Expression and Immunogenicity of Newcastle disease virus Gene Ⅶ Hemagglutinin-neuraminidase (HN) Protein in Rice (Oryza sativa) Cells
SHI Qian-Qian1, CHEN Jin-Xuan1, CHEN Heng-Yao1, KOU Shao-Kang1, CHU Hong-Yan1, ZHANG Lei1, PAN Shi-Yuan1, ZHANG Er-Qin1*, ZHANG Gai-Ping1,2,3,4*
1 College of Veterinary Medicine/International Joint Research Center of National Animal Immunology, Henan Agricultural University, Zhengzhou 450046, China; 2 Longhu Laboratory of Advanced Immunology, Zhengzhou 450046, China; 3 College of Modern Agriculture, Peking University, Beijing 100871, China; 4 Key Laboratory of Animal Immunology, Henan Academy of Agricultural Sciences, Zhengzhou 450002, China
Abstract：Newcastle disease is an acute, febrile and highly contagious disease caused by Newcastle disease virus (NDV), in which genotype Ⅶ is widespread. Hemagglutinin-neuraminidase protein (HN) is a structural protein of viruses and an important immunogenic protein for host cell receptors to recognize and promote cell membrane fusion. Rice (Oryza sativa) cells are a promising economic and effective bioreactor platform for the production of biological drugs. The rice α-amylase 3D system contains an inducer promoter, which has strong activation activity under the condition of sugar starvation and can express foreign proteins efficiently and at a high level. In this study, NDV gene Ⅶ HN sequence was screened from NCBI, HN gene was optimized according to rice codon preference, and the recombinant plant expression vector pCAMBIA1300-HN was constructed. HN gene was transferred into rice callus by Agrobacterium-mediated method, and the positive callus was identified by hygromycin screening and PCR. HN protein expression was induced in the positive callus, and then identified by Western blot. HN protein was purified by SP cationic chromatography and Hiloard 75 pg gel chromatography, and emulsified with Al(OH)3 and 50V adjuvant at doses of 5 μg and 10 μg, respectively. BALB/c female mice (Mus musculus) aged 6 weeks were immunized, the titer of antibody was detected by hemagglutination inhibition (HI) test and neutralizing test (NT), and the level of interleukin-2 (IL-2) after 2 weeks of immunization was detected. The results showed that the recombinant plant expression vector pCAMBIA1300-HN was successfully constructed, and 85 positive calluses were identified. HN protein was successfully expressed and purified in rice cells. After immunizing mice, the antibody levels of each group except PBS group achieved immune protection (HI≥24), and neutralized antibodies were produced, which had virus neutralization effect. In this study, HN protein of Newcastle disease was expressed in rice cells, and a purification method of HN protein was established. After immunizing animals with HN protein at low dose, the immune effect was good. This study provides a reference for the preparation of Newcastle disease subunit vaccine in the future.
[1] 程小果, 王慧, 程宁, 等. 2016. 新城疫的合理免疫及常见疫苗使用误区[J]. 动物医学进展, 37: 116-120.(Cheng X G, Wang H, Cheng N, et al.2016. Rational immunization of Newcastle disease and common vaccine use misunderstandings[J]. Advances in Veterinary Medicine, 37(06): 116-20.) [2] 丁壮, 金宁一, 王兴龙, 等. 2002. 鸡新城疫病毒HN基因亚单位疫苗诱导免疫保护的实验研究[J]. 动物医学进展: (01): 49-51.(Ding Z, Jin N Y, Wang X L, et al. 2022. Immunoprotection of chickens from ND by vaccination with expressing HN subunit protein of NDV[J].Progress In Veterinary Medicine, (01): 49-51.) [3] 刘万华, 王晶钰, 武宁, 等. 2013. 鸡新城疫病毒的分离鉴定与交叉免疫保护试验[J]. 动物医学进展, 34(03): 62-67.(Liu W H, Wang J Y, Wu N, et al.2013. Isolation and identification of Newcastle disease virus in chickens and cross-immune protection assay[J]. Advances in Veterinary Medicine, 34(03): 62-7.) [4] 刘秀梵, 胡顺林, 胡增垒, 等. 2012. 新城疫重组病毒灭活疫苗(A-VII株)的研制和临床试验[C]. 中国畜牧兽医学会禽病学分会第十六次学术研讨会论文集, 34-35.(Liu X F, Hu S L, Hu Z L, et al.2012. Development and clinical trials of Newcastle disease recombinant virus inactivated vaccine (A-Ⅶ strain)[C]. Proceedings of the 16th Symposium of the Chinese Society of Animal Husbandry and Veterinary Medicine, Poultry Branch, 34-35.) [5] 魏家阳, 李丽, 王国康, 等. 2023. 新城疫病毒Mukteswar株反向遗传平台的建立[J]. 动物医学进展, 44(02): 30-35.(Wei J Y, Li L, Wang G K, et al.2023. Establishment of reverse genetic platform of Newcastle disease virus strain Mukteswar[J]. Progress In Veterinary Medicine, 44(02):30-35.) [6] 于淼, 王洪利, 姜平. 2014. 鸡新城疫病毒基因Ⅶ型和LaSota株灭活疫苗免疫保护效力观察[J]. 畜牧与兽医, 46(02): 56-58.(Yu M, Wang H L, Jiang P.2014. Observation on the immune protection efficacy of inactivated chicken Newcastle disease virus genotype Ⅶ and LaSota strain inactivated vaccine[J]. Animal Husbandry and Veterinary Medicine, 46(02): 56-58.) [7] 张申立, 许倩茹, 杨继飞, 等. 2022. 新城疫病毒HN蛋白在水稻中的表达及半定量快速检测抗体方法的建立[J]. 生物工程学报, 38(05): 1981-1993.(Zhang S L, Xu Q R, Yang J F, et al.2022. Expression of NDV HN protein in rice and development of a semi-quantitative rapid method for detection of antibodies[J]. Chinese Journal of Biotechnology, 38(05): 1981-1993.) [8] 赵建伟, 姜昊妍, 杨锐, 等. 2020. 表面展示基因Ⅶ型新城疫病毒F蛋白胞外区细菌样颗粒的构建与鉴定[J]. 中国兽医学报, 40(12): 2298-2304.(Zhao J W, Jiang H Y, Yang R, et al.2020. Construction and identification of bacterial-like particles in the extracellular region of Newcastle disease virus F protein of type Ⅶ Newcastle disease virus[J]. Chinese Journal of Veterinary Medicine, 40(12): 2298-304.) [9] Alamares J G, Li J, Iorio R M.2005. Monoclonal antibody routinely used to identify avirulent strains of Newcastle disease virus binds to an epitope at the carboxy terminus of the hemagglutinin-neuraminidase protein and recognizes individual mesogenic and velogenic strains[J]. Journal of Clinical Microbiology, 43(8): 4229-4233. [10] Berinstein A, Vazquez-Rovere C, Asurmendi S, et al.2005. Mucosal and systemic immunization elicited by Newcastle disease virus (NDV) transgenic plants as antigens[J]. Vaccine, 23(48-49): 5583-5589. [11] Boroujeni N A, Khatouni S B, Motamedi M J, et al.2022. Root-preferential expression of Newcastle virus glycoproteins driven by NtREL1 promoter in tobacco hairy roots and evaluation of oral delivery in mice[J]. Transgenic Research, 31(2): 201-213. [12] Corbin J M, Kailemia M J, Cadieux C L, et al.2018. Purification, characterization, and N-glycosylation of recombinant butyrylcholinesterase from transgenic rice cell suspension cultures[J]. Biotechnology and Bioengineering, 115(5): 1301-1310. [13] Corbin J M, Mcnulty M J, Macharoen K, et al.2020. Technoeconomic analysis of semicontinuous bioreactor production of biopharmaceuticals in transgenic rice cell suspension cultures[J]. Biotechnology and Bioengineering, 117(10): 3053-3065. [14] Dimitrov K M, Afonso C L, Yu Q, et al.2017. Newcastle disease vaccines-A solved problem or a continuous challenge?[J]. Veterinary Microbiology, 206: 126-136. [15] Gallili G E, Ben-Nathan D1998. Newcastle disease vaccines[J]. Biotechnology Advances, 16(2): 343-366. [16] Ganar K, Das M, Sinha S, et al.2014. Newcastle disease virus: Current status and our understanding[J]. Virus Research, 184: 71-81. [17] Huang J, Wu L, Yalda D, et al.2002. Expression of functional recombinant human lysozyme in transgenic rice cell culture[J]. Transgenic Research, 11(3): 229-239. [18] Huang T K, Plesha M A, Falk B W, et al.2009. Bioreactor strategies for improving production yield and functionality of a recombinant human protein in transgenic tobacco cell cultures[J]. Biotechnology and Bioengineering, 102(2): 508-520. [19] Karaca K, Sharma J M, Winslow B J, et al.1998. Recombinant fowlpox viruses coexpressing chicken type Ⅰ IFN and Newcastle disease virus HN and F genes: Influence of IFN on protective efficacy and humoral responses of chickens following in ovo or post-hatch administration of recombinant viruses[J]. Vaccine, 16(16): 1496-1503. [20] Karki U, Fang H, Guo W, et al.2021. Cellular engineering of plant cells for improved therapeutic protein production[J]. Plant Cell Reports, 40(7): 1087-1099. [21] Lee Y J, Sung H W, Choi J G, et al.2008. Protection of chickens from Newcastle disease with a recombinant baculovirus subunit vaccine expressing the fusion and hemagglutininneuraminidase proteins[J]. Journal of Veterinary Science, 9(3): 301-308. [22] Liu H, De Almeida R S, Gil P, et al.2018. Can genotype mismatch really affect the level of protection conferred by Newcastle disease vaccines against heterologous virulent strains?[J]. Vaccine, 36(27): 3917-3925. [23] Mahon P J, Mirza A M, Musich T A, et al.2008. Engineered intermonomeric disulfide bonds in the globular domain of Newcastle disease virus hemagglutinin-neuraminidase protein: Implications for the mechanism of fusion promotion[J]. Journal of Virology, 82(21): 10386-10396. [24] Mayers J, Mansfield K L, Brown I H.2017. The role of vaccination in risk mitigation and control of Newcastle disease in poultry[J]. Vaccine, 35: 5974-5980. [25] Meng C, Qiu X, Yu S, et al.2016. Evolution of Newcastle disease virus quasispecies diversity and enhanced virulence after passage through chicken air sacs[J]. Journal of Virology, 90(4): 2052-2063. [26] Miller P J, Decanini E L, Afonso C L.2010. Newcastle disease: Evolution of genotypes and the related diagnostic challenges[J]. Infection, Eenetics and Evolution, 10(1): 26-35. [27] Miller P J, King D J, Afonso C L, et al.2007. Antigenic differences among Newcastle disease virus strains of different genotypes used in vaccine formulation affect viral shedding after a virulent challenge[J]. Vaccine, 25(41): 7238-7246. [28] Moyer T J, Zmolek A C, Irvine D J.2016. Beyond antigens and adjuvants: Formulating future vaccines[J]. The Journal of Clinical Investigation, 126(3): 799-808. [29] Nausch H, Buyel J F.2021. Cryopreservation of plant cell cultures-diverse practices and protocols[J]. New Biotechnology, 62: 86-95. [30] Palya V, Kiss I, Tatár-Kis T, et al.2012. Advancement in vaccination against Newcastle disease: Recombinant HVT NDV provides high clinical protection and reduces challenge virus shedding with the absence of vaccine reactions[J]. Avian Diseases, 56(2): 282-287. [31] Peng L H, Gu T W, Xu Y, et al.2022. Gene delivery strategies for therapeutic proteins production in plants: Emerging opportunities and challenges[J]. Biotechnology Advances, 54: 107845. [32] Putri D D, Handharyani E, Soejoedono R D, et al.2017. Pathotypic characterization of Newcastle disease virus isolated from vaccinated chicken in West Java, Indonesia[J]. Veterinary World, 10(4): 438-444. [33] Shahriari A G, Bagheri A, Afsharifar A, et al.2019. Induction of immune response in animal model using recombinant anti-NDV vaccine[J]. Iranian Journal of Biotechnology, 17(1): e2215. [34] Sultan H A, Elfeil W K, Nour A A, et al.2021. Efficacy of the Newcastle disease virus genotype Ⅶ.1.1-matched vaccines in commercial broilers[J]. Vaccines (Basel), 10(1): 29. [35] Tekoah Y, Shulman A, Kizhner T, et al.2015. Large-scale production of pharmaceutical proteins in plant cell culture-the Protalix experience[J]. Plant Biotechnology Journal, 13(8): 1199-1208. [36] Ul-Rahman A, Ishaq H M, Raza M A, et al.2022. Zoonotic potential of Newcastle disease virus: Old and novel perspectives related to public health[J]. Reviews in Medical Virology, 32(1): e2246. [37] Wang J, Yang J, Ge J, et al.2016. Newcastle disease virus-vectored West Nile fever vaccine is immunogenic in mammals and poultry[J]. Virology Journal, 13: 109. [38] Wang Q, Shen X, Qiu T, et al.2021. Evaluation and application of an efficient plant DNA extraction protocol for laboratory and field testing[J]. Journal of Zhejiang University. Science. B, 22(2): 99-111. [39] Xu J, Ge X, Dolan M C.2011. Towards high-yield production of pharmaceutical proteins with plant cell suspension cultures[J]. Biotechnology Advances, 29(3): 278-299.
