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张淑雅, 李端友, 刘莹莉, 贺甜甜, 聂品, 谢海侠. 一种利用λ Red重组技术在杀鱼爱德华氏菌基因组添加标签的方法[J]. 水生生物学报, 2023, 47(6): 895-902. DOI: 10.7541/2023.2022.0461
引用本文: 张淑雅, 李端友, 刘莹莉, 贺甜甜, 聂品, 谢海侠. 一种利用λ Red重组技术在杀鱼爱德华氏菌基因组添加标签的方法[J]. 水生生物学报, 2023, 47(6): 895-902. DOI: 10.7541/2023.2022.0461
ZHANG Shu Ya, LI Duan You, LIU Ying Li, HE Tian Tian, NIE Pin, XIE Hai Xia. EPITOPE TAGGING OF CHROMOSOMAL GENE IN EDWARDSIELLA PISCICIDA[J]. ACTA HYDROBIOLOGICA SINICA, 2023, 47(6): 895-902. DOI: 10.7541/2023.2022.0461
Citation: ZHANG Shu Ya, LI Duan You, LIU Ying Li, HE Tian Tian, NIE Pin, XIE Hai Xia. EPITOPE TAGGING OF CHROMOSOMAL GENE IN EDWARDSIELLA PISCICIDA[J]. ACTA HYDROBIOLOGICA SINICA, 2023, 47(6): 895-902. DOI: 10.7541/2023.2022.0461

一种利用λ Red重组技术在杀鱼爱德华氏菌基因组添加标签的方法

EPITOPE TAGGING OF CHROMOSOMAL GENE IN EDWARDSIELLA PISCICIDA

  • 摘要: 在杀鱼爱德华氏菌病原学研究中, 在动物中制备杀鱼爱德华氏菌蛋白抗体耗时长, 且获得的多克隆或多肽抗体在宿主细胞中特异性差, 背景信号强。为解决这一问题, 对在大肠杆菌(Escherichia coli)和沙门氏菌(Salmonella)中建立起来的λ Red基因编辑方法进行调整和优化, 建立了在杀鱼爱德华氏菌(Edwardsiella piscicida)基因组基因上添加HA标签序列的方法, 为使用标签抗体研究杀鱼爱德华氏菌基因功能提供便利。λ Red重组系统利用同源线性DNA片段与基因组DNA进行重组。即以质粒pSU315为模板, 在引物上引入目的基因的特异性序列, 扩增FRT序列和抗生素抗性基因; 以获得的PCR产物转化携带pKD46的杀鱼爱德华氏菌, 在pKD46表达的λ噬菌体的3个重组蛋白(Exo、Beta和Gam)作用下, PCR产物与杀鱼爱德华氏菌基因组发生同源重组, 获得引入了抗生素抗性的靶基因缺失或靶基因携带标签序列的菌株; 接着利用pKD46的温敏型特性, 消除引入的pKD46; 最后向杀鱼爱德华氏菌引入文章构建的表达Flp重组酶的质粒pKD46-flp, 在FLP作用下, 两个FRT位点之间发生重组, 消除抗生素抗性基因和一个FRT位点, 获得携带一个FRT位点序列、靶基因缺失或靶基因添加标签序列的杀鱼爱德华氏菌菌株。该遗传操作平台的建立为杀鱼爱德华氏菌基因功能研究提供便利条件, 亦为其他水产病原细菌遗传操作方法的建立提供借鉴。

     

    Abstract: Edwardsiella piscicida, belonging to the family Enterobacteriaceae, is a Gram-negative intracellular pathogen. It causes hemorrhagic septicemia in more than 20 important species of farmed fish, especially flounder (Paralichthys olivaceus) and turbot (Scophthalmus maximus), leading to huge economic losses. In dissecting the host-pathogen interaction, antibodies against virulent proteins of E. piscicida are often required. It always takes a long time to prepare antibodies in mice or rabbits, and the polyclonal or polypeptide antibodies obtained are often not specific, especially when applied in eukaryotic cells. To resolve this problem, the λ Red recombination method established in E. coli and Salmonella was optimized to introduce epitope tagging to chromosomal genes in E. piscicida, by using linear DNA fragments for homologous recombination with genomic DNA. Plasmid (pKD3/pKD4, pSU312/pSU313, pSU314/pSU315, or pSUB7) was used as the template, primers are composed of gene specific sequence plus FRT sequence. The DNA fragment obtained by PCR was introduced into E. piscicida transformed with pKD46. With the supplementation of L-arabinose, pKD46 expresses three recombinant proteins (Exo, Beta, Gam) of λ phage, under which the DNA fragment introduced recombine homologically with E. piscicida genome, resulting in target gene deletion or a commercial tag insertion. To eliminate the antibiotic gene introduced in this process, pKD46 expressing Flp recombinase (pKD46-flp) was constructed. By homologous recombination, the antibiotic gene between two FRT sites and one FRT site are eliminated. By culturing at 37℃, the pKD46-flp introduced is cured, obtaining an epitope tagging of chromosomal gene of E. piscicida. A commercial monoclonal antibody against the tag (HA, FLAG, or His) could then be used to monitor the expression or translocation of virulent proteins from E. piscicida. The method established in this study facilitates the dissection on host-pathogen interaction, and will shed light on the genetic operation on other aquatic bacterial pathogens.

     

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