猪圆环病毒2型间接ELISA抗体检测试剂盒的研制及初步应用

以大肠杆菌原核表达系统表达的猪圆环病毒2型Cap蛋白为抗原,建立猪圆环病毒2型间接ELISA检测方法,优化ELISA反应条件,研制猪圆环病毒2型ELISA抗体检测试剂盒。与商品化试剂盒相比,该检测试剂盒敏感性、特异性和符合率分别为95.12%、92.86%和94.55%;同时与猪圆环病毒1型（PCV1）、猪瘟病毒（CSFV）、猪繁殖与呼吸综合征病毒（PRRSV）等多种病毒阳性血清无交叉反应。试剂盒具有较好的重复性,在-20 ℃至少可保存1年以上,将其应用于临床血清样品的检测,结果安徽、广东、广西采样猪场的PCV2阳性率分别为100%、48.39%、100%。     

猪圆环病毒2型阻断ELISA抗体检测试剂盒的研制与应用

为了进一步提高对猪圆环病毒2型(PCV2)检测的特异性,利用PCV2Cap蛋白及酶标记Cap单克隆抗体,建立了一种检测猪血清中PCV2Cap蛋白抗体的阻断ELISA方法,并将其组装成试剂盒。对阻断ELISA试剂盒进行了敏感性、特异性、重复性、符合率和保存期测定。结果显示,其敏感性高,特异性强,批内批间重复性好,与商品化试剂盒符合率高,保存期长且性质相对稳定。先后制备了5个批次的阻断ELISA试剂盒,用于检测PCV2不同疫苗免疫猪血清、规模猪场PMWS发病猪及同群健康猪血清、不同日龄猪群血清.结果显示,PCV2油佐剂免疫组血清抗体水平相对较高,PWMS发病猪血清抗体水平高于同群健康猪,猪群中育肥猪血清抗体水平最高。     

猪圆环病毒2型ELISA抗体检测试剂盒的研制及初步应用

用PCV2 Cap蛋白单克隆抗体预包被酶标板,将杆状病毒表达系统表达的PCV2 Cap蛋白作为检测用抗原,利用捕获包被法建立了间接ELISA法用于猪圆环病毒2型抗体的检测.通过优化ELISA条件,研制试剂盒并应用于圆环病毒2型疫苗的免疫效果检验.利用研制的试剂盒与IFA、商品化进口和国产同类试剂盒对178份猪血清进行平行检测,总符合率分别为96.63％、97.75％和84.27％,与其他几种常见猪病毒抗体阳性血清无交叉反应.试剂盒批内、批间变异系数分别为2.35％ ～4.06％和4.87％ ～ 6.54％,2～8℃保存15个月稳定,适合于对不同来源猪圆环病毒2型疫苗人工免疫猪血清抗体进行检测.     

猪圆环病毒3型ORF2基因的截短表达及间接ELISA抗体检测方法的建立

试验旨在建立一种快速的猪圆环病毒3型(PCV3)抗体检测方法。采用PCR方法对PCV3 ORF2基因主要抗原区域进行扩增,并利用原核表达系统进行截短表达,以纯化后的重组蛋白作为包被抗原,建立间接ELISA抗体检测方法。结果显示:PCR扩增了片段大小为348 bp的ORF2基因主要抗原区域,PCR扩增产物克隆至pET-32a原核表达载体,转入大肠杆菌BL21,获得了以包涵体形式表达的重组蛋白,纯化后经Western blot鉴定表明具有良好的反应活性。以重组蛋白作为包被抗原建立了检测PCV3抗体的间接ELISA方法,对PCV3阳性血清的最低检出效价可达1∶20 480;用该方法检测猪圆环病毒2型(PCV2)、猪繁殖与呼吸综合征病毒(PRRSV)、猪瘟病毒(CSFV)、猪细小病毒(PPV)、伪狂犬病毒(PRV)、乙型脑炎病毒(JEV)、猪流行性腹泻病毒(PEDV)、猪传染性胃肠炎病毒(TGEV)等猪常见疫病阳性血清,结果均为阴性;批内重复性试验和批间重复性试验的变异系数均小于5%。应用该方法检测四川地区533份不同日龄临床猪血清样品,PCV3阳性感染率达14.82%。表明建立的ELISA方法具有良好的敏感性、特异性、重复性和临床适用性,为我国商品化PCV3血清学抗体检测试剂盒的研制奠定了基础。     

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。     

猪圆环病毒2型ELISA抗体检测试剂盒的研制及应用

利用纯化的重组杆状病毒表达猪圆环病毒2型（PCV2）Cap蛋白作为检测抗原,建立一种间接ELISA方法用于血清抗体检测。昆虫细胞株（Sf-21）接种重组杆状病毒,对蛋白表达参数及纯化工艺进行了优化,制备了5批重组蛋白抗原,表达量占总蛋白的18.7%左右;Ni2＋亲和层析柱纯化获得重组蛋白纯度为90.1%;重组蛋白可被特异性抗体识别,具有良好的免疫活性。用建立的ELISA法对30份已知阴性血清样本检测临界OD405nm值为0.343;ELISA与IPMA对150份血清进行平行检测符合率为95.3%,特异性为91.2%,敏感性为96.5%;与其他几种常见猪病毒参考血清无交叉反应。组装的试剂盒批内和批间变异系数分别为4.0%～4.8%和8.9%～9.9%;置-20℃保存12个月稳定。试剂盒对人工感染猪血清检测结果,接种后第5周抗体阳转,第8周～9周抗体水平达到高峰;对来自黑龙江、吉林、河北、上海等地猪场1085份血清抗体检测,阳性率为73.6%。研制的ELISA试剂盒为临床PCV2血清抗体检测及疫苗免疫效果的评价提供了技术手段。     

猪圆环病毒2型抗体间接ELISA检测方法的建立

为了更加快速灵敏地检测血清中猪圆环病毒2型(Porcine circovirus type 2,PCV2)抗体的水平,我们通过优化反应条件建立了快速检测猪圆环病毒2型抗体的间接ELISA方法。结果显示,本研究研制的试剂盒检测3份PCV2抗体阳性血清的阳性滴度为1∶12 800～1∶25 600,而韩国JBT PCV2试剂盒检测的阳性滴度为1∶3200～1∶6400;对猪繁殖与呼吸综合征病毒、猪瘟病毒、猪伪狂犬病毒、猪口蹄疫病毒等10种其他病原体的20份阳性血清的检测结果均为阴性,表明本研究研制的试剂盒具有良好的特异性;批内重复性试验变异系数(C.V%)为2.26%～7.60%,批间重复试验的变异系数(C.V%)为1.67%～6.41%,变异系数均小于10%,呈现良好的可重复性。因此,我们成功建立了敏感性高、特异性强及重复性好的猪圆环病毒2型抗体间接ELISA检测方法。     

应用原核表达的猪圆环病毒Ⅱ型Cap蛋白建立一种间接ELISA诊断方法

本试验在最佳诱导条件下获得猪圆环痛毒Ⅱ型重组Cap蛋白的基础上，利用HisBind蛋白质纯化试剂盒对表达产物进行纯化，通过Westernblot检测证明表达产物具有良好的抗原性。以纯化后的重组融合蛋白作为诊断抗原，对ELISA反应条件进行优化，初步建立了检测PCV2抗体的间接ELISA方法。用该法对广东、广西一些地区收集到的380份血清样品进行检测，检测结果阳性率为86．1％，从中随机取90份猪血清样品与国产商品化试剂盒检测结果对比，符合率为95．6％，表明本实验建立的间接ELISA方法具有较高的敏感性和特异性，适于大规模检测PCV2血清抗体的流行病学调查。     

猪圆环病毒2型Cap蛋白表达及其间接ELISA诊断试剂盒研制

根据已知猪圆环病毒2型(PCV2)序列设计引物,PCR扩增出579 bp去除信号肽的ORF2基因片段,将其克隆入表达载体pGEX-4T-1的BamHI和XhoI位点构建重组质粒,转化Rossetta(DM3)进行诱导表达,经检测表达蛋白以包涵体形式存在。用GST蛋白纯化试剂盒纯化目的蛋白,SDS-PAGE检测只存在一条约45.3 ku的目的条带,纯度达到90%以上。Western blot分析表明纯化的目的蛋白具有良好的免疫反应性。以纯化的蛋白为抗原,建立了间接ELISA诊断方法及组装了试剂盒,确定了最适的抗原包被浓度为0.625μg/mL,抗体稀释浓度为1∶100,确定临界值为0.371。该试剂盒与猪圆环病毒1型(PCV1)、猪细小病毒(PPV)、猪伪狂犬病毒(PRV)、猪瘟病毒(CSFV)、猪繁殖与呼吸综合征病毒(PRRSV)、猪乙型脑炎病毒(JEV)血清无交叉反应性;与北京世纪元亨公司ELISA试剂盒相比其特异性、敏感性、符合率分别为90%、93.3%、91%;对同份血清样品批内、批间重复性试验变异系数均小于10%;稳定性试验变异系数小于5%,在4℃条件下保存期可达12个月;对240份临床血清检出率为80%。结果表明,研制的ELISA诊断试剂盒具有良好的特异性、敏感性,重复性、稳定性,完全达到国内外同类产品质量要求,可大规模应用于临床检测。     

猪圆环病毒2型ORF2基因截断表达及ELISA抗体检测方法的初步建立

为建立以重组PCV2Cap蛋白为包被抗原的间接ELISA检测方法,构建了猪圆环病毒2型（PCV2）ORF2基因原核表达质粒pET28a-ORF2。SDS-PAGE显示,在0.1mmol/L IPTG和37℃条件下诱导4h,重组Cap蛋白高效表达。Western blotting证实该蛋白能够被PCV2阳性血清特异性识别。以纯化的蛋白为抗原建立了检测PCV2抗体的间接ELISA方法。结果表明,抗原最适包被质量浓度为2mg/L;血清最佳稀释度为1∶100;酶标二抗最适浓度为1∶2 000,该方法的敏感性为86.96%,特异性为100%。用该方法对河南省152份猪血清样品进行检测,与间接免疫荧光（IFA）的符合率为85.53%（130/152）,与商品化的韩国金诺PCV2ELISA试剂盒的符合率为88.16%（134/152）。本试验成功建立了PCV2血清抗体间接ELISA方法,具有较高的敏感性和特异性,可用于大规模的血清学检测。     

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。     

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.     

Mechanism of Action of Probiotic Function of Lactobacilli

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.     

Evaluation of serum concentrations of biomarkers of skeletal metabolism and results of radiography as indicators of severity of osteochondrosis in foals

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.