南方农业学报2017,Vol.48Issue(7):1310-1316,7.DOI:10.3969/j.issn.2095-1191.2017.07.30
凡纳滨对虾抗菌肽crunstinA在毕赤酵母菌中的表达
Expression of Litopenaeus vannamei antimicrobial peptide crunstinA in Pichia pastoris
摘要
Abstract
[Objective]Expression 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. [Method]LvcrustinA 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. [Result]The 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. [Conclusion]Expression plasmid pYE-GAPa and P. pastoris strain GS115 can recombine into LvcrustinA. LvcrustinA can help L. vannamei resist infection of V. parahaemolyticus.关键词
凡纳滨对虾/抗菌肽/毕赤酵母菌/真核表达Key words
Litopenaeus vannamei/antimicrobial peptides/Pichia pastoris/eukaryotic expression分类
农业科技引用本文复制引用
马春霞,彭金霞,何苹萍,雷爱莹,马宁,王瑞,黎铭..凡纳滨对虾抗菌肽crunstinA在毕赤酵母菌中的表达[J].南方农业学报,2017,48(7):1310-1316,7.基金项目
广西自然科学基金项目(2015GXNSFAA139067) (2015GXNSFAA139067)
广西水产畜牧科技推广应用项目(桂渔牧科201528012) (桂渔牧科201528012)