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《中国预防兽医学报》2015,(11)
为筛选牛源多杀性巴氏杆菌(Pm)交叉保护性抗原,本研究以A型Pm CQ2株基因组为模板,对其omp A、omp H、plp E、pm0979、P6和pfh B基因分别进行扩增,克隆于相应表达载体并进行原核表达,同时对表达产物进行纯化及western blot鉴定。将纯化的重组蛋白免疫小鼠,分别测定其对2 LD50剂量的A型和B型Pm的攻毒保护效果。结果表明,重组蛋白r Omp A、r Omp H、r Plp E、r Pm0979、r P6和r Pfh B对A型Pm CQ2株的攻毒保护率分别为20%、60%、70%、40%、20%和10%,对B型Pm CVCC450株的攻毒保护率分别为0、30%、40%、20%、10%和0,表明重组蛋白r Plp E、r Omp H和r Pm0979可以针对不同血清型的Pm提供较好的交叉保护作用。本研究为牛源Pm新型疫苗的研制奠定了基础。 相似文献
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试验为了研究牛冠状病毒NS32蛋白的免疫原性,以NS32作为研究对象,通过原核表达分析其抗原性。首先,从牛冠状病毒基因组中克隆出NS32编码基因,进一步将其克隆到原核表达载体上;其次,通过纯化重组的His-NS32蛋白进一步免疫小鼠,测定其特异性抗体水平评估NS32蛋白免疫原性。本试验成功地利用反转录PCR克隆出NS32基因,其片段大小为837 bp,序列同源性分析结果显示,该基因在不同牛冠状病毒分离株间高度保守。通过原核表达系统纯化蛋白并成功获得目的蛋白HiS-NS32,SDS-PAGE显示其大小约为32 ku。将纯化的蛋白免疫小鼠,免疫小鼠能够产生针对NS32蛋白的高水平特异性抗体,证明牛冠状病毒非结构蛋白NS32同样具有较高的免疫原性。本试验通过表达NS32蛋白证实了其免疫原性,为后期的NS32蛋白研究奠定了基础。 相似文献
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为了验证EgM123表达蛋白的免疫原性功能,建立犬细粒棘球绦虫EgM123基因的原核表达和蛋白纯化体系,为EgM123蛋白单克隆抗体制备与基因重组疫苗的研制提供研究基础。本试验通过构建EgM123基因原核蛋白表达载体及其蛋白表达条件优化、利用亲和层析方法对表达蛋白进行纯化、SDS-PAGE（聚丙烯酰氨凝胶电泳）和Western blotting（蛋白质印迹）试验对表达蛋白的分子量和免疫原性进行检测,并通过间接酶联免疫吸附（ELISA）方法对所表达的EgM123蛋白的抗原功能进行验证。本试验通过菌液聚合酶链式反应（PCR）扩增和双酶切验证成功构建EgM123基因原核蛋白表达载体;优化出最佳诱导条件为37℃、表达时间为7h下表达量最高;Western blotting显示其在28KDa处均有特异性条带;ELISA显示纯化后的蛋白能很好的与EgM123多克隆抗体结合。上述结果表明EgM123表达蛋白极具免疫原性,可用于免疫BALB/c小鼠制备EgM123单克隆抗体。 相似文献
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以牛分枝杆菌Vallee株基因组为模板,利用PCR方法扩增该菌株的环丙烷一分枝茵酸合酶pcaA基因,并克隆到原核表达载体pET-32a（＋）中,通过大肠杆菌BL21（DE3）表达重组融合蛋白,并利用Ni-亲和层析的方法纯化出重组蛋白。通过鉴定其免疫活性。试验结果表明,成功扩增出了牛分枝杆菌中pcaA基因并纯化出了重组的融合蛋白,通过免疫印记发现,重组蛋白可与牛结核阳性血清发生特异性反应,为进一步研究牛分枝杆菌的致病机理奠定基础。 相似文献
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以牛分枝杆菌DNA为模板,克隆了牛分枝杆菌MPB70基因,构建了克隆载体pGEM-MPB70和表达载体pET30a-MPB70,经IPTG诱导在大肠杆菌BL-21中表达,用SDS-PAGE和免疫印迹分析表达产物并进行蛋白纯化。试验结果表明,牛分枝杆菌MPB70基因体外扩增产物与预期值相符,约582 bp;所构建表达质粒pET30a-MPB70经测序,结果与预期一致;SDS-PAGE分析表明,该融合蛋白以包涵体的形式表达,其分子质量约为29 kD,蛋白表达量约占菌体总蛋白的20%;该蛋白经电洗脱纯化后,纯度达85%以上;免疫印迹分析表明,原核表达的融合蛋白可与兔抗牛分枝杆菌多克隆抗体结合,并且具特异的免疫反应性。 相似文献
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目的研究布鲁氏菌外膜蛋白OMP25的克隆、测序和表达,并对其进行免疫特性检测。方法采用PCR技术对牛布鲁氏菌外膜蛋白OMP25基因进行了扩增和克隆测序,并成功构建了原核表达质粒pGEX-OMP25。将其转入E.coliBL21,经IPTG诱导表达,用电洗脱纯化后获得了高纯度的目的蛋白。纯化后的蛋白与弗氏佐剂乳化后免疫家兔得到了较高效价的抗血清。结论通过ELISA、Western-blotting分析及特异性检测试验,证明目的蛋白不仅具有抗原性且有良好的免疫反性。 相似文献
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试验旨在确定牛支原体P48基因的免疫原性，为进一步筛选牛支原体免疫保护性基因奠定基础。本研究以牛支原体新疆分离株为研究对象，运用Overlap PCR方法扩增得到点突变后的牛支原体新疆分离株P48基因，构建原核表达载体pET-32a （+）-P48，转化大肠杆菌BL21（DE3）感受态细胞，在诱导剂ITPG的诱导下获得重组蛋白P48，纯化后的重组P48蛋白免疫BALB/c小鼠制备多克隆抗体，运用Western blotting和ELISA方法验证其反应原性和免疫原性。结果表明，试验成功构建原核表达载体pET-32a （+）-P48，重组蛋白P48大小约为66 ku，纯化后的牛支原体P48重组蛋白免疫小鼠后可产生良好的免疫反应，血清抗体滴度达到较高水平（D450 nm值为1.126）。Western blotting结果显示，抗牛支原体P48重组蛋白的鼠血清与牛支原体P48重组蛋白及牛支原体全菌蛋白抗原均能产生明显的抗原抗体反应，表明P48重组蛋白具有良好的免疫原性与反应原性，可作为牛支原体新型疫苗的候选基因，且牛支原体新疆分离株P48基因与国内外5株牛支原体P48基因的同源性很高，亲缘关系较近。 相似文献
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本研究对乳房链球菌GapC蛋白基因进行了克隆、重组表达、蛋白纯化和免疫原性试验。应用PCR技术直接从临床分离的乳房链球菌菌株基因组中扩增出GapC蛋白基因,并将其克隆至pET28a（＋）上,转化大肠杆菌BL21（DE3）中表达。经DNA序列测定分析,扩增出的基因与GenBank发表的乳房链球菌GapC基因序列AF421900的同源性为99．8％。氨基酸同源性为100％;与GenBank发表的无乳链球菌GapC基因序列AF421899的同源性为91．4％;与停乳链球菌GapC基因序列AF375662的同源性为88．9％。表达的融合蛋白通过MagneHis^TM蛋白纯化试剂盒纯化,纯化蛋白免疫小鼠3次,制备GapC抗血清。本研究表达和纯化了乳房链球菌GapC蛋白,并制备出鼠抗血清,为下一步开展GapC重组蛋白的应用研究莫定了基础。 相似文献
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为了阐明牛分枝杆菌侵入宿主细胞和在肺泡巨噬细胞长期存活的机理，本研究以牛分枝杆菌的DNA为模板，通过PCR的方法扩增克隆牛分枝杆菌哺乳动物细胞侵袭蛋白4E（Mammalian cell-entry protein 4E，mce4E）基因，将所扩增基因克隆于原核表达载体pET30a（＋）并进行测序，结果显示该基因与GenBank上所公布的牛分枝杆菌和人分枝杆菌mce4E基因同源性为100％。将重组表达载体转入宿主菌BL21进行诱导表达，表达蛋白经SDS-PAGE分析和Western-blotting免疫印迹鉴定，结果证明目的蛋白获得高效表达，表达量占菌体总量的53．3％，分子量约为45ku；利用Ni-NTA琼脂糖柱对表达蛋白进行纯化，纯化率大于95％；纯化的蛋白经过透析复性、圆二色谱（CD）测定，结果表明重组蛋白为典型的α螺旋型结构，经Jascow32软件分析计算，mce4E蛋白含有39．1％的α螺旋，60．9％的无规卷曲，无β-折叠和转角，为进一步开展牛分枝杆菌致病机理的研究和寻找新的药物作用靶位点奠定了基础。 相似文献
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魏静 《四川畜牧兽医学院学报》2009,(4):28-32
在现代法律秩序中,商会自治规范是制定法的基础和必要的补充,甚至在某些方面替代了制定法;商会自治规范主要包括商会组织规范、行为规范、惩罚规范以及争端解决规范等;其效力仅及于其内部成员;商会自治规范和制定法之间存在冲突,但也存在整合的基础。 相似文献
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本文概述了猪的毛色类型、猪的毛色遗传模式,着重综述了猪毛色基因分子基础的研究进展,指出存在问题并就未来发展方向做了思考。 相似文献
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REASONS FOR PERFORMING STUDY: Centesis of the bicipital bursa using an 8.9 cm long spinal needle has been reported but the alternative of employing a 3.8 cm long hypodermic needle requires validation. OBJECTIVE: To compare the efficacy of 2 different methods of centesis of the bicipital bursa and to evaluate the usefulness of ultrasonographic imaging to determine the location of solution administered when centesis of the bursa is attempted. METHODS: For Trial 1, 6 clinicians, who had no previous experience of centesis of the bicipital bursa, attempted to inject a solution composed of an aqueous radiopaque contrast medium and physiological saline solution (PSS) into the bicipital bursae of 2/12 horses using the previously described distal approach to inject one bursa and a proximal approach to inject the contralateral bursa. The bicipital tendon and bursa were examined ultrasonographically before and after injection; and both shoulders were examined radiographically to identify the location of the medium. In Trial 2, another 6 clinicians, also with no previous experience of centesis, repeated Trial 1, using 6 horses, but the radiopaque contrast medium was mixed with air instead of PSS. RESULTS: Accuracy of centesis using the proximal approach was 39% and that of the distal approach 28%. Ultrasonographic examination of the shoulder allowed the location of solution and air to be accurately predicted in all 12 shoulders examined. CONCLUSIONS: Clinicians who have had no previous experience performing centesis of the bicipital bursa are unlikely to be successful in centesis using either approach. Radiographic examination after injecting a radiopaque contrast medium may be necessary to assess the success of centesis especially if bursal fluid is not obtained during centesis. Injecting air along with the radiopaque contrast medium provides more accurate ultrasonographic confirmation of centesis and better radiographic definition than does injection without air. 相似文献
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Effects of size of ingestively masticated fragments of plant tissues on kinetics of digestion of NDF
Ingestively masticated fragments were collected and sized via sieving. Different sizes of esophageal masticate and ruminal digesta fragments, and ground fragments of larger masticated pieces were incubated in vitro, and undigested NDF remaining at intervals of up to 168 h of incubation was determined. The ruminal age-dependent time delay (tau) for onset of digestion of NDF was positively correlated (P < 0.004) with the mean sieve aperture estimated to retain 50% of the fragments between successive sieve apertures (MRA). Degradation rate of potentially degradable NDF (PDF) and level of indigestible NDF were not related (P > 0.10) to MRA of masticated and ground fragments. Estimates of tau were positively related to MRA, with slopes of bermudagrass < corn silage < ruminal fragments of corn silage. It was concluded that fragment size-, and consequently, ruminal age-dependent onset of PDF degradation of a mixture of different fragment sizes results in an age-dependent rate of degradation of the more rapidly degrading of two subentities of PDF. Models are proposed that assume a tau before onset of simultaneous degradation of PDF from two pools characterized as having gamma-modeled age-dependency and age-constant rates. The ruminal age-dependent pool seems to be associated with the faster-degrading pool, and its rate parameter increases with range in MRA in the population of fragments. Conceptually, the ruminal age-dependent rate parameter for PDF degradation seems to represent a composite of several effects: 1) effects of the size-dependent tau; 2) range in MRA of the population of ingestively masticated fragments; and 3) subentities of PDF that degrade via more rapid age-dependent rates compared with subentities of PDF that degrade via age-constant rates. The estimated fractional rates of ruminative comminution of ingestively masticated fragments (0.060 to 0.075/h) were of a magnitude similar to the mean fractional rates of PDF digestion (0.030 to 0.085/h), which implies that ruminative comminution may be first-limiting to fractional rate of PDF digestion. The in vivo roles of ingestive and ruminative mastication of fragments on PDF degradation must be considered in any kinetic system for estimating PDF digestion in the rumen. These results and others in the literature suggest that the rate of surface area exposure rather than intrinsic chemical attributes of PDF may be first-limiting to degradation rate of PDF in vivo. 相似文献
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乳酸杆菌作为益生菌广泛用于人和动物.本文综述了乳酸杆菌改善宿主健康的机制.乳酸杆菌可通过产生抗菌物质如乳酸、过氧化氢、细菌素,或者通过竞争营养或肠道黏附位点来抑制致病菌;通过诱导黏附素的分泌或阻止细胞凋亡而增强肠道的屏障功能,从而保护肠道.文章重点讨论了乳酸杆菌表面成分(表面蛋白、脂磷壁酸和肽聚糖)与肠道受体(C型凝集素受体、Toll样受体和Nod样受体),阐述了他们结合后启动免疫调节信号,调控肠道免疫功能以发挥改善健康作用的机制. 相似文献
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Billinghurst RC Brama PA van Weeren PR Knowlton MS McIlwraith CW 《American journal of veterinary research》2004,65(2):143-150
OBJECTIVE: To determine whether serum concentrations of biomarkers of skeletal metabolism can, in conjunction with radiographic evaluation, indicate severity of osteochondrosis in developing horses. ANIMALS: 43 Dutch Warmblood foals with varying severity of osteochondrosis. PROCEDURE: 24 foals were monitored for 5 months and 19 foals were monitored for 11 months. Monthly radiographs of femoropatellar-femorotibial and tibio-tarsal joints were graded for osteochondral abnormalities. Serial blood samples were assayed for 8 cartilage and bone biomarkers. At the end of the monitoring period, foals were examined for macroscopic osteochondrosis lesions. RESULTS: Temporal relationships were evident between certain serum biomarkers and osteochondrosis severity in foals during their first year. Biomarkers of collagen degradation (collagenase-generated neoepitopes of type-II collagen fragments, type-I and -II collagen fragments [COL2-3/4C(short)], and cross-linked telopeptide fragments of type-I collagen) and bone mineralization (osteocalcin) were positive indicators of osteochondrosis severity at 5 months of age. In foals with lesions at 11 months of age, osteochondrosis severity correlated negatively with COL2-3/4C(short) and osteocalcin and positively with C-propeptide of type-II procollagen (CPII), a collagen synthesis marker. Radiographic grading of osteochondrosis lesions significantly correlated with macroscopic osteochondrosis severity score at both ages and was strongest when combined with osteocalcin at 5 months and CPII at 11 months. CONCLUSIONS AND CLINICAL RELEVANCE: The ability of serum biomarkers to indicate osteochondrosis severity appears to depend on stage of disease and is strengthened with radiography. In older foals with more permanent lesions, osteochondrosis severity is significantly related to biomarker concentrations of decreased bone formation and increased cartilage synthesis. 相似文献