流行性乙型脑炎病毒NS1基因克隆及其蛋白表达纯化

doi:10.16431/j.cnki.1671-7236.2018.08.034

Abstract

Abstract:

In order to express non-structural protein NS1 of epidemic encephalitis type B,NS1 gene of Japanese encephalitis virus (JEV) P3 strain was amplified by PCR method,and the prokaryotic expressing vectors were constructed by homologous recombination that fusion expressed His or GST tag in this study.The correct plasmids tested by PCR,enzyme digestion and sequencing were transformed into E.coli BL21,and NS1 fusion proteins were induced by IPTG.The proteins were identified using SDS-PAGE electrophoresis and Western blotting,and purified by urea modified and renaturation and affinity chromatography of nickel.The results indicated that two prokaryotic expression vectors of NS1 pET-42b-NS1 and pGEX-KG-NS1 were constructed successfully,and the length of NS1 gene of P3 strain was 1 056 bp,which expressed about 40 ku protein in the form of inclusion body,the degeneration and renaturation effect of urea was better,and NS1 protein had high purity and concentration by nickel affinity chromatography.JEV NS1 gene could express the protein with high purity through prokaryotic expression system,and which laid a foundation for the later biological function research.

Key words: JEV; NS1 gene; cloning; protein expression; protein purification

CLC Number:

S852.65⁺9.6

ZHANG Xiaozhong, LIU Yifan, GU Siying, YU Na, DUAN Yanxiang, LI Youwen. Cloning, Expression and Purification of NS1 Gene of Epidemic Encephalitis Type B[J]. , 2018, 45(8): 2320-2326.

References

[1] 陈溥言.兽医传染病学[M].第6版.北京:中国农业出版社,2015.CHEN F Y.Veterinary Lemology[M].6th Edition.Beijing:China Agriculture Press,2015.(in Chinese)
[2] 李波,毛远丽,貌盼勇.乙型脑炎病毒致病机理及临床诊疗的研究进展[J].国际病毒学杂志,2008,25(2):42-45.LI B,MAO Y L,MAO P Y.The research progress of pathogenic mechanism and clinical diagnosis and treatment of Japanese encephalitis virus[J].International Journal of Virology,2008,25(2):42-45.(in Chinese)
[3] 朱碧波.乙型脑炎病毒诱导miR-15b的表达调控及功能研究[D].武汉:华中农业大学,2014.ZHU B B.Study on the regulation and function of miR-15b during Japanese encephalitis virus infec-tion[D].Wuhan:Huazhong Agricultural University,2014.(in Chinese)
[4] 叶静.乙型脑炎病毒非结构蛋白NS5与宿主蛋白相互作用的研究[D].武汉:华中农业大学,2014.YE J.Study on interaction of Japanese encephalitis virus NS5 protein with host proteins[D].Wuhan:Huazhong Agricultural University,2014.(in Chinese)
[5] CHANG C Y,LI J R,CHEN W Y,et al.Disruption of in vitro endothelial barrier integrity by Japanese encephalitis virus-infected astrocytes[J].Glia,2015,63(11):1915-1932.
[6] CHEN H L,CHANG J K,TANG R B,et al.Current recommendations for the Japanese encephalitis vac-cine[J].Journal of the Chinese Medical Association,2015,78(5):271-275.
[7] LIN Y L,CHEN L K,LIAO C L,et al.Immunization with Japanese encephalitis virus nonstructural protein NS1 elicits protective immunity in mice[J]. Journal of Virology,1998,72(1):191-200.
[8] 陈园生,梁晓峰,王晓军.中国2000-2004年流行性乙型脑炎流行病学特征分析[J].中国计划免疫,2006,12(3):196-198.CHEN Y S,LIANG X F,WANG X J.Epidemiological analysis of Japanese B encephalitis in China during 2000-2004[J].China's Immunization,2006,12(3):196-198.(in Chinese)
[9] GAO N,CHEN W,ZHENG Q,et al.Co-expression of Japanese encephalitis virus prM-E-NS1 antigen with colony-stimulating factor enhances humoral and anti-virus immunity after DNA vaccination[J].Immunology Letters,2010,129(1):23-31.
[10] CHEN S T,LIU R S,WU M F,et al.CLEC5A regulates Japanese encephalitis virus-induced neuroinflammation and lethality[J].PLoS Pathogens,2012,8(4):e1002655.
[11] 马士恒,陈宇萍,王占国.乙型脑炎研究进展[J].中国人兽共患病杂志,2001,17(1):95-97.MA S H,CHEN Y P,WANG Z G.Reviewed of encephalitis B[J].Chinese Journal of Zoonoses,2001,17(1):95-97.(in Chinese)
[12] LIN C W,HUANG H D,SHIU S Y,et al.Functional determinants of NS2B for activation of Japanese encephalitis virus NS3 protease[J].Virus Research, 2007,127(1):88-94.
[13] 胡仲达.猪乙型脑炎病的诊断与防制[J].现代农业科技,2010,39(1):329-330.HU Z D.The pig encephalitis b disease diagnosis and prevention[J].Modern Agricultural Science and Technology,2010,39(1):329-330.(in Chinese)
[14] FAN Y C,CHEN J M,CHIU H C, et al.Partially neutralizing potency against emerging genotype ivirus among children received formalin-inactivated Japanese encephalitis virus vaccine[J].PLoS Neglected Tropical Diseases,2012,6(9):e1834.
[15] CHEN C J,KUO M D,CHIEN L J,et al.RNA-protein interactions:Involvement of NS3,NS5,and 3' noncoding regions of Japanese encephalitis virus genomic RNA[J].Journal of Virology,1997,71(5):3466-3473.
[16] JIANG R,YE J,ZHU B,et al.Roles of TLR3 and RIG-Ⅰ in mediating the inflammatory response in mouse microglia following Japanese encephalitis virus infection[J].Research Journal of Immunology,2014,61(5):1-11.
[17] JIANG Y,ZHU Y,WANG X, et al.Temporal regulation of hif-1 and NF-kappab in hypoxic hepatocarcinoma cells[J].Oncotarget,2015,6(11):9409-9419.
[18] 李金娜,宋书婷,陈宏伟,等.山羊痘病毒毒力因子KLP2的表达及多克隆抗体的制备[J].中国畜牧兽医,2017,44(10):2851-2857.LI J N,SONG S T,CHEN H W,et al.Expression of goatpox virus virulence factor KLP2 and preparation of polyclonal antibody[J].China Animal Husbandry & Veterinary Medicine,2017,44(10):2851-2857.(in Chinese)
[19] 杨洋,李文卉,张念章,等.多头带绦虫蛋白激酶A催化亚基基因的克隆及原核表达[J].中国畜牧兽医,2017,44(10):2875-2880.YANG Y,LI W H,ZHANG N Z, et al.cloning and prokaryotic expression of Long with tapeworm protein kinase A catalytic subunit gene[J].China Animal Husbandry & Veterinary Medicine,2017,44(10):2875-2880.(in Chinese)
[20] 陈慧芹,王小平,罗树红,等.羊口疮病毒蛋白ORFV035的表达、纯化和多克隆抗体的制备[J].生物技术通报,2017,33(7):150-154.CHEN H Q,WANG X P,LUO S H, et al.Sheep aphtha ORFV035 virus protein expression,purification and preparation of polyclonal antibody[J].Biotechnology Bulletin,2017,33(7):150-154.(in Chinese)
[21] 文强,唐明美,陈玲,等.结核分枝杆菌包涵体蛋白Rv3480c的克隆、表达与纯化[J].遵义医学院学报, 2017,40(4):401-409.WEN Q,TANG M M,CHEN L,et al.Cloning,expression and purification of the Rv3480c inclusion body protein of Mycobacterium tuberculosis[J].Journal of Zunyi Medical College,2017,40(4):401-409.(in Chinese)
[22] 唐威华,张景六,王宗阳,等.SDS-PAGE法测定His-tag融合蛋白分子量产生偏差的原因[J].植物生理学报,2000,26(1):64-68.TANG W H,ZHANG J L,WANG Z Y, et al.The cause of deviation made in determining the molecular weight of His-tag fusion proteins by SDS-PAGE[J].Acta Phytophysiologica Sinica,2000,26(1):64-68.(in Chinese)
[23] 章正瑛.生物化学实验指导[M].上海:第二军医大学出版社,2007.ZHANG Z Y.Instruction of Biochemistry Experi-ment[M].Shanghai:Second Military Medical University Press,2007.(in Chinese)

Cloning, Expression and Purification of NS1 Gene of Epidemic Encephalitis Type B

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