拟穴青蟹抗脂多糖因子基因工程表达重组载体的构建及其表达研究.docVIP

  拟穴青蟹抗脂多糖因子基因工程表达重组 载体的构建及其表达研究# 陈荣元，刘海鹏** 5 10 15 20 25 30 35 40 （厦门大学近海海洋环境科学国家重点实验室，福建厦门 361102） 摘要：抗菌肽是广泛分布于生物体内的一种具有广谱抗菌活性的小分子多肽。研究发现，抗 菌肽在生物体抗病原免疫防御过程中发挥重要作用，特别是对于那些仅具有先天性免疫系统 的低等生物（如虾、蟹等）。本研究在获得拟穴青蟹（Sylla paramamosain）两个抗脂多糖 因子基因的基础上：构建 pPIC9k 真核表达载体 pPIC9k/Sp-ALF1 和 pPIC9k/Sp-ALF2；将重 组表达载体导入毕赤酵母(Pichia pastoris)，以甲醇诱导重组酵母克隆子，分泌表达上清 中检测到 14kDa 左右的诱导重组表达产物，与预期目的蛋白抗脂多糖因子的成熟肽大小基本 相符。另外，重组蛋白表达试验发现该重组克隆子在甲醇诱导 36h 后即可达到较高的表达产 量。综上，该研究成功构建了拟穴青蟹抗脂多糖因子真核表达系统，为该抗菌肽的功能鉴定 及其开发应用奠定了良好基础。 关键词：拟穴青蟹抗脂多糖因子，pPIC9k，毕赤酵母，真核表达 中图分类号：请查阅《中国图书馆分类法》 Construction of recombinant expression vector of anti-lipopolysaccharide factor from Sylla paramamosain and its recombinant expression in yeast CHEN Rongyuan, LIU Haipeng (State Key Lab of Marine Environmental Science, Xiamen University, Xiamen, Fujian, 361102) Abstract: Antimicrobial peptide (AMP) is a kind of small molecule with wide spectrum of antimicrobial activity which is present in almost all multicellular organisms. It is well known that AMPs play vital role in host defense against pathogen, especially in invertebrates such as shrimp and crab. Previously, we have isolated two isoforms of anti-lipopolysaccharide factor (ALF), designated as Sp-ALF1 and Sp-ALF2, from mud crab Sylla paramamosain. In order to obtain recombinant Sp-ALFs protein, the yeast recombinant expression system was developed. The coding sequences for mature Sp-ALF1 and Sp-ALF1, respectively, were cloned into pPIC9k vector. Then the recombinant vector was transfected into yeast, Pichia pastoris, followed by MD plate screening and methanol induction. A 14 kDa induced recombinant product was detected in the supernatant, which was considerable to the predicted protein mass of Sp-ALFs. Further, we found the expression of recombinant Sp-ALFs proteins reached a high level at 36 h post methanol induction. In summary, the yeast expression system for Sp-ALF1 and Sp-ALF2 were successfully developed which will be useful for the functional study of these two molecules