摘要
根据NCBI上果胶裂解酶(PL)基因在黑曲霉基因组上的分布特点设计特异引物,通过重组PCR扩增出黑曲霉编码PL的结构基因。将PL基因连接到大肠杆菌表达载体pET-28a上,得到重组质粒pET-28a-PL,转化大肠杆菌(E.coli)BL21后,用IPTG进行诱导表达。结果表明,PL基因片段序列全长1 431bp,与已知PL基因(XM_001397206.2)核苷酸和氨基酸序列同源性均为99%。表达产物经12%SDS-PAGE电泳,得到了一条大小约为50kD的条带,与预期分子量相符,证明真菌PL基因在E.coli BL21中可以有效表达。
According to the pectin lyase （PL） gene sequence of Aspergillus niger from NCBI, spe cific primer pairs were designed targeting the conserved regions,and the structure gene encoding PL was amplified by overlap PCR. The PL fragment was cloned into the plasmid pET-28a to con- struct a recombinant plasmid pET-28a-PL, and then pET-28a-PL was transformed into E. coli BL21 to express fusion protein by IPTG induction. The results indicated that the amplified PL fragment was 1 431 bp in length, which shared 99M identity with the known PL gene （access number XM_001397206. 2） at nucleotide or amino acid level. SDS-PAGE analysis proved that E. coli BL21 harboring the plasmid pET-28a-PL could express a protein with molecular weight of 50 kD,in accordance with the expected size of PL.
出处
《河南农业科学》
CSCD
北大核心
2013年第1期82-85,共4页
Journal of Henan Agricultural Sciences
基金
科技部"十二五"国家科技支撑计划项目(2012BAD19B04)
关键词
黑曲霉
果胶裂解酶
重组PCR
原核表达
Aspergillus niger
pectin lyase
overlap PCR
prokaryotic expression
