|
|
|
| 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.
|
|
Received: 29 November 2021
|
|
|
|
|
|
[1] 李桂黎, 任冉阳, 胡凌, 等. 2015. 兔病毒性出血病病毒VP60蛋白基因研究进展[J]. 动物医学进展, 36(2): 93-96.
(Li G L, Ren R Y, Hu L, et al.2015. Research progress of VP60 protein gene of Rabbit hemorrhagic disease virus[J]. Progress in Veterinary Medicine, 36(2): 93-96.)
[2] 刘怀然, 陈洪岩, 关云涛, 等. 2002. 兔出血症病毒衣壳蛋白VP60基因克隆与序列分析[J]. 中国预防兽医学报, (05): 2-5.
(Liu H R, Chen H Y, Guan Y T, et al.Cloning and sequence analysis for capsid protein VP60 gene of Rabbit hemorrhagic disease virus[J]. Chinese Journal of Preventive Veterinary Medicine, 2002(05): 2-5.)
[3] 刘胜江, 薛华平, 浦伯清, 等. 1984. 兔的一种新病毒病—兔病毒性出血症[J]. 畜牧与兽医, (06): 253-255.
(Liu S J, Xue H P, Pu B Q, et al. 1984. A new viral disease of rabbits-rabbit hemorrhagic disease[J]. Animal Husbandry and Veterinary Medicine, (06): 253-255.)
[4] 王芳, 陈萌萌, 宋艳华, 等. 2016. 兔出血症病毒新毒株RHDV2的流行与控制(综述)[J]. 江苏农业科学, 44(9): 1-3.
(Wang F, Chen M M, Song Y H, et al.2016. Epidemic and control of a new strain of Rabbit hemorrhagic disease virus RHDV2 (Review)[J]. Jiangsu Agricultural Sciences, 44(9): 1-3.)
[5] 邬旭龙, 彭彬, 姜睿姣, 等. 2016. 以原核表达的非洲猪瘟病毒pK205R蛋白为包被抗原的间接ELISA抗体检测方法的建立[J]. 中国预防兽医学报, 38(10): 800-803.
(Wu X L, Peng B, Jiang R J, et al.2016. Development of an indirect ELISA for detection of antibody against African swine fever virus (ASFV) with prokaryotic expressed ASFV pK205R protein as the coating antigen[J]. Chinese Journal of Preventive Veterinary Medicine, 38(10): 800-803.)
[6] 肖璐, 于吉锋, 林毅, 等. 2021. 中国首例兔出血症病毒2型(RHDV2/b/GI.2)的鉴定及病理学观察[J]. 中国畜牧兽医, 48(01): 348-355.
(Xiao L, Yu J F, Lin Y, et al.2021. First Detection of a Type 2 Rabbit hemorrhagic disease virus (RHDV2/b/GI.2) in China and its pathological observation[J]. China Animal Husbandry and Veterinary Medicine, 48(01): 348-355.)
[7] 杨泽晓, 张彦明, 林祥梅, 等. 2008. 醋酸甲羟孕酮(MPA)人工抗原的合成与单克隆抗体的制备[J]. 上海交通大学学报(农业科学版), 026(002): 119-123.
(Yang Z X, Zhang Y M, Lin X H, et al.2008. Preparation of artificial antigen for medroxyprogesterone acetate (MPA) and McAb against MPA[J]. Journal of Shanghai Jiaotong University (Agricultural Science), 026(002): 119-123.)
[8] 郑东, 谌东华, Tao Xue, 等. 2000. 低温电镜术证实了兔出血症病毒亚基因组颗粒的存在[J]. 电子显微学报, 19(5): 661-666.
(Zhen D, Chen D H, Tao X, et al.2000. Visualization of RHDV particles packaging genomic and subgenomic RNAs by electron cryomicroscopy[J]. Journal of Chinese Electron Microscopy Society, 19(5): 661-666.)
[9] 赵林, 李天宪, 宋宝云, 等. 1992. 兔的一种新病毒. Ⅲ. 兔出血症病毒形态的超微结构和抗原性的研究[J]. 微生物学报, 32(05): 359-363.
(Zhao L, Li T X, Song B Y, et al.1992. A new virus of rabbit. Ⅲ. Study on morphological superstructure and antigenicity of Rabbit hemorrhagic disease virus (RHDV)[J]. Acta microbiologica Sinica, 32(5): 359-363.)
[10] 周碧君, 刘刚, 曾新华, 等. 2000. 兔瘟的诊断及感染脏器含毒量的测定[J]. 贵州农业科学, 28(006): 9-11.
(Zhou B J, Liu G, Zeng X H, et al.2000. Diagnosis of rabbit plague and determination of virus content in affected organs[J]. Guizhou Agricultural Sciences, 28(006): 9-11.)
[11] Abrantes J, Loo W, Pendu J L, et al.2012. Rabbit haemorrhagic disease (RHD) and Rabbit haemorrhagic disease virus (RHDV): A review[J]. Veterinary Research, 43(1): 1-19.
[12] Abrantes J, Lopes A M.2021. A review on the methods used for the detection and diagnosis of Rabbit hemorrhagic disease virus (RHDV)[J]. Microorganisms, 9(5): 972-972.
[13] Castanon S, Martin-Alonso J M, Marinm S, et al.2002. The effect of the promoter on expression of VP60 gene from Rabbit haemorrhagic disease virus in potato plants[J]. Plant Science, 162(1): 87-95.
[14] Cooke B D .2002. Rabbit haemorrhagic disease: Field epidemiology and the management of wild rabbit populations[J]. Revue Scientifique et Technique, 21(2): 347-358.
[15] Gil F, Titarenko E, Terrada E, et al.2010. Successful oral prime-immunization with VP60 from Rabbit haemorrhagic disease virus produced in transgenic plants using different fusion strategies[J]. Plant Biotechnology Journal, 4(1): 135-143.
[16] Le G G, Zwingelstein F, Boucher S, et al.2011. Detection of a new variant of Rabbit haemorrhagic disease virus in France[J]. The Veterinary Record, 168(5): 137-138.
[17] Peacey M, Wilson S, Baird A, et al.2007. Versatile RHDV virus-like particles:Incorporation of antigens by genetic modification and chemical conjugation[J]. Biotechnology and Bioengineering, 98(5): 968-977.
[18] Wang X, Xu F, Liu J, et al.2013. Atomic model of Rabbit hemorrhagic disease virus by cryo-electron microscopy and crystallography[J]. Plos Pathogens, 9(1): e1003132. |
|
|
|
