Seroprevalence of Ehrlichia canis, Ehrlichia equi, and Ehrlichia risticii in sick dogs from North Carolina and Virginia

Ehrlichia canis, E. equi, and E. risticii seroprevalence was determined by microimmunofluorescent antibody testing (IFA) in a sequential population of 1,845 sick dogs admitted during a 1-year period to the North Carolina State University Veterinary Teaching Hospital. A seroreactor was defined by a reciprocal IFA titer of > or =80 to E. canis, E. equi, or E. risticii antigens. Of the 48 IFA seroreactors, 44 dogs were seroreactive to E. canis, 21 to E. equi, and 0 to E. risticii. Seventeen dogs reacted to both E. canis and E. equi antigens. There was concordance of E. canis IFA and western immunoblot (WI) test results for 36/44 dogs. Because of cross-reactivity of E. canis sera with E. equi antigens, WI was of less utility to confirm E. equi exposure. After elimination of E. canis seroreactors, there was concordance of 2/4 E. equi IFA and WI test results. Based upon a retrospective review of medical records, ehrlichiosis was diagnosed in 10/48 (21%) IFA seroreactive dogs, 9 of which were confirmed positive by WI. Of the remaining 38 IFA seroreactors, 29 also were confirmed by E. canis or E. equi WI. These results indicate that (1) ehrlichiosis was not diagnosed in the majority of serologically confirmed cases, (2) based upon E. canis and E. equi WI analysis, IFA testing was not specific (21% false positive), (3) E. canis sera cross-react with E. equi antigens, and (4) serologic evidence of E. risticii infection was lacking in the dog population studied.     

Thrombolytic properties of a conjugate of liposomal urokinase with a monoclonal antibody specific for cross-linked fibrin in the model of rabbit artery thrombosis

AIM: To observe the targeting thrombolytic effect of a monoclonal antibody specific for cross-linked fibrin connected with liposomal urokinase（UK） in the model of rabbit artery thrombosis.METHODS: Preparation of thrombolytic solutions: with the method of controllable dialysis eradicator in liposomat,empty liposomes,liposomally entrapped urokinase and liposomally entrapped urokinase linked with D-dimer antibody were made.Experiment in vivo: a rabbit thrombosis model of abdominal aorta was induced by ferric chloride.When the blood pressure fall to the lowest,5 different solutions were separately imported (PBS,maximal-level UK,Ab/Lip/UK,Ab/UK-Lip,Ab-UK-Lip) and observed for 40 min continuously.RESULTS: The varieties of 5 groups blood pressures were analyzed with q-test.Significant differences were observed among Ab-UK-Lip group,maximal-level UK group and others (P<0.01).There were marked differences between Ab-UK-Lip group and others in wet weight of thrombuses (P<0.05).However,the quantity of UK in Ab-UK-Lip group was 1/3 of that in maximal-level of UK group.CONCLUSION: The thrombolytic effect on Ab-UK-Lip group is similar to that on maximal-level of UK group,but the dosage of UK is less,and the side-effect is light.This shows,as a homing-device of liposomal urokinase,the D-dimer antibody has targeting effect and the agent of Ab-UK-Lip will be attached importance by-and-by.     

新城疫病毒糖蛋白致病作用的研究

本实验以分子生物学和现代免疫学方法,对新城疫病毒（ＮＤＶ）中国毒株Ｆ４８Ｅ９和ＬａＳｏｔａ等毒株的糖蛋白的致病作用进行了探讨。结果表明,ＮＤＶ Ｆ和ＨＮ蛋白为糖蛋白,强弱毒株之间Ｆ蛋白裂解位点的氨基酸序列有明显的不同,并决定了毒株的毒力,但ＮＤＶ的致病作用尚需ＨＮ、Ｆ蛋白的协同作用,单一的Ｆ或ＨＮ蛋白不能构成ＮＤＶ的致病作用。     

Development and validation of an improved enzyme-linked immunosorbent assay for the detection of thyroglobulin autoantibodies in canine serum samples

An enzyme-linked immunosorbent assay to detect thyroglobulin autoantibodies (TGAB) in canine serum was developed and validated. The test result for each sample was derived from the optical density readings (OD) and expressed as an Ab-score(%) calculated from three in-house calibrators. The assay specifically detected TGAB as judged from lack of response in the assay after samples had been incubated with specific antigen. Intra- and interassay coefficients of variation ranged from 2.0–4.9% and 4.6–9.9%, respectively. The detection limit, an Ab-score of 5.6%, was close to the median Ab-score of 10% observed in healthy dogs (n = 132). The median Ab-score of dogs with primary hypothyroidism and lymphocytic thyroiditis (n = 11), skin diseases (n = 35), and non-thyroidal diseases (n = 63) was 340%, 12%, and 8%, respectively. The prevalence of TGAB in hypothyroid dogs with lymphocytic thyroiditis (sensitivity) was 91% (95% confidence limits: 59%–99%). In dogs with dermatological diseases without lymphocytic thyroiditis the prevalence of TGAB was 3% corresponding to a specificity of 97% (95% confidence limit: 85%–100%). In dogs with non-thyroidal diseases and healthy dogs the prevalence of TGAB was 5% and 6%, respectively. The diagnostic accuracy of serum TGAB was evaluated by subjecting the data from 11 dogs with lymphocytic thyroiditis and 35 control dogs without lymphocytic thyroiditis to receiver-operating characteristic curve analysis. The area under the receiver-operating characteristic curve (W = 0.966; 95% confidence limit 87%–100%) was significantly higher than that of a worthless test (0.5) (P < 0.0001), thereby indicating that serum TGAB measurements distinguished between dogs with and without lymphocytic thyroiditis.     

抗猪流行性腹泻病毒单克隆抗体的研制及其特性鉴定

以新生乳猪增殖猪流行性腹泻病毒（ＰＥＤＶ）,通过蔗糖密度梯度离心将其纯化,获得较为完整的病毒颗粒。病毒ＥＬＩＳＡ效价为１∶３×１０５,蛋白含量为３．９６ｇ／Ｌ。将提纯的ＰＥＤＶ免疫ＢＡＬＢ／ｃ小鼠,取免疫鼠脾细胞与ＳＰ２／０骨髓瘤细胞进行融合,融合率为９５．６％。用间接ＥＬＩＳＡ进行筛选,共检出３８个阳性孔,阳性率为２８．８％。对其中１５个阳性值较高的孔进行３次再克隆,阳性率达１００％,最后获得１５株稳定分泌特异性抗ＰＥＤＶ单抗（ＭｃＡｂ）的杂交瘤细胞。ＥＬＩＳＡ阻断试验与交叉试验证明,其分泌的抗体具有高度特异性。腹水和细胞培养上清的ＥＬＩＳＡ效价分别为１∶１０３～１∶１０６和１∶１２８～１∶５１２。经鉴定,１５株ＭｃＡｂ的分子类型均为ＩｇＧ,均无免疫沉淀特性。杂交瘤细胞的染色体数目为９０～１１０,平均９５。尤为重要的是,这些杂交瘤细胞所分泌的抗体,有的对ＰＥＤＶ具有中和作用,能够阻断病毒对猪只的侵害作用。     

家蚕同源异型结构域蛋白基因Bmvis的克隆及组织表达和亚细胞定位

同源异型结构域蛋白(homeobox,HOX)基因是对生物体的生长发育从时间和空间上进行调控的一类基因。Vis(vismay)属于同源异型结构域蛋白转化生长影响因子(transforming growth interacting factor,TGIF)家族成员,在脊椎动物的研究中表明TGIF是转化生长因子-β(transforming growth factor β,TGF-β)信号转导途径中的转录抑制子,但在果蝇(Drosophilamelanogaster)的研究中发现其是一种转录激活子。基于为探究家蚕(Bombyx mori)中vis基因的功能提供基础信息的目的,在电子克隆的基础上通过RT-PCR从家蚕5龄幼虫精巢cDNA中克隆了长度为847 bp的vis基因(Bmvis)片段(GenBank登录号:JF412700)。蛋白结构分析表明Bmvis在HOX结构域的螺旋区和TALE区段内高度保守,同时在HOX结构域外的C末端大约20个氨基酸也是高度保守的。进化分析显示昆虫的Vis与Achi(achintya)聚为一类,但Vis与Achi根据物种分类关系聚在一起。对Bmvis在家蚕5龄第3天幼虫组织中表达的RT-PCR检测显示其仅在精巢中表达。将Bmvis进行原核表达后获得抗Bmvis多克隆抗体,亚细胞定位显示Bmvis在细胞核和细胞质中均有表达,但主要分布在细胞核中。     

抗盐酸克伦特罗单链抗体基因的克隆、序列分析及表达

提取分泌抗盐酸克伦特罗单克隆抗体杂交瘤细胞株的总RNA,通过RT-PCR技术,扩增VH和VL,用一段柔性肽链-(G4S)3将VH和VL连接成ScFv。测序后经NCBI Blast分析,所得ScFv具有重组功能性鼠抗体可变区基因的特征。将所得目的基因与pET-22b( )连接,转化E.coli BL21,用IPTG诱导表达,表达蛋白经SDS-PAGE分析,37℃培养5 h表达量较大,25℃诱导表达以可溶性为主。     

噬菌体抗体库技术研究进展

噬菌体抗体库技术是利用PCR扩增出抗体的全套可变区基因,将抗体分子DNA片断如Fab或单链抗体(ScFv)与噬菌体外壳蛋白基因PⅢ或PVⅢ连接,使融合蛋白表达于噬菌体颗粒的表面,经过“吸附-洗脱-扩增”过程富集筛选特异性抗体。这一技术将抗体基因型和表型联系在一起,使识别抗原的能力和噬菌体的可扩增性统一起来,较好的模拟了体内的抗体产生的过程,成为一种高效的筛选体系。本文就噬菌体抗体库技术的原理、构建、筛选及其应用研究进展做一综述。     

用免疫荧光技术检测细胞培养物中兔出血症病毒

用本实验室制备的兔出血症病毒（rabbit hemorrhagic disease virus,RHDV）高免血清,按常规方法提纯出抗体（IgG）,用异硫氰酸荧光素（FITC）标记IgG。在细胞培养时,培养瓶中放入玻片,当细胞长成单层时,按常规方法接种病毒液,培养24、48、96、120h时取出玻片,用荧光抗体染色,在荧光显微镜下观察不同代次的细胞毒。经观察：兔肾上皮细胞（RK）毒培养到36—48h,羊睾丸细胞（ST）毒培养到72—96h时可观察到特异性荧光,随着培养时间的延长,荧光亮度增强,胞浆内充满特异性荧光。用肝组织强毒病料触片,呈特异性荧光,对照细胞培养48h无荧光出现,证实了两株细胞培养物中有大量的兔出血症病毒存在,从而成功的分离培养出了兔出血症病毒细胞毒。     

检测南方水稻黑条矮缩病毒胶体金免疫试纸条的建立

由白背飞虱Sogatella furcifera(Horváth)传播的南方水稻黑条矮缩病毒(Southern rice blackstreaked dwarf virus,SRBSDV)是目前我国南方水稻上危害最严重的病毒,为开发简便、快速、准确的SRBSDV病毒检测技术和检测试剂,以感染SRBSDV的植物粗提液为免疫原,利用杂交瘤技术制备了2株抗SRBSDV的单抗(14A8和15G6),并利用制备的单抗建立了可快速、特异、灵敏地检测SRBSDV的胶体金免疫试纸条。结果表明,2株制备单抗的抗体类型及亚类均为Ig G1、kappa链,单抗腹水的间接ELISA效价均达到10~(-7);Western blot分析表明,2株单抗均与SRBSDV的外壳蛋白亚基有特异反应,而不与水稻黑条矮缩病毒(Rice black-streaked dwarf virus,RBSDV)反应。以制备14A8和15G6单抗分别为捕获抗体和胶体金标记抗体,开发成能在5 min内准确、特异地检测水稻植物和白背飞虱传毒介体体内SRBSDV的胶体金免疫试纸条;灵敏度分析表明,该检测试纸条的检测水稻病叶的灵敏度达到1∶6 400倍(g/m L),检测单头携毒白背飞虱的灵敏度达到1∶51 200倍(单头/μL)。田间样品检测结果表明,该试纸条的检测结果与RT-PCR的符合率达到100%。建立的SRBSDV胶体金免疫试纸条可对南方水稻黑条矮缩病毒进行快速、特异、灵敏的诊断和检测。