EB病毒gp125蛋白的酵母表达及在鼻咽癌检测中的应用

Expression of gp125 Protein of Epstein-Barr Virus in Pichia pastoris and Application in Detection of Nasopharyngeal Carcinoma

【目的】采用毕赤酵母系统构建表达EB病毒gp125重组蛋白,并探讨其在鼻咽癌血清学诊断中的应用。【方法】以EBV DNA为模版,采用PCR法扩增gp125的编码基因BALF4(2574 bp),与毕赤酵母表达载体pPIC9连接,转化酵母菌GS115,甲醇诱导重组蛋白表达。重组蛋白经SDS-PAGE、免疫印迹法鉴定后,包被聚丙乙烯板,制备ELISA试剂检测鼻咽癌患者和正常人群gp125-IgA抗体。【结果】在毕赤酵母菌中高效地表达了分泌型的gp125重组蛋白,相对分子质量为110 000,免疫印迹证实目的蛋白有免疫原性,gp125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78.0%和92.0%。【结论】采用毕赤酵母系统高效分泌表达了gp125重组蛋白,初步评估了gp125蛋白在鼻咽癌血清筛选中的诊断价值。

[Objective] To analyze the application of recombinant gp125 proteins in serological diagnosis of nasopharyngeal carcinoma （NPC）.[Methods] DNA was extracted from the B95-8 cells was used as the templates in a polymerase chain reaction （PCR）.gp125 gene （2574 bp） was generated and inserted into pPIC9 vector.The recombinant plasmids were transformed into GS115 yeast by electroporation.The yeast transformants induced by methanol expressed recombinant proteins.The recombinant proteins synthesized were coated to microplate for detection of EBV-IgA antibody in NPC patients by ELISA.[Results] We have successfully secretly expressed the recombinant gp125 protein in the Pichia pastoris.The molecular weight of product was approximately 110 000.The recombinant protein gp125 in the culture supernatant showed good immunoreactivity with IgA antibodies to EBV by Western blot.A novel ELISA was established using Pichia pastoris-expressed gp125 proteins.Serum samples were collected from patients with NPC and healthy controls and using this ELISA tested.The sensitivity and the specificity of gp125 tests in the NPC sera were 78.0％ and 92.0％,separately.[Conclusion] The recombinant proteins gp125 were highly secretly expressed in pichia pastoris,the diagnostic value of the recombinant protein in screening for NPC patients was primary evaluated and the valuable pPIC9-gp125 yeast strain was obtained.