新城疫病毒基因Ⅲ型强毒JS/7/05/Ch的拯救

›› 2013, Vol. 40 ›› Issue (10): 37-41.

新城疫病毒基因Ⅲ型强毒JS/7/05/Ch的拯救

宋庆庆, 朱杰, 丁平云, 段志强, 屠颉, 刘晓文, 刘秀梵

扬州大学农业部畜禽传染病学重点开放实验室, 江苏扬州 225009

收稿日期:2013-04-15 出版日期:2013-10-20 发布日期:2013-12-19
通讯作者: 刘秀梵 E-mail:xfliu@yzu.edu.cn
作者简介:宋庆庆(1985-),男,山东人,博士生,研究方向:分子病原。
基金资助:
国家自然科学基金(31172338);现代农业产业技术体系建设专项资金(nycytx-41-G07);公益性行业(农业)专项经费(201003012)。

Rescue of Genotype Ⅲ Velogenic Newcastle Disease Virus Strain JS/7/05/Ch

SONG Qing-qing, ZHU Jie, DING Ping-yun, DUAN Zhi-qiang, TU Jie, LIU Xiao-wen, LIU Xiu-fan

Key Laboratory of Animal Infectious Diseases, Ministry of Agriculture, Yangzhou University, Yangzhou 225009, China

Received:2013-04-15 Online:2013-10-20 Published:2013-12-19

摘要/Abstract

摘要： 新城疫病毒Ⅰ系苗Mukteswar株为中等毒力,野外分离株JS/7/05/Ch的基因组核苷酸与其具有高于99%的相似性,但其毒力却明显强于前者。为了给基因Ⅲ型NDV的遗传进化机制研究提供基础,本研究成功构建了JS/7/05/Ch株的反向遗传平台。首先根据GenBank上公布的基因Ⅲ型新城疫病毒JS/7/05/Ch株基因组全序列设计并合成8对引物,RT-PCR扩增目的片段后,分别依次亚克隆至pCR2.1载体中构建含有JS/7/05/Ch全长基因组cDNA的克隆质粒pJS/7/05/Ch,然后再通过特异性酶切位点将JS/7/05株全长基因组cDNA转移到TVT7R(0.0)转录载体中,成功构建出含JS/7/05/Ch株基因组全长cDNA的转录质粒pTVT/JS705。然后将该质粒与3个辅助质粒pCI-NP、pCI-P和pCI-L共转染BSR-T7/5细胞,转染60 h后将该细胞及其上清接种鸡胚;血凝(HA)试验和RT-PCR结果表明JS/7/05/Ch株新城疫病毒被成功拯救。

关键词: 新城疫病毒; 基因Ⅲ型; 反向遗传技术; 拯救

Abstract: The genome of JS/7/05/Ch isolate shared more than 99% nucleotide identity with that of Mukteswar strain. However, the pathogenicity of JS/7/05/Ch was much stronger than that of Mukteswar strain. In order to provide a good foundation for the further related research, we built the rescue system of JS/7/05/Ch in this study. Based on the genomic sequence of JS/7/05/Ch strain, eight pairs of primers were designed to amplify the genomic fragments by RT-PCR and cloned into pCR2.1 vector, to construct the plasmid pJS/7/05/Ch which contained the full-length cDNA of NDV. Then the full-length cDNA was transferred to TVT7R(0.0) vector to construct full-length NDV infectious clone, pTVT/JS705, using specific enzymes. The infectious clone together with three helper plasmids (pCI-NP, pCI-P and pCI-L) were cotransfected into BSR-T7/5 cell, and the transfection supernatant was inoculated into SPF embryonated eggs to rescue the virus. The results of HA and RT-PCR indicated that JS/7/05/Ch strain of NDV was successfully rescued.

Key words: Newcastle disease virus; genotype Ⅲ; reverse genetics technique; rescue

中图分类号:

S852.65

宋庆庆, 朱杰, 丁平云, 段志强, 屠颉, 刘晓文, 刘秀梵. 新城疫病毒基因Ⅲ型强毒JS/7/05/Ch的拯救[J]. , 2013, 40(10): 37-41.

SONG Qing-qing, ZHU Jie, DING Ping-yun, DUAN Zhi-qiang, TU Jie, LIU Xiao-wen, LIU Xiu-fan. Rescue of Genotype Ⅲ Velogenic Newcastle Disease Virus Strain JS/7/05/Ch[J]. , 2013, 40(10): 37-41.

参考文献

1 仇旭升, 孙庆, 姚春峰, 等. 两株基因Ⅲ型强毒新城疫的全基因组测序及其与Ⅰ系苗的亲缘性分析[J]. 微生物学报, 2009, 49(3):302~308.
2 石跃, 刘怀然, 刘培欣,等. 一株野鸭源基因Ⅲ型新城疫病毒主要生物学特性及基因组序列测定分析[J]. 中国预防兽医学报, 2011, 33(5):391~394.
3 刘玉良, 吴艳涛, 黄勇, 等. 鹅源新城疫病毒NP、P和L基因的克隆与P基因的表达鉴定[J]. 微生物学通报, 2004, 31(2):37~40.
4 孙丰廷, 郑杰, 张发明, 等. 几株鸡新城疫病毒的分离鉴定及其基因型分析[J]. 中国畜牧兽医, 2012, 39(8):76~79.
5 胡顺林, 吴艳涛, 刘文博, 等. 鹅源新城疫病毒单克隆抗体的研制及其与不同NDV毒株的反应性[J]. 中国兽医科技,2005,35(5):341~345.
6 胡顺林, 孙庆, 吴艳涛, 等. 用反向遗传技术致弱基因Ⅶd型鹅源新城疫病毒ZJ1株[J]. 微生物学通报, 2007, 47(2):197~200.
7 姚春峰, 仇旭升, 刘文博, 等. 新城疫分离毒HN蛋白的抗原性初步分析及分子特性研究[J]. 微生物学通报, 2008, 35(1):87~93.
8 Aldous E W, Alexander D J. Detection and differentiation of Newcastle disease virus (avian paramyxovirus type 1). Avian Pathol, 2001, 30(2): 117~128.
9 Aldous E W, Mynn J K, Banks J, et al. A molecular epidemiological study of avian paramyxovirus type 1 (Newcastle disease virus) isolates by phylogenetic analysis of a partial nucleotide sequence of the fusion protein gene. Avian Pathol, 2003, 32(3): 239~256.
10 De Leeuw O, Peeters B. Complete nucleotide sequence of Newcastle disease virus: Evidence for the existence of a new genus within the subfamily Paramyxovirinae. J Gen Virol, 1999, 80 (Pt1): 131~136.
11 Duncan R, Murphy F A, Mirkovic R R. Characterization of a novel syncytium-inducing baboon reovirus. Virology, 1995, 212(2): 752~756.
12 Iyer S G, Dobson N. A successful method of immunization against Newcastle disease of fowls. Vet Rec, 1940, 52: 891~894.
13 Johnson K N, Zeddam J L, Ball L A. Characterization and construction of functional cDNA clones of Pariacoto virus, the first alphanodavirus isolated outside Australasia. J Virol, 2000, 74(11): 5123~5132.
14 Kattenbelt J A, Stevens M P, Gould A R. Sequence variation in the Newcastle disease virus genome. Virus Res, 2006, 116(1~2): 168~184.
15 Kim L M, King D J, Curry P E, et al. Phylogenetic diversity among low-virulence Newcastle disease viruses from waterfowl and shorebirds and comparison of genotype distributions to those of poultry-origin isolates. J Virol, 2007, 81(22): 12641~12653.
16 Liu X F, Wan H Q, Ni X X, et al. Pathotypical and genotypical characterization of strains of Newcastle disease virus isolated from outbreaks in chicken and goose flocks in some regions of China during 1985—2001. Arch Virol, 2003, 148(7): 1387~1403.
17 Miller P J, Decanini E L, Afonso C L. Newcastle disease: Evolution of genotypes and the related diagnostic challenges. Infect Genet Evol, 2010, 10(1): 26~35.
18 Peeters B P, de Leeuw O S, Koch G, et al. Rescue of Newcastle disease virus from cloned cDNA: Evidence that cleavability of the fusion protein is a major determinant for virulence. J Virol, 1999, 73(6): 5001~5009.
19 Romer-Oberdorfer A, Mundt E, Mebatsion T, et al. Generation of recombinant lentogenic Newcastle disease virus from cDNA. J Gen Virol, 1999, 80 (Pt11): 2987~2995.
20 Wehmann E, Czegledi A, Werner O, et al. Occurrence of genotypes Ⅳ, Ⅴ, Ⅵ and Ⅶ a in Newcastle disease outbreaks in Germany between 1939 and 1995. Avian Pathol, 2003, 32(2): 157~163.
21 Wu S, Wang W, Yao C, et al. Genetic diversity of Newcastle disease viruses isolated from domestic poultry species in Eastern China during 2005—2008. Arch Virol, 2011, 156(2):253~261.

[1]	郗珊珊, 贾伟娟, 何云江, 孟庆磊, 陈云娇, 王学理. 新城疫病毒样颗粒研究进展[J]. 中国畜牧兽医, 2022, 49(4): 1507-1515.
[2]	商雨, 李林涛, 李丽, 徐英英, 冯贺龙, 汪宏才, 张蓉蓉, 曾哲, 王红琳, 罗青平, 邵华斌, 温国元. 表达禽腺病毒血清4型Fiber2蛋白的重组耐热新城疫病毒的构建及免疫效果评估[J]. 中国畜牧兽医, 2022, 49(2): 650-659.
[3]	刘欢, 张蒙蒙, 崔一芳, 刘静, 张靖菊, 陈登金, 李秀波, 李存, 徐飞. 复方季铵盐泡沫消毒剂成泡性能的优化及对病毒的杀灭效果评价[J]. 中国畜牧兽医, 2022, 49(12): 4866-4877.
[4]	李丽, 王国康, 商雨, 徐英英, 冯贺龙, 汪宏才, 张蓉蓉, 曾哲, 罗青平, 邵华斌, 曾驰, 温国元. 基于F和HN蛋白的基因Ⅶ型新城疫病毒嵌合疫苗的构建及免疫效力评估[J]. 中国畜牧兽医, 2022, 49(11): 4346-4355.
[5]	王淼, 陈雪阳, 王兴明, 梁雄燕, 杨玉莹. 两株ALV-K重组病毒感染性克隆的构建及病毒拯救[J]. 中国畜牧兽医, 2022, 49(10): 3764-3770.
[6]	曾建宇, 董振远, 贾文凤, 张国中, 薛佳. 新城疫病毒M蛋白单克隆抗体的制备与鉴定[J]. 中国畜牧兽医, 2021, 48(9): 3423-3431.
[7]	殷娟斌, 周亚花, 殷相平, 张志东. 携带绿色荧光蛋白基因重组猫泛白细胞减少症病毒的拯救[J]. 中国畜牧兽医, 2020, 47(6): 1853-1860.
[8]	熊晓妍, 覃振斌, 莫红芳, 何东贤, 虞霖田, 许家荣. VA5免疫增强剂对鸽新城疫病毒灭活疫苗的增效作用研究[J]. 中国畜牧兽医, 2020, 47(12): 4032-4040.
[9]	李佳佳, 宋建领. 一株蛋鸡新城疫病毒的分离鉴定及全基因组序列分析[J]. 中国畜牧兽医, 2020, 47(1): 221-228.
[10]	布合丽倩穆·依明, 赛福丁·阿不拉, 艾克拜尔·依明, 胡安古丽·努尔别克, 阿克珠利·努尔包, 李昊. 中药牛皮消多糖体外抗新城疫病毒作用的研究[J]. 中国畜牧兽医, 2019, 46(9): 2737-2744.
[11]	董炳梅, 孙培娇, 苗立中, 沈志强, 韩文瑜. 鸡新城疫病毒LaSota株在BHK-21细胞上增殖条件的优化[J]. 《中国畜牧兽医》, 2019, 46(3): 858-864.
[12]	许丽文, 王秋玲, 王芳芳, 刘诚刚, 马得莹. 人工感染鸭源新城疫病毒对绍鸭β-防御素和细胞因子基因表达的影响[J]. 《中国畜牧兽医》, 2019, 46(2): 536-547.
[13]	孙化露, 毕云英, 于泽坤, 李思菲, 刘阳, 李明义, 崔现兰. 1株基因Ⅶ型新城疫病毒的生物学特性研究及全基因组序列分析[J]. 《中国畜牧兽医》, 2018, 45(10): 2840-2848.
[14]	刘文俊, 黄运茂, 阳佑天, 许丹宁, 曹楠, 周德荣, 田允波. 鹅新城疫病毒RT-LAMP可视化检测方法的建立[J]. 《中国畜牧兽医》, 2018, 45(1): 22-31.
[15]	张慧敏, 木哈买提江·铁格斯, 史军, 吴桂灵, 古丽扎提·阿地理, 加尔肯, 苏战强, 冉多良, 沙依兰·卡依扎, 兰玲, 姚刚, 刘建华. 一株新疆野鸟源新城疫病毒的分离鉴定、F基因克隆及遗传进化分析[J]. 《中国畜牧兽医》, 2017, 44(9): 2755-2760.