Abstract Abstract Rabbit haemorrhagic disease virus (RHDV) belongs to the genus Lagovirus of Caliciviridae. VP60 protein is one of the capsid proteins of RHDV, and also is main immunogenic protein which the major antigenic epitopes mainly locate in the N-terminal region. In this study, the VP60 truncated gene was cloned by employing the method RT-PCR and then subcloned to pMD20-T. After complete sequence analysis, the prokaryotic expression plasmid pET-RHDV-VP60 was successfully constructed and then inserted into the expression Escherichia coli Rosetta(DE3). The target protein was highly expressed under the induction with 1 mmol/L isopropyl β-D-1 -thiogalactopyranoside (IPTG), and the Balb/C mice were immunized with purified expression products, then the polyclonal antibody was prepared. Subsequently, the immunogenicity of recombinant protein was analyzed using indirect enzyme linked immunosorbent assay (iELISA) and Western-blot. Twenty clean rabbits were randomly divided into 4 groups and subcutaneously injected once, using commercial vaccine (5 rabbits), the RHDV GS/YZ inactivated by 0.4% formaldehyde (5 rabbits), recombinant protein mixed with adjuvant (5 rabbits) and PBS as negative controls (5 rabbits)，respectively . The dosage of recombinant protein was 500 μg mixed with equal amounts of freund's complete adjuvant per injection, with 14 days interval, the recombinant protein was 250 μg mixed with equal amounts of freund's incomplete adjuvant per injection, After 21 days, all the rabbits attacked by the RHDV GS/YZ that HA titer was 210 and observed the results after 14 days. The results showed that the homology of nucleotide sequences of VP60 truncated gene from RHDV GS/YZ strain with 32 RHDV strains in GenBank was from 91.9% to 98.6%, and had 98.6% nucleotide sequence identity with the RHDV HB strain(accession number: KU207100.1) and RHDV WX strain (accession number: AF402614.1). The homology of amino acid sequence of RHDV GS/YZ strain with 32 RHDV strains in GenBank was from 96.7% to 99.1%, and had 99.1% amino sequence identity with the RHDV WX strain (accession number：AF402614.1) and RHDV Mexico 89 strain (accession number：AF295785.1). The target gene was successfully expressed in Escherichia coli, and the recombinant protein expression in the form of inclusion body. SDS-PAGE analysis showed that the molecular weight of the recombinant protein was approximately 50 kD. The titer of polyclonal antibody was 1∶32 000 by iELISA and Western-blot demonstrated that the recombinant protein could bind with antibody induced by RHDV GS/YZ and the polyclonal antibody induced by recombinant protein could also combine with vaccine of RHDV - Pasteurella multocida and wild viruses of RHDV GS/YZ. So it had good immunogenicity. The immune protection experiment showed that the rabbits in the RHDV GS/YZ inactivated by 0.4% formaldehyde group, commercial vaccine group and recombinant protein mixed with adjuvant survived facing the challenge without any clinical signs of RHD while those in the negative control group died within 96 h post-infection and exhibited typical clinical symptoms. The titer of hemagglutination inhibition of commercial vaccine was 25~28, the self-made inactivated vaccine was 25~26, and the recombinant protein mixed with equal amounts of freund's adjuvant was 24~25. The results showed that the proteins expressed by Rosetta (DE3) could be ideal candidate for RHDV vaccines.
AN Kai,KONG Hui-Ping,GENG Xiao-Yong, et al. Prokaryotic Expression and Protective Efficacy of VP60 Truncated Gene in RHDV GS/YZ Strain[J]. , 2018, 26(5): 861-870.
5 参考文献 扈荣良. 等. 2014. 现代动物病毒学[M]. 北京: 中国农业出版社, p. 1215. (Hu R L, et al. 2014. Modern Animal Virology[M]. Beijing: China Agriculture Press, p.1215.) 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, 16(6): 253-255. (Liu S J, Xue H P, Pu B Q, et al. 1984. A new viral disease of rabbit-rabbit viral hemorrhagic disease[J]. Veterinary Medicine, 16(6): 253-255.) 祖立闯, 沈志强, 李娇, 等. 2012. 兔病毒性出血症病毒VP60蛋白在诊断与亚单位疫苗应用中的研究进展[J]. 畜牧与兽医, 44(9): 100-103. (Zu L C, Shen Z Q, Li J, et al. 2012.The research progress of VP60 protein of rabbit hemorrhagic disease virus in the diagnosis and subunit vaccine[J]. Veterinary Medicine, 44(9): 100-103.) 肖敏, 包世俊, 薛慧文, 等. 2017. 小反刍兽疫病毒甘肃株F基因的克隆、原核表达及多克隆抗体的制备[J]. 农业生物技术学报, 25(3): 477-484.(Xiao M, Bao S J, Xue H W, et al. 2017. Cloning and Prokaryotic Expression of F Gene of Peste des petits ruminants virus GS Strain and Preparation of Polyclonal Antibody[J]. Journal of Agricultural Biotechnology, 25(3): 477-484.) 徐为燕. 1994. 兔病毒性出血症研究进展及其在国际上的评价[J]. 南京农业大学学报, 17(3): 47-52.(Xu W Y. 1994. Progress of researchs on rabbit viral hemorrhagic disease and its valuation[J]. Journal of Nanjing Agricultural University, 17(3): 47-52.) 严维巍, 崔治中, 王永坤. 2003. 兔出血症病毒中国株衣壳蛋白基因的克隆和序列分析[J]. 中国病毒学,?18 (2) :129-133.( Yan W W, Cui Z Z, Wang Y K. 2003. Cloning and sequence analysis of capsid protein gene of rabbit hemorrhagic disease virus strain in China[J]. Virologica Sinica, 18 (2) :129-133.) 张夏兰, 王红宁, 张昌菊, 等. 2007. 兔病毒性出血症基因工程疫苗的研究进展[J]. 中国预防兽医学报，29(10): 821-824. (Zhang X L, Wang H N, Zhang C J, et al. 2007. Rabbit viral research progress in genetic engineering vaccine[J]. Chinese Journal of Preventive Veterinary Medicine 2007 29(10): 821-824.) 于作. 2015. 表达兔出血症病毒VP60基因的重组犬2型腺病毒的构建及其免疫效力评价[D]. 博士学位论文, 哈尔滨兽医研究所, 导师: 曲连东. p. 49. (Yu Z. 2015. Construction and Immunogenicity Study on Recombinant Canine Adenovirus Type 2 Expressing the vp60 Gene of Rabbit Hemorrhagic Disease Virus[D]. Thesis for Ph.D, Harbin Veterinary Research Insititute, Supervisor: Qu L D. p. 49.) 祖立闯, 王金良, 沈志强. 2010. 兔病毒性出血症病毒VP60蛋白的截短表达与活性检测[J]. 中国畜牧兽医学会畜牧兽医生物技术学分会暨中国免疫学会兽医免疫分会学术研讨会. (Zu L C, Wang J L, Shen Z Q. 2010. Truncated expression and activity detection of VP60 protein of rabbit hemorrhagic disease virus[J]. Symposium on animal husbandry and Veterinary Biotechnology of China animal husbandry and Veterinary Association and Symposium of Veterinary Immunology section of Chinese Immunology Society.) 杨丽梅, 马力, 张志美, 等. 2013. 兔出血症病毒ZB分离株VP60基因克隆、截短表达与活性鉴定[J] 动物医学进展, 34(9): 57-61.(Yang L M, Ma L, Zhang Z M, et al. 2013. Cloning, truncated expression and activity identification of VP60 gene of rabbit hemorrhagic disease virus ZB isolate[J]. Progress In Veterinary Medicine, 34(9): 57-61.) 任冉阳. 2015. 兔瘟病毒的VP60截段基因原核表达及抗体检测金标试纸的初步研究[D]. 硕士学位论文, 四川农业大学, 导师: 杨泽晓. pp. 43-44. (Ren R Y. 2015. Preliminary study on detection of RHDV VP60 truncated gene prokaryotic expression and antibody test[D]. Thesis for M.S, Sichuan Agricultural University, Supervisor: Yang Z X. pp. 43-44.) 程英杰. 2013. 兔病毒性出血症基因工程疫苗研究[D]. 硕士学位论文, 甘肃农业大学, 导师: 吴润晓. pp. 17-18. (Cheng Y J. 2013. Genetic engineering vaccines against rabbit hemorrhagic disease virus [D]. Thesis for M.S, Gansu Agricultural University, Supervisor: Wu R. pp. 17-18.) Hukowska-Szematowicz B, Tokarz-Deptula B, Deptu?a W. 2013. Analysis of genetic variability and phylogenetic analysis of selected Czech and French strains of rabbit haemorrhagic disease virus (RHDV)[J]. Journal of Applied Genetics, 54(2): 235-248. Oka T, Katayama K, Ogawa S, et al. 2005. Proteolytic processing of sapovirus ORF1 Polyprotein[J]. Journal of Virology, 79(12): 7283-7290. Abrantes J, WVD Loo, Pendu JL, et al. 2012. Rabbit Haemorrhagic Disease (RHD) and Rabbit Haemorrhagic Disease Virus (RHDV): a review[J]. Veterinary Research, 43(1): 1-19. Viaplana E, Plana J, Villaverde A. 1997. Antigenicity of VP60 structural protein of rabbit hemorrhagic disease virus[J]. Archives of Virology, 142(9): 1843-1848. Farnos O, Boue O, Parra F, et al. 2005. High-level expression and immunogenic properties of the recombinant rabbit hemorrahagic disease virus VP60 capsid protein obtained in pichia pastoris[J]. Journal of Biotechnology, 117(3): 215-224. Forrester NL, Trout RC, Turner SL, et al. 2006. Unravelling the paradox of rabbit haemorrhagic disease virus emergence, using phylogenetic analysis; possibke implications for rabbit conservation strategies[J]. Biological Conservation, 131(2): 296-306. Morales M, Barcena J, Ramirez MA, et al. 2004. Synthesis in vitro of rabbit hemorrhagic disease virus subgenomic RNA by internal initiation on (-)sense genomic RNA: mapping of a subgenomic promoters[J]. Journal of Biological Chemistry,?279(17): 17013-17018. Moss SR, Turner SL, Trout RC, et al. 2002. Molecular epidemiology of rabbit haemorrhagic disease virus [J]. Journal of General Virology, 83(10): 2461-2467. Perez-Filgueira D, Resino-Talavan P, Cubillos C, et al. 2007. Development of a low-cost, insect larvae-derived recombinant subunit vaccine against RHDV[J].Virology, 364(2): 422-430. Spibey N, Mccabe VJ, Greenwood NM, et al. 2012. Novel bivalent vectored vaccine for control of myxomatosis and rabbit haemorrhagic disease[J]. Veterinary Record, 170(12): 309-314. Wang X, Qiu L, Hao H, et al. 2012. Adenovirus-based oral vaccine for rabbit hemorrhagic disease[J]. Vet Immunol Immunopathol, 145(1-2): 277-282.
