猪血凝性脑脊髓炎病毒单克隆抗体的制备与鉴定  

Preparation and Identification of Monoclonal Antibody against Porcine Hemagglutinating Encephalomyelitis Virus

作　　者：陈克研[1] 赵传博[1] 贺文琦[1] 陆慧君[2] 高巍[1] 常志广[1] 王丽[1] 高丰[1] 

机构地区：[1]吉林大学畜牧兽医学院,长春130062 [2]吉林大学人兽共患病与教育部重点实验室人畜共患病研究所,长春130062

出　　处：《中国生物制品学杂志》2009年第9期907-910,共4页Chinese Journal of Biologicals

基　　金：国家自然科学基金资助项目(30671551);吉林省科技发展计划重点资助项目(20060206-2);吉林省科技发展计划项目(20090154);吉林省国际合作项目(20080722)

摘　　要：目的制备猪血凝性脑脊髓炎病毒(HEV)单克隆抗体,并进行鉴定。方法通过差速离心和蔗糖密度梯度离心法对猪HEV进行纯化,免疫BALB/c小鼠后,取其脾细胞与骨髓瘤SP2/0细胞进行融合,经间接ELISA和血凝抑制试验(HI)筛选能稳定分泌抗HEV单克隆抗体的杂交瘤细胞株,并对单抗进行生物学鉴定。结果筛选出4株能稳定分泌抗HEV单抗的杂交瘤细胞株2H2、2A1、1E2、4D4。经鉴定,4株杂交瘤细胞株诱生小鼠腹水抗HEV的ELISA效价可达1∶12800～1∶51200,其中3株HI效价可达1∶24～1∶28,另1株为0。4株杂交瘤细胞分泌的单抗与猪传染性胃肠炎病毒(TGEV)、猪流行性腹泻病毒(PEDV)和猪伪狂犬病病毒(PRV)均不发生交叉反应;杂交瘤细胞染色体数为83～103;除2A1单抗为IgG2b外,其他3株均为IgG1;Westernblot分析表明,2H2、2A1和4D4能识别HEV的血凝素-酯酶蛋白(HE),1E2可识别纤突蛋白(S)。结论已成功制备出抗HEV的单克隆抗体,为HEV快速检测试剂的研制奠定了基础。Objective To prepare and identify the monoclonal antibody （McAb） against porcine hemagglutinating encephalomyelitis virus （HEV）. Methods HEV was purified by differential and sucrose density gradient centrifugations and used for immunization of BALB / c mice. The spleen cells of immunized mice were fused with myeloma SP2 / 0 cells, and the hybridoma cell strains secreting McAb against HEV stably were screened by indirect ELISA and HI test for biological identification. Results Four hybridoma cell strains secreting McAbs against HEV, i.e. 2H2, 2A1, 1E2 and 4D4, were screened. The ELISA titers of McAbs secreted by the four strain were 1 ： 12 800 - 1 ： 51 200. However, the HI titers of McAbs secreted by three of the four strains reached 1 ： 24 - 1 ： 2^8, while that by the other one was 0. The McAbs secreted by the four strains showed no cross reaction with porcine transmissible gastroenteritis virus （TGEV）, porcine epidemic diarrhea virus （PEDV） or porcine pseudorabies virus （PPV）. The numbers of chro- mosome of the hybridoma cell strains were 83 - 103. The McAb secreted by 2A1 was IgG2b, while those by the other three strains were IgG1. Western blot proved that the McAbs secreted by 2H2, 2A1 and 4D4 recognized the hemagglutinin-esterase （HE） protein of HEV, and that secreted by 1E2 recognized spike （S） protein. Conclusion The McAbs against porcine HEV was successfully prepared, which laid a foundation of developing a rapid determination method for HEV.

关 键 词：血凝性脑脊髓炎病毒 单克隆抗体 酶联免疫吸附试验 

分 类 号：S852.659.6[农业科学—基础兽医学]