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目的:构建编码融合基因NT4-Apoptin-HA2-TAT的重组腺相关病毒表达载体。方法:利用限制性内切酶切相应载体后将Apoptin和HA2-TAT连入pUC19/NT4质粒,再将融合基因NT4-Apoptin-HA2-TAT亚克隆至腺相关病毒的穿梭质粒内,与辅助质粒pAAV/Ad、腺病毒质粒pFG140共同转染HEK-293细胞,通过同源重组获得NT4-Apoptin-HA2-TAT重组腺相关病毒载体,收集病毒上清,Dot blot法测定其滴度。MTT比色法观察NT4-Apoptin-HA2-TAT重组腺相关病毒表达载体,对HepG2细胞存活率的影响。结果:经酶切及测序证实克隆出NT4-Apoptin-HA2-TAT融合基因;得到高滴度的(3.14×1015pfu/L)重组腺相关病毒表达载体。NT4-Apoptin-HA2-TAT重组腺相关病毒表达载体,对HepG2细胞有强烈的诱导凋亡作用,与对照组比较,处理组细胞的存活率明显降低。结论:通过分子克隆体外重组技术成功制备了NT4-Ap-optin-HA2-TAT重组腺相关病毒载体,为下一步的Apoptin应用于基因治疗奠定了基础。
Objective: To construct a recombinant adeno-associated virus expression vector encoding fusion gene NT4-Apoptin-HA2-TAT. METHODS: Apoptin and HA2-TAT were ligated into pUC19 / NT4 plasmid by restriction enzyme digestion. The fusion gene NT4-Apoptin-HA2-TAT was subcloned into the shuttle plasmid of adeno-associated virus and ligated with helper plasmid The recombinant adeno-associated virus vector NT4-Apoptin-HA2-TAT was transfected into HEK-293 cells by homologous recombination. The virus supernatants were collected and the titers were determined by Dot blot. MTT colorimetric assay NT4-Apoptin-HA2-TAT recombinant adeno-associated virus expression vector, the survival of HepG2 cells. Results: NT4-Apoptin-HA2-TAT fusion gene was confirmed by restriction enzyme digestion and sequencing. A high titer recombinant adeno-associated virus (3.14 × 1015pfu / L) expression vector was obtained. NT4-Apoptin-HA2-TAT recombinant adeno-associated virus expression vector, HepG2 cells have a strong induction of apoptosis, compared with the control group, the survival rate of treated cells was significantly reduced. Conclusion: The recombinant adeno-associated virus vector NT4-Ap-optin-HA2-TAT was successfully prepared by molecular cloning in vitro, which laid the foundation for the further application of Apoptin in gene therapy.