烟曲霉脱乙酰几丁质酶cDNA的克隆及其在大肠杆菌中表达的研究

将烟曲霉的一种胞外脱乙酰几丁质酶（AF CSN）纯化，并测定该酶N-端氨基酸的序列。用此序列推测的寡核苷酸引物扩增得到脱乙酰几丁质酶（csn）的cDNA，其全长为804bp，编码一种251个氨基酸的单肽，对csn的序列比较分析表明，它与茄病镰刀菌在两个区域具有同源性，这两个区域分别有重复天冬氨酸及苏氨酸残基，这说明它与细菌具有相同的演化起源。cns与已知的细菌脱乙酰几丁质酶没有同源性，Southern杂交分析显示，cns在烟曲霉基因组中的单拷贝的。用质粒栽体pET28a(t),csn cDNA被导入E.coli中得到csn的融合蛋白，在IPTG诱导下，E.coli细胞中可获得大量的约28kDa的成熟蛋白。

cDNA Cloning and Expression of Chitosanase from Aspergillus fumigatus

An extracellular chitosanase (AF CSN) was purified from Aspergillus fumigatus and N terminal amino acids of the enzyme were sequenced. Oligonucleotide primer based on the sequence was used to PCR amplify of the chitosanase cDNA. The cloned full length cDNA (csn), 804 bp in size, encoded a single peptide of 251 amino acids. The blast search results of the cDNA sequence showed that the sequence shared the homology with chitosanase of Fusarium solani in two region, specifically but showed no homology with known bacterial chitosanases. The two region have repeated aspartic acid (DIDCD) and threonin (TWTPT) amino acid residue, respectively. This suggests that the chitosanase might have an evolutional origin distinct from bacterial chitosanase. Southern blot analysis results indicated that csn is present as a single copy in the genome of A. fumigatus. The recombinant protein corresponding to the mature protein was generated in E. coli using a plasmid vector pET28a(+). When csn cDNA corresponding to the mature protein was introduced into E. coli and expressed, approximately 28kDa protein was produced in a large amount after IPTG induction.