[1]徐　飞,冯继伟,李　鹏,等.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[J].新乡医学院学报,2022,39(2):101-105.[doi:10.7683/xxyxyxb.2022.02.001]
　XU Fei,FENG Jiwei,LI Peng,et al.Prokaryotic expression of envelope protein of epidemic encephalitis B virus strain SA14-14-2 and preparation of its specific rabbit anti-sera[J].Journal of Xinxiang Medical University,2022,39(2):101-105.[doi:10.7683/xxyxyxb.2022.02.001]
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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
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《新乡医学院学报》[ISSN:1004-7239/CN:41-1186/R]

卷:
39
期数:
2022年2
页码:
101-105
栏目:
基础研究
出版日期:
2022-02-05

文章信息/Info

Title:
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1; 冯继伟1; 李　鹏2; 孟玉娇1; 陈玉明1; 王龙康1; 王寅彪1
(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）
Author(s):
XU Fei1; FENG Jiwei1; LI Peng2; MENG Yujiao1; CHEN Yuming1; WANG Longkang1; WANG Yinbiao1
(1.School of Public Health,Xinxiang Medical University,Xinxiang 453003,Henan Province,China；2.School of Life Sciences and Basic Medicine,Xinxiang University,Xinxiang 453003,Henan Province,China)
关键词:
流行性乙型脑炎病毒; 囊膜蛋白; 原核表达; 兔抗血清
Keywords:
epidemic encephalitis B virus; envelope protein; prokaryotic expression; rabbit anti-sera
分类号:
R183.5
DOI:
10.7683/xxyxyxb.2022.02.001
文献标志码:
A
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。
Abstract:
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.

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更新日期/Last Update: 2022-02-05