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1.
设计1对特异性引物对羊布鲁菌16M总DNA进行外膜蛋白omp10的PCR扩增,得到了一个大小为330 bp的目的基因片段(去掉17个氨基酸编码的信号肽),测序证实它与国外报道的羊布鲁菌omp10基因完全一致.将其克隆到表达载体PET-30a中,经酶切、PCR扩增和测序分析,表明重组表达载体构建成功.将此重组质粒转化入大肠埃希菌BL21(DE3)中,IPTG诱导表达,该基因以包涵体的形式在大肠埃希菌中表达,经过包涵体的变性、复性和亲和层析纯化,成功获得大小为14.2 ku的融合蛋白,与理论推测的蛋白分子质量一致;Western blot和间接ELISA试验证明,纯化之后的OMP10重组蛋白可以被布鲁菌阳性血清识别.  相似文献   

2.
新疆绵羊布鲁氏菌外膜蛋白OMP2b的克隆与表达   总被引：2，自引：0，他引：2  
表达新疆绵羊布鲁氏菌外膜抗原蛋白OMP3b的表达蛋白OMP2b，探索其作为诊断抗原和亚单位疫苗的可能性。采用PCR方法，从新疆绵羊布鲁氏菌基因组DNA中扩增出omp2b基因片段，将该片段克隆于原核表达载体PET-28a（＋），构建成重组质粒，经IPTG诱导，SDS-PAGE检测。结果表明，获得长约1083bp的PCR片段，序列分析结果与已知绵羊布鲁氏菌外膜蛋白OMP2b同源性达89.72％；SDS-PAGE检测表达产物，在相对分子质量39ku处有表达带。获得了新疆绵羊布鲁氏菌外膜蛋白OMP36的表达蛋白omp2b基因片段，并在大肠杆菌中实现了表达。  相似文献   

3.
根据已报道的绵羊种布鲁氏茵外膜蛋白基因omp2b的核酸序列设计引物，从新疆绵羊种布鲁氏菌基因组中扩增到了omp2b基因。将该片段克隆到PBS—T载体上，并对所得到的重组质粒进行酶切分析、PCR鉴定，证明所得到的片段为阳性重组子。与报道的绵羊种布鲁氏菌omp2b序列有89．72％的同源性，并在N端存在高度保守区。至此得到omp2b基因的全长克隆，通过对所克隆的基因和其他布鲁氏菌外膜蛋白omp2b的进化树分析，发现其和牛种布鲁氏茵的亲缘关系最近，同源性较高。  相似文献   

4.
用RT-PCR技术对猪瘟兔化弱毒C株、石门强毒株(Shimen)、近期地方流行的属于第1群毒株的新疆毒株(XJ)和第2群代表毒株甘肃临洮(LT)株E2基因的主要抗原区进行扩增，均得到约561bp的基因片段；经克隆测序及序列分析，该基因编码187个氨基酸残基，预计蛋白分子质量为21.7ku。将这4个基因分别克隆到原核表达载体pGEX-4T-1中，经酶切、PCR扩增和测序分析确证其正确插入到表达载体中，阅读框是正确的，从而构建成重组表达载体。重组质粒转化至宿主菌BL21(DE3)中，用异丙基硫代-β-半乳糖苷(IPTG)进行诱导表达，对表达的蛋白用SDSPAGE电泳、免疫印迹和薄层凝胶扫描分析。结果表明，E2基因的主要抗原区可以在大肠埃希氏菌中表达，表达的蛋白多以包涵体形式存在，表达产物的分子质量约为50．0ku，与理论推测的分子质量一致；薄层凝胶扫描分析表明。表达蛋白约占菌体蛋白总量的20．1％；免疫印迹结果证明，表达的E2蛋白可被猪瘟阳性血清所识别。  相似文献   

5.
为在大肠杆菌中表达马耳他布鲁菌omp25基因并鉴定重组蛋白的抗原性,从马耳他布鲁菌中用聚合酶链反应技术(PCR)扩增得到布鲁菌omp25基因片段,并将目的基因插入原核表达载体pET-32a中,构建重组质粒pET-32a-omp25转入大肠杆菌Rosetta中表达,用SDS-PAGE和Western-blot检测表达蛋白。结果显示成功构建了重组质粒pET-32a-omp25,并在大肠杆菌Rosetta中获得了重组蛋白,重组蛋白与布鲁菌阳性血清发生特异性反应。表明重组质粒pET-32a-omp25可以在大肠杆菌Rosetta中成功表达,并且重组蛋白可与布鲁菌阳性血清发生特异性反应,说明该重组蛋白有良好的免疫原性,该研究为以后疫苗的研制及布鲁菌病的检测打下良好的基础。  相似文献   

6.
本试验以布鲁氏菌外膜蛋白22（omp22）基因作为研究对象,通过基因序列克隆、表达载体构建、大肠杆菌原核表达与亲和纯化,对蛋白的表达与纯化进行了研究。结果显示,omp22基因的核苷酸序列含有639 bp,编码212个氨基酸残基,预测分子质量为22 ku,电泳结果显示重组omp22蛋白的分子质量为47 ku,与理论值相符。本试验结果为进一步研究重组omp22蛋白的免疫刺激与免疫保护作用奠定了基础。  相似文献   

7.
克隆羊外膜蛋白Omp25基因,在大肠杆菌中表达、纯化,并对Omp25蛋白的抗原性进行分析.以布鲁氏菌染色体DNA模板,扩增Omp25基因,双酶切后克隆至pET32a上,在大肠杆菌ER2566 (DE3)中诱导表达,组氨酸结合树脂柱纯化,Western blotting鉴定Omp25蛋白的免疫原性.将Omp25克隆至载体pET32a,提取的重组质粒经PCR鉴定、双酶切鉴定和测序分析确定目的基因成功插入到了克隆载体中.将重组质粒转化于大肠杆菌ER2566 (DE3)中表达获得HIS融合蛋白,SDS-PAGE分析证明,表达产物为43 000的融合蛋白.Western blotting分析表明,所表达的蛋白具有免疫原性.结果表明,成功地表达并纯化了Omp25蛋白,而且纯化的蛋白具有一定的免疫原性.本试验为进一步研究Omp25蛋白的功能以及寻找布鲁氏菌的诊断性蛋白奠定了基础.  相似文献   

8.
以PCR扩增猪带缃虫TS61抗原基因，与载体pUC18连接后进行测序；并构建了该基因的pGEX-1λT原核表达载体，制备了其原核表达产物，进行浓度梯度SDS－PAGE和免疫印迹分析，对该基因的碱基序列及其编码蛋白进行了同源性比较。结果表明，猪带绦虫TS61抗原基因含893对碱基，编码含70个氨基酸残基的多肽，分子质量为8.0ku，等电点为4.19。在基因库和欧洲分子学实验室数据库中均未发现该基因的同源序列。重组质粒在大肠埃希氏菌中表达的融合蛋白分子质量为34ku，该蛋白抗原能被兔抗猪囊尾蚴超免疫血清所识别。  相似文献   

9.
以表达新疆绵羊种布鲁氏菌外膜抗原蛋白OMP36的表达蛋白OMP2b,探索其作为诊断抗原和分子疫苗的可能性。采用PCR扩增技术,从新疆绵羊种布鲁氏菌基因组DNA中扩增出OMP2b基因片段,将该片段克隆于原核表达载体PE-28a(+),构建成重组质粒,IPTG诱导表达,SDS-PAGE检测有无蛋白的表达。结果表明,获得长约1 083bp的PCR片段,序列分析结果与已知绵羊种OMP2b同源性达89.72%;SDS-PAGE检测表达产物,在相对分子量39 ku获得了新疆绵羊种布鲁氏菌外膜蛋白OMP36的表达蛋白OMP2b基因片段,并在大肠杆菌中实现了表达。  相似文献   

10.
本研究根据GenBank牛瑟氏泰勒虫P33表面蛋白基因设计1对引物,利用全血基因组DNA提取试剂盒提取牛瑟氏泰勒虫全血基因组DNA,PCR扩增得到了大小为786 bp的基因片断,测序分析证明,它与已报道序列的核苷酸同源性可达99%.将该基因与pET-28a(+)载体进行连接,经酶切和PCR鉴定,证明成功构建了含有目的基因片段的重组表达载体pET-28a-P33.将此质粒转化至大肠杆菌Rosetta(DE3)中,用IPTG进行诱导.结果证实,该基因在大肠杆菌中用IPTG诱导4 h时表达量达到高峰,表达产物为分子质量约30.3 ku的融合蛋白,其表达量占菌体总蛋白的43%,以包涵体形式存在;Western blotting试验证明,表达蛋白P33可被牛瑟氏泰勒虫阳性血清所识别,表明该融合蛋白具有较好的反应原性.  相似文献   

11.
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   

12.
Human and equine athletes are reported to have a high prevalence of gastric disease, and anecdotal evidence suggests a similar phenomenon applies to racing sled dogs. To investigate the prevalence of gastric disease in racing sled dogs, we conducted 2 gastroscopy studies on dogs competing in the annual Iditarod Sled Dog Race. A pilot study of dogs that were either dropped from the 2000 Iditarod Sled Dog Race because of illness or that finished the race indicated that, approximately 5 days after competing, 10 of 28 dogs (35%) had endoscopic evidence of gastric ulceration, erosion, or hemorrhage. The next year, an endoscopic study of 73 dogs participating in the 2001 Iditarod race was performed in order to evaluate a larger population of dogs. Data from 70 of these dogs could be used; 34 (48.5%) had ulceration, erosion, gastric hemorrhage, or some combination of these findings. When this group of 70 dogs was compared retrospectively to a control group of 87 dogs presented to the Texas A&M University (TAMU) Veterinary Medical Teaching Hospital, the Iditarod sled dogs had a significantly higher prevalence (P = .049) of gastric lesions. These findings suggest that, similar to athletes of other species, elite canine athletes have an increased prevalence of gastric disease compared to the canine population at large.  相似文献   

13.
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   

14.
Tropical Animal Health and Production -  相似文献   

15.
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   

16.
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   

17.
Our particular attention in this article was given to natural mediators for macrophages isolated from the sites of tissue injury. A number of chemotactic factors, which may satisfy many criteria making them acceptable as inflammatory leucocyte chemotactic factors, has been separated. Among them, our laboratory has isolated three macrophage (monocyte) chemotactic factors (MCF-a, -b and -c). Their purification, characterization and functional specificity are discussed.  相似文献   

18.
Comparison of different tests to diagnose feline infectious peritonitis   总被引：1，自引：0，他引：1  
  相似文献   

19.
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   

20.
Serum biochemical values and the wet weight to dry weight ratios of tissues were determined in yaks in Shandan county of Gansu province. The liver, kidney, heart and muscle contents of seven elements in yaks were also analysed. Most serum biochemical values were similar to those of cattle, camels and sheep, but the calcium concentration was considerably above the normal range for other ruminants. The liver contained the highest concentrations of copper, manganese and cobalt and the kidney of selenium, iron and calcium.Abbreviations Alb albumin - ALT alanine aminotransferase - AST aspartate aminotransferase - BUN blood urea nitrogen - Chol cholesterol - Glob globulin - -GT -glutamyl transpeptidase - IP inorganic phosphorus - TP total protein  相似文献   

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