Cloning of vp4 Gene from Porcine Rotavirus JL94 Isolate and Expression in Baculovirus System

SONG Yan; SHI Dong-fang; FAN Chen; LIU Sheng-wang; LI Yi-jing

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February 2005

SONG Yan, SHI Dong-fang, FAN Chen, LIU Sheng-wang, LI Yi-jing and Cloning of vp4 Gene from Porcine Rotavirus JL94 Isolate and Expression in Baculovirus System[J]. Virologica Sinica, 2005, 20(1): 61-64.

Citation: SONG Yan, SHI Dong-fang, FAN Chen, LIU Sheng-wang, LI Yi-jing, . Cloning of vp4 Gene from Porcine Rotavirus JL94 Isolate and Expression in Baculovirus System .VIROLOGICA SINICA, 2005, 20(1) : 61-64.

猪轮状病毒vp4基因的克隆及其在昆虫细胞中的表达

宋岩 ¹ ,
师东方 ¹ ,
樊琛 ¹ ,
刘胜旺 ² ,
魏华 ³ ,
李一经 ^1*

1.
1.黑龙江哈尔滨东北农业大学动物医学院病原分子生物学实验室 2.中国农业科学院哈尔滨兽医研究所生物技术国家重点实验室 3.江苏南京东南大学医学院微生物学教研室

摘要

本研究扩增猪轮状病毒中国分离株JL94株VP4蛋白主要抗原编码区基因(1 756 bp),将测序结果与国外分离株进行比较;将该基因片段同载体pMel BacA连接后,与杆状病毒DNA共转染入昆虫细胞Sf9,经蚀斑筛选纯化重组病毒并再感染Sf9细胞获得vp4 基因的表达,对表达的VP4 蛋白进行Western blot分析和血清中和抗体试验。结果表明:JL94株VP4主要抗原编码区基因与国外分离株CRW 8 株、Gottfried株该基因片段氨基酸同源性分别为96.43%和67%,说明JL94株与CRW 8 株属同一VP4 血清型,而与Gottfried株属不同血清型。JL94 株VP4主要抗原编码区氨基酸最大变异处位于aa81 aa207。vp4 基因在昆虫细胞中表达量占细胞总蛋白的20%,Western Blot证实表达蛋白有良好的生物学活性。所表达的蛋白免疫小鼠产生中和抗体,阻断JL94 在MA104 细胞上引起的细胞病变。
- 猪轮状病毒
- , vp4基因
- , 主要抗原编码区基因
- , 克隆
- , 真核表达

Cloning of vp4 Gene from Porcine Rotavirus JL94 Isolate and Expression in Baculovirus System

SONG Yan ¹ ,
SHI Dong-fang ¹ ,
FAN Chen ¹ ,
LIU Sheng-wang ² ,
LI Yi-jing ³ ,
^1*

Abstract

A pair of primers was designed to amplify vp4 gene of major antigen site (1-756 bp) of JL94 isolate. The sequence was analyzed in comparison with the VP4 amino acid sequences of two reference porcine rotavirus. The amino acid sequences were 96.43% and 67% correspondingly. Notably, the most divergence of amino acid sequence is located in a region delimited by aa81-aa207. The vp4 gene was inserted into expression plasmid pMel BacA. pMel BacA and Bac-N-blue DNA were cotransinfected insect cell Sf9. After 3 time plaque, reconstitution virus affected Sf9 and expresed in the cell. It indicated that the 30kDa product of the vp4 gene was 20% of total cell protein. Western blotting and neutralization test continmed that product had a nice biological activity. This study provides the basis for PRV identification, molecular epdiemiology investigation, and research of diagnostic reagent and genetic engineering vaccine
- Porcine rotavirus（PRV）
- , vp4 gene
- , Clone
- , Major antigen site
- , Eukaryotic expression

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通讯作者: 陈斌, bchen63@163.com

1.
沈阳化工大学材料科学与工程学院沈阳 110142

Cloning of vp4 Gene from Porcine Rotavirus JL94 Isolate and Expression in Baculovirus System

Abstract: A pair of primers was designed to amplify vp4 gene of major antigen site (1-756 bp) of JL94 isolate. The sequence was analyzed in comparison with the VP4 amino acid sequences of two reference porcine rotavirus. The amino acid sequences were 96.43% and 67% correspondingly. Notably, the most divergence of amino acid sequence is located in a region delimited by aa81-aa207. The vp4 gene was inserted into expression plasmid pMel BacA. pMel BacA and Bac-N-blue DNA were cotransinfected insect cell Sf9. After 3 time plaque, reconstitution virus affected Sf9 and expresed in the cell. It indicated that the 30kDa product of the vp4 gene was 20% of total cell protein. Western blotting and neutralization test continmed that product had a nice biological activity. This study provides the basis for PRV identification, molecular epdiemiology investigation, and research of diagnostic reagent and genetic engineering vaccine