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构建携带hTERT I540-PTD融合基因表达框的穿梭质粒 被引量：2

Construction of shuttle plasmids containing expression cassettes of hTERT I540-PTD fusion genes
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摘要 目的: 设计hTERTI540 PTD融合基因表达框并构建携带该表达框的穿梭质粒.方法: 根据hTERTI540表位、HIV 1TAT PTD和PTD4的氨基酸序列设计融合疫苗,然后根据哺乳动物偏爱密码子反翻译成基因序列.分段合成寡聚脱氧核糖核酸,磷酸化、退火后先后克隆至pSP72的BamHI EcoRI和XbaI BanHI之间.将经双酶切鉴定获得的阳性重组子进行基因测序.然后将hTERTI540 PTD融合基因表达框亚克隆至pDC315.结果: 测序结果表明hTERTI540 PTD和hTERTI540 PTD融合表达框的基因序列与设计完全一致.穿梭质粒pDC315 I540 PTD和pDC315 I540 PTD4经EcoRI酶切后均清晰可见 112bp电泳条带,与预测相符.结论: 成功获得了携带hTERTI540 PTD和hTERTI540 PTD4表达框的穿梭质粒pDC315 I540 PTD和pDC315 I540 PTD4,为制备以重组腺病毒为载体的I540 PTD基因疫苗、比较蛋白转导能力的强弱对基因疫苗免疫效能的影响奠定了基础. AIM: To design and construct shuttle plasmids containing expression cassettes of hTERT I540-PTD fusion genes.METHODS: The fusion genes were designed based on the preferred codons of mammalian and the amino acid sequences of hTERT I540 epitope,HIV-1 TAT-PTD and PTD4,and were chemically synthesized in 6 oligonucleotides.The whole expression cassettes were then constructed by phosphorylation,annealing,and ligating into pSP72 in turns.After confirmed by restriction enzyme digestion and DNA sequencing,the fusion genes were subcloned into shuttle plasmid pDC 315.RESULTS: DNA sequencing results verified that the sequences of I540-PTD4 and I540-PTD were consistent with those we had designed.Both the pDC315-I540-PTD and pDC315-I540-PTD4 yielded a fragment of 112 bp following EcoRI digestion as we predicted.CONCLUSION: Shuttle plasmids containing expression cassettes of hTERT I540-PTD and hTERT I540-PTD4 have been successfully constructed,which provides a premise of the preparation of I540-PTD DNA vaccines using adenoviruses as gene vector and for further investigation of the relationship between the transduction ability of PTDs and the levels of antigen-specific CTLs primed by the DNA vaccines.
作者 郭晓彤 张积仁 刘文超 任军 李立文
机构地区 南方医科大学珠江医院肿瘤中心 第四军医大学西京医院肿瘤科 第四军医大学西京医院全军骨科研究所
出处 《第四军医大学学报》 北大核心 2005年第6期505-508,共4页 Journal of the Fourth Military Medical University
基金 陕西省攻关课题(2000K14 G11)
关键词 癌症疫苗 端粒酶逆转录酶 肿瘤 免疫疗法 cancer vaccines telomerase reverse transcriptase neoplasms immunotherapy
分类号 R730 [医药卫生—肿瘤]
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参考文献13

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共引文献1

同被引文献31

  • 1郭晓彤,张积仁,李立文.蛋白转导功能域在肿瘤疫苗领域的应用前景[J].第四军医大学学报,2004,25(21):2014-2016. 被引量：2
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第四军医大学学报
2005年 第6期

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