鲢抗菌肽Hepcidin的基因克隆和表达及抑菌活性分析  

Cloning,expression and antimicrobial activity of Hepcidin from Hypophthalmichthys molitrix

作　　者：周卫军[1,2] 刘振兴[2] 柯浩[2] 马艳平[2] 郝乐[2] 徐明芳[1] 

机构地区：[1]暨南大学生物工程学系实验室,广东广州510630 [2]广东省农业科学院动物卫生研究所,广东省兽医公共卫生公共实验室,广东广州510640

出　　处：《南方水产科学》2014年第3期58-64,共7页South China Fisheries Science

基　　金：广州市农业科技攻关项目(GZCQC1202FG04002-01);广东省海洋渔业科技推广专项(A201201C01);广州市科技计划项目(201300000064);广东省科技计划项目(2008B020700006)

摘　　要：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-1IPTG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。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.

关 键 词：鲢 基因克隆 原核表达 抑菌效果 

分 类 号：Q785[生物学—分子生物学]