磷脂酰肌醇特异性磷脂酶C基因的克

发布时间：2018-04-25 08:45

  本文选题：磷脂酰肌醇特异性磷脂酶C + 大肠杆菌　； 参考：《齐鲁工业大学》2016年硕士论文

【摘要】：细菌磷脂酰肌醇特异性磷脂酶C(Phosphatidylinositol-specific phospholipase C,简称PI-PLC)是一个小型的、具有晶体结构的水溶性酶,可以催化水解磷酸肌醇磷酸二酯键,水解底物为水溶性肌醇磷酸盐以及脂溶性二酰基甘油(DAG)。研究表明,细菌酶PI-PLC能够水解大多数致病性寄生虫细胞膜表面糖基(GPI)锚定蛋白,使寄生虫失去入侵宿主细胞和在宿主细胞内增殖的能力。因此,细菌酶PI-PLC显示出显著的抗感染特性。由于大多数产PI-PLC的野生型菌株为致病菌,产量低,且分离纯化困难。本论文以来源于一株蜡状芽孢杆菌的PI-PLC基因作为研究对象,分别在大肠杆菌和乳酸乳球菌中进行异源表达,并初步探索了PI-PLC的抗鸡球虫效果。研究内容如下:根据Genbank上公布的一段蜡状芽胞杆菌PI-PLC基因序列,对其进行优化设计并合成PI-PLC基因,构建重组表达载体p ET28a-PIPLC。将构建好的表达载体转化到大肠杆菌BL21中,经IPTG诱导,重组菌BL21/p ET28a(+)-PIPLC表现出明显的PI-PLC活性,SDS-PAGE测得重组蛋白相对分子量约35k Da。优化诱导条件:以4%接种量,37℃,200 r/min,培养重组大肠杆菌OD600=0.4时,添加IPTG至终浓度为1 mmol/L,诱导6 h后,测得培养基上清液中PI-PLC的浓度为0.688mg/L。产乳酸细菌(LAB)通常被认为是安全菌株而广泛应用于食品行业中。作为经济有效的菌株之一,乳酸乳球菌常作为粘膜免疫活体疫苗运载工具。因此,将PI-PLC基因克隆到大肠杆菌-乳酸乳球菌穿梭载体pAMJ399上,电转化至乳酸乳球菌中进行诱导表达。SDS-PAGE分析显示:重组蛋白以可溶性蛋白的形式分泌于胞外,分子质量约35kDa,在PI-李斯特菌显色平板上显示出显著的酶活性。结果表明,PI-PLC在乳酸乳球菌中成功表达。初步优化表达条件,以2%转接量,在含有1%红霉素抗性的GM17培养基中,于32℃,静置培养24 h,测得培养基上清液中PI-PLC的浓度为1.092 mg/L。将乳酸乳球菌表达的PI-PLC用于抗球虫效果试验,对实验小鸡进行攻虫试验,攻虫量为5×10~4卵囊/鸡。实验结果发现,PI-PLC对促进感染球虫的雏鸡增重有一定的效果,但是效果不明显;通过盲肠病变计分分值和卵囊值来看,PI-PLC组小鸡盲肠内球虫数量显著减少,这表明PI-PLC对抵抗球虫感染起到了明显的作用;综合来看,PI-PLC有抗球虫效果,且抗球虫效果接近良好。
[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，

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