摘要
对牛凝乳酶基因进行密码子优化,并首次在高效表达宿主巨大芽孢杆菌中表达。通过克隆牛凝乳酶基因,将片段与穿梭质粒pHIS1525连接,连接产物转化大肠杆菌XL-10,筛选出阳性克隆子,所获重组质粒经酶切、测序鉴定,证实含目的片段。通过原生质体转化的方法转化到表达宿主巨大芽孢杆菌WH320。用木糖诱导表达,离心取上清经镍柱亲和层析(Ni-NTA)和聚丙烯酰氨凝胶电泳(SDS-PAGE)分离纯化并鉴定,测定重组牛凝乳酶的酶学性质,表明在巨大芽孢杆菌中得到有效表达。为我国利用生物工程技术的方法自主生产凝乳酶提供了基础。。
To improve expression efficiency of recombinant bovine chymosin in Bacillus megaterium which was never done before, one DNA encoding bovine chymosin gene was designed and synthesized by using optimized codons. The gene segment was cloned into the shuttle plasmid pHIS1525,then transformed into Escherichia coli XL-10.Screening the positive cloning, the recombinant plasmid confirmed by doubleenzyme digestion and gene sequencing. Transformed into the protoplasts of Bacillus megaterium strains WH320. The recombinant protein was produced under xylose induction. Centrifuge the sample, leave the soluble to purification of protein by Ni-NTA chromatography and SDS- PAGE, and determination of chymosin activity. By synthesizing the modified bovine chymosin gene, active chymosin was expressed in Bacillus megaterium WH320, which provided the basis for developing bio-ergineering technology for producing chymosin in China.
出处
《中国乳品工业》
CAS
北大核心
2013年第4期4-6,共3页
China Dairy Industry
关键词
重组凝乳酶
巨大芽孢杆菌
酶学性质
recombinant chymosin
Bacillus megaterium
enzymatic properties
