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为研究草鱼呼肠孤病毒(GCRV)HZ08株S10基因节段编码蛋白的可能功能,采用PCR方法扩增草鱼呼肠孤病毒HZ08株S10基因节段,并把该基因节段克隆至表达载体pET-32a(+),获得的重组表达载体pET-32a-S10转化到大肠杆菌BL21(DE3)菌株,用IPTG诱导表达,表达产物通过SDS-PAGE分析鉴定后,再通过变性、过Ni柱纯化、透析复性纯化获得目的蛋白。然后用纯化的重组蛋白免疫昆明小白鼠,制得多克隆抗体,用间接ELISA方法测定抗体效价,用Western blot和IFA(间接免疫荧光试验)鉴定抗体特异性。结果表明,SDS-PAGE分析表达的重组蛋白约为53 ku,大小与预期相符,目的蛋白主要存在于包涵体中;过Ni柱纯化、透析复性纯化后的重组蛋白纯度可达97.4%;间接ELISA测得制备的多克隆抗体效价约为1∶106,Western blot和IFA结果显示,制备的多克隆抗体能识别HZ08毒株,表明S10编码蛋白为GCRV-HZ08株的结构蛋白。
In order to study the possible function of S10 protein segment in grass carp reovirus (GCRV) HZ08 strain S10, the S10 gene fragment of grass carp reovirus HZ08 strain was amplified by PCR and subcloned into the expression vector pET -32a (+). The recombinant plasmid pET-32a-S10 was transformed into E. coli BL21 (DE3) strain and induced by IPTG. The expressed product was identified by SDS-PAGE analysis, Dialyzed and purified to obtain the target protein. The purified recombinant protein was used to immunize Kunming mice to obtain polyclonal antibodies. The antibody titers were measured by indirect ELISA and the antibody specificity was identified by Western blot and IFA (indirect immunofluorescence assay). The results showed that the recombinant protein expressed by SDS-PAGE was about 53 ku, and its size was in line with the expectation. The target protein mainly existed in the inclusion bodies. Purification by Ni-Ni column and purification by dialysis could reach 97.4% purity. The titer of the prepared polyclonal antibody was about 1:106 by ELISA. The results of Western blot and IFA showed that the prepared polyclonal antibody recognized the HZ08 strain, indicating that S10 encoded protein was the structural protein of GCRV-HZ08 strain.