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.
出处
《南方水产科学》
CAS
CSCD
北大核心
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)
关键词
鲢
基因克隆
原核表达
抑菌效果
Hepcidin
Hypophthalmichthys molitrix
Hepcidin
gene clone
prokaryotic expression
inhibitory effect
