森林脑炎病毒prM-E蛋白在昆虫细胞中的表达及免疫活性测定

目的为了表达森林脑炎病毒prME蛋白,为森林脑炎快速诊断试剂的研制奠定基础。方法经过RTPCR扩增、重组转移载体构建、细菌内转座和昆虫细胞转染,以杆状病毒昆虫细胞表达系统成功地表达了森林脑炎病毒MDJ01株prME蛋白。结果从感染细胞上清中电镜观察到重组蛋白形成的球型颗粒,说明重组病毒感染细胞后产生病毒样表达颗粒(viruslikeparticlesVLPs),并且分泌至细胞外。免疫印迹试验和间接免疫荧光试验表明,表达的重组蛋白能够与抗森林脑炎病毒抗体特异结合,具有良好的抗原性。ELISA和间接免疫荧光染色证实,重组prME蛋白可以作为抗原用于检测患者血清特异性抗体。结论在昆虫细胞中表达的prME具有良好的抗原性,本研究为森林脑炎快速特异诊断试剂研制奠定了基础。

Expression of tick-borne encephalitis virus prM-E protein in insect cells and studies on its antigenicity

Objective To express the prM-E protein in Sf9 cells, and lay a basis for further study on the function of the viral proteins and development of specific diagnostic reagents. Methods The recombinant prM-E protein of tick-borne encephalitis virus was expressed in insect cell Sf9 by RT-PCR amplification of prM-E gene, construction of donor plasmid of Bac-to-Bac baculovirus expression system, homologous recombination of donor plasmid with bacmid DNA at the site of Tn7 and transfection of insect cell Sf9. Results Recombinant subviral particles, about 30 nm in diameter, consisting of prM-E were observed by electron microscope in the supernatant of infected cells, which indicated that infected cells released virus-like particles(VLPs) into the culture medium. The results of Western-blot and the indirect immunofluorescence assay(IFA)showed that the recombinant proteins retained antigenic and conformational structures similar to those of native virus proteins. Using the recombinant prM-E as antigens to detect samples of patient sera by ELISA and IFA, all of 16 sera from patients with tick-borne encephalitis were positive and all of 6 sera from other patients were negative. Conclusions The prM-E protein expressed in insect cells retains good antigenicity.