白色噬琼胶菌QM38 β-琼胶酶基因agaD02的克隆与表达

Cloning and expression of the beta-agarase gene agaD02 from Agarivorans albus QM38

从白色噬琼胶菌(Agarivorans albus)QM38基因组中扩增到一段编码β-琼胶酶的DNA序列,命名为agaD02。序列相似性分析的结果表明,基因agaD02和弧菌Vibrio sp.JT0107及Vibrio sp.PO-303的琼胶酶基因具有同源性,其相似性程度分别为98.7%和97.4%,而同GenBank中的其他序列同源性很低。对此基因的序列进行了分析,结果表明,其开放阅读框长度为2 868 bp,编码的蛋白质包含955个氨基酸,在蛋白数据库中的编号为ABM90422,推测其分子质量为106 ku,等电点为4.87。利用网上数据库资源和生物学软件对预测的琼胶酶蛋白序列进行了同源性分析和结构分析。设计两端含酶切位点的特定引物,克隆agaD02基因,构建入表达载体pET24a(+),转化大肠杆菌BL21(DE3),转化后提取质粒进行酶切和电泳验证,转化后的大肠杆菌经诱导可产生胞外琼胶酶。

The β-agarase gene, agaD02, was amplified by PCR using genomic DNA ofAgarivorans albus strain QM38 as a template. The agaD02 gene consists of an open reading frame of 2868 bp encoding β-agarase, a protein of 955 amino acids and a molecular weight of 106 kD. The PI of the agarase is 4.87. The accession numbers for agaD02 in GenBank was EF199908. We found that the agaD02 gene shared 98.7% sequence identity to the agaB gene of Vibrio sp. JT0107 and 97.4% identity to the agaE gene of Vibrio sp. PO-303. The deduced amino acid sequence of the agaD02 gene was compared with entries in the DDBJ database. According to the sequence of agaD02 gent, a pair of primers were designed and synthesized. After PCR amplification, the product was cloned into pMD19-T simple vector using TA cloning. The recombinants were sequenced and identified by restrictive endonuclease digestion. The target gene sequences were then subcloned into a highly efficient eukaryotic expression vector pET24a（＋）.After confirmation by double restrictive endonuclease digestion, the expression vectors were transformed into E. coli BL21 （DE3）. The recombinants E. coli BL21 （pET24a-agaD02） were induced by IPTG on Petri dish and were stained with Lugol iodide solution after being cultured for 24 h at 37 ℃. A clear zone was observed around the colony of the recombinants E. coli, confirming that the pET24a-agaD02 expression vectors were successfully constructed.