大黄鱼抗菌肽hepcidin在巴斯德毕赤酵母中的表达及其产物的抑菌活性

Expression of large yellow croaker(Pseudosciana crocea)hepcidin in Pichia pastoris and antibacterial activity of the recombinant protein

Hepcidin是一类具有独特性质的小分子抗菌肽,克隆得到大黄鱼(Pseudosciana crocea)的hep-cidin基因(Lyc-Hepc),cDNA序列全长585个核苷酸,编码一个由85个氨基酸组成的蛋白,包含24个氨基酸的信号肽、35个氨基酸的前导肽和26个氨基酸的成熟肽,成熟肽具有8个保守的半胱氨酸.将Lyc-Hepc 183bp的编码前导肽和成熟肽的前体肽(25aa-85aa)基因片段克隆到毕赤酵母表达载体pPICZαA中,然后电击转化巴斯德毕赤酵母SMD1168,经高浓度Zeocin筛选获得多个单克隆.挑取4个克隆进行培养,提取酵母基因组进行PCR检测,结果表明目的基因片段已成功整合到酵母菌SMD1168基因组中,获得酵母工程菌株SMD1168/pPICZα-Lyc-Hepc.SDS-PAGE和WesternBlot分析表明酵母工程菌株在0.5%甲醇诱导培养72h后,Lyc-Hepc在培养上清中得到了分泌型表达.体外抑菌实验表明重组Lyc-Hepc蛋白能够抑制2株水产养殖病害菌嗜水气单胞菌和副溶血弧菌的生长.研究结果表明大黄鱼hepcidin可能在抗细菌免疫中发挥了作用.

Hepcidin is a micromolecule antimicrobial peptide with unique characteristic. In this study, we cloned a hepcidin gene from Pseudosciaena crocea（Lyc-Hepc）. The complete cDNA of Lyc-Hepc is 585 nucleotides （nt） en- coding a protein of 85 amino acids （aa）. The deduced Lyc-Hepc contains a 24-aa signal peptide, a 35-aa prodo- main and a 26-aa mature polypeptide, which possesses a typical arrangement of eight conserved cysteine residues. We cloned a 183 bp sequence encoding the prodomain and mature peptide of Lyc-Hepc by polymerase chain reac- tion （PCR）, then inserted this DNA fragment into the yeast expression vector pPICZcxA. The constructed recombi- nant plasmid pPICZc^Lyc-Hepc was linerized and transfected into Pichia pastoris SMD1168 by electroporation. We screened the positive clone from the YPDS pahe containing 1 000μg/cm3 zeocin and extracted the genome of posi- tive clone as template for PCR to prove the integration of the Lyc-Hepc gene into the yeast genome. Then we got the engineering yeast strain SMD1168/pPICZa-Lyc-Hepc. Subsequently, the expression of recombinant Lyc-Hepc in- duced by methanol was detected in the supernatant by SDS-PAGE and Western Blot analysis. Antibacterial activity analysis showed that the recombinant Lyc-Hepc lyticus and Aeromonas hydrophila, which are 2 that Lyc-Hepc may have a role in anti-bacterial exhibits obvious antimicrobial activities against Vibrio parahaemo- important bacteria strains for aquatic disease. The results suggest immune for the large yellow croaker.