摘要
从黑曲霉(Aspergillus niger)nl-1中克隆获得木聚糖酶基因xynB.该基因全长745 bp,含有67 bp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肠杆菌Top10 F'、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4 h,重组菌株pTrc-99a-xynB[BL21 condon plus(DE3)]表达量最高,胞内酶活达到299 IU/L.重组质粒pTrc-99a-xyn B(S)在不同宿主胞内和胞外均能分泌目的蛋白,诱导10 h,胞外酶活pTrc-99a-xynB(S)[BL21 condon plus(DE3)]达到347 IU/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.
The xynB gene of endo-1,4-xylanase was cloned from Aspergillus niger.The full-length gene contained 745 bp,including an intron of 67 bp,which has high similarity with other xylanase genes.The xynB cDNA fragment encoding mature peptide and the xynB cDNA were inserted into the expression vectors pTrc-99a and pET-20b,respectively.Then the recombinant plasmids were transformed into Escherichia coli TOP10F,DH10B and two kinds of BL21,respectively.The xynB gene was expressed in the recombinant stains by inducing with IPTG.Its expression products existed as both soluble proteins and inclusion bodies.And the recombinant protein expressed in pTrc-99a-xynB [BL21 condon plus（DE3）] was highest,which reached to 299 IU/L in 4 h,and moreover,the extracelluar xylanase activity was detected in the strains with pTrc-99a-xynB（S）.A maximum activity of 347 IU/L was obtained from extracelluar extract of pTrc-99a-xynB（S） [BL21 condon plus（DE3）] after 10 h.But the pET-20b-xynB（S） could not express in the hosts of two kinds of BL21.The molecular mass of the recombinant protein with the vector pTrc-99a was about 45×103 according to SDS-PAGE,but 30×103 with pET-20b.Compared to the vector pET-20b,pTrc-99a and the native signal peptide were more effective.
出处
《应用与环境生物学报》
CAS
CSCD
北大核心
2011年第1期100-103,共4页
Chinese Journal of Applied and Environmental Biology
基金
国家自然科学基金项目(No.30871990)
江苏省自然科学基金项目(No.BK2008423)资助~~
关键词
木聚糖酶
xynB基因
大肠杆菌
重组表达
信号肽
黑曲霉
xylanase
xynB gene
Escherichia coli
recombinant expression
signal peptide
Aspergillus niger
