Preparation and Identification of Canine Parvovirus NY Strain VP2 Protein Polyclonal Antibody     

MAO Qian-qian  ZHOU Ling  TANG Qing-hai  BU Bin  TANG Cun-duo  JIAO Zhu-jin  YAO Lun-guang  KAN Yun-chao  YANG Jian-wei  CUI Shang-jin 《中国畜牧兽医》2016,43(7):1659-1666

This study was aimed to prepare canine parvovirus (CPV) VP2 protein polyclonal antibody.The recombinant expression vector pET28a-CPV-VP2 was constructed and transfromed into E.coli BL21 (DE3),the expression of recombinant proteins was induced by IPTG from which the fusion protein was identified by SDS-PAGE.The target protein was purified and emulsify with adjuvant,the prepared immunogen was inoculated into rabbit by subcutaneous injections to prepare of VP2 protein specific polyclonal antibody.The immuno-activity,titers,neutralization titers of the prepared polyclonal antibody were determined by immunoperoxidase monolayer assay (IPMA).The results showed that the expressed recombinant protein VP2 (rVP2) existed in the form of inclusion body with a molecular weight of 72 ku.The prepared polyclonal antibody titer was 1 600 dilution,the virus titer was 10⁷ TCID₅₀/mL,the neutralizing titer was 1∶2 884.The antibodies showed specific reaction with CPV.In conclusion,rVP2 specific polyclonal antibody showed wonderful immunocompetence,specificity and neutralizing activity,providing foundation for the development of genetic vaccine and clinical therapeutic method.  相似文献   

倍他米松ELISA检测方法的建立     

郭璐  张晶  沙芳芳  赵兴然  王敬  王向红 《动物医学进展》2016,(12):50-54

为建立倍他米松(BET)ELISA检测方法,先使BET与琥珀酸酐反应,再采用活化酯法与牛血清白蛋白(BSA)偶联制备免疫原;其次,利用免疫动物获得多克隆抗体,经Sephrose 4B-proteinA方法对抗体进行纯化;最后,建立倍他米松ELISA检测方法。结果表明,免疫抗原偶联成功,获得了亲和力较高的多克隆抗血清,间接竞争ELISA测定其滴度为102 400、IC15为6.60×10-5 ng/mL和IC50为0.97ng/mL。该方法适用于残留在动物性食品中的BET现场大批量检测。  相似文献   

玉米黄花叶病毒运动蛋白的原核表达纯化与抗血清制备     

马秋萌  刘玉姿  吴迪  李洪蕊  王颖  韩成贵 《植物保护学报》2023,50(2):343-349

为实现对玉米黄花叶病毒（maize yellow mosaic virus,MaYMV）的血清学检测,丰富该病毒的检测方法,将编码MaYMV运动蛋白（movement protein,MP）的基因连接到原核表达载体pDBHis-MBP上,将构建成功的原核表达质粒转化到大肠杆菌Escherichia coli中诱导表达融合蛋白,将纯化后的融合蛋白对新西兰大白兔Oryctolagus cuniculus进行免疫并制备MaYMV MP多克隆抗血清,并采用Western blot对抗血清的效价、灵敏度和特异性进行检测。结果显示,利用成功构建的原核表达载体经诱导表达获得分子量大小约为62 kD的融合蛋白,纯化后对新西兰大白兔进行免疫获得MaYMV MP抗血清,该抗血清的效价为1∶128 000,灵敏度为1∶32,且该抗血清能够特异性地检测到本氏烟Nicotiana benthamiana中瞬时表达的MaYMV MP,而不与马铃薯卷叶病毒属Polerovirus及黄症病毒属Luteovirus的其他病毒发生血清学交叉反应,证明该抗血清具有良好的特异性。表明本研究制备的MaYMV MP抗血清能特异性...  相似文献   

建鲤组织蛋白酶L 的原核表达、纯化鉴定及多克隆抗体的制备     

李树红  陈志光  李冉  李新  李松  陈秀华  蒋然然  李美良  马璐阳 《中国水产科学》2015,22(5):849-857

对原核表达的重组建鲤组织蛋白酶L(Cathepsin L,CAT L)蛋白进行尿素洗涤和Ni-NTA亲和层析纯化,该目的蛋白经300 mmol/L咪唑洗脱为单一峰,SDS-PAGE结合TSK-GEL G2000SWxl凝胶过滤高效液相色谱分析表明重组CAT L获得了高度纯化,分子量约28 k D,纯度超过95%。Z-Phe-Arg-MCA底物测活法显示该重组CAT L表现为半胱氨酸蛋白酶活性,能与其内源抑制因子Cystatin以1︰1的摩尔比结合,具有生物学活性。以纯化的重组CAT L蛋白免疫Balb/C小鼠获得抗血清,经ELISA法检测获得的CAT L抗血清效价高于1︰512000;Western blotting鉴定结果表明该抗体具有良好的特异性,能够识别原核表达的重组CAT L蛋白。免疫组织化学分析结果表明,该抗体还能识别建鲤小肠、肝胰脏、脾、背肌和心肌组织表达的内源性CAT L蛋白。因此可利用该抗体从蛋白水平检测CAT L在鱼类不同组织中的表达和分布情况。  相似文献   

基于抗原表位策略的苹果茎痘病毒抗血清的制备     

李丽丽  杨洪一  董雅凤  高源 《植物病理学报》2010,40(4):357-363

苹果茎痘病毒(Apple stem pitting virus,ASPV)是一种严重危害果树生产的潜隐性病毒,但缺乏实用的ASPV抗血清。应用不同生物信息学软件对ASPV外壳蛋白不同区域的抗原指数、蛋白二级结构中α螺旋、β片层、β转角、无规则卷曲结构、亲水性、可塑性(Flexibility)和表面可及性(Surface probability)进行了分析。根据抗原表位主要分布在β转角结构和无规则卷曲区域、亲水性和表面可及性参数较高的特点,综合分析预测ASPV外壳蛋白抗原表位区可能位于N端6-20、100-114、400-414位氨基酸残基附近。通过合成2条多肽(CRGYEEGSRPNQRVLP、CTGGKIGPKPVLSIRK),与载体蛋白偶联后免疫动物,最后得到了2份抗血清(编号为1468和1469)。制备的抗血清可与ASPV外壳蛋白基因原核表达产物产生免疫反应。应用此抗血清检测苹果样品,抗血清1468检测结果与RT-PCR检测结果一致,而抗血清1469仅能检测到部分样品。  相似文献   

Application of a putative fatty-acid binding protein to discriminate serologically the two European quarantine root-knot nematodes, Meloidogyne chitwoodi and M. fallax, from other Meloidogyne species     

Christophe Tastet  Florence Val  Michel Lesage  Lionel Renault  Laurent Marché  Michel Bossis  Didier Mugniéry 《European journal of plant pathology / European Foundation for Plant Pathology》2001,107(8):821-832

Two major proteins, Mcf-A67 and Mcf-B66, were identified by mini two-dimensional polyacrylamide gel electrophoresis in order to distinguish the two European quarantine root-knot nematodes, Meloidogyne chitwoodi and M. fallax, from eight other species. These quarantine proteinic markers have been microsequenced after enzymatic digestion. The internal amino acid sequences exhibit similarities to members of a family of low molecular weight intracellular lipid-binding proteins. Moreover, to explore a simple, rapid, and inexpensive way to identify the two quarantine nematodes, dot blot hybridizations were performed using an antiserum (A67) produced from the longest amino-acid sequence of the protein Mcf-A67. Although several proteins stained on the M. chitwoodi and M. fallax western blot membranes, the two nematodes were easily distinguished from other root-knot nematodes, on dot blot assays with soluble proteins extracted from a single female. Because of its specificity and sensitivity, the use of the A67 antiserum to improve the diagnosis of the two European quarantine root-knot nematodes is discussed.  相似文献   

In vitro characterization andin vivo detection ofRigidoporus lignosus,the causal agent of white root disease inHevea brasiliensis,by ELISA techniques     

M. Louanchi  P. Robin  T. Michels  M. H. Balesdent  D. Despréaux 《European journal of plant pathology / European Foundation for Plant Pathology》1996,102(1):33-44

The aim of these studies is to develop a method for early detection ofRigidopoms lignosus (Basidiomycete, Polyporaceae), the causative agent of white root disease of rubber tree. Two polyclonal sera were produced against soluble mycelial proteins of twoR. lignosus isolates, one from Africa (FCI2), the other from Asia (FID2). The specificity of the antisera was tested using isoelectric focusing (IEF)/Western-blot and DAS-ELISA. The two sera recognized all 20R. lignosus isolates from various geographical origins. The banding patterns obtained by Western-blot enabled a distinction to be made between isolates from Africa and those from Asia. In DAS-ELISA and Western-blot analyses, strong cross reactions were observed withR. ulmarius. Only slight reactions were observed in Western-blot analysis toR. lineatus andP. noxius, both causative agents of root rot inHevea. These cross reactions were not observed under our DAS-ELISA analysis conditions. Finally, no cross reactions were obtained with 9 otherPolyporaceae orHevea root pathogen species. The sensitivity threshold of the DAS-ELISA method was 5 ng ml^–1 ofR. lignosus protein. An initial approach to using the DAS-ELISA test for the detection ofR. lignosus in infected plants was carried out on artificially inoculated root samples. The DAS-ELISA protocol enabled detection ofR. lignosus in the root systems of diseased plants. Moreover, no cross reaction was observed with healthy plant extracts.  相似文献   

兽用生物制品的运输、贮存与使用     

张振兴  李玉峰  蒋文明 《经济动物学报》2006,10(2):63-65

对兽用生物制品的命名作了概述,并对免疫预防用制品、诊断用制品、治疗用制品的运输、贮存与使用分别进行了阐述,同时对改善我国兽用生物制品的监管提出了建议。  相似文献   

Prokaryotic Expression and Polyclonal Antibody Preparation of σC Protein of Novel Duck Reovirus DH13 Strain     

LI Wenjun  HUANG Huilan  YANG Huihu  XIE Zimin  CUI Huiyi  HUANG Shujian  ZHANG Xuelian 《中国畜牧兽医》2007,47(9):2953-2959

The aims of the experiment was to optimize the prokaryotic expression system of σC protein,prepare polyclonal antibody against σC protein of novel duck reovirus (NDRV),and evaluate the titer of the antibody.The σC gene of NDRV-DH13 strain was amplified by RT-PCR,ligated into pET-30a(+) and pET-32a(+) expression vector,constructed prokaryotic expression plasmid,which were transformed into E.coli BL21(DE3) and the expression of the σC protein were induced by IPTG.The proteins expression were analyzed by SDS-PAGE.The recombinant protein without His tag was purified by digestion,and the recombinant protein with His-tagged was purified by Ni-NTA column.Then the polyclonal antibody was obtained from rabbits which had been immunized by the purified protein without His tag.Anti-His-labeled mAb and NDRV-σC positive serum were used as primary antibodies to evaluate antibodies specificity,the antibodies titer was detected by indirect ELISA (iELISA).SDS-PAGE results showed that the molecular weight of the expression on recombinant proteins were 34 and 37 ku respectively,the proteins were highly expression.Western blotting showed that they had the specific reaction and the prepared antibodies had higher affinity with σC protein,the titer were about 1:25 600 by iELISA detection.This study successfully constructed and optimized the prokaryotic expression system of the polyclonal antibody against σC protein,laid a foundation for the further study of σC protein and the research of genetically engineered vaccine.  相似文献   

苏云金杆菌HBF-1菌株在土壤中毒蛋白含量的ELISA检测   总被引：1，自引：0，他引：1  

吴瑞华  李国勋  王容燕  王金耀  冯书亮 《中国生物防治》2007,23(3):263-268

以苏云金芽孢杆菌HBF-1菌株中分离纯化的高特异性杀虫蛋白作为抗原，免疫新西兰白兔制备出多克隆抗体，其效价可达到16000。应用制备的特异性抗体建立了间接酶联免疫吸附测定法（ELISA），检测不同土壤样品中Bt毒蛋白含量。初步结果表明，该方法能够检测土壤中Bt毒蛋白的含量，因土壤理化特性及组成成分的不同，检测到的毒蛋白含量也略有不同，河沙中的蛋白检测量最低，只有12．53％；另一种沙壤混合物中检测量达到76.16％。  相似文献