兔出血症病毒2型VP60蛋白特异性单克隆抗体的制备  

Preparation of Monoclonal Antibody Specific to VP60 Protein of Rabbit hemorrhagic virus Type 2

作　　者：曾红梅 庞雪晴 唐诗 杨泽晓[1,2] 罗燕[1,2] 王印 姚学萍[1,2] 李丽[3] ZENG Hong-Mei;PANG Xue-Qing;TANG Shi;YANG Ze-Xiao;LUO Yan;WANG Yin;YAO Xue-Ping;LI Li(College of Veterinary Medicine,Sichuan Agricultural University,Chengdu 611130,China;Key Laboratory of Animal Disease and Human Health of Sichuan Province,Chengdu 611130,China;Sichuan Animal Disease Prevention and Control Center,Chengdu 611130,China)

机构地区：[1]四川农业大学动物医学院,成都611130 [2]动物疫病与人类健康四川省重点实验室,成都611130 [3]四川省动物疫病预防控制中心,成都611130

出　　处：《农业生物技术学报》2022年第10期2053-2060,共8页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(31402222)。

摘　　要：兔病毒性出血症(rabbit hemorrhagic disease,RHD)又称兔瘟,是由兔出血症病毒(Rabbit hemorrhagic disease virus,RHDV)感染引起的兔(Oryctolagus cuniculus)急性出血性传染病。为制备RHDV2的特异性单克隆抗体(McAb),进而探索其与经典兔瘟病毒(RHDV)鉴别诊断技术,本研究原核表达纯化RHDV2 VP60蛋白(pET-32a-R2VP60蛋白)和RHDV VP60蛋白(pET-32a-R1VP60蛋白),以p ET-32aR2VP60蛋白免疫BALB/c小鼠(Mus musculus),并分别以p ET-32a-R1VP60蛋白与pET-32a-R2VP60蛋白为包被原建立特异性的RHDV2单克隆抗体筛选间接ELISA方法,采用常规杂交瘤细胞融合技术,通过克隆、亚克隆、腹水制备、效价测定、Western blot分析、血凝-血凝抑制试验以及亚型鉴定成功筛选出一株抗RHDV2的杂交瘤细胞株,命名为2A1。结果显示,2A1融合细胞的染色体约为100条,分泌IgG3亚型McAb,轻链为Kappa亚型;杂交瘤2A1的抗体分泌稳定,腹水纯化McAb效价高,特异性良好,其间接ELISA效价高达1∶128000。Western blot分析显示McAb 2A1仅与RHDV2病毒粒子和p ET-32a-R2VP60蛋白反应,与RHDV1病毒粒子和pET-32a-R1VP60蛋白反应阴性。血凝(hemagglutination,HA)与血凝抑制(hemagglutination inhibition,HI)实验表明2A1纯化McAb可有效抑制RHDV2的血凝作用,HI效价高达10log2。本研究制备的RHDV2特异性McAb 2A1,为RHDV2特异快速的血清学诊断方法建立和试剂盒研发提供了科学材料。Rabbit hemorrhagic disease(RHD)is an acute hemorrhagic infectious disease caused by Rabbit hemorrhagic disease virus(RHDV).RHDV2 VP60 protein(pET-32a-R2VP60 protein)and RHDV VP60protein(pET-32a-R1VP60 protein)were expressed and purified by prokaryotic expression in order to prepare specific monoclonal antibody(McAb)of RHDV2 and explore the differential diagnosis technology between RHDV2 and classical rabbit hemorrhagic disease virus(RHDV).BALB/C mice were immunized with p ET-32a-R2VP60 protein,and specific monoclonal antibodies against RHDV2 were screened by indirect ELISA using pET-32a-R1VP60 protein and pET-32a-R2VP60 protein,respectively.Conventional hybridoma cell fusion technique was used.By cloning,subcloning,ascites preparation,titer determination,Western blot analysis,hemagglutination-hemagglutination inhibition test and subtype identification,a hybridoma cell strain resistant to RHDV2 was successfully screened out,named 2A1.The results showed that 2A1 fusion cell had about 100 chromosomes,secreted IgG3 and Kappa subtype McAb.Hybridoma 2A1 antibody secretion was stable,ascites purification McAb titer was high,good specificity,indirect ELISA titer was up to1∶128000.Western blot analysis showed that McAb 2A1 reacted to RHDV2 virion and p ET-32A-R2VP60protein,but negatively with RHDV1 virion and pET-32a-R1VP60 protein.Hemagglutination and hemagglutination inhibition tests showed that 2A1 purified McAb could effectively inhibit hemagglutination of RHDV2 with HI value up to 10lg2.This study provides scientific materials and lays a foundation for the establishment of specific and rapid serological diagnosis methods and kit development of RHDV2-specific McAb 2A1.

关 键 词：兔出血症病毒2(RHDV2) VP60蛋白 单克隆抗体 Western blot 

分 类 号：S855.3[农业科学—临床兽医学]