稳定表达猪传染性胃肠炎病毒N蛋白的ST细胞株的构建及其亚细胞定位

为研究猪传染性胃肠炎病毒(TGEV)N蛋白的表达对宿主细胞功能的影响,本研究通过构建TGEV N基因的重组表达质粒p EGFP-N,将其转染于猪睾丸(ST)细胞中,并在活细胞状态下直接观察GFP-N蛋白在ST细胞中的表达与定位。结果显示,转染后4 h~5 h出现荧光蛋白表达,24 h达到高峰,TGEV的N蛋白主要定位于细胞质中,进一步的激光共聚焦共定位显示TGEV N蛋白主要定位于ST细胞的内质网上面。以G418筛选4周后,转染细胞形成稳定的细胞株,RT-PCR和western blot验证TGEV N蛋白的m RNA及其蛋白在ST细胞中稳定表达,提示本研究建立了稳定表达的TGEV N蛋白的ST细胞株,为进一步研究TGEV N蛋白对ST细胞的生理功能的影响及其在TGEV致病过程中的作用奠定了基础。     

稳定表达T7RNA聚合酶PK-15细胞系的建立

为构建稳定表达T7 RNA聚合酶(RNAP)的猪肾细胞系(PK-15),本研究采用PCR方法,以E.coli BL21(DE3)细菌基因组为模板扩增T7 RNAP基因,将其克隆到逆转录病毒载体pLXSN中,构建重组质粒pLXSN-T7.采用脂质体将pLXSN-T7转染至PT67细胞中,经G418筛选培养获得重组逆转录病毒rMLV-T7,将其接种于PK-15细胞进行G418加压筛选和纯化;应用PCR、间接免疫荧光试验及T7启动子控制下的红色荧光蛋白的重组表达质粒(pET-RED)进行瞬时表达,检测T7 RNAP的表达及活性,结果表明筛选所得的细胞系PK/T7的不同代次均具有转录活性.本研究建立稳定表达T7 RNAP的细胞系PK/T7,为实现细胞内T7启动的转录和猪源RNA病毒等的反向遗传操作提供了有效的方法.     

鸡B-F2基因真核表达系统的建立与鉴定

鸡B-F2基因编码的主要组织相容性复合体(MHC)I类分子α链在递呈内源性抗原中起重要作用。本研究应用RT-PCR方法从鸡外周血单核细胞中扩增了B-F2基因片段,其大小为1 068 bp。进一步将其定向插入真核表达载体,构建重组质粒pEGFP-N1-B-F2。经脂质体法转染COS7,所表达的α链定位于在细胞的浆膜,同时转染P815细胞,经G418的筛选(0.6 mg/mL)得到稳定表达细胞株。该细胞株经培养10代次后,仍在RT-PCR中能获得相应DNA片段。上述结果表明,B-F2基因在动物真核表达系统得到表达,建立的P815(B-F2)细胞株为进一步研究MHC I类分子α链在免疫应答中作用奠定了基础。     

稳定表达乙型脑炎病毒NS1蛋白细胞系的建立

为建立稳定表达乙型脑炎病毒(JEV) NS1蛋白的真核细胞系,本研究将编码JEV NS1蛋白的人工合成基因克隆到真核表达载体pCAGG-TK-neo中,构建了重组质粒pCAGG-opti-NS1.重组质粒经脂质体转染RK-13细胞,以含G418的选择性培养基选择培养,经细胞克隆纯化,以间接免疫荧光试验(IEA)筛选表达目的基因的细胞.结果表明,转染的RK-13细胞经G418加压及IFA筛选后,获得表达JEV NS1蛋白的阳性RK-13细胞系.经RT-PCR、western blot和IFA鉴定,该细胞系在传代至第15代后仍然可以稳定表达NS1蛋白.本实验获得了能够稳定表达JEV NS1蛋白的细胞系,为进一步开展JEV相关的研究奠定了基础.     

稳定表达猪IRF 7基因的PK-15细胞系建立及其抗病毒效果评价

为探讨猪IRF7基因对某些猪源病毒在PK-15细胞系中增值的影响,利用Piggy Bac真核转座子系统将猪源IRF7基因转染入PK-15细胞中,通过嘌呤霉素和绿色荧光进行双重筛选获得阳性转化细胞,再经克隆纯化得到单个阳性细胞克隆株。通过观察细胞荧光和荧光定量PCR鉴定,最终获得了稳定表达IRF7基因的PK-15细胞系。在Poly I:C诱导下,该细胞系能够显著上调IFN-β的表达。初步研究了该细胞系对病毒复制的影响,结果表明过表达IRF-7基因能够显著抑制猪伪狂犬病病毒(PRV)和猪水泡性口炎病毒(VSV)的复制。本研究为进一步探索猪IRF7基因的功能与作用机制提供有效工具。     

绵羊NYD-SP27基因在BHK-21细胞中的稳定表达及其特征鉴定

旨在构建稳定表达绵羊NYD-SP27基因的BHK-21细胞株,对NYD-SP27基因的表达特征进行分析,为进一步研究NYD-SP27基因功能奠定基础。本研究克隆绵羊NYD-SP27基因,并将其插入到真核表达载体pDsRed1-C1中,利用猪捷申病毒2A肽(P2A)将NYD-SP27蛋白和红色荧光蛋白连接以实现共表达,将重组质粒pDsRed-P2A-Flag-NYDSP转染至BHK-21细胞中,经G418筛选获得稳定转基因细胞株,利用RT-PCR、间接免疫荧光(Indirect immunofluorescence assay,IFA)和Western blot对NYD-SP27基因的表达进行鉴定。结果表明,NYD-SP27基因稳定整合至宿主细胞基因组;NYD-SP27蛋白能够在BHK-21细胞中正常表达,分子大小约为63ku;在NYD-SP27蛋白和红色荧光蛋白翻译过程中,P2A成功自我剪切。本研究成功构建了绵羊NYD-SP27基因的真核表达载体,建立了稳定表达绵羊NYD-SP27基因的BHK-21细胞系。     

猪FcγRⅠ真核表达载体的构建及稳定表达细胞系的建立

本研究旨在建立稳定表达猪FcγRⅠ的Marc-145细胞系。从猪肺泡巨噬细胞中提取总RNA,应用RT-PCR技术获得猪FcγRⅠ和γ链cDNA,并分别构建PIREShyg3-γ和pcDNA3.1-FcγRⅠ真核表达质粒;用脂质体共转染Marc-145细胞,经潮霉素B(300μg.mL-1)和G418(400μg.mL-1)共筛选获得稳定表达猪FcγRⅠ的细胞系;运用RT-PCR、玫瑰花环和流式细胞术对细胞系进行鉴定。结果表明成功构建了猪FcγRⅠ和γ链真核表达载体,建立了稳定表达猪FcγRⅠ的细胞系,且表达于转染细胞表面的poFcγRⅠ受体分子能与猪IgG特异结合。本研究为进一步研究奠定了基础。     

FADS1基因对猪不饱和脂肪酸含量的影响研究

【目的】研究脂肪酸去饱和酶1(fatty acid desaturases 1,FADS1)基因对不饱和脂肪酸代谢的调控作用,为揭示其分子机制提供参考。【方法】利用PCR扩增猪FADS1基因的CDS区,将目的基因与pcDNA3.1(-)骨架载体连接获得重组表达载体pcDNA3.1-FLAG-FADS1,将重组表达载体瞬时转染宁乡猪肾成纤维细胞,转染后24、48 h分别收集细胞,利用实时荧光定量PCR和Western blotting检测FADS1基因的表达情况,经过遗传霉素(geneticin, G418)筛选之后获得稳定表达FADS1基因的细胞系,命名为1-4^#。利用甘油三酯检测试剂盒检测野生型和1-4^#细胞甘油三酯含量变化;利用气相色谱质谱法(gas chromatography-mass spectrometer, GC-MS)检测野生型和1-4^#细胞不饱和脂肪酸含量变化情况。【结果】成功获得过表达载体pcDNA3.1-FLAG-FADS1,在转录水平和蛋白水平检测均有过表达效果。通过G418筛选出1株稳定过表达F...     

适应H_9亚型禽流感病毒增殖的重组R-MDCK细胞系的建立

克隆鸡ST3GalⅠ基因并将其插入真核表达质粒p IRES中,构建重组质粒p IRES-ST3GalⅠ,鉴定正确的真核表达载体转染MDCK细胞,用G418抗性筛选稳定表达的重组细胞R-MDCK。PCR及RT-PCR鉴定表达阳性的单克隆细胞株。将不同H_9亚型禽流感病毒毒株按相同滴度接种R-MDCK细胞和空白MDCK细胞,通过HA试验和TCID_(50)试验,检测不同H_9亚型禽流感病毒毒株在R-MDCK细胞上的增殖效果。结果发现,所有H_9亚型禽流感病毒毒株在RMDCK细胞HA和TCID_(50)结果均显著高于空白MDCK细胞。ST3GalⅠ基因在第12代R-MDCK细胞中仍能稳定表达,R-MDCK细胞可显著提升H_9亚型低致病性禽流感病毒培养的滴度,可以替代鸡胚用于H_9亚型低致病性禽流感病毒的生产。     

猪圆环病毒2型orf2基因在奶山羊胎儿成纤维细胞中的表达与生物活性鉴定

为获得稳定表达猪圆环病毒2型(PCV-2)orf2基因的奶山羊胎儿成纤维细胞,将PCV-2 orf2基因与筛选基因neo表达框串联插入到pBC1质粒,得到重组的真核表达载体pBC1-orf2-neo。然后采用脂质体介导法将重组质粒转染至奶山羊胎儿成纤维细胞(gFFs),经G418筛选获得具有稳定抗性的阳性gFFs细胞株。RT-PCR分析显示,在获得的阳性gFFs细胞株中能够扩增出689bp的orf2基因和1 953bp的neo表达框,与预期结果相符。Western blot分析显示,获得了约37 000的表达产物,且能够与兔抗PCV-2Cap蛋白抗体发生反应。结果表明,orf2基因成功整合到山羊胎儿成纤维细胞染色体上并能够正确表达,表达产物具有良好的反应原性,这为构建分泌Cap蛋白的奶山羊乳腺生物反应器奠定了理论和物质基础。     

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.     

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