蛙皮素与死亡素杂合抗菌肽在大肠杆菌中的融合表达

发布时间：2018-06-02 02:47

  本文选题：抗菌肽 + 融合表达　； 参考：《陕西科技大学》2017年硕士论文

【摘要】：抗菌肽作为生物免疫系统的主要组成部分,是生物体的免疫作用产生的抗菌谱广、难以产生耐药性的一类带正电荷的两亲性小分子多肽。目前在医药、食品及畜牧业领域应用广泛。将两种或多种抗菌肽片段杂合形成一种新型的杂合肽,能够弥补天然抗菌肽的缺点,增加抗菌肽的抑菌活性。本研究将蛙皮素抗菌肽与死亡素抗菌肽的有效作用部位拼接,获得新型的杂合肽MT。根据杂合抗菌肽MT基因与大肠杆菌密码子的偏好性设计了该杂合肽的基因序列,对该基因进行生物信息学分析;利用SOE-PCR技术设计了三段引物,经过两轮PCR扩增获得目的基因MT,将目的基因克隆至大肠杆菌表达载体pGEX-6P-1构建重组表达载体pGEX-6P-1-MT;将构建的重组表达载体转化到表达菌株E.coli BL21(DE3)中构建了基因工程菌pGEX-6P-1-MT/BL21(DE3),通过对IPTG浓度、诱导时间、温度以及培养基等发酵条件的优化,利用GST琼脂糖凝胶层析柱对融合蛋白进行纯化,肠激酶裂解后对杂合抗菌肽的抑菌活性进行初步分析。本论文研究结果如下:(1)生物信息学手段对杂合抗菌肽MT的理化参数、二级结构、亲水性等进行预测得出:氨基酸总数为29,分子量大小为3.35 kDa,等电点pI为10.57,含有α螺旋与β折叠结构,亲水性图谱结果显示该杂合肽具有两亲性,具有抗菌潜力。根据大肠杆菌密码子偏好性,优化了该基因序列,最终得到具有抗菌潜力的目的基因MT序列。(2)利用重叠延伸扩增(SOE-PCR)技术,用合成的三段引物通过两轮PCR成功合成了杂合抗菌肽MT的基因。(3)目的基因MT与表达载体p GEX-6P-1用EcoR I和BamH I双酶切,然后进行重组载体的构建,成功获得了重组表达载体pGEX-6P-1-MT。(4)超声破碎细胞进行SDS-PAGE电泳发现,融合蛋白以可溶形式存在于菌体上清,Western-blot证明目的蛋白表达,用GST琼脂糖凝胶纯化柱对杂合肽纯化,纯化后获得融合蛋白,占菌体总量的50.1%,蛋白的表达量为49.5 mg/L,肠激酶切割后得到分子量为3.35 kDa大小的MT目的蛋白。(5)通过对杂合抗菌肽MT工程菌诱导表达条件的优化,获得了杂合抗菌肽MT工程菌的优化后条件分别为:IPTG浓度0.8 mM;诱导时间3 h;诱导温度37℃;TB培养基。(6)采用抑菌圈法对杂合抗菌肽MT的抑菌活性进行研究,发现该杂合肽对大肠杆菌DH5α、金黄色葡萄球菌、枯草芽孢杆菌均有抑制作用,抑菌圈大小分别为5 mm、6 mm和10 mm,对枯草芽孢杆菌的抑制效果强于前两者。最小抑菌浓度分别20μM、16.5μM、9μM。本研究成功实现了杂合肽MT在大肠杆菌pGEX-6P-1表达载体中的可溶性表达,通过纯化获得了纯度较高、具有抗菌活性的MT目的蛋白,在食品、医疗、饲料领域等有一定的应用价值。
[Abstract]:As the main component of biological immune system, antimicrobial peptide is a kind of amphiphilic polypeptide with positive charge which is difficult to produce due to its wide spectrum of antimicrobial activity. It is widely used in medicine, food and animal husbandry. Two or more antimicrobial peptides were hybridized to form a new hybrid peptide, which can make up for the shortcomings of natural antimicrobial peptides and increase the antibacterial activity of antimicrobial peptides. In this study, a novel hybrid peptide MTP was obtained by splicing the effective sites of the antimicrobial peptide of frog skin and the antibacteriopeptide of death. According to the preference between the MT gene and the codon of Escherichia coli, the gene sequence of the hybrid peptide was designed, and the gene was analyzed by bioinformatics, and a three-piece primer was designed by using SOE-PCR technique. After two rounds of PCR amplification, the target gene was cloned into E. coli expression vector pGEX-6P-1 to construct the recombinant expression vector pGEX-6P-1-MTand the recombinant expression vector was transformed into the expression strain E.coli BL21DE3. The optimal fermentation conditions, such as induction time, temperature and culture medium, were optimized. The fusion protein was purified by GST agarose gel chromatography and the antibacterial activity of the hybrid antibacterial peptide was preliminarily analyzed after the decomposition of enterokinase. The results of this study are as follows: (1) Physicochemical parameters, secondary structure of hybrid antimicrobial peptide MT by bioinformatics, The hydrophilic prediction showed that the total number of amino acids was 29, the molecular weight was 3.35 kDa, the isoelectric point Pi was 10.57, and had 伪 helix and 尾 folding structure. The hydrophilic map showed that the hybrid peptide had amphiphilic properties and antibacterial potential. According to the preference of Escherichia coli codon, the gene sequence was optimized, and the target gene MT sequence with antibacterial potential was obtained. The target gene MT and the expression vector p GEX-6P-1 were digested with EcoR I and BamH I by two rounds of PCR, and the recombinant vector was constructed. The recombinant expression vector pGEX-6P-1-MT.f4 was successfully obtained by SDS-PAGE electrophoresis. It was found that the fusion protein was expressed in the supernatant of the cell in Western-blot. The fusion protein was purified by GST agarose gel column. After purification, the fusion protein was obtained, accounting for 50.1% of the total mycelium. The protein expression was 49.5 mg / L, and the target protein of MT with molecular weight of 3.35 kDa was obtained after enterokinase cleavage. The optimized conditions of the engineering strain of hybrid antimicrobial peptide MT were obtained as follows: concentration of 1: IPTG 0.8 mm; induction time 3 h; induction temperature 37 鈩，

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