Development Of Indirect ELISA Based On The EBV Fusion Protein P54-ztaN Which Expressed In E.coli And Its Application In The Diagnosis Of Nasopharyngeal Carcinoma

Objective:To construct BMRF1-BZLF1N fusion gene and its prokaryotic expression vector and express it in E.coli BL21(DE3). Development of ELISA based on antigen of this fusion protein to establish a new serological parameter for NPC diagnosis.Methods:The B95-8 cells were cultured till the logarithmic phase.Total RNA in B95-8 cells were extracted, and RNA were retranscripted into cDNA. Both BMRF1 and BZLF1N were amplified from B95-8 cells cDNA, and were cloned into pGEM-T Easy vector. By Gene SOEing technique, a fusion gene fragment containing BamHâ… and Xhoâ… sites was constructed by splicing BMRF1 gene and BZLF1N gene,then cloned into pGEX5T plasmid to construct pGEX5T-BMRF1-BZLF1N recombinant plasmid.The pGEX5T-BMRF1-BZLF1N recombinant plasmid was identified by restriction endonuclease BamHâ… and Xhoâ… digestion, specific PCR and sequence analysis. The confirmed recombinant plasmid was transformed into E.coli BL21(DE3), and the expression of recombinant protein was analyzed by SDS-PAGE and Western blot. The recombinant protein was purified using the GST-glutathione affinity system. The purified recombinant protein was applied in an indirect ELISA by coating the 96-well plate to detect its IgG antibody in NPC patients, healthy controls, other head and neck malignancies patient's serum.Results:1233bp BMRF1 fragment and 519bp BZLF1N fragment were obtained by RT-PCR and were successfully cloned into pGEM-T Easy vector respectively. By Gene SOEing technique, BMRF1-BZLF1N fusion gene (linked by gene sequences encoding elastin peptides) was acpuired successfully. BMRF1-BZLF1N fusion gene was inserted into the prokaryotic expression vector pGEX-5T, the prokaryotic expression system E.coli BL21(pGEX5T-BMRF1-BZLF1N) system was constructed successfully. A recombinant protein, about 88kDa, was expressed in E.coli BL21(pGEX5T-BMRF1-BZLF1N) system. By Western blotting, the proteins expressed can be detected by corresponding antibody. We acquired high pure fusion protein by using the GST-glutathione affinity system. The mean OD450 of NPC, healthy control and other head and neck malignancies were 1.083, 0.303 and 0.396 respectively. The mean OD450 of NPC group was significant higher than that of healthy control and other head and neck malignancies, P<0.05. The group of preradiotherapy and the group of radiotherapy also showed the different ELISA OD450 values, the mean OD450 were 1.083 and 0.676 respectively. There was significant difference between two groups. The ROC curve indicate that when select OD450(0.433) as cutoff value, the ELISA test employing the recombinant protein as coating antigen to detect antibody produced in NPC showed 86% in sensitivity and 83% in specificity .Conclusion:The BMRF1-BZLF1N fusion gene of EBV could be highly expressed in E.coli BL21. The fusion protein p54-ztaN with immunoreactivity could be used as diagnostic antigen. The indirect ELISA which using the fusion protein p54-ztaN as antigen could be used as the new parameter in diagnosis of NPC. The indirect ELISA also had the potential to be used as the parameter to monitor the effect of NPC radiotherapy.