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| 引用本文: | 邱楚雯,刘 梅,王宝杰,蒋克勇,孙姝娟,孟晓林,骆作勇,王 雷.中国明对虾血蓝蛋白C末端片段在毕赤酵母中的表达及其抗菌活性[J].海洋科学,2013,37(6):1-7. |
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| 中国明对虾血蓝蛋白C末端片段在毕赤酵母中的表达及其抗菌活性 |
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邱楚雯1,2, 刘 梅1, 王宝杰1, 蒋克勇1, 孙姝娟1, 孟晓林1,2, 骆作勇1,2, 王 雷1
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1.中国科学院 海洋研究所;2.中国科学院大学
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| 摘要: |
| 为研究中国明对虾(Fenneropenaeus chinensis)血蓝蛋白C末端(FcHC-C)的抗菌功能,将血蓝蛋白基因FcHC的2个C末端基因片段连接到毕赤酵母表达载体pPIC9K中,构建酵母表达载体pPIC9K/FcHC-C。该载体经SalI酶切后,采用PEG法转化毕赤酵母(Pichia pastoris GS115)。转化子经过PCR鉴定后,阳性克隆通过含有G418的YPD平板筛选,获得高拷贝重组子。重组毕赤酵母利用甲醇诱导表达目的基因。经Tricine-SDS-PAGE和Western blot分析结果表明,利用酵母工程菌成功表达了血蓝蛋白C末端片段(rFcHC-C1和rFcHC-C2)。抑菌活性鉴定实验结果显示,重组蛋白rFcHC-C1和rFcHC-C2作为阴离子抗菌肽具有抗真菌和抗细菌的活性。 |
| 关键词: 中国明对虾(Fenneropenaeus chinensis) 血蓝蛋白 抗菌肽 毕赤酵母(Pichia pastoris) 重组表达 |
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| 基金项目:国家863计划资助项目(2006AA100311) |
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| Recombinant expression and antimicrobial activity analysis of hemocyanin C-terminal fragments from Fenneropenaeus chinensis in Pichia pastoris |
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| Abstract: |
| Two C-terminal coding sequences of Fenneropenaeus chinensis hemocyanin FcHC were cloned into the Pichia pastoris expression vector pPIC9K to produce the pPIC9K/FcHC-C yeast expression vectors. The constructed vectors pPIC9K/FcHC-C1 and pPIC9K/FcHC-C2 were linearized by Sal I enzyme, and transformed into P. pastoris GS115 using PEG-mediated transformation method. PCR identified transformants were screened by G418 selected YPD plates. The P. pastoris transfomants of pPIC9K/FcHC-C expressed the two hemocyanin C-terminal gene fragments by methanol induction. The results of Tricine-SDS-PAGE and Western blotting showed that the recombinant FcHC-C1 and FcHC-C2 peptides (rFcHC-C1 and rFcHC-C2) were expressed successfully. An antimicrobial assay showed that rFcHC-C1 and rFcHC-C2 have antifungal and antibacterial activities as anionic AMPs. |
| Key words: Fenneropenaeus chinensis hemocyanin antimicrobial peptides (AMPs) Pichia pastoris recombinant expression |
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