WT1抗原多表位与热休克蛋白70刺激表位融合基因疫苗的构建、表达及免疫原性研究  

Construction of Fusion Gene Vaccine of WT1 Multi-epitope Fused with Stimulating Epitope of Mycobacterium Tuberculosis Heat Shock Protein 70 and Its Expression and Immunogenicity

作　　者：田卫伟[1] 乔振华[1] 杨林花[1] 王宏伟[1] 覃艳红[1] 边思成[1] 

机构地区：[1]山西医科大学第二医院血液科,山西太原030001

出　　处：《中国实验血液学杂志》2011年第2期485-490,共6页Journal of Experimental Hematology

摘　　要：本研究用基因工程方法构建一种由WT1抗原多表位与结核分枝杆菌热休克蛋白70(mHSP70)刺激表位组成的融合基因疫苗并检测其表达和病疫原性。根据文献资料,筛选WT1抗原有良好免疫原性的3个受HLA0201与1个受HLA2402限制性的细胞毒性T细胞(CTL)表位及2个辅助性T细胞(Th)表位,加入通用外源性T细胞刺激表位Pan-DR-Th(PADRE)后,以不同的间隔序列相连,蛋白酶体切割软件优化多表位构成,合成1条由732 bp组成的多表位WT1基因片段,并插入真核表达质粒pcDNA3.1(+)中。通过PCR技术从结核分支杆菌基因组(mycobacterium tuberculosis heat shock protein 70,mHSP70)中扩增包含mHSP70刺激表位的DNA片段,亚克隆至pcDNA3.1(+),测序正确后将含有多表位的WT1基因片段亚克隆入该载体上游,构建融合基因疫苗pcDNA3.1-WT1-mHSP70407-426。用RT-PCR方法检测该基因在293T细胞表达,并免疫C57BL/6小鼠,酶联免疫吸附斑点法(ELISPOT)检测该疫苗的细胞免疫学反应。结果表明:通过蛋白酶体切割工具PAPROC及NETCHOP3.1预测,复合多表位基因工程疫苗各表位可被正确裂解,PCR和酶切鉴定结果表明,构建的重组真核表达质粒pcDNA3.1-WT1-mHSP70407-426含有正确编码的融合基因片段,并在真核细胞获得了正确表达。将该基因疫苗免疫小鼠后,可诱导特异性的CTL应答。结论:成功构建含有WT1多表位与热休克蛋白70刺激表位融合基因疫苗。This study was purposed to construct a fusion DNA vaccine containing WT1 multi-epitope and stimulating epitope of mycobacterium tuberculosis heat shock protein 70 and to detect its expression and immunogenicity.On the basis of published data,a multi-epitope gene（Multi-WT1） containing three HLA ＊0201-restricted CTL epitopes： one HLA＊2402-restricted CTL epitope,two Th epitopes and one universal Th Pan-DR epitope（PADRE） was constructed.DNA-coding sequence was modified by Computer-Aided Design（CAD） to optimize proteasome-mediated epitope processing through the introdcution of different amino acid spacer sequences.The synthetic nucleotide sequence was then inserted into an eukaryotic vector to construct the plasmid pcDNA3.1-WT1.For enhancing CTL activity,HSP70 fragment including stimulatory domain P407-426 was amplified by PCR from mycobacterial HSP70 gene and cloned into pcDNA3.1（＋）.Then Multi-WT1 was fused to the N-terminal of pcDNA3.1-mHSP70407-426 to make the multi-epitope fusion gene vaccine pcDNA3.1-WT1-mHSP70407-426.HEK-293T cells were transfected with this vaccine and the expressed product was identified by RT-PCR.Enzyme-linked immunospot assay（ELISPOT） was used to evaluate the immunological responses elicited by vaccine.The results showed that the most of WT1 epitopes could be correctly cleaved which was confirmed by software Net Chop 3.1 and PAPROCⅠanalysis.RT-PCR showed correct expression of target gene in HEK293T cells and ELISPOT showed specific T-cell responses.It is concluded that the eukaryotic expression vector PcDNA3.1-WT1-mHSP70407-426 fusion gene has been successfully constructed and the immunity response is also elicited,which is a good candidate for further research of DNA vaccine.

关 键 词：WT1 热休克蛋白70 多表位 基因疫苗 

分 类 号：Q78[生物学—分子生物学] R392.27[医药卫生—免疫学]