| 凡纳滨对虾抗菌肽crunstinA在毕赤酵母菌中的表达 |
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| 引用本文： | 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). |
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| 作者姓名： | 马春霞 彭金霞 何苹萍 雷爱莹 马宁 王瑞 黎铭 |
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| 作者单位： | 1. 广西兽医研究所,南宁,530001;2. 广西水产科学研究院/广西遗传育种与健康养殖重点实验室,南宁,530021 |
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| 基金项目： | 广西自然科学基金项目，广西水产畜牧科技推广应用项目 |
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| 摘 要： | 【目的】明确凡纳滨对虾抗菌肽crunstin A(Lvcrustin A)在毕赤酵母菌中的表达情况,为研发出一种基于重组蛋白crustin A的新型水产药物打下基础。【方法】采用全基因合成法(PAS)合成Lvcrustin A基因,然后将Lvcrustin A基因克隆至真核表达载体p YE-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p YE-GAPα-Lvcrustin A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Lvcrustin A的表达情况,并通过保护试验验证融合蛋白Lvcrustin A对副溶血弧菌的抵抗作用。【结果】以构建的重组质粒p YE-GAPα-Lvcrustin A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Lvcrustin A,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Lvcrustin A能被抗His抗体识别。保护试验结果表明,融合蛋白Lvcrustin A对副溶血弧菌具有良好的抵抗力。【结论】利用表达质粒p YE-GAPα和酵母菌GS115可成功重组表达获得Lvcrustin A,且获得的Lvcrustin A具有促进凡纳滨对虾抵抗副溶血弧菌感染的作用。
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| 关 键 词： | 凡纳滨对虾 抗菌肽 毕赤酵母菌 真核表达 |
Expression of Litopenaeus vannamei antimicrobial peptide crunstinA in Pichia pastoris |
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| MA Chun-xia,PENG Jin-xia,HE Ping-ping,LEI Ai-ying,MA Ning,WANG Rui,LI Ming.Expression of Litopenaeus vannamei antimicrobial peptide crunstinA in Pichia pastoris[J].Journal of Southern Agriculture,2017,48(7). |
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| Authors: | MA Chun-xia PENG Jin-xia HE Ping-ping LEI Ai-ying MA Ning WANG Rui LI Ming |
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| 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. |
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| Keywords: | Litopenaeus vannamei antimicrobial peptides Pichia pastoris eukaryotic expression |
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