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1.
羊流产衣原体主要外膜蛋白基因的克隆与序列分析   总被引：6，自引：0，他引：6  
将自行分离、传代培养的内蒙古地区山羊流产衣原体按常规方法分离纯化，提取衣原体基因组DNA作为模板，按照国外发表的衣原体主要外膜蛋白（MOMP）基因两端序列设计合成一对引物，用PCR方法扩增出－1.17Kb的DNA片段。利用引物上预先设计的限制性内切酶位点，将扩增片段经限制性内切酶切割后连接到pUC19质粒相应位点上，转化大肠杆菌DH5α，筛选重组子。经PCR检测和内切酶分析鉴定含MOMP基因的重组子质粒。对克隆处段进行全序列分析，结果证明得到MOMP全编码序列的基因克隆。本株衣原体MOMP编码区由1170个核苷酸组成。序列比较发现本株衣原体的MOMP基因与国外的羊流产衣原体S26／3株的MOMP基因完全相同，与B577株的MOMP基因仅有一个核苷酸的同义变异。  相似文献   

2.
对具有角膜混浊表型的B6-Co突变系小鼠突变候选基因Map3k1进行克隆及测序分析,寻找该突变系小鼠Map3kl基因的突变位点.以小鼠Map3k1基因的mRNA、全长及上游5 kb序列设计引物,分别以基因组DNA和mRNA为模板,采用PCR和RT-PCR技术分段扩增目的基因,将目的片段连接在T栽体上,转化至感受态细胞,...  相似文献   

3.
猪氟烷基因PCR-RFLP检测新引物设计与条件优化   总被引：1，自引：0，他引：1  
本文在测定4个不同品种猪包含突变位点的氟烷基因序列的基础上,重新设计了PCR引物,并对反应体系和反应条件进行了优化。实验结果表明:新设计的引物在PCR扩增中对DNA模板量要求不高,PCR反应对温度变化不敏感,具有较高的稳定性,结合限制性酶切技术,能快速准确地鉴别猪氟烷基因的三种基因型,可应用于猪氟烷基因的分子检测。  相似文献   

4.
猪带绦虫六钩蚴TSOL18真核表达载体pVTSOL18m的构建   总被引：1，自引：0，他引：1  
根据Kozak原理设计引物，以重组原核表达载体pGEX—TSOL18为模板扩增TSOL18基因，用EcoRⅠ、XhoⅠ酶切后与经相同处理的pVAX1载体连接并转化JM109感受态细胞，经测序证明读码框正确后，再根据PCR点突变的方法设计引物。将目的基因NruⅠ位点中的碱基（第347位）经三次PCR反应突变，最终获得含突变位点的TSOL18m基因。EcoRI、XhoI再次酶切后与pVAX1载体连接，成功构建了含点突变的重组pVAX1表达载体，为TSOL18基因重组犬2型腺病毒活载体疫苗的研制奠定了基础。  相似文献   

5.
根据已发表的猪瘟病毒（classical swine fever virus，CSFV）Shimen株的全基因组序列，设计2对引物P1／P2和B1／B2，在B1和B2的5’端分别加上EcoR 1和BamH 1位点，以CSFV—Shimen株脾毒为材料。一步法提取总RNA。并以此为模板采用反转录PCR（RT—PCR）和套式PCR（nPCR）。成功地扩增出约1．2kb的E2基因。将PCR产物回收后与pMD18-T载体连接并转化。经PCR和酶切鉴定获得重组质粒E2-T。将E2-T经EeoR 1和BamH 1酶切消化、回收目的基因后。与经BamH 1／EcoR 1酶切的逆转录病毒载体pBABE—puro连接并转化，经酶切鉴定获得重组质粒pBABE—puro—E2。序列测定表明，目的基因的插入位置、方向和读码框完全正确。  相似文献   

6.
为使大肠杆菌热敏毒素LT的毒素活性丧失的同时仍保留其较强的免疫原性，实验依据LT基因的同源序列设计并合成5条引物，采用突出末端PCR法和重组PCR法，将克隆于质粒pEWD299上的LT基因，分别引入BamHI、Ndel和xhol等位点，扩增出带有上述RE位点且含有m7和m112突变点的约1100bp和约800bp的DNA片段和不含突变点的约300bp的DNA片段，使控制LT毒力活性中心的第7位和第112位氨基酸的碱基分别发生诱变，各DNA片段经分熟、纯化、RE酶切和DNA连接酶连接后，插入经BamHI和XhoI双酶切的线性化的高效表达载体pGEX-4T-1的多克隆位点区，使LTm基因置于pTac强启动子下且与Thrombin蛋白基因融合表达，将连接产物转化入JM105菌株，挑出可疑阳性菌落，提取质粒，经酶切鉴定、PCR鉴定和DNA序列分析，证明读框正确，序列正确，获得了pGEX-4T-1(LT7t GEX-4T-1(LTm112两个重组表达质粒。为运用基因工程手段大量生产人 动物大肠杆菌流行性腹泻的疫苗抗原和幽门螺杆菌疫苗的粘膜免疫佐剂，完成了基因水平的工作。  相似文献   

7.
猪肺炎支原体黏附因子基因R1R2区的克隆及表达   总被引：4，自引：0，他引：4  
根据GenBank登录的猪肺炎支原体232株P97基因和J株黏附因子基因设计了1对引物，以我国猪肺炎支原体Z株(强毒株)基因组DNA为模板，通过PCR方法扩增了该株黏附因子基因的部分序列。经序列分析后，重新设计了1对带有EcoRI和HindⅢ酶切位点的引物，并经引物的定点突变，PCR扩增了Z株黏附因子的R1R2区。扩增产物经双酶切后克隆到表达载体pET-32(a) 中。该重组质粒经酶切鉴定后，将其具有正确阅读框架的重组质粒转化到大肠杆菌BL21(DE3)感受态细胞，37℃下经IPTG诱导表达，得到相对分子质量约29000的融合蛋白，表达量约为11％。  相似文献   

8.
为了进一步评价E.coli OmpF的功能,我们构建了E.coli外膜蛋白F的缺失突变株。首先根据E.coli F-M-2的测序后的OmpF基因序列设计PCR敲除引物,引物5’端分别有50bp和48bp的拟敲除基因的同源臂,3’端为扩增引物,以pKD3为模板,扩增两侧含FRT位点的氯霉素抗性基因,然后利用pKD46的λ-Red重组系统替换E.coli F-M-2基因组上的OmpF基因,再利用表达FLP重组酶的质粒pCP20将FRT位点之间的氯霉素抗性基因删除,最后用鉴定引物进行鉴定并测序。结果成功地构建了云南撒坝猪致病性E.coli OmpF缺失突变株。  相似文献   

9.
犬2型腺病毒E3区缺失性表达载体的构建及外源基因表达   总被引：2，自引：0，他引：2  
为构建可用于同源重组或体外重组的犬腺病毒（canime adenovirus,CAV)E3区缺失性表达载体，在克隆的E3区两末端设计并合成了2对突变引物，在引物中分别引入1个BgⅠⅡ酶切位点，以PBE3质粒为模板，经2次点突变后，得到含有2个BgⅠⅡ位点的重组质粒PBE3^M。该质粒经BgⅠⅡ酶切后自连得到E3缺失的质粒PBE3△。在PBE3△质粒的BgⅠⅡ位点加入接头后，产生含多个酶切位点的质粒PBE3L，经相应的酶切鉴定，证明突变，缺失和接头连接正确后，利用PBE3L分别构建了带有和不带有外源性启动子的绿色荧光蛋白基因的重组表达质粒，并分别进行转染以检测其表达。结果显示，PBE3LCGFP质粒在转染DK细胞后，于36h即可观察到荧光，72-96h无明显差别，传3代后仍可见表达荧光的细胞；而PBE3LGFP质粒转染DK细胞后经检测无表达。结果表明，构建的E3区缺失性载体不能利用E3区自身的启动子进行目的的基因的表达，外源性启动子的加入是必要的。  相似文献   

10.
利用 PCR技术从含有霍乱毒素 B亚单位 ( CTB)基因的质粒 p UCT1中扩增出不包括信号肽的 3 0 9bp的 CTB全基因 ,并通过点突变技术消除了 CTB阅读框内的终止密码子 ( TAA→GAA) ,以便于在 CTB基因的下游融合其他的外源基因。用限制性内切酶 Bam H 和 Eco R 对 PCR产物进行双酶切 ,以 T4DNA连接酶将其定向连接于经同样酶切处理的质粒 p ET-2 8b( )的多克隆位点上 ,构建成重组质粒 p ECTB,转化至BL2 1 ( DE3 )中。经 Bam H 和 Eco R 双酶切分析和 PCR扩增检测 ,证明重组质粒 p ECTB中含有 CTB基因。核苷酸序列分析表明 ,克隆的 CTB基因在重组质粒中的连接向位和阅读框架是正确的  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 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   

18.
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   

19.
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   

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