副猪嗜血杆菌Flp-FRT双基因敲除方法的建立与优化

doi:10.11843/j.issn.0366-6964.2022.01.022

摘要/Abstract

摘要： 本研究旨在利用Flp-FRT位点特异性重组系统建立副猪嗜血杆菌（Glaesserella parasuis）的无抗性标记基因敲除方法，为G.parasuis的毒力因子、致病机制和基因工程疫苗研究提供有力的遗传操作工具。首先利用λ噬菌体cI857/P_RM/P_R基因表达调控系统调控Flp重组酶的表达，实现抗性突变体中抗性基因的消除；然后将Flp表达质粒电转化引入G.parasuis CF7066，再自然转化介导同源重组的自杀性质粒获得有抗性标记的基因缺失突变株，调节Flp酶表达并筛选无抗突变株，提高培养温度消除表达Flp酶的温敏质粒；进一步通过筛选突变FRT位点以保证基因敲除的效率。结果表明成功构建了温敏穿梭质粒pSHG5C-Flp，证实了λ cI857/P_RM/P_R表达系统可调控Flp重组酶在G.parasuis中表达并切除抗性突变株ΔnanH：：Kan^R中的抗性基因；然后用Flp重组酶表达质粒pSHG5C-Flp电转化G.parasuis CF7066，再用自杀性质粒pKF-ΔnanH和pKF-Δapd2对其进行两次连续自然转化，依次在自然转化的初代转化子中筛选并获得了无抗的单基因缺失株ΔnanH和双基因缺失株ΔnanHΔapd，提高培养温度至42℃培养消除了Flp重组酶表达质粒。尽管该系统实现了G.parasuis两个基因的连续敲除，但敲除第2个基因时的效率明显下降，这很可能源于细菌细胞中Flp重组酶表达的积累，对转化质粒介导的同源重组产生了干扰。因此，作者筛选了突变的FRT位点、构建自杀性质粒pKFM-Δapd进行自然转化，显著提高了自然转化后获得突变株的效率，从而保证了该敲除系统的有效性和稳定性。本研究首次报道了可用于G.parasuis的无抗性标记双基因缺失突变系统，为其分子致病基因与基因工程疫苗的研究奠定了基础。

关键词: 副猪嗜血杆菌, 表达调控, Flp-FRT, 基因敲除, 无抗缺失

Abstract: This study aimed to establish an antibiotic-resistance-free gene knockout method for Glaesserella parasuis based on the Flp-FRT site-specific recombination system, thus providing a genetic manipulation tool for the research on virulence factors, pathogenic mechanism, genetic engineering vaccines of G. parasuis. Firstly, the cI857/P_RM/P_R expression regulation system from λ bacteriophage was used to control the expression of Flp recombinase to realize resistance gene excision from the resistance-marker mutants of G. parasuis. Then, the Flp expression plasmid was introduced into the G. parasuis CF7066 by electroporation, and the obtained recombinant strain was naturally transformed with the suicide plasmid containing the homologous DNA sequence flanked with FRT sites to generate the marker mutants. Following the regulation expression of Flp recombinase, the markerless mutants were screened by colony PCR and the Flp expression plasmid was cured by improving the culturing temperature to 42 ℃. Furthermore, this knockout system was improved by mutation of unilateral FRT site. Initially，the Flp recombinase carried by temperature-sensitive shuttle plasmid pSHG5C-Flp was verified to be able to express under the control of λ repressor and promotor of cI857/P_RM/P_R and to excise the resistance gene in the mutant of ΔnanH::Kan^R. Subsequently, the G. parasuis CF7066 was electrotransformed with pSHG5C-Flp in advance, then it was naturally transformed with the suicide plasmids of pKF-ΔnanH and pKF-Δapd2 successively. Correspondingly, the markerless single gene and double gene deletion mutants of ΔnanH and ΔnanHΔapd were generated from the primary transformants of the primary generation, and the Flp recombinase expression plasmid pSHG5C-Flp also was cured. Although we achieved successive two gene knockout, the knockout efficiency for the second gene was obviously declined, which was probably due to the accumulating Flp recombinase that probably interfered the homologous recombination mediated by the plasmid DNA from natural transformation. Thus, we screened the mutated one FRT site and introduced it into the plasmid of pKFM-Δapd2 to conduct natural transformation. As a result, the introduction of mutated FRT site into the suicide plasmid guaranteed the occurrence of homologous recombination and deletion mutation, which increased the effectiveness and stability of this method. This study provided a novel markerless and double gene knockout system for G. parasuis and paved a way for the research on molecular pathogenetic mechanism and genetic engineering vaccines.

Key words: Glaesserella parasuis, expression regulation, Flp-FRT, gene knockout, markerless deletion

中图分类号:

S852.613

肖静, 肖坤雪, 王翘楚, 陈焕春, 蔡旭旺, 徐晓娟. 副猪嗜血杆菌Flp-FRT双基因敲除方法的建立与优化[J]. 畜牧兽医学报, 2022, 53(1): 219-230.

XIAO Jing, XIAO Kunxue, WANG Qiaochu, CHEN Huanchun, CAI Xuwang, XU Xiaojuan. Development and Optimization of Double-Gene Knockout Method for Glaesserella parasuis based on the Flp-FRT System[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(1): 219-230.

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