Expression And Purification Of GST-LMP-1 Extracellular Domain Fusion Protein Of EB Virus

Nasopharyngeal carcinoma(NPC)is malignant tumor derived from nasopharyngeal epithelium cell.Its incidence is high in certain regions such as south-east Asia,especially in the south of China.The serum detection of EBV infected patients and tissue biopsy have been provided insight to the contribution of EBV and to the development of NPC.The presence of the Epstein-Barr virus(EBV)-derived latent membrane protein-1(LMP-1)gene in nasopharyngeal swabs from patients with NPC was reported.This newly PCR technique has yielded a sensitivity rate of 94.7%and a specificity rate of 100%for NPC diagnosis. LMP-1 can stimulate or inhibit signaling pathways,resulting in transformation of rodent fibroblast cell lines,blockade of differentiation in epithelial cells,upregulation of antiapoptotic proteins,production of cytokines,upregulation of cell surface markers,upregulation of DNA methyltransferase activity,and downregulation of cell adhesion molecules and cyclin-dependent kinases.Overall,this results in greater transformation and survival in LMP 1-expressing cells.Recombinant EBV experiments have shown that LMP-1 is required for immortalization of resting B lymphocytes,and experiments in rodent fibroblast cell lines have shown that LMP1 is relevant to EBV-induced cellular transformation in non-lymphoid cells.Development of an epitope-based vaccination strategy designed to enhance.Epstein-Barr virus(EBV)-specific CD8-cytotoxic T lymphocytes(CTLs)is increasingly being considered as a preferred approach for the treatment of EBV associated relapsed Hodgkin disease (HD)and nasopharyngeal carcinoma(NPC).EBV-encoded latent membrane proteins,LMP1 and LMP2,are the only target antigens available for therapeutic augmentation of CTL responses in patients with HD and NPC.Structural LMP-1 protein was expressed in E.coli to detect and analyze anti-LMP1 antibodies in NPC patients' sera of EBV associated malignancies by Western-blot and ELISA.Methods:The plasmid pMD18-LMP 1 containing the recombinant extracellular peptide gene of LMP 1 of EBV was digested with BamH I and EcoR I, and cloned into expression vector pGEX-4T-2.The constructed vector which had been identified by enzyme digestion and nucleotide sequence analysis was transformed into BL21(DE3).After induced with IPTG,the recombinant protein was purified with GST affinity chromatography,and confirmed by Western blot.The anti-LMP-1 antibodies were detected by ELISA of 17 patients' sera with nasopharyngeal cancer.Results: The recombinant plasmid was constructed correctly by gene sequencing.SDS-PAGE and Western blot analysis showed that the recombinant protein was about 32.2 ku.The purified protein could be used to detect specific antibodies in the sera of patients with NPC by Western blot.While 17 sera of NPC patients and 10 sera of health adult being detected,13 sera of patients are positive for these antibodies and these Abs were absent for the sera of the health adult.The positive ratio of anti-LMP1 antibodies in patients with nasopharyngeal cancer is 76.5% by Western blot and 64.7%by ELISA.Conclusion:The recombinant extracellular peptide gene of LMP 1 of EBV could be expressed with high performance,and the antigenicity of the purified protein was certificated by Western blot and ELISA in sera of patients with NPC which were associated with EBV.