Ⅰ型鸭肝炎病毒VP3基因的克隆及原核表达

Cloning and Prokaryotic Expression of VP3 Gene of Duck Hepatitis VirusⅠ

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1mmol/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-ⅠVP3基因,片段大小为711bp。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47ku;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。

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 1mmol/L IPTG.The expression product was analyzed by SDS-PAGE and Western blotting.The results showed that VP3 gene was successfully cloned with 711bp in length.Sequences analysis results indicated that VP3 gene of DHV-1 ZJ-V strain had 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,could be expressed in the E.coli Rosetta（DE3）cell and recognized by positive serum against DHV-I ZJ-V strain,showing good immunogenicity.