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根据犬新孢子虫NcSAG1-NcSRS2融合基因序列,设计了1对含有Kozak序列、终止密码子、BamHⅠ和XhoⅠ酶切位点的引物,以含有NcSAG1-NcSRS2融合基因的质粒pGEX-tNcP43-P36为模板,经PCR扩增获得NcSAG1-NcSRS2融合基因片段,用BamHⅠ和XhoⅠ双酶切该片段,回收得到含有以上2个酶切位点黏端的NcSAG1-NcSRS2融合基因,将此基因片段克隆至相同酶切回收后的pcDNA3.1（＋）真核表达载体中,获得重组质粒pcNCSAG1-SRS2。经PCR鉴定、限制性内切酶酶切分析和克隆片段序列测定、比较,证实了重组质粒的正确性。将构建好的真核表达质粒转染到COS-7细胞中进行瞬时表达,经免疫荧光检测,证实了该载体能在细胞内进行蛋白表达。 相似文献
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《贵州畜牧兽医》2015,(6)
利用实验室构建的含有犬瘟热病毒H基因的p MD18-H质粒,根据其序列设计带有Bam HⅠ和HindⅢ酶切位点的引物,对H基因ORF进行PCR扩增,得到约1 949 bp的片段。将该片段克隆到p MD18-T载体内,用Bam HⅠ和HindⅢ进行双酶切鉴定、质粒PCR鉴定,筛选出阳性克隆。将阳性克隆再用Bam HⅠ和HindⅢ进行双酶切,纯化回收CDV H基因ORF片段;将原核表达载体p ET32a(+)用同样的方法酶切,回收载体片段,并用T4连接酶将以上两回收片段连接,构建原核表达载体质粒p ET32-H,后经Bam HⅠ和HindⅢ双酶切、质粒PCR、测序进行系列鉴定。结果表明,p ET32-H原核表达质粒构建成功,其中插入片段大小为1 842 bp,可编码607个氨基酸残基。该实验为下一步H蛋白的原核表达及单克隆抗体的制备奠定了基础。 相似文献
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为了获得口蹄疫病毒3AB基因重组伪狂犬病毒载体,试验以伪狂犬病毒TK/gⅠ缺失疫苗株为亲本株提取病毒基因组DNA,用限制性内切酶AscⅠ酶切基因组获得含完整PK基因的8.7 kb片段,将此片段克隆入pPolyⅡ载体的AscⅠ多克隆位点获得中间载体P8-AA;用限制性内切酶SacⅠ+NdeⅠ酶切质粒P8-AA,切去PK基因中的2 218 bp片段,在质粒P8-AA的PK基因缺失区插入已构建好的质粒pEGFP-3AB中含绿色荧光蛋白(EGFP)基因和3AB基因的完整表达盒,转染于猪肾细胞。结果表明:成功构建出可以用于同源重组的转移载体P8-EGFP-3AB,并且在猪肾细胞中成功表达了绿色荧光蛋白基因。 相似文献
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本研究旨在获得猪GDF-11的特异性抗体,以便于进一步研究猪GDF-11的功能.对猪GDF-11的编码区进行了克隆、表达载体的构建和转化、原核诱导表达和纯化以及将GDF-11注射到小鼠中并分离抗体.从猪胚肾中分离总RNA,经RT-PCR扩增得到猪GDF-11基因的编码区,插入pMD18-T载体中.再经限制性内切酶BarnH Ⅰ和EcoRⅠ双酶切,将回收的GDF-11酶切片段插入原核表达载体pGEX-4T-2中,获得重组原核表达质粒pGEX-4T-2-GDF-11.重组质粒经测序鉴定后转化大肠杆菌BL21(DE3),并经IPTG诱导产生了65 ku的GST-GDF-11融合蛋白.融合蛋白经凝血酶酶切,分离纯化出42 ku左右的GDF-11蛋白;然后注射小鼠,制备了多克隆抗体,其滴度最高可达1:25 600,而且特异性好,表明得到了猪GDF-11的多克隆抗体. 相似文献
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将携带有化学合成的SREBP-1c基因的质粒先用Xba Ⅰ酶切,Klenow补平突出末端,纯化回收后再用Hind Ⅲ酶切,切胶回收目的基因片段;并将其克隆至用EcoR Ⅴ/HindⅢ酶切回收的pShuttle-EGFP-CMV(-)TEMP穿梭载体上.经酶切鉴定后,重组穿梭质粒和腺病毒pAdxsi质粒分别用I-Ceu Ⅰ+I-Sce Ⅰ双酶切处理,T4DNA连接酶连接,获得重组质粒pAdxsi-GFP-SREBP1c,酶切鉴定后经Pac Ⅰ酶切线性化转染HEK293细胞,经包装扩增后用TCID50测定病毒滴度.以重组腺病毒感染犊牛原代肝细胞,采用qRT-PCR和Western blot检测细胞中SREBP1c mRNA和蛋白的表达量.结果显示,重组腺病毒酶切鉴定正确,滴度可达1.2×1010,腺病毒感染犊牛原代肝细胞中SREBP-1c mRNA和蛋白的表达显著升高.结果表明,本试验成功构建了SREBP-1c基因过表达重组腺病毒,且SREBP-1c在犊牛原代肝细胞中高度表达. 相似文献
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SRY基因是哺乳动物性别分化与控制的主导基因,参与有序的、多层次调控性别分化和控制的过程。本研究从牛血中提取总RNA,设计特异性引物,利用RT-PCR技术扩增出SRY基因HMG box基因片段,将该片段与pMD18-T载体连接并转化到大肠杆菌(E.coliTop)10中,提取质粒并用EcoRⅠ、AvrⅡ酶消化,将消化后的目的基因纯化并回收,与经同样的酶消化后并纯化回收的载体pPIC9K连接并转化到E.coliTop10,获得重组表达质粒pPIC9K-SRYHMG box,经PCR、酶切、测序验证了此载体构建成功,为下一步使用毕赤酵母表达SRY蛋白打下基础。 相似文献
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轻链可变区和重链可变区基因产物等摩尔浓度混合后,用重叠延伸拼接法扩增出单链抗体基因,再用带限制性内切酶位点的引物(NcoⅠ和HindⅢ)扩增出带有特定酶切位点的单链抗体基因片段.NcoⅠ和HindⅢ双酶切单链抗体与表达载体pET-22b( ),用回收试剂盒回收酶切后的单链抗体与表达载体pET-22b( )基因片段,用连接试剂盒将单链抗体与表达载体pET-22b( )基因片段连接,并将连接产物转化感受态细胞JM109.氨苄青霉素筛选阳性重组子.以T7promoter/T7terminator primer进行茼落PCR鉴定,测定正确后,37℃摇菌扩增,测菌浓度至D600为0.6时,加入IPTG诱导表达,对表达产物进行SDS-PAGE初步检测.SDS-PAGE检测显示其相对分子质量是31 000,与预期结果一致,证明单链抗体基因在大肠杆菌中得到表达. 相似文献
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SRY基因是哺乳动物性别分化与控制的主导基因,参与有序的、多层次调控性别分化和控制的过程.本研究从牛血中提取总RNA,设计特异性引物,利用RT-PCR技术扩增出SRY基因HMG box基因片段,将该片段与pMD18-T载体连接并转化到大肠杆菌(E.coli Top)10中,提取质粒并用EcoRⅠ、AvrⅡ酶消化,将消化后的目的基因纯化并回收,与经同样的酶消化后并纯化回收的载体pPIC9K连接并转化到E.coliTop10,获得重组表达质粒pPIC9K-SRYHMG box,经PCR、酶切、测序验证了此载体构建成功,为下一步使用毕赤酵母表达SRY蛋白打下基础. 相似文献
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分别以pEGFP-Mito和pAN4质粒为模板,扩增出MTS-EGFP和EcoRⅠ基因片段,利用快速体外基因重组技术-重组PCR,将两者连接得到MTS-EGFP-EcoRⅠ片段,构建克隆载体pTRE2hyg-EE,经AgeⅠ和NotⅠ双酶切得到MTS-EGFP-EcoRⅠ融合基因产物,连接克隆于真核表达质粒pEGFP-Mito后获得pMDD-Z质粒。琼脂糖凝胶电泳结果显示,MTSEGFP和EcoRⅠ基因片段大小分别是831 bp和860 bp。测序结果显示,插入pTRE2hyg--EE克隆载体中的MTS-EGFPEcoRⅠ片段大小1 672 bp,且基因序列正确,没发生点突变。琼脂糖凝胶电泳结果显示,pMDD-Z质粒的双酶切产物分别为1 587 bp的插入片段和4 024 bp的载体片段。测序结果显示,pMDD-Z质粒中含1 659 bp的开放阅读框,编码553个氨基酸,从N端至C端,分别为线粒体信号导肽、绿色荧光蛋白和限制性内切酶EcoRⅠ,且未发生移码突变。 相似文献
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Krizsan SJ Nyholm L Nousiainen J Südekum KH Huhtanen P 《Journal of animal science》2012,90(9):3162-3173
The objective of this study was to compare the application of different in vitro and in situ methods in empirical and mechanistic predictions of in vivo OM digestibility (OMD) and their associations to near-infrared reflectance spectroscopy spectra for a variety of forages. Apparent in vivo OMD of silages made from alfalfa (n = 2), corn (n = 9), corn stover (n = 2), grass (n = 11), whole crops of wheat and barley (n = 8) and red clover (n = 7), and fresh alfalfa (n = 1), grass hays (n = 5), and wheat straws (n = 5) had previously been determined in sheep. Concentrations of indigestible NDF (iNDF) in all forage samples were determined by a 288-h ruminal in situ incubation. Gas production of isolated forage NDF was measured by in vitro incubations for 72 h. In vitro pepsin-cellulase OM solubility (OMS) of the forages was determined by a 2-step gravimetric digestion method. Samples were also subjected to a 2-step determination of in vitro OMD based on buffered rumen fluid and pepsin. Further, rumen fluid digestible OM was determined from a single 96-h incubation at 38°C. Digestibility of OM from the in situ and the in vitro incubations was calculated according to published empirical equations, which were either forage specific or general (1 equation for all forages) within method. Indigestible NDF was also used in a mechanistic model to predict OMD. Predictions of OMD were evaluated by residual analysis using the GLM procedure in SAS. In vitro OMS in a general prediction equation of OMD did not display a significant forage-type effect on the residuals (observed - predicted OMD; P = 0.10). Predictions of OMD within forage types were consistent between iNDF and the 2-step in vitro method based on rumen fluid. Root mean square error of OMD was least (0.032) when the prediction was based on a general forage equation of OMS. However, regenerating a simple regression for iNDF by omitting alfalfa and wheat straw reduced the root mean square error of OMD to 0.025. Indigestible NDF in a general forage equation predicted OMD without any bias (P ≥ 0.16), and root mean square error of prediction was smallest among all methods when alfalfa and wheat straw samples were excluded. Our study suggests that compared with the in vitro laboratory methods, iNDF used in forage-specific equations will improve overall predictions of forage in vivo OMD. The in vitro and in situ methods performed equally well in calibrations of iNDF or OMD by near-infrared reflectance spectroscopy. 相似文献
12.
大庆市羊东毕吸虫病流行病学调查 总被引：1，自引：0，他引：1
对大庆市羊东毕吸虫病的流行病学进行了调查研究。结果表明，大庆有3种东毕吸虫，即程氏东毕吸虫（O.cheni)、土耳其斯坦东毕吸虫（O.turkestanica)和土耳斯坦东毕吸虫结节变种（O.turkestanican var.thuberculata)；中间宿主螺类有1种，即卵萝卜螺；东毕吸虫病在大庆分布很广，所调查的4区2县均有发生，羊东毕吸虫平均感染率分别为50．33％，平均死亡率为21．12％，大庆羊急性病例发病时间为8月上旬至10月下旬，慢性病例为11月上旬至翌年2月中旬。 相似文献
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14.
中国圈养野生动物疫苗使用调查 总被引：1，自引：0，他引：1
2005年对中国动物园协会单位中的18家动物园饲养野生动物疫苗免疫情况进行调查。受调查动物园在野生动物疫苗使用和动物种类方面具有一定的代表性。调查结果显示动物园动物现共使用18类36种疫苗,预防31种疫病。其中哺乳动物使用14类24种疫苗,预防24种疫病;禽类使用4类12种疫苗,预防7种疫病。使用范围最广的有禽流感、犬瘟热疫苗等、新城疫疫苗、猫瘟热疫苗等。共有24目58科动物接种疫苗,其中食肉动物类1目8科,食草动物类3目9科,杂食动物类2目3科,禽类18目38科。研究结果提示应加强动物园之间疫病信息交流、防疫资源的利用、疫病监测和研究,加大对动物园动物疫病的研究投入,逐步建立动物园动物统一的防疫规程。 相似文献
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Information gathered from cases of fowl cholera (FC) in commercial turkey flocks through case records, flock records, and telephone and mail surveys was used to estimate disease costs. The cost to the Georgia commercial turkey industry in 1986 from preventive measures, treatment of outbreaks, and production losses from the disease was estimated at $634,545. The cost of FC per kg of live production was estimated to be $0.015. 相似文献
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猪伪狂犬病是由伪狂病病毒(Pseudorabies virus PRV)引起的急性传染病。其特征是成年猪常为隐性感染，可有流产、死胎、呼吸系统症状，新生仔猪除神经症状外还可侵害消化系统，并伴大批死亡。本病已经给全世界的养猪业造成严重的损失，我国许多地区已报道该病。为进一步探讨该病的控制、净化措施，我们对12个养猪场共856分血清采用美国嗍公司的ELISA试剂盒进行了抗体检测。 相似文献
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W A Vestre 《Modern veterinary practice》1984,65(2):93-97
In 5 cases of glaucoma (2 from trauma, 2 from narrowed drainage angles, 1 secondary to lens extraction), cyclocryotherapy was used to control intraocular pressure. In all cases the intraocular pressure decreased, with the usual result being a cosmetic and painless but blind eye. 相似文献
18.
Occurrence of MRSA in pigs and in humans involved in pig production--preliminary results of a study in the northwest of Germany 总被引：1，自引：0，他引：1
Meemken D Cuny C Witte W Eichler U Staudt R Blaha T 《DTW. Deutsche tier?rztliche Wochenschrift》2008,115(4):132-139
In 2007, 678 pigs of all age groups out of 347 different farms from Lower Saxony and Northrhine-Westphalia and 86 persons occupationally exposed to pigs were investigated for their nasal colonisation with methicillin-resistant Staphylococcus areus (MRSA) by the Field Station for Epidemiology of the University of Veterinary Medicine Hannover and the Robert Koch-Institute. At the individual animal level, a frequency of positive results of 13% (n = 85 positive animals) and at the herd level, a frequency of positive results of 18% (n = 62 positive herds) were found. All isolates were assigned to the Multilocus Sequence Typing Type ST398. Within MRSA-positive herds, there were more MRSA-negative than MRSA-positive animals. Among the occupationally exposed persons (veterinarians, laboratory personnel and meat inspection personnel), 20 persons (23%) showed a nasal colonisation with MRSA ST398. A quite strong association between the intensity of the contact to pigs with the frequency of nasal colonisation in the occupationally exposed persons was detected. None of the animals or the humans nasally colonised by MRSA ST398 showed any clinical symptoms of a staphylococcal infection. Conclusions are drawn on the herd and intra-herd prevalence of the nasal colonisation of pigs with MRSA ST398 in pigs, but especially on which questions need to be addressed by further research. 相似文献
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为了解西藏拉萨市家禽禽流感抗体水平,2017年8月随机选择免疫过H5禽流感疫苗的8个规模养殖场、2个散养户、3个活禽交易市场,共13个群体,采集1 041份家禽血清样品进行H5、H7、H9亚型禽流感HI抗体检测。结果显示：H5亚型禽流感免疫抗体合格率为45.73%,H7、H9亚型抗体阳性率分别为21.13%、52.64%;养殖场、散养户和活禽交易市场的不同亚型抗体水平存在较大差异,规模化养殖场的H7亚型抗体阳性率最高（29.92%）,活禽市场的H5、H9亚型阳性率最高（78.16%、79.31%）,散养户的所有亚型抗体阳性率均最低。结果表明,拉萨市H5亚型禽流感疫苗免疫效果较差,特别是散养户,疫病发生风险较高,H7、H9亚型疑似自然感染率较高,应进一步查明原因,及时采取相应的措施,防止疫情发生。 相似文献
20.
N. Hawa D. G. Rae S. Younis W. Mahadi R. Ibrahim W. Al-Wahab 《Tropical animal health and production》1988,20(3):130-136
Summary Parvaquone was tested in cattle infected withTheileria annulata when they were presented at clinics in the vicinity of Baghdad. Out of over 200 cases presented with suspected theileriosis between July 1984 and July 1985, the drug was used in 45 cases where theileriosis was confirmed by microscopic examination of blood and lymph node biopsy smears. Twenty seven of the cases were considered mild and 18 cases severe. Weights of the cattle were estimated and parvaquone was administered by intramuscular injection at a nominal dose of 20 mg/kg. A single treatment with parvaquone was used in 25 cases and 20 cases were treated twice but there was no correlation between severity of disease and the number of treatments given. Twelve cases (27%) also received antibacterial therapy. All cases were in exotic cattle or cattle born from exotic (imported) cattle and 64% of the cases were in cattle under six months of age. Temperatures dropped immediately after treatment and the majority were normal (below 39.5°C) by two to three days after the first treatment. Of the 45 cases treated 43 recovered. This compares very favourably with a previously reported mortality of 66% in untreated imported cattle in Iraq.
Eficacia Del Parvaquone En El Tratamiento De La Theilerosis Natural En Ganado En Iraq
Resumen Se trató ganado infectado conTheileria annulata en forma natural con parvaquone, en clínicas veterinarias localizadas en la vecindad de Baghdad. De 200 casos admitidos con sospecha de theileriosis, entre julio 1984 y julio 1985, la droga se usó en 45 casos confirmados mediante el examen microscópico de frotis de sangre y biopsias de ganglios linfáticos. Veintisiete de los casos se consideraron moderados y 18 casos severos. Se estimaron los pesos de los animales enfermos, suministrandoles parvaquone vía intramuscular en dosis nominales de 20 mg/kg (rango 16–35 mg/kg). Se utilizó un solo tratamiento con parvaquone en 25 casos clínicos, mediante la aplicación de una sola inyección y se aplicaron dos inyecciones en 20 casos (doble tratamiento), sin que se encontrara correlación alguna entre la severidad de la enfermedad, recuperación y número de tratamientos. Doce casos (27%) recibieron también algún tratamiento antibacteriano. Todos los casos de theileriosis se presentaron en ganado exótico o nacido de ganado exótico (importado) y 64% de los casos ocurrieron en ganado bajo los seis meses de edad. La temperatura bajó inmediatamente después del tratamiento, normalizándose dos o tres días después de tratamiento (bajo 39.5°C). De los 45 casos tratados 43 se recuperaron. Se informa una mortalidad previa de 60% en ganado importado no tratado contra theileriosis en Iraq.
Efficacite De La Parvaquone Pour Le Traitement De La Theileriose Naturelle Des Bovins En Irak
Résumé La parvaquone a été essayée pour le traitement de bovins infectés parTheileria annulata lorsqu'ils étaient présentés aux cliniques des environs de Bagdad. Sur plus de 200 cas présentés pour suspicion de theilériose entre juillet 1984 et juillet 1985, le médicament a été utilisé dans 45 cas où la theilériose était confirmée par l'examen microscopique du sang et de biopsies de ganglions lymphatiques, 27 cas étaient considérés comme des atteintes modérées et 18 comme des cas graves. Les poids des animaux étaient estimés à l'oeil et la parvaquone administrée par voie intra-musculaire à la dose normale de 20 mg/kg (soit 16 à 35 mg/kg). Un traitement unique à la parvaquone a été pratiqué dans 25 cas et il a du être renouvelé dans 20 cas mais il n'y avait pas corrélation entre la gravité des symptômes et le nombre de traitements requis. Dans 12 cas (27%) une thérapie antibactérienne a également été mise en oeuvre. Tous les malades étaient des animaux importés ou issus d'animaux importés et dans 64% des cas les animaux étaient âgés de moins de 6 mois. Les températures ont chuté immédiatement après le traitement et, pour la plupart, étaient normales (moins de 39°5) deux à trois jours après le traitement—sur 45 animaux traités, 43 ont guéri. Ces résultats apparaissent très favorables par rapport à la mortalité de 66% rapportée antérieurement et affectant les bovins non traités en Irak.相似文献