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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.
Objective: To design a hTERTI540 PTD fusion gene expression cassette and construct a shuttle plasmid carrying the expression cassette. Methods: Design the fusion vaccine based on the amino acid sequences of the hTERTI540 epitope, HIV 1 TAT PTD and PTD4, and then reverse-translate the gene sequence according to mammalian preference codons. Fragments of synthetic oligodeoxyribonucleotides were phosphorylated and annealed and were subsequently cloned between BamHI EcoRI and XbaI BanHI of pSP72. The positive recombinants identified by double enzyme digestion were sequenced. The hTERTI540 PTD fusion gene was then expressed. Box was subcloned into pDC315. Results: Sequencing results showed that the gene sequences of the hTERTI540 PTD and hTERTI540 PTD fusion expression cassettes were completely consistent with the design. The shuttle plasmids pDC315 I540 PTD and pDC315 I540 PTD4 were clearly cleavable with 112 bp electrophoretic bands after EcoRI digestion. The predictions are consistent. Conclusions: The shuttle plasmids pDC315 I540 PTD and pDC315 I540 PTD4 harboring the hTERTI540 PTD and hTERTI540 PTD4 expression cassettes were successfully obtained, which was used to prepare the I540 PTD gene vaccine based on the recombinant adenovirus and compare the transduction ability of the protein. The foundation of the effect of genetic vaccine immunity is laid.