透明颤菌血红蛋白基因(vgb)表达载体的构建及其诱导表达

利用PCR技术将透明颠菌血红蛋白基因(vhb)克隆到融合表达裁体pET28a，在大肠杆菌BL21(DE3)表达。重组蛋白分别在30℃和37℃诱导表达，30℃诱导获得可溶性表达。结果表明：在30℃，1．5mmol／L和2．5mmol／LIPTG诱导下，诱导4—8h透明颠菌血红蛋白可获得高表达——透明颠菌血红蛋白的表达量分别占菌体总蛋白的18．7％和27．7％。     

日本血吸虫丝氨酸-苏氨酸蛋白磷酸酶基因的表达及其免疫保护试验

为研究日本血吸虫丝氨酸-苏氨酸蛋白磷酸酶（SjPP）的免疫功能，构建了原核表达重组质粒pET28a（＋）-SjPP，转化大肠埃希氏菌BL21（DE3）进行诱导表达，并用重组蛋白进行了小鼠免疫保护试验，免疫剂量为每次20μg／只，共免疫3次。结果表明，pET28a（＋）-siPP／BL21（DE3）在0．1mmol／L IPTG诱导2h时，每1g菌体可产生17．8mg以包涵体形式存在的重组蛋白；重组蛋白免疫小鼠后诱导产生了高效价的血清特异性IgG抗体，并获得了部分减虫率、肝组织减卵率和显著的粪便减卵率，具有一定的免疫保护作用。     

水疱性口炎病毒糖蛋白基因主要抗原表位区在大肠埃希氏菌中的高效表达及纯化

利用PCR技术扩增编码水疱性口炎病毒印第安纳型（VSV—Ind）糖蛋白基因的主要抗原表位区，通过引入其两端的EcoRⅠ和XhoⅠ酶切位点，定向插入到pET30c质粒T7启动子下游的多克隆区，构建重组质粒pET30c—vsvG，并转化至宿主菌BL21（DE3）株中。用1mmol／LIPTG诱导后，SDS—PAGE电泳表明，重组蛋白在大肠埃希氏菌中得到了高效表达，经过4h表达即可达到高峰；融合蛋白的相对分子质量为57ku。重组蛋白含有六聚组氨酸尾，主要以包涵体形式存在，Ni柱纯化后的蛋白经Western—blotting鉴定，具有良好的抗原性和特异性。     

牛乳源性金黄色葡萄球菌nEBPS蛋白表达鉴定及多克隆抗体制备

利用PCR方法扩增乳源致病性金黄色葡萄球菌nEBPS全基因，将其定向克隆至原核表达载体pET30a（＋）中，鉴定正确后，转化BL21表达菌，经IPTG诱导获得了以可溶性表达的重组蛋白。通过NiNTAPurificationSystem纯化重组蛋白，纯化蛋白质量浓度为2．16g／L。纯化蛋白经免疫印迹检测显示重组蛋白能够被牛源金黄色葡萄球菌阳性血清识别，具有良好的反应性。将纯化的重组蛋白免疫新西兰白兔制备多克隆抗体，间接EI，1SA测定抗体效价为1：25600，凝集试验测定抗体效价为1：128。     

桑树低温诱导蛋白基因（Wap25）的原核表达

以pGEX-4T-2为载体构建了桑树低温诱导蛋白基因（wap25）的重组表达质粒,转化受体菌Ecoli BI21。经IPTG诱导后,基因产物在大肠杆菌中得到了有效表达。为了提高表达效果,我们从不同表达载体、诱导时间和诱导温度等方面对目的蛋白的表达条件进行了优化,用SDS-PAGE电泳检测,结果表明30℃条件下经0．5mmol／L的IPTG诱导8h为最优诱导条件。     

抗菌肽V13KL原核表达载体的构建及其表达产物的鉴定

将V13KL编码基因片段克隆到原核表达载体pET30b（＋），并在目的蛋白N端设计肠激酶酶切位点，构建出pET30b（＋）-VK13L重组质粒，转化大肠杆菌表达菌株BL21（DE3），优化诱导条件，使得抗菌肽得到了高效表达。经Tricine-SDS-PAGE和Westernblotting鉴定，目的蛋白的相对分子质量与预期一致。结果表明，V13KL基因成功克隆并且获得表达，在诱导体系中加入1mmol／LIPTG诱导6h便可检测到蛋白表达。     

重组牛α-干扰素的原核表达与纯化

为在大肠杆菌表达系统中高效表达牛α-干扰素(bovine interferon-α,Bo IFN-α),将经密码子优化后的牛α-干扰素成熟蛋白基因合成并克隆到表达载体p Cold II中,转化大肠杆菌BL21(DE3)中诱导表达,获得可溶性的表达蛋白。对重组菌表达条件进行优化,结果显示在IPTG浓度为0.64 mmol/L,诱导温度为15℃,诱导时间为9 h的条件下,可溶性目的蛋白的表达量最高。重组蛋白经镍柱纯化后的纯度为99.2%,表达量为36 mg/L菌液。重组蛋白在MDBK细胞上抗水泡性口炎病毒的活性为1×106U/mg。     

细胞黏附分子1（ICAM-1）ScFv基因原核载体的构建及表达

应用PCR技术扩增ICAM-1ScFv基因,经纯化测序,得到约750bp的核苷酸片段,并构建了原核重组表达质粒pET20b-ICAM-1ScFv。将重组质粒转化至表达菌BL21（DE3）中,诱导后的SDS-PAGE分析显示,pET20b-ICAM-1ScFv表达量占菌体蛋白总量的18.4%。本试验将重组蛋白的表达条件进行优化,结果表明,ICAM-1ScFv蛋白在pET20b表达载体中的最佳表达条件为37℃诱导5h。     

鸡β-防御素-1基因(Gal-1)在大肠杆菌中的融合表达与纯化

内源性抗菌多肽防御素是机体先天性免疫系统的重要组成部分，可帮助机体防御外界病原微生物的入侵。鸡β防御素-1（Gallinaein-1，Gal-1）对许多致病菌都具有杀菌作用，具有很好的研究开发价值。本研究利用PCR技术从重组质粒pGEM-TEasy-gal-1中克隆出Gal-1基因成熟肽区，将该基因插入原核表达载体pET-32a（＋）中，构建重组质粒pET-32a（＋）-gal-1，然后用重组质粒转化大肠杆菌BL-21。挑选阳性克隆菌，用浓度为0．5mmol／L的IPTG进行诱导表达。SDS-PAGE电泳显示在目标位置约25kD处出现了条带并用Werstern Blot进行了鉴定。表明Gal-1基因在大肠杆菌以融合形式得到了表达。经灰度扫描显示重组蛋白表达量占细菌总蛋白的15．54％。用50％Ni-NTA纯化树脂进行层析纯化，在洗脱液E中出现单一条带。Gal-1多肽在pET-32a（＋）质粒表达体系中成功表达为鸡防御素多肽下一步的研究奠定基础。     

DAB389(Gly4Ser)2αMSH重组融合蛋白的构建及表达

利用含有白喉毒素N端序列的基因作上游引物、含有αMSH全序列和（Gly4Ser）2互补序列的基因作下游引物，以pET28a／DAB389（Gly4Set）2EFG为模板，PCR扩增DAB389（Gly4Ser）2αMSH基因片段，用限制性内切酶EcoR Ⅰ和NcoⅠ酶切，并插入原核表达载体pET28a的相应位点，构建了重组表达载体pET28a／DABm（Gly4Ser）2αMSH，在大肠杆菌中表达重组融合蛋白DAB389（Gly4Ser）2αMSH，转化菌经IPTG、30C诱导5h，用SDS—PAGE和Western blot鉴定表达的重组蛋白。结果表明扩增的片段与理论值一致。重组质粒的DNA序列分析正确。SDS—PAGE表明，重组蛋白相对分子质量为45870，且表达量达菌体总蛋白量的29．2％。Western blot分析显示，重组蛋白能特异地与抗白喉抗体结合。成功构建表达了重组DABm（Gly4Ser）2αMSH的工程菌株，表达产物具有生物学活性。     

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.     

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.     

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.     

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 -