CAJ | 학술논문

目的：拟在大肠杆菌中高效表达纯化乙型脑炎病毒EDⅢ蛋白，希望能为乙型脑炎（乙脑）病毒（ JEV）感染血清的诊断试剂盒提供候选抗原。方法针对乙脑病毒的EDⅢ蛋白设计特异的PCR扩增引物，对提取的乙脑病毒RNA进行RT-PCR扩增，获取目的基因并构建重组质粒，将重组质粒转入E．coli BL21中并在IPTG作用下表达目的蛋白。将表达的乙型脑炎病毒EDⅢ抗原系列稀释后与对照抗原同时点制在ELISA微孔板中，采用Array-ELISA技术同时检测临床JEV感染患者及正常人血清，并与间接免疫荧光方法的检测结果进行比较。结果成功克隆了JEV EDⅢ蛋白基因段，并在E．coli BL21中表达，成功获得EDⅢ蛋白。该重组蛋白可用于Array-ELISA方法，进行JEV感染血清的检测，并且与传统免疫学检测方法---间接免疫荧光法结果一致。结论该重组抗原可作为JEV感染血清检测的候选抗原。
목적：의재대장간균중고효표체순화을형뇌염병독EDⅢ단백，희망능위을형뇌염（을뇌）병독（ JEV）감염혈청적진단시제합제공후선항원。방법침대을뇌병독적EDⅢ단백설계특이적PCR확증인물，대제취적을뇌병독RNA진행RT-PCR확증，획취목적기인병구건중조질립，장중조질립전입E．coli BL21중병재IPTG작용하표체목적단백。장표체적을형뇌염병독EDⅢ항원계렬희석후여대조항원동시점제재ELISA미공판중，채용Array-ELISA기술동시검측림상JEV감염환자급정상인혈청，병여간접면역형광방법적검측결과진행비교。결과성공극륭료JEV EDⅢ단백기인단，병재E．coli BL21중표체，성공획득EDⅢ단백。해중조단백가용우Array-ELISA방법，진행JEV감염혈청적검측，병차여전통면역학검측방법---간접면역형광법결과일치。결론해중조항원가작위JEV감염혈청검측적후선항원。
Objective To express and purify Japanese encephalitis virus ( JEV) EDⅢprotein and evaluate the possibility of using it as a candidate antigen in JEV diagnostic kit .Methods PCR primers spe-cific for the gene encoding JEV EDⅢprotein were designed and used to amplify the gene fragment by RT-PCR.The cloned gene fragment was then inserted into pET-30a (+) to construct the recombinant expression plasmid.The transformed E.coli BL21 carrying expression plasmid were induced by IPTG to express JEV EDⅢ protein.The expressed JEV EDⅢprotein and a control antigen of tick-borne encephalitis virus protein were deposited in small spots to set up ELISA microarray .The serum samples from patients with Japanese encephalitis and healthy people were detected by Array-ELISA.The results obtained by Array-ELISA were compared with those by using indirect immunofluorescence assay .Results The gene fragment encoding JEV EDⅢprotein was successfully cloned and expressed in E.coli BL21.The recombinant protein could be used in Array-ELISA assay for the detection of serum samples from patients with Japanese encephalitis and healthy subjects .The results were consistent with those by using indirect immunofluorescence assay.Conclusion The recombinant JEV EDⅢprotein can be used as a candidate antigen for the diagnosis of JEV infection .