梅花鹿结核病间接ELISA方法的建立

为了建立鹿结核病的快速检测方法,试验采用亲和层析方法纯化梅花鹿血清IgG,并免疫兔,制备兔抗梅花鹿IgG抗体,辣根过氧化物酶(HRP)进行标记,通过ELISA和Western-blot对HRP标记的兔抗梅花鹿IgG抗体进行鉴定。同时合成结核分枝杆菌38 ku基因,并与pET-22b(+)质粒连接,转化到大肠杆菌BL21(DE3)感受态细胞中,诱导表达和纯化38 ku蛋白,以38 ku蛋白作为包被抗原,以HRP标记的兔抗梅花鹿IgG抗体作为二抗,建立间接ELISA方法。利用建立的间接ELISA方法对结核菌素皮肤试验检出的20份阴性血清和10份阳性血清进行验证。结果表明：对梅花鹿血清IgG进行纯化,得到大小分别约为50 ku和25 ku的蛋白质;制备的HRP标记的兔抗梅花鹿IgG抗体可特异性识别梅花鹿血清IgG,在抗体浓度稀释比例为1∶20 000时仍有很好的结合效果;38 ku蛋白作为抗原的最佳包被浓度为1μg/mL,被检梅花鹿血清的最佳稀释比例为1∶100,HRP标记的兔抗梅花鹿IgG抗体的最佳稀释比例为1∶10 000;间接ELISA方法能够准确区分结核菌素皮肤试验确定的阳性和阴性梅...     

马冠状病毒抗体的间接ELISA检测方法的建立

将含有马冠状病毒N蛋白(ECV-N)的重组杆状病毒rBac-ECV-N感染Sf9昆虫细胞,以表达的ECV-N蛋白作为抗原,以酶标记的鼠抗马IgC特异性单抗为二抗,建立了检测马冠状病毒抗体的ELISA方法.研究结果表明,ECV-N蛋白最佳包被浓度为10μg/mL初提重组蛋白,马血清以1:100稀释,酶标记的抗马IgG单克隆抗体工作浓度1:5 000.可以检出抗马冠状病毒血清抗体.利用该方法对来自3个国家的1064份马血清进行了检测,检测结果表明:国内711份马血清中检测出13份阳性;澳大利亚335份马血清中检测出31份阳性;瑞典18份马血清均为阴性.该间接ELISA方法的建立,为马冠状病毒感染的预防和控制,出入境检验检疫提供了一种新的检测方法.     

猫血清IgG的纯化及酶标兔抗猫二抗的制备

采用辛酸-硫酸铵法和Sephadex G-150分子筛层析法纯化猫血清IgG,SDS-PAGE电泳检测;用纯化的IgG免疫新西兰兔,制备兔抗猫IgG抗血清,并用上述方法提纯兔抗猫IgG,改良过碘酸钠法标记辣根过氧化物酶,即获得兔抗猫IgG的酶标抗体。     

牛病毒性腹泻-黏膜病病毒斑点酶联免疫吸附检测法的建立

采用辛酸-硫酸铵法提取兔抗BVDV高免血清中的免疫球蛋白IgG,在混合硝酸纤维素膜上进行间接酶联免疫吸附试验,建立了检测BVDV抗原的斑点酶联免疫吸附检测法.结果显示,抗体最佳包被量为400μg/mL,酶标记羊抗兔IgG的最佳工作浓度是1:500倍稀释,抗原最小检出量是5.36μg/mL.应用建立的检测方法对78份腹泻...     

检测微小隐孢子虫抗体的间接ELISA方法的建立

以在E．coli高效表达的微小隐孢子虫子孢子表面抗原CPl5／60为抗原，以辣根过氧化物酶(HRP)标记的山羊抗鼠IgG为二抗，建立了检测微小隐孢子虫抗体的间接ELISA方法。经检测筛选出最佳反应条件为1μg／孔纯化的E．coli表达的CPl5／60抗原包被酶标板，用10％的兔血清进行封闭，以正常E．coli裂解上清液稀释待检血清。结果表明应用CPl5／60重组蛋白作为诊断C．parvum抗原具有特异性高、抗原易纯化和成本低等特点。     

双抗夹心ELISA检测兔出血症病毒抗原方法的建立及初步评估

为建立一种快速、特异性强的用于检测兔出血症病毒(Rabbit hemorrhagic disease virus,RHDV)抗原的双抗夹心ELISA方法,本研究通过抗体配对实验,确定以抗兔出血症病毒结构蛋白VP60单克隆抗体9H9为捕获抗体,辣根过氧化物酶标记的9H9为检测抗体建立双抗夹心ELISA方法。通过优化,确定最佳条件为:0.5μg/m L稀释后的9H9作为捕获抗体,底物显色液37℃包被2 h 4℃过夜,1%明胶溶液37℃封闭2 h,用含10%胎牛血清的PBST按5μg/m L稀释后的辣根过氧化物酶标记的9H9作为检测抗体,37℃作用15 min。建立的ELISA方法与杯状病毒科其他病毒无交叉反应,应用本方法和RT-PCR方法对85份兔肝脏样品进行检测,证明双抗夹心ELISA方法具有良好的特异性和敏感性,有望作为临床快速检测的一种简便方法。     

免疫小鼠血清中猪圆环病毒2型抗体ELISA检测试剂盒的研究

为研制一种小鼠血清中猪圆环病毒2型（Porcine circovirus type 2,PCV2）抗体检测试剂盒,以PCV2 Cap蛋白兔多抗作为包被用抗体,捕获纯化的PCV2 Cap蛋白,制备抗原检测板,辣根过氧化物酶(HRP)标记羊抗小鼠IgG作为二抗,建立检测小鼠血清中PCV2抗体ELISA检测方法并制备试剂盒。采用所制备的ELISA试剂盒对50份已知阴性血清样本进行检测,临界OD450nm值为0335;采用ELISA试剂盒与IFA分别对100份血清检测,总符合率为96%,检测小鼠血清敏感性效价达1∶16000,与其他常见的猪病毒小鼠阳性血清无交叉反应,2～8 ℃保存15个月稳定,批内变异系数为2.28%～4.17%,批间变异系数为4.27%～6.02%,具有良好的敏感性、特异性、稳定性及重复性;采用ELISA试剂盒对不同来源疫苗免疫组小鼠血清进行检测,均显示良好效果。研究表明,本研究制备的ELISA试剂盒可用于小鼠血清中PCV2抗体效价检测。     

以重组mE2蛋白为抗原建立检测猪瘟病毒抗体间接ELISA方法 …

以在Ｅ．ｃｌｏｉ高效表达的猪瘟病毒Ｅ２基因主要抗原编码区为抗原，以辣根过氧化物酶标记的兔抗猪ＩｇＧ为二抗，建立了检测猪瘟病毒抗体的间接ＥＬＩＳＡ方法。经检测筛选出最佳反应条件为；１．２μｇ／孔纯化的Ｅ．ｃｏｌｉ表达的ｍＥ２重组蛋白包被酶标板，用１０％的兔血清或马血清进行封闭，以正常Ｅ．ｃｏｌｉ裂解上清液稀释待检血清。     

犬瘟热病毒F1基因的原核表达与初步应用

将犬瘟热病毒(CDV)F1基因定向克隆入pET-28a( )载体．构建了原核表达质粒pET-28F1。pET-28F1转化宿主菌BL21(DE3)plysS．在IPTG的诱导下．可高效表达目的基因，表达量达菌体蛋白总量的23．52％。Western blot分析表明，所表达蛋白能与抗CDV阳性血清发生特异性的抗原-抗体反应。以E．coli表达的CDV F1蛋白为抗原．HRP标记的兔抗犬IgG为二抗．建立了检测抗CDV抗体的间接ELISA方法。试验表明．应用CDV F1蛋白作为抗原检测抗CDV抗体具有特异性高、抗原易纯化、成本低等特点。     

牛病毒性腹泻-黏膜病病毒双抗体夹心Dot-ELISA检测方法的建立

采用辛酸-硫酸铵法提取兔抗牛病毒性腹泻-黏膜病毒(BVDV)高免血清中的免疫球蛋白IgG,辣根过氧化物酶标记提纯后IgG,建立了检测BVDV抗原的双抗体夹心斑点酶联免疫吸附试验(Dot-ELISA)法。结果显示,抗体最佳包被量为300μg/mL,酶标记抗体的最适浓度为1∶50倍稀释,以5%牛血清作为封闭液、封闭45min效果最佳,抗原最小检出量是1.34μg/mL。应用建立的检测方法对河北省内78份腹泻奶牛血样进行了检测,阳性检出率为57.69%;经卡方检验分析,建立的Dot-ELISA与琼脂扩散(AGP)法相比较,阳性检出率差异显著。试验证明,该方法具有简便快速、特异性强、重复性高的优点,适合于基层兽医的检测诊断。     

商会自治的基石——商会自治规范研究

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。     

癸氧喹酯干混悬剂含量测定的改进

采用高效液相色谱法测定癸氧喹酯干混悬剂的含量,在2-250μg/mL范围内,峰面积的常用对数与进样量浓度的常用对数呈良好的线性关系,R^2=1(n=5),平均回收率为99.24%~99.51%,RSD在0.05%~0.28%。此方法分析时间短,样品前处理简便、定量结果准确,重现性好,结果满意,为其质量控制提供了依据。     

猪毛色遗传的研究进展

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。     

Comparison of 2 methods of centesis of the bursa of the biceps brachii tendon of horses

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.     

不同样本商品蜂蜜中硒含量的测定

用硝酸和高氯酸消化蜂蜜,使硒游离出来,在微酸性环境下,硒和2,3-二氨基萘(DAN)生成有较强荧光的物质,用环己烷萃取,在激发波长378nm,荧光波长518nm处测定其荧光强度。蜂蜜中硒含量范围:0.10～0.82μg/g。表明:蜂蜜应视为天然富硒营养品。     

乳酸杆菌益生作用机制的研究进展

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。     

中华人民共和国农业部公告 第267号

为贯彻落实《兽药生产质量管理规范》(简称《兽药GMP》),进一步推动兽药GMP实施进程,我部制定了《兽药生产质量管理规范检查验收办法》,现予公告。本公告自2003年6月1日起施行。附件:兽药生产质量管理规范检查验收办法二○○三年四月十日第一章 总则 第一条 为推动《兽药生产质量管理规范》(以下简称兽药GMP)的实施,规范兽药GMP检查验收工作,制定本办法。 第二条 农业部负责全国兽药GMP管理和检查验收工作;负责制修订兽药GMP检查验收管理规定;负责兽药GMP检查员队伍建设和监督管理工作,负责国际兽药贸易中GMP互认工作。 …     

鸡败血支原体垂直传播模式及其阻断方法

以国际标准强毒Ｒ株人工感染非免疫产蛋鸡,定时扑杀,分别从鼻窦、眶下孔、气管、肺、气囊、卵巢和输卵管分离ＭＧ,并收集感染鸡所产蛋分离ＭＧ。结果表明,人工感染４８小时后上、下呼吸道及肺已被全面感染,９６小时气囊已被感染,１２０小时输卵管已能分离到ＭＧ,卵巢始终分离不到ＭＧ。人工感染鸡自１４４小时便能在其所产蛋中分离出ＭＧ。药物治疗能在７２小时内消除感染,油乳剂苗则需２４天后逐渐降低蛋内ＭＧ分离率,药物卵内注射、种蛋药浴、高温处理均能杀死卵内ＭＧ,但以研制的种蛋浸泡剂药浴效果为最好。     

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.