烟曲霉菌壳聚糖酶基因的克隆及在大肠杆菌中的表达

根据GenBank中发布的烟曲霉菌壳聚糖酶（Aspergillus fumigatus chitosanase,EC3.2.1.132）基因序列人工合成8条DNA长链及4条引物链。DNA链的设计上在不改变壳聚糖酶氨基酸组成的前提下选择大肠杆菌使用频率高的密码子。PCR拼接法扩增壳聚糖酶基因并克隆入pGEM_T easy载体进行序列分析，进一步亚克隆入表达载体pGEX_3X。重组质粒pGEX_Csn转化E.coli DH5α，IPTG诱导表达，亲和层析及Factor Xa酶解纯化重组Csn。所得重组壳聚糖酶具有降解壳聚糖的生物活性，其活性受温度及pH值的影响。

Cloning and expression of an Aspergillus fumigatus chitosanase gene

According to published DNA sequence of Aspergillus fumigatus chitosanase(Csn) gene, 8 long single DNA strands each about 100bp and 4 DNA primers were designed and synthesised. By PCR, 8 DNA strands were connected into a complete chitosanase gene of 624bp. This chitosanase gene was not identical with its wild type, some point mutations were introduced into its DNA sequence by special design of those 8 DNA strands. These mutaions did not change amino acid composition of the chitosanase, however, the codens were changed into E. coli favorites. The Csn gene was cloned into plasmid pGEM-Teasy and verified by DNA sequence analysis. Thereafter, Csn gene was subcloned into a fusion-protein expressing vector pGEX-3X. Recombinant plasmid pGEX-Csn was transformed into E. coli DH5alpha and the transformant was induced expressing with 0.5 mmol/L IPTG. Expressing product was analyzed by SDS-PAGE, fusion protein GST-Csn was purified by affinity chromatography. By factor X a digestion GST-Csn was cleaved and GST was taken out by another chromatography. The biological activity of recombinant chitosanase(rCsn) was also detected, as a result the recombinant Csn had a strong ability of degrading chitosan, which was much higher than lysozyme. Its chitosan-degradation activity could be influenced by pH and temperature.