基因Ⅰ型乙型脑炎病毒病毒样颗粒的制备及其对小鼠的免疫原性分析

doi:10.11843/j.issn.0366-6964.2020.022

畜牧兽医学报 ›› 2020, Vol. 51 ›› Issue (6): 1391-1398.doi: 10.11843/j.issn.0366-6964.2020.022

基因Ⅰ型乙型脑炎病毒病毒样颗粒的制备及其对小鼠的免疫原性分析

施磊¹, 周亚红¹, 田占成^1*, 高闪电¹, 独军政¹, 关贵全¹, 刘光远¹, 罗建勋¹, 殷宏^1,2*

1. 中国农业科学院兰州兽医研究所家畜疫病病原生物学国家重点实验室甘肃省动物寄生虫病重点实验室, 兰州 730046;
2. 江苏省动物重要疫病与人兽共患病防控协同创新中心, 扬州 225009

收稿日期:2019-12-03 出版日期:2020-06-25 发布日期:2020-06-23
通讯作者: 田占成,主要从事动物寄生虫与分子生物学研究,E-mail:tianzhancheng@caas.cn;殷宏,主要从事兽医微生物及其分子生物学研究,E-mail:yinhong@caas.cn
作者简介:施磊(1980-),男,甘肃张掖人,硕士生,主要从事虫媒传播病毒分子病毒学研究,E-mail:1040717960@qq.com
基金资助:
国家自然科学基金项目（31201899）；中国农业科学院农业科技创新工程项目（CAAS-ASTIP-2016-LVRI）

Generation of Genotype Ⅰ Japanese Encephalitis Virus Virus-like Particles and Evaluation of Its Immunogenicity in Mice

SHI Lei¹, ZHOU Yahong¹, TIAN Zhancheng^1*, GAO Shandian¹, DU Junzheng¹, GUAN Guiquan¹, LIU Guangyuan¹, LUO Jianxun¹, YIN Hong^1,2*

1. State Key Laboratory of Veterinary Etiological Biology/Key Laboratory of Veterinary Parasitology of Gansu Province, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, China;
2. Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou 225009, China

Received:2019-12-03 Online:2020-06-25 Published:2020-06-23

摘要/Abstract

摘要： 降低猪场的流行性乙型脑炎（Japanese encephalitis，JE）感染率对于阻断人的JE暴发至关重要，鉴于我国JE防控形势的紧迫性和现有基因Ⅲ型商品化乙型脑炎病毒疫苗对部分基因Ⅰ型乙型脑炎病毒（genotype Ⅰ Japanese encephalitis virus，GⅠ JEV）流行毒株免疫保护的不确定性，急需研制安全高效、成本低廉的新型猪用GI JEV疫苗。本研究在杆状病毒载体pFastBac^TM多克隆酶切位点插入GⅠ JEV的prME基因表达盒，测序验证后的阳性重组质粒转染DH10Bac^TM感受态细胞后获得Bacmid-prME重组杆粒，进一步将其转染至Sf9昆虫细胞后，获得Bac-prME重组杆状病毒。Western blot、间接免疫荧光及电镜观察GⅠ JEV病毒样颗粒组成蛋白表达及病毒样颗粒形成情况，通过小鼠免疫试验初步评价GⅠ JEV病毒样颗粒的免疫原性。Sf9昆虫细胞中prME基因表达盒编码的蛋白得到高效表达，且组装形成病毒样颗粒。病毒样颗粒免疫原性评估结果表明：GⅠ JEV病毒样颗粒可诱导免疫小鼠产生高滴度的乙型脑炎病毒中和抗体。本研究利用杆状病毒昆虫细胞表达系统获得了GⅠ JEV病毒样颗粒，为研制安全高效的乙型脑炎病毒病毒样颗粒亚单位疫苗提供物质基础。

关键词: 乙型脑炎病毒, 杆状病毒, 病毒样颗粒, 免疫原性

Abstract: Decreasing the circulation of the Japanese encephalitis virus (JEV) in pigs is critical for blocking JEV infection in human. In view of the urgency of JE prevention and control in China and the uncertainty of the commercial Genotype Ⅲ JEV attenuated vaccine to the immuno-protection of partial Genotype Ⅰ JEV (GⅠ JEV) endemic strains, it is urgent to develop a safe, effective and low-cost GⅠ JEV vaccine for pigs. The expression cassette of JEV prME gene was cloned into the baculovirus shuttle vector to generate a recombinant bacmid (Bacmid-prME), the recombinant baculovirus was produced by transfecting Sf9 cells with Bacmid-prME. The accurate expression of the recombinant prME protein was confirmed by Western blot and immunofluorescence analysis, and the formation of Virus-Like Particles (VLPs) was verified through observing under Electronic microscopy. The immunogenicity of GⅠ JEV VLPs was further evaluated in mice. The recombinant prME protein was efficiently expressed in Sf9 cells and assembled into VLPs. GⅠ JEV VLPs induced the higher neutralizing antibody titer in mice. In our study, GⅠ JEV VLPs obtained in the baculovirus expression system provides a material basis for the development of a safe and effective JEV subunit vaccine.

Key words: Japanese encephalitis virus, baculovirus, virus-like particles, immunogenicity

中图分类号:

S852.659.6

施磊, 周亚红, 田占成, 高闪电, 独军政, 关贵全, 刘光远, 罗建勋, 殷宏. 基因Ⅰ型乙型脑炎病毒病毒样颗粒的制备及其对小鼠的免疫原性分析[J]. 畜牧兽医学报, 2020, 51(6): 1391-1398.

SHI Lei, ZHOU Yahong, TIAN Zhancheng, GAO Shandian, DU Junzheng, GUAN Guiquan, LIU Guangyuan, LUO Jianxun, YIN Hong. Generation of Genotype Ⅰ Japanese Encephalitis Virus Virus-like Particles and Evaluation of Its Immunogenicity in Mice[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(6): 1391-1398.

参考文献 30

[1]	CAMPBELL G L, HILLS S L, FISCHER M, et al. Estimated global incidence of Japanese encephalitis:a systematic review[J]. Bull World Health Organ, 2015, 89(10):766-774E.
[2]	VAN DEN HURK A F, RITCHIE S A, JOHANSEN C A, et al. Domestic pigs and Japanese encephalitis virus infection, Australia[J]. Emerg Infect Dis, 2014, 14(11):1736-1738.
[3]	MIN J G, XUE M. Progress in studies on the overwintering of the mosquito Culex tritaeniorhynchus[J]. Southeast Asian J Trop Med Public Health, 1996, 27(4):810-817.
[4]	SOLOMON T, VAUGHN D W. Pathogenesis and clinical features of Japanese encephalitis and West Nile virus infections[J]. Curr Top Microbiol Immunol, 2002, 267:171-194.
[5]	HEGDE N R, GORE M M. Japanese encephalitis vaccines:Immunogenicity, protective efficacy, effectiveness, and impact on the burden of disease[J]. Hum Vaccin Immunother, 2017, 13(6):1320-1337.
[6]	成依依, 周红宁. 我国流行性乙型脑炎病毒基因型研究进展[J]. 中国病原生物学杂志, 2018, 13(12):1413-1419.CHENG Y Y, ZHOU H N. Advances in the study of Japanese encephalitis virus genotyping in China[J]. Journal of Pathogen Biology, 2018, 13(12):1413-1419. (in Chinese)
[7]	PARANJPE S, BANERJEE K. Phylogenetic analysis of the envelope gene of Japanese encephalitis virus[J]. Virus Res, 1996, 42(1-2):107-117.
[8]	GAO X Y, LIU H, LI M H, et al. Insights into the evolutionary history of Japanese encephalitis virus (JEV) based on whole-genome sequences comprising the five genotypes[J]. Virol J, 2015, 12:43.
[9]	GAO X Y, LIU H, WANG H Y, et al. Southernmost Asia is the source of Japanese encephalitis virus (genotype 1) diversity from which the viruses disperse and evolve throughout Asia[J]. PLoS Negl Trop Dis, 2013, 7(9):e2459.
[10]	PAN X L, LIU H, WANG H Y, et al. Emergence of genotype I of Japanese encephalitis virus as the dominant genotype in Asia[J]. J Virol, 2011, 85(19):9847-9853.
[11]	LIU X Y, YU Y X, LI M G, et al. Study on the protective efficacy of SA14-14-2 attenuated Japanese encephalitis against different JE virus isolates circulating in China[J]. Vaccine, 2011, 29(11):2127-2130.
[12]	WEI J C, WANG X, ZHANG J J, et al. Partial cross-protection between Japanese encephalitis virus genotype Ⅰ and Ⅲ in mice[J]. PLoS Negl Trop Dis, 2019, 13(8):e0007601.
[13]	CAO L, FU S H, GAO X Y, et al. Low protective efficacy of the current Japanese encephalitis vaccine against the emerging genotype 5 Japanese Encephalitis virus[J]. PLoS Negl Trop Dis, 2016, 10(5):e0004686.
[14]	DONALDSON B, LATEEF Z, WALKER G F, et al. Virus-like particle vaccines:immunology and formulation for clinical translation[J]. Expert Rev Vaccines, 2018, 17(9):833-849.
[15]	张艳芳, 杜瑞坤, 黄少梅, 等. 乙型脑炎病毒样颗粒疫苗的制备及其免疫保护效率的初步评价[J]. 病毒学报, 2016, 32(2):150-155.ZHANG Y F, DU R K, HUANG S M, et al. Generation of Japanese encephalitis virus-like particle vaccine and preliminary evaluation of its protective efficiency[J]. Chinese Journal of Virology, 2016, 32(2):150-155. (in Chinese)
[16]	HUA R H, LI Y N, CHEN Z S, et al. Generation and characterization of a new mammalian cell line continuously expressing virus-like particles of Japanese encephalitis virus for a subunit vaccine candidate[J]. BMC Biotechnol, 2014, 14:62.
[17]	于瑞明, 田占成, 独军政, 等. 基因Ⅰ型乙脑病毒EDⅢ结构域蛋白的原核表达及其抗体制备与鉴定[J]. 中国兽医科学, 2017, 47(12):1475-1480.YU R M, TIAN Z C, DU J Z, et al. Identification of EDⅢ protein from the genotype Ⅰ Japanese encephalitis virus[J]. Veterinary Science in China, 2017, 47(12):1475-1480. (in Chinese)
[18]	周亚红, 田占成, 于瑞明, 等. IFN-λ1作为乙型脑炎病毒DNA疫苗佐剂的免疫效应研究[J]. 中国兽医科学, 2019, 49(7):841-846.ZHOU Y H, TIAN Z C, YU R M, et al. Immune effect of IFN-λ1 as adjuvant for Japanese encephalitis virus DNA vaccine[J]. Chinese Veterinary Science, 2019, 49(7):841-846. (in Chinese)
[19]	HSU S T, CHANG L C, LIN S Y, et al. The effect of vaccination with a live attenuated strain of Japanese encephalitis virus on stillbirths in swine in Taiwan[J]. Bull World Health Organ, 1972, 46(4):465-471.
[20]	FAN Y C, CHIU H C, CHEN L K, et al. Formalin inactivation of Japanese encephalitis virus vaccine alters the antigenicity and immunogenicity of a neutralization epitope in envelope protein domain III[J]. PLoS Neg Trop Dis, 2015, 9(10):e0004167.
[21]	冯瑶, 岳辉喜, 刘洪明, 等. 基因Ⅰ型猪乙型脑炎灭活疫苗免疫原性评价[J]. 畜牧兽医学报, 2017, 48(5):889-895.FENG Y, YUE H X, LIU H M, et al. The immunogenicity evaluation of inactivated vaccines of swine Japanese encephalitis virus genetype I[J]. Acta Veterinaria et Zootechnica Sinica, 2017, 48(5):889-895. (in Chinese)
[22]	MANSFIELD K L, HERNÁNDEZ-TRIANA L M, BANYARD A C, et al. Japanese encephalitis virus infection, diagnosis and control in domestic animals[J]. Vet Microbiol, 2017, 201:85-92.
[23]	ZLATKOVIC J, STIASNY K, HEINZ F X. Immunodominance and functional activities of antibody responses to inactivated West Nile virus and recombinant subunit vaccines in mice[J]. J Virol, 2011, 85(5):1994-2003.
[24]	KOJIMA A, YASUDA A, ASANUMA H, et al. Stable high-producer cell clone expressing virus-like particles of the Japanese encephalitis virus e protein for a second-generation subunit vaccine[J]. J Virol, 2003, 77(16):8745-8755.
[25]	HEINZ F X, ALLISON S L. Structures and mechanisms in flavivirus fusion[J]. Adv Virus Res, 2000, 55:231-269.
[26]	KONISHI E, FUJII A, MASON P W. Generation and characterization of a mammalian cell line continuously expressing Japanese encephalitis virus subviral particles[J]. J Virol, 2001, 75(5):2204-2212.
[27]	YAMAJI H, SEGAWA M, NAKAMURA M, et al. Production of Japanese encephalitis virus-like particles using the baculovirus-insect cell system[J]. J Biosci Bioeng, 2012, 114(6):657-662.
[28]	GASTAMINZA P, DRYDEN K A, BOYD B, et al. Ultrastructural and biophysical characterization of hepatitis C virus particles produced in cell culture[J]. J Virol, 2010, 84(21):10999-11009.
[29]	KUHN R J, ZHANG W, ROSSMANN M G, et al. Structure of dengue virus:implications for flavivirus organization, maturation, and fusion[J]. Cell, 2002, 108(5):717-725.
[30]	REN X J, FU S H, DAI P F, et al. Pigsties near dwellings as a potential risk factor for the prevalence of Japanese encephalitis virus in adult in Shanxi, China[J]. Infect Dis Poverty, 2017, 6(1):100.

基因Ⅰ型乙型脑炎病毒病毒样颗粒的制备及其对小鼠的免疫原性分析

Generation of Genotype Ⅰ Japanese Encephalitis Virus Virus-like Particles and Evaluation of Its Immunogenicity in Mice

RichHTML

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献 30

相关文章 15

编辑推荐

Metrics

本文评价

[1]	程天印, 吴聪颖, 刘雨珂, 段德勇. 褐黄血蜱HSP70-b2及其类14-Mer肽的作用研究[J]. 畜牧兽医学报, 2023, 54(6): 2570-2580.
[2]	刘青, 吴少鹏, 石彬, 邵红霞, 钱琨, 叶建强, 秦爱建. 鸡CTLA-4蛋白在昆虫细胞中的表达及其单克隆抗体的制备[J]. 畜牧兽医学报, 2023, 54(6): 2596-2604.
[3]	李硕, 张韵, 白满元, 赵瑞翀, 宋河涛, 穆素雨, 滕志东, 董虎, 马娥宁, 孙世琪, 郭慧琛, 尹双辉. 生物矿化的口蹄疫病毒样颗粒疫苗的免疫原性评价[J]. 畜牧兽医学报, 2023, 54(4): 1598-1607.
[4]	张家祺, 贾浠宁, 周群, 宋鑫, 朱晨曦, 李格格, 张斌. 基于E2蛋白BVDV-1病毒样颗粒的构建及对小鼠的免疫效力评估[J]. 畜牧兽医学报, 2023, 54(12): 5143-5153.
[5]	李畅, 周平, 温平, 杨克礼, 刘威, 郭锐, 刘泽文, 袁芳艳, 高婷, 宋浩菲, 田永祥, 周丹娜. 一株猪圆环病毒2d基因型毒株的全基因组序列及其衣壳蛋白的小鼠免疫原性分析[J]. 畜牧兽医学报, 2023, 54(10): 4289-4299.
[6]	岳亚辉, 邢小勇, 武小椿, 温峰琴, 张宏燕, 龙翠琴, 张立, 马海云, 包世俊. 滑液支原体WVU1853株热不稳定延伸因子的表达及黏附特性分析[J]. 畜牧兽医学报, 2022, 53(2): 520-528.
[7]	李亚军, 茹毅, 郝荣增, 蒋成辉, 王伟, 张越, 张贵才, 刘华南, 卢炳州, 杨洋, 陶世宇, 杨锐, 宋向东, 陈娇, 余四九, 郑海学. CHO细胞表达牛病毒性腹泻病毒E2蛋白及免疫原性分析[J]. 畜牧兽医学报, 2022, 53(12): 4315-4324.
[8]	韩伟建, 张俊娟, 张义明, 王家鑫, 李丽敏. 骨髓源肥大细胞通过甘露糖受体识别FMDV-VLPs的细胞因子应答[J]. 畜牧兽医学报, 2022, 53(11): 3917-3926.
[9]	朱静静, 戴政列, 汪涵, 李向臣, 赵阿勇, 周晓龙, 杨松柏. 乙型脑炎病毒感染PK15细胞的lncRNA差异表达谱分析[J]. 畜牧兽医学报, 2022, 53(1): 272-281.
[10]	刘君雯, 吴琼娟, 邢刚, 占松鹤, 魏建忠, 孙裴, 刘雪兰, 李郁. 猪丹毒丝菌CbpB蛋白对小鼠的免疫效果分析[J]. 畜牧兽医学报, 2021, 52(6): 1689-1699.
[11]	刘如月, 范京惠, 刘涛, 苑军辉, 李清艳, 翟向和, 左玉柱. 携带猪流行性腹泻病毒S1抗原表位的乙肝核心抗原病毒样颗粒的构建与鉴定[J]. 畜牧兽医学报, 2021, 52(6): 1760-1764.
[12]	陈胜利, 郝华芳, 季文恒, 储岳峰. 动物支原体相关蛋白的免疫原性研究进展[J]. 畜牧兽医学报, 2021, 52(5): 1230-1237.
[13]	吕芬芬, 马晓慧, 汪丽, 马陈, 张宝江, 苏艳. 马链球菌马亚种3种抗原蛋白对小鼠免疫的免疫原性分析[J]. 畜牧兽医学报, 2021, 52(3): 752-762.
[14]	杜加茹, 陈浩南, 王方雨, 张运尚, 樊剑鸣. 乙型脑炎病毒感染小鼠原代神经元细胞的miRNA表达谱差异分析[J]. 畜牧兽医学报, 2020, 51(9): 2319-2323.
[15]	周晓慧, 肖景景, 张鑫宇, 张泉, 夏晓莉, 孙怀昌. 非洲猪瘟病毒强免疫原性重组CD2v抗原的制备与初步应用[J]. 畜牧兽医学报, 2020, 51(10): 2472-2480.