EB病毒EBNA1基因酵母工程菌的构建、表达及临床应用

Construction,expression and clinical application of Pichia pastoris for EBNA1 gene expression

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。

Objective To construct the pichia pastoris strain for effective expression of epstein - barr vires nuclear antigen 1 gene（ EBNA1 ）, and explore the application of recombinant EBNA1 protein in serological diagnosis of nasopharyngeal carcinoma （NPC）. Methods DNA extracted from the B95 -8 cells was used as the templates in a polymerase chain reaction （PCR）. EBNA1 759 bp （ aa 390 - aa 641 ） was generated and inserted into pPICZaA vector. The recombinant plasmids were transformed into GS115 yeast by electroporation. The yeast transformants were induced by methanol to express recombinant proteins. After i- dentification of SDS - PAGE and western blot, the synthesized recombinant protein was coated to mieroplate for detection of EBV - IgA antibody in NPC patients and normal persons by ELISA. Results Secretory recombinant EBNA1 protein was successful- ly expressed in the pichia pastoris. The molecular weight of products was approximately 28 000. The recombinant protein EBNA1 in the culture supernatant showed a good immtmoreactivity with IgA antibodies to EBV by Western blot. A novel ELISA was es- tablished using pichia pastoris - expressed EBNA1 protein. The sensitivity and specificity of EBNA1 as coating antigen in detec- tion of the serum specimens from NPC patients and normal persons were 81.4% and 95.8% respectively. Conehmion The se- cretory recombinant protein EBNA1 was highly exp.ressed in pichia pastoris. The diagnostic value of the recombinant protein in screening of NPC patients were primarily evaluated and the valuable pPICZa - EBNA1 yeast strain was obtained.