宇佐美曲霉E001菌株XynⅡ基因的克隆和融合表达

Cloning and fusion expression of the Xylanase XynⅡgene from Aspergillus usamii E001

以宇佐美曲霉(Aspergillus usamii)E001株基因组DNA为模板,PCR扩增了编码木聚糖酶Ⅱ(Xyla-nases,XynⅡ)成熟肽的DNA片段。构建重组克隆质粒pUCm-T-XynⅡ,并以其为摸板,PCR扩增XynⅡ成熟肽的cDNA片段(555 bp),连接于表达质粒pGEX-5X-3的BamHⅠ和EcoRⅠ酶切位点间,构建重组表达质粒pGEX-5X-3-XynⅡ,转化E.coliBL21(DE3),IPTG诱导表达,并对表达产物进行检测。结果表明,XynⅡDNA中存在1个内含子,长度为51 bp;融合蛋白GST-XynⅡ主要以包涵体形式存在,相对分子质量约为50 ku,IPTG诱导1,3和5 h融合蛋白GST-XynⅡ的表达量分别约占菌体总蛋白量的12.6%,25.3%和32.1%。表明宇佐美曲霉E001菌株XynⅡ基因融合表达成功。

The DNA fragment of encoding xylanase Ⅱ （Xyn Ⅱ ）mature peptide was amplified with chromosome DNA isolated from Aspergillus usamii E001 by PCR. The recombinant cloning plasmid pUCm-T-Xyn Ⅱ was constructed,and the cDNA fragment （555 bp in length） by encoding Xyn Ⅱ mature peptide was obtained from the pUCm-T-xyn Ⅱ by PCR. The PCR product was digested with BamH Ⅰ and EcoR Ⅰ ,and then inserted into the expressive vector pGEX-5X-3. The recombinant plasmid pGEX-5X-3- Xyn Ⅱ was transformed into Escherichia coli BL21 （DE3） and inducing expressed by IPTG. The expressed products were examined. The results showed that the DNA sequence only contained one intron, and its length was 51 bp. The fusion protein mainly existed in the form of inclusion body,and its relative molecular weight was about 50 ku. The expression levels of pGEX-5X-3-Xyn Ⅱ in E. coli BL21 induced by IPTG for 1 h,3 h and 5 h were about 12.6% ,25.3% and 32.1% ,respectively. Based on this study,the protein engineering modification of the E001 Xyn Ⅱ and its high expression in Pichia pastoris will be further studied .