NT4-Ant-Shepherdin[79-87]融合基因重组腺相关病毒载体的构建及鉴定

Construction and identification of recombinant adeno-associated virus vector harboring fusion gene NT4-Ant-Shepherdin[79-87]

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。

AIM： To construct a recombinant adeno-associated virus vector harboring fusion gene NT4-Ant-Shepherdin[79-87] and investigate Survivin as a anticancer therapeutic target by use of Shepherdin [79-87]. METHODS： The gene of Ant-Shepherdin [79-87] was obtained by PCR and T-vector method. After inserted in PBV220-NT4 vector and digested with restricted enzyme, The fusion gene of NT4-Ant-Shepherdin[79-87] was sub-cloned into the shuttle plasmid of adeno-associated virus; the products were cotransferred into HEK-293 cell line with helper plasmid pAAV-Ad and adeno-plasmid pFGI40. The recombinant adeno-associated virus was produced by homologous recombination of above 3 plasmids in HEK-293 cells and its titer was meas- ured by Dot-blot hybridization. The effect of rAAV-NT4-Ant- Shepherdin[79-87] on A549 cell line was measured by a colorimetric 3-（4, 5-dimethyl-2-thiazolyl）-2, 5-diphenyl-2H-tetrazolium bromide （MTT） assay. RESULTS： DNA sequencing results verified that the sequence of Ant-Shepherd- in[79-87] was consistent with that we had designed. After transformed E. coil DH5α, a fragment of 321 bp was confirmed. High titer of recombinant adeno-associated virus was obtained by homologous recombination in HEK-293 cell lines （3.4×10^13pfu/L）, rAAV-NT4-Ant-Shepherdin[79-87] had strong induce apoptosis effect on A549 cells. CONCLUSION： The recombinant adeno-associated virus vector encoding fusion gene NT4-Ant-Shepherdin[79-87] is success-fully constructed in this experiment by molecular cloning and in vitro recombination techniques, which provided the basis of further research of Survivin for cancer gene therapy.