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
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摘要: 目的明确凡纳滨对虾抗菌肽crunstinA(LvcrustinA)在毕赤酵母菌中的表达情况,为研发出一种基于重组蛋白crustinA的新型水产药物打下基础.方法采用全基因合成法(PAS)合成LvcrustinA基因,然后将LvcrustinA基因克隆至真核表达载体pYE-GAPα,所得重组质粒转化感受态细菌TOP10,以质粒提取试验盒提取的重组质粒pYE-GAPα-LvcrustinA电转至毕赤酵母菌GS115;以SDS-PAGE电泳检测和Western blotting鉴定分析融合蛋白LvcrustinA的表达情况,并通过保护试验验证融合蛋白LvcrustinA对副溶血弧菌的抵抗作用.结果以构建的重组质粒pYE-GAPα-LvcrustinA电转毕赤酵母菌GS115,获得的重组菌株在30℃下摇床(220 r/min)培养24 h即可获得融合蛋白LvcrustinA,且随培养时间的延长,其表达量逐渐增多.Western blotting鉴定结果显示,纯化的融合蛋白LvcrustinA能被抗His抗体识别.保护试验结果表明,融合蛋白LvcrustinA对副溶血弧菌具有良好的抵抗力.结论利用表达质粒pYE-GAPα和酵母菌GS115可成功重组表达获得LvcrustinA,且获得的LvcrustinA具有促进凡纳滨对虾抵抗副溶血弧菌感染的作用.Abstract: 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.