本文關(guān)鍵詞： 肝素酶 克隆 表達(dá) 優(yōu)化 純化 酶學(xué)性質(zhì)　出處：《浙江工商大學(xué)》2012年碩士論文　論文類型：學(xué)位論文

【摘要】：肝素酶(heparanase, Hpa)是近年來研究較多的一類-D糖苷內(nèi)切酶(endo-D-glucuronidase),它既存在于正常組織,如胎盤和淋巴器官,對胚胎的形成,神經(jīng)系統(tǒng)的發(fā)育,新生血管的形成,炎癥反應(yīng)等都發(fā)揮著正常的生理功能；同時(shí)也表達(dá)于多種惡性腫瘤細(xì)胞的表面,能夠促進(jìn)細(xì)胞的侵襲和轉(zhuǎn)移,誘導(dǎo)腫瘤血管生成,降低腫瘤患者的生存率。肝素用于治療和預(yù)防血栓已經(jīng)有很長的歷史,但是研究表明肝素有一定的副作用,例如影響血小板的穩(wěn)定,從而導(dǎo)致手術(shù)后大出血。而分子量在4000-8000的低分子量肝素(LMWH)則能夠有效地治療血栓,但是沒有明顯的副作用。目前我國LMWH主要依靠進(jìn)口或者來自合資企業(yè)生產(chǎn),因此LMWH的生產(chǎn)研究具有重要的工業(yè)應(yīng)用前景。而肝素酶的重要用途之一便是用于生產(chǎn)LMWH。 針對以上問題,本課題組構(gòu)建了高效表達(dá)可溶性肝素酶的重組大腸桿菌系統(tǒng),獲得了純度高,活力高,性質(zhì)穩(wěn)定的肝素酶,極大降低了肝素酶的生產(chǎn)成本,使得肝素酶用于LMWH的生產(chǎn)成為可能。本課題組已完成了以大腸桿菌表達(dá)系統(tǒng)克隆表達(dá)黃桿菌肝素酶基因的工作,并以該基因作為起始材料,利用酶活測定方法篩選出高活力菌株；然后對其發(fā)酵培養(yǎng)基進(jìn)行優(yōu)化；最后對重組蛋白進(jìn)行表達(dá)、純化并研究其酶學(xué)性質(zhì)。主要研究結(jié)果如下所述： 提取黃桿菌肝素酶的全基因組,設(shè)計(jì)合適的引物(引入HindⅢ和EcoR Ⅰ兩個(gè)酶切位點(diǎn)),利用PCR技術(shù)擴(kuò)增出肝素酶的ORF;同時(shí)用上述兩種酶雙酶切pET-28a載體和肝素酶基因,在T4連接酶的作用下連接上述目的基因與載體片段。將所得到的克隆載體轉(zhuǎn)化大腸桿菌BL21菌株,在含Kan的固體LB培養(yǎng)基上篩選陽性可克隆子。經(jīng)PCR、酶切鑒定成功后測序確定重組表達(dá)載體構(gòu)建成功。 以適當(dāng)濃度的IPTG誘導(dǎo)表達(dá)肝素酶,用SDS-PAGE檢測不同的誘導(dǎo)時(shí)間下肝素酶的表達(dá)量,從而確定最佳的誘導(dǎo)時(shí)間。結(jié)合酶活測定方法,得到表達(dá)肝素酶最高活性的菌株。 采用兩水平部分因子實(shí)驗(yàn)設(shè)計(jì)研究了發(fā)酵培養(yǎng)基各組分對肝素酶活力的影響,得出主要影響因子為蛋白胨、酵母粉和葡萄糖。三者在95%的概率水平上對菌體產(chǎn)酶都有正效應(yīng)。然后采用BBD確定各主要影響因子的最佳作用濃度,得到優(yōu)化的發(fā)酵培養(yǎng)基組成(g/L)：蛋白胨11.95,酵母粉7.4,葡萄糖2.74,Na2HPO4·12H2O10, MgSO4·7H2O0.5, NH4Cl3, KH2P042。在優(yōu)化發(fā)酵培養(yǎng)基中供試菌株的酶活力是未優(yōu)化發(fā)酵培養(yǎng)基中的1.95倍。 發(fā)酵液經(jīng)鎳柱親和層析純化得到電泳純肝素酶,SDS-PAGE電泳結(jié)果顯示,經(jīng)純化得到一條單一的目的蛋白條帶,經(jīng)分析得該酶的分子量為42.9kDa。對純化后肝素酶的酶學(xué)性質(zhì)進(jìn)行研究,發(fā)現(xiàn)該酶的最適反應(yīng)溫度為30℃,最適反應(yīng)pH為7.0。該酶熱穩(wěn)定性較差,當(dāng)溫度高于60℃時(shí),30min可完全喪失酶活。當(dāng)pH范圍在7-10之間時(shí),1h內(nèi)酶活基本不受影響,偏離此范圍則對酶活影響較大。對比8種不同金屬離子對優(yōu)化后酶的活性影響,發(fā)現(xiàn)Ca2+和Cu2+對該酶有比較明顯的激活作用,Mn2+和Pb2+對該酶有明顯的抑制作用。
[Abstract]:Heparanase (heparanase, Hpa) is a kind of -D endoglycosidase much studied in recent years (endo-D-glucuronidase), which exists in normal tissues such as placenta and embryo formation of lymphoid organs, nervous system development, angiogenesis, inflammation and other play a normal physiological function and surface; expressed in various malignant tumor cells, can promote cell invasion and metastasis, tumor induced angiogenesis, reduce the survival rate of cancer patients. Heparin for the treatment and prevention of thrombosis has a long history, but the research shows that heparin has certain side effects, such as platelet stability, resulting in massive hemorrhage after surgery while the molecular weight of low molecular weight heparin 4000-8000 (LMWH) can effectively treat thrombosis, but no obvious side effects. At present LMWH mainly rely on imports or from joint ventures Therefore, the production research of LMWH has an important industrial application prospect. One of the important uses of heparanase is to produce LMWH..
To solve the above problems, the research group to construct a recombinant Escherichia coli system for efficient expression of soluble heparin enzyme, has high purity, high activity, heparanase stable, greatly reduces the production cost of heparanase, the heparin enzyme for LMWH production possible. The research group has completed the expression of Flavobacterium heparinase the gene expressed in Escherichia coli and the gene cloning system, as the starting material, determination of the activity of high activity strains were screened by enzyme; then the fermentation medium was optimized; finally the recombinant protein expression, purification and characterization. The main results are as follows:
鎻愬彇榛勬潌鑿岃倽绱犻叾鐨勫叏鍩哄洜緇，

本文編號(hào)：1535576