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(xì)菌磷脂酰肌醇特異性磷脂酶C(Phosphatidylinositol-specific phospholipase C,簡(jiǎn)稱(chēng)PI-PLC)是一個(gè)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�；视�(DAG)。研究表明,細(xì)菌酶PI-PLC能夠水解大多數(shù)致病性寄生蟲(chóng)細(xì)胞膜表面糖基(GPI)錨定蛋白,使寄生蟲(chóng)失去入侵宿主細(xì)胞和在宿主細(xì)胞內(nèi)增殖的能力。因此,細(xì)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(lái)源于一株蠟狀芽孢桿菌的PI-PLC基因作為研究對(duì)象,分別在大腸桿菌和乳酸乳球菌中進(jìn)行異源表達(dá),并初步探索了PI-PLC的抗雞球蟲(chóng)效果。研究?jī)?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(duì)其進(jìn)行優(yōu)化設(shè)計(jì)并合成PI-PLC基因,構(gòu)建重組表達(dá)載體p ET28a-PIPLC。將構(gòu)建好的表達(dá)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(cè)得重組蛋白相對(duì)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(shí),添加IPTG至終濃度為1 mmol/L,誘導(dǎo)6 h后,測(cè)得培養(yǎng)基上清液中PI-PLC的濃度為0.688mg/L。產(chǎn)乳酸細(xì)菌(LAB)通常被認(rèn)為是安全菌株而廣泛應(yīng)用于食品行業(yè)中。作為經(jīng)濟(jì)有效的菌株之一,乳酸乳球菌常作為粘膜免疫活體疫苗運(yùn)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(jìn)行誘導(dǎo)表達(dá)。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(dá)。初步優(yōu)化表達(dá)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(cè)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(dá)的PI-PLC用于抗球蟲(chóng)效果試驗(yàn),對(duì)實(shí)驗(yàn)小雞進(jìn)行攻蟲(chóng)試驗(yàn),攻蟲(chóng)量為5×10~4卵囊/雞。實(shí)驗(yàn)結(jié)果發(fā)現(xiàn),PI-PLC對(duì)促進(jìn)感染球蟲(chóng)的雛雞增重有一定的效果,但是效果不明顯;通過(guò)盲腸病變計(jì)分分值和卵囊值來(lái)看,PI-PLC組小雞盲腸內(nèi)球蟲(chóng)數(shù)量顯著減少,這表明PI-PLC對(duì)抵抗球蟲(chóng)感染起到了明顯的作用;綜合來(lái)看,PI-PLC有抗球蟲(chóng)效果,且抗球蟲(chóng)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(hào)：1800612