基于NS4蛋白的蓝舌病病毒间接ELISA抗体检测方法的建立及初步应用

旨在建立蓝舌病病毒（BTV）血清学ELISA抗体检测方法,本研究以原核表达并纯化的BTV NS4重组蛋白为包被抗原,通过反应条件优化,建立了一种BTV重组NS4蛋白的间接ELISA抗体检测方法。SDS-PAGE结果显示,获得大小约52 ku的NS4重组融合蛋白,主要在上清中存在,Western blot显示,纯化后的重组蛋白具有良好的抗原性。通过方阵试验进行了ELISA反应条件优化,确定了重组蛋白抗原最佳包被量为3.0 μg·孔^-1;血清最佳稀释倍数为1：200,酶标二抗最佳工作浓度为1：4 000,临界值分别为0.29和0.35。上述以NS4蛋白作为包被抗原建立的BTV抗体间接ELISA方法检测敏感性可达1：1 600;批内和批间重复性变异系数均小于10%;检测76份重庆地区牛群血清样品,阳性符合率为98%,阴性符合率为100%。本研究建立的间接ELISA方法为临床BTV血清抗体检测及BTV血清流行病学调查奠定了基础。     

非洲猪瘟病毒p30基因的原核表达及间接ELISA抗体检测方法的建立

为建立检测血清中非洲猪瘟病毒(African swine fever virus,ASFV)抗体的间接ELISA方法,本试验将ASFVp30基因进行原核表达,采用SDS-PAGE和Western blotting方法对重组蛋白进行表达鉴定和免疫原性分析,随后以纯化的重组蛋白为包被抗原,经条件优化、特异性试验、敏感性试验和重复性试验,建立一种血清中ASFV抗体的检测方法。结果显示,ASFVp30基因成功克隆到原核表达载体pET-32a(+)中,获得pET-32a-p30重组质粒;转化大肠杆菌BL21(DE3)感受态细胞进行诱导表达,得到P30重组蛋白,重组蛋白大小约为42ku,主要以包涵体形式存在;Western blotting结果显示,纯化后的蛋白具有良好的免疫原性;以纯化的P30重组蛋白为包被抗原,建立了检测ASFV抗体的间接ELISA方法,通过方阵试验对间接ELISA方法进行优化,最终确定了抗原最佳包被浓度为1.2μg/mL,待检血清最佳稀释倍数为1∶100,最佳封闭液为1%BSA,酶标抗体最佳稀释度为1∶4 000,以此建立的ASFV间接ELISA方法临界值为0.322。本方法仅与ASFV阳性血清发生特异性反应,与猪瘟病毒、猪繁殖与呼吸综合征病毒、口蹄疫病毒、伪狂犬病病毒、猪圆环病毒2型及猪流行性腹泻病毒阳性血清均无交叉反应,具有较强的特异性。该方法检测ASFV阳性血清灵敏度可达到1∶1 600;批内重复性和批间重复性变异系数均10%。本试验建立的间接ELISA方法具有良好的特异性、灵敏度和重复性,可初步应用于ASFV抗体的检测。     

猪血凝性脑脊髓炎病毒重组N蛋白间接ELISA检测方法的建立与应用

为建立检测猪血凝性脑脊髓炎病毒(porcine hemagglutinating encephalomyelitis virus,PHEV)血清抗体的间接ELISA方法,构建了高效表达PHEV核衣壳蛋白(N)的重组质粒(pET28a-N),Western blot检测该重组蛋白具有较好的免疫原性。以纯化的N重组蛋白作为包被抗原,通过方阵试验确定了抗原的最适包被浓度为2mg/L,酶标二抗的最佳稀释倍数为1∶10 000,建立了用于PHEV抗体检测间接ELISA方法。此外,用该法(N-ELISA)对天津市地区采集到的200份猪血清样品进行检测,阳性检出率为83.5%,与前期工作中建立的全病毒作为包被抗原的间接ELISA诊断方法 (HEV-ELISA)检测结果的符合率为90.91%。结果表明:以重组N蛋白为包被抗原所建立的用于PHEV血清抗体检测的方法能够用于检测PHEV感染及相关的流行病学调查。     

犬冠状病毒N蛋白的原核表达及其多克隆抗体的制备

本研究旨在克隆犬冠状病毒(canine coronavirus,CCV)N基因,体外表达N蛋白,并制备抗该蛋白质的多克隆抗体,用于CCV的诊断及其抗原的检测。参考GenBank中CCV的N基因序列(登录号:KY063618.2),选择CCV流行毒株的N基因,通过对该基因密码子进行优化和基因合成,最后选择一段有效基因构建重组表达质粒pET-B2M-N,将成功构建的重组质粒转化大肠杆菌DH5α感受态细胞,挑取阳性克隆提取质粒,转化大肠杆菌BL21(DE3)感受态细胞,通过0.5mmol/L IPTG 30℃进行诱导表达。结果表明,优化诱导条件后成功表达出大小约为49ku的重组蛋白。将重组蛋白与弗氏佐剂按一定比例混合,每隔2周免疫G767、G768两只日本大耳白兔数次,用间接ELISA检测G768抗体效价可达1∶512 000,选用G768抗体进行抗体纯化,纯化后浓度可达10mg/mL,用间接ELISA、Western blotting和间接免疫荧光试验对N蛋白纯化后制备的兔多克隆抗体进行检测分析,表明表达的重组N蛋白免疫原性良好,制备的多克隆抗体具有良好的反应原性。本研究为犬冠状病毒抗原抗体检测以及靶标CCV诊断试剂盒的建立奠定了一定的基础。     

猪流行性腹泻病毒重组N蛋白间接ELISA检测方法的建立

为建立检测猪流行性腹泻病毒(PEDV)血清抗体的间接ELISA方法,本研究以原核表达的重组N蛋白作为检测抗原,优化ELISA反应条件,建立了PEDV间接ELISA抗体检测方法。该方法仅对PEDV血清检测为阳性,对猪传染性胃肠炎、猪轮状病毒感染猪的阳性血清检测结果均为阴性;其批内和批间重复性试验的变异系数均小于10%。采用建立的ELISA方法检测了130份临床样品,其中78份血清样品为PEDV抗体阳性。表明建立的以重组N蛋白为包被抗原检测PEDV血清抗体的方法可以用于检测PEDV感染及相关的流行病学调查。     

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.     

猪丁型冠状病毒重组N蛋白间接ELISA抗体检测方法的建立

猪丁型冠状病毒(PDCoV)是2014年检测出的一种新型猪源肠道冠状病毒,对养殖业造成严重的经济损失。为建立快速检测PDCoV的血清学方法,本研究以原核表达的PDCoVN重组蛋白为包被抗原,通过反应条件优化,建立了一种PDCoV重组N蛋白的间接ELISA抗体检测方法。结果显示,原核表达的PDCoV重组N蛋白约为28ku,westernblot证实表达蛋白具有良好的反应原性;以其作为包被抗原建立的PDCoV抗体间接ELISA方法仅对PDCoV血清检测为阳性,与猪流行性腹泻病毒、猪传染性胃肠炎病毒、猪繁殖与呼吸系统综合征病毒等7种主要猪源病毒阳性血清均无特异性反应,具有良好的特异性。该方法检测灵敏度较高、重复性好,批内和批间重复性变异系数均小于10%;采用该ELISA方法对我国2014年~2016年间采集的不同地区猪场的100份临床血清样品进行了检测,阳性率高于45.9%,表明我国猪群中存在PDCoV感染。本实验建立的ELISA方法可以用于检测临床样品中的PDCoV抗体,是PDCoV流行病学调查的一种有效工具,对该病的防控具有重要意义。     

绵羊肺炎支原体P6058aa-928aa蛋白的原核表达及间接ELISA方法的建立

【目的】 建立基于绵羊肺炎支原体表面脂蛋白P60的间接ELISA方法。【方法】 利用生物信息学软件对P60蛋白进行抗原性分析,筛选出抗原性最佳的区域,扩增目的基因后构建重组表达载体并进行基因测序,将测序正确的质粒转化大肠杆菌BL21(DE3)感受态细胞,用IPTG诱导抗原性最佳蛋白表达并进行诱导时间优化;利用SDS-PAGE分析该蛋白分子质量大小及其表达形式。以重组蛋白作为抗原,绵羊肺炎支原体阳性血清作为抗体,通过Western blotting方法分析其反应原性。以重组蛋白为包被抗原,建立抗原性最佳蛋白的间接ELISA抗体检测方法,用该方法对20份阴性血清进行检测,计算阴阳临界值;检测该方法的特异性、敏感性和重复性,并用建立的间接ELISA方法与间接血凝法对92份绵羊临床血清样本进行检测。【结果】 经DNAStar分析,P60蛋白第58－928位氨基酸区域的平均抗原指数在0.8~1.7之间,亲水指数为0~2.0,证明该区域(P60_58aa-928aa)抗原性和亲水性较强;通过PCR扩增得到P60_58aa-928aa基因,并构建重组表达载体,通过基因测序证明该重组表达载体与预期结果一致。SDS-PAGE结果显示,IPTG诱导浓度为1 mmol/L,诱导10 h时蛋白表达量较高,蛋白分子质量为42 ku,在大肠杆菌中以包涵体的形式表达;Western blotting结果表明,构建的重组蛋白具有良好的反应原性。对ELISA各条件进行优化结果显示:抗原包被浓度为0.5 μg/mL,一抗最佳稀释浓度为1∶100,最佳孵育时间为1.5 h,酶标二抗最佳稀释倍数为1∶4 000,判定阴阳性血清的临界值为0.36。对口蹄疫病毒(FMDV)、小反刍兽疫病毒(PPRV)、布氏杆菌(Brucella)、牛支原体(Mb)、丝状支原体山羊亚种(Mmc)的标准阳性血清的检测均为阴性,证明该方法特异性较强;敏感性试验结果表明,血清稀释度为1∶2 048时仍为阳性;批内重复试验和批间重复试验的变异系数均<10%,证明该方法具有较好的重复性。利用所建立的间接ELISA方法与间接血凝法对92份羊血清检测结果表明,二者的阳性符合率为81.6%,阴性符合率为92.6%,总符合率为88.0%。【结论】 本研究建立的间接ELISA方法可用于临床样本的检测,为绵羊肺炎支原体的疫苗免疫效果评价和疾病诊断提供了较为可靠的方法。     

犬瘟热病毒重组核蛋白间接ELISA方法的建立

将已构建成功的重组质粒pGEX-4T-1-N转化大肠杆菌BL21株,在最佳诱导条件下获得犬瘟热病毒(CDV)重组N蛋白。将表达产物纯化后进行SDS-PAGE和W estern-b lot分析,与CDV标准阳性血清呈阳性反应。本研究初步建立了以纯化的N蛋白为包被抗原的间接ELISA检测方法,经初步试验证实,该方法敏感、特异。试验结果表明,大肠杆菌中表达的CDV N蛋白在免疫原性上与天然核衣壳蛋白具有较高相似性,可作为诊断用抗原。     

欧洲禽源H1N1亚型猪流感病毒抗体间接ELISA检测方法的建立

本研究利用原核表达并纯化的重组蛋白pCold TF-HA1作为包被抗原,建立欧洲禽源H1N1亚型猪流感病毒抗体的间接ELISA检测方法。通过反应条件的优化,确定了间接ELISA最佳工作条件:抗原最佳包被浓度为800 ng/孔,血清稀释度为1:200,封闭时间为1 h,一抗血清最佳作用时间为2 h,酶标二抗最佳稀释度为1:40 000,二抗最佳作用时间为1 h,TMB显色时间为7 min。阴/阳性血清的判断结果分别为OD_(450)≤0.255和OD_(450)≥0.292,介于两者之间为可疑。符合性试验结果显示,建立的间接ELISA方法与血凝抑制(haemagglutination inhibition,HI)检测的阳性率分别为53.27%和49.53%,两者总复合率达96.27%。特异性试验结果显示,该方法与古典H1N1亚型和H3N2亚型猪流感阳性血清无交叉反应。通过重复性试验表明,同一批制备的重组蛋白包被96孔酶标板,检测OD_(450)值的批内变异系数为1.97%~7.85%,批间变异系数为1.94%~6.93%。总之,本研究建立的禽源H1N1亚型猪流感病毒抗体间接ELISA方法,具有敏感性较高、特异性强、重复性好的特点,为猪流感抗体检测提供了一种准确、快捷、简便、有效的技术手段,可用于禽源H1N1亚型猪流感抗体检测和流行病学调查。     

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.