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目的:优化蜱传脑炎病毒包膜糖蛋白胞外区的密码子,提高其在真核系统中的表达量,为蜱传脑炎病毒感染血清的检测提供更为有效的检测抗原。方法:对蜱传脑炎病毒MDJ01株E蛋白胞外区进行真核密码子优化,获得优化的胞外区蛋白基因序列OPT-EC;将抗原基因与海肾萤光素酶基因Rluc融合构建重组质粒pc DNA3.1-Rluc-OPT-EC、pc DNA3.1-Rluc-EC,用重组质粒转染COS7细胞获得融合抗原Rluc-OPT-EC和Rluc-EC,并将融合抗原用于临床感染血清样本的免疫共沉淀检测以评价抗原的灵敏性和特异性。结果:真核密码子优化显著提高了Rluc-OPT-EC的表达量;OPT-EC的检测灵敏度可达86%以上,其对乙型脑炎病毒(JEV)感染血清、黄热病度(YFV)感染血清、禽流感病毒(AIV)感染血清和正常人血清的检测特异度高于90%,但是在西尼罗病毒(WNV)感染血清和登革病毒(DV)感染血清的检测中发生了交叉反应。结论:优化后的E蛋白胞外区蛋白作为TBEV感染的检测抗原,能够有效地区分JEV、YFV感染,但不能区分WNV和DV感染。
OBJECTIVE: To optimize the codons of extracellular domain of tick-borne encephalitis virus envelope glycoprotein and improve its expression in eukaryotic system so as to provide a more effective detection antigen for the detection of tick-borne encephalitis virus-infected sera. Methods: The extracellular domain of tick-borne encephalitis virus MDJ01 strain was eukaryotic codon optimized, and the optimized OPT-EC gene sequence was obtained. The antigen gene was fused with renilla luciferase gene Rluc Plasmids pcDNA3.1-Rluc-OPT-EC and pc DNA3.1-Rluc-EC were transfected into COS7 cells with recombinant plasmids to obtain the fusion antigens Rluc-OPT-EC and Rluc-EC. Co-immunoprecipitation of samples to assess antigen sensitivity and specificity. Results: The expression of Rluc-OPT-EC was significantly enhanced by eukaryon codon usage. The detection sensitivity of OPT-EC was over 86%. The sensitivity of OPT-EC to sera of Japanese encephalitis virus (JEV), yellow fever (YFV ) Infected sera, bird flu virus (AIV) sera, and normal human sera were over 90% specific but detected in sera of West Nile virus (WNV) and sera infected with dengue virus (DV) Cross reaction. CONCLUSION: The optimized E protein extracellular domain protein as the detection antigen for TBEV infection can distinguish JEV and YFV infection effectively, but it can not distinguish between WNV and DV infection.