Expression of Litopenaeus vannamei antimicrobial peptide crunstinA in Pichia pastoris

ObjectiveExpression of antimicrobial peptides crunstinA(LvcrustinA) isolated from Litopenaeus van-namei in Pichia pastoris was studied in order to lay a foundation for developing new aquaculture medicines based on re-combinant protein crunstinA. MethodLvcrustinA gene was synthesized by PCR-based accurate synthesis(PAS) method. LvcrustinA gene was cloned into eukaryotic expression vector pYE-GAPα. The recombinant plasmid obtained transformed into competent bacteria TOP10. Recombinanted plasmid pYE-GAPα-Lvcrustin-A extracted by plasmid extraction kit was transformed into P. pastoris GS115. Expression of fusion protein LvcrustinA was analyzed by SDS-PAGE electrophoresis detection and Western blotting identification. Resistance effects of fusion protein LvcrustinA to Vibrio parahaemolyticus were tested by protection experiment. ResultThe established recombinant plasmid pYE-GAPa-LvcrustinA was trans-formed into P. pastoris strain GS115, the obtained recombinant strain could be cultured in shaking table(220 r/min) for 24 h under 30 ℃ to produce fusion protein LvcrustinA. As the culture time extended, the expression increaseed. Western blotting identification indicated that purified fusion protein LvcrustinA could be identified by His antibody. Protection ex-periment results showed that fusion protein LvcrustinA was resistant to V. parahaemolyticus. ConclusionExpression plasmid pYE-GAPa and P. pastoris strain GS115 can recombine into LvcrustinA. LvcrustinA can help L. vannamei resist infection of V. parahaemolyticus.