鲢抗菌肽 Hepcidin 的基因克隆和表达及抑菌活性分析

利用同源克隆的方法从鲢（Hypophthalmichthys molitrix）的肝脏中克隆出抗菌肽 Hepcidin cDNA（GenBank 登入号 KF312213）后，通过 pET32a（＋）构建含抗菌肽 Hepcidin 的重组质粒的 pET-Hep/ Rosetta 菌株，在37℃、28℃和16℃下分别用0.5 mmol·L -1和1.0 mmol·L -1 IPTG 诱导表达，产物用 Ni SepharoseTM 亲和层析柱纯化并进行体外抑菌试验。鲢 Hepcidin cDNA 总长度755 bp，ORF 为282 bp，5′UTR 为108 bp，3′UTR 为365 bp，编码93氨基酸，信号肽24个氨基酸，前域42个氨基酸和成熟肽27个氨基酸。pET-Hep/ Rosetta 在28℃和16℃主要是可溶性表达，37℃主要是包涵体表达。纯化的产物经15％ SDS-PAGE 电泳验证为目的产物。目的产物、pET32/Rosetta 产物以及氟苯尼考的体外抑菌试验表明，目的表达产物对无乳链球菌（ Streptococcus agalactiae）、金黄色葡萄球菌（Staphylococcus aureus）、嗜水气单胞菌（Aeromonas hydrophila）等具有很好的抑菌效果。

Cloning,expression and antimicrobial activity of Hepcidin from Hypophthalmichthys molitrix

The full-length Hepcidin cDNA sequence GenBank No. KF312213 was amplified from the liver of chub（Hypophthalmichthys molitrix）by semi-Nested PCR[rapid amplification of cDNA ends（RACE）]. According to prodomain and mature peptide of the Hepcidin cDNA sequence,we designed an upstream primer with EcoR I restriction site and downstream primers with Sal I restriction site,and cloned the target gene into the expression vector pET-32a（ ＋ ）. The recombined plasmid was transformed into the expression stain-E. coli Rosetta and expressed induciblely at different temperatures（37 ℃,28 ℃ and 16 ℃）and of different IPTG concentrations（0. 5 mmol· L -1 and 1. 0 mmol·L -1 ）. The protein was purified by Ni SepharoseTM affinity chromatography column. The full-length of the Hepcidin cD-NA gene was 755 bp,which included a 282-bp ORF encoding a 93-amino acid prepropeptide. The prepropeptide contained a signal peptide（24 amino acids）,a prodomain（42 amino acids）and a mature peptide（27 amino acids）. The purified product displayed a single protein band through 15% SDS-PAGE electrophoresis. The purified production of pET-Hep/ Rosetta had obvious antibacterial effect on Streptococcus agalactiae,Staphylococcus aureus and Aeromonas hydrophila,but the control group showed no inhibitory effect.