堆形艾美球虫广东株3-1E基因在毕赤酵母中的表达

以重组质粒pGEM-3-1E为模板，扩增了序列两端分别含有EcoRⅠ和XbaⅠ酶切位点的堆形艾美球虫（Eimeria acervulina）广东株3-1E基因（长度为529bp），将3-1E基因克隆至巴斯德毕赤酵母分泌型表达载体pPICZαC中，构建了酵母表达质粒pPICZαC-3-1E。转化毕赤酵母X-33得到含有3-1E基因的重组酵母，甲醇诱导产生的目的蛋白经SDS-PAGE分析和免疫印迹检测，表明毕赤酵母成功表达了3-1E基因。     

鼠层粘蛋白α5链G结构域E3和E8基因在毕赤酵母中的表达

为探索层粘连蛋白G结构域在甲醇型酵母（Pichia Yeast）中的表达，用PCR的方法获得了鼠层粘蛋白α5链G结构域的E3（LG4-5）和E8（LG1-3）基因片段，并将其克隆到P．pastoris分泌型表达载体pPIC9中，构建了pPIC9-E3和pPIC9-E8等2个毕赤酵母重组质粒。将测序正确的质粒用电转化的方法转化酵母表达受体菌GS115。通过表型和PCR的方法筛选出P.pastoris重组子。用甲醇诱导表达后．进行SDS-PAGE和Western-blot鉴定。结果表明，鼠层粘蛋白α5链G结构域的E3和E8片段在毕赤酵母中获得了成功的表达。     

E.tenella河北株EtMIC-2基因的克隆及毕赤酵母系统表达

为表达鸡柔嫩艾美耳球虫(E.tenella)河北株EtMIC-2蛋白,本试验采用RT-PCR方法克隆出E.tenella河北株EtMIC-2基因,连接到毕赤酵母分泌表达载体pPIC9中,构建重组表达载体pPIC9-EtMIC-2。将其线化后转化到巴斯德毕赤酵母GS115中,甲醇诱导表达,饱和硫酸铵4℃沉淀浓缩后,用His选择镍-亲和层析柱纯化EtMIC-2蛋白。采用SDS-PAGE和Western blot方法验证其蛋白的表达。结果表明,构建的重组表达载体pPIC9-EtMIC-2在毕赤酵母菌中表达出相对分子质量约为45 000的目的蛋白,表达的EtMIC-2蛋白能被E.tenella阳性血清识别,具有良好的免疫反应原性。为进一步研究EtMIC-2蛋白功能及构建DNA疫苗奠定基础。     

鸡IL-18酵母表达载体的构建及表达条件的优化

应用PCR技术从含有罗曼鸡白介素18全长基因质粒pMD18-T-ChIL-18中扩增出鸡IL-18成熟肽基因,亚克隆于毕赤酵母表达载体pPICZαA上,构建了重组质粒pPICZαA-ChIL-18。经酶切、PCR和测序鉴定正确后,电转化入毕赤酵母菌X-33,筛选多拷贝单克隆进行诱导表达,并进一步分析了培养液pH值、诱导剂浓度、诱导时间和诱导温度对重组菌表达水平的影响,从而获得最优化的表达条件。结果表明,重组毕赤酵母菌能够表达鸡IL-18,且在培养液pH6.0,甲醇诱导浓度1.5%,诱导温度26℃的条件下诱导96 h,目的蛋白的表达量最高,可占菌体总蛋白的60.2%。本文为鸡IL-18的规模化发酵生产奠定了基础。     

毕赤酵母(Pichia pastoris )分泌表达牛白细胞介素2

利用舍有强启动子PAOX1和α-因子信号肽序列的巴斯德毕赤酵母载体pPICZαA，构建出舍牛白细胞介素2(BoIL-2)基因的重组质粒BoIL2-pPICZαA。线性化的重组表达栽体转化到巴斯德毕赤酵母X-33及KM71H中，筛选Zeoein高抗性酵母菌株，甲醇诱导目的蛋白表达。经SDS-PAGE和Western blot检测表明，BoIL-2以融合蛋白形式在胞内表达，但没能分泌到胞外。通过BoIL-2在巴斯德毕赤酵母中的表达，重点讨论了信号肽、基因的偏爱性等对外源基因分泌表达的影响。     

毕赤酵母工程菌株表达人溶菌酶-鲎素融合蛋白发酵条件的优化

能分泌表达重组人溶茵酶-鲎素融合蛋白的巴斯德毕赤酵母(Pichia pastoris)工程菌(X33/pPICZαA-human lysozyme-Tachyplesin Ⅰ构建的).除了基因本身内在特性外,表达条件对外源基因的表达量也有着极显著的影响.本文在摇瓶水平上通过发酵液起始pH、甲醇诱导浓度、诱导时间这3个因...     

猪瘟病毒E2基因在毕赤酵母中的表达

猪瘟是严重危害养猪业的一种烈性传染病,病死率高,被世界动物卫生组织(OIE)列为必须报告的动物疫病。E2蛋白是猪瘟病毒中最主要的保护性抗原,因此,围绕E2的基因工程疫苗研究已成为热点。本研究以含有猪瘟E2基因的重组质粒pMD18-T-E2为模板,设计一对特异性引物扩增去除跨膜区的E2基因,将PCR产物插入巴斯德毕赤酵母表达载体pPICZαA中,构建重组质粒pPICZαA-E2,将该质粒用SacⅠ酶切线性化后,电穿孔导入巴斯德毕赤酵母X-33中,经Zeocin筛选得到高拷贝转化子,通过甲醇诱导表达、SDS-PAGE和Western bolt试验验证,结果表明E2蛋白在酵母中获得成功表达。     

微小牛蜱Bm86基因的真核表达

采用RT-PCR技术从微小牛蜱饥饿幼蜱破解物中扩增到Bm86基因，将其与巴斯德毕赤酵母分泌型表达载体pPlC9K重组构建了重组表达载体pPIC9K-Bm86，测序正确后将其用SacⅠ内切酶线性化后采用电穿孔法转化巴斯德毕赤酵母菌Gs115，经G418抗性筛选高拷贝重组菌株后用甲醇诱导表达，SDSPAGE和Western-blotting分析结果表明，诱导表达的培养上清液中表达出具有反应活性的68ku重组Bm86蛋白，目的蛋白约占培养上清液中蛋白总量的32％以上，诱导96h目的蛋白的表达量为0．36mg／mL。     

土耳其斯坦东毕吸虫磷酸丙糖异构酶基因的克隆及在毕赤酵母中的表达

利用RT-PCR技术从土耳其斯坦东毕吸虫（Orientobilharzia turkestanicum）成虫总RNA中扩增磷酸丙糖异构酶基因（TPI），鉴定后将目的片段与毕赤酵母表达载体pPIC9k连接，构建重组表达质粒pPIC9k-TPI，并将其电击转化到毕赤酵母GS115中，重组菌株经甲醇诱导后表达的TPI蛋白，经SDS-PAGE、Western-blotting检测，并利用葡聚糖凝胶层析柱纯化。结果显示，成功地克隆了土耳其斯坦东毕吸虫TPI；重组毕赤酵母表达了分子质量为43ku的TPI蛋白；葡聚糖凝胶层析过滤得到单一的TPI蛋白。     

猪圆环病毒2型Cap蛋白毕赤酵母分泌表达及发酵条件优化

为实现猪圆环病毒2型(PCV2)Cap蛋白在酵母中的高效表达,本研究将去除信号肽并经序列优化后的PCV2 ORF2片段插入pPICZαA表达载体,将重组表达质粒克隆转化巴斯德毕赤酵母(Pichia pastoris)X33感受态细胞中,应用Zeocin抗性和PCR法对转化子进行初步筛选和表型鉴定,利用"双膜法"筛选出PCV2 Cap表达量相对较高的酵母。采用正交试验对优选的酵母进行发酵试验,分析培养基pH、甲醇诱导时间、甲醇诱导浓度和诱导时酵母浓度对PCV2重组Cap蛋白(rCap)分泌表达的影响。结果显示酵母分泌表达PCV2 rCap的优化条件为:培养基pH为5.0,发酵液OD600nm至1.0,加入终浓度1.0%甲醇诱导96 h。优化后的PCV2 rCap分泌量可达207.58μg/m L。中和试验结果显示,发酵产物PCV2 rCap免疫小鼠产生的抗体可有效中和PCV2。本研究为利用酵母表达PCV2 Cap进行疫苗研制奠定了基础。     

Phase I evaluation of CCNU (lomustine) in tumor-bearing cats

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.     

Furosemide continuous rate infusion in the horse: evaluation of enhanced efficacy and reduced side effects

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.     

Prevalence of gastric lesions in racing Alaskan sled dogs

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.     

The Epidemiology of Gastrointestinal Nematodes of Dairy Cattle in Central Kenya

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.     

Diversity of Ectoparasites in Sheep Flocks in Sa~o Paulo,Brazil

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.     

Evaluation of biohydrogenation rate of canola vs. soya bean seeds as unsaturated fatty acids sources for ruminants in situ

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.     

Serum biochemical values and mineral element contents of tissues in yaks

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     

Avian brood parasitism — a growing research area in behavioral ecology (part Ⅱ)

正We are pleased to publish the second special issue on avian brood parasitism and to be responsible guest editors for the two special issues of Chinese Birds (Vol. 3, No. 4, 2012 and Vol. 4, No. 1, 2013), entitled "Avian Brood Parasitism - A Growing Research Area in Behavioral Ecology". The first issue was published in December 2012. The goal of the two special issues is to publish accumulated knowledge and some of the recent developments in     

Internal Parasites and Haematological Values in Cattle Slaughtered in Buea Subdivision of Cameroon

Tropical Animal Health and Production -