本文選題：人葡萄球菌 + 脂肪酶��； 參考：《天津科技大學(xué)》2010年碩士論文

【摘要】：葡萄球菌是人類和動物的臨床條件致病菌。目前為止,已有來自10種不同葡萄球菌種的13個胞外脂肪酶在基因水平上得到了鑒定,其中3個分離自表皮葡萄球菌,2個分離自金黃色葡萄球菌,各有1個分別來自豬葡萄球菌(Staphylococcus hyicus),溶血葡萄球菌(S. haemolyticus),腐生葡萄球菌(S. saprophyticus),木糖葡萄球菌(S. xylosus),模仿葡萄球菌(S. simulans),頭葡萄球菌(S. capitis),肉葡萄球菌(S. carnosus)與沃氏葡萄球菌(S. warneri)。對這13個已知的葡萄球菌脂肪酶進行DNA與蛋白質(zhì)序列比較發(fā)現(xiàn),它們的DNA與蛋白質(zhì)序列具有高度的同源性。 本論文對人葡萄球菌GIMT1.079(S. hominis)分泌型脂肪酶(SHoL)進行定性檢測后,根據(jù)已知報道的葡萄球菌脂肪酶的保守序列設(shè)計簡并引物,擴增出人葡萄球菌脂肪酶基因的保守序列,再在測出的已知序列的5’端和3’端設(shè)計嵌套的特異性引物,利用改良的hiTAIL-PCR (Thermal Asymmetric Interlaced PCR)技術(shù)擴增已知序列的側(cè)翼序列。 將三次測序結(jié)果進行拼接后,得到人葡萄球脂肪酶基因序列并提交GenBank,登陸號為：GU207403。人葡萄球菌脂肪酶基因編碼754個氨基酸,根據(jù)序列比對分析,其中包含35個氨基酸的信號肽,332個氨基酸的前肽和387個氨基酸的成熟肽,SD序列(AGAGGTG)在起始密碼子ATG上游7個堿基處,在靠近N末端處35個氨基酸的信號肽中具有典型的高度保守的SIRK序列,成熟肽部分包括Ser483、Asp674和His714組成的催化活性位點,與其它葡萄球菌脂肪酶一樣,靠近絲氨酸殘基的部分是兩個保守的甘氨酸；P-loop (-[AG]-x4-G-K-[ST]-)的保守序列也存在于該序列中；與其它葡萄球菌脂肪酶成熟肽同源性達53%-88%。總之,人葡萄球菌GIMT1.709的測序序列與其它葡萄球菌脂肪酶序列具有高度的相似性。 為了進一步驗證所克隆的后選脂肪酶基因是否編碼脂肪酶,將人葡萄球菌脂肪酶成熟肽基因(mSHoL)克隆到pET-28a(+)表達載體上,并在大腸桿菌(Escherichia coli)BL21中進行了表達。SDS-PAGE分析發(fā)現(xiàn)重組蛋白的分子量約為42kDa,測定了陽性克隆BL21/pET-28a(+)-mSHoL細胞裂解液的脂肪酶活力,達到0.35U/mL。
[Abstract]:Staphylococcus is a pathogenic bacteria in human and animal clinical conditions. So far, 13 extracellular lipases from 10 different Staphylococcus species have been identified at the gene level, of which 3 were isolated from Staphylococcus epidermidis and 2 from Staphylococcus aureus. One from Staphylococcus hyicuschus, one from Staphylococcus haemolyticus, one from Staphylococcus saprophyticus, one from Staphylococcus xylosus, one from Staphylococcus hyicustii, one from Staphylococcus hyicuschus, one from Staphylococcus hyicusa, one from Staphylococcus haemolyticus, one from Staphylococcus saprophyticus, one from Staphylococcus xylosus, one from S. The DNA and protein sequences of the 13 known staphylococcal lipases were compared and their DNA and protein sequences were found to be highly homologous. In this paper, the secretory lipase of staphylococcus GIMT 1.079 Shominis was detected qualitatively. According to the conserved sequence of staphylococcal lipase, degenerate primers were designed to amplify the conserved sequence of staphylococcal lipase gene. Then nested specific primers were designed at the 5 'and 3' ends of the known sequences, and the flanking sequences of the known sequences were amplified by modified hiTAIL-PCR thermal Asymmetric Interlaced. After three times of sequencing, the gene sequence of human grape globular lipase was obtained and submitted to GenBank. the landing number was: GU207403. The human staphylococcal lipase gene encodes 754 amino acids. According to sequence alignment analysis, it contains 35 amino acid signal peptide, 332 amino acid prepeptide and 387 amino acid mature peptide SD sequence, which is located at 7 bases upstream of the start codon ATG. A typical highly conserved SIRK sequence was found in the 35 amino acid signal peptides near the N terminal. The mature peptides included a catalytic active site composed of Ser483, Asp674 and His714, similar to other staphylococcal lipases. The conserved sequence of two conserved glycine P-loop-[AG] -x4-G-K- [St]-is also present in the region near the serine residue, and the homology with other staphylococcal lipase mature peptides is 53-88. In conclusion, the sequencing sequence of human staphylococcus GIMT1.709 is highly similar to that of other staphylococcal lipases. In order to further verify whether the cloned lipase gene encodes lipase, the mature peptide gene of staphylococcus staphylococcus lipase, mSHoL, was cloned into pET-28a () expression vector. SDS-PAGE analysis showed that the molecular weight of the recombinant protein was about 42 kDa. the lipase activity of the positive clone BL21 / pET-28a (mSHoL) cell lysate was determined to reach 0.35 UP / mL. the recombinant protein was expressed in E. coli Escherichia coli)BL21 and the molecular weight of the recombinant protein was about 42 kDa. the lipase activity of the positive clone BL21 / pET-28a (mSHoL) was determined.
【學(xué)位授予單位】：天津科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R378

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