摘要
以蛋白酶高产菌株Ｂ．ｓｕｂｔｉｌｉｓＭＬ为供体菌，提取其染色体ＤＮＡ，经限制性内切酶ＢａｍＨＩ完全酶切后，插入枯草杆菌载体ｐＮＱ１２２的相同酶切位点，连接后转化中性碱性蛋白酶基因双缺陷型菌株Ｂ．ｓｕｂｔｉｌｉｓＤＢ１０４。从含有氯霉素的酪蛋白平板上获得了２０个具有蛋白酶活性的克隆。测定了克隆菌的蛋白酶活性和抗性，对来自部分阳性克隆的重组质粒进行了Ｓｏｕｔｈｅｒｎ杂交分析，其中３个与已知的中性蛋白酶基因有很强的同源性。经对这３个阳性克隆的蛋白酶活性抑制试验以及分泌蛋白的聚丙烯酰胺凝胶电泳分析，可确定存在着中性蛋白酶基因的表达产物。从这３个阳性克隆中获得的重组质粒，被分别命名为ｐＭＰＲ４、ｐＭＰＲ８和ｐＭＰＲ１６。
Information regarding the cloning of neutral protese gene from a high-yield protese-producing strain of Bacillus subtilis ML is presented. The Chromosomal DNA of B. subtilis ML was digested with restriction enzyme BemH I and the resulted fragments wers inserted into the BamH1 site of pNQ122 of B. subtilis cloning vector. the recombinant DNA molecules were used to transform the B. subtilis DB104,an alkaline and neutral protease genes double delection mutant strain.Twenty positive clones containing protease genes were isolated. The Southern hybridization, protease inhibition test, and SDS-pAGE analysis of the expressing proteases showed that there were three positive clones containing npr gene. The recombinant plasmids from the three clones were named as PNPR4,pNPR8,and pNPR16,respectively.
出处
《皮革科学与工程》
CAS
1995年第2期1-8,共8页
Leather Science and Engineering
关键词
枯草芽孢杆菌
中性蛋白酶
基因
基因克隆
制草
Bacillus subtilis, neutral protease gene, gene cloning, gene express,southern hybridization
