后亚克隆至表达载体pGEX-KG,经PCR、酶切、测序鉴定挑选阳性克隆,转化至表达菌株BL21 (DM3)中,进行体外诱导表达,SDS-PAGE和Western blot分析蛋白表达及其活性.结果显示,成功构建表达重组载体pGEX-KG-VP73,对该重组载体进行诱导,有效表达了41 ku重组蛋白,表达量约占菌体总蛋白的17％,Western blot分析证实该蛋白具有良好反应原性.  相似文献   

4.
新城疫病毒F基因在大肠埃希菌中的表达及其抗原性分析   总被引：2，自引：0，他引：2  
构建新城疫病毒融合蛋白F基因的原核表达载体,并将其在宿主菌BL21(DE3)感受细胞中表达.以含有融合蛋白F基因的重组质粒pMD19T-F为模板,设计特异性引物,应用PCR技术扩增获得F基因的F1片段,定向插入原核表达载体pET-28a,构建重组质粒pET-F1,将构建成功的重组质粒pET-F1转化宿主菌BL21(DE3)感受态细胞经IPTG诱导,将其在宿主菌细胞中表达,表达产物用SDS-PAGE和Western blot方法检测.结果显示,成功克隆出了新城疫病毒F1基因片段序列852 bp.构建的pET-F1载体经PCR、双酶切、测序鉴定均无误,转化表达宿主菌后经SDS-PAGE和Western blot检测结果显示,目的基因被成功表达,并具有良好的反应原性.成功构建了新城疫病毒的融合蛋白F基因原核表达载体,转化宿主细胞后成功表达了融合蛋白F1重组蛋白片段.  相似文献   

5.
构建截短的弓形虫表面抗原2(SAG2)原核表达系统,并探讨其抗原活性。PCR扩增去掉信号肽和C-末端的SAG2基因片段,插入原核表达载体pGEX-4T-3后转化到大肠杆菌DH5α,并用IPTG诱导。SDS-PAGE和Western blot鉴定重组SAG2t的表达及其免疫反应原性。每升菌液约纯化出4mg rSAG2t蛋白;Western blot显示,ME49株感染的鼠血清和rSAG2t免疫的鼠血清均可强烈识别表达的截短SAG2蛋白。原核表达体系实现了截短的SAG2蛋白的可溶性表达,并具有良好的完全抗原活性。  相似文献   

6.
为了重组表达乳房链球菌表面脱氢酶GAPC基因,为免疫学研究提供目标蛋白,用PCR方法从石河子分离株的基因组DNA中扩增出GAPC基因,用T/A克隆法将其插入PBST载体,并构建原核表达载体pET-32a（＋）-GAPC。用BL21（DE3）/pET系统表达Trix-GAPC融合蛋白,SDS-PAGE和Western blot分析鉴定表达产物。结果表明,PCR扩增产物经测序,证实与GenBank中乳房链球菌（AF421900.1）GAPC的基因序列同源性为99%。SDS-PAGE显示,经IPTG诱导后BL21（DE3）/pET-32a（＋）-GAP总蛋白中出现一条分子质量为55ku的新蛋白带。Western blot分析显示,GAPC蛋白可与乳房链球菌多克隆抗血清发生特异性反应。该研究已成功表达了GAPC,为GAPC在细菌致病中作用的研究以及相关疫苗的制备奠定了基础。  相似文献   

7.
根据GenBank发表的细粒棘球绦虫G1型Eg95序列合成Eg95抗原基因(HM345604.1),设计并合成一对引物,采用PCR技术扩增Eg95抗原基因,亚克隆到pET-28a原核表达载体,并在大肠埃希菌BL21(DE3)中表达重组蛋白。经IPTG诱导,SDS-PAGE分析,Eg95抗原蛋白可以在大肠埃希菌中高效表达,表达重组蛋白的分子质量约为16.5ku。Western blot结果表明,该蛋白可与阳性血清发生特异反应,具有很好的反应原性。  相似文献   

8.
采用RT—PCR方法从EAVBucyrus株扩增了截短的GL、M和全长的N基因片段,将三者分别克隆到表达载体pGEX-6p-1中,测序验证后转化Rosetta（DE3）宿主菌中经IPTG诱导表达,诱导后菌体裂解物经SDS-PAGE分析。试验结果表明,重组菌表达出约35000、34000和38000的特异性条带,通过条件优化及切胶纯化获得较高浓度的目的蛋白。经Western-blot和间接ELISA分析,纯化的蛋白只与抗马动脉炎病毒阳性血清发生特异性反应,证实该蛋白具有良好的反应原性和特异性。  相似文献   

9.
为了制备猪流行性腹泻病毒（Porcine epidemic diarrhea virus,PEDV）N蛋白,通过PCR扩增获得PEDV的N基因片段与载体pET-32a（+）相连构建原核表达载体pET-32a（+）-PEDV-N。将构建成功的质粒转化到BL21（DE3）感受态细胞,经IPTG（1 mmol/mL）37℃诱导表达后,采用SDS-PAGE检测重组蛋白的表达情况,对影响重组蛋白表达的诱导时间进行分析。利用Western blot方法分别检测重组蛋白的免疫原性。结果表明：PCR产物通过琼脂糖凝胶电泳检测发现条带位置正确,重组质粒pET-32a（+）-PEDV-N测序正确,含有重组质粒pET-32a（+）-PEDV-N的表达菌株BL21（DE3）在诱导表达后,经SDS-PAGE检测证明有重组蛋白表达,利用Western blot结果表明该重组蛋白与PEDV多克隆抗体发生特异性反应,说明成功制备了PEDV-N重组蛋白。  相似文献   

10.
本研究扩增和克隆了堆形艾美耳球虫巨噬细胞移动抑制因子,并在大肠杆菌中表达出MIF重组蛋白。根据GenBank中的MIF mRNA序列,设计特异引物,采用PCR方法以堆形艾美耳球虫孢子化卵囊cDNA文库为模板扩增获得MIF基因,将MIF基因片段连接到原核表达载体pET-28a（＋）,构建重组表达质粒pET28a—MIF,并在大肠杆菌BL21（DE3）中诱导表达,对表达产物进行Western blot分析。结果从堆形艾美耳球虫孢子化卵囊cDNA文库中扩增出MIF基因片段,长度为709bp;经序列分析,扩增片段的核苷酸序列与GenBank中登录的MIF序列的同源性为99．5％;经SDS-PAGE检测表明重组质粒pET（28a）-MIF在大肠杆菌中以包涵体形式表达;Western blot分析证明堆形艾美耳球虫抗血清可与重组表达蛋白特异性结合。本研究结果为进一步研究该基因的功能奠定了基础。  相似文献   

11.
试验选用96头平均体重14.82 kg左右的杜×长×大断奶仔猪,随机分成4组,每组3栏,每栏8头(公母各半)。对照组饲喂基础日粮,试验1、2、3组分别添加1%80目白术、0.2%白术多糖和1%微米白术。试验期30 d。结果表明:在生长性能方面,与对照组相比,1%微米白术添加组可显著提高日增重(P0.05)、降低饲料增重比和腹泻率,而且效果优于1%80目白术组和0.2%白术多糖组,在肠道形态和肠道微生态区系方面,与对照组相比,日粮添加1%80目白术、0.2%白术多糖、1%微米白术均可不同程度的提高十二指肠和空肠的绒毛高度,加深十二指肠和空肠的隐窝深度,并且增加肠道微生态区系的多样性,其中以1%微米白术添加组的效果最佳。  相似文献   

12.
Sissay, M.M., Uggla, A. and Waller, P.J., XXXX. Prevalence and seasonal incidence of nematode parasites and fluke infections of sheep and goats in eastern Ethiopia. Tropical Animal Health and Production, XXXX. A 2-year abattoir survey was carried out to determine the prevalence, abundance and seasonal incidence of gastro-intestinal (GI) nematodes and trematodes (flukes) of sheep and goats in the semi-arid zone of eastern Ethiopia. During May 2003 to April 2005, viscera including liver, lungs and GI tracts were collected from 655 sheep and 632 goats slaughtered at 4 abattoirs located in the towns of Haramaya, Harar, Dire Dawa and Jijiga in eastern Ethiopia. All animals were raised in the farming areas located within the community boundaries for each town. Collected materials were transported within 24 h to the parasitology laboratory of Haramaya University for immediate processing. Thirteen species belonging to 9 genera of GI nematodes (Haemonchus contortus, Trichostrongylus axei, T. colubriformis, T. vitrinus, Nematodirus filicollis, N. spathiger, Oesophagostomum columbianum, O. venulosum, Strongyloides papillosus, Bunostomum trigonocephalum, Trichuris ovis, Cooperia curticei and Chabertia ovina), and 4 species belonging to 3 genera of trematodes (Fasciola hepatica, F. gigantica, Paramphistomum {Calicohoron} microbothrium and Dicrocoelium dendriticum) were recorded in both sheep and goats. All animals in this investigation were infected with multiple species to varying degrees. The mean burdens of adult nematodes were generally moderate in both sheep and goats and showed patterns of seasonal abundance that corresponded with the bi-modal annual rainfall pattern, with highest burdens around the middle of the rainy season. In both sheep and goats there were significant differences in the mean worm burdens and abundance of the different nematode species between the four geographic locations, with worm burdens in the Haramaya and Harar areas greater than those observed in the Dire Dawa and Jijiga locations. Similar seasonal variations were also observed in the prevalence of flukes. But there were no significant differences in the prevalence of each fluke species between the four locations. Overall, the results showed that Haemonchus, Trichostrongylus, Nematodirus, Oesophagostomum, Fasciola and Paramphistomum species were the most abundant helminth parasites of sheep and goats in eastern Ethiopia.  相似文献   

13.
1-(2-Chloroethyl)3-cyclohexyl-1-nitrosourea (CCNU) is an alkylating agent in the nitrosourea subclass. A prospective evaluation of CCNU was done to determine the maximally tolerated dosage of CCNU in tumor-bearing cats. Response data were obtained when available. Twenty-five cats were treated with CCNU at a dosage of 50-60 mg/m3 body surface area. Complete hematologic data were available for 13 cats. Neutropenia was the acute dose-limiting toxicity. The median neutrophil count at the nadir was 1,000 cells/microL (mean, 2,433 cells/microL; range, 0-9,694 cells/microL). The time of neutrophil nadir was variable, occurring 7-28 days after treatment, and counts sometimes did not return to normal for up to 14 days after the nadir. Based on these findings, a 6-week dosing interval and weekly hematologic monitoring after the 1st treatment with CCNU are recommended. The nadir of the platelet count may occur 14-21 days after treatment. The median platelet count at the nadir was 43,500 cells/microL. No gastrointestinal, renal, or hepatic toxicities were observed after a single CCNU treatment, and additional studies to evaluate the potential for cumulative toxicity should be performed. Five cats with lymphoma and 1 cat with mast cell tumor had measurable responses to CCNU. Phase II studies to evaluate antitumor activity should be completed with a dosing regimen of 50-60 mg/m3 every 6 weeks.  相似文献   

14.
Continuous rate infusion (CRI) of furosemide in humans is considered superior to intermittent administration (IA). This study examined whether furosemide CRI, compared with IA, would increase diuretic efficacy with decreased fluid and electrolyte fluctuations and activation of the renin-angiotensin-aldosterone system (RAAS) in the horse. Five mares were used in a crossover-design study. During a 24-hour period, each horse received a total of 3 mg/kg furosemide by either CRI (0.12 mg/kg/h preceded by a loading dose of 0.12 mg/kg IV) or IA (1 mg/kg IV q8h). There was not a statistically significant difference in urine volume over 24 hours between methods; however, urine volume was significantly greater after CRI compared with IA during the first 8 hours ([median 25th percentile, 75th percentile]: 9.6 L [8.9, 14.4] for CRI versus 5.9 L [5.3, 6.0] for IA). CRI produced a more uniform urine flow, decreased fluctuations in plasma volume, and suppressed renal concentrating ability throughout the infusion period. Potassium, Ca, and Cl excretion was greater during CRI than IA (1,133 mmol [1.110, 1,229] versus 764 mmol [709, 904], 102.7 mmol [96.0, 117.2] versus 73.3 mmol [65.0, 73.5], and 1,776 mmol [1,657, 2.378] versus 1,596 mmol [1,457, 1,767], respectively). Elimination half-lives of furosemide were 1.35 and 0.47 hours for CRI and IA, respectively. The area under the excretion rate curve was 1,285.7 and 184.2 mL x mg/mL for CRI and IA, respectively. Furosemide CRI (0.12 mg/kg/h) for 8 hours, preceded by a loading dose (0.12 mg/kg), is recommended when profound diuresis is needed acutely in horses.  相似文献   

15.
Genetic variations in chromosome Y are enabling researchers to identify paternal lineages, which are informative for introgressions and migrations. In this study, the male‐specific region markers, sex‐determining region‐Y (SRY), amelogenin (AMELY) and zinc finger (ZFY) were analysed in seven Turkish native goat breeds, Angora, Kilis, Hair, Honaml?, Norduz, Gürcü and Abaza. A SNP in the ZFY gene defined a new haplotype Y2C. All domestic haplogroups originate from Capra aegagrus, while the finding of Y1A, Y1B, Y2A and Y2C in 32, 4, 126 and 2 Turkish domestic goats, respectively, appears to indicate a predomestic origin of the major haplotypes. The occurrence of four haplotypes in the Hair goat and, in contrast, a frequency of 96% of Y1A in the Kilis breed illustrate that Y‐chromosomal variants have a more breed‐dependent distribution than mitochondrial or autosomal DNA. This probably reflects male founder effects, but a role in adaptation cannot be excluded.  相似文献   

16.
The epidemiology of H. placei and of other gastrointestinal nematodes in yearling dairy cattle was examined on two farms in Kiambu District, central Kenya during each of 13 one-month periods from April 1993 to April 1994. On each farm, 32 newly weaned dairy calves were given a single dose of albendazole and then placed on experimental pastures. Twelve of the animals were designated for bi-monthly slaughter (n = 2) and analysis of worm population characteristics and 20 were designated for blood and faecal collection and for weighing. Two parasite-free tracer calves were grazed alongside the weaner calves each month throughout the study period and were also slaughtered for analysis of worm populations. Faecal egg counts, haematological and serum pepsinogen determinations, herbage larval counts, and animal live weight changes were recorded monthly. The study revealed that Haemonchus placei, Trichostrongylus axei, Cooperia spp. and Oesophagostomum radiatum were responsible for parasitic gastroenteritis and that H. placei was the predominant nematode present in the young cattle on both farms. Faecal egg counts from resident cattle and necropsy worm counts revealed that pasture larval levels were directly related to the amount of rainfall. The total worm burdens in the animals were highest during the rainy season (March–June and October–December) and lowest during the dry seasons (July–September and January–February). The very low recovery of immature larvae of H. placei from the tracer calves indicated that arrested development is not a feature of the life cycle of this parasite in central Kenya. The maintenance of the parasite population depended on continuous cycling of infection between the host and the pasture. The agroclimatic conditions of the study area were such that, in general, favourable weather conditions for the development and survival of the free-living stages of gastrointestinal nematodes existed all year round.  相似文献   

17.
The occurrence of ectoparasites in sheep flocks is frequently reported but seldom quantified. Sheep production used to be a predominantly family activity in the state of Sa~o Paulo (Brazil), but it began to become a commercial activity in the past decade. Thus, information about the ectoparasites existing in sheep flocks has become necessary. The present data were obtained by means of questionnaires sent to all sheep breeders belonging to the `Associaça~o Paulista de Criadores de Ovinos' (ASPACO; Sa~o Paulo State Association of Sheep Breeders). Response reliability was tested by means of random visits paid to 10.6% of the respondents. Most of the properties (89.5%) reported the presence of one or more ectoparasites. Screw-worm (Cochliomyia hominivorax) was the most frequent ectoparasite (72.5%), followed by bot fly larvae (Dermatobia hominis, 45.0%), ticks (Amblyomma cajennense) and Boophilus microplus, 31.3%) and finally lice (Damalinia ovis, 13.8%). Combined infestations also occurred, the most common one being screw-worm with bot fly larvae (36.0%) followed by bot fly larvae with ticks (13.9%), screw-worm with ticks (9.3%), bot fly larvae with lice (6.9%), and ticks with lice (5.0%). The most common triple combination was screw-worm, bot fly larvae and ticks (12.8%). Breeds raised for meat or wool were attacked by bot fly larvae and ticks more often than other breeds. Lice were only absent from animals of indigenous breeds. The relationships among these ectoparasites are discussed in terms of sheep breeds, flock size, seasonality and the ectoparasitic combinations on the host.  相似文献   

18.
The purpose of this study was to determine the frequency of different tumor types within a large cohort of cats with intracranial neoplasia and to attempt to correlate signalment, tumor size and location, and survival time for each tumor. Medical records of 160 cats with confirmed intracranial neoplasia evaluated between 1985 and 2001 were reviewed. Parameters evaluated included age, sex, breed, FeLV/FIV status, clinical signs, duration of signs, number of tumors, tumor location(s), imaging results, treatment, survival times, and histopathologic diagnosis. Most of the cats were older (11.3 +/- 3.8 years). Primary tumors accounted for 70.6% of cases. Metastasis and direct extension of secondary tumors accounted for only 5.6 and 3.8% of cases, respectively. Twelve cats (7.5%) had 2 or more discrete tumors of the same type, whereas 16 cats (10.0%) had 2 different types of intracranial tumors. The most common tumor types were meningioma (n = 93, 58.1%), lymphoma (n = 23, 14.4%), pituitary tumors (n = 14, 8.8%), and gliomas (n = 12, 7.5%). The most common neurological signs were altered consciousness (n = 42, 26.2%), circling (n = 36, 22.5%), and seizures (n = 36, 22.5%). Cats without specific neurological signs were common (n = 34, 21.2%). The tumor was considered an incidental finding in 30 (18.8%) cats. In addition to expected relationships (eg, meninges and meningioma, pituitary and pituitary tumors), we found that lesion location was predictive of tumor type with diffuse cerebral or brainstem involvement predictive of lymphoma and third ventricle involvement predictive of meningioma.  相似文献   

19.
Comparison of different tests to diagnose feline infectious peritonitis   总被引：1，自引：0，他引：1  
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20.
An experiment was conducted to study disappearance of C14 to C18 fatty acids, lag times and biohydrogenation (BH) rates of C18 fatty acids of ground soya bean and canola seeds in situ. Three ruminally fistulated Dallagh sheep were used to determine ruminal BH of unsaturated fatty acids (UFAs). Differences in the disappearance of fatty acids through the bags and lag times were observed between the oilseeds. We saw that the longer the incubation time of the oilseeds in the rumen, the lower the content of C18:2 and C18:3. Significantly higher lag times for both C18:2 and C18:3 were observed in ground canola compared to ground soya bean. BH rates of C18:2 and C18:3 fatty acids in soya bean were three times higher than those of canola. These results suggest that the fatty acid profile of fat source can affect the BH of UFAs by rumen micro‐organisms. So that UFAs of canola had higher ability to escape from ruminal BH. It seems that fatty acid profile of ruminant products is more affected by canola seed compared to soya bean seed.  相似文献   

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