EB病毒EBNA1基因酵母工程菌的构建、表达及临床应用

发布时间：2018-03-08 18:36

本文选题：鼻咽癌　切入点：EB病毒核抗原　出处：《中国卫生检验杂志》2014年17期 　论文类型：期刊论文

【摘要】：目的建立能有效表达EB病毒核抗原1(EBNA1)基因的酵母工程菌株,并探讨EBNA1重组蛋白在鼻咽癌血清学诊断中的应用。方法以EBV DNA为模板,采用PCR法扩增目的基因片段EBNA1,与毕赤酵母表达载体pPICZaA连接,转化酵母菌GS115,甲醇诱导重组蛋白表达。表达产物经SDS-PAGE、免疫印迹法鉴定后,纯化目的蛋白作为包被抗原,制备ELISA试剂检测鼻咽癌患者和正常人群EBNA1-IgA抗体。结果在毕赤酵母菌中成功高效地表达了分泌型的EBNA1重组蛋白,相对分子质量为28 000,免疫印迹证实目的带有免疫原性,目的蛋白经纯化后作为包被抗原检测鼻咽癌患者和健康对照的敏感度和特异度分别为81.4%和95.8%。结论采用毕赤酵母系统高效分泌表达了EBNA1重组蛋白并对其在鼻咽癌血清筛选中的诊断价值进行了初步评估,获得了在鼻咽癌筛选中有一定诊断价值的pPICZa-EBNA1生产酵母工程菌株。
[Abstract]:Objective to establish a yeast engineering strain that can effectively express EBV nuclear antigen 1EBNA1 gene, and to explore the application of EBNA1 recombinant protein in serological diagnosis of nasopharyngeal carcinoma. Methods EBV DNA was used as a template. The target gene fragment EBNA1 was amplified by PCR and ligated with Pichia pastoris expression vector pPICZaA, then transformed into yeast GS115, and methanol induced the expression of recombinant protein. The expressed product was identified by SDS-PAGE.After being identified by Western blotting, the target protein was purified as coating antigen. ELISA reagent was prepared to detect EBNA1-IgA antibody in patients with nasopharyngeal carcinoma and normal population. Results the secreted EBNA1 recombinant protein was successfully expressed in Pichia pastoris with a relative molecular weight of 28000. The objective of immunoblotting was immunogenicity. Objective the sensitivity and specificity of purified protein for detecting nasopharyngeal carcinoma patients and healthy controls were 81.4% and 95.8 respectively. Conclusion the recombinant EBNA1 protein was secreted by Pichia pastoris and expressed in nasopharyngeal carcinoma blood. The diagnostic value of screening was preliminarily evaluated. An engineering strain of yeast producing pPICZa-EBNA1 with diagnostic value in nasopharyngeal carcinoma screening was obtained.
【作者单位】：中山大学附属第三医院检验科;海南省人民医院检验科;
【基金】：教育部第44批留学回国人员科研启动基金(教外司留(2012)940)
【分类号】：R739.63

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