摘要
目的可溶性表达具有良好抗原性的腮腺炎病毒核蛋白(nuclear protein,NP),并建立特异性的腮腺炎病毒IgG抗体血清学检测方法。方法提取腮腺炎病毒RNA,用RT-PCR法扩增NP基因全长,并连接至pET-32a(+)载体,构建重组表达质粒pET-32a(+)-NP,鉴定正确后转化大肠埃希菌BL21(DE3)菌株,异丙基-β-D-硫代半乳糖苷(isopropyl-β-D-1-thiogalactopyranoside,IPTG)诱导表达,可溶性成分经饱和硫酸铵沉淀粗提后,再利用镍柱进行亲和层析纯化,获得高纯度的NP蛋白,并用Western blot方法进行鉴定。包被纯化后NP抗原建立间接酶联免疫吸附测定(enzyme-linked immunosorbent assays,ELISA)检测方法 (NP ELISA法),并检测了348份健康儿童血清中抗腮腺炎病毒IgG抗体水平,与维润腮腺炎病毒ELISA IgG抗体检测试剂盒(维润ELISA法)进行平行比较。结果本研究建立的NP ELISA法和维润ELISA法符合率为78.45%(273/348);与维润ELISA法比较灵敏性为72.58%(135/186),特异性为85.19%(138/162)。两种方法检测348份健康儿童血清腮腺炎病毒IgG抗体的阳性率分别为45.69%(159/348)和53.45%(186/348)。结论利用原核表达的方法成功制备出具有腮腺炎抗原特性的NP抗原,并建立了腮腺炎病毒IgG抗体ELISA检测方法,为腮腺炎病毒人群血清流行病学调查奠定了基础,但需进一步使用中和试验评估两种方法的敏感性和特异性。
Objective To express mumps virus nuclear protein（NP）with good antigenicity and established a specific serological test method for mumps virus IgG antibody. Methods Mumps virus RNA was extracted to amplify the full length of NP protein coding region by RT-PCR,PCR product was inserted into pET-32a（＋）vector to construct a recombinant expression plasmid pET-32a（＋）-NP,and transformed to E.coli BL21（DE3）,and expression was induced by IPTG.The ammonium sulfate precipitated crude extract was purified by nickel column affinity chromatography to obtain high purity NP protein and identified by Western Blot.An indirect ELISA with purified NP antigen（NP ELISA）was established,and the anti-mumps virus IgG in serum of 348 children aged 1-3years were detected,with parallel comparison detection by Virion/Serion ELISA kit. Results The total agreement rate of the two methods was 78.45%（273/348）.The sensitivity was 72.58%（135/186）and the specificity was 85.19%（138/162）.The positive rates of mumps IgG antibodies for 348 serum samples by NP ELISA and Virion/Serion ELISA were 45.69%（159/348）and53.45%（186/348）,respectively. Conclusions Recombined antigen of mumps virus nuclear protein was successfully prepared in the prokaryotic expression system and an ELISA detection method of mumps IgG antibody was established.
出处
《中国病毒病杂志》
CAS
2017年第3期185-190,共6页
Chinese Journal of Viral Diseases
基金
十二五国家科技重大专项(2013ZX10004-803)
内蒙古自治区研究生科研创新项目(S20161013211)
