新型鸭肝炎病毒全基因组测序及其VP1基因的克隆表达分析
摘要
鸭病毒性肝炎(Duck Virus Hepatitis, DVH)又称鸭肝炎,是由鸭病毒性肝炎病毒(Duck Hepatitis virus, DHV)引起的一种急性、高度致死性的传染病,以肝炎为主要特征,主要发生于三周龄以下雏鸭。目前主要采用传统的弱毒疫苗和高免卵黄抗体进行Ⅰ型DHV的预防和治疗。然而,近几年来,国内许多地区频频出现免疫鸭群爆发鸭肝炎的报道,严重制约了养鸭业的发展。
1.韩国新型DHV JFX-08株的分离及RT-PCR鉴定
从山东省某疑似鸭肝炎发病区分离到1株病毒JFX08;血清中和试验结果表明该分离毒与传统的Ⅰ型鸭肝炎病毒无交叉保护;分离毒回归3日龄雏鸭,可复制出鸭肝炎的典型症状和病理变化。根据已公布的韩国新型(基因C型)DHV的序列设计合成一对引物,进行RT-PCR扩增,结果得到大小约414bp的目的条带;测序分析发现,分离毒与传统Ⅰ型DHV之间的核苷酸序列相似性较低,而与韩国新型DHV之间的核苷酸序列相似性高达93.2%~94.0%;进化分析结果表明,该分离毒与韩国新型DHV的遗传距离最近,属基因C型DHV。
2.新型DHV JFX-08分离株全基因组序列测序与分析
根据已发表的DHV基因组序列设计多对特异的引物,按常规RT-PCR方法扩增毒株的中间相应区域,采用5’和3’cDNA末端快速扩增(Rapid amplification of cDNA ends, RACE)方法扩增病毒RNA的5’和3’末端序列。对分离株JFX08的全基因组进行了序列测定分析。结果表明,JFX08的基因组全长为7793nt,包括652nt的5’UTR、6753nt的ORF、366nt的3’UTR以及19nt的poly(A)尾巴。JFX08的ORF编码2251个氨基酸,各基因均与韩国新型的氨基酸序列相似性最高,与Ⅰ型DHV和台湾新型DHV的相似性均较低。其中VP1较Ⅰ型DHV存在2个氨基酸的插入和较多氨基酸位点的突变,高变区主要集中在180-194位和213-219位。JFX08的非编码区核苷酸序列之间存在大量碱基突变和插入/缺失。
3.新型DHV VP1基因的克隆表达及其多克隆抗体的制备
采用RT-PCR方法,扩增基因C型DHV JFX08株的VP1基因,与pMD18-T载体进行连接,构建新型DHV VP1基因克隆重组质粒。然后将VP1基因定向插入到pET-32a(+)表达载体中,筛选原核表达载体pET-32a-VP1,进行IPTG诱导表达。SDS-PAGE电泳分析表明,基因C型DHV-VP1基因可在大肠杆菌中稳定高效的表达。Western-blot检测表明,表达产物可与基因C型DHV阳性血清发生特异性反应。将纯化的重组蛋白免疫4周龄SPF鸡,以纯化的VP1重组蛋白和基因C型DHV全病毒分别包板,制备的抗血清ELISA效价可分别达1:25 600、1:51 200,表明该重组蛋白具有良好的免疫原性。
Duck Viral Hepatitis (DVH) caused by duck hepatitis virus (DHV) is a highly mortal disease and spreads rapidly in three-week-old ducklings characterized primarily by Hepatitis. Now, the traditional attenuated vaccines and the highⅠ-yolk antibody were the main methods for the prevention and treatment of DHV. However, many domestic areas frequently reported that immuned ducks outbreaks of duck hepatitis in recent years, which was restricted the development of ducks industry seriously.
1. Isolataion and RT-PCR Identification of a Korea new type Duck Hepatitis Virus
One strain of duck hepatitis virus was isolated from Shandong province. The result of serum neutralization test showed that there was no cross-protection occured in strain JFX08 and typeⅠduck hepatitis virus (DHV-Ⅰ). Clinical symptom and pathological lesion could be reproduced in 3-day ducklings challenged by the isolated strain. A pair of primers was designed and synthesized according to the sequence of duck hepatitis virus and a reverse transcriptase polymerase chain reaction method was developed for strain JFX08.Nucleotide sequence identity between strain JFX08 and N-DHV from Korean(genotype C) was 93.2%~94.0%. However, it was lower between strain JFX08 and DHV-Ⅰor N-DHV from Taiwan than N-DHV from Korean. Phylogenetic and evolutionary analysis showed that our isolated strain JFX08 belonged to genotype C, which was on different branches from genotype A and B.
2. Sequencing and analysis of the Genome of New Duck Hepatitis Virus JFX-08
The genome of duck hepatitis virus strain JFX08 isolated from China has been sequenced and analyzed. The results showed that the full length of the genome was 7793nt; including 652nt of 5’untranslated region (UTR), an open reading frame of 6753nt encoded a polypeptide of 2251 amino acids, 366nt of 3’untranslated region. The pairwise percent identity of the amino acid sequences at ORF region between strain JFX08 and Korea new type DHV were higher than between strain JFX08 and I type DHV or Taiwanese new type DHV. There were 2 amino acids insertion and many variant sites and the hypervariable regions were found in 180-194aa and 213-219aa of VP1 gene. A lot of variant sites and insertion/ deletion were found in untranslated region. Phylogenetic and evolutionary analysis based on polyprotein, VP0, VP3, VP1, 2C and 3D indicated that strain JFX08 had a very close relationship with Korea new type DHV, which affiliated with genotype C.
3. Cloning and Expression of VP1 Gene of Duck Hepatitis Virus Genotype C and Preparation of Its Polyclonal Antibody
The VP1 gene of strain JFX08 of duck hepatitis virus genotype C was amplified by reverse transcription-polymerase chain reaction (RT-PCR). To obtain expression plasmid pET-32a -VP1, the VP1 gene was cloned into pMD18-T and pET-32a(+)vectors. SDS-PAGE analysis revealed that the recombinant VP1 protein of Genotype C of duck hepatitis virus was expressed in Escherichia coli BL21 (DE3) at a high level after being induced with Isopropylthio-β-D-galactoside (IPTG) stably. Western-blot revealed that the recombinant protein was recognized specifically by antisera against the Genotype C of duck hepatitis virus. 4-week-old SPF chickens were immunized with the purified recombinant protein. ELISA established by the purified recombinant protein and DHV Genotype C virus revealed that the titer of antiserum was 1:25 600 and 1:51 200 respectively, which indicated that the recombinant protein had good immunogenicity.
1.韩国新型DHV JFX-08株的分离及RT-PCR鉴定
从山东省某疑似鸭肝炎发病区分离到1株病毒JFX08;血清中和试验结果表明该分离毒与传统的Ⅰ型鸭肝炎病毒无交叉保护;分离毒回归3日龄雏鸭,可复制出鸭肝炎的典型症状和病理变化。根据已公布的韩国新型(基因C型)DHV的序列设计合成一对引物,进行RT-PCR扩增,结果得到大小约414bp的目的条带;测序分析发现,分离毒与传统Ⅰ型DHV之间的核苷酸序列相似性较低,而与韩国新型DHV之间的核苷酸序列相似性高达93.2%~94.0%;进化分析结果表明,该分离毒与韩国新型DHV的遗传距离最近,属基因C型DHV。
2.新型DHV JFX-08分离株全基因组序列测序与分析
根据已发表的DHV基因组序列设计多对特异的引物,按常规RT-PCR方法扩增毒株的中间相应区域,采用5’和3’cDNA末端快速扩增(Rapid amplification of cDNA ends, RACE)方法扩增病毒RNA的5’和3’末端序列。对分离株JFX08的全基因组进行了序列测定分析。结果表明,JFX08的基因组全长为7793nt,包括652nt的5’UTR、6753nt的ORF、366nt的3’UTR以及19nt的poly(A)尾巴。JFX08的ORF编码2251个氨基酸,各基因均与韩国新型的氨基酸序列相似性最高,与Ⅰ型DHV和台湾新型DHV的相似性均较低。其中VP1较Ⅰ型DHV存在2个氨基酸的插入和较多氨基酸位点的突变,高变区主要集中在180-194位和213-219位。JFX08的非编码区核苷酸序列之间存在大量碱基突变和插入/缺失。
3.新型DHV VP1基因的克隆表达及其多克隆抗体的制备
采用RT-PCR方法,扩增基因C型DHV JFX08株的VP1基因,与pMD18-T载体进行连接,构建新型DHV VP1基因克隆重组质粒。然后将VP1基因定向插入到pET-32a(+)表达载体中,筛选原核表达载体pET-32a-VP1,进行IPTG诱导表达。SDS-PAGE电泳分析表明,基因C型DHV-VP1基因可在大肠杆菌中稳定高效的表达。Western-blot检测表明,表达产物可与基因C型DHV阳性血清发生特异性反应。将纯化的重组蛋白免疫4周龄SPF鸡,以纯化的VP1重组蛋白和基因C型DHV全病毒分别包板,制备的抗血清ELISA效价可分别达1:25 600、1:51 200,表明该重组蛋白具有良好的免疫原性。
Duck Viral Hepatitis (DVH) caused by duck hepatitis virus (DHV) is a highly mortal disease and spreads rapidly in three-week-old ducklings characterized primarily by Hepatitis. Now, the traditional attenuated vaccines and the highⅠ-yolk antibody were the main methods for the prevention and treatment of DHV. However, many domestic areas frequently reported that immuned ducks outbreaks of duck hepatitis in recent years, which was restricted the development of ducks industry seriously.
1. Isolataion and RT-PCR Identification of a Korea new type Duck Hepatitis Virus
One strain of duck hepatitis virus was isolated from Shandong province. The result of serum neutralization test showed that there was no cross-protection occured in strain JFX08 and typeⅠduck hepatitis virus (DHV-Ⅰ). Clinical symptom and pathological lesion could be reproduced in 3-day ducklings challenged by the isolated strain. A pair of primers was designed and synthesized according to the sequence of duck hepatitis virus and a reverse transcriptase polymerase chain reaction method was developed for strain JFX08.Nucleotide sequence identity between strain JFX08 and N-DHV from Korean(genotype C) was 93.2%~94.0%. However, it was lower between strain JFX08 and DHV-Ⅰor N-DHV from Taiwan than N-DHV from Korean. Phylogenetic and evolutionary analysis showed that our isolated strain JFX08 belonged to genotype C, which was on different branches from genotype A and B.
2. Sequencing and analysis of the Genome of New Duck Hepatitis Virus JFX-08
The genome of duck hepatitis virus strain JFX08 isolated from China has been sequenced and analyzed. The results showed that the full length of the genome was 7793nt; including 652nt of 5’untranslated region (UTR), an open reading frame of 6753nt encoded a polypeptide of 2251 amino acids, 366nt of 3’untranslated region. The pairwise percent identity of the amino acid sequences at ORF region between strain JFX08 and Korea new type DHV were higher than between strain JFX08 and I type DHV or Taiwanese new type DHV. There were 2 amino acids insertion and many variant sites and the hypervariable regions were found in 180-194aa and 213-219aa of VP1 gene. A lot of variant sites and insertion/ deletion were found in untranslated region. Phylogenetic and evolutionary analysis based on polyprotein, VP0, VP3, VP1, 2C and 3D indicated that strain JFX08 had a very close relationship with Korea new type DHV, which affiliated with genotype C.
3. Cloning and Expression of VP1 Gene of Duck Hepatitis Virus Genotype C and Preparation of Its Polyclonal Antibody
The VP1 gene of strain JFX08 of duck hepatitis virus genotype C was amplified by reverse transcription-polymerase chain reaction (RT-PCR). To obtain expression plasmid pET-32a -VP1, the VP1 gene was cloned into pMD18-T and pET-32a(+)vectors. SDS-PAGE analysis revealed that the recombinant VP1 protein of Genotype C of duck hepatitis virus was expressed in Escherichia coli BL21 (DE3) at a high level after being induced with Isopropylthio-β-D-galactoside (IPTG) stably. Western-blot revealed that the recombinant protein was recognized specifically by antisera against the Genotype C of duck hepatitis virus. 4-week-old SPF chickens were immunized with the purified recombinant protein. ELISA established by the purified recombinant protein and DHV Genotype C virus revealed that the titer of antiserum was 1:25 600 and 1:51 200 respectively, which indicated that the recombinant protein had good immunogenicity.
引文
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