EB病毒BALF4基因在大肠杆菌中的表达纯化与鉴定

Expression,purification and identification of BALF4 gene of Epstein-Barr virus in Eschrichia coli

目的采用大肠杆菌表达EB病毒(Epstein-Barr virus,EBV)壳抗原BALF4(S)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法扩增目的基因片段BALF4(S),与原核表达载体PGEX-5X-1连接,构建PGEX-5X-BALF4(S)工程菌株,在大肠杆菌BL21(DE3)中表达GST/BALF4(S)重组融合蛋白。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BALF4(S)-IgA抗体。结果在大肠杆菌中成功地表达了GST/BALF4(S)重组融合蛋白,相对分子质量为63000,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者的灵敏度和特异度分别为78%和90%。结论本文采用大肠杆菌系统成功表达了GST/BALF4(S)重组融合蛋白,对重组抗原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应用价值的PGEX-5X-BALF4(S)工程菌株。

Objective To express BALF4（S） gene of Epstein-Barr in E.coli and analyze the application of recombinant BALF4（S） 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）.BALF4（S） gene 1008bp（aa287-623） was generated and inserted into PGEX-5X-1 vector.The recombinant plasmid was transformed into E.coli BL21（DE3）.The expression of GST / BALF4（S） fusion protein was induced by IPTG,identified by both SDS-PAGE and Western blotting,and then purified with glutathione-sepharose beads.The purified recombinant protein was coated to microplate for detection of EBV-IgA antibody in NPC patients and normal controls by ELISA.Results The GST / BALF4（S） fusion protein was successfully expressed in E.coli.The molecular weight of product was approximately 63000.The recombinant fusion protein GST / BALF4（S） showed good immunoreactivity with IgA antibodies to EBV by Western blot.The sensitivity and the specificity of GST / BALF4（S） tests in the NPC patients were 78% and 90% separately.Conclusion The recombinant protein GST / BALF4（S） was expressed in E.coli,the diagnostic value of the recombinant protein in screening of NPC patients were primary evaluated and the valuable PGEX-5X-BALF4（S） strain was obtained.