| 摘要: |
| 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。 |
| 关键词: 琼胶酶 白色噬琼胶菌(Agarivorans albus) 克隆 序列分析 表达 |
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| 基金项目:山东省自然科学基金项目 (ZR2009EQ009); 国家海洋局海洋生物活性物质与现代分析技术重点实验室开放基金项目(MBSMAT-2009-07);山东大学自主创新基金资助 |
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| Cloning and expression of the beta-agarase gene agaD02 from Agarivorans albus QM38 |
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WANG Jing,MA Ji-fei,MIAO Ting-ting,LI Zhao-jie,DU Zong-jun
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| Abstract: |
| The β-agarase gene, agaD02, was amplified by PCR using genomic DNA of Agarivorans 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 gene, 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. |
| Key words: aragase Agarivorans albus clone sequence analysis expression |
