人组织型纤溶酶原激活剂原核表达载体的构建及其在大肠杆菌中的初步表达

发布时间：2018-06-26 05:17

  本文选题：组织型纤溶酶原激活剂 + 原核表达载体　； 参考：《重庆医科大学》2012年硕士论文

【摘要】：目的:通过构建人组织型纤溶酶原激活剂(humantissue-typepLasminogenactivatortPA)原核表达载体，检测tPA目的蛋白是否能在大肠杆菌中进行初步表达，并进一步探索最佳表达条件，希望经纯化、复性后获得有活性的目的蛋白tPA，以满足生产需要和临床应用。 方法:选用pET-28a为原核表达载体，从野生型tPA重组mRNA中克隆得到EST-tPA（含有人tPA基因全长的质粒），以此质粒为模板，通过设计上下游引物，于tPA的C末端加入RGDS四肽序列，利用PCR技术克隆扩增目的基因片段RGDS-tPA，胶回收并纯化。NcoI和XhoI分别双酶切目的基因tPA及载体pET-28a。NcoI和XhoI黏性末端互补,体外连接酶切产物,重组体转化感受态细胞XL-1,筛选、酶切鉴定重组质粒和测序鉴定。制备感受态细胞BL21（DE3），将pET28a-tPA转化入感受态细胞BL21（DE3）。聚丙烯酰胺凝胶电泳（SDS-PAGE）显示rtPA在大肠杆菌中的表达。 结果:琼脂糖凝胶电泳获得目的基因片段rtPA的条带约1700bp,鉴定为阳性重组体,测序证实为正确的pET28a-tPA序列；聚丙烯酰胺凝胶电泳鉴定获得目的蛋白片段rtPA的条带约53KDa,为非活性的包涵体。表达条件经优化后，，rtPA在37℃下培养4小时表达量和表达水平较好；在25℃下培养8小时表达量和表达水平最佳；在17℃下培养12小时表达量和表达水平较前两者下降。 结论:（1）人组织型纤溶酶原激活剂原核表达载体构建成功,可在大肠杆菌中有效表达。（2）rtPA在大肠杆菌中表达时与诱导的时间、环境温度、IPTG的浓度有关，长时间低温诱导可能会使rtPA外源蛋白表达量和表达水平有所下降。
[Abstract]:Objective: to construct a prokaryotic expression vector of human tissue type plasminogen activator (human tissue plasminogen activator), to detect whether the target protein of tPA can be expressed in Escherichia coli, and to explore the best expression conditions. The active target protein tPA was obtained after renaturation to meet the needs of production and clinical application. Methods: using pET-28a as prokaryotic expression vector, EST-tPA (full length plasmid containing human tPA gene) was cloned from wild-type tPA recombinant mRNA. The plasmid was used as template, and the sequence of RGDS tetrapeptide was added to the C-terminal of tPA by designing upstream and downstream primers. The target gene fragment RGDS-tPAwas cloned and amplified by PCR technique. The target gene tPA and vector pET-28a.NcoI and XhoI were digested by gel and purified respectively. The ligated products were ligated in vitro and transformed into XL-1 cells. The recombinant plasmid was identified by enzyme digestion and sequenced. BL21 (DE3) was prepared and pET28a-tPA was transformed into BL21 (DE3). Polyacrylamide gel electrophoresis (SDS-PAGE) showed the expression of rtPA in Escherichia coli. Results: the target gene fragment rtPA was obtained by agarose gel electrophoresis and identified as a positive recombinant. The sequence was confirmed to be the correct pET28a-tPA sequence. Polyacrylamide gel electrophoresis identified the target protein fragment rtPA as an inactive inclusion body with a band of about 53KDa. The expression of rtPA was better at 37 鈩

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