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

ntimicrobial 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 as well as their applied use in aqaculture.