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2025年8月3日 星期日
  2018, Vol. 26 Issue (5): 861-870    
  研究论文与报告 本期目录 | 过刊浏览 | 高级检索 |
兔出血症病毒GS/YZ分离株VP60截段基因的原核表达及免疫效力评价
安凯1,孔惠萍2,邢小勇3,温峰琴3,包世俊3
1. 甘肃农业大学
2. 甘肃农业大学 动物医学院
3. 甘肃农业大学 动物医学学院
Prokaryotic Expression and Protective Efficacy of VP60 Truncated Gene in RHDV GS/YZ Strain
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摘要 摘 要 兔(Lepus)出血症病毒(Rabbit hemorrhagic disease virus, RHDV)属杯状病毒科(Caliciviridae)兔病毒属(Lagovirus)。VP60为RHDV的衣壳蛋白与免疫原性蛋白,且其抗原表位区主要集中在N端。本研究应用反转录(reverse transcription-PCR, RT-PCR)扩增获得VP60截段基因并将其亚克隆至pMD20-T,构建原核表达质粒pET-RHDV-VP60。转化大肠杆菌(Escherichia coli) Rosetta(DE3)后经异丙基-β-d-硫代半乳糖苷(isopropyl β-D-1-thiogalactopyranoside, IPTG)诱导表达,表达产物纯化后免疫Balb/C小鼠(Mus musculus)制备多抗,进而应用间接酶联免疫吸附试验(indirect enzyme linked immunosorbent assay, iELISA)和Western-blot对重组蛋白免疫原性进行分析。用混有弗氏佐剂的重组蛋白、商品苗、RHDV GS/YZ灭活苗、磷酸缓冲盐溶液(phosphate buffer saline, PBS)分别注射易感兔,14 d后重组蛋白组加强免疫1次,21 d后用HA(hemagglutination)效价为210的RHDV GS/YZ肝脏悬液全部攻毒,观察14 d,并记录结果。结果表明:RHDV GS/YZ株VP60截段基因与RHDV HB株(登录号:KU207100.1)和RHDV WX株(登录号:AF402614.1)同源性最高,达98.6%;RHDV GS/YZ株VP60截段氨基酸序列与与RHDV WX株(登录号:AF402614.1)和RHDV Mexico 89株(登录号:AF295785.1)同源性最高,达99.1%;RHDV GS/YZ株VP60截段片段在Rosetta(DE3)中成功获得表达,且以包涵体形式存在;iELISA测定制备的多克隆抗体效价为1∶32 000;Western-blot结果显示,重组蛋白具有良好的免疫原性;免疫保护实验表明,免疫组在攻毒后全部存活,保护率为100%,商品苗血凝抑制效价为25~28,自制灭活苗为25~26,重组蛋白加佐剂为24~25,而只注射了PBS的对照组在攻毒后的96 h全部死亡,表明大肠杆菌表达的VP60截段蛋白在混有弗氏佐剂后能够完全抵抗RHDV强毒的攻击。研究结果为RHD亚单位疫苗的研发及其快速诊断方法的建立提供了资料基础。
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安凯
孔惠萍
邢小勇
温峰琴
包世俊
关键词 兔出血症病毒(RHDV)VP60截段基因原核表达免疫原性免疫效力    
Abstract:Abstract Rabbit haemorrhagic disease virus (RHDV) belongs to the genus Lagovirus of Caliciviridae. VP60 protein is one of the capsid proteins of RHDV, and also is main immunogenic protein which the major antigenic epitopes mainly locate in the N-terminal region. In this study, the VP60 truncated gene was cloned by employing the method RT-PCR and then subcloned to pMD20-T. After complete sequence analysis, the prokaryotic expression plasmid pET-RHDV-VP60 was successfully constructed and then inserted into the expression Escherichia coli Rosetta(DE3). The target protein was highly expressed under the induction with 1 mmol/L isopropyl β-D-1 -thiogalactopyranoside (IPTG), and the Balb/C mice were immunized with purified expression products, then the polyclonal antibody was prepared. Subsequently, the immunogenicity of recombinant protein was analyzed using indirect enzyme linked immunosorbent assay (iELISA) and Western-blot. Twenty clean rabbits were randomly divided into 4 groups and subcutaneously injected once, using commercial vaccine (5 rabbits), the RHDV GS/YZ inactivated by 0.4% formaldehyde (5 rabbits), recombinant protein mixed with adjuvant (5 rabbits) and PBS as negative controls (5 rabbits),respectively . The dosage of recombinant protein was 500 μg mixed with equal amounts of freund's complete adjuvant per injection, with 14 days interval, the recombinant protein was 250 μg mixed with equal amounts of freund's incomplete adjuvant per injection, After 21 days, all the rabbits attacked by the RHDV GS/YZ that HA titer was 210 and observed the results after 14 days. The results showed that the homology of nucleotide sequences of VP60 truncated gene from RHDV GS/YZ strain with 32 RHDV strains in GenBank was from 91.9% to 98.6%, and had 98.6% nucleotide sequence identity with the RHDV HB strain(accession number: KU207100.1) and RHDV WX strain (accession number: AF402614.1). The homology of amino acid sequence of RHDV GS/YZ strain with 32 RHDV strains in GenBank was from 96.7% to 99.1%, and had 99.1% amino sequence identity with the RHDV WX strain (accession number:AF402614.1) and RHDV Mexico 89 strain (accession number:AF295785.1). The target gene was successfully expressed in Escherichia coli, and the recombinant protein expression in the form of inclusion body. SDS-PAGE analysis showed that the molecular weight of the recombinant protein was approximately 50 kD. The titer of polyclonal antibody was 1∶32 000 by iELISA and Western-blot demonstrated that the recombinant protein could bind with antibody induced by RHDV GS/YZ and the polyclonal antibody induced by recombinant protein could also combine with vaccine of RHDV - Pasteurella multocida and wild viruses of RHDV GS/YZ. So it had good immunogenicity. The immune protection experiment showed that the rabbits in the RHDV GS/YZ inactivated by 0.4% formaldehyde group, commercial vaccine group and recombinant protein mixed with adjuvant survived facing the challenge without any clinical signs of RHD while those in the negative control group died within 96 h post-infection and exhibited typical clinical symptoms. The titer of hemagglutination inhibition of commercial vaccine was 25~28, the self-made inactivated vaccine was 25~26, and the recombinant protein mixed with equal amounts of freund's adjuvant was 24~25. The results showed that the proteins expressed by Rosetta (DE3) could be ideal candidate for RHDV vaccines.
Key wordsRabbit hemorrhagic disease virus(RHDV)    VP60 truncated gene    Prokaryotic expression    Immunogenicity    Protective efficacy
收稿日期: 2017-10-13      出版日期: 2018-05-02
基金资助:国家自然科学基金
通讯作者: 包世俊     E-mail: bsjdy@126.com
引用本文:   
安凯 孔惠萍 邢小勇 温峰琴 包世俊. 兔出血症病毒GS/YZ分离株VP60截段基因的原核表达及免疫效力评价[J]. , 2018, 26(5): 861-870.
链接本文:  
http://journal05.magtech.org.cn/Jwk_ny/CN/     或     http://journal05.magtech.org.cn/Jwk_ny/CN/Y2018/V26/I5/861
 
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