论文部分内容阅读
目的:设计构建融合基因NT4-Ant-Shepherdin[79-87]的重组腺相关病毒表达载体。方法:应用非对称引物/模板法,PCR技术制备Ant-Shepherdin[79-87]cDNA片段,通过连接pGEM-T-Easy载体及PBV220-NT4质粒,转化感受态细胞,获得NT4-Ant-Shepherdin[79-87]融合基因,连接腺相关病毒穿梭质粒pSSHG-CMV,采用磷酸钙沉淀法三质粒共转染HEK-293细胞获取重组腺相关病毒,Dot-blot法测定重组病毒滴度,MTT比色法观察重组腺相关病毒对A549细胞存活率的影响。结果:合成Ant-Shepherdin[79-87]基因,连接PBV220-NT4,经克隆、酶切,琼脂糖凝胶电泳证实获得321bp的NT4-Ant-Shepherdin[79-87]目的基因;得到高滴度的(3.4×1013pfu/L)重组腺相关病毒表达载体;带有融合基因NT4-Ant-Shepherdin[79-87]的重组腺相关病毒对A549细胞具有强烈的诱导凋亡作用。结论:成功构建了NT4-Ant-Shepherdin[79-87]融合基因的重组腺相关病毒表达载体,为进一步研究针对Survivin的靶向抗肿瘤作用奠定了基础。
Objective: To construct a recombinant adeno-associated virus expression vector with fusion gene NT4-Ant-Shepherdin [79-87]. METHODS: Ant-Shepherdin [79-87] cDNA fragment was prepared by asymmetric primer / template method and PCR technique. The competent cells were transformed by ligating pGEM-T-Easy vector and PBV220-NT4 plasmid. NT4- 79-87] fusion gene was ligated with adeno-associated virus shuttle plasmid pSSHG-CMV. Recombinant adeno-associated virus was co-transfected into HEK-293 cells by calcium phosphate precipitation method. The recombinant virus titer and MTT colorimetric assay Method to observe the effect of recombinant adeno-associated virus on A549 cell survival rate. Results: Ant-Shepherdin [79-87] gene was synthesized and ligated with PBV220-NT4. The 321 bp NT4-Ant-Shepherdin [79-87] gene was confirmed by cloning, digestion and agarose gel electrophoresis. High titer (3.4 × 1013pfu / L) recombinant adeno-associated virus expression vector; recombinant adeno-associated virus with fusion gene NT4-Ant-Shepherdin [79-87] had a strong induction of apoptosis on A549 cells. CONCLUSION: The recombinant adeno-associated virus expression vector with NT4-Ant-Shepherdin [79-87] fusion gene was successfully constructed, which lays the foundation for further study on the targeted anti-tumor effect of Survivin.