發(fā)布時間：2018-04-25 08:45

  本文選題：磷脂酰肌醇特異性磷脂酶C + 大腸桿菌��； 參考：《齊魯工業(yè)大學(xué)》2016年碩士論文

【摘要】：細菌磷脂酰肌醇特異性磷脂酶C(Phosphatidylinositol-specific phospholipase C,簡稱PI-PLC)是一個小型的、具有晶體結(jié)構(gòu)的水溶性酶,可以催化水解磷酸肌醇磷酸二酯鍵,水解底物為水溶性肌醇磷酸鹽以及脂溶性二酰基甘油(DAG)。研究表明,細菌酶PI-PLC能夠水解大多數(shù)致病性寄生蟲細胞膜表面糖基(GPI)錨定蛋白,使寄生蟲失去入侵宿主細胞和在宿主細胞內(nèi)增殖的能力。因此,細菌酶PI-PLC顯示出顯著的抗感染特性。由于大多數(shù)產(chǎn)PI-PLC的野生型菌株為致病菌,產(chǎn)量低,且分離純化困難。本論文以來源于一株蠟狀芽孢桿菌的PI-PLC基因作為研究對象,分別在大腸桿菌和乳酸乳球菌中進行異源表達,并初步探索了PI-PLC的抗雞球蟲效果。研究內(nèi)容如下:根據(jù)Genbank上公布的一段蠟狀芽胞桿菌PI-PLC基因序列,對其進行優(yōu)化設(shè)計并合成PI-PLC基因,構(gòu)建重組表達載體p ET28a-PIPLC。將構(gòu)建好的表達載體轉(zhuǎn)化到大腸桿菌BL21中,經(jīng)IPTG誘導(dǎo),重組菌BL21/p ET28a(+)-PIPLC表現(xiàn)出明顯的PI-PLC活性,SDS-PAGE測得重組蛋白相對分子量約35k Da。優(yōu)化誘導(dǎo)條件:以4%接種量,37℃,200 r/min,培養(yǎng)重組大腸桿菌OD600=0.4時,添加IPTG至終濃度為1 mmol/L,誘導(dǎo)6 h后,測得培養(yǎng)基上清液中PI-PLC的濃度為0.688mg/L。產(chǎn)乳酸細菌(LAB)通常被認為是安全菌株而廣泛應(yīng)用于食品行業(yè)中。作為經(jīng)濟有效的菌株之一,乳酸乳球菌常作為粘膜免疫活體疫苗運載工具。因此,將PI-PLC基因克隆到大腸桿菌-乳酸乳球菌穿梭載體pAMJ399上,電轉(zhuǎn)化至乳酸乳球菌中進行誘導(dǎo)表達。SDS-PAGE分析顯示:重組蛋白以可溶性蛋白的形式分泌于胞外,分子質(zhì)量約35kDa,在PI-李斯特菌顯色平板上顯示出顯著的酶活性。結(jié)果表明,PI-PLC在乳酸乳球菌中成功表達。初步優(yōu)化表達條件,以2%轉(zhuǎn)接量,在含有1%紅霉素抗性的GM17培養(yǎng)基中,于32℃,靜置培養(yǎng)24 h,測得培養(yǎng)基上清液中PI-PLC的濃度為1.092 mg/L。將乳酸乳球菌表達的PI-PLC用于抗球蟲效果試驗,對實驗小雞進行攻蟲試驗,攻蟲量為5×10~4卵囊/雞。實驗結(jié)果發(fā)現(xiàn),PI-PLC對促進感染球蟲的雛雞增重有一定的效果,但是效果不明顯;通過盲腸病變計分分值和卵囊值來看,PI-PLC組小雞盲腸內(nèi)球蟲數(shù)量顯著減少,這表明PI-PLC對抵抗球蟲感染起到了明顯的作用;綜合來看,PI-PLC有抗球蟲效果,且抗球蟲效果接近良好。
[Abstract]:Bacterial phosphatidylinositol specific phospholipase C(Phosphatidylinositol-specific phospholipase C (PI-PLC) is a small, crystalline water-soluble enzyme that catalyzes the hydrolysis of inositol phosphate diester bonds. The hydrolyzed substrate was water soluble inositol phosphate and liposoluble diacylglycerol. It has been shown that the bacterial enzyme PI-PLC can hydrolyze the glycosylated GPI-based protein on the surface of most pathogenic parasites and make the parasites lose the ability to invade host cells and proliferate in host cells. Therefore, bacterial enzyme PI-PLC showed significant anti-infection characteristics. Because most of the wild-type strains producing PI-PLC are pathogenic bacteria, the yield is low, and it is difficult to isolate and purify. In this study, the PI-PLC gene from a Bacillus cereus strain was expressed in E. coli and Lactococcus lactis, respectively, and the effect of PI-PLC against coccidiosis was preliminarily explored. The main contents are as follows: according to the sequence of PI-PLC gene of Bacillus cereus published on Genbank, the PI-PLC gene was optimized and synthesized, and the recombinant expression vector pET28a-PIPLC was constructed. The constructed expression vector was transformed into Escherichia coli BL21 and induced by IPTG, the relative molecular weight of the recombinant protein was about 35kDa. the recombinant strain BL21/p ET28a (PIPLC) showed obvious PI-PLC activity and SDS-PAGE showed that the relative molecular weight of the recombinant protein was about 35k Da. The optimal induction conditions were as follows: when the recombinant Escherichia coli OD600=0.4 was cultured with 4% inoculation at 37 鈩，

本文編號：1800612