發(fā)布時間：2018-06-26 05:17

  本文選題：組織型纖溶酶原激活劑 + 原核表達(dá)載體　； 參考：《重慶醫(yī)科大學(xué)》2012年碩士論文

【摘要】：目的:通過構(gòu)建人組織型纖溶酶原激活劑(humantissue-typepLasminogenactivatortPA)原核表達(dá)載體，檢測tPA目的蛋白是否能在大腸桿菌中進(jìn)行初步表達(dá)，并進(jìn)一步探索最佳表達(dá)條件，希望經(jīng)純化、復(fù)性后獲得有活性的目的蛋白tPA，以滿足生產(chǎn)需要和臨床應(yīng)用。 方法:選用pET-28a為原核表達(dá)載體，從野生型tPA重組mRNA中克隆得到EST-tPA（含有人tPA基因全長的質(zhì)粒），以此質(zhì)粒為模板，通過設(shè)計(jì)上下游引物，于tPA的C末端加入RGDS四肽序列，利用PCR技術(shù)克隆擴(kuò)增目的基因片段RGDS-tPA，膠回收并純化。NcoI和XhoI分別雙酶切目的基因tPA及載體pET-28a。NcoI和XhoI黏性末端互補(bǔ),體外連接酶切產(chǎn)物,重組體轉(zhuǎn)化感受態(tài)細(xì)胞XL-1,篩選、酶切鑒定重組質(zhì)粒和測序鑒定。制備感受態(tài)細(xì)胞BL21（DE3），將pET28a-tPA轉(zhuǎn)化入感受態(tài)細(xì)胞BL21（DE3）。聚丙烯酰胺凝膠電泳（SDS-PAGE）顯示rtPA在大腸桿菌中的表達(dá)。 結(jié)果:瓊脂糖凝膠電泳獲得目的基因片段rtPA的條帶約1700bp,鑒定為陽性重組體,測序證實(shí)為正確的pET28a-tPA序列；聚丙烯酰胺凝膠電泳鑒定獲得目的蛋白片段rtPA的條帶約53KDa,為非活性的包涵體。表達(dá)條件經(jīng)優(yōu)化后，，rtPA在37℃下培養(yǎng)4小時表達(dá)量和表達(dá)水平較好；在25℃下培養(yǎng)8小時表達(dá)量和表達(dá)水平最佳；在17℃下培養(yǎng)12小時表達(dá)量和表達(dá)水平較前兩者下降。 結(jié)論:（1）人組織型纖溶酶原激活劑原核表達(dá)載體構(gòu)建成功,可在大腸桿菌中有效表達(dá)。（2）rtPA在大腸桿菌中表達(dá)時與誘導(dǎo)的時間、環(huán)境溫度、IPTG的濃度有關(guān)，長時間低溫誘導(dǎo)可能會使rtPA外源蛋白表達(dá)量和表達(dá)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

本文編號：2069278