水稻源禽流感病毒H9N2亚型HA蛋白两种纯化方法的建立

doi:10.3969/j.issn.1674-7968.2021.07.001

Abstract
Figure/Table
References (0)
Related Citation (15)

Download: PDF (3788 KB) HTML (1 KB)
Export: BibTeX | EndNote (RIS)

Abstract Hemagglutinin (HA) protein is a kind of glycoprotein on the surface of Avian influenza virus, which is a main protective antigen inducing neutralizing antibody. Avian influenza virus H9N2 HA gene was successfully expressed in rice (Oryza sativa) endosperm. In order to establish a purification method for H9N2 Avian influenza virus HA protein expressing in rice, two methods were used in this study to purify HA protein. Method 1: Rice powder expressing HA protein was added into the extract in a ratio of 1∶5 mass to volume, stirred at room temperature, centrifuged and supernatant was taken. After filtration, crude HA protein was firstly captured by Q anion chromatography. Subsequently, HA protein was purified by HA protein antibody affinity chromatography, eluent was collected and the target protein was detected. Method 2: Crude HA protein solution was obtained in the same way as method 1. According to packing property of the Butyl hydrophobic, ammonium sulfate was added to HA protein solution, which was then stirred, centrifuged and filtered for a second stage of purification. The collected eluent was concentrated and changed, and purified by SP-cationic chromatography, and the eluent was collected to obtain the HA protein. The HA protein purified by the 2 methods was tested by SDS-PAGE and Western blot, and the HA protein was sent to the company for sequencing. The results showed that the purity of HA protein was about 90% by Q anion chromatography combined with HA affinity chromatography. The method of purification was relatively simple, but monoclonal antibodies need to be cultured and purified. This method was suitable for rapid and small-scale protein purification in laboratory. The purity of HA protein was about 90% by Q anion chromatography, Butyl hydrophobic chromatography and SP cationic chromatography. This method is suitable for establishing the purification process of HA protein and is used to purify HA protein on a large scale. The HA protein obtained by these two methods was sequenced and the result was consistent with the known amino acid sequence. In this study, two methods were established for the first time to obtain high purity recombinant H9N2 subtype HA protein from rice, it will provide basic data for the preparation of avian influenza H9N2 subunit vaccine in the future.

Key words： Transgenic rice HA protein Ion-exchange chromatography Hydrophobic chromatography Affinity chromatography

Received: 27 November 2020

ZTFLH:

S851.3

Corresponding Authors: *zhangerqin76@163.com;zhanggaiping2003@163.com

	Service

	E-mail this article
	Add to my bookshelf
	Add to citation manager
	E-mail Alert
	RSS
	Articles by authors
	NIU Xiang-Xiang
	LI Xue-Yang
	XU Qian-Ru
	LI Qing-Mei
	WANG Ya-Nan
	ZHANG Shen-Li
	MA Qiang
	ZHANG Er-Qin
	ZHANG Gai-Ping

Cite this article:

NIU Xiang-Xiang,LI Xue-Yang,XU Qian-Ru, et al. Establishment of Two Purification Methods of Avian influenza virus H9N2 Subtype HA Protein from Rice (Oryza sativa)[J]. 农业生物技术学报, 2021, 29(7): 1237-1247.

URL:

http://journal05.magtech.org.cn/Jwk_ny/EN/10.3969/j.issn.1674-7968.2021.07.001 OR http://journal05.magtech.org.cn/Jwk_ny/EN/Y2021/V29/I7/1237

[1] 安娜.2013.植物源重组人碱性/酸性成纤维细胞生长因子表达研究[D].博士学位论文, 武汉大学, 导师: 杨代常.pp 18-20.
(An N.2013.Expression of plant-derived recombinant human basic/acid fibroblast growth factor[D].Thesis for Ph.D., Wuhan University, Supervisor: Yang D C, pp.18-20.)
[2] 陈陆, 刘守川, 赵军, 等.2011.不同H9N2亚型禽流感病毒分离株致病力研究及HA抗原性变异分析[J].中国农业科学, 44(24): 5100-5107.
(Chen L, Liu S C, Zhao J, et al.2011.Characteristics of pathogenic and HA antigenic variation of H9N2 subtybe Avian influenza viruses isolated from 1998 to 2008 in China[J].Scientia Agricultura Sinica, 44(24): 5100-5107.)
[3] 丁一, 魏际华, 张宁, 等.2017.水稻胚乳细胞生物反应器研究进展[J].中国稻米, 23(03): 6-12.
(Ding Y, Wei J H, Zhang N, et al.2017.Research progress in rice endosperm cell bioreactor[J].China Rice, 23(03): 6-12.)
[4] 侯力嘉, 吴胜昔, 梁望旺, 等.2020.H9N2禽流感病毒HA蛋白的原核表达与鉴定[J].黑龙江畜牧兽医, 10:58-61.
(Hou L J, Wu S X, Liang W W, et al.2020.Prokaryotic expression and identification of the HA protein of Avian influenza A virus H9N2[J].Heilongjiang Animal Husbandry and Veterinary, 510:58-61.)
[5] 蒋世强, 潘能庆, 鄢航.等.2017.重组蛋白纯化研究进展[J].现代农业科技,(04): 250-251, 254.
(Jiang S Q, Pan N Q, Yan H, et al.2017.Progress in purification of recombinant protein[J].Modern Agricultural Science and Technology, 4: 250-251, 254.)
[6] 李令臣, 侯力嘉, 吴胜昔, 等.2019.禽流感病毒HA蛋白的原核表达与鉴定[J].重庆理工大学学报(自然科学), 33(05): 137-141.
(Li L C, Hou L J, WU S X, et al.2019.Prokaryotic expression and identification of HA protein of Avian influenza virus[J].Journal of Chongqing University of Technology (Natural Science), 33(05): 137-141.)
[7] 李甜甜, 蒋大伟, 姬鹏超,等.2020.传染性法氏囊病毒VP2蛋白的原核表达及其免疫效果评价[J].河南农业科学, 49(9):136-142.
(Li T T,Jiang D W, Ji P C, et al.2020.Prokaryotic expression of VP2 protein of infectious Bursal disease virus and its immunization evaluation[J].Journal of Henan Agricultural Sciences,49(9):136-142.)
[8] 路秀玲, 苏志国.2002.人血清白蛋白纯化技术研究进展[J].生物工程学报, 18(6): 761-766.
(Lu X L, Su Z G.2002.Research progress on purification technology of human serum albumin[J].Chinese Journal of Bioengineering, 18(6): 761-766.)
[9] 潘学, 陈新武, 季雅宁, 等.2019.H9N2亚型禽流感病毒样颗粒疫苗的制备研究[J].中国动物传染病学报, 27(1):1-7.
(Pan X, Chen X W, Ji Y N, et al.2019.Chracterization of virus-like particle vaccine against H9N2 subtype Avian influenza virus infection[J].Chinese Journal of Animal Infectious Diseases, 27(1): 1-7).
[10] 申雪静, 张二芹, 许倩茹, 等.2019.狂犬病病毒G蛋白在水稻中的表达及遗传稳定性鉴定[J].农业生物技术学报, 27(02): 204-211.
(Shen X J, Zhang E Q, Xu Q R, et al.2019.Expression of Rabies virus G protein in rice and identification of its genetic stability[J].Journal of Agricultural Biotechnology, 27(02): 204-211.)
[11] 宿春虎, 王慧, 程小果.2017.H9N2亚型禽流感的流行现状与防控措施[J].家禽科学, 12: 29-31.
(Su C H, Wang H, Cheng X G.2017.Epidemic Status and prevention and control measures of H9N2 subtype avian Influenza[J].Poultry Science, 12: 29-31.)
[12] 王聚财, 刘运超, 陈玉梅, 等.2017.HPV45-L1和HPV58-L1蛋白植物表达载体的构建及水稻转基因植株的鉴定[J].华北农学报, 32(02): 55-60.
(Wang J C, Liu Y C, Chen Y M, et al.2017.Construction of plant expression vectors for HPV45-L1 and HPV58-L1 protein and identification of transgenic rice plants[J].Acta Agriculturae Boreali-Sinica, 32(02): 55-60.)
[13] 王善辉, 汪鹏旭, 吕颜枝, 等.2015.H9N2亚型禽流感病毒HA-M2融合基因的真核表达及免疫原性研究[J].中国家禽, 37(13): 18-21.
(Wang S H, Wang P X, Lu Y Z, et al.2015.Eukaryotic expression and immunogenicity of the fusion gene of H9N2 subtype Avian influenza virus HA-M2[J].China Poultry, 37(13): 18-21.)
[14] 查夕馨.2019.重组H9N2亚型禽流感病毒冷适应和NS截短致弱活疫苗的研制[D].硕士学位论文, 扬州大学, 导师: 彭大新, pp.1-3.
(Cha X X.2019.Development of live vaccine for cold adaptation and NS truncation of recombinant H9N2 subtype Avian influenza virus[D].Thesis for M.S, Yangzhou University, Supervisor: Peng D X, pp.1-3.)
[15] 张民秀, 谢芝勋, 黄莉, 等.2017.H9N2亚型禽流感病毒HA基因在昆虫细胞中的表达[J].基因组学与应用生物学, 36(3): 859-864.
(Zhang M X, Xie Z X, Huang L, et al.2017.Expression of HA gene of H9N2 subtype Avian influenza virus in insect cells[J].Genomics and Applied Biology, 36(3): 859-864.)
[16] 张烨, 于在江, 唐启惠, 等.2010.禽流感病毒H9N2血凝素基因和神经氨酸酶基因在真核酵母中的表达[J].中国生物工程杂志, 30(7): 33-38.
(Zhang Y, Yu Z J, Tang Q H, et al.2010.Expression of the hemagglutinin and neuramidinase gene of Influenza virus H9N2 in P.methanolica[J].China Biotechnology, 30(7): 33-38.)
[17] 赵翔玥, 张二芹, 许倩茹, 等.2020.禽流感病毒H9N2亚型HA蛋白在水稻胚乳中的稳定、高效表达及活性鉴定[J].华北农学报, 35(4): 230-238.
(Zhao X Y, Zhang E Q, Xu Q R, et al.2020.Stable and efficient expression of HA protein of Avian influenza virus H9N2 subtype in rice endosperm and identification of activity[J].Acta Agriculturae Boreali-Sinica, 35(4): 230-238.)
[18] Adlhoch C, Fusaro A, Kuiken T, et al.2020.Avian Influenza overview february-may 2020[J].Scientific Report, 18(6): 6194-6241.
[19] An N, Ou J, Jiang D, et al.2013.Expression of a functional recombinant human basic fibroblast growth factor from transgenic rice seeds[J].International Journal of Molecular Sciences, 14(2): 3556-3567.
[20] Arai Y, Ibrahim M S, Elgendy E M, et al.2019.Genetic compatibility of reassortants between avian H5N1 and H9N2 influenza viruses with higher pathogenicity in mammals[J].Journal of Virology, 93(4): 1969-1986.
[21] Buyel J F, Twyman R M, Fischer R, et al.2015.Extraction and downstream processing of plant-derived recombinant proteins[J].Biotechnology Advances, 33(6Pt1): 902-913.
[22] Chen Z, Dai H, Liu Z, et al.2014.Quantitation of the residual DNA from rice-derived recombinant human serum albumin[J].Analytical Biochemistry, 450: 4-10.
[23] Fujiwara Y, Yang L, Takaiwa F, et al.2016.Expression and purification of recombinant mouse interleukin-4 and -6 from transgenic rice seeds[J].Molecular Biotechnology, 58(4): 223-231.
[24] Ge F, Li X, Ju H, et al.2016.Genotypic evolution and antigenicity of H9N2 Influenza virus in Shanghai, China[J].Archives of Virology, 161: 1437-1445.
[25] Ge J, An Q, Gao D, et al.2016.Construction of recombinant baculoviruses expressing hemagglutinin of H5N1 avian Influenza and research on the immunogenicity[J].Scientific Reports, 6: 24290.
[26] Gu M, Xu L, Wang X, et al.2017.Current situation of H9N2 subtype avian Influenza in China[J].Veterinary Research, 48(1): 49.
[27] Low D, O' Leary R, Pujar N S, et al.2007.Future of antibody puriﬁcation[J].Journal of Chromatography B, 848(1): 48-63.
[28] Ma F, Zhang E, Li Q, et al.2020.A plant-produced recombinant fusion protein-based newcastle disease subunit vaccine and rapid differential diagnosis platform[J].Vaccines, 8(1):122.
[29] Nochi T, Takagi H, Yuki Y, et al.2007.Rice-based mucosal vaccine as a global strategy for cold-chain-and needle -free vaccination.[J].Proceedings of the National Academy of Sciences of the USA, 104(26) :10986-10991.
[30] Peacock T P, James J, Sealy J E, et al.2019.A global perspective on H9N2 avian influenza virus[J].Viruses, 11(7): 620.
[31] Potdar V, Hinge D, Satav A, et al.2019.Laboratory-confirmed avian influenza a(H9N2)virus infection, India, 2019[J].Emerging Infectious Diseases, 25(12): 2328-2330.
[32] Pua T L, Chan X Y, Loh H S, et al.2017.Purification and immunogenicity of hemagglutinin from highly pathogenic Avian influenza virus H5N1 expressed in Nicotiana benthamiana[J].Human Vaccines & Immunotherapeutics, 13(2): 306-313.
[33] Sjöstrand D, Diamanti R, Lundgren C, et al.2017.A rapid expression and purification condition screening protocol for membrane protein structural biology[J].Protein Science, 26(8): 1653-1666.
[34] Suárez M, Sordo Y, Prieto Yet al.2017.A single dose of the novel chimeric subunit vaccine E2-CD154 confers early full protection against classical Swine fever virus[J].Vaccine, 35(34): 4437-4443.
[35] Sun Y, Liu J.2015.H9N2 influenza virus in China: A cause of consern[J].Protein Cell, 6(1):18-25.
[36] Ulmer J B, Valley U, Rappuoli R, et al.2006.Vaccine manufacturing: Challenges and solutions[J].Nature Biotechnol, 24(11): 1377-1383.
[37] Wilken L R, Nikolov Z L.2012.Recovery and purification of plant-made recombinant proteins[J].Biotechnology Advances, 30: 419-433.
[38] Zhang Q, Yu H, Zhang F Z, et al.2013.Expression and purification of recombinant human serum albumin from selectively terminable transgenic rice[J].Journal of Zhejiang University Science B, 14(10): 867-874.
[39] Zhao J, Yu H Y, Zhao Y, et al.2019.Soluble expression, rapid purification, biological identification of chicken interferon-alpha using a thioredoxin fusion system in E.coli and its antiviral effects to H9N2 Avian influenza virus[J].Preparative Biochemistry & Biotechnology, 49(2): 192-201.