Ⅰ型鸭肝炎病毒VP3基因的克隆及原核表达

›› 2013, Vol. 40 ›› Issue (10): 33-37.

Ⅰ型鸭肝炎病毒VP3基因的克隆及原核表达

李露^1,2, 程英杰^2,3, 李传峰², 陈宗艳², 孟春春², 何后军¹, 刘光清²

1. 江西农业大学动物科技学院, 江西南昌 330045;
2. 中国农业科学院上海兽医研究所, 上海 200241;
3. 甘肃农业大学动物医学院, 甘肃兰州 730070

收稿日期:2013-01-06 出版日期:2013-10-20 发布日期:2013-12-19
通讯作者: 刘光清 E-mail:liugq@shvri.ac.cn何后军。E-mail:hehoujun@163.com
作者简介:李露(1989-),女,江西人,硕士生,研究方向:禽病毒分子生物学。
基金资助:
国家自然科学基金项目(31101848,31270194);公益性农业科研专项(201003012);"863"计划 (2011AA10A200);中央级公益性科研院所基本科研业务费专项(2012JB06,2012JB13)。

Cloning and Prokaryotic Expression of VP3 Gene of Duck Hepatitis Virus Ⅰ

LI Lu^1,2, CHENG Ying-jie^2,3, LI Chuan-feng², CHEN Zong-yan², MENG Chun-chun², HE Hou-jun¹, LIU Guang-qing²

1. College of Animal Science and Technology, Jiangxi Agricultural University, Nanchang 330045, China;
2. Shanghai Veterinary Research Institute, CAAS, Shanghai 200241, China;
3. College of Veterinary Medicine, Gansu Agricultural University, Lanzhou 730070, China

Received:2013-01-06 Online:2013-10-20 Published:2013-12-19

摘要/Abstract

摘要： 采用RT-PCR方法从Ⅰ型鸭肝炎病毒ZJ-Ⅴ株中扩增VP3基因,运用DNAStar软件对VP3序列与参考序列的同源性进行比对,并进行了遗传进化分析,然后将VP3基因克隆至原核表达载体pET-32a(+),获得重组表达质粒pET-VP3,转化宿主菌E.coli Rosetta(DE3),经1 mmol/L IPTG诱导表达后用SDS-PAGE和Western blotting方法对表达产物进行检测。结果显示,成功克隆了DHV-Ⅰ VP3基因,片段大小为711 bp。序列分析结果表明,ZJ-Ⅴ株VP3与其他A型DHV分离株的同源性较高,为95.1%~97.7%;而与C型DHV分离株的同源性较低,为71.3%~72.6%;与B型DHV分离株的VP3基因同源性最差,为70.3%~70.5%。SDS-PAGE和Western blotting检测结果显示,重组VP3基因在大肠杆菌细胞(DE3)中获得了良好表达,其分子质量约为47 ku;与抗Ⅰ型鸭肝炎病毒ZJ-Ⅴ株阳性血清发生特异性免疫反应,表明重组VP3蛋白具有良好免疫原性。

关键词: Ⅰ型鸭肝炎病毒; VP3基因; 原核表达; 序列分析

Abstract: The VP3 gene of duck hepatitis virus type 1 (DHV-1) was amplified by reverse transcription-polymerase chain reaction (RT-PCR), compared with the reference sequences and analyzed genetically using DNAStar software. The acquired VP3 gene was cloned into the prokaryotic expression vector pET32a(+). The resultant pET-VP3 was transformed into E.coli Rosetta(DE3) cell and induced with 1mM IPTG. The expression product was analyzed by SDS-PAGE and Western blotting. The results showed that the VP3 gene was successfully cloned with 711bp in length. Sequences analysis results indicated that the VP3 gene of DHV-1 ZJ-V strain has a higher sequence identify with the other DHV genotype A isolates ranging from 95.1% to 97.7%, a lower sequence identify with the other DHV genotype C isolates ranging from 71.3% to 72.6%, and the lowest sequence identify with the other DHV genotype B isolates ranging from 70.3% to 70.5%. the results of SDS-PAGE and Western blotting revealed that the recombinant VP3 protein with a relative molecular mass of 47ku, can be expressed in the E.coli Rosetta(DE3) cell and recognized by positive serum against DHV-I ZJ-V strain, showing good immunogenicity.

Key words: duck hepatitis virusⅠ; VP3 gene; prokaryotic expression; sequence analysis

中图分类号:

Q785

李露, 程英杰, 李传峰, 陈宗艳, 孟春春, 何后军, 刘光清. Ⅰ型鸭肝炎病毒VP3基因的克隆及原核表达[J]. , 2013, 40(10): 33-37.

LI Lu, CHENG Ying-jie, LI Chuan-feng, CHEN Zong-yan, MENG Chun-chun, HE Hou-jun, LIU Guang-qing. Cloning and Prokaryotic Expression of VP3 Gene of Duck Hepatitis Virus Ⅰ[J]. China Animal Husbandry & Veterinary Medicine, 2013, 40(10): 33-37.

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