肿瘤抑素T_7肽及其衍生物T_7-NGR载体构建及表达

Cloning and expression of the tumstatin active peptides- T_7 and its derivant-T_7-NGR

目的为了提高作用靶向性,将导向肽NGR与肿瘤抑素T_7肽的C端连接,获得T_7肽及其衍生物T_7-NGR的载体。方法通过PCR和合成基因序列方法分别构建了肿瘤抑素T_7肽及T_7-NGR的克隆载体pMD-T_7和pMD-T_7N,经酶切和测序鉴定后,将2种载体分别双酶切,经低熔点琼脂糖凝胶电泳分离后,切下目的片段与酶切回收的质粒载体pET28a在低熔点琼脂糖中直接进行连接反应。阳性克隆经酶切和测序鉴定,转化感受态E.coliBL21(DE3),IPTG诱导表达。结果酶切和测序鉴定结果正确,分别成功构建表达载体pET-T_7和pET-T_7N,经IPTG诱导,完成了表达条件的优化。Tricine-SDS-PAGE凝胶电泳结果显示,当IPTG浓度为1mmol/L时,25℃诱导8h,分别获得了T_7肽和T_7-NGR的表达产物。结论已经成功构建T_7肽和T_7-NGR的表达载体,获得了表达产物,为下一步的小肽活性实验奠定了基础。

Objective To enhance the active targeting by establishing a link of NGR sequence with tumstatin active peptides- T7′S C-terminal and acquire,the derivant TT-NGR. Methods The cloning vector pMD-T7 and pMD-TTN were constructed by PCR and gene synthesis methods respectively and identified by digestion and DNA sequencing. After digesting the plasmids were isolated by low melting point agarose electrophoresis, the target-fragment was cut off and mixed with the recovery of the digested vector pET28a. Expression vector pET-T7 and pET-T7N were constructed in low melting point agarose,identified by digestion and DNA sequencing,then it was transformed into competent E.coli BL21 （DE3）,induced by IPTG. Results Identification results showed that pET-T7 and pET-T7N were successful acquired. Tficine-SDS-PAGE gel electrophorasis results showed that when IPTG concentration of 1 mmol/L with the induction at 25 ℃ for 8 h,T7 peptides and TT-NGR peptides did achieve the optimal expression. Conclusion The expression vectors of the two peptides are successfully constructed, and producing an expressing product. This study establishes the foundation for further experiments of peptide activity.