發(fā)布時間：2018-03-18 09:04

  本文選題：白眉蝮蛇　切入點：5　出處：《昆明理工大學(xué)》2010年碩士論文　論文類型：學(xué)位論文

【摘要】：白眉蝮蛇(Agkistrodon blomhoffii Ussurensis)屬于蝰蛇科蝮蛇亞科,是我國劇毒蛇類之一,在國內(nèi)廣泛分布河北、甘肅、上海、遼寧、臺灣等省市,在國外朝鮮半島也有分布。白眉蝮蛇毒已作為生產(chǎn)溶血栓藥物“蛇毒溶栓酶”的主要原料,但蛇毒中其他活性組分的分離純化、生物學(xué)特性及其經(jīng)濟價值還未得到全面深入研究。 5’-核苷酸酶(5'-nucleotidase)是催化核苷酸分子中的磷酸鍵水解的一種特異性水解磷酸酶。5’-核苷酸酶能特異地水解5’-核苷酸酶和5’-脫氧核苷酸酶連接戊糖的5’-磷酸,生成對應(yīng)的核苷或脫氧核苷和無機磷酸。5’-核苷酸酶是一種常規(guī)分子生物學(xué)酶試劑,因其活性的變化與很多疾病密切相關(guān),還可以用作許多疾病的診斷對照試劑,比如：肝膽疾病、冠心病、肺癌、消化道腫瘤、甲亢、骨病、喉癌等。5’-核苷酸酶廣泛分布于細菌、植物細胞和蛇毒毒液中。1951年Hepple首次從蛇毒中分離純化出5’-核苷酸酶。至今為止已發(fā)現(xiàn)二十八種蛇毒5’-核苷酸酶,但尚未有白眉蝮蛇蛇毒5’-核苷酸酶相關(guān)研究報道。 建立白眉蝮蛇毒5’-核苷酸酶的分離純化方法,并對其理化性質(zhì)進行研究,將為白眉蝮蛇毒的深入開發(fā)、積累5’-核苷酸酶基礎(chǔ)數(shù)據(jù)等方面都具有重要意義。 本論文采用離子交換DEAE Sephadex A-25柱層析、凝膠過濾SephadexG-75柱層析,從白眉蝮蛇蛇毒分離純化得到了一個5’-核苷酸酶。采用SDS-聚丙烯酰胺凝膠電泳(SDS-Polyacrylamide Gel Electrophoresis, SDS-PAGE)鑒定了其純度并計算了其分子量,測定了其比活、最適pH、溫度等理化性質(zhì),考察了常見金屬離子對其活性的影響,還觀察了簡單糖對其活性的保護作用以及其對ADP誘導(dǎo)的血小板聚集過程的影響。 經(jīng)SDS-PAGE此5’-核苷酸酶分子量為70kDa,用高碘酸硝酸銀阿利辛藍染色法鑒定白眉蝮蛇蛇毒5’-核苷酸酶為糖蛋白。白眉蝮蛇蛇毒5，，-核苷酸酶的最適溫度為55℃,最適pH為pH 9.0。以AMP為底物測得純化所得酶蛋白的5，-核苷酸酶活力為562.44U。從目前的實驗結(jié)果看來,此5’-核苷酸酶對熱穩(wěn)定酶,耐堿性環(huán)境。Cu2+、Fe3+濃度范圍為(0.05 mmol/L.0.3mmol/L)能抑制白眉蝮蛇蛇毒5’-核苷酸酶活性,Fe3+強烈抑制白眉蝮蛇蛇毒5’-核苷酸酶活性。Mg2+、Ca2+、K+濃度范圍為(0.05 mmol/L.0.3mmol/L)均能促進白眉蝮蛇蛇毒5’-核苷酸酶活性,其中Ca2+促進效果更好。低溶度(0.05 mmol/L.0.25mmol/L)β-巰基乙醇對白眉蝮蛇蛇毒5’-核苷酸酶活性有一定促進作用。低濃度(0.05mmol/L.0.25mmol/L)EDTA對白眉蝮蛇蛇毒5’-核苷酸酶活性有促進作用,而0.3 mmol/L EDTA對白眉蝮蛇蛇毒5’-核苷酸酶有明顯抑制作用�？赡苁且驗榈腿芏菶DTA能作為金屬螯合劑先與抑制白眉蝮蛇蛇毒5’-核苷酸酶的金屬離子結(jié)合比如Cu2+、Fe3+,隨著EDTA溶度增加與促進白眉蝮蛇蛇毒5’-核苷酸酶金屬離子結(jié)合比如Mg2+、、Ca2+、K+等。導(dǎo)致白眉蝮蛇蛇毒5’-核苷酸酶活性降低。 本文還研究了單糖和雙糖對酶蛋白的保護作用,實驗中采用了葡萄糖、果糖、海藻糖、蔗糖四種。經(jīng)測定,葡萄糖、果糖、海藻糖對白眉蝮蛇蛇毒5’-核苷酸酶均具有一定的保護作用。糖對白眉蝮蛇蛇毒5’-核苷酸酶保護作用從未有發(fā)現(xiàn),具體機制尚待研究。 白眉蝮蛇蛇毒5’-核苷酸酶對血小板聚集有誘導(dǎo)作用,促進ADP誘導(dǎo)的血小板聚集。白眉蝮蛇蛇毒5’-核苷酸酶可以作為血小板聚集的誘導(dǎo)劑。值得注意的是,這一實驗現(xiàn)象與目前文獻報道其他蛇毒5’-核苷酸酶活性相反。其機制還待進一步深入探討。
[Abstract]:Halysussuriensis (Agkistrodon blomhoffii Ussurensis) belongs to the Viperidae Agkistrodon subfamily, is one of China's poisonous snakes, widely distributed in the domestic Hebei, Gansu, Shanghai, Liaoning, Taiwan and other provinces and cities in foreign countries, the Korean Peninsula is also distributed. The main raw material from Agkistrodon halys ussuriensis venom thrombolytic drugs have been produced as "fibrolase", but the venom in the separation of other active components of the purification, biological characteristics and economic value has not been fully investigated.
5 '- nucleotidase (5'-nucleotidase) is the catalytic nucleotide molecule in phosphate bond hydrolysis of a specific phosphatase hydrolysis.5' - nucleotidase can specifically hydrolyze 5 '- and 5' - nucleotidase DNA enzyme linked pentose 5 '- phosphate, generate the corresponding nucleoside or deoxyriboside and inorganic phosphate.5' - nucleotidase is a common molecular biological enzyme reagent, due to changes in its activity is closely related to many diseases, the diagnosis of many diseases can also be used as control agents, such as: liver disease, coronary heart disease, lung cancer, gastrointestinal cancer, hyperthyroidism, bone disease, laryngeal.5 '- nucleotidase widely distributed in bacteria, plants cells and venom.1951 Hepple was first isolated from the venom of 5' - nucleotidase. So far, twenty-eight species have been found from 5 '- nucleotidase, but not the snake venom of Agkistrodon 5' nucleoside acid enzyme Research reports.
A white browed Viper 5 '- nucleotidase purification method, and to study its physicochemical properties, for further development of Agkitrodon acutus venom, the accumulation of 5' - nucleotidase based data and other aspects are of great significance.
This paper adopts DEAE Sephadex A-25 ion exchange chromatography, gel filtration chromatography on SephadexG-75, purified from the snake venom of Agkistrodon a 5 '- nucleotidase. Using SDS- polyacrylamide gel electrophoresis (SDS-Polyacrylamide Gel Electrophoresis, SDS-PAGE) identified its purity and its molecular weight was calculated, the determination of the specific activity, the most optimum pH, temperature and other physical and chemical properties, the effects of metal ions on the activity, also observed the protective effect of simple sugars on the activity and its effects on the platelet aggregation induced by ADP.
The SDS-PAGE of the 5 '- nucleotidase molecular weight is 70kDa, with periodate silver nitrate alcian blue staining to identify the snake venom of Agkistrodon 5' - nucleotidase was a glycoprotein. Halysussuriensis venom 5 - an optimum temperature of 55 DEG C nucleotide enzyme, the optimum pH for pH 9.0. with AMP as substrate measured purification the enzyme 5 - nucleotidase activity was 562.44U. from the current view of the experimental results, the 5 '- nucleotidase of thermostable enzymes, alkaline environment.Cu2+, Fe3+ concentration range (0.05 mmol/L.0.3mmol/L) can inhibit the snake venom of Agkistrodon 5' - nucleotidase activity, Fe3+ strongly inhibited the snake venom of Agkistrodon 5 '- nucleotidase activity.Mg2+, Ca2+, K+ concentration range (0.05 mmol/L.0.3mmol/L) could promote the snake venom of Agkistrodon 5' - nucleotidase activity, of which Ca2+ promotes better. Low solubility (0.05 mmol/L.0.25mmol/L) beta mercaptoethanol on Agkitrodon acutus snake venom 5 'nucleoside Have a certain role in promoting acid enzyme activity. Low concentration of EDTA (0.05mmol/L.0.25mmol/L) on the snake venom of Agkistrodon 5 '- nucleotidase activity promoted, while the 0.3 mmol/L EDTA of snake venom of Agkistrodon 5' - nucleotidase is inhibited. Probably because of the low solubility of EDTA can be used as a metal chelator and inhibition of the snake venom of Agkistrodon 5 '- nucleotidase binding metal ions such as Cu2+, Fe3+, EDTA with the solubility was increased and the promotion of the snake venom of Agkistrodon 5' - nucleotidase binding metal ions such as Mg2+, Ca2+, K+, etc. to the snake venom of Agkistrodon 5 '- nucleotidase activity decreased.
This paper also studied the protective effect of monosaccharide and disaccharide on enzyme, was used in the experiment of glucose, fructose, trehalose, sucrose four. The determination of glucose, fructose, trehalose on the snake venom of Agkistrodon 5 '- nucleotidase has certain protective effect on sugar. Agkitrodon acutus snake venom 5' - nucleotidase protection the role has never found that the specific mechanism remains to be investigated.
The snake venom of Agkistrodon 5 '- nucleotidase on platelet aggregation induced by, promote platelet aggregation induced by ADP. The snake venom of Agkistrodon 5' - nucleotidase as a platelet aggregation inducer. It is worth noting that this phenomenon and the reported other venom 5 '- nucleotidase activity and its mechanism to be opposite. To further study.

【學(xué)位授予單位】：昆明理工大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R341

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