基因Ⅱ型草鱼呼肠孤病毒病毒样颗粒的构建与鉴定

为研究II型草鱼呼肠孤病毒(grass carp reovirus, GCRV)病毒样颗粒(viruses-like particles, VLPs)疫苗,本试验利用杆状病毒-昆虫细胞表达系统(baculovirus expression vector system, BEVS)将编码VP35蛋白的GCRV-S11基因克隆入杆状病毒载体pFastBacHTA^TM,然后将鉴定正确的重组质粒转化至DH10Bac感受态细胞,筛选得到重组穿梭质粒Bacmid-VP35。将穿梭质粒Bacmid-VP35以及实验室前期构建的重组穿梭质粒Bacmid-VP3、Bacmid-VP4分别转染sf9昆虫细胞获得重组杆状病毒pFHB-VP35、pFHB-VP3以及pFHB-VP4。利用Bac-PAK快速滴定试剂盒测定重组杆状病毒滴度,并通过间接免疫荧光(IFA)和Western Blot鉴定重组蛋白的表达情况。结果显示,本试验获得了较高滴度的重组杆状病毒,并且重组蛋白在杆状病毒感染的sf9昆虫细胞中正确表达。将成功表达的重组杆状病毒pFHB-VP35、pFHB-VP3以及pFHB-VP4共感染sf9细胞组装GCRV-VLPs,通过透射电镜(EM)进行检测。结果显示,GCRV的3个蛋白在sf9昆虫细胞中可以完成自我组装,形成与天然病毒结构形似的VLPs,直径大小为65-72nm。本试验结果为进一步研制安全、高效的GCRV-VLPs疫苗奠定基础。

Construction and Identification of genotype II grass carp reovirus virus-like particles

In order to construct genotype II grass carp reovirus (GCRV) virus-like particulars (VLPs) based vaccines, the immunogenic GCRV-VP3/VP4/VP35 protein was assembled into GCRV-VLPs using baculovirus expression system (BEVS). The structural protein VP35 encoded by the segment S11 of GCRV-II was inserted into baculovirus vector pFastBacHTA^TM using BEVS, and the identified recombinant vector was transformed into DH10Bac competent cells to screen recombinant plasmid Bacmid-VP35. Recombinant baculovirus pFHB-VP35 was obtained by transfecting recombinant plasmid Bacmid-VP35, recombinant baculovirus pFHB-VP3 and pFHB-VP4 was obtained by transfecting recombinant plasmid Bacmid-VP3 and Bacmid-VP4 which have been successfully constructed in our laboratory. Then the titers of recombinant baculovirus were determined by Bac-PAK rapid titer kit and the expression of proteins was identified by IFA and Western blot. The results showed that high titers of pFHB-VP35, pFHB-VP3, and pFHB-VP4 were obtained, and the corresponding proteins were successfully expressed in infected Sf9 cells. GCRV-VLPs were assembled co-infection of Sf9 cells with pFBH-VP35, pFBH-VP3, and pFBH-VP4, and the GCRV-VLPs were identified by electron microscopy (EM). The results showed that the three proteins could self-assemble in Sf9 cells and formed VLPs with diameter of 65-72 nm similar with natural viruses. This study lays a foundation for development of novel vaccines for preventing the disease caused by GCRV genotype II.