Preparation of Monoclonal Antibody Specific to VP60 Protein of Rabbit hemorrhagic virus Type 2
ZENG Hong-Mei1, PANG Xue-Qing1, TANG Shi1, YANG Ze-Xiao1,2,*, LUO Yan1,2, WANG Yin1,2, YAO Xue-Ping1,2, LI Li3
1 College of Veterinary Medicine, Sichuan Agricultural University, Chengdu 611130, China; 2 Key Laboratory of Animal Disease and Human Health of Sichuan Province, Chengdu 611130, China; 3 Sichuan Animal Disease Prevention and Control Center, Chengdu 611130, China
Abstract：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 VP60 protein (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 pET-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 to 1∶128 000. Western blot analysis showed that McAb 2A1 reacted to RHDV2 virion and pET-32A-R2VP60 protein, 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.
曾红梅, 庞雪晴, 唐诗, 杨泽晓, 罗燕, 王印, 姚学萍, 李丽. 兔出血症病毒2型VP60蛋白特异性单克隆抗体的制备[J]. 农业生物技术学报, 2022, 30(10): 2053-2060.
ZENG Hong-Mei, PANG Xue-Qing, TANG Shi, YANG Ze-Xiao, LUO Yan, WANG Yin, YAO Xue-Ping, LI Li. Preparation of Monoclonal Antibody Specific to VP60 Protein of Rabbit hemorrhagic virus Type 2. 农业生物技术学报, 2022, 30(10): 2053-2060.
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