人β.doc

1 人β作者:庹晓晔, 柴家科, 蒋伟, 常东, 盛志勇【关键词】人β防御素 3;, 细菌膜穿透增加蛋白; , 融合表达; , 毕赤酵母 Fusion expression of human β defensin 3 and bactericidal/permeability increasing protEin in P. pastoris 【 Abstract 】 AIM: To investigate the possibility of fusion expression of human β defensin 3 (hBD3) and bactericidal/permeability increasing protEIn (BPI) in P. pastoris. METHODS: DNA fragments encoding hBD3 mature peptide and BPI were linked via a Linker sequence and cloned into yeast expression vector pPICZ αB followed by introduction into P. pastorisX33 by electrical transduction. Positive clones identified by genomic PCR and phenotype analysis were induced by methanol for protein expression. In supernatants, binant protein was purified by phenyl sepharose high performance and source 30Q ionexchange columns. Western Blot against hBD3 2 and BPI was performed respectively to identify the binant protein. RESULTS: binant pICZ α BhBD3BPI was proved correct by restriction analysis and sequencing. X33pICZ α BhBD3BPI clone expressed the binant fusion protein in SDSPAGE electrophoresis after induction for 24 h. The protein product was both hBD3 and BPIpositive. A purity of 89% was reached after purification procedures. CONCLUSION: Its feasible to express hBD3 fused to BPI in P. pastoris yeast expression system. 【 Keywords 】 human β defensin 3; bactericidal/permeability increasing protein; fusion expression; P. pastoris yeast 【摘要】目的:探讨采用酵母表达系统进行人β防御素3( hBD3 ) 与细菌膜穿透增加蛋白(BPI) 融合表达的可行性. 方法:将 hBD3 成熟肽基因通过 Linker 蛋白与 BPI 基因串联,克隆于酵母表达载体 pPICZ αB 中,电转导入 X33 毕赤酵母菌,经重组酵母基因组 PCR 和表型鉴定获得阳性克隆, 对阳性克隆进行甲醇诱导表达,上清进行目的蛋白纯化和 Western Blot 鉴定. 结果: 重组载体经酶切和测序证实序列正确, 重组 X33pICZ α BhBD3BPI 克隆经甲醇诱导 24h后,上清 3 SDSPAGE 电泳显示有目的蛋白表达, Western Blot 分析表明重组蛋白抗人 hBD3 和 BPI 均阳性,该目的蛋白依次通过疏水色谱、离子交换色谱纯化,蛋白纯度达到 89%. 结论: 采用毕赤酵母系统融合表达 hBD3 和 BPI 是可行的. 【关键词】人β防御素 3; 细菌膜穿透增加蛋白; 融合表达;毕赤酵母 0 引言抗菌肽是生物体内经诱导产生的一种具有生物活性的小分子多肽,具有强碱性、热稳定性以及广谱抗菌等特点, 是解决细菌抗生素耐药性的希望[1]. 某些抗菌肽对部分真菌、原虫、病毒及癌细胞等也具有强力的杀伤作用, 因而这类活性多肽被命名为多肽抗生素[ 2-4 ]. 防御素是抗菌肽的一种,其中人β防御素 3( hBD3 )