鸡马立克病疫苗株CVI988/Rispens meq基因编辑及缺失毒株的构建与鉴定

doi:10.11843/j.issn.0366-6964.2020.08.021

畜牧兽医学报 ›› 2020, Vol. 51 ›› Issue (8): 1970-1976.doi: 10.11843/j.issn.0366-6964.2020.08.021

鸡马立克病疫苗株CVI988/Rispens meq基因编辑及缺失毒株的构建与鉴定

杨森^1,2,3, 滕蔓^2,3, 刘金玲^2,3, 周子誉^1,2,3, 郑鹿平^2,3, 楚钰淑^2,3,4, 丁轲¹, 余祖华^1*, 罗俊^2,3*

1. 河南科技大学动物科技学院, 动物疫病与公共卫生重点实验室, 洛阳 471003;
2. 河南省农业科学院动物免疫学重点实验室, 农业部动物免疫学重点实验室, 河南省动物免疫学重点实验室, 郑州 450002;
3. 河南省农业科学院, 中英禽病国际研究中心, 郑州 450002;
4. 河南农业大学牧医工程学院, 郑州 450002

收稿日期:2020-02-08 出版日期:2020-08-25 发布日期:2020-08-19
通讯作者: 罗俊,主要从事动物病毒学研究,Tel:0371-65056056,E-mail:luojun593@aliyun.com;余祖华,主要从事动物传染病学研究,Tel:0379-64280348,E-mail:yzhd05@163.com
作者简介:杨森(1995-),男,河南驻马店人,硕士,主要从事动物病毒学研究,Tel:0371-65756056,E-mail:896457574@qq.com
基金资助:
国家自然科学基金（U1604232）；中原千人计划-中原基础研究领军人才；河南省重点研发与推广专项（192102110072）；河南省农业科学院杰出青年科技基金（2019JQ01）；河南省农业科学院科研发展专项资金（2019CY012）

Construction of meq Deleted strain by Gene Editing of Marek's Disease Vaccine Strain CVI988/Rispens via the CRISPR/Cas9 System and Identification

YANG Sen^1,2,3, TENG Man^2,3, LIU Jinling^2,3, ZHOU Ziyu^1,2,3, ZHENG Luping^2,3, CHU Yushu^2,3,4, DING Ke¹, YU Zuhua^1*, LUO Jun^2,3*

1. Key Laboratory of Animal Disease and Public Safety, College of Animal Science and Technology, Henan University of Science and Technology, Luoyang 471003, China;
2. Key Laboratory of Animal Immunology, Ministry of Agriculture&Henan Provincial Key Laboratory of Animal Immunology, Henan Academy of Agricultural Sciences, Zhengzhou 450002, China;
3. UK-China Centre of Excellence for Research on Avian Diseases, Henan Academy of Agricultural Sciences, Zhengzhou 450002, China;
4. College of Animal Science and Veterinary Medicine, Henan Agricultural University, Zhengzhou 450002, China

Received:2020-02-08 Online:2020-08-25 Published:2020-08-19

摘要/Abstract

摘要： meq是鸡马立克病病毒（MDV）最重要的致瘤基因，在马立克病（MD）肿瘤发生中发挥关键作用。同时，它在疫苗株和强毒株之间具有明显的序列差异性。本文利用CRISPR/Cas9基因编辑技术，以MDV疫苗株CVI988/Rispens meq基因为靶点，设计合成gRNA，克隆构建pX459-gRNA质粒，转染CEF并感染CVI988/Rispens，然后对meq基因编辑的病毒噬斑进行克隆纯化，经过PCR扩增、测序分析及IFA鉴定，成功构建1株meq基因编辑的缺失毒株CVI988Δmeq-C7，为后续筛选和鉴定抗MD疫苗株MEQ单抗及鉴别诊断研究奠定了基础。

关键词: 马立克病, CVI988/Rispens, meq基因, CRISPR/Cas9, 基因编辑

Abstract: Meq gene is commonly regarded as the main oncogene that plays critical role in the tumorigenesis of Marek's disease (MD). Evolutionarily, there is an obvious sequence difference between the meq genes of vaccine and pathogenic strains with differ virulence. In this study, we have designed the specific guide RNAs (gRNAs) targeting to the meq gene of the vaccine strain CVI988/Rispens, to make the plasmid pX459-gRNA. The CEF cells were transfected with pX459-gRNA plasmids and were infected with CVI988/Rispens virus to produce meq-gene mutated viral particles. Utilizing the CRISPR/Cas9-based gene editing technique, together with the viral plaque purification, PCR analysis, DNA sequencing and indirect immunofluorescence assay (IFA), we successfully generated a meq-deleted mutant named as CVI988Δmeq-C7. Our data provides an important basis for further screening of the monoclonal antibodies against MD vaccine MEQ protein and the development of differential diagnostic reagents.

Key words: Marek's disease, CVI988/Rispens, meq gene, CRISPR/Cas9, gene editing

中图分类号:

S852.659.1

杨森, 滕蔓, 刘金玲, 周子誉, 郑鹿平, 楚钰淑, 丁轲, 余祖华, 罗俊. 鸡马立克病疫苗株CVI988/Rispens meq基因编辑及缺失毒株的构建与鉴定[J]. 畜牧兽医学报, 2020, 51(8): 1970-1976.

YANG Sen, TENG Man, LIU Jinling, ZHOU Ziyu, ZHENG Luping, CHU Yushu, DING Ke, YU Zuhua, LUO Jun. Construction of meq Deleted strain by Gene Editing of Marek's Disease Vaccine Strain CVI988/Rispens via the CRISPR/Cas9 System and Identification[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(8): 1970-1976.

参考文献 30

[1]	SAIF Y M. 禽病学[M]. 苏敬良, 高福, 索勋, 译. 12版. 北京:中国农业出版社, 2012:129-151.SAIF Y M. Diseases of poultry[M]. SU J L, GAO F, SUO X, trans. 12th ed. Beijing:China Agriculture Press, 2012:129-151. (in Chinese)
[2]	DAVISON F, NAIR V. Use of Marek's disease vaccines:could they be driving the virus to increasing virulence?[J]. Expert Rev Vaccines, 2005, 4(1):77-88.
[3]	EBINA H, MISAWA N, KANEMURA Y, et al. Harnessing the CRISPR/Cas9 system to disrupt latent HIV-1 provirus[J]. Sci Rep, 2013, 3:2510.
[4]	SUENAGA T, KOHYAMA M, HIRAYASU K, et al. Engineering large viral DNA genomes using the CRISPR-Cas9 system[J]. Microbiol Immunol, 2014, 58(9):513-522.
[5]	BI Y W, SUN L, GAO D D, et al. High-efficiency targeted editing of large viral genomes by RNA-guided nucleases[J]. PLoS Pathog, 2014, 10(5):e1004090.
[6]	BIERLE C J, ANDERHOLM K M, WANG J B, et al. Targeted mutagenesis of guinea pig cytomegalovirus using CRISPR/Cas9-mediated gene editing[J]. J Virol, 2016, 90(10):6989-6998.
[7]	XU A T, QIN C, LANG Y, et al. A simple and rapid approach to manipulate pseudorabies virus genome by CRISPR/Cas9 system[J]. Biotechnol Lett, 2015, 37(6):1265-1272.
[8]	YUAN M, ZHANG W S, WANG J, et al. Efficiently editing the vaccinia virus genome by using the CRISPR-Cas9 system[J]. J Virol, 2015, 89(9):5176-5179.
[9]	YUEN K S, CHAN C P, WONG N H, et al. CRISPR/Cas9-mediated genome editing of Epstein-Barr virus in human cells[J]. J Gen Virol, 2015, 96(3):626-636.
[10]	LIANG X, SUN L Q, YU T, et al. A CRISPR/Cas9 and Cre/Lox system-based express vaccine development strategy against re-emerging pseudorabies virus[J]. Sci Rep, 2016, 6:19176.
[11]	ZOU Z, HUANG K, WEI Y M, et al. Construction of a highly efficient CRISPR/Cas9-mediated duck enteritis virus-based vaccine against H5N1 avian influenza virus and duck tembusu virus infection[J]. Sci Rep, 2017, 7(1):1478.
[12]	PENG Z Y, OUYANG T, PANG D X, et al. Pseudorabies virus can escape from CRISPR-Cas9-mediated inhibition[J]. Virus Res, 2016, 223:197-205.
[13]	TANG Y D, LIU J T, WANG T Y, et al. Live attenuated pseudorabies virus developed using the CRISPR/Cas9 system[J]. Virus Res, 2016, 225:33-39.
[14]	PAN Q, WANG J, GAO Y L, et al. The natural large genomic deletion is unrelated to the increased virulence of the novel genotype fowl adenovirus 4 recently emerged in China[J]. Viruses, 2018, 10(9):494.
[15]	BORCA M V, HOLINKA L G, BERGGREN K A, et al. CRISPR-Cas9, a tool to efficiently increase the development of recombinant African swine fever viruses[J]. Sci Rep, 2018, 8(1):3154.
[16]	OSTERRIEDER N, KAMIL J P, SCHUMACHER D, et al. Marek's disease virus:from miasma to model[J]. Nat Rev Microbiol, 2006, 4(4):283-294.
[17]	WITTER R L, SCHAT K. Marek's disease[M]//SAIF Y M. Diseases of Poultry. 11th ed. Ames:Iowa State University Press, 2003:407-464.
[18]	SILVA R F, DUNN J R, CHENG H H, et al. A MEQ-deleted Marek's disease virus cloned as a bacterial artificial chromosome is a highly efficacious vaccine[J]. Avian Dis, 2010, 54(2):862-869.
[19]	LI Y P, SUN A J, SU S, et al. Deletion of the meq gene significantly decreases immunosuppression in chickens caused by pathogenic Marek's disease virus[J]. Virol J, 2011, 8:2.
[20]	DE BOER G F, GROENENDAL J E, BOERRIGTER H M, et al. Protective efficacy of Marek's disease virus (MDV) CVI-988 CEF65 clone C against challenge infection with three very virulent MDV strains[J]. Avian Dis, 1986, 30(2):276-283.
[21]	RISPENS B H, VAN VLOTEN H, MASTENBROEK N, et al. Control of Marek's disease in the Netherlands. I. Isolation of an avirulent Marek's disease virus (strain CVI 988) and its use in laboratory vaccination trials[J]. Avian Dis, 1972, 16(1):108-125.
[22]	NAIR V. Evolution of Marek's disease-a paradigm for incessant race between the pathogen and the host[J]. Vet J, 2005, 170(2):175-183.
[23]	SPATZ S J, PETHERBRIDGE L, ZHAO Y G, et al. Comparative full-length sequence analysis of oncogenic and vaccine (Rispens) strains of Marek's disease virus[J]. J Gen Virol, 2007, 88(4):1080-1096.
[24]	ROSS N L J. T-cell transformation by Marek's disease virus[J]. Trends Microbiol, 1999, 7(1):22-29.
[25]	SCHAT K A, BUCKMASTER A, ROSS L J N. Partial transcription map of Marek's disease herpesvirus in lytically infected cells and lymphoblastoid cell lines[J]. Int J Cancer, 1989, 44(1):101-109.
[26]	CHANG K S, OHASHI K, ONUMA M. Suppression of transcription activity of the MEQ protein of oncogenic Marek's disease virus serotype 1(MDV1) by L-MEQ of non-oncogenic MDV1[J]. J Vet Med Sci, 2002, 64(12):1091-1095.
[27]	YAO Y X, BASSETT A, NAIR V. Targeted editing of avian herpesvirus vaccine vector using CRISPR/Cas9 nuclease[J]. Intl J Vaccine Technol, 2016, 1:1-7.
[28]	TANG N, ZHANG Y Y, PEDRERA M, et al. A simple and rapid approach to develop recombinant avian herpesvirus vectored vaccines using CRISPR/Cas9 system[J]. Vaccine, 2018, 36(5):716-722.
[29]	ZHANG Y Y, LUO J, TANG N, et al. Targeted editing of the pp38 gene in Marek's disease virus-transformed cell lines using CRISPR/Cas9 System[J]. Viruses, 2019, 11(5):391.
[30]	ZHANG Y Y, TANG N, LUO J, et al. Marek's disease virus-encoded microRNA 155 ortholog critical for the induction of lymphomas is not essential for the proliferation of transformed cell lines[J]. J Virol, 2019, 93(17):e00713-19.

鸡马立克病疫苗株CVI988/Rispens meq基因编辑及缺失毒株的构建与鉴定

Construction of meq Deleted strain by Gene Editing of Marek's Disease Vaccine Strain CVI988/Rispens via the CRISPR/Cas9 System and Identification

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]	张晨俭, 李隐侠, 丁强, 刘伟佳, 王慧利, 何南, 吴家顺, 曹少先. CRISPR/Cas9技术高效制备山羊SOCS2基因编辑胚胎[J]. 畜牧兽医学报, 2024, 55(1): 129-141.
[2]	费晓钰, 石超群, 刘雪明, 苏峰, 姜运良. CRISPR/Cas9系统介导的猪MRC1修饰基因降低PCV2复制的研究[J]. 畜牧兽医学报, 2023, 54(3): 934-946.
[3]	刘铃, 王丹丹, 崔凯, 马月辉, 蒋琳. 猪繁殖与呼吸综合征抗病育种研究进展[J]. 畜牧兽医学报, 2023, 54(2): 434-442.
[4]	余祖华, 贾艳艳, 何雷, 廖成水, 李静, 魏颖, 陈建, 陈松彪, 尚珂, 丁轲. gga-miR-155对MDCC-MSB1细胞转录组的影响[J]. 畜牧兽医学报, 2023, 54(2): 663-672.
[5]	陈俊贞, 权冉, 付强, 葛丽娟, 袁圆圆, 张成远, 李建林, 史慧君. 热休克蛋白HSP90B1影响牛病毒性腹泻病毒复制的研究[J]. 畜牧兽医学报, 2023, 54(2): 683-693.
[6]	李林燕, 滕蔓, 刘金玲, 郑鹿平, 柴书军, 丁轲, 余祖华, 罗俊. 马立克病病毒单克隆抗体库构建及gB蛋白特异性单抗的筛选与鉴定[J]. 畜牧兽医学报, 2023, 54(11): 4712-4723.
[7]	张硕, 周雨潇, 吴海波, 索伦. 长效CRISPR/Cas9基因编辑结局的动态追踪研究[J]. 畜牧兽医学报, 2023, 54(10): 4196-4208.
[8]	邓敏儿, 李娜, 郭亚琼, 冯耀宇, 肖立华. CRISPR/Cas9系统在寄生原虫基因编辑中的应用[J]. 畜牧兽医学报, 2023, 54(1): 69-79.
[9]	赵为民, 王慧利, 曹少先, 郭日红, 王泽平, 陈哲, 徐奎, 付言峰, 李碧侠, 任守文, 程金花. 猪CD163基因的单碱基编辑研究[J]. 畜牧兽医学报, 2022, 53(4): 1041-1050.
[10]	李兆龙, 张惠芳, 丰志华, 方舟. 携带CRISPR/Cas9的重组腺联病毒对伪狂犬病病毒感染小鼠的治疗效应[J]. 畜牧兽医学报, 2022, 53(3): 834-846.
[11]	邹惠影, 李俊良, 朱化彬. 引导编辑系统的研究与应用进展[J]. 畜牧兽医学报, 2022, 53(11): 3721-3730.
[12]	罗俊, 刘金玲, 郑鹿平, 罗琴, 滕蔓. 家禽疱疹病毒CRISPR/Cas9基因编辑最新研究进展[J]. 畜牧兽医学报, 2022, 53(10): 3335-3344.
[13]	王沛, 王萌, 李婷婷, 郑晓楠, 梁勤立, 陈小庆. 弓形虫4个假定蛋白基因缺失株的构建及其基本生物功能学研究[J]. 畜牧兽医学报, 2022, 53(10): 3598-3608.
[14]	李琛, 何文峰, 赵丽娜, 凡启, 杨国庆, 刘慧敏. PK-15细胞的ISG15基因敲除促进PRV的复制[J]. 畜牧兽医学报, 2022, 53(10): 3621-3630.
[15]	王圣楠, 王丹丹, 田文杰, 浦亚斌, 潘登科, 邢向阳, 马月辉, 蒋琳. ZBED6基因对巴马香猪脾发育的作用机制分析[J]. 畜牧兽医学报, 2021, 52(9): 2394-2405.