CAJ | 학술논문

根据Ⅰ型鸭肝炎病毒（Duck hepatitis virus,DHV-Ⅰ）ZJ-Ⅴ株基因组序列（GenBank登录号：EF382778）,设计了一对扩增RNA聚合酶基因（RNA-dependent RNA polymerase,RdRp）的特异性引物,以ZJ-Ⅴ株基因组为模板,扩增DHV-Ⅰ的RdRp基因。运用DNAStar软件对ZJ-Ⅴ株病毒与GenBank上已发表的其他不同DHV毒株的RdRp基因序列进行同源性比较和遗传进化分析,结果表明,ZJ-Ⅴ株与A型DHV-ⅠR85952株（DQ226541）的同源性为97.1%,亲缘关系最近,与2株台湾新型（B型）DHV之间为76.9%和77.0%,与C-GY株和4株韩国新型（C型）DHV株之间为76.2%~76.5%,亲缘关系较远。将RdRp基因克隆至原核表达载体pET-32a（＋）,获得重组表达质粒pET-RdRp,转化宿主菌E.coli BL21（DE3）,用IPTG进行诱导表达。SDS-PAGE分析结果显示RdRp蛋白分子量约为65 kDa,Western blot检测证明RdRp蛋白能与Ⅰ型鸭病毒性肝炎阳性血清产生特异性免疫反应,具有良好的免疫学活性。本文为进一步研究DHV-Ⅰ的复制机理奠定了基础。
근거Ⅰ형압간염병독（Duck hepatitis virus,DHV-Ⅰ）ZJ-Ⅴ주기인조서렬（GenBank등록호：EF382778）,설계료일대확증RNA취합매기인（RNA-dependent RNA polymerase,RdRp）적특이성인물,이ZJ-Ⅴ주기인조위모판,확증DHV-Ⅰ적RdRp기인。운용DNAStar연건대ZJ-Ⅴ주병독여GenBank상이발표적기타불동DHV독주적RdRp기인서렬진행동원성비교화유전진화분석,결과표명,ZJ-Ⅴ주여A형DHV-ⅠR85952주（DQ226541）적동원성위97.1%,친연관계최근,여2주태만신형（B형）DHV지간위76.9%화77.0%,여C-GY주화4주한국신형（C형）DHV주지간위76.2%~76.5%,친연관계교원。장RdRp기인극륭지원핵표체재체pET-32a（＋）,획득중조표체질립pET-RdRp,전화숙주균E.coli BL21（DE3）,용IPTG진행유도표체。SDS-PAGE분석결과현시RdRp단백분자량약위65 kDa,Western blot검측증명RdRp단백능여Ⅰ형압병독성간염양성혈청산생특이성면역반응,구유량호적면역학활성。본문위진일보연구DHV-Ⅰ적복제궤리전정료기출。
In this study,a pair of specific primer,according to the complete genomic sequence of duck hepatitis virus Ⅰ（DHV-Ⅰ） ZJ-Ⅴ strain,was designed to amplify RNA-dependent RNA polymerase gene（RdRp）.Then the sequence of RdRp was compared with some reference sequences of DHV-I published in GenBank,and the results showed that ZJ-Ⅴ strain had closer genetic relationship with DHV-Ⅰgenotype A,compared with genotype B and C.To express RdRp in E.coli,RdRp gene was inserted into pET-32a（＋） vector.And the recombinant plasmid pET-RdRp was transfected into BL21（DE3）cells.The expression product was analyzed with SDS-PAGE and Western blot.The results showed that RdRp was expressed successfully,about 65 kDa.The paper laid the foundation for further study on the replication mechanism of DHV-Ⅰ.