本文關(guān)鍵詞：洱源熱泉噬菌體裂解酶基因多樣性及裂解酶基因mmplysin的克隆表達　出處：《昆明理工大學》2017年碩士論文　論文類型：學位論文

  更多相關(guān)文章： 裂解酶多樣性分析 裂解酶mmplysin 基因克隆 誘導表達

【摘要】：噬菌體是感染細菌、古菌及真菌等微生物的病毒。由于其基因組小、機構(gòu)相對簡單、便于基因操作等優(yōu)點,噬菌體自被發(fā)現(xiàn)以來,就被作為基因復制及表達調(diào)控研究的模型。噬菌體裂解酶為噬菌體感染宿主后表達釋放的一類細胞壁水解酶,通過水解細胞壁肽聚糖而使細菌裂解,并釋放出子代噬菌體。傳統(tǒng)的抗生素治療會導致耐藥性菌株出現(xiàn),而噬菌體裂解酶能高效、專一地裂解細菌,使其有望取代傳統(tǒng)的抗生素治療。本文以來源于洱源熱泉環(huán)境的混合樣品ya為材料,將噬菌體混合液超速離心后收集噬菌體,提取噬菌體DNA后交由諾禾致源公司完成測序。測序結(jié)果通過FastQC進行測序質(zhì)量分析和IDBA-UD序列拼接。此后對拼接好的序列與NCBI中基因庫進行序列檢索分析,獲得裂解酶共有1006條序列,分別為amidase、endopeptidase、transglycosylase、glucosaminidase,這四種裂解酶對應的contig數(shù)分別是422、306、250和28條,其中具有完整ORF數(shù)目分別是94、65、66和8條。將其中堿基序列長度為500-1500 bp翻譯成氨基酸序列,并對amidase、endopeptidase、transglycosylase、glucosaminidase 這四種裂解酶的保守結(jié)構(gòu)域進行分析,為后續(xù)尋找新的更高效的噬菌體裂解酶以及噬菌體裂解酶的裂解機制奠定基礎。此外,本文對亞棲熱菌噬菌體MMP17的裂解酶基因mmplysin進行克隆表達和活性分析。裂解酶基因mmplysin序列長度為633 bp,保守結(jié)構(gòu)域顯示來自M23肽酶家族。mmplysin經(jīng)過PCR擴增、連接、轉(zhuǎn)化和誘導重組后,與pET28a構(gòu)建成重組質(zhì)粒pET28a-mmplysin。經(jīng)誘導表達后,得到重組蛋白mmplysin。該重組蛋白在28℃,誘導5h后得最大表達量,重組蛋白的分子量23 kDa左右。該重組蛋白mmplysin最適酶活溫度為56-65℃,從37℃到75℃均有酶活,高于大部分已報道的噬菌體裂解酶的最適作用溫度;其在pH7-8時活性最強;金屬離子對酶的影響研究結(jié)果顯示,Mn2+,Ca2+,K+存在時導致mmplysin酶活性降低,而Zn2+,Cu2+,Mg2+對酶有激活作用。通過裂解酶mmplysin對不同細菌的抑菌實驗發(fā)現(xiàn),重組蛋白mmplysin對噬菌體MMP17宿主菌TG17有明顯的抑菌作用,對沙門氏菌、金黃色葡萄球菌和大腸桿菌都具有一定的抑制作用。在耐藥性肺炎克雷伯菌的抑菌實驗中,發(fā)現(xiàn)其對部分菌株同樣具有一定的抑制作用。裂解酶對耐藥性肺炎克雷伯菌的抑制作用的原子力顯微鏡觀察表明,酶液作用后會在細胞壁表面形成穿孔從而使得細胞質(zhì)的流出導致細胞的死亡。本文研究了噬菌體裂解酶的多樣性,對裂解酶mmplysin的特征進行研究,為后續(xù)高溫裂解酶在實際生活中的應用提供了一定的實驗依據(jù)。
[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，

本文編號：1417109