论文部分内容阅读
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 the mumps virus nucleoprotein (NP) with good antigenicity and to establish a specific serological test for mumps virus IgG. Methods mumps virus RNA was extracted and the full length of NP gene was amplified by RT-PCR and ligated into pET-32a (+) vector to construct recombinant plasmid pET-32a (+) - NP. The strain BL21 (DE3) was induced by isopropyl-β-D-thiogalactopyranoside (IPTG), and the soluble components were precipitated by saturated ammonium sulfate precipitation, The column was purified by affinity chromatography to obtain high-purity NP protein and identified by Western blot. The purity of anti-mumps IgG in serum of 348 healthy children was tested by ELISA (enzyme-linked immunosorbent assay, ELISA) Mumps virus ELISA IgG antibody detection kit (Victoria Run ELISA) for parallel comparison. Results The coincidence rate of NP ELISA and Veretin ELISA established in this study was 78.45% (273/348). Compared with Veretin ELISA, the sensitivity was 72.58% (135/186) and the specificity was 85.19% (138/162 ). The positive rates of IgG antibodies against mumps virus in 348 healthy children by two methods were 45.69% (159/348) and 53.45% (186/348), respectively. Conclusion The NP antigen with mumps antigen characteristic was successfully prepared by prokaryotic expression, and the mumps virus IgG antibody ELISA assay was established, which laid the foundation for the epidemiological investigation of mumps virus in human population. However, The test assesses the sensitivity and specificity of both methods.