基于SOE-PCR的兩種魚類hepcidin基因串聯(lián)及其在畢赤酵母中的表達 Hepcidin抗菌肽是由生物肝臟細胞表達的一類具有抗菌作用和鐵代謝調(diào)節(jié)功能的堿性小分子肽，它們在機體免疫系統(tǒng)中發(fā)揮了重要作用，被認為是抗生素的理想替代品。通過重組DNA表達技術(shù)制備抗菌肽無疑是一條良好的途徑。為擴大hepcidin的抗菌譜和提高其表達量，通過SOE-PCR將斑點叉尾鮰Ictalurus punctatus hepcidin成熟肽的cDNA“mCH"與尼羅羅非魚Oreochromis niloticus hepcidin成熟肽的cDNA“mTH"進行串聯(lián)，并在兩端分別添加EcoRⅠ和NotⅠ酶切位點，以pPIC9K為真核表達載體，成功構(gòu)建了“pPIC9K-mCH-mTH"重組表達載體；電轉(zhuǎn)化進畢赤酵母GS115中，經(jīng)不同濃度G418和選擇性培養(yǎng)基篩選以及對酵母基因組DNA的PCR鑒定，得到高拷貝酵母轉(zhuǎn)化子；在30 ℃、以1%的甲醇誘導(dǎo)表達不同時間后，經(jīng)Tricine-SDS-PAGE分析，表明發(fā)酵培養(yǎng)72 h時目的蛋白的表達量zui高，達77 mg/L。發(fā)酵上清經(jīng)SP-Sepharose陽離子交換層析純化后獲得高純度的目的蛋白。抑菌實驗顯示含目的蛋白的發(fā)酵上清和經(jīng)純化后的重組目的蛋白對革蘭氏陽性和革蘭氏陰性細菌都有良好的抑菌效果。本研究結(jié)果為hepcidin抗菌肽的產(chǎn)業(yè)化生產(chǎn)奠定了良好的基礎(chǔ)。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.