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当前位置: 首页 > 文章 > 凡纳滨对虾抗菌肽crunstinA在毕赤酵母菌中的表达 南方农业学报 2017 (7) 1310-1316
Position: Home > Articles > Expression of Litopenaeus vannamei antimicrobial peptide crunstin A in Pichia pastoris Journal of Southern Agriculture 2017 (7) 1310-1316

凡纳滨对虾抗菌肽crunstinA在毕赤酵母菌中的表达

作  者：
马春霞;彭金霞;何苹萍;雷爱莹;马宁;王瑞;黎铭
单  位：
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;广西兽医研究所
关键词：
凡纳滨对虾;抗菌肽;毕赤酵母菌;真核表达
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。
译  名：
Expression of Litopenaeus vannamei antimicrobial peptide crunstin A in Pichia pastoris
作  者：
MA Chun-xia;PENG Jin-xia;HE Ping-ping;LEI Ai-ying;MA Ning;WANG Rui;LI Ming;Guangxi Veterinary Research Institute;Guangxi Academy of Fishery Sciences/Guangxi Key Laboratory Aquatic Genetic Breeding and Healthy Aquaculture;
关键词：
Litopenaeus vannamei;;antimicrobial peptides;;Pichia pastoris;;eukaryotic expression
摘  要：
【Objective 】Expression of antimicrobial peptides crunstin A(Lvcrustin A) isolated from Litopenaeus van-namei in Pichia pastoris was studied in order to lay a foundation for developing new aquaculture medicines based on recombinant protein crunstin A. 【Method】Lvcrustin A gene was synthesized by PCR-based accurate synthesis(PAS) method.Lvcrustin A gene was cloned into eukaryotic expression vector p YE-GAPα. The recombinant plasmid obtained transformed into competent bacteria TOP10. Recombinanted plasmid p YE-GAPα-Lvcrustin-A extracted by plasmid extraction kit was transformed into P. pastoris GS115. Expression of fusion protein Lvcrustin A was analyzed by SDS-PAGE electrophoresis detection and Western blotting identification. Resistance effects of fusion protein Lvcrustin A to Vibrio parahaemolyticus were tested by protection experiment. 【 Result 】 The established recombinant plasmid p YE-GAPa-Lvcrustin A was transformed 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 Lvcrustin A. As the culture time extended, the expression increaseed. Western blotting identification indicated that purified fusion protein Lvcrustin A could be identified by His antibody. Protection experiment results showed that fusion protein Lvcrustin A was resistant to V. parahaemolyticus. 【Conclusion】Expression plasmid p YE-GAPa and P. pastoris strain GS115 can recombine into Lvcrustin A. Lvcrustin A can help L. vannamei resist infection of V. parahaemolyticus.

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