CAJ | 학술논문

目的对EB病毒(EBV)潜伏膜蛋白2(LMP2)中富含T、B细胞多表位肽段基因进行原核表达,并分析该多表位蛋白的抗原特性.方法利用计算机在线软件预测EBV LMP2蛋白的CTL表位、Th表位.选取富含CTL表位和Th表位的肽段,兼顾其上下游已预测的B细胞表位,组成含多个T、B细胞表位的EBV LMP2多表位,该多表位基因序列经原核密码子优化后全序列合成,并克隆入原核表达载体pET32a(+)得到pET32a(+)/EBV-LMP2多表位重组质粒,经IPTG诱导在E.coli BL21(DE3)表达并纯化,经SDS-PAGE和Western blot分析鉴定;采用EBV膜蛋白家兔免疫血清和鼻咽癌患者血清进行Western blot分析其抗原特性;并利用EBV-LMP2多表位蛋白免疫BALB/c小鼠,分别采用LDH和ELISA方法检测小鼠脾细胞特异性CTL杀伤效应及血清特异性抗体IgG水平,以分析该表位蛋白的免疫原性.结果 LMP2(aa195-232)和LMP2(aa419-436)肽段富含CTL、Th和B细胞表位,将其串联后作为EBV LMP2多表位,该多表位基因在大肠杆菌中获得了表达.表达产物的相对分子质量(Mr)约27×103,与预期Mr相符;经Western blot证实EBV-LMP2多表位具有抗原特异性,可被EBV膜蛋白家兔免疫血清和鼻咽癌患者血清特异性抗体识别;小鼠免疫结果显示EBVLMP2多表位可诱导机体产生特异性的CTL杀伤效应,随着效靶比(1:5,1:10,1:25)增加,CTL杀伤活性逐渐增强(12.52%±2.59%,21.80%±1.08%,23.68%±3.74%),同时产生了特异性血清IgG抗体反应(A490=0.258±0.040),与对照组比较差异均具有统计学意义(P＜0.05).结论本研究没计的EBV-LMP2多表位具有良好的抗原性和一定的免疫原性.
목적 대EB병독(EBV)잠복막단백2(LMP2)중부함T、B세포다표위태단기인진행원핵표체,병분석해다표위단백적항원특성.방법 이용계산궤재선연건예측EBV LMP2단백적CTL표위、Th표위.선취부함CTL표위화Th표위적태단,겸고기상하유이예측적B세포표위,조성함다개T、B세포표위적EBV LMP2다표위,해다표위기인서렬경원핵밀마자우화후전서렬합성,병극륭입원핵표체재체pET32a(+)득도pET32a(+)/EBV-LMP2다표위중조질립,경IPTG유도재E.coli BL21(DE3)표체병순화,경SDS-PAGE화Western blot분석감정;채용EBV막단백가토면역혈청화비인암환자혈청진행Western blot분석기항원특성;병이용EBV-LMP2다표위단백면역BALB/c소서,분별채용LDH화ELISA방법검측소서비세포특이성CTL살상효응급혈청특이성항체IgG수평,이분석해표위단백적면역원성.결과 LMP2(aa195-232)화LMP2(aa419-436)태단부함CTL、Th화B세포표위,장기천련후작위EBV LMP2다표위,해다표위기인재대장간균중획득료표체.표체산물적상대분자질량(Mr)약27×103,여예기Mr상부;경Western blot증실EBV-LMP2다표위구유항원특이성,가피EBV막단백가토면역혈청화비인암환자혈청특이성항체식별;소서면역결과현시EBVLMP2다표위가유도궤체산생특이성적CTL살상효응,수착효파비(1:5,1:10,1:25)증가,CTL살상활성축점증강(12.52%±2.59%,21.80%±1.08%,23.68%±3.74%),동시산생료특이성혈청IgG항체반응(A490=0.258±0.040),여대조조비교차이균구유통계학의의(P＜0.05).결론 본연구몰계적EBV-LMP2다표위구유량호적항원성화일정적면역원성.
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.