【作者】 杨春晖；

【导师】 王海燕；

【作者基本信息】 四川大学 ， 遗传学， 2006， 硕士

【摘要】 短小芽孢杆菌UN-31-C-42是一株产生具有脱毛功能的碱性蛋白酶的菌株。该菌株的碱性蛋白酶基因编码区已被克隆，本研究通过TAIL—PCR(Thermalasymmetric interlaced PCR)扩增得到碱性蛋白酶基因编码区上游的启动子片段Papr，经测序、序列拼接及比对分析等对Papr进行了鉴定。该启动子片段长797bp，在片段的5’端存在一个开放阅读框，可能为磷酸甘油酸变位酶基因的部分编码区，故碱性蛋白酶基因启动子的长度为378 bp。通过对启动子序列的分析，推测出了该启动子的保守序列及转录起始位点。对启动子的顺序缺失研究证实基因起始密码子上游160bp的DNA片段包含完整的启动功能片段。根据Papr序列设计引物，从短小芽孢杆菌UN-31-C-42基因组中扩增获得了包含启动子、信号肽、前肽、成熟肽及终止子的完整的碱性蛋白酶基因片段WAp。将WAp插入大肠杆菌—芽孢杆菌穿梭质粒载体pSUGV4中，构建了碱性蛋白酶基因表达质粒pSUBpWAp。将pSUBpWAp分别转入大肠杆菌JM109、枯草芽孢杆菌WB600以及短小芽孢杆菌UN-31-C-42中进行表达。包含了质粒pSUBpWAp的大肠杆菌JM109细胞内外均检测不到碱性蛋白酶活性。在枯草芽孢杆菌WB600中的表达则可在胞外检测到碱性蛋白酶活性，证明启动子Papr可在枯草芽孢杆菌中启动碱性蛋白酶基因的表达，产生的碱性蛋白酶活性最高达到466.5 U／ml，与包含Bp53启动子的碱性蛋白酶基因表达质粒pSUBpAp比较，酶活提高2倍。pSUBpWAp在短小芽孢杆菌UN-31-C-42中也能表达产生碱性蛋白酶活性，但宿主菌的蛋白酶产量并未提高。利用细菌的16s rDNA通用引物从短小芽孢杆菌UN-31-C-42基因组中扩增到16s rDNA片段，将该片段插入载体pMD18-T中，构建了重组质粒pMD16s。从载体pSUGV4中扩增到卡那霉素抗性基因片段，与碱性蛋白酶基因一同插入质粒pMD16s的16s rDNA片段中，构建整合型质粒。拟将该质粒整合至短小芽孢杆菌基因组，提高其蛋白酶产量。更多还原

【Abstract】 Bacillus pumilus UN-31-C-42 is a strain which produces alkaline protease with dehairing function. The native promoter of alkaline protease gene, Papr, was cloned by TAIL-PCR from B. pumilus UN-31-C-42. Through blast analysis, the sequence of promoter Papr was confirmed and the conserved sequences were deduced. The promoter was 378 bp in length, and a phosphoglycerate mutase gene was located at the upstream region of promoter Papr. Deletion analysis of Papr revealed that the promoter of 160 bp could initiate the transcription of alkaline protease gene.The alkaline protease gene, including promoter, the coding region of prepeptide, propeptide and mature peptide, and terminator, was inserted into pSUGV4, an Escherichia coli-Bacillus subtilis shuttle vector. The recombinant plasmid pSUBpWAp was transformed into E. coli JM109, B. subtilis WB600 and B. pumilus UN-31-C-42, respectively.Expression of alkaline protease gene in pSUBpWAp in E. coli JM109 did not result in detectable extracellular and intracellular protease activity. But it expressed active extracellular protease in B. subtilis WB600 and B. pumilus UN-31-C-42. The enzymatic activity of recombinants in B. subtilis WB600 was 466.5 U/ml. It was twice more than that of plasmid pSUBpAp which was under the control of promoter Bp53. The enzymatic activity of pSUBpWAp in B. pumilus UN-31-C-42 achieved 3960 U/ml, but it was still lower than the host strain.The 16s rDNA was cloned from genome of B. pumilus UN-31-C-42 by PCR. The fragment of 16s rDNA was inserted into vector pMD18-T, constructing recombinant plasmid pMD16s. The fragments of alkaline protease gene and kanamycin-resistant gene were inserted into 16s rDNA in pMD 16s. The recombinant will be integrated in the chromosome of B. pumilus UN-31-C-42 to increase the yield of alkaline protease.更多还原