新型脂肪酶基因的克隆与表达

摘要

Objective Select novel lipase with high activity from Aspergillus Niger through homologous alignment.Methods Through alignment in NCBI with the screened lipase gene of Aspergillusfumigatus,afl-1,a putative lipase gene in Aspergillus Niger was searched and amplified for the gene cloning.A pair of primers was designed and the gene was amplified with PCR after the extraction of Aspergillus Niger genomic DNA.After recovery the PCR products were directly cloned into pMD18T for amplification,and with the certain of sequencing,the gene was subcloned into the expression vector pET28a to obtain an expression plasmid.The expression plasmid was transformed into Escherichia coli BL21 (DE3) for expression and activity assay.Results A novel lipase gene,an1-1,isolated from Aspergillus Niger,was cloned and expressed in E.coli for the first time.After induced at 15℃,massive soluble protein was collected.The recombinant lipase showed an activity of 2365 U/L toward C4 p-Nitrophenyl ester.Conclusion Homologous sequence alignment can improve the efficiency of gene screening.A new kind of lipase gene was cloned from Aspergillus Niger,which could be expressed as soluble protein with biological activity.%目的 通过同源比对筛选黑曲霉中新型活性脂肪酶.方法 抽提黑曲霉基因组,依据从烟曲霉中获得的高活性脂肪酶基因序列,NCBI网站序列比对后搜索到与烟曲霉af1-1同源性较高的黑曲霉脂肪酶基因序列an1-1,设计引物进行PCR扩增,产物胶回收后连接至PMD 18T载体,测序验证正确后将目的序列连接至表达载体PET28a,转化Escherichia coli BL21 (DE3)中表达和测定活性.结果 克隆了一种来源于黑曲霉新型脂肪酶基因an1-1,15℃表达条件下主要为可溶性表达,对C4底物(对硝基苯酚丁酸酯)活性达2365 U/L.结论 同源序列比对能有效提高筛选效率,并从黑曲霉中筛选到一种新的脂肪酶基因,在大肠杆菌中可高表达并具有生物学活性.