人α-防御素基因克隆及其毕赤酵母表达的研究

Cloning and expression of human α-defensin gene in Pichia Pastoris yeast

为探讨人α-防御素(HNP2)酵母表达,根据HNP2氨基酸序列和巴斯德毕赤酵母密码偏爱性,设计HNP2基因序列片段,构建pGAPZαA-HNP2酵母表达载体,转化E.coliJM109,扩增重组子,经限制性核酸内切酶BlnI线性化,转毕赤酵母.PCR扩增挑选阳性转化子,SDS-PAGE分析酵母表达,利用阳离子交换树脂纯化HNP2,开展抗菌试验.结果显示转入巴斯德毕赤酵母HNP2基因得到表达,表达的HNP2具有显著的抗菌作用.

This study focused on the expression of human α-defensin （HNP2） gene in Pichia yeast system. Human HNP2 cDNA was cloned based on the published sequence and the biased codon usage of Pichia yeast. The modified HNP2 cDNA was inserted into yeast expression vector pGAPZα The plasmid pGAPZαA-NP2 was produced in E. coli HB109 and used for yeast transformation. Pichia pastoris SMD1168 was transformed with BinⅠ digested pGAPZαA-HNP2 and positive yeast clones were selected with PCR. The expression of HNP2 and the production of the recombinant HNP2 protein was purified with ion-exchange column were detected with SDS-PAGE and Western blotting, and quantified with Bradford and then used for biological activity assay. Our results demonstrated that the recombinant HNP2 had antimicrobial activity. These findings have provided valuable tools for further studies on the function and medicinal application of HNP2.