基于SOE-PCR的两种鱼类hepcidin基因串联及其在毕赤酵母中的表达

Connection of hepcidin genes from two fish species and their expression in Pichia pastoris

摘要:

Hepcidin抗菌肽是由生物肝脏细胞表达的一类具有抗菌作用和铁代谢调节功能的碱性小分子肽，它们在机体免疫系统中发挥了重要作用，被认为是抗生素的理想替代品。通过重组DNA表达技术制备抗菌肽无疑是一条良好的途径。为扩大hepcidin的抗菌谱和提高其表达量，通过SOE-PCR将斑点叉尾鮰Ictalurus punctatus hepcidin成熟肽的cDNA“mCH”与尼罗罗非鱼Oreochromis niloticus hepcidin成熟肽的cDNA“mTH”进行串联，并在两端分别添加EcoRⅠ和NotⅠ酶切位点，以pPIC9K为真核表达载体，成功构建了“pPIC9K-mCH-mTH”重组表达载体；电转化进毕赤酵母GS115中，经不同浓度G418和选择性培养基筛选以及对酵母基因组DNA的PCR鉴定，得到高拷贝酵母转化子；在30 ℃、以1%的甲醇诱导表达不同时间后，经Tricine-SDS-PAGE分析，表明发酵培养72 h时目的蛋白的表达量最高，达77 mg/L。发酵上清经SP-Sepharose阳离子交换层析纯化后获得高纯度的目的蛋白。抑菌实验显示含目的蛋白的发酵上清和经纯化后的重组目的蛋白对革兰氏阳性和革兰氏阴性细菌都有良好的抑菌效果。本研究结果为hepcidin抗菌肽的产业化生产奠定了良好的基础。

Abstract:

Hepcidin are small cationic peptides with antibacterial activity expressed mainly in the liver of living organisms, and they play important roles in the host’s immune response against microbial invasion and regulation of iron metabolism. Thus, they are considered to be good substitutes for traditional antibiotics. It is a good choice that the antimicrobial peptides are prepared by recombinant DNA expression. In the present study, two hepcidin mature peptide cDNAs from channel catfish (Ictalurus punctatus) (mCH) and tilapia (Oreochromis niloticus) (mTH) were connected by SOE-PCR in order to obtain more recombinant hepcidin with broad antimicrobial spectrum, and EcoR I and Not I sites were added to 5′- and 3′- ends of the fragment, respectively. The recombinant eukaryotic expression vector “pPIC9K-mCH-mTH” was successfully constructed, and transformed into Pichia pastoris GS115. The transformants containing multicopy gene insertion were selected by using different concentrations of G418 and other specific mediums, and identified by PCR for yeast genomic DNA. Expression was induced by adding 1% methanol at 30 oC for different times. Tricine-SDS-PAGE analysis demonstrated that the most appropriate expression time was 72 h, at which a high expression yield (77 mg/L) for the target protein was exhibited. The highly purified target protein was obtained from the fermentation supernatant by SP-Sepharose cation exchange chromatography. Bacteriostatic activity assay demonstrated that the fermentation supernatant containing the target protein and purified recombinant target protein had bacteriostatic activities against gram-positive and gram-negative bacterium. The present result provides the important initial value for industrial production of hepcidin antimicrobial peptide.