山地乌骨鸡β防御素Gal-2基因的克隆及酵母表达载体的构建

为了构建山地乌骨鸡β防御素Gal-2基因酵母表达载体,试验根据GenBank中鸡β防御素Gal-2基因序列设计引物,采用RT-PCR技术从山地乌骨鸡骨髓细胞中扩增出Gal-2基因,经PCR和酶切鉴定后测序,并采用DNAStar软件进行生物信息学分析;再根据毕赤酵母偏爱密码子和山地乌骨鸡β-防御素Gal-2成熟肽编码基因序列设计并合成1对引物,采用PCR技术从重组质粒pMD18-T-Gal-2扩增出其成熟肽编码基因,并将其插入载体pPICZaA,构建重组质粒pPICZaA-Gal-2,将pPICZaA-Gal-2线性化,通过电击转入毕赤酵母菌株GS115,用1%甲醇诱导表达120 h,表达产物纯化后进行Tricine-SDS-PAGE电泳鉴定。结果表明:山地乌骨鸡β防御素Gal-2基因克隆成功;山地乌骨鸡β-防御素Gal-2成熟肽编码基因重组真核表达载体构建成功;山地乌骨鸡Gal-2基因在酵母中得到表达。     

禽流感病毒NA基因在巴斯德毕赤酵母系统中的表达

采用PCR扩增禽流感病毒(AIV)NA基因，克隆入pMD18-T载体中，再亚克隆入含有AOX1启动子和α分泌信号肽序列的巴斯德毕赤酵母(Pichia pastoris)表达载体pPIC9k中，构建了重组转移载体pPIC9kNA。经电穿孔转化酵母宿主菌GSll5和筛选高拷贝重组转化子及筛选His^ Mut^ 表型转化子后，摇瓶培养，10mL／L甲醇诱导表达后，经SDS-PAGE、Western-blotting、双向琼脂扩散试验、神经氨酸酶试验分析证明，获得了几株高效表达重组表达株，并且该重组NA蛋白具有免疫学活性。     

鸡β-防御素-2基因的克隆及原核表达载体的构建

鸡β-防御素是一类广谱抗菌阳离子小肽的总称,一般具有3个分子内二硫键。防御素对细菌、真菌乃至某些被膜病毒具有广谱杀伤作用。试验根据已发表的Gal-2基因序列,设计并合成了一对引物。以骨髓中的总RNA为模板,利用RT-PCR技术扩增目的片段并克隆到pGEM-TEasy载体上,经筛选鉴定获得阳性重组子pGEM-T-Gal-2。再经EcoRI、XbaI双酶切,插入pMAL-c2x质粒构建表达载体。利用PCR技术和酶切反应筛选转化子成功获得pMAL-c2x-Gal-2重组质粒。     

Gal-13成熟肽基因在毕赤酵母中的表达与抑菌活性分析

扩增鸡β-防御素-13(Gal-13)成熟肽基因序列,构建酵母重组表达载体pPICZαA-Gal-13,通过SacⅠ限制性内切酶线性化处理后,电击转化毕赤酵母X-33,并对利用甲醇诱导后的重组菌株表达产物进行表达规律分析和抑菌活性研究。结果表明,扩增到的Gal-13成熟肽基因序列为108bp,共编码36个氨基酸;筛选到的重组菌株均为Mut+表型,其诱导表达上清经Tricine-SDS-PAGE凝胶电泳后,发现其在约5 900的位置出现目的条带,抑菌试验发现其对沙门菌(CVCC534)和金黄色葡萄球菌(ATCC25923)具有抑菌活性。     

山地乌骨鸡β防御素Gal -2基因的克隆及酵母表达载体的构建

为了构建山地乌骨鸡β防御素Gal -2基因酵母表达载体,试验根据GenBank中鸡β防御素Gal -2基因序列设计引物,采用RT - PCR技术从山地乌骨鸡骨髓细胞中扩增出Gal -2基因,经PCR和酶切鉴定后测序,并采用DNAStar软件进行生物信息学分析；再根据毕赤酵母偏爱密码子和山地乌骨鸡β -防御素Gal -2...     

番鸭IFN—α基因在毕赤酵母中的分泌表达

利用PCR从质粒pGEM-T Easy／MDIFN扩增出番鸭IFN-α基因cDNA，将其插入到舍AOXI启动子和α分泌信号肽序列的Pichia pastoris表达载体pPICZαC3．6kb中，测序，待其结果正确后，将重组质粒转化至酵母X-33，PCR法筛选出Mut^ 表型的转化子置于BMMY培养基中培养。用5mL／L甲醇诱导表达4d后，经SDS-PAGE分析可见1条约25ku的清晰蛋白条带。试验结果表明pPICZαC3．6kb／MD IFN质粒构建成功，并且实现重组酵母高效分泌表达。     

猪带绦虫TS018基因真核表达载体的构建及其表达

采用PCR从重组克隆载体pGEM—TSO18中扩增出猪带绦虫六钩蚴TSO18基因，与毕赤酵母分泌性表达载体pPIC9k相连接，构建重组表达载体pPIC9k—TSO18，转化大肠埃希氏菌JM109，经测序证实，基因序列完全正确。制备重组质粒pPIC9k—TSO18，用SalⅠ线性化，并电转化毕赤酵母(Pichia pastoris)GS115，使重组表达载体与酵母染色体发生同源整合。采用G418抗性梯度法筛选得到高拷贝重组菌株，以甲醇进行诱导表达，SDS—PAGE和Western-blotting分析结果表明，诱导表达的培养上清中表达出具有反应活性的16ku重组蛋白，目的蛋白约占培养上清液中蛋白总量的80％以上，诱导72h目的蛋白表达量为0．5mg／mL。     

粤北三黄鸡β-防御素基因的表达及其生物活性研究

采用RT-PCR 方法从三黄鸡肝脏中扩增β-防御素 Gal-6基因的 cDNA 片段,构建重组表达质粒pGEX-6p-1-GAL-6,将重组质粒转化至大肠杆菌BL21进行原核表达。结果表明,成功获得201 bp的鸡β-防御素基因,SDS-PAGE 电泳分析表明,原核表达的重组鸡融合蛋白分子质量约为32 kD。此外,表达的该重组蛋白对金黄色葡萄球菌有较高抗菌活性。     

牛白细胞介素-2在毕赤酵母中的表达

将编码牛白细胞介素-2(BoIL2)成熟肽的cDNA克隆到巴斯德毕赤酵母(Pichia pastoris)表达载体pPICZB中，构建出含BoIL2基因的重组质粒BoIL2-pPICZB。将经Sac Ⅰ酶切后线性化的BoIL2-pPICZB电转化到巴斯德毕赤酵母X-33中，转化子经高浓度Zeoein抗性筛选鉴定后，用1％甲醇诱导目的蛋白表达。经SDS-PAGE及Western blotting检测，表明BoIL2在酵母中获得了胞内表达；通过金属螯合亲和层析(MCAC)获得纯化的重组蛋白；培养小鼠CTLL2细胞进行活性检测，证实所表达的重组BoIL2具有生物活性。     

环状抗菌肽cyclopsychotride在毕赤酵母X-33中的表达及其活性鉴定

为提高重组外源蛋白在毕赤酵母中的高效表达,试验采用毕赤酵母(Pichia pastoris)偏嗜性密码子改变抗菌肽cyclopsychotride的碱基序列,通过SOEing法合成后连接到pPICZα-C质粒上,从而获得分泌型重组酵母表达载体pPICZα-C-cyclopsychotride,再通过限制性内切酶BlnⅠ线性化,经电穿孔法转入毕赤酵母细胞X-33中。结果表明:经PCR检测后及Zeocin抗性筛选,获得高拷贝转化子;通过琼脂孔穴扩散法检测,酵母表达上清液对革兰阳性菌和革兰阴性菌有抑菌活性;抗菌肽cy-clopsychotride经沸水、酸碱及胃蛋白酶处理后仍具有抗菌活性。     

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.     

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

Tropical Animal Health and Production -     

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.     

Chemotaxis of macrophage in inflammation

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.     

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