牛源荚膜A型多杀性巴氏杆菌疫苗候选株的筛选及鉴定

为了筛选生长快、毒力强、免疫原性好、副反应小的牛源荚膜A型多杀性巴氏杆菌（Pasteurella multocida，Pm）灭活疫苗菌株，本试验选取6株来自不同地区致犊牛肺炎死亡的牛源荚膜A型多杀性巴氏杆菌分离株，测定了培养基生长曲线、小鼠毒力、菌体脂多糖（LPS）含量及各菌株灭活菌苗免疫小鼠和家兔后的抗体效价，并进行了攻毒保护试验。结果显示，分离株Pm2、Pm3、Pm5生长速度较快、毒力较强、LPS含量较多，均含有与毒力和免疫相关的ptfA和fimA基因；免疫小鼠及家兔未发现明显不良反应，在二免后14 d血清抗体达1：64~1：128，强毒攻毒后全部存活，而PBS对照组全部死亡。本试验结果表明，Pm2、Pm3、Pm5均可作为多杀性巴氏杆菌灭活菌苗的候选菌株，其中Pm3作为首选株。     

Generation of mouse monoclonal antibodies specific to tilapia immunoglobulin using fish immunoglobulin/BSA complex for monitoring of the immune response in Nile tilapia Oreochromis niloticus

The simple immunoprecipitation method was used to isolate tilapia immunoglobulin (Ig) for immunization in order to produce monoclonal antibodies (MAbs) specific to tilapia Ig. First, the tilapia antiserum against bovine serum albumin (BSA) was prepared by peritoneal injection of BSA into tilapia, and the tilapia anti‐BSA antiserum was used to precipitate BSA to form the Ig/BSA immune complex. The Ig/BSA immune complex was then injected into Swiss mice for hybridoma production. After fusion, three hybridoma clones producing MAbs specific to the tilapia antibody were selected by dot blot and Western blot. All MAbs (101A, 59G, and 11A) were bound specifically to the heavy chain of immunoglobulin M (IgM). The MAbs 101A and 59G demonstrated twofold higher affinity than MAb 11A and the commercialized antibody. However, MAbs 11A could also bind to the heavy chain of IgM in Asian seabass, Lates calcarifer, as well. These MAbs can be used to monitor the immune responses of individual fish by indirect ELISA upon exposure to various antigens.     

粮食污染物残留免疫分析技术研究与示范

随着人们对粮食安全问题的重视,政府部门不断加强对粮食安全的监测工作。针对粮食生产、加工、储运过程中的化学污染物安全性问题,研究粮食污染物快速监测技术,研究污染物免疫分析方法通过利用抗原、抗体的特异性反应来检测农药含量,设计并筛选吡虫啉、多溴联苯醚、功夫菊酯、氰戊菊酯4种半抗原,制备相关高特异性的抗体,分析吡虫啉、多溴联苯醚、功夫菊酯与气相色谱法(GC)相比,稻谷、大米、玉米样品通过相关性分析均高度相关,操作简便、快速。     

抗番鸭细小病毒卵黄抗体的制备

[目的]有效预防和治疗番鸭细小病毒病。[方法]用番鸭细小病毒连续免疫产蛋母鸡3次,收集鸡蛋,并制备出高免卵黄抗体。通过琼脂扩散试验对制备的卵黄抗体进行效价测定,用雏番鸭进行中和与治疗试验。[结果]制备的卵黄抗体琼扩效价达到1∶32,能有效中和番鸭细小病毒对雏番鸭的致病性,对发病的雏番鸭有很好的治疗作用。[结论]该研究为番鸭细小病毒的预防和治疗提供了一种有效的生物制品。     

2018年中国部分地区猪瘟病毒2.1d亚型新流行株的基因组特征分析

为了解中国猪瘟病毒（classical swine fever virus，CSFV）的分子流行病学及遗传变异情况，本研究应用RT-PCR方法对2018年采集自河南、河北、山东、黑龙江和辽宁5个省份的350份疑似CSFV感染的病料进行E2和NS5B基因扩增，并对PCR产物进行测序和序列分析。结果显示，350份样品中21份为CSFV阳性，共获得14株CSFV的E2基因序列和7株CSFV的部分NS5B基因序列。E2全基因、NS5B部分基因序列分析表明，21份阳性样品均属于近年来在中国流行的CSFV 2.1d亚型，且新发现的2.1d亚型CSFV与中国较早2.1d亚型CSFV毒株间同源性差异不大，新发现的2.1d亚型CSFV分离株在E2基因的6个氨基酸（R³¹、S³⁴、W¹⁸²、K²⁰⁵、K³⁰³、A³³¹）上具有相同的分子特征，E2蛋白中15个位点上的半胱氨酸均未发生变异。韩国2.1d亚型CSFV在E2蛋白上具有3个独特的氨基酸（N⁹⁷、K¹⁵⁹、R²⁰⁵）特征，并且发现了韩国毒株YC11WB可能作为2.1b和2.1d亚型CSFV过渡毒株的证据，流行于中国和韩国的2.1d亚型CSFV可能分别来自于本国早期2.1b亚型CSFV的衍化。本研究证实，2018年中国及周边国家CSFV较为活跃，且流行毒株依然以2.1d亚型为主，为中国科学防控CSFV提供了依据。     

鸭坦布苏病毒E蛋白具有独特交叉反应性中和表位的鉴定

利用抗鸭坦布苏病毒(DTMUV)中和单抗1G2,结合E蛋白多肽扫描技术,鉴定了最小抗原表位²²⁷GSSAGTWQN²³⁵。Western blot显示,残基²³¹G或²³³W的突变后,表位完全失去了与1G2抗体识别的功能,表明²³¹G或²³³W是抗体1G2识别的关键氨基酸位点。通过免疫荧光分析(IFA)显示,MAb 1G2与JEV、WNV和ZIKV的E蛋白发生交叉反应,表明该表位是黄病毒的交叉反应性表位。蛋白质和病毒模型显示,表位定位于成熟病毒粒子可接近的表面,在E蛋白结构域Ⅱ的hi环中。本研究首次证明,中和抗体1G2靶向交叉反应表位定位在E蛋白结构域Ⅱ的hi环,该表位的鉴定有助于提高对黄病毒血清诊断中存在交叉反应问题的认识和理解。     

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

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。     

福州市周边蛋鸡场禽白血病亚群的血清流行病学检测

应用ELISA方法对分别采集自福州市周边30个蛋鸡场的597羽海兰蛋鸡、512羽罗曼蛋鸡、486羽伊莎蛋鸡和553羽特佳蛋鸡共2148羽的血样进行禽白血病病毒(ALV)亚群ALV-A/B和ALV-J抗体检测.结果表明:样品ALV-A/B抗体阳性率由高至低依次为:特佳蛋鸡群11.39%(63/553)、罗曼蛋鸡群11.32%(58/512)、海兰蛋鸡群8.54%(51/597)和伊莎蛋鸡群7.61%(37/486);样品ALV-J抗体阳性率由高至低依次为:特佳蛋鸡群5.24%(29/553)、罗曼蛋鸡群4.10%(21/512)、伊莎蛋鸡群3.51%(13/486)和海兰蛋鸡群3.18%(19/597);而样品ALV-A/B抗体总阳性率为9.73%(209/2148),高于ALV-J抗体总阳性率3.82%(82/2148),其中有0.70%(15/2148)的ALV-A/B和ALV-J抗体双阳性样品;蛋鸡场ALV-A/B抗体阳性率为70.00%(21/30),高于ALV-J抗体阳性率36.7%(11/30),其中有16.7%(5/30)的ALVA/B和ALV-J抗体双阳性样品.结论:福州市周边蛋鸡场不同品系和不同鸡群间存在不同程度的ALV流行,相比ALV-J,ALV-A/B流行更普遍,且存在ALV-A/B和ALV-J抗体双阳性样品,应予重视.     

陕西省重大动物疫病秋防免疫抗体的检测与分析

猪瘟、口蹄疫、禽流感和新城疫四种重大动物疫病对畜禽业的危害非常严重,目前疫苗免疫是预防和控制动物疫病最有效的途径,免疫抗体检测能很好的反映出免疫效果的好坏。为了及时了解陕西省重大动物疫病免疫状况,2010年11月份在全省11个市(区)的21个县随机采集牛、羊、猪、鸡秋防免疫血清共2359份,按照农业部规定的检测方法,对其进行了重大动物疫病免疫抗体水平的检测,检测结果均达到或超过农业部规定的合格标准,说明陕西省今年秋防动物免疫效果总体较好。     

Preparation of Cathepsin L1 Monoclonal Antibody Against Fasclala gigantica and Establishment of Double Antibody Sandwich ELISA

【Objective】 This study was intend to obtain cathepsin L1(rFgCat L1) specific monoclonal antibody and construct the double antibody sandwich ELISA.【Method】 Five BALB/c mice were immunized with 1 mg/mL rFgCat L1 protein for four times.Mouse splenocytes were isolated and fused with SP2/0 cells to construct hybridoma cells.Strong positive hybridoma cell lines were screened, 1×10⁶ cells were injected intraperitoneally per mouse to prepare monoclonal antibodies.Antibody titer and antigenic epitope were detected using ELISA method, antibody subtype and specificity were identified using Western blotting method.The double antibody sandwich ELISA was constructed by combining the anti-rFgCat L1 polyclonal antibody, and its sensitivity and specificity were tested.The positive and negative critical value was screened by 20 negative sera with positive control, and the constructed double antibody sandwich ELISA was verified by 47 goat positive sera and 47 dairy cow positive sera.【Result】 After immunization, the antibody titers in serum of 4 mice were all more than 10⁴.After isolated mouse with the highest immune response spleen cells were fused with SP2/0 cells total of 8 of them were positive cell lines were obtained after selective culture.5D5 and 7G6 were identified as strong positive strains with stable antibody secretion.After multiple subcloning screens and subcultures, the antibodies secreted in the cell supernatant were stable, with titers of 2⁹ and 2¹⁰ respectively, with ascites titers of 10⁷ and 10⁸.Western blotting and antibody subtype identification kits identified that the two antibodies were IgG1 type and the light chain was kappa type, both of which could specifically bind FgESP.According to the same antigen site was recognized by the two kinds of antibodies, the antigen titer of the two monoclonal antibodies were comparied, 7G6 was used as the coating antibody, and anti-rFgCat L1 was used as the enzyme-labeled secondary antibody.The optimized condition of method was that 7G6 was coated at a concentration of 2 μg/mL, the dilution concentration of anti-rFgCat L1 polyclonal antibody was 25 μg/mL, the dilution of Don-HRP-conjugated was 1∶4 000, 5% skimmed milk powder was selected as the blocking solution and the color development time was 25 min.The method was proved that could recognize the lowest antigen concentration of 0.625 μg/mL, also could specifically recognize antigen of Fasciola fasciatus.The constructed sandwich ELISA method was used for antigen detection of 47 dairy cow positive serum and 47 goat positive serum infective samples kept in the laboratory and the positive antigen rate were 72.3% and 78.7%, respectively.【Conclusion】 Anti-rFgCat L1 monoclonal antibody was successfully prepared and the double-sheet sandwich ELISA method for fascioliasis was constructed, which provided a good theoretical basis and material basis for the development of low-cost and rapid diagnostic kits.