本文選題：赤子愛勝蚓　切入點(diǎn)：蚓激酶　出處：《安徽工程大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：蚓激酶(Lumbrukinase,LK)是從蚯蚓粗提物中分離得到的一類具有纖溶活性的蛋白質(zhì)。其不但能激活纖維蛋白溶酶原轉(zhuǎn)變?yōu)槔w溶酶,刺激血管內(nèi)皮細(xì)胞釋放組織纖溶酶原激活物(t-PA),間接促進(jìn)纖維蛋白的溶解,且能夠直接水解纖維蛋白。此外,蚓激酶擁有良好的溶栓效果,可以改善微循環(huán),增加血管彈性,降低血液粘度,抑制血栓再次形成,因而廣泛應(yīng)用于臨床,且越來越多地用在心腦血管、內(nèi)分泌,呼吸系統(tǒng)等疾病的預(yù)防和治療中。論文主要研究了野生型赤子愛勝蚓(Eisenia foetida)蚓激酶EFLK在大腸桿菌和巴斯德畢赤酵母中的表達(dá),對(duì)重組畢赤酵母的發(fā)酵條件進(jìn)行了優(yōu)化,并對(duì)蚓激酶進(jìn)行分離純化及酶學(xué)特性的初步探究。主要工作如下:(1)從野生蚯蚓中提取基因組,利用分子生物學(xué)手段進(jìn)行物種鑒定及系統(tǒng)發(fā)育樹的構(gòu)建,確定該野生蚯蚓屬于赤子愛勝蚓。(2)提取赤子愛勝蚯蚓總RNA,利用反轉(zhuǎn)錄試劑盒獲得單鏈cDNA作為為模板,克隆得到蚓激酶基因Eflk(GenBank登錄號(hào):KY452025)。利用生物信息學(xué)軟件分析蚓激酶基因Eflk全長為738 bp,編碼245個(gè)氨基酸,其中含有一個(gè)由7個(gè)氨基酸組成的信號(hào)肽。蚓激酶EFLK與粉正蚓的蚓激酶F-Ⅲ-2(GenBank登錄號(hào):AB045719)相似性最高達(dá)96.21%。經(jīng)DS2.5軟件建模及分子對(duì)接,確定蚓激酶EFLK 的活性中心為:Arg15-Phe19-Pro20-Glu66-Asn113。(3)構(gòu)建重組質(zhì)粒pET-28a(+)-Eflk,并將其導(dǎo)入大腸桿菌BL21(DE3)中,成功實(shí)現(xiàn)蚓激酶EFLK在大腸桿菌的表達(dá)。在培養(yǎng)基中添加山梨醇、蔗糖、甘氨酸和乙醇可有效提高酶活,最高達(dá)到15.6 U/mL。(4)構(gòu)建重組質(zhì)粒pPIC9K-Eflk,經(jīng)Sac I線性化后導(dǎo)入巴斯德畢赤酵母GS115中,成功實(shí)現(xiàn)蚓激酶EFLK在酵母中的表達(dá)。在重組菌GS115-pPIC9K-EflK發(fā)酵上清液中檢測到了纖溶活性,其中發(fā)酵第72 h達(dá)到最高酶活為224.8 U/mL。(5)利用單因素法和水平正交法優(yōu)化畢赤酵母發(fā)酵條件,經(jīng)單因素實(shí)驗(yàn)優(yōu)化確定4個(gè)關(guān)鍵因素:初始pH、誘導(dǎo)溫度、接種量和甲醇添加量。就此進(jìn)行4因素3水平正交實(shí)驗(yàn),得到最佳發(fā)酵條件:初始pH5.5、誘導(dǎo)溫度30℃、接種量OD_(600)=2.0、甲醇添加量1%,優(yōu)化后發(fā)酵72 h酶活最高達(dá)到259.4 U/mL。(6)利用Ni~+柱純化得到蚓激酶EFLK蛋白,比酶活為4545.84 U/mg。酶學(xué)性質(zhì)研究表明,蚓激酶EFLK最適pH為8.0,酶液在pH 7.4條件下37 ℃保溫12 h時(shí)蚓激酶活力快速損失20%,第48 h蚓激酶殘余活力為67.33%左右。蚓激酶EFLK在低溫(低于40 ℃)條件下較為穩(wěn)定。酶液在最適pH 8.0條件下4 ℃保存48 h蚓激酶活力損失41.63%,仍保持較高的活力。
[Abstract]:LumbrukinaseLK is a kind of fibrinolytic protein isolated from earthworm extract, which not only activates plasminogen transformation into plasminogen, Stimulating vascular endothelial cells to release tissue plasminogen activator t-PAN indirectly promotes fibrinolysis and directly hydrolyzes fibrin. In addition, lumbrokinase has a good thrombolytic effect, which can improve microcirculation and increase vascular elasticity. It is widely used in clinic, and is more and more used in cardiovascular, cerebrovascular, endocrine, etc. In the prevention and treatment of respiratory diseases, the expression of wild type Eisenia foetida EFLK in Escherichia coli and Pichia pastoris was studied, and the fermentation conditions of recombinant Pichia pastoris were optimized. The main work is as follows: extracting genome from wild earthworm, using molecular biological methods to identify species and construct phylogenetic tree. It was determined that the wild earthworm belongs to Aisheng vermis. 2) the total RNAs of Aisheng earthworm were extracted, and the single-stranded cDNA was obtained by reverse transcription kit as template. Eflk(GenBank accession number: KY452025 was cloned. The total length of Eflk was 738 BP, encoding 245 amino acids, using bioinformatics software. It contains a signal peptide consisting of seven amino acids. The similarity between EFLK and F- 鈪，

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