流行性乙型脑炎病毒SA14-14-2株囊膜蛋白的原核表达及其兔抗血清的制备  

Prokaryotic expression of envelope protein of epidemic encephalitis B virus strain SA14-14-2 and preparation of its specific rabbit anti-sera

作　　者：徐飞 冯继伟 李鹏[2] 孟玉娇 陈玉明 王龙康 王寅彪[1] XU Fei;FENG Jiwei;LI Peng;MENG Yujiao;CHEN Yuming;WANG Longkang;WANG Yinbiao(School of Public Health,Xinxiang Medical University,Xinxiang 453003,Henan Province,China;School of Life Sciences and Basic Medicine,Xinxiang University,Xinxiang 453003,Henan Province,China)

机构地区：[1]新乡医学院公共卫生学院,河南新乡453003 [2]新乡学院生命科学与基础医学学院,河南新乡453003

出　　处：《新乡医学院学报》2022年第2期101-105,共5页Journal of Xinxiang Medical University

基　　金：河南省重点研发与推广专项(编号:212102310335,202102110094);河南省本科高校省级大学生创新创业训练计划项目(编号:S202110472042);新乡医学院2019年大学生自然科学类科研课题资助(编号:xyxskyz201924)。

摘　　要：目的表达流行性乙型脑炎病毒(EEBV)的囊膜蛋白(E蛋白),制备该蛋白特异性兔抗血清,为建立诊断EEBV感染的检测方法奠定基础。方法将pET32a-E质粒转化大肠杆菌BL21(DE3)细胞,以终浓度为0.5 mmol·L^(-1)的异丙基-β-D-硫代半乳糖苷于37℃下诱导蛋白表达8 h,在8 mol·L^(-1)尿素变性条件下,采用镍离子金属螯合亲和层析法对重组E蛋白进行纯化。透析复性后,以E蛋白为免疫原免疫新西兰兔制备兔抗血清,采用Western blot、酶联免疫吸附试验和免疫荧光分析法检测免疫血清中EEBV特异性抗体。结果成功表达了重组E蛋白,经纯化、复性后可从1 L培养物中获得23.8 mg具有生物活性的E蛋白,该蛋白能与抗EEBV单抗发生特异反应。免疫制备的兔抗血清能与E蛋白发生特异反应,抗体效价高达1×51 200;兔抗血清能与EEBV感染的细胞发生特异反应。结论成功表达了EEBV E蛋白并制备了特异性强、亲和力高的兔抗血清,为建立诊断EEBV感染的免疫学检测方法、有效防控流行性乙型脑炎奠定了基础。Objective To express the envelope protein(E protein)of epidemic encephalitis B virus(EEBV)and prepare its rabbit anti-sera in order to provide the foundation for the establishment of detection methods for the diagnosis of EEBV infection.Methods pET32a-E plasmid was transformed into Escherichia coli BL21(DE3)cells.The protein expression was induced by isopropyl-β-D-thiogalactoside with a final concentration of 0.5 mmol·L^(-1) at 37℃for 8 hours,the recombinant E protein was purified by using nickel-charged nitrilotriacetic acid affinity chromatography under the condition of denaturation with 8 mmol·L^(-1) of urea.After renaturation by dialysis,New Zealand rabbits were immunized with E protein as immunogen to prepare rabbit anti-sera.The specific antibodies of EEBV in immune serum were detected by Western blot,enzyme-linked immunosorbent assay and immunofluorescence analysis.Results The recombinant E protein was successfully expressed.After purification and renaturation,23.8 mg of bioactive E protein was harvested from 1 liter of bacterial cell culture.The protein could react specifically with anti-EEBV monoclonal antibody.The rabbit anti-sera prepared by immunization could react specifically with E protein,and the antibody titer reached 1×51 200;The rabbit anti-sera could react specifically with EEBV infected cells.Conclusion Recombinant E protein was successfully expressed and rabbit anti-sera against E protein with strong specificity and high affinity was produced,which provide the foundation for the development of immunoassays to detect EEBV infection and effective prevention and control of epidemic encephalitis B.

关 键 词：流行性乙型脑炎病毒 囊膜蛋白 原核表达 兔抗血清 

分 类 号：R183.5[医药卫生—流行病学]