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Objective:To explore the Epstein-Barr virus (EBV) latent infection membrane protein (LMP) 1 or LMP2 specific T cell immune response and clinical significance in stage III-IVa nasopharyngeal carcinoma (NPC), aiming to provide ideas and evidence for immunotherapy in NPC.Methods:Fifty-nine NPC patients admitted to the Affiliated Tumor Hospital of Xinjiang Medical University from February 2018 to October 2020 for primary treatment were collected. Peripheral blood monocytes (PBMCs) were stimulated by LMP antigen. Intracellular cytokine staining and flow cytometry were applied to study the expression levels of IL-2, IL-13, interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) from CD4 + T and CD8 + T cells, and then analyzed in conjunction with clinical factors. Results:The positive rates of total PBMCs to LMP1 and LMP2 in NPC patients were different. The positive rate of LMP1 specific CD4 + T cells was statistically higher in stage T 3-T 4 NPC than that in stage T 1-T 2 (51.0% vs. 10.0%, P=0.042). There were also differences in the expression of cytokines between LMP1 and LMP2, CD4 +T cells and CD8 +T cells. Survival analysis showed the 2-year and 3-year overall survival (OS) rates were 91.5% and 88.2%, and the 2-year and 3-year progression-free survival (PFS) rates were 83.3% and 75.3%. Univariate analysis suggested that smoking history, male and LMP1 stimulated IL-13 positive expression in CD4 + T cells affected the disease progression ( P=0.026, 0.045 and 0.006); multivariate analysis showed LMP1 stimulated IL-13 positive expression in CD4 + T cells and smoking history were the independent prognostic factors affecting PFS ( P=0.017, 0.019). Conclusions:LMP1 and LMP2 generate specific T-cell immune response in PBMCs of NPC patients, with differential expression in two T-cell subsets. LMP1 and LMP2 specific T cell immune response is associated with primary tumor size and metastatic lymph node volume. LMP1 stimulated IL-13 positive expression in CD4 + T cells and smoking history affects the disease progression.
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Objective: Regulatory T cells (Tregs) are critical factors that impair antitumor immunity. Epstein–Barr virus (EBV)-encoded latent membrane protein 1 (LMP1) is one of the most pathogenic factors in nasopharyngeal carcinoma (NPC). However, the role of EBV-encoded LMP1 in regulating Treg generation in NPC remains unclear. Materials and Methods: The in vitro stability of activated Tregs (aTregs) influenced by LMP1 was analyzed by flow cytometry. The inhibitory effects of LMP1-HONE1 antigen-induced aTregs on tumor-associated antigen (TAA)-specific T cells were analyzed in vitro and in vivo. Finally, the expression of LMP1, Foxp3, and enhancer of zeste homolog 2 (EZH2) were analyzed in samples from 86 NPC patients by immunohistochemistry and immunofluorescence. Results: LMP1 upregulated the expression of EZH2, which increased the stability of aTregs in vitro. EZH2 inhibitor, DZnep, depleted LMP1-HONE1 antigen-induced aTregs in vitro and led to potent TAA-specific T cell antitumor immunity in vivo. In NPC tissues, LMP1 expression level was positively correlated with the number of EZH2+ Tregs, which was positively correlated with clinical stage and overall survival. Conclusions: EZH2 is essential for maintaining the stability and inhibitory functions of aTregs that are induced by EBV-encoded LMP1 in NPC
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Non-Hodgkin’s Lymphoma is a haematological malignancy with various etiological factors and one among them is Epstein Barr Virus. The expression of Epstein-Barr virus in Non-Hodgkin’s lymphoma can be identified by immunohistochemistry for detection of Epstein-Barr virus latent membrane protein (LMP-1). In our study, we are trying to clarify the extent of expression LMP-1 in our population. This can be used as a prognostic marker and for therapeutic interventions targeting EBV encoded proteins. We wanted to determine the proportion of LMP1 (EBV marker) expression in Non-Hodgkin’s Lymphoma and evaluate the association of LMP1 expression in B cell and T cell type of NHL.METHODSThis is a cross sectional analytical study conducted at Department of Pathology, Govt. Medical College, Kottayam, from December 2017 to May 2019. A total of 67 cases were studied. All of them were histopathologically diagnosed Non-Hodgkin’s Lymphoma specimens received in the Department of Pathology, Govt. Medical College, Kottayam, during the study period of 18 months. NHL was subtyped into B cell type and T cell type. LMP-1 immunohistochemistry was done on all cases to assess its expression. Then analysis was done using SPSS software and strength of association between LMP 1 positivity and cell type was calculated using Chi square test and Fisher’s exact test.RESULTSMean age of Non-Hodgkin’s lymphoma is 57.82 +/- 7.4 in this study. Minimum age is 2 years and maximum age is 89 years. The present study had 10.4 % LMP 1 positive cases. Of which there were 6% moderately stained positive cases, 3% of weakly stained cases and 1.5% cases of intensely stained cases. Among NHL 86.6% cases were B cell lymphoma and 13.4% cases of T cell lymphoma. And they had a LMP 1 positivity of 10.3% and 11.1 % respectively. But there was no significant association between LMP 1 positivity and cell type according to our study.CONCLUSIONSThe present study was done to determine the proportion of LMP 1 expression in Non-Hodgkin’s Lymphoma and to find out whether there is any association between LMP1 expression in B cell and T cell type of NHL. LMP 1 was positive in 10.4% of NHL and there was no association between LMP 1 positivity in B cell and T cell Lymphoma. This suggest that EBV might play a role in pathogenesis of NHL.
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Latent membrane protein 1 (LMP1) as a product of human herpesvirus 4 encoding is closely related to the occurrence and development of various tumors. However, the articles related to hematological tumors are rare. This review focuses on the correlation between LMP1 and hematological tumors and the progress of treatment.
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AIM: To investigate the effects of TNF receptor-associated death domains (TRADD) of latent membrane protein 1 (LMP1) on the proliferation of nasopharyngeal cancer SP18 cells.METHODS: The SP18-LMP1 cells and SP18-LMP1TRADD cells, which expressed LMP1 and LMP1 TRADD proteins, respectively, were established.The proliferation of SP18 cells affected by LMP1TRADD was detected by cell counting to analyze the cell growth curve, and by colony formation assay, soft agar formation assay, and flow cytometry.Moreover, the expression profile of differential genes between SP18-LMP1 cells and SP18-LMP1TRADD cells was analyzed by gene chips.RESULTS: The cell growth curve, and the results of colony formation and soft agar formation displayed that the growth velocity and colony forming ability of SP18-LMP1 cells were stronger than those of SP18-LMP1TRADD cells (P<0.01).The results of flow cytometry analysis showed that the proliferation index of SP18-LMP1 cells was higher than that of SP18-LMP1TRADD cells (P<0.01).Sixty-three differentially expressed genes associated with cell proliferation were screened out, in which 33 genes were up-regulated and 30 were down-regulated in the SP18-LMP1TRADD cells.CONCLUSION: TRADD active region is an important functional site of LMP1 to promote the proliferation of SP18 cells.LMP1 may improve the cell proliferation index and induce the proliferation of SP18 cells through TRADD.
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Background:Epstein-Barr virus (EBV)is associated with various human lymphoid and epithelial malignancies such as Burkitt's lymphoma,nasopharyngeal carcinoma and gastric carcinoma. LMP2A,a virus-encoded latent membrane protein is expressed in a portion of EBV-associated gastric carcinoma (EBVaGC)and has been shown to be related with the tumorigenesis and progression of EBVaGC. Aims:To explore the effect of LMP2A silencing on growth of EBVaGC cells in vitro by using a lentivirus-mediated RNA interference (RNAi)to inhibit LMP2A gene expression. Methods:A lentivirus vector pGCSIL-LMP2A-shRNA-LV and a negative control vector were constructed and transfected into the EBVaGC cell line GT38. Real-time PCR and Western blotting were used to determine the inhibitory effect of the lentivirus vector;CCK-8 assay,colony formation assay and flow cytometry were employed to assess the cell growth,cell cycle and apoptosis of GT38 cells. Results:In GT38 cells transfected with LMP2A-shRNA-LV,the expression level of LMP2A mRNA was decreased by 65. 4%,and that of LMP2A protein was reduced by 50. 8%;the cell proliferation was inhibited,the colony formation ability was suppressed,the percentage of cells in G0 / G1 phase and apoptotic rate were increased when compared with those transfected with negative control vector or without transfection (P < 0. 05). Conclusions:Lentivirus-mediated RNAi is effective for silencing LMP2A gene expression,subsequently inhibiting the growth of EBVaGC cells,inducing G0 / G1 phase arrest and enhancing cell apoptosis in vitro. LMP2A is supposed to be a potential target for gene therapy of EBVaGC.
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Objective To prepare an attenuated Listeria vaccine Lmdd-LMP2A expressing the Ep-stein-Barr virus latent membrane protein 2A ( EBV-LMP2A) and evaluate its specific anti-tumor effects on nasopharyngeal carcinoma.Methods The gene fragment encoding EBV-LMP2A was amplified by PCR analysis and then subcloned into the shuttle vector p1565.PCR restriction enzyme digestion and DNA se-quencing were performed to identify the recombinant shuttle vector p1565-LMP2A.The p1565-LMP2A vector was then transformed into competent strains of the attenuated Listeria monocytogenes ( Lmdd) .The recombi-nant attenuated Listeria vaccine strain Lmdd-LMP2A was verified by Western blot assay.The histological sections of spleen and liver tissues were stained by Haematoxylin and eosin ( H&E) for analysis of inflamma-tion.A tumor-bearing HLA-A2 transgenic mouse model was established by subcutaneous injection of CNE-1/HLA-A2/LMP2A nasopharyngeal carcinoma cell line.The prepared Lmdd-LMP2A vaccine was inoculated into the mice via tail intravenous injection for the evaluation of specific CTL induction and the in vivo anti-tumor effects.Results The shuttle vector p1565-LMP2A and the recombinant attenuated Listeria vaccine strain Lmdd-LMP2A with stable expression of LMP2A protein were successfully constructed.The immunized mice showed mild inflammations with no structural damage and necrosis as indicated by H&E staining.The growth of tumors in tumor-bearing HLA-A2 transgenic mice was significantly inhibited by the immunization of Lmdd-LMP2A vaccine as compared with mice without inoculation.The survival time of mice was prolonged with the immunization of Lmdd-LMP2A vaccine.Conclusion The prepared attenuated Listeria vaccine Lm-dd-LMP2A showed specific anti-tumor effects with the safety advantage, suggesting the possibility of using it for anti-tumor therapy in clinic.
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Objective To explore the clinical significance of the serum Epstein-Barr virus determined nuclear antigen 1 (EBNA1)/latent membrane protein 1 (LMP1) in patients with extranodal NK/T-cell lymphoma,nasal type (ENKL).Methods The serum EBNA1 and LMP1 were detected by realtime PCR in 36 ENKL patients hospitalized in Beijing Tongren Hospital from August 2010 to August 2013.Twenty healthy volunteers were recruited as controls.Results The median serum EBNA1 was 1.9 × 104 (ranged from 0 to 11.0 × 104) copies/μl in ENKL patients and 8.0 (ranged from 0 to 43.8) copies/μl in healthy volunteers.The median serum LMP1 was 3.9 × 103 (ranged from 118.3 to 24.0 × 103) copies/μl in ENKL patients and 3.3 (ranged from 0 to 33.3) copies/μl in healthy volunteers.Both EBNA1 and LMP1 were higher in ENKL patients than healthy volunteers (all P ＜ 0.01).The median EBNA1 and LMP1 in ENKL patients posttreatment were 1.0 × 103 (ranged from 0 to 2.0 × 103) copies/μl and 300.8(ranged from 0 to 825.7) copies/μl respectively,which were both significantly decreased than pretreatment (all P ＜ 0.05).The EBNA1 and LMP1 were decreased in effective treatment group versus ineffective treatment group (P ＜0.05).The serum EBNA1 and LMP1 were positively correlated with lactic dehydrogenase (LDH) level (r =0.364,0.546 ; P =0.040,0.012).Conclusions (1) The measurement of EBNA1/LMP1 may be useful in evaluating the therapeutic effect.(2)The serum EBNA1/LMP1 may reflect the tumor load in ENKL patients.
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Objective To investigate the distribution characteristics of the Epstein-Barr virus (EBV)in lymphomas.Methods 438 cases of lymphomas were reclassified according to the WHO classification of lymphoma (2008).ALK1,CD3,CD5,CD7,CD10,CD15,CD20,CD23,CD30,CD43,CD56,CD68,CD79,CD99,CyclinD1,EMA,IgD,TdT,Vs38C and LMP-1 were detected by in situ hybridization of EBER and immunohistochemistry.Results In B cell lymphoma,T and NK cell lymphoma and Hodgkin' s lymphoma (HL),the positive rates of EBER were 5.4 ％ (14/261),16.5 ％ (19/115) and 59.7 ％ (37/62),respectively,and the positive rates of LMP-1 were 5.4 ％ (14/261),5.2 ％ (6/115) and 59.7 ％ (37/62).In DLBCL patients,EBER expression in the older group was significantly higher than that in the younger one [13.2 ％ (7/53) vs 1.2 ％ (1/81),P ＜ 0.05].The expression of EBER and LMP-1 were inconsistent in T and NK cell lymphomas,and the positive rate of EBER was significantly higher than that of LMP-1 (P ＜ 0.05).EBER was all positive in 5 cases of NK/T cell lymphoma-nasal type.The expression of EBER and LMP-1 were consistent in HL.Conclusion The EBV infection was associated with the classification of the lymphoma.The EBV infection was the highest in NK/T cell lymphoma-nasal type,and the next was in HL.Due to the consistency of EBER and LMP-1 expression in the HL,economically,LMP-1 may replace EBER as the indicator of EBV.EBV might play an important role in the occurrence and development of NK/T cell lymphoma-nasal type,HL and so on.
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Objective To assess the diagnostic value of the latent membrane protein 1(LMP-1)gene detection for diagnosing na-sopharyngeal carcinoma(NPC).Methods The related researches on the detection of LMP-1 gene in the nasopharyngeal swab secre-tion for diagnosing NPC by PCR were entirely collected by using the computer retrieval or manual inquiring.Two estimators screened the literature according to the criteria of inclusion and exclusion.The quality evaluation was performed by adopting the QUADAS scale.The heterogeneity test was conducted by using Meta-Disc 1.4 software.According to the heterogeneous character-istics,the corresponding effect model was selected for calculating the pooled sensitivity and specificity and the summary receiver op-erating character(SROC)curve was drawn.Results 139 related articles were retrieved,in which 6 articles were finally included. 394 cases of NPC were definitely diagnosed by the pathological golden standard,802 cases were in the control group.The pooled sensitivity and specificity of the LMP-1 gene for diagnosing NPC were 0.90[95%CI (0.87,0.93)]and 0.98[95%CI (0.96,0.99)] respectively.The area under SROC(AUC)was 0.973 7.Conclusion LMP-1 gene has the higher diagnostic value for NPC and could be used for screening and auxiliary diagnosis of NPC.
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Objective To investigate the expression of Epstein-Barr virus(EBV)-latent membrane protein 1 (LMP1) in chronic atrophic gastritis (CAG) with intestinal metaplasia and discuss its effect on gastric cancer.Methods Immunohistochemistry was used to examine the expression of EBV-LMP1 in 45 cases of chronic superficial gastritis(CSG),63 cases of CAG with intestinal metaplasia and 36 cases of gastric cancer.Results There was no expression of EBV-LMP1 in CSG and gastric cancer,while the positive rate of EBV-LMP1 in CAG with intestinal metaplasia was 36.5％ (23/63) and EBV-LMP1 was mainly stained in the cell nucleus.The expression of EBV-LMP1 in CAG with intestinal metaplasia was significantly higher than that in CSG and gastric cancer,and there was significant difference (P =0.000).Conclusions EBV-LMP1 is expressed in CAG with intestinal metaplasia.The expression of EBV-LMP1 is significantly higher than that in CSG and gastric cancer indicating that EBV infection in gastric carcinogenesis may play an important role in the early stages.
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Aims: To determine the effect of cytokines, namely transforming growth factor-beta one (TGF-β1), and interleukin-6 (IL-6) on the expression of 88 cancer-related microRNAs (miRNAs) in TW01 nasopharyngeal carcinoma (NPC) cells with or without the presence of Epstein-Barr virus latent membrane protein 1 (LMP1). Methodology: TW01 and TW01-LMP1 cells were treated with cytokines. MicroRNAs were isolated from treated and untreated TW01/TW01-LMP1 cells and were subjected to RT-PCR array of 88 cancer-related microRNAs. The threshold cycle (Ct) data were analysed and fold-change in the level of gene expression was calculated based on ΔΔCt using two endogenous controls, SNORD 47 and SNORD 44. Data obtained from each treatment were compared with the data obtained from the respective control group (untreated TW01/ TW01-LMP1). Results: TGF-β1 down-regulated miR-143 in TW01 NPC cells. In TW01 cells that expressed the EBV LMP1 gene (TW01-LMP1), approximately 97% of the 88 miRNAs were up-regulated by TGF-β1. Among them was miR-181c a well-known repressor of NOTCH2/4 and KRAS and has important role in cell differentiation. IL-6 up-regulated approximately 65% of the miRNAs in TW01 cells but in less than four-fold. In TW0- LMP1 cells, eight miRNAs; namely, miR-15b, miR-155, miR-16, miR-215, miR-23b, miR-25, miR-9 and miR-98 were significantly up-regulated by IL-6. Among these, miR- 15b, miR-155 and miR-25 had been reported to be elevated in NPC tissues. Conclusion: This study provides a preliminary perspective on the effects of cytokines on the expression of miRNAs in TW01 NPC cells.
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Objective: Epstein–Barr virus (EBV) latent membrane protein 1 (LMP1) is known to plays important role in B cells growth and transformation. LMP1 is considered to be a functional homologue of the CD40 receptor and they can activate many overlapping signaling pathways. In this study, we compared the function of CD40 with that of LMP1 in B cells transformation. Materials and methods: Expression of CD40L was observed in infected B cells with LMP1 mutated EBV. To observe the expression reverse transcription-PCR were performed. Results: This expression of CD40L did not support proliferation and transformation of B cell. Even in vitro proliferation and transformation of B cell infected with LMP1 deleted EBV supplemented with CD40L were also not observed. Conclusion: Despite many similarities shared between CD40 and LMP1, CD40-CD40L interaction didn’t complement on LMP1 mediated B cells transformation in vitro.
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Epstein-Barr virus (EBV) latent infection transforms B lymphocytes into proliferating lymphoblastoid cell lines (LCLs). EBV latent infection membrane protein 1 (LMP1) is required for EBV-mediated B lymphocyte transformation, and LMP1-induced NF-kappaB activation is essential for LCL survival. Previously, it was reported that the level of reactive oxygen species (ROS) and the expression of apoptosis signal-regulating kinase 1 (ASK1) are elevated in EBV-positive Burkitt's lymphoma (BL) cells, the potential role of ASK1 in LMP1-induced NF-kappaB activation was thus investigated in this study. In EBV-positive BL cells, ASK1 was highly expressed and activated. In addition, TRAF6-ASK1 interaction was significantly increased in EBV-positive BL cells. Interestingly, the expression of LMP1 alone facilitated ASK1 activation. The expression of a dominant negative ASK1 mutant (ASK1KM) strongly blocked LMP1-induced NF-kappaB activation. Furthermore, LMP1-induced NF-kappaB activation was significantly reduced in ASK1 knock out (ASK1-/-) mouse embryonic fibroblasts (MEFs). Taken together, these results demonstrate that ASK1 is activated by LMP1 and is critical for LMP1-induced NF-kappaB activation.
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Animales , Ratones , Linfocitos B , Linfoma de Burkitt , Línea Celular , Fibroblastos , Herpesvirus Humano 4 , Activación de Linfocitos , MAP Quinasa Quinasa Quinasa 5 , Proteínas de la Membrana , FN-kappa B , Especies Reactivas de OxígenoRESUMEN
Objective: To investigate the effects of Epstein-Barr virus-latent membrane protein 1 (LMP1) and p38 mediating epithelial-mesenchymal transition of nasopharyngeal carcinoma (NPC), and to elucidate its relationship with tumor metastasis. Methods: The expression levels of E-cadherin, vimentin, LMP1 and p38 in NPC tissues and cell lines were detected by immunohistochemistry, immunocytochemistry, RT-PCR and Western blotting. The invasion and migration abilities of NPC cell lines were examined by Transwell assay. Results: The positive expression rates of E-cadherin in different NPC tissues and lymph node metastases were decreased (P<0.01), whereas the positive expression rates of vimentin and LMP1 were increased (P<0.001). The difference in the positive expression rate of p38 was not statistically significant (P<0.05).The expression of LMP1 was negatively associated with the expression of E-cadherin in NPC tissues (P<0.05). In cervical lymph node metastasis, the expression of LMP1, which had no association with the expression of E-cadherin (P<0.05), was associated with the expression of vimentin (P<0.05). The expressions of E-cadherin mRNA and protein were suppressed in CNE1-GL and CNE2Z cells, whereas the expressions of vimentin mRNA and protein were increased. The expression level of LMP1 protein was high in CNE1-GL and CNE2Z cells, and the expression level of p38 protein was high in CNE1-GL cells. Transwell assay showed that LMP1 could promote the migration and invasion abilities of NPC cells (P<0.001). Conclusion: LMP1 can induce EMT and enhance the metastatic potential of nasopharyngeal carcinoma. Copyright© 2011 by Tumor.
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AIM: To investigate the differential expression profile between nasopharyngeal carcinoma cell line CNE1 and its steady EBV-LMP1-transfected cell line CNE1-LMP1, and to explore the regulatory effect of LMP1 on oncomiRs expression in CNE1 cell line. METHODS: A microRNA array that targets 132 of the most well studied oncomiRs was used to detect the expression profile of CNE1 and CNE1-LMP1. qRT-PCR assay were used to verify the expression data detected by microarray. RESULTS: Among the restricted 132 miRNAs, 30 were detectable. Among which, 30 were expressed in CNE1-LMP1, 19 in CNE1 and 11 were specifically expressed in CNE1-LMP1. Among the 19 shared miRNAs, the expression level of 6 miRNAs (hsa-miR-19b, hsa-miR-17-3p, hsa-miR-22, hsa-miR-149, hsa-miR-150 and hsa-miR-188) elevated over two folds in CNE1-LMP1. No decrease in miRNA expression more than two folds was observed. qRT-PCR confirmed the expression difference of these six miRNAs (P<0.01). Among the 11 specifically expressed miRNAs in CNE1-LMP1, hsa-miR-122a showed the highest expression level surpassing the internal control sample. CONCLUSION: Our data suggest that LMP1 may play an important role in regulating the expression of miRNAs in tumor, which may be another important pathway employed by LMP1 in the development of nasopharyngeal carcinoma.
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AIM: To investigate the molecular mechanism of Epstein-Barr virus encoded latent membrane protein 1 regulated cellular proliferation in nasopharyngeal epithelial cells. METHODS: The nasopharyngeal epithelial cells NP69 were infected with RV-pLNSX (the empty vector) and RV-LMP1 retroviruses, respectively. Therefore, the NP69-pLNSX and NP69-LMP1 cell lines were established. Sequentially, cellular proliferation of NP69-pLNSX and NP69-LMP1 cells was compared to draw the cellular growth curve. The experiments of plate clone formation and forming of soft agar colony were conducted. Meanwhile, the differential expression of proteins were identified between NP69-pLNSX and NP69-LMP1 cell lines by proteomic methods, and the expression levels of partial identified proteins were verified. RESULTS: (1) LMP1 was able to accelerate cellular proliferation of nasopharyngeal epithelial cell NP69 (n=3, P<0.05). (2) Twenty two proteins (9 up-and 13 down-regulated) of LMP1 mediated regulation were identified from infected NP69 cell lines, and the differential expression of partial identified proteins was confirmed by Western blotting and fluorescent real-time quantitative RT-PCR. CONCLUSION: LMP1 probably mediates the regulation of vimentin protein and keratin 19 protein expression to promote cellular proliferation in NP69 cells.
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Objective To express in prokaryotic system and to analyze the antigenic specificity of EB virus(EBV) latent membrane protein 2(LMP2) multi-epitopes gene rich of T cell and B cell epitopes.Methods Using on-line prediction service, T cell epitopes and B cell epitopes of EB virus latent membrane protein 2 were predicted. The genes rich of CTL and th cell epitopes were selected as the candidate gene sequences, while B cell epitopes around them were taken into account. The finial selected multi-epitope gene was synthesized after being optimized according to prokaryotic codon bias and inserted into prokaryotic expression vector pET32a( + ) to get the recombinant plasmid: pET32a( + )/EBV-LMP2 multi-epitopes. After transformed into E. coli BL21 (DE3) and induced by IPTG, the target multi-epitopes gene can be expressed as Trx-His fusion protein. The expression products can be identified by SDS-PAGE and Western blot. Moreover, rabbit serum antibody to EBV membrane protein and nasopharyngeal carcinoma(NPC) patient serum were used respectively to detect the antigenic specificity of the multi-epitopes. Meanwhile, 6-8 weeks female BALB/c mice were immunized with EBV-LMP2 multi-epitope at 2 week intervals, three times in all, Trx-His protein and PBS were set as the control groups. At the second week after the last immunization, the mice were sacrificed. LDH and indirect ELISA were taken to detect the specific spleen CTL activityand specific IgG in serum, which reflected the immunogenicity of the EBV-LMP2 multi-epitope. Results Two amino acid sequences which locate at the LMP2 (aa195 -232 ) and LMP2 (aa419-436 ) were selected and connected in series to be the target gene. The recombinant plasmid containing EBV-LMP2 multi-epitope gene successfully constructed and the target protein was expressed in E. coli BL21 ( DE3 ). The relative molecular mass(Mr) of The expression products is about 27 × 103 , which matches up to the expected Mr. The antigenic specificity of the multi-epitopes protein was identified by Western blot and the multi-epitopes protein also can be detected by rabbit serum antibody to EBV membrane protein and NPC patient serum respectively. In the result of the animal experiment, EBV-LMP2 multi-epitope was able to induce the specific CTL activity in BALB/c mice. With the increasing of the effector: target ( E: T) 1: 5,1: 10, 1: 25, the CTL activity was also increased wih( 12.52% + 2.59% ), (21.80% + 1.08% ), (23.68% + 3.74% ) respectively; EBV-LMP2 multi-epitope was able to induce LMP2-specific antibody response(A490 =0.258 +0.040) as compared with Trx-His protein(A490 =0.095 +0.011) and PBS(A490 =0.068 +0.014,P＜0.05＝. Conclusion The EBV-LMP2 multi-epitopes gene was designed successfully and expressed precisely in prokaryotic expression system with good antigenicity and immunogenicity.
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Objective:To evaluate the expression of vascular endothelial growth factor C(VEGF-C),Epstein-Barr virus(EBV) latent membrane protein 1(LMP1),cyclooxygenase-2 (COX-2) in nasopharyngeal carcinoma(NPC).Method:LMP1, COX-2 and VEGF-C were detects by immunohistochemical staining for 57 case NPC tissue.Result:The positives rates of LMP1,COX-2 and VEGF-C detected by immunohistochemical staining were 49.1%(28/57), 75.4%(43/57)and 59.6%(34/57), respectively. The expression of LMP1, COX-2 and VEGF-C were correlated to each1 other in NPC(P<0.05).Conclusion:LMP1 and COX-2 may induce expression of VEGF-C directly or LMP1 induce expression of VEGF-C by induce COX-2 expression, may contribute to lymph metastasis and develop NPC.
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To find Epstein-Barr virus (EBV) strains with genetic variations of EBV latent membrane protein 1 (EBV-LMP1) from nasopharyngeal carcinoma (NPC), the full-length DNA of LMP1 genes from 21 NPC biopsies obtained in Hunan province in southern China was amplified and sequenced. Our sequences were compared to those previously reported by the Clustal V method. Results showed that all 21 sequences displayed two amino acid changes most frequently in LMP1 of CD4+ T cell epitopes at codons 144 (F arrow right I, 21/21) and 212 (G arrow right S, 19/21) or (G arrow right N, 2/21). We also show that type A EBV strain is prevalent in the cases of NPC from Hunan province with a 30-bp 18/21 deletion, and we highlight that this deletion resulted in loss of one of the CD4+ T cell-restricted epitopes. The other 3 sequences without this deletion all had a change at codon 344 (G arrow right D). Furthermore, in the major epitope sequence of CD8+ T cells restricted by HLA-A2, all 21 sequences showed changes at codons 126 (L arrow right F) and 129 (M arrow right I). Our study discovered that one of the 21 sequence variations harbored a new change at codon 131 (W arrow right C), and 5/21 specimens showed another novel change at codon 115 (G arrow right A) in the major epitope sequence of CD8+ T cells restricted by HLA-A2. Our study suggests that these sequence variations of NPC-derived LMP1 may lead to a potential escape from host cell immune recognition, protecting latent EBV infection and causing an increase in tumorigenicity.