介导p73去阻抑肽表达的重组腺伴病毒的构建和鉴定

目的构建编码融合基因NT4-p53(N37)-HA2-TAT的重组腺伴病毒表达载体,为恶性肿瘤基因治疗的实验研究奠定基础。方法采用互补引物二次PCR法以及T载体克隆法获得p53(N37)基因克隆,酶切后将其连同穿膜肽HA2-TAT片段一起连入pUC19/NT4质粒,再将融合基因NT4-p53(N37)-HA2-TAT亚克隆至腺伴病毒的穿梭质粒pSSHG-CMV中,构建重组质粒pSSHG-CMV/NT4-p53(N37)-HA2-TAT并进行酶切鉴定;应用磷酸钙沉淀法,pSSHG-CMV/NT4-p53(N37)-HA2-TAT、辅助质粒pAAV-Ad,腺病毒全基因组质粒pFG140三种质粒共转染HEK293细胞,包装出重组腺伴病毒rAAV/NT4-p53(N37)-HA2-TAT并用斑点杂交法测定重组病毒的滴度;MTT比色法、流式细胞仪观察重组腺伴病毒rAAV/NT4-p53(N37)-HA2-TAT对HepG2细胞的抑制作用。结果克隆出p53(N37)基因,经酶切及测序证实结果正确;得到高滴度的(2×1013pfu/L)重组腺伴病毒表达载体并对HepG2细胞有明显的抑制作用,且这一作用是通过诱导肿瘤细胞凋亡实现的。结论通过分子克隆体外重组技术成功制备了rAAV/NT4-p53(N37)-HA2-TAT复制缺陷型重组腺伴病毒,为下一步开展在p53突变或缺失肿瘤中针对p73的靶向性肿瘤基因治疗研究奠定了基础。

Construction and identification of recombinant adeno-associated virus vector mediating the expression of the derepressing p73 peptide-p53(N37)

Objective To construct a recombinant adeno-associated virus vector mediating the expression of the derepressing p73 peptide-p53(N37) so as to lay a foundation for further research on gene therapy of malignant tumors. Methods The p53(N37) gene was obtained by self-complementary primer PCR and T-vector cloning techniques;then the p53(N37) gene and HA2-TAT segment were cloned into pUC19/NT4 vector after digested with restriction enzyme.The fusion gene of NT4-p53(N37)-HA2-TAT was subcloned into the shuttle plasmid of adeno-associated pSSHG-CMV,and recombinant plasmid pSSHG-CMV/NT4-p53(N37)-HA2-TAT was constructed and identified by enzyme cutting analysis.The rAAV/NT4-p53(N37)-HA2-TAT was produced by using calcium phosphate coprecipitation method and HEK 293 cell line was co-transfected by pSSHG-CMV/NT4-p53(N37)-HA2-TAT,aid plasmid pAAV-Ad and adenovirus genome plasmid pFG140.The virus titer was determined by dot-blot hybridization.The effect of rAAV/NT4-p53(N37)-HA2-TAT on HepG2 cell line was measured by a methyl thiazolyl tetrazolium(MTT) assay and flow cytometry. Results The p53(N37) gene was confirmed by restriction enzyme digestion and DNA sequencing.High titer of recombinant adeno-associated virus was obtained by homologous recombination in HEK293 cells(2×1013pfu/L).The rAAV/NT4-p53(N37)-HA2-TAT had a notable lethal effect on HepG2 cells and this effect was realized by inducing the apoptosis of HepG2 cells. Conclusion The recombinant adeno-associated virus vector mediating the expression of the derepressing p73 peptide-p53(N37) has been successfully constructed by molecular cloning and in vitro recombination techniques in this experiment,which lays a foundation for further research on gene therapy of malignant tumors.