肽抗生素hPAB-β在毕赤酵母中的表达与活性鉴定

Expression of human peptide antibiotics hPAB-β in Pichia pastoris and its biological activity analysis

目的构建表达人源肽抗生素hPAB-β的重组毕赤酵母,为大量制备hPAB-β奠定基础。方法用PCR及引物延伸法得到天然及优化的hPAB-β序列,克隆入pPIC9K质粒,转化DH5α大肠埃希菌,用酶切和核苷酸测序鉴定。重组质粒经SalⅠ酶线性化,整合入毕赤酵母GS115中,在DNA,mRNA及蛋白水平鉴定阳性重组子。结果构建的含天然及优化序列的重组质粒酶切均可得到相应大小的插入片段,与预期相符,测序结果表明序列和阅读框均正确。线性化的重组质粒转入GS115后,得到7个不同的高拷贝阳性转化子,均能表达重组肽并具有良好的抑菌活性。结论本研究成功构建含天然及优化hPAB-β序列的重组毕赤酵母工程菌,能够用于肽抗生素hPAB-β的制备。

Objective To construct the recombinant Pichia pastoris of peptide antibiotics hPAB-β for making a foundation of largescale preparation of hPAB-β.Method Natural and optimized sequences of hPAB-β were obtained by using PCR and primer extension. The obtained hPAB-β sequences were cloned into pPIC9K plasmid, and then transformed the recombinant plasmids into DH5α. After identification by enzyme digestion and nucleotide sequencing, the recombinant plasmids were digested by SalⅠ. The digested recombinant plasmids were integrated into GS115 and the positive recons were identified in levels of DNA, mRNA, and protein. Results An expected fragment was obtained by digesting recombinant plasmids with Xho Ⅰ/Not Ⅰ. Nucleotide sequencing confirmed that the recombinant plasmids were contained the correct target sequence. Seven multicopy positive transformants were obtained after transforming the recombinant plasmids to GS115. All of the multicopy transformants could express the recombination peptide possessing favorable bacteriestasis activity. Conclusion The recombinant Pichia pastoris containing natural and optimized sequence of hPAB-β have been successfully constructed and can be used to prepare the peptide antibiotics hPAB-β.