摘要
目的构建NT4-p53(N37)-HA2-TAT融合基因表达盒并进行序列分析。方法应用互补引物二次PCR技术及T载体克隆法克隆p53(N37)基因,筛选阳性克隆、酶切鉴定并测序。扩增阳性重组质粒后用限制性内切酶切取p53(N37)和HA2-TAT片段连入pUC19/NT4质粒。结果克隆了p53(N37)基因,经酶切及测序证实结果正确;重组质粒pUC19/NT4-p53(N37)-HA2-TAT经限制性内切酶及琼脂糖凝胶电泳鉴定,结果显示酶切片段大小和理论值完全一致。结论通过基因克隆体外重组技术成功制备了含有NT4-p53(N37)-HA2-TAT表达盒的pUC/19质粒,为进一步开展肿瘤的基因治疗奠定了基础。
Objective To construct the expression box of fusion gene NT4-p53 （N37） -HA2-TAT. Methods The DNA fragment encoding p53 （N37）apoptotic peptide was amplified using self-complementation PCR method, and cloned into the pGEM-T Easy. pGEM- T/p53 （N37）was confirmed by restriction enzyme analysis and sequencing respectively. After digested with restriction enzyme, the inter- est gene of p53 （N37） and cell-penetrating peptide HA2-TAT was subcloned into the plasmid pUC19/NT4. Results The gene of p53 （ N37 ） was successfully confirmed by the digestion of restriction enzyme and sequencing. The digestion of restriction enzyme and agarose gel electrophoresis showed that the fragment length of recombinant plasmid pUC19/NT4-p53 （N37）-HA2-TAT was conformed to the theoretical value. Concluzion The plasmid pUC19 containing the expression box of NT4-p53 （N37）-HA2-TAT can be successfully constructed by molecular cloning and recombination techniques in vitro, which will guide further study on gene therapy of cancer.
出处
《山西医科大学学报》
CAS
2009年第12期1057-1061,F0003,共6页
Journal of Shanxi Medical University
