【作者】 赵爱珍；

【导师】 韩文瑜；

【作者基本信息】 吉林大学 ， 预防兽医学， 2005， 博士

【摘要】 联合应用表型测定和PCR 技术判定屎肠球菌AS1.2025 具有产生enterocin A 的能力,并从其染色体DNA 中克隆到包含结构基因在内的三种enterocin A 合成相关基因。成功构建了三种enterocin A 重组表达质粒,表达的MBP-enterocin A、GST-enterocin A 及His-tag-enterocin A 呈现抗李斯特氏菌活性。进行了MBP-enterocin A 和His-tag-enterocin A 的抑菌谱、稳定性及作用模式的研究,结果表明,环境因素如温度和pH 值及化学物质如蛋白酶和还原剂均不同程度影响重组enterocin A 的活性;重组enterocin A 表现特异的抗李斯特氏菌活性和杀菌的作用模式。通过PCR 突变技术成功构建了5种MBP-enterocin A突变体重组表达质粒,并对MBP-enterocin A及其突变体的抑菌活性进行比较分析,结果表明:二硫键为重组enterocin A 活性的关键因素,而亮氨酸不为活性的关键氨基酸;enterocin A 的N-端氨基酸序列为活性的关键因素,并也表明了enterocin A 前体也具有活性。更多还原

【Abstract】 Characterization of Enterococcus faecium AS 1.2025 demonstrated the strain was gram-positive, facultatively anaerobic coccus. It did not produce gas from glucose.Furthermore, it was able to grow at 10℃and 45℃, at pH 9.6, and in the presence of 6.5%NaCl. At the same time ,its feature of producing bacteriocins was identificated. Bioassays are employed to assess the inhibitory effect of enterocins produced by Enterococcus faecium AS 1.2025 on indicator microorganisms by agar overlay test and agar diffusion test. Antagonistic activities of Enterococcus faecium against Gram-positive bacteria strains and Gram-negative bacteria strains could be detected on agar plates by overlay test, while no bacterocin activity in preparations from culture supernatants by agar diffusion test. Three pairs of specific primers were designed for amplification of related enterocin gene fragment according to the nucleotide sequence of enterocin A ,enterocin B and enterocin P published in GenBank by PCR. PCR results showed that only 409-bp A containing part of the enterocin A structural gene and immunity gene was amplified from chromosomal DNA of the selected strain. The PCR fragment was directly sequenced to confirm the gene of interest. The other fragments were not amplificated by PCR. These information suggested that Enterococcus faecium AS 1.2025 has the property of enterocins-producing. Based on this result, one pair of specific primers was designed for amplification of enterocin A-related genes responsible for production, immunity and regulation. A 2,180-bp fragment entAIF was obtained from chromosomal DNA of Enterococcus faecium AS 1.2025 by PCR. The PCR product was cloned into pGEM -T ,resulting in plasmid pGAIF. DNA sequencing analysis of pGAIF revealed that the presence of four open reading frames (ORFs),three of which were enterocin A-related genes. The full length of entA198 nucleotides., encoded the prepeptide of 65 amino acid residues, containing an N-terminal leader sequence of 18 amino acid residues, which was removed during the maturation process to give the mature bacteriocin enterocin A. The second ORF entI, spanning 312 bp, was located 2 nucleotides downstream of ent A and encoded a 103-amino-acid protein conferring immunity to enterocin A. Sequence alignment showed that genes entA and entI from different E. faecium strains shared 99.87% and 99.76% identity, respectively; Enterocin A prepeptide exhibited complete identity, while immunity protein diversity of 1 amino acid residue . The full length of ORF3 was1179 bp, very similar to nucleotide sequence of a probable transposase gene from Enterococcus faecalis. The gene entF consist nucleotides, encoding the induction factor prepeptide. The homologies among deduced amino acid sequence of EntFfrom different strains were 100%. In order to facilitate a biotechnological production and purification of enterocin A, three heterologous E.coli expression systems were used. The DNA fragments OAH,BAE1and BAE2 were amplified from pGAIF by PCR using different specific primers and cloned into vectors pMAL-p2x, pGEX-3X and pET-28(+),respectively. The three constructed expression vectors, namely pMA, pXA and pTA, respectively, were transformed into E.coli host strains. SDS-PAGE analysis of protein preparations indicated high -level expression of three fusion proteins, which were shown to be soluble . Inhibitory activity of fusion proteins were detected by agar diffusion test. The lysates containing MBP-enterocin A or His-tag-enterocin A displayed strong antilisterial activity, while GST-enterocin A weak antagonistic activity against Listeria. Antilisterial activity characterization and limitations of activity of the recombinant enterocin A fusion proteins were investigated with regard to substitution of native enterocin A and possible use as preservatives. MBP-enterocin A and His-tag-enterocin A were purified from the clarified sample of the cell lysate by affinity chromatography. MBP-enterocin A possessed inhibitory activity against a narrow range of Gram-positive bacteria, mainly active against Listeria spp such as food-borne pathogen Listeria monocytogenes. His-tag-enterocin A show a relatively high heat stability,and retain detectable activity after heat treatment of 100℃for 10更多还原