桦褐孔菌纤维素酶基因的克隆与原核表达

成功获得了桦褐孔菌(Inonotus obliquus)JL 01菌株的内切葡聚糖酶(IO-EG)和β-葡萄糖苷酶(IO-BGL)的cDNA全长序列.eg2基因cDNA序列全长(ORF)为1149 bp,编码382个氨基酸,bgl2基因cDNA序列全长(ORF)为2583 bp,编码860个氨基酸;成功构建了30a-eg2和30a-bgl2大肠杆菌表达菌株,诱导培养后,SDS-PAGE电泳分析表明两个菌株均表达蛋白,且蛋白以包涵体形式存在,为桦褐孔菌高效外源基因表达系统的建立提供了基础.

Cloning and Prokaryotic Expression of Two Cellulase cDNAs from Inonotus obliquus JL01

Primers for genes encoding endo-l,4-β-D-glucanase (eg) and β-glucosidase (bgl) were designed based on GenBank data and used to generate full-length cDNA sequences of genes encoding EG2 (eg2) and BGL2 (bgl2) in I.obliquus,strain JL 01.The cDNAs of eg2 and bgl2 contained ORFs of 1149 and 2583 bp,respectively,encoding 382 and 860 amino acids,respectively.PCR products generated by these primers were cloned in vector pMD-18T and positive clones were used to construct recombinant plasmids with pET30a,which were then transformed into E.coli strain BL21 for enzyme expression.SDS-PAGE analysis indicated that EG2 and BGL2 proteins existed mainly in the form of inclusion bodies within E.coli cells.