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pPICZαB中,电转导入X-33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X-33-pICZαB-hBD3-BPI克隆经甲醇诱导24h后,上清SDS-PAGE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.
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 pPICZαB followed by introduction into P. pastor/s-X-33 by electrical transduction. Positive clones identified by genomic PCR and phenotype analysis were induced by methanol for protein expression. In supernatants, recombinant protein was purified by phenyl sepharose high performance and source 30Q ion-exchange columns. Western Blot against hBD3 and BPI was performed respectively to identify the recombinant protein. RESULTS： Recombinant pICZαB-hBD3-BPI was proved correct by restriction analysis and sequencing. X-33-pICZαB-hBD3-BPI clone expressed the recombinant fusion protein in SDS-PAGE electrophoresis after in- duction for 24 h. The protein product was both hBD3- and BPI- positive. A purity of 89% was reached after purification procedures. CONCLUSION： It＇s feasible to express hBD3 fused to BPI in P. pastoris yeast expression system.
出处
《第四军医大学学报》
北大核心
2007年第7期648-650,共3页
Journal of the Fourth Military Medical University
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
human β-defensin 3
bactericidal/permeability increasing protein
fusion expression
P. pastoris yeast
