表达猪β防御素-2和cecropin P1抗菌肽枯草芽孢杆菌的构建及特性研究

发布时间：2018-09-01 10:52

【摘要】：枯草芽孢杆菌(Bacillus subtilis)是动物肠道中的一种具有代表性的益生菌添加剂,猪β防御素-2(pBD-2)和抗菌肽cecropin P1是猪肠道中重要的两种抗菌肽,动物肠道中的益生菌和抗菌肽在维持肠道菌群平衡,调节机体免疫以及防御病原菌方面起重要作用。利用基因工程方法制备表达抗菌肽的益生菌将有利于畜禽的生长和提高抗病力。本研究试图制备表达猪pBD-2和cecropin P1抗菌肽的枯草芽孢杆菌,以提高仔猪抗病力和促进仔猪的生长。为提高抗菌肽的表达水平,根据枯草芽孢杆菌偏爱密码子对β防御素-2和cecropin P1基因进行密码子优化,并将优化后的β防御素-2和cecropin P1的基因插入到pMK4原核表达载体Lac启动子下游,构建成β防御素-2和cecropin P1基因的融合表达载体pMK-BD-2/CP1,经转化枯草芽孢杆菌,筛选出表达融合抗菌肽的枯草芽孢杆菌工程菌。经发酵实验证明构建的工程菌能够表达猪β防御素-2和cecropin P1融合抗菌肽,表达的融合抗菌肽对工程菌的生长没有明显影响。融合抗菌肽经肠激酶酶切处理后释放的pBD-2、cecropin P1对多种细菌都具有明显的抗菌作用,包括革兰氏阴性菌(大肠杆菌、鼠伤寒沙门氏菌、副猪嗜血杆菌)和革兰氏阳性菌(金黄色葡萄球菌)。动物实验证实,在仔猪基础日粮中添加此工程菌可明显促进断奶仔猪的生长和提高仔猪抗病力。这项研究不仅为该工程菌在养猪业的应用打下了必要的基础。同时也为其它重组抗菌肽益生菌的制备提供了重要科学依据。
[Abstract]:Bacillus subtilis (Bacillus subtilis) is a representative probiotic additive in animal intestines. Porcine 尾 -defensin -2 (pBD-2) and antimicrobial peptide cecropin P1 are two important antimicrobial peptides in pig intestine. Probiotics and antimicrobial peptides in animal intestine play an important role in maintaining the balance of intestinal flora, regulating body immunity and defending pathogenic bacteria. The preparation of probiotics expressing antimicrobial peptides by genetic engineering will be beneficial to the growth of livestock and poultry. The purpose of this study was to prepare Bacillus subtilis expressing pBD-2 and cecropin P1 antimicrobial peptides in order to improve the disease resistance of piglets and promote the growth of piglets. In order to improve the expression of antimicrobial peptides, 尾 -defensin -2 and cecropin P1 genes were codon optimized according to the preference codon of Bacillus subtilis. The optimized 尾 defensin 2 and cecropin P1 genes were inserted into the downstream of Lac promoter of pMK4 prokaryotic expression vector, and the fusion expression vector pMK-BD-2/CP1, of 尾 defensin 2 and cecropin P1 gene was transformed into Bacillus subtilis. An engineering strain of Bacillus subtilis expressing a fusion of antimicrobial peptides was screened out. The fermentation experiments showed that the recombinant strain could express porcine 尾 -defensin -2 and cecropin P1 fusion antimicrobial peptides, and the expressed fusion antimicrobial peptides had no significant effect on the growth of engineering bacteria. PBD-2,cecropin P1 released by recombinant antimicrobial peptide digested by enterokinase has obvious antimicrobial activity against a variety of bacteria, including Gram-negative bacteria (E. coli, Salmonella typhimurium). Haemophilus parasuis and Gram-positive bacteria (Staphylococcus aureus). Animal experiments showed that adding this engineering bacterium to the basal diet of piglets could significantly promote the growth of weaned piglets and improve the disease resistance of piglets. This study not only laid a foundation for the application of the engineering bacteria in pig industry. It also provides an important scientific basis for the preparation of other recombinant antimicrobial peptide probiotics.
【学位授予单位】：燕山大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S816

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