深海古菌Thermococcus siculi HJ21高温普鲁兰酶基因的克隆及表达

Cloning and Expression of a New Thermostable Pullulanase Gene from a Deep-sea Archaeaon Strain Thermococcus siculi HJ21

根据NCBI上公布的普鲁兰酶基因的保守序列设计简并引物,以Thermococcus siculi HJ21基因组DNA为模板进行PCR,得到T.siculi HJ21普鲁兰酶的基因,测序后通过Blast(NCBI)数据库比对和分析表明扩增基因有一个4056bp、编码1351个氨基酸的开放阅读框,为一新的普鲁兰酶基因。将该基因插入表达载体pET28a,并转化Escherichia coli BL21(DE3),经IPTG诱导,测定普鲁兰酶比活力。重组转化子的细胞破碎液有高温普鲁兰酶活性,SDS-PAGE电泳结果显示出分子质量约为150kD的特异性条带。

The gene of pullulanase was amplified from Thermococcus siculi HJ21 with degenerate primes designed based on the NCBI published conserved sequence information. The DNA sequencing and BLAST （NCBI） analysis showed that this DNA sequence was a new pullulanase gene with an open reading frame （ORF） of 4056 bp in length encoding 1351 amino acids.The gene was cloned into the expression vector,pET28a,producing a hybrid plasmid pET28a-pull. Subsequently,pET28a-pull was introduced into Escherichia coli BL21（DE3）. The lysate of the transformant cells showed thermostable pullulanase activity. The SDS-PAGE analysis showed a band with apparent molecular weight of 150 kD.