【摘要】 本文根据GenBank登录的对虾传染性皮下及造血组织坏死病毒(IHHNV)的全基因组序列(NC-002190),设计出主要结构蛋白基因的相应引物,从厦门发病的对虾中,提取DNA,以此为模板,利用PCR扩增目的基因.再将目的基因分别与pGEX-6p1载体和pET-28a载体连接,构建重组表达载体pGEX-IV、pET-IV,重组子经酶切和测序鉴定后导入表达型大肠杆菌BL21,IPTG诱导表达,SDS-PAGE检测,大小约为62KDa和41KDa,与预测大小一致.将pET-IV表达重组蛋白经纯化,免疫小鼠,W estern免疫印迹反应结果表明其抗体均可与pGEX-IV和pET-IV表达的蛋白发生特异性反应.本研究的结果为对虾IHHNV的快速检测及其免疫提供了研究基础.更多还原

【Abstract】 The infectious hypodermal & haematopoietic necrosis virus,pathogenic for penaeid shrimp,is an icosahedral unenveloped particle,22nm in diameter,with an ssDNA linear genome,and proposed to be a member of the parvoviridae.According to the complete genome sequences of Infectious Hypodermal & Haematopoietic Necrosis Virus(IHHNV) published on GenBank,one pair of primers were designed to amplify the main structure protein genes of IHHNV respectively.These amplified products were 989bp.They were constructed to the expression vector pET-28a or pGEX-6p1 and named as pET-IV and pGEX-IV.The recombinant plasmids were transformed into E.coli BL21(DE3) after the identification of the nuclear restriction enzymes cutting and sequencing.After IPTG induction and SDS-PAGE analysis,the fusion proteins of about 41KDa and 62KDa in molecule weight were detected.The isolated recombinant proteins were inoculated into the Kun-ming mouse,before detected by the western blot tests with Kun-ming mouse polyclonal antiserum.The results of this research should make a scientific bases for rapid detection and immunization of IHHNV further.更多还原