EB病毒重组抗原Rta蛋白的表达及用于鼻咽癌筛查ELISA方法的建立

Expression of Rta Recombinant Antigen of Epstein-Barr Virus and Establishment of ELISA Method for Nasopharyngeal Carcinoma Screening

目的制备和表达EB病毒重组抗原Rta蛋白,并建立筛查鼻咽癌的ELISA方法,为鼻咽癌的早期筛查提供科学依据。方法设计引物扩增BRLF1编码区全长序列,构建重组质粒p PICZαA-BRLF1,电转入毕赤酵母GS115,筛选His+Mut+表型转化子并用甲醇进行诱导表达和纯化;建立Ig G/Rta抗体的ELISA检测技术,用于300例鼻咽癌患者和200例健康对照者血清的筛查,对检测结果进行评价。结果成功构建p PICZαA-BRLF1重组质粒;目的蛋白在毕赤酵母GS115中高效分泌表达(50%),蛋白电泳显示在66.0 k Da处有相应条带;同时Western-blot也证明其有良好的免疫活性;利用该重组Rta蛋白建立的ELISA检测方法对鼻咽癌检测阳性率显著高于Ig A/VCA试剂盒(P<0.01)。结论本研究构建了EB病毒重组抗原Rta蛋白的真核表达载体,表达外源蛋白产量高,免疫活性好,可用于Ig G/Rta ELISA检测技术研究,适合于鼻咽癌的大规模筛查和早期诊断。

Objective To prepare and express the Rta recombinant antigen of Epstein-Barr virus and to establish an ELISA method in order to facilitate the screening of nasopharyngeal carcinoma （NPC）. Methods The target gene BRLF1 which coded for Rta was ampliflied by PCR with a specific primer. The recombinant vector pPICZctA-BRLF1 was electronically transformed into pichia pastoris GS115 to express Rta protein so as to screen the His＋Mut＋ phenotype transformant before proteins were purified and the immunogenicity of the product was proved by Western blot. ELISA was established with the IgG/Rta anti- body and was applied to the detection of 300 patients with NPC and 200 healthy people to assess the capability of detection. Resuits The pPICZαA-BRLF1 recombinant plasmid was successfully constructed and the target protein was secreted and expressed with high-performance （50%）. Protein electrophoresis demonstrated a corresponding band in 66 KDa. Western blot showed a good immune-competence with the IgG monoclonal antibody of Rta. The Rta recombinant antigen was used for establishment of the ELISA method. The positive rate of this method was significantly higher than that of IgA/VCA Kit for screening NPC （P〈0.01）. Conclusion A eukaryotic expression vector of the Rta recombinant antigen of Epstein-Barr virus has been constructed that can ensure the high-performance of foreign proteins with good immunogenicity for the development of IgG/Rta ELISA methods. The detection capability can be improved by combining other antigens and this method is applicable to large-scale screening and diagnosis of NPC.