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  被引量:3

Cloning and expression of VP1 gene of Duck hepatitis virus genotype C and preparation of its polyclonal antibody

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作  者:赵立娜[1,2] 崔言顺[1] 任建亭 李建亮[1] 黄兵[2] 李玉峰[2] 马秀丽[2] 

机构地区:[1]山东农业大学动物医学院,山东泰安271018 [2]山东省农业科学院家禽研究所,山东省家禽疫病诊断与免疫重点实验室,山东济南250023 [3]山东省东营市广饶县中等专业中学,山东广饶257300

出  处:《浙江农业学报》2011年第2期258-262,共5页Acta Agriculturae Zhejiangensis

基  金:山东省自然基金项目(Z2006D06);家畜疫病病原生物学国家重点实验室开放课题;山东省优秀中青年科学家科研奖励基金(BS2009YY019)

摘  要: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DHVVP1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。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-galactoside(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 immunogenicity.

关 键 词:鸭肝炎病毒 基因C型 VP1基因 克隆表达 多克隆抗体 

分 类 号:S858.32[农业科学—临床兽医学]

 

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