人葡萄球菌GIMT1.079脂肪酶基因的克隆与表达

发布时间：2018-04-30 07:55

本文选题：人葡萄球菌 + 脂肪酶　；参考：《天津科技大学》2010年硕士论文

【摘要】：葡萄球菌是人类和动物的临床条件致病菌。目前为止,已有来自10种不同葡萄球菌种的13个胞外脂肪酶在基因水平上得到了鉴定,其中3个分离自表皮葡萄球菌,2个分离自金黄色葡萄球菌,各有1个分别来自猪葡萄球菌(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),头葡萄球菌(S. capitis),肉葡萄球菌(S. carnosus)与沃氏葡萄球菌(S. warneri)。对这13个已知的葡萄球菌脂肪酶进行DNA与蛋白质序列比较发现,它们的DNA与蛋白质序列具有高度的同源性。本论文对人葡萄球菌GIMT1.079(S. hominis)分泌型脂肪酶(SHoL)进行定性检测后,根据已知报道的葡萄球菌脂肪酶的保守序列设计简并引物,扩增出人葡萄球菌脂肪酶基因的保守序列,再在测出的已知序列的5’端和3’端设计嵌套的特异性引物,利用改良的hiTAIL-PCR (Thermal Asymmetric Interlaced PCR)技术扩增已知序列的侧翼序列。将三次测序结果进行拼接后,得到人葡萄球脂肪酶基因序列并提交GenBank,登陆号为：GU207403。人葡萄球菌脂肪酶基因编码754个氨基酸,根据序列比对分析,其中包含35个氨基酸的信号肽,332个氨基酸的前肽和387个氨基酸的成熟肽,SD序列(AGAGGTG)在起始密码子ATG上游7个碱基处,在靠近N末端处35个氨基酸的信号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组成的催化活性位点,与其它葡萄球菌脂肪酶一样,靠近丝氨酸残基的部分是两个保守的甘氨酸；P-loop (-[AG]-x4-G-K-[ST]-)的保守序列也存在于该序列中；与其它葡萄球菌脂肪酶成熟肽同源性达53%-88%。总之,人葡萄球菌GIMT1.709的测序序列与其它葡萄球菌脂肪酶序列具有高度的相似性。为了进一步验证所克隆的后选脂肪酶基因是否编码脂肪酶,将人葡萄球菌脂肪酶成熟肽基因(mSHoL)克隆到pET-28a(+)表达载体上,并在大肠杆菌(Escherichia coli)BL21中进行了表达。SDS-PAGE分析发现重组蛋白的分子量约为42kDa,测定了阳性克隆BL21/pET-28a(+)-mSHoL细胞裂解液的脂肪酶活力,达到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.
【学位授予单位】：天津科技大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R378

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