蜱传脑炎病毒包膜糖蛋白胞外区的真核表达优化及其在血清学检测中的效果评价

Codon Optimization of the Envelope Extracellular Region of Tick-Borne Encephalitis Virus Could Improve its Sensitivity in Antibody Detection Assay

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：We optimized the envelope extracellular region codens of tick-borne encephalitis virus （TBEV） to improve the expression level in eukaryotic expression system.Selecting the specific diagnostic antigens is crucial for developing highly sensitive serological methods.Methods：We optimized the envelope extracellular region codens of TBEV and expressed it as a recombinant protein in COS7 cells.Using Renilla luciferase（Rluc） as a reporter,we evaluated the sensitivity and specificity of optimized ecto-membrance（OPT-EC） protein for antibody detection by luciferase immunoprecipitation system（LIPs）.Results：By optimizing the sequence codens,the expression level of OPT-EC was improved significantly in eukaryotic expression system.The sensitivity of OPT-EC in LIPs assay reached to 86％.However,even the specificity of OPT-EC was more than 90％ when detecting JEV,YFV and AIV infected sera and sera from healthy people without any contact to TBEV,strong cross-reactions were observed when detecting WNV and DV infected sera.Conclusion：The OPT-EC antigen was sensitive in TBEV antibody detection and could distinguish TBE from JEV and YFV infection,but could not distinguish TBE from DV and WNV infection.