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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 use E. coli to express Epstein-Barr virus (EBV) shell antigen BALF4 (S) recombinant protein and to explore its application in serological diagnosis of nasopharyngeal carcinoma. Methods EBV DNA was used as a template to amplify the target gene fragment of BALF4 (S) by PCR and ligated with prokaryotic expression vector PGEX-5X-1 to construct PGEX-5X-BALF4 (S) GST / BALF4 (S) recombinant fusion protein was expressed. The expressed product was identified by SDS-PAGE and Western blotting, then purified the target protein as coating antigen and ELISA kit was used to detect BALF4 (S) -IgA antibody in nasopharyngeal carcinoma patients and normal controls. Results The recombinant fusion protein of GST / BALF4 (S) was successfully expressed in Escherichia coli. The molecular weight of the recombinant fusion protein was 63000. Immunoblotting confirmed the immunogenicity of the target protein. The purified protein was used as antigen for the detection of nasopharyngeal carcinoma patients and The sensitivity and specificity of healthy controls were 78% and 90%, respectively. Conclusion The recombinant fusion protein GST / BALF4 (S) was successfully expressed in Escherichia coli system. The diagnostic value of recombinant antigen in serum screening of nasopharyngeal carcinoma was preliminarily evaluated, and it has obtained some value in the screening of nasopharyngeal carcinoma PGEX-5X-BALF4 (S) engineered strain.