猪流感病毒血凝素基因原核表达及其在间接ELISA中的初步应用

利用RT-PCR方法扩增猪流感病毒A/Swine/Henan/11/2005(H1N1)血凝素(HA)基因,克隆于pMD18-T载体进行测序。以pMD18-HA为模板、利用带酶切位点的引物再次扩增HA基因的开放阅读框(ORF),克隆到表达载体pET32a中,经双酶切、测序及PCR鉴定得到阳性重组表达质粒pET-HA,将质粒转化到表达宿主菌BL21(DE3)中,经终浓度为1 mmol/L的IPTG诱导,SDS-PAGE结果显示,HA蛋白获得了高效表达,经Western-blot检测证实表达产物具有良好的免疫学活性,在间接ELISA中的初步应用表明具有良好的抗原反应性。本研究为以重组HA蛋白为抗原建立H1亚型猪流感抗体的ELISA检测方法奠定了基础。     

H7亚型禽流感病毒HA1基因的克隆与原核表达

利用PCR技术亚克隆了H7亚型禽流感病毒的HA1基因,并将PCR产物连接到克隆载体pMD 18-T Simple vector,转化大肠杆菌。测序结果表明所克隆的片断大小为1035bp。将HA1基因克隆至原核表达载体pGEX-6P-1,经酶切和PCR鉴定,证明成功构建了重组表达载体pGEX-HA1。将构建好的融合表达载体在IPTG的诱导下在大肠杆菌中得到了表达。融合蛋白GST-HA1的分子量为63ku。Western-Blot和ELISA鉴定结果表明,融合蛋白与H7亚型禽流感阳性血清发生特异性反应,而与H5、H9亚型抗血清不发生反应。     

猪源H9N2亚型流感病毒HA基因的序列测定及真核表达

利用RT-PCR扩增猪流感病毒A/Swine/Zhejiang/7/2004(H9N2)(SW/ZhJ7/04)HA基因,测序后将其克隆到真核表达载体pCAGGS上,经酶切分析、PCR鉴定和测序验证,得到重组表达质粒pCAGGS-HA,之后将其转染293T细胞,48h后收集转染细胞样品,进行SDS-PAGE分析,结果显示HA蛋白获得了表达。经Western blot检测,结果表明pCAGGS-HA能够在体外瞬时表达具有免疫学活性的HA蛋白,通过免疫小鼠,对其免疫效果进行了初步研究。     

猪繁殖与呼吸综合征病毒RdRp基因的克隆与原核表达

将猪繁殖与呼吸综合征病毒RNA依赖性的RNA聚合酶(RdRp)基因进行克隆、表达,并获得纯化重组蛋白,为进一步研究猪繁殖与呼吸综合征病毒RdRp的功能奠定基础.以RdRp基因cDNA为模板,用PCR扩增出RdRp基因片段,应用定向克隆策略将扩增后片段与高效表达载体pET32a(+)重组,阳性克隆重组质粒经酶切及测序鉴定,IPTG诱导高效表达.PCR法扩增出720 bp的片段,酶切及测序鉴定获得正确的重组质粒,在大肠埃希菌LB21中表达得到分子质量约为46 ku的融合蛋白.成功克隆及表达了猪繁殖与呼吸综合征病毒RdRp基因.     

禽流感病毒H7亚型血凝素基因的原核表达及鉴定

参考已发表的禽流感病毒(AIV)H7亚型血凝素(HA)基因序列设计引物，经RT-PCR扩增了AIV-H7亚型的HA1基因片段，再将该片段定向插入到原核表达载体pGEX-4T-2中，转化大肠埃希氏菌。重组表达栽体经酶切和测序鉴定后，用IPTG诱导表达。用Western-blotting和ELISA试验鉴定表达产物的抗原性。结果表明，融合表达蛋白能与AIV H7N1、H7N2、H7N7和H1N1亚型标准抗血清反应，而与H5N1、H9N2等其他13个亚型的抗血清无交叉反应。     

兔出血症病毒主要结构蛋白基因的克隆、原核表达及其免疫原性鉴定

本试验采用RT-PCR方法扩增了兔出血症病毒(rabbit haemorrhagic disease virus,RHDV)衣壳蛋白VP60基因。PCR产物纯化后连接T载体,提取质粒后,进行序列测定,将测序正确的纯化产物双酶切,克隆入原核表达载体pET28b(+)中,构建原核表达载体。将鉴定正确的重组质粒转化表达宿主菌E.coli Rosetta^TM,用IPTG诱导培养重组表达菌。经SDS-PAGE分析,在分子质量62.0 ku处可见明显的表达带,经Western blotting检测,约在62.0 ku处出现特异性的反应带。结果表明,表达蛋白具有良好的抗原性。     

牛瑟氏泰勒虫P23表面蛋白基因的克隆与原核表达

构建牛瑟氏泰勒虫P23基因片段原核重组表达质粒,表达重组蛋白。采用PCR方法扩增牛瑟氏泰勒虫P23表面蛋白基因,将扩增产物与pMD18-T-Simple载体连接,测序,验证扩增产物。将P23基因片段克隆到载体pGEX-4T-3上,经酶切分析、PCR鉴定后,IPTG诱导表达,最后用SDS-PAGE和Western blotting分析鉴定表达产物。结果表明克隆的P23基因片段为684 bp,重组质粒pGEX-4T-3/P23构建成功;SDS-PAGE显示目的蛋白相对分子质量约为58 ku;Western blotting分析结果显示,与牛瑟氏泰勒虫阳性血清发生反应,而与牛瑟氏泰勒虫阴性血清无反应,表明牛瑟氏泰勒虫P23表面蛋白具有良好的抗原性和特异性,提示可以利用融合蛋白来建立检测抗体的间接ELISA诊断方法。     

刚地弓形虫丝裂原活化蛋白激酶1基因克隆、表达及抗原性分析

克隆、表达刚地弓形虫(Toxoplasma gondii)丝裂原活化蛋白激酶1(TgMAPK1)基因片段,并分析其抗原性。提取弓形虫GT1株速殖子总RNA,逆转录合成cDNA。RT-PCR扩增TgMAPK1基因。扩增产物经双酶切后连接入pET28a(+)载体,重组质粒转化大肠埃希菌(Escherichia Coli)DH5α,阳性菌落经PCR和双酶切鉴定,并测序。将测序正确的重组质粒pET28a(+)-TgMAPK1转化至E.coli BL21并加入异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)结合考马斯亮蓝染色检测表达产物。以鼠抗弓形虫血清为一抗,蛋白质印迹(Western blotting)分析重组蛋白的抗原性。RT-PCR扩增产物约为1 599bp。菌落PCR、双酶切和测序结果显示,重组质粒pET28a(+)-TgMAPK1构建成功。SDS-PAGE结果显示,经IPTG诱导获得相对分子质量约58 000的包涵体重组蛋白。Western blotting分析证实其能被鼠抗弓形虫血清识别。刚地弓形虫GT1株TgMAPK1基因片段可在原核表达系统中表达,且该重组蛋白具有抗原性。     

鸡柔嫩艾美尔球虫ZJ株3-1E基因的原核表达及鉴定

本实验对E．tenellaZJ株的3-1E基因进行了克隆和表达。根据已报道的柔嫩艾美尔球虫3-1E基因序列设计引物,以孢子化卵囊总RNA为模板,用RT—PCR方法扩增得到一条特异片段,将扩增产物克隆至pUCM—T,转化感受态菌DH5仅,经酶切鉴定获得阳性重组质粒并对其进行测序。测序结果与参考序列比较,核苷酸同源性为99．5％。然后将重组质粒和表达载体pET-30a分别以EcoRⅠ、SalⅠ酶切后构建重组表达载体pET-30a-3—1E,并将其转化入大肠杆菌BL21中,提取质粒经酶切和PCR鉴定正确后,用IPTG诱导表达。表达产物经SDS—PAGE和Westernblot检测显示,3-1E基因在大肠杆菌中成功表达;融合蛋白的分子量约为27ku,诱导6h的蛋白表达量可达到47、024％。     

H5亚型禽流感病毒HA基因杆状病毒载体的构建

经过PCR反应,以特异性引物扩增了禽流感病毒DK/Zhejiang/11/00(H5N1)去除信号肽的HA基因,克隆到杆状病毒转移载体pFastBac-HTA中,阳性重组质粒转座DH10Bac感受态细胞,通过蓝白斑筛选、PCR鉴定获得阳性克隆,碱裂解法提取阳性质粒,转染sf 9昆虫细胞,获得含H5亚型禽流感病毒HA基因的重组杆状病毒H5HA-Bac HTA。利用SDS-蛋白酶K方法提取重组病毒DNA,PCR反应证实HA基因片段已重组到杆状病毒基因组中。以适宜剂量的重组杆状病毒接种sf 9昆虫细胞,待绝大部分细胞产生细胞病变后收获细胞。细胞裂解产物经间接免疫荧光、SDS-PAGE及Western-Blot试验检测,结果表明:H5亚型禽流感病毒HA基因在重组杆状病毒H5HA-Bac-HTA感染sf 9细胞后获得高效表达,且具有良好的蛋白活性及特异免疫反应原性。     

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.     

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 -