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OALib Journal期刊
ISSN: 2333-9721
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木聚糖酶基因xynb在不同大肠杆菌表达系统中的表达比较
DOI: 10.3724/SP.J.1145.2011.00100, PP. 100-103
Keywords: 木聚糖酶,xynb基因,大肠杆菌,重组表达,信号肽,黑曲霉
Abstract:
从黑曲霉（aspergillusniger）nl-1中克隆获得木聚糖酶基因xynb.该基因全长745bp，含有67bp内含子，与公布的黑曲霉xynb基因有较高的同源性.将pcr扩增的木聚糖酶成熟肽基因和含有信号肽的基因分别连接到表达载体ptrc-99a和pet-20b上获得重组质粒ptrc-99a-xynb、ptrc-99a-xynb（s）、pet-20b-xynb和pet-20b-xynb（s），并转化到大肠杆菌top10f’、dh10b和两种bl21宿主中获得重组菌株.通过iptg诱导，xynb基因在重组菌株中获得特异性表达.表达产物以胞内可溶性蛋白和不溶性包涵体形式存在.诱导4h，重组菌株ptrc-99a-xynb[bl21condonplus（de3）]表达量最高，胞内酶活达到299iu/l.重组质粒ptrc-99a-xynb（s）在不同宿主胞内和胞外均能分泌目的蛋白，诱导10h，胞外酶活ptrc-99a-xynb（s）[bl21condonplus（de3）]达到347iu/l.而pet-20b-xynb（s）在两种bl21宿主中均不表达.经sds-page分析，以ptrc-99a和pet-20b作为表达载体时，重组蛋白相对分子质量分别约为45×103和30×103.与pet-20b相比，ptrc-99a以及自身信号肽的引导更有利于重组木聚糖酶的表达.图6参12
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