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目的:制备重组EB病毒(Epstein-Barr virus,EBV)壳抗原(viral capsid antigen,VCA)BALF4蛋白,并探讨其在鼻咽癌检测中的应用价值。方法:采用PCR法从EBV DNA序列中扩增BALF4基因,酵母表达载体pPICZaA连接,重组pPICZαA-BALF4载体转染GS115酵母菌,甲醇诱导重组BALF4蛋白表达并纯化。重组BALF4蛋白经SDS-PAGE、Western blotting鉴定后作为包被抗原,制备ELISA试剂,检测鼻咽癌患者EBV-IgA抗体。结果:成功构建pPICZαA-BALF4表达载体,并在GS115酵母菌中高效地表达重组BALF4蛋白。重组BALF4蛋白相对分子质量为52000,经Western blotting证实重组BALF4蛋白具有免疫原性。重组BALF4蛋白作为包被抗原,检测鼻咽癌患者和正常对照血清(各300份)中EBV-IgA抗体的灵敏度和特异性分别为81%和94%。结论:成功制备重组EB病毒壳抗原BALF4蛋白,该蛋白在鼻咽癌血清筛选具有较高的敏感度及特异性。
OBJECTIVE: To prepare BALF4 protein of Epstein-Barr virus (EBV) viral capsid antigen (VCA) and to explore its value in the detection of nasopharyngeal carcinoma. Methods: The BALF4 gene was amplified by PCR from the EBV DNA sequence. The yeast expression vector pPICZaA was ligated. The recombinant pPICZαA-BALF4 vector was transfected into yeast of yeast strain GS115. The recombinant BALF4 protein was induced by methanol and purified. The recombinant BALF4 protein was identified by SDS-PAGE and Western blotting as coating antigen to prepare ELISA reagent for detection of EBV-IgA antibody in patients with nasopharyngeal carcinoma. Results: The pPICZαA-BALF4 expression vector was successfully constructed and recombinant BALF4 protein was expressed efficiently in GS115 yeast. Recombinant BALF4 protein molecular weight of 52000, confirmed by Western blot recombinant BALF4 protein is immunogenic. The sensitivity and specificity of recombinant BALF4 protein as coating antigen for detection of EBV-IgA antibody in nasopharyngeal carcinoma patients and normal control sera (300 copies each) were 81% and 94%, respectively. CONCLUSION: BALF4 protein of recombinant Epstein-Barr virus (Epstein-Barr virus) shell antigen was successfully prepared and it has high sensitivity and specificity in screening serum of NPC patients.