[1]刘娅梅,乔永峰,顾一奇,等. 猪源伪狂犬病病毒变异株AH02LA株细菌人工染色体的构建与鉴定[J].中国预防兽医学报,2018,(10):880-885.[doi:10.3969/j.issn.1008-0589.201606026]
 LIU Ya-mei,QIAO Yong-feng,GU Yi-qi,et al. Construction and characterization of bacterial artificial chromosome of a pseudorabies virus strain AH02LA isolated from a dead piglet[J].Chinese journal of preventive veterinary medicine,2018,(10):880-885.[doi:10.3969/j.issn.1008-0589.201606026]
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猪源伪狂犬病病毒变异株AH02LA株细菌人工染色体的构建与鉴定

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《中国预防兽医学报》[ISSN:1008-0589/CN:23-1417/S]

卷:
期数:
2018年10
页码:
880-885
栏目:
出版日期:
2018-11-15

文章信息/Info

Title:
 

Construction and characterization of bacterial artificial chromosome of a pseudorabies virus strain AH02LA isolated from a dead piglet

文章编号:
1008-0589(2018)10-0880-06
作者:
 

刘娅梅1乔永峰1顾一奇1 2柳 畅1 2许梦微1 2王志胜1郭容利1

张传建1侯继波1王继春1*

 

(1. 江苏省农业科学院 兽医研究所/国家兽用生物制品工程技术研究中心,江苏 南京210014;

2. 南京农业大学 动物医学院,江苏 南京210095)

Author(s):
 

LIU Ya-mei1 QIAO Yong-feng1 GU Yi-qi12 LIU Chang12 XU Meng-wei12 WANG Zhi-sheng1

GUO Rong-li1 ZHANG Chuan-jian1 HOU Ji-bo1 WANG Ji-chun1*

 

(1. National Research Center of Veterinary Biological Engineering and Technology, Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China; 2. College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China)

关键词:
 :伪狂犬病病毒变异株猪源细菌人工染色体生长特性
Keywords:
 

 pseudorabies virus variant strain pig origin bacterial artificial chromosome growth kinetics

分类号:
S852.61
DOI:
10.3969/j.issn.1008-0589.201606026
文献标志码:
A
摘要:
 

为构建猪源伪狂犬病病毒(PRV)变异株AH02LA的细菌人工染色体(BAC),本研究将转移载体pHA2-pUC19-H1-H2的DNA与PRV AH02LA株基因组DNA共转染鸡胚成纤维细胞(CEF)进行同源重组,利用转移载体中的GFP 为筛选标记,获得携带GFP 基因的gE/gI 基因缺失突变株PRV-AH02LA(gE-/gI-)-pHA2。提取纯化的该重组病毒DNA转化E.coli DH10B,对获得的阳性克隆命名为BACPRV-G。提取BACPRV-G的DNA,将含gE、gI和其上下游同源片段的DNA与BACPRV-G的DNA共转染CEF,获得gE、gZ基因恢复的病毒命名为PRV- AH02LARec。比较亲本株PRV AH02LA、基因缺失株和恢复株的生长动力学,结果表明,恢复株的生长特性与亲本株无显著差异。本研究成功构建了PRV变异株AH02LA的 BAC,并证实其为全基因组的感染性克隆,为PRV变异株的致病机理研究和新型疫苗研制提供了重要的平台。

 

Abstract:
 

To construct the bacterial artificial chromosome (BAC) of a pseudorabies virus (PRV) mutant strain AH02LA and to characterize its growth properties, chicken embryo fibroblasts (CEFs) were co-transfected with the transfer vector plasmid pHA2-pUC19-H1-H2 and AH02LA genomic DNA for homologous recombination. Green fluorescence plaques were picked under fluorescence microscopy to purify the recombinant virus, named PRV-AH02LA(gE-/gI-)-pHA2. The recombinant virus DNA was transformed into E. coli DH10B. The obtained positive clone was confirmed by PCR and restriction fragment length polymorphism (RFLP) analysis, and named BACPRV-G. The BACPRV-G DNA was extracted to transfect CEFs, and green fluorescence plaques were observed under fluorescence microscopy. CEFs were co-transfected with BACPRV-G DNA and PCR product containing gE, gI, as well as upstream and downstream homologous DNA fragments to recover AH02LA virus, named PRV-AH02LARec. The growth kinetics of PRV-AH02LA, PRV-AH02LA(gE-/gI-)-pHA2 and PRV-AH02LARec were compared and no significant difference in the growth characteristics was observed among the three strains. In this study, the BAC of AH02LA was successfully constructed and confirmed as an infectious whole-genome clone, which could be used as an important platform to study the pathology of PRV variants and the construction of novel vaccines.

参考文献/References:

 

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(本文编辑:李 娜)

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更新日期/Last Update: 2018-11-28