EB病毒潜伏期膜蛋白2A重组腺病毒的制备及表达

Generation and expression of Epstein-Barr Virus Latent membrane protein 2A recombinant adenovirus

目的 :构建EB病毒潜伏期膜蛋白 2A重组腺病毒 ,研究EB病毒相关肿瘤治疗性疫苗。方法 :利用RT PCR扩增出EB病毒B95 8株潜伏期膜蛋白 2A(LMP2A)全部编码蛋白的基因 ,并克隆至pGEM T载体中。并将LMP2AcDNA插入E1、E3区替代的腺病毒载体pAX1CW ,选择正确的克隆pAX1CW LMP2A与Ad5DNA 末端肽复合体共转染 2 93细胞 ,通过同源重组获得复制缺陷型的重组腺病毒。提取重组腺病毒转染的 2 93细胞DNA ,通过酶切初步鉴定。选择阳性克隆感染CV1细胞 ,通过流式细胞仪和激光共聚集显微镜分析LMP2A蛋白表达情况。结果 :挑选同源重组 17个克隆中有 9个为阳性克隆 ,扩增到的病毒滴度为 2 3× 10 8pfu/ml。用MOI =10 0的重组腺病毒感染CV1细胞 ,48h后在激光共聚焦显微镜下可见LMP2A蛋白表达于CV1细胞膜上 ,经流式细胞仪检测LMP2A蛋白表达阳性细胞数为 94 4 %。结论 :重组腺病毒能有效地介导LMP2A基因的表达 ,为进一步研究该基因功能及其工程疫苗打下了基础。

Objective:To generate Epstein-Barr virus(EBV) Latent Membrane Protein 2A(LMP2A) recombinant adenovirus,and provide for further investigation on the therapy vaccine against EBV associated malignancies.Methods:Full length cDNA of encoding LMP2A of EBV had been amplified by reverse transcription-PCR and cloned into pGEM-T vector.The encoding cDNA of LMP2A was inserted into E1,E3-substituted adenovirus vector pAX1CW,then the LMP2A recombinant adenovirus vector was contransfected into 293 cells togetherwith EcoT221 digested Ad5-TPC.The LMP2A recombinant adenovirus was generated by homologous recombination,and primarily identificated by ClaI enzyme digestion.The expression of LMP2A on CV1 cells infected with recombinant adenovirus analyzed by fluorescence-activated cell sorting(FACS) and confocal microscope.Results:The replication-deficient LMP2A recombinant adenovirus was generated efficiently with the titers of 2.3×10 8 pfu/ml.The LMP2A could be seen on CV1 cells membrane with confocal microscope 48 h post infected with recombinant adenovirus and the percentage of CV1 cells expressing LMP2A was 94.4% by means of FACS analysis.Conclusion:These suggested that LMP2A could be expressed efficiently by recombinant adenovirus mediated transfer,and it was the foundation of further researching in its function and developing the suitable genetic engineering vaccine against EBV associated malignancies.