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1.
采用蛋白酶K法从母牛肝中提取染色体DNA,将其纯化后作为PCR扩增模板。以引物设计软件从牛β-酪蛋白基因上游600bp至第1个外显子设计引物,引物长度19nt,跨度637bp。扩增产物电泳结果显示,在分子量Marker657bp带附近,出现一明显亮带,这一结果与设计产物大小基本一致。用低温冷冻法纯化PCR产物,煮沸裂解法提取载体pBluescriptⅡKs+质粒,EcoRV酶切质粒DNA,T4DNA连接酶连接PCR扩增片段和质粒DNA。将重组质粒DNA转化到JM101大肠杆菌中,经筛选、酶切、测序鉴定,结果表明所克隆的片段为牛β-酪蛋白基因上游调控序列。  相似文献   

2.
产气荚膜梭菌β-毒素基因的克隆与核苷酸序列分析   总被引:1,自引:0,他引:1  
为了给产气荚膜梭菌(Clostridiumperfringens)β毒素基因工程亚单位苗和细菌毒素多价基因工程苗的研制提供基因材料,用聚合酶链式反应(PCR)技术,从B型产气荚膜梭菌染色体基因组中扩增了930bp的β-毒素基因。用限制性核酸内切酶BamHI和EcoRI对PCR产物进行双酶切处理,然后通过T4DNA连接酶将其定向连接于事先经同样的双酶切处理的载体质粒pET-28C(+)的多克隆位点,转化至受体菌BL21(DE3)中。经BamHI和EcoRI双酶切分析和PCR扩增检测,证明重组质粒pECB2中含有产气荚膜梭菌的β-毒素基因。经核苷酸序列分析,明确了克隆的β-毒素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   

3.
猪生殖和呼吸综合征中国分离株ORF7基因的克隆及鉴定   总被引:2,自引:0,他引:2  
本研究利用对应于ATCCVR-2332及LVORF7基因保守序列的1对均为28个碱基的引物10011PCS、1010PCR对PRRSV中国分离株B13进行RT-PCR,结果扩增出了1条包括完整ORF7基因的510bp的DNA片段。纯化此扩增产物,并对其进行EcoRI/PstI双酶切,与用EcoRI、PstI双酶切及碱性磷酸酶处理的PUC18载体连接。转化大肠杆菌。结果得到了1个B13ORF7基因与PUC18载体的重组质粒PUC18B13ORF710。通过EcoRI/PstI及PCR证明此重组质粒即为B13ORF7基因与PUC18载体的重组质粒。从而为ORF7基因及所表达的核衣壳蛋白进一步研究奠定了基础。  相似文献   

4.
猪生殖与呼吸综合征病毒ORF6片段的cDNA克隆及鉴定   总被引:1,自引:0,他引:1  
根据已发表的PRRSV基因组序列,设计并合成了1对特异扩增ATCC VR-2332株的ORF6的基因的引物,以ATCC VR-2332基因组为模板,通过RT-PCR扩增出586bp的cDNA产物。将该cDNA片段经T-A连接插入pGEM-Teasy载体中,用重组质粒转化大肠埃希氏菌JM109,获得重组质粒转化菌。对重南粒DNA进行PCR及酶切鉴定,证明插入的DNA片段与PCR扩增的DNA片段大小相  相似文献   

5.
副结核分枝杆菌DNA探针的制备与应用   总被引:1,自引:0,他引:1  
以溶菌酶、SDS、高氯酸钠等处理提取副结核分析杆菌C2株染色体DNA,经限制性内切酶Pst I消化后,以质粒pBluescript SK为载体,通过T4DNA连接酶连接,转入E。coli DH5a受体菌中,构建了副结核菌C2株的DNA基因文库。应用反向杂交试验,从基因文库中筛选出4个重组克隆:PTP12、PTP19、PTP38。对这4个重组克隆进行酶切、电泳分析,结果表明4个插入片段长度分别为:4  相似文献   

6.
参考禽副粘病毒的融合蛋白基因(F基因)cDNA序列,设计并合成了1对引物,以他离到对鹅有致病力的Ⅰ型禽副粘病毒GPMV/QY97-1株的核酸(RNA)为模板,用逆转录聚合酶链反应(RT-PCR)对其融合蛋白基因3'端进行扩增,获得了预计的884bp左右的片段。将该片段克隆进pGEM-T Easy质粒载体,获得重组质料T-GPMVF3',采用限制性内切酶和PCR方法对该重组质粒进行鉴定,证明所克隆的  相似文献   

7.
基因探针和PCR对鲤吉陶单极虫的检测   总被引:3,自引:1,他引:3  
采用液氮冷冻研磨法破碎吉陶单极虫(Thelohaneluskitauei)孢子,按常规法提取其基因组DNA。取DNAEcoRI消化产物,采用低熔点琼脂糖回收法制备大(3.0~15.0kb)、小(0.5~3.0kb)片段,将其克隆于pBluescriptksEcoRI位点,经α-互补现象、酶切鉴定和虫体DNA生物素标记探针筛选,构建了含23个克隆的基因文库,克隆片段介于0.9~6.8kb之间;经阳性克隆标记筛选及特性分析,制备了长度为0.9kb(19号质粒克隆片段)的探针,该探针具有较强的敏感性和特异性。对该探针两端DNA序列进行分析,设计出1对引物。上游引物序列为:5′TTTCGAACCCGCACAACAAG3′,下游引物序列为:5′TGAGTGGATAG-TATCGCTGC3′。应用该对引物对虫体进行了检测  相似文献   

8.
产蛋下降综合征(EDS-76)病毒DNA酶切图谱分析   总被引:4,自引:0,他引:4  
选用EcoRI、PstⅠ、PvuⅡ、HindⅢ、BglⅠ和BglⅡ6种限制性内切酶,对国内4株EDS-76病毒鸡源分离株和国外标准株AV-127及1株鹅源腺病毒的DNA进行酶切图谱的比较,结果发现4种鸡源分离株与AV-127株用上述6种酶切的片段数分别为4、8、10、10、6和7条。各酶切图形、片段大小亦十分相似,表明均为EDS-76病毒。EcoRI-HindⅢ和PstI-EcoRI的双酶切也得到同样结果。鹅源分离株的酶切片段数分别为6、7、9、7、4和10条,酶切图形和片段大小均与AV-127有很大不同,表明该分离株可能为1株血清型相同而基因组不同的EDS-76鹅腺病毒。  相似文献   

9.
将含产气荚膜梭菌α毒素基因的质粒pKMA100用限制性核酸内切酶BamHI和HindⅢ双酶切,经1.5%琼脂糖凝胶电泳分离、透析袋电洗脱法回收0.95kbα毒素基因片段,再将载体pET-28b(+)用BamHI和HindⅢ双酶切,然后将处理好的pET-28b(+)与0.95kb的α毒素基因片段通过T4DNA连接酶进行粘性末端连接,转化至受体菌BL21(DE3)中。经BamHI和HindⅢ双酶切分析和核苷酸序列分析,证明重组质粒pXETA1含有产气荚膜梭菌α毒素基因。确定了α毒素基因的全部核苷酸序列,并证明其具有正确的阅读框架。  相似文献   

10.
新城疫病毒F48E9株HN基因的克隆与酶切分析   总被引:9,自引:0,他引:9  
为深入探讨新城疫病毒(NDV)的结构与功能的关系,扩增和克隆了F48E9株NDV的HN基因。首先以提取的F48E9株NDV基因组RNA为模板逆转录合成第一链cDNA,然后用HN基因特异性引物作PCR扩增HNcDNA,结果得到1条分子量约1.8kb的DNA带,与NDVHN基因的大小一致。Southernblot杂交进一步证实其为HN特异性cDNA。随后采用平端连接法将其克隆到pSV·Sportl质粒中,应用限制性内切酶EcoRI、ScaⅠ、MluⅠ、ApaⅠ、PstⅠ、和SmaⅠ对NDVF48E9株HNcDNA重组质粒作单酶或双酶切,结果表明,NDVF48E9株HN基因较HitchnerB1株的HN基因缺少1个MluⅠ位点(705bp左右),多1个PstⅠ位点(802bp左右)。  相似文献   

11.
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   

12.
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   

13.
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   

14.
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   

15.
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   

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17.
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.  相似文献   

18.
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  相似文献   

19.
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  相似文献   

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