WT1蛋白CTL表位肽基因载体的构建与鉴定  

Construction and identification of genetic vector containing CTL epitope of WT1 protein

作　　者：彭霞[1] 樊卫平[1] 张凯[1] 苑晓娟[1] 刘玲玲[1] 王亮[1] 

机构地区：[1]山西医科大学微生物免疫教研室,山西太原030001

出　　处：《中国生物制品学杂志》2013年第4期448-451,共4页Chinese Journal of Biologicals

基　　金：国家自然基金专项(81041081)

摘　　要：目的构建WT1(Wilms’tumor gene 1)蛋白CTL表位肽基因载体,并检测其在293T细胞中的转录。方法设计分别含WT1-126肽、WT1-235肽以及这两种肽的基因载体,并加入Th通用表位Pan-DR-Th(PADRE),应用蛋白酶体切割软件PAProC和NetChop优化各表位和间隔序列,DNA疫苗在线预测工具DyNAVacS优化真核密码子后,人工合成核苷酸序列,分别插入pUC57载体,构建pUC57-WT1质粒,测序鉴定后,酶切回收各目的片段,亚克隆至真核表达载体pcDNA3.1(+)中,构建重组真核表达质粒,转染293T细胞,RT-PCR检测各目的基因在293T细胞中的转录。采用无内毒素质粒大量提取试剂盒提取各重组质粒,采用紫外分光光度计测定质粒的纯度和浓度。结果各重组真核表达质粒经双酶切及测序鉴定证实构建正确;重组质粒携带的目的基因可在293T细胞中成功转录;各重组质粒DNA的纯度均合格,浓度在864.6～883.9μg/ml之间。结论成功构建了WT1蛋白CTL表位肽基因载体,并能在真核细胞中正常转录,为下一步在小鼠体内探讨特异性不同的CTLs群发挥抗肿瘤作用的机制奠定了基础。Objective To construct the genetic vector containing CTL epitopes of WT1（Wilms＇ tumor gene 1） protein and determine its transcription in 293T cells.Methods Genetic vector containing WT1-126 peptide,WT1-235 peptide and both of the two peptides were designed respectively,in each of which universal epitope Pan-DR-Th（PADRE） was introduced.The sequences of various epitopes and their gaps were optimized by using proteasome cutting software PAProC and NetChop,while the eukaryotic codon by DNA vaccine online prediction tool DyNAVacS,based on which the nucleotide sequences were synthesized and inserted into vector pUC57.After the constructed recombinant plasmid pUC57-WT1 was identified by sequencing,the target gene fragments were recovered and subcloned into eukaryotic expression vector pcDNA3.1（＋）.293T cells were transfected with the constructed recombinant plasmids and determined for transcription of various target genes by RT-PCR.The recombinant plasmids were extracted by endotoxin-free large-scale plasmid extraction kit,and determined for purity and concentration by ultraviolet spectrophotometer.Results Restriction analysis and sequencing proved that the eukaryotic expression vectors were constructed correctly,in which the target genes were transcribed in 293T cells successfully.The purities of DNAs in various recombinant plasmids were qualified,while the concentrations were 864.6 ~ 883.9 μg / ml.Conclusion The genetic vector containing CTL epitopes of WT1 protein were successfully constructed and normally transcribed in eukaryotic cells,which laid a foundation of further study on anti-tumor mechanism of various specific CTLs.

关 键 词：WT1蛋白 细胞毒性T淋巴细胞 表位肽 DNA疫苗 

分 类 号：Q782[生物学—分子生物学]