NT4-p53(N15)-Ant融合基因的克隆和鉴定

目的构建NT4-p53(N15)-Ant融合基因表达盒并进行序列分析。方法应用互为模板的引物PCR技术及T载体克隆法克隆p53(N15)-Ant基因,筛选阳性克隆、酶切鉴定并测序。扩增阳性重组质粒后限制性内切酶切取p53(N15)-Ant片段连入pBV220/NT4质粒。结果克隆了p53(N15)-Ant基因,经酶切及测序证实结果正确;重组质粒pBV220/NT4p53(N15)Ant经限制性内切酶及琼脂糖凝胶电泳,结果显示酶切片段大小和理论值一致。结论通过分子克隆体外重组技术成功制备了含有NT4-p53(N15)-Ant表达盒的pBV220质粒,为进一步开展肿瘤的基因治疗奠定了基础。

Construction and identification of fusion gene of NT4-p53 (N15)-Ant

Objective To construct the expression box of fusion gene NT4-p53(N15)-Ant.Methods The gene of p53(N15)-Ant was obtained by PCR of two primers templating with each other and T-vector cloning method.The positive clone was identified and analyzed by the restriction enzymes and sequencing respectively.After digested with restriction enzyme,the interest gene of p53(N15)-Ant was subcloned into the plasmid pBV220/NT4.Results The gene of p53(N15)-Ant was confirmed by the digestion of restriction enzyme and sequencing.The recombinant plasmid pBV220/NT4p53(N15)Ant was identified by the digestion of restriction enzyme and agarose gel electrophoresis and the results conformed theoretical values.Conclusion The plasmid pBV220 containing the expression box of NT4-p53(N15)-Ant was successfully constructed by molecular cloning and recombination techniques in vitro,which will guide further study on gene therapy of cancer.