蜱传脑炎病毒prM-E蛋白的真核表达及其在荧光素酶免疫共沉淀技术中的应用?

为了探索一种新型的血清学技术的荧光素酶免疫共沉淀技术（LIPS），本研究利用真核表达系统制备了蜱传脑炎病毒结构蛋白prM?E与荧光素酶融合蛋白，并评价了其在LIPS系统中的检测效果。首先以蜱传脑炎病毒RNA为模板，采用RT?PCR技术扩增prM?E抗原基因，连接到pNLF1?secN质粒上构建真核表达载体pNLF?prM?E，使prM?E蛋白与荧光素酶串联融合表达，并将该质粒转染到Cos7细胞中。结果显示，转染重组质粒pNLF?prM?E的细胞上清中可检测到荧光素酶的高表达；进而用间接免疫荧光试验（ IFA）检测到转染的细胞与蜱传脑炎病毒抗体特异性结合；在此基础上，用LIPS方法对蜱传脑炎病毒感染患者血清进行检测，从20份患者血清中检出19份阳性，7份对照血清均为阴性。结果表明该重组抗原具有良好的特异性和灵敏性，可以用于LIPS系统中的候选检测抗原。

EXPRESSION OF TICK-BORNE ENCEPHALITIS VIRUS PRM-E PROTEIN IN EUKARYOTIC SYSTEM AND ITS APPLICATION IN LUCIFERASE IMMUNOPRECIPITATION SYSTEM

In order to establish a novel serologic test—Luciferase immunoprecipitation system ( LIPS) , full?length prM?E of TBE virus was expressed by eukaryotic expression system and its effect was evaluated by LIPS technology. Firstly, using TBEV′s RNA as template, prM?E gene of TBEV was amplified by RT?PCR, eukaryotic expression vector pNLF?prM?E was constructed through inserting prM?E gene into pNLF1?secN vector. Fusion protein prM?E?luciferase was expressed by transfecting Cos7 cells. The expression of fusion protein was measured by testing values of luciferase in supernatant of Cos7 cells. Immunofluorescence assay ( IFA) was employed to identify the specificity of the recombinant protein and LIPS was used to detect the serum of TBEV infected patients. High expression level of luciferase was tested in the supernatants of the transfected cells. The specific binding of prM?E with TBEV antibody was confirmed by IFA. The LIPS assay detected 19 positive results out of 20 sera of the TBEV infected patients, 7 control sera all showed negative results. The results indicated that the expressed prM?E had good sensitivity and specitivity, which could be used as a candidate diagnostic antigen for LIPS assay for TBEV infection.