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1.
鸡白细胞介素2分子单克隆抗体的研制   总被引：1，自引：0，他引：1  
以含有鸡白细胞介素2（ChlL-2）基因的重组真核表达质粒pcDNA3．1-ChlL-2免疫6周龄BALB／c小鼠,末次加强免疫后取其脾细胞与骨髓瘤细胞Sp2／0-Ag-14融合,应用间接ELISA筛选阳性克隆。共获得2株特异性分泌抗鸡IL-2分子的杂交瘤细胞株,分别命名为1H10和1E6,其腹水ELISA效价分别为1：6400和1：3200,亚类鉴定结果均为IgM。重组质粒pcDNA3．1-ChlL-2转染COS-7细胞,以上述单克隆抗体（mAb）检测表达产物,结果表明,2株mAb均能与表达产物反应;Western blot分析结果显示,2株mAb均与原核表达鸡IL-2蛋白发生特异性反应。本研究所制备的抗鸡IL-2分子mAb可为建立简单、快速的鸡IL-2检测方法提供可能,并为鸡IL-2生物学特性和禽类细胞免疫机理的研究提供材料。  相似文献   

2.
研究旨在制备DRV p18蛋白单克隆抗体。试验通过原核表达并纯化DRV p18重组蛋白,经BCA蛋白浓度测定后免疫BALB/c小鼠;通过细胞融合技术制备杂交瘤细胞,获得的p18单克隆抗体通过ELISA、IFA和Western blot试验进行鉴定,并将杂交瘤细胞腹腔注射小鼠获得单抗腹水,通过Western blot试验验证其特异性。结果显示：纯化的重组p18蛋白浓度为1.37 mg/mL,经过三轮亚克隆获得4株分泌单克隆抗体的杂交瘤细胞株1E3、2C4、4A2和4D4,亚型鉴定1E3和4D4重链为IgG1型、2C4重链为IgM型、4A2为IgG2b型,轻链均为κ型;4株单克隆抗体均能使感染DRV的BHK-21细胞发出特异性荧光,并在18 ku处存在特异性条带;4D4腹水ELISA效价为1∶409 600,Western blot效价达1∶102 400,IFA效价为1∶51 200;4D4与DRV反应,而不与其他禽源常见病毒反应。研究成功制备了抗DRV p18蛋白的单克隆抗体,为进一步研探究p18蛋白的功能奠定基础。  相似文献   

3.
为制备猪圆环病毒4型(PCV4)Cap蛋白单克隆抗体,以原核表达的重组Cap蛋白免疫6～8周龄小鼠,三次免疫后取免疫小鼠脾细胞与SP2/0骨髓瘤细胞进行融合。利用亚克隆技术和间接免疫荧光(IFA)方法筛选到2株阳性单克隆杂交瘤细胞株,分别命名为5H9和2F3。间接免疫荧光和Western blot试验表明,2株单抗均能特异性识别293T细胞中特异表达的Cap蛋白,且5H9和2F3的IFA效价分别为1∶12 800和1∶6 400。亚类鉴定结果表明,两株单抗重链均属于IgG1,轻链类型为kappa型。本研究制备的抗PCV4 Cap特异性单抗,为PCV4检测方法的建立和流行病学调查提供了有效的生物材料,也为该病毒的分离鉴定与Cap蛋白功能的探究奠定了基础。  相似文献   

4.
为制备犬瘟热病毒(CDV)F蛋白融合信号肽(FSP)的单克隆抗体(MAb)，本研究通过原核表达系统表达CDV HLJ1-13株重组FSP蛋白(rFSP)。将其免疫BALB/c小鼠，通过间接ELISA方法筛选到1株能够稳定分泌抗FSP蛋白MAb的杂交瘤细胞株G8D6E3E5，亚类鉴定结果显示该株MAb重链为Ig G2a型，轻链为κ型。构建一系列表达部分重叠的重组FSP片段，经western blot鉴定该株MAb识别的抗原区域为aa1～aa55，通过合成一系列重叠多肽，经ELISA进一步鉴定该株MAb识别的抗原表位为21QQHSTRSTET30。采用western blot检测该株MAb与r FSP的反应原性；采用间接免疫荧光试验(IFA)检测该株MAb与天然FSP的反应原性。Western blot结果显示，CDV Snyder Hill和HLJ1-13株r FSP蛋白样品在34 ku左右均出现特异性条带；IFA结果显示，感染CDV Snyder Hill株48 h后的vero细胞出现红色荧光。以上结果表明，该株MAb既能够与原核表达的CDV...  相似文献   

5.
本研究制备并鉴定了口蹄疫病毒非结构蛋白2C的单克隆抗体(mAb).以纯化的原核表达2C蛋白免疫BALB/c小鼠,取其脾细胞与小鼠骨髓瘤细胞融合,所获得的杂交瘤细胞经间接ELISA法筛选,有限稀释法克隆,直至所克隆的细胞能够100%的分泌抗体,用Dot-ELISA以及IFA对mAbs的特异性等进行鉴定.共获得4株抗重组蛋白2C的mAbs,其亚类鉴定结果3株为IgG1,1株为IgG2b,叠加ELISA初步判定4株单抗所识别表位相同或相近.Dot-ELISA显示这些mAbs与重组2C蛋白和(2C3AB)蛋白以及口蹄疫病毒O/China99感染的BHK-21细胞中的2C抗原均可特异性结合,IFA结果显示所得单抗能与FMDV感染细胞中的2C蛋白特异性结合.本研究获得的针对2C蛋白的特异性mAb,为进一步研究2C蛋白的结构和功能以及诊断方法奠定了基础.  相似文献   

6.
猪伪狂犬病毒gE蛋白单克隆抗体制备与鉴定   总被引：2，自引：0，他引：2  
为获得猪伪狂犬病毒(PRV)gE蛋白单克隆抗体,选择原核表达的重组gE蛋白免疫6周龄BALB/c雌性小鼠,将其脾细胞与SP2/0进行融合,经间接ELISA筛选阳性杂交瘤细胞,结果获得了2株能稳定分泌抗PRV gE蛋白的杂交瘤细胞,命名为E3B8和E5C11。间接ELISA检测2株杂交瘤细胞的培养上清液抗体效价为1∶6.4×10~3,腹水的抗体效价分别达到1∶3.28×10~6和1∶6.55×10~6。2株杂交瘤细胞的染色体数分别为105和108。E3B8亚类鉴定重链为IgG1,轻链为κ链;E5C11亚类鉴定重链为IgG2b,轻链为κ链。Western blot检测显示2株单克隆抗体腹水均能与PRV重组gE蛋白发生特异性反应,间接免疫荧光试验(IFA)检测显示2株单克隆抗体均能与PRV分离毒株感染的BHK-21细胞发生特异性反应,交叉反应性检测显示2株单克隆抗体与常见病毒不发生交叉反应。表明制备的2株gE蛋白单克隆抗体效价高、特异性强,为gE蛋白结构与功能分析以及PRV免疫诊断试剂盒的开发奠定了基础。  相似文献   

7.
鸡CD4基因片段原核表达及其单抗制备   总被引：1，自引：0，他引：1  
将PCR扩增的鸡CD4基因片段克隆到pET-32a原核表达载体,构建获得原核表达重组载体pET-32a-CD4.重组载体导入大肠杆菌BL21(DE3),经过IPTG诱导表达,Ni-NTA亲和树脂纯化获得融合蛋白His-CD4.以此融合蛋白为抗原免疫BALB/c小鼠,用杂交瘤细胞技术制备了1株能够稳定传代并分泌抗CD4多肽单抗的杂交瘤细胞株,命名为3E12.经检测该抗体亚类为IgG2b,单抗腹水的间接ELISA效价为1:105,Western blot结果显示单抗与CD4多肽能特异性地结合.  相似文献   

8.
旨在制备禽腺病毒血清4型（FAdV-4）纤突蛋白（Fiber2）的特异性单克隆抗体（MAb）,本研究将原核表达的可溶性重组蛋白NusA-Fiber2作为免疫原免疫BALB/c雌鼠,筛选获得3株能稳定分泌抗FAdV-4 Fiber2蛋白MAb的杂交瘤细胞株2G5、2G8、4C2,取细胞株2G5制备腹水并纯化,利用间接免疫荧光试验（immunofluorescence assay,IFA）和Western blot鉴定该单抗的特异性。用制备的单抗包被酶标板,通过一系列的优化,建立了FAdV-4 Fiber2双抗夹心ELISA检测方法。通过逐步截短分析鉴定出单克隆抗体识别的抗原表位区域。结果表明：成功获取3株单克隆细胞株2G5、2G8、4C2。MAb 2G5可与原核表达纯化的Fiber2蛋白及FAdV-4特异性反应。建立的双抗夹心ELISA检测方法具有很好的特异性、灵敏性和重复性。该MAb识别的表位序列是N端aa1—33。本研究成功制备了具有良好Western blot和IFA反应原性的单克隆抗体,为Fiber2蛋白功能研究和FAdV-4新型表位疫苗商品化研发奠定了基础。  相似文献   

9.
以原核表达纯化的猪(嗜血)支原体MSG1蛋白免疫BALB/c小鼠,运用细胞融合技术筛选分泌针对MSG1蛋白抗体的融合细胞。通过间接ELISA方法筛选获得2株能稳定分泌抗体的融合细胞株,分别命名为1A7和3G6,Western blot结果证明这2株细胞分泌的抗体能够与重组MSG1蛋白发生特异性反应。细胞上清和腹水中的ELISA抗体效价分别为1∶4 096、1∶1 024和1∶1 638 400、1∶51 200,其单抗亚类鉴定均属于IgG1,轻链为κ型。抗原识别位点分析结果表明,2株单抗所识别的抗原位点相同。猪(嗜血)支原体MSG1蛋白特异性单克隆抗体的制备成功,为制备免疫诊断试剂盒和致病机制的研究奠定了基础。  相似文献   

10.
为制备禽传染性支气管炎病毒(IBV) S2蛋白的单克隆抗体(MAb)，本研究通过原核表达系统表达并纯化重组S2蛋白，将其免疫BALB/c小鼠并通过间接ELISA方法筛选到4株能够稳定分泌S2蛋白MAb的杂交瘤细胞株，分别命名为1A7、4F12、4A7、4D1。亚类鉴定结果显示4株MAb的重链均为Ig G1，轻链均为κ。采用间接ELISA法检测上清及腹水效价，结果显示MAb细胞上清效价均不低于1∶12 800，腹水效价均不低于1∶51 200。采用western blot检测制备的S2蛋白MAb与IBV感染鸡胚尿囊液后天然表达的S2蛋白的反应原性；采用间接免疫荧光试验(IFA)检测IBV感染非洲绿猴肾细胞(Vero)后天然表达的S2蛋白的反应原性。Western blot结果显示，感染IBV的鸡胚尿囊液中出现了90 ku左右的特异性条带；IFA结果显示，感染IBV的Vero细胞中出现绿色荧光。以上结果表明，制备的S2蛋白MAb能够与天然表达的S2蛋白反应，反应原性较强。利用间接ELISA检测MAb与S2蛋白的亲和力，结果显示4株MAb对S2蛋白均有良好的亲和力。利用一系列表达部分重叠的重...  相似文献   

11.
试验选用96头平均体重14.82 kg左右的杜×长×大断奶仔猪,随机分成4组,每组3栏,每栏8头(公母各半)。对照组饲喂基础日粮,试验1、2、3组分别添加1%80目白术、0.2%白术多糖和1%微米白术。试验期30 d。结果表明:在生长性能方面,与对照组相比,1%微米白术添加组可显著提高日增重(P0.05)、降低饲料增重比和腹泻率,而且效果优于1%80目白术组和0.2%白术多糖组,在肠道形态和肠道微生态区系方面,与对照组相比,日粮添加1%80目白术、0.2%白术多糖、1%微米白术均可不同程度的提高十二指肠和空肠的绒毛高度,加深十二指肠和空肠的隐窝深度,并且增加肠道微生态区系的多样性,其中以1%微米白术添加组的效果最佳。  相似文献   

12.
Sissay, M.M., Uggla, A. and Waller, P.J., XXXX. Prevalence and seasonal incidence of nematode parasites and fluke infections of sheep and goats in eastern Ethiopia. Tropical Animal Health and Production, XXXX. A 2-year abattoir survey was carried out to determine the prevalence, abundance and seasonal incidence of gastro-intestinal (GI) nematodes and trematodes (flukes) of sheep and goats in the semi-arid zone of eastern Ethiopia. During May 2003 to April 2005, viscera including liver, lungs and GI tracts were collected from 655 sheep and 632 goats slaughtered at 4 abattoirs located in the towns of Haramaya, Harar, Dire Dawa and Jijiga in eastern Ethiopia. All animals were raised in the farming areas located within the community boundaries for each town. Collected materials were transported within 24 h to the parasitology laboratory of Haramaya University for immediate processing. Thirteen species belonging to 9 genera of GI nematodes (Haemonchus contortus, Trichostrongylus axei, T. colubriformis, T. vitrinus, Nematodirus filicollis, N. spathiger, Oesophagostomum columbianum, O. venulosum, Strongyloides papillosus, Bunostomum trigonocephalum, Trichuris ovis, Cooperia curticei and Chabertia ovina), and 4 species belonging to 3 genera of trematodes (Fasciola hepatica, F. gigantica, Paramphistomum {Calicohoron} microbothrium and Dicrocoelium dendriticum) were recorded in both sheep and goats. All animals in this investigation were infected with multiple species to varying degrees. The mean burdens of adult nematodes were generally moderate in both sheep and goats and showed patterns of seasonal abundance that corresponded with the bi-modal annual rainfall pattern, with highest burdens around the middle of the rainy season. In both sheep and goats there were significant differences in the mean worm burdens and abundance of the different nematode species between the four geographic locations, with worm burdens in the Haramaya and Harar areas greater than those observed in the Dire Dawa and Jijiga locations. Similar seasonal variations were also observed in the prevalence of flukes. But there were no significant differences in the prevalence of each fluke species between the four locations. Overall, the results showed that Haemonchus, Trichostrongylus, Nematodirus, Oesophagostomum, Fasciola and Paramphistomum species were the most abundant helminth parasites of sheep and goats in eastern Ethiopia.  相似文献   

13.
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.  相似文献   

14.
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.  相似文献   

15.
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.  相似文献   

16.
Genetic variations in chromosome Y are enabling researchers to identify paternal lineages, which are informative for introgressions and migrations. In this study, the male‐specific region markers, sex‐determining region‐Y (SRY), amelogenin (AMELY) and zinc finger (ZFY) were analysed in seven Turkish native goat breeds, Angora, Kilis, Hair, Honaml?, Norduz, Gürcü and Abaza. A SNP in the ZFY gene defined a new haplotype Y2C. All domestic haplogroups originate from Capra aegagrus, while the finding of Y1A, Y1B, Y2A and Y2C in 32, 4, 126 and 2 Turkish domestic goats, respectively, appears to indicate a predomestic origin of the major haplotypes. The occurrence of four haplotypes in the Hair goat and, in contrast, a frequency of 96% of Y1A in the Kilis breed illustrate that Y‐chromosomal variants have a more breed‐dependent distribution than mitochondrial or autosomal DNA. This probably reflects male founder effects, but a role in adaptation cannot be excluded.  相似文献   

17.
The purpose of this study was to determine the frequency of different tumor types within a large cohort of cats with intracranial neoplasia and to attempt to correlate signalment, tumor size and location, and survival time for each tumor. Medical records of 160 cats with confirmed intracranial neoplasia evaluated between 1985 and 2001 were reviewed. Parameters evaluated included age, sex, breed, FeLV/FIV status, clinical signs, duration of signs, number of tumors, tumor location(s), imaging results, treatment, survival times, and histopathologic diagnosis. Most of the cats were older (11.3 +/- 3.8 years). Primary tumors accounted for 70.6% of cases. Metastasis and direct extension of secondary tumors accounted for only 5.6 and 3.8% of cases, respectively. Twelve cats (7.5%) had 2 or more discrete tumors of the same type, whereas 16 cats (10.0%) had 2 different types of intracranial tumors. The most common tumor types were meningioma (n = 93, 58.1%), lymphoma (n = 23, 14.4%), pituitary tumors (n = 14, 8.8%), and gliomas (n = 12, 7.5%). The most common neurological signs were altered consciousness (n = 42, 26.2%), circling (n = 36, 22.5%), and seizures (n = 36, 22.5%). Cats without specific neurological signs were common (n = 34, 21.2%). The tumor was considered an incidental finding in 30 (18.8%) cats. In addition to expected relationships (eg, meninges and meningioma, pituitary and pituitary tumors), we found that lesion location was predictive of tumor type with diffuse cerebral or brainstem involvement predictive of lymphoma and third ventricle involvement predictive of meningioma.  相似文献   

18.
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.  相似文献   

19.
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.  相似文献   

20.
Comparison of different tests to diagnose feline infectious peritonitis   总被引：1，自引：0，他引：1  
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