洱源热泉噬菌体裂解酶基因多样性及裂解酶基因mmplysin的克隆表达

发布时间：2018-01-13 02:36

  本文关键词：洱源热泉噬菌体裂解酶基因多样性及裂解酶基因mmplysin的克隆表达　出处：《昆明理工大学》2017年硕士论文　论文类型：学位论文

  更多相关文章： 裂解酶多样性分析 裂解酶mmplysin 基因克隆 诱导表达

【摘要】：噬菌体是感染细菌、古菌及真菌等微生物的病毒。由于其基因组小、机构相对简单、便于基因操作等优点,噬菌体自被发现以来,就被作为基因复制及表达调控研究的模型。噬菌体裂解酶为噬菌体感染宿主后表达释放的一类细胞壁水解酶,通过水解细胞壁肽聚糖而使细菌裂解,并释放出子代噬菌体。传统的抗生素治疗会导致耐药性菌株出现,而噬菌体裂解酶能高效、专一地裂解细菌,使其有望取代传统的抗生素治疗。本文以来源于洱源热泉环境的混合样品ya为材料,将噬菌体混合液超速离心后收集噬菌体,提取噬菌体DNA后交由诺禾致源公司完成测序。测序结果通过FastQC进行测序质量分析和IDBA-UD序列拼接。此后对拼接好的序列与NCBI中基因库进行序列检索分析,获得裂解酶共有1006条序列,分别为amidase、endopeptidase、transglycosylase、glucosaminidase,这四种裂解酶对应的contig数分别是422、306、250和28条,其中具有完整ORF数目分别是94、65、66和8条。将其中碱基序列长度为500-1500 bp翻译成氨基酸序列,并对amidase、endopeptidase、transglycosylase、glucosaminidase 这四种裂解酶的保守结构域进行分析,为后续寻找新的更高效的噬菌体裂解酶以及噬菌体裂解酶的裂解机制奠定基础。此外,本文对亚栖热菌噬菌体MMP17的裂解酶基因mmplysin进行克隆表达和活性分析。裂解酶基因mmplysin序列长度为633 bp,保守结构域显示来自M23肽酶家族。mmplysin经过PCR扩增、连接、转化和诱导重组后,与pET28a构建成重组质粒pET28a-mmplysin。经诱导表达后,得到重组蛋白mmplysin。该重组蛋白在28℃,诱导5h后得最大表达量,重组蛋白的分子量23 kDa左右。该重组蛋白mmplysin最适酶活温度为56-65℃,从37℃到75℃均有酶活,高于大部分已报道的噬菌体裂解酶的最适作用温度;其在pH7-8时活性最强;金属离子对酶的影响研究结果显示,Mn2+,Ca2+,K+存在时导致mmplysin酶活性降低,而Zn2+,Cu2+,Mg2+对酶有激活作用。通过裂解酶mmplysin对不同细菌的抑菌实验发现,重组蛋白mmplysin对噬菌体MMP17宿主菌TG17有明显的抑菌作用,对沙门氏菌、金黄色葡萄球菌和大肠杆菌都具有一定的抑制作用。在耐药性肺炎克雷伯菌的抑菌实验中,发现其对部分菌株同样具有一定的抑制作用。裂解酶对耐药性肺炎克雷伯菌的抑制作用的原子力显微镜观察表明,酶液作用后会在细胞壁表面形成穿孔从而使得细胞质的流出导致细胞的死亡。本文研究了噬菌体裂解酶的多样性,对裂解酶mmplysin的特征进行研究,为后续高温裂解酶在实际生活中的应用提供了一定的实验依据。
[Abstract]:Bacteriophage is a virus that infects bacteria, archaea and fungi. Because of its small genome, simple mechanism and easy gene manipulation, bacteriophage has been discovered since. Phage lyase is a kind of cell wall hydrolase which is expressed and released by phage infected host, which cleavage bacteria by hydrolyzing peptidoglycan of cell wall. Traditional antibiotic therapy will lead to the emergence of drug-resistant strains, and bacteriophage lyase can be highly efficient and specific to the lysis of bacteria. It is expected to replace the traditional antibiotic therapy. In this paper, the mixed sample ya from Eryuan hot spring environment was used as the material, the bacteriophage mixture was centrifuged to collect bacteriophage after ultracentrifugation. The phage DNA was extracted and sequenced by Novosource. The sequencing result was analyzed by FastQC and IDBA-UD sequence was spliced together. Then the spliced sequence was matched with NCBI. The gene bank was sequenced and analyzed. A total of 1006 cleavage enzymes were obtained, namely amidase endopeptidase transglycosylase. Glucosaminidase, the corresponding contig number of these four enzymes was 422,306,250 and 28, respectively, and the number of complete ORF was 94. The length of the nucleotide sequence was 500-1500 BP translated into amino acid sequence, and the amidase endopeptidase was transformed into amino acid sequence. The conserved domains of transglycosylase glucosaminidase were analyzed. In addition, it lays a foundation for further searching for new and more efficient phage lyase and the lytic mechanism of phage lyase. The lyase gene mmplysin of the bacteriophage MMP17 was cloned and expressed and its activity was analyzed. The mmplysin sequence of the lyase gene was 633bp. The conserved domain showed that the M23 peptidase family. Mmplysin was amplified by PCR, ligated, transformed and induced to recombine. The recombinant plasmid pET28a-mmplysin was constructed with pET28a. After induced expression, the recombinant protein mmplysin was obtained. The recombinant protein was expressed at 28 鈩，

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