基因C型鸭肝炎病毒VP1基因的克隆表达及其多克隆抗体的制备

摘要

The VP1 gene of strain JFX08 of Duck hepatitis virus(DHV) 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-galacteside (IPTG) stably.Western-blot revealed that the recombinant protein was recognized specifically by antisera against the genotype C of Duck hepatitis virus.Four-week-old SPF chickens were immunized with the purified recombinant protein.ELISA established by the purified recombinant protein and DHV genotype C revealed that the titer of antiserum was 1∶ 25 600 and 1∶ 51 200 respectively, which indicated that the recombinant protein had good immtmogenicity.%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效价分别可达1∶25 600,1∶51 200,表明该重组蛋白具有良好的免疫原性.