H-2K^b限制性WT1蛋白CTL表位肽基因疫苗的构建与鉴定

Construction and identification of the H-2K^b restricted genetic vaccine containing CTL epitope of WT1 protein

目的构建H-2Kb限制性WT1蛋白CTL表位肽的真核表达载体并检测其在293T细胞中的表达,并鉴定疫苗抗肿瘤效应。方法筛选鉴定H-2Kb限制性WT1蛋白CTL表位9肽,设计并合成肽疫苗核酸序列,插入p UC57载体,经酶切和测序鉴定,亚克隆至真核表达载体pc DNA3.1(+)及p EGFP-N1载体,转染293T细胞,RT-PCR鉴定目的基因的转录,共聚焦显微镜鉴定目的基因的表达。构建pc DNA3.1(+)-WT1免疫小鼠,取小鼠脾细胞进行体外特异CTL杀伤FBL3细胞试验。结果构建的基因疫苗经测序、双酶切及PCR鉴定确认无误;其携带的目的基因可在293T细胞中表达。免疫小鼠后特异CTL对FBL3杀伤结果高于对照组(P<0.05)。结论成功构建了H-2Kb限制性WT1蛋白CTL表位肽基因疫苗,载体疫苗能在真核细胞中成功表达,疫苗特异CTL具有体外杀伤肿瘤细胞功能,为提高WT1肽疫苗的抗肿瘤效应提供了新思路。

This study performed to construct the H-2K6 restricted genetic vaccine containing CTL epitope of WT1 （Wilms＇ tumor gene 1） protein and detect its expression in 293T ceils and its anti-tumor effects in mouse. The epitopes were designed, and the nucleotide sequences of the epitope were synthesized and inserted into vector pUC57. After being identified by endonuclease digestion and sequence analysis, the correct target fragments were subcloned into eukaryotic expression vector peDNA3.1 （＋） and pEGFP-N1. The constructed recombinant plasmids were transfected into 293T cells. Then, the transcription and expression of the target WT1 gene were identified by RT-PCR and conlbcal microscopy, respectively. After injection with the WT1 DNA vaccine, mice were killed and the cytotoxic activity test of the specific CTLs in spleen cells to FBL3 was performed. Data showed that the recombinants were correctly constructed and the target genes could be successfully expressed in 293T cells. The cytotoxic activity of the experimental group was stronger than that of control group （P〈0.05）. Taken together, H-2Kh restricted genetic vaccine containing CTL epitope of WT1 has successfully constructed and normally expressed in eukaryotic cells. The specific CTLs show high cytotoxic activity to tumor cells. The study may lay a foundation for further research of improving the anti tumor effects of WT1 Dentide vaccine.