新加坡石斑鱼虹彩病毒ORF39L基因克隆、表达及抗体的制备

Cloning,expression and antibody preparation of ORF39L gene in Singapore grouper iridovirus

为研究新加坡石斑鱼虹彩病毒(Singapore grouper iridovirus,SGIV)核心基因ORF39L在病毒侵染过程中的作用,制备了SGIV ORF39L表达蛋白的抗体;通过生物信息学软件预测SGIV ORF39L的抗原表位基因,并以所选抗原表位基因片段39Ag1和39Ag2构建原核表达质粒pET32a-39Ag1和pET32a-39Ag2;将质粒转入大肠杆菌BL21(DE3)中成功表达融合蛋白pET32a-VP39Ag1和pET32a-VP39Ag2;用所得融合蛋白分别免疫小鼠,获得了高效特异抗体39Ab1和39Ab2;利用制备的抗体进行间接免疫荧光试验,结果显示,VP39参与了SGIV的装配。本研究结果为进一步研究ORF39L基因在SGIV感染过程中的作用机制提供了数据资料。

ORF39 L antigen epitope gene as a core gene in Singapore grouper iridovirus（ SGIV） in family Iridoviridae was analyzed and predicted with bioinformatics software and the selected antigen epitope gene fragment of 39Ag1 and 39Ag2 were constructed by prokaryotic recombinant expression plasmid p ET32a-39Ag1 and p ET32a-39Ag2 which transformed into Escherichia coli BL21（ DE3） in order to prepare the antibody of ORF39 L expression protein for study of the role of ORF39 L in SGIV infection. SDS-PAGE analyses proved that the transformed E. coli BL21 expressed recombinant protein pET32 a-VP39Ag1 and pET32 -VP39Ag2. The polyclonal antibodies 39Ab1 and 39Ab2 were prepared by immunizing mice with purified recombinant protein proved to be specific against SGIV VP39. Furthermore,an indirect immunofluorescence assay by the prepared polyclonal antibody revealed that VP39 was involved in the assembly of the SGIV in the process of infection.