Recombinant Expression of a Hepcidin Variant AS-hepc2 from Black Porgy (Acanthopagrus schlegelii B.) in Pichia pastoris and Its Antimicrobial Activity
- 环境生态－已发表论文 
鱼类抗菌肽HEPCIdIn具有广谱抗菌活性.利用PCr技术扩增获得了黑鲷抗菌肽HEPCIdIn(AS-HEPC2)的前体肽和成熟肽CdnA序列,片段大小约250bP.将其与毕赤酵母(PICHIA PASTOrIS)PPIC9k质粒连接,构建了分泌表达载体PPIC9k/AS-HEPC2.电击法转化重组表达载体,经过g418筛选和选择性培养基以及PCr鉴定,得到的克隆子都为MuT+.以甲醇诱导PPIC9k/AS-HEPC2,分泌表达上清中获得10ku左右的表达产物,与预期的目的蛋白黑鲷HEPCIdIn的前体肽和成熟肽的大小相符.表达试验证明,随时间的延长,表达产物量增多,至120H达到高峰.表达产物具有很强的热稳定性.用抑菌圈法测定重组蛋白PrO-AS-HEPC2的抗菌活性,发现能抑制革兰氏阳性菌和革兰氏阴性菌的生长.It is well known that hepcidin is an important antimicrobial peptide and widely expressed in various fish.A 250 bp sequence coding the prodomain and mature peptide of AS-hepc2 from black porgy(Acanthopagrus schlegelii B.)was inserted into the vector pPIC9K through EcoR I and Not I sites and an expressed plasmid of pPIC9K/AS-hepc2 was constructed.The Sal I-digested plasmid pPIC9K/AS-hepc2 was transformed into the Pichia pastoris GS115 strain by electroporation.The positive His+ transformants were screened using G418 and further identified by culturing on Minimal Dextrose(MD)agar plates or Minimal Methanol(MM)agar plates.The selected Mut+ transformants were induced for expressing Pro-AS-hepc2 with 0.5% methanol every 24 h.The quantity of the expressed product increased with the induction time lasting and attained to the maximum after 120 h.The recombinant protein expressed in P.pastoris was approximately 10 ku,corresponding to the theoretical molecular mass of this target peptide.The recombinant Pro-AS-hepc2 showed obvious antimicrobial activity against several microorganisms tested,including two strains of Gram-negative bacteria and four strains of Gram-positive bacteria.