假单胞菌的聚-β-羟基丁酸酯聚合酶基因的克隆与表达

Cloning and Expression of Poly-β-hydroxybutyrate Polymerase Gene from Pseudomonas koreensis

根据Gen Bank上公布的假单胞菌属的phb C基因设计引物。以假单胞菌UVCN18基因组DNA为模板,成功克隆了菌株假单胞菌UVCN18中的PHB聚合酶基因phb C。生物信息学方法分析表明,该基因全长1 704 bp,编码567个氨基酸。通过Blast P序列对比发现,该基因所表达的氨基酸属于PHA聚合酶家族。将获得的基因序列提交到Gen Bank,得到登录号为KT716020。采用MEGA5.1软件构建系统发育树,显示该基因编码的氨基酸序列与菌种恶臭假单胞菌所表达的氨基酸序列的同源性较高。同时构建工程菌大肠杆菌BL21-p ET-28a(+)-phb C,经IPTG诱导过量表达,表达产物进行SDS-PAGE和Western blotting分析鉴定结果与PHB聚合酶分子质量63 ku理论相符合,证明实现了异源蛋白表达。

Based on Gen Bank published Pseudomonas phb C gene primers were designed. In strain Pseudomonas koreensis UVCN18. genomic DNA as a template, successfully cloned strain Pseudomonas koreensis UVCN18 the PHB polymerase gene. Bioinformatics analysis showed that the gene full length 1 704 bp, encoding 567 amino acids. By comparison of Blast P sequences, the gene expression of the amino acid belongs to PHA polymerases family. The gene sequences obtained submitted to Gen Bank, accession number obtained KT716020. And using MEGA5.1 software phylogenetic tree,showing homology of the gene encoding the amino acid sequence of Pseudomonas putida strain the amino acid sequence of a higher expression. While building engineering bacteria E. coli BL21-p ET-28 a（＋）-phb C, overexpression induced by IPTG. Expression products by SDS-PAGE and Western blotting analysis and identification, results of PHB polymerase molecular weight 63 ku consistent theory, it proved to achieve the expression of heterologous proteins.