单增李斯特菌hly基因缺失株的构建及重要特性分析
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摘要
为构建单增李斯特菌(LM) hly基因缺失株,并分析其生物学特性和免疫保护效果,本研究利用同源重组技术获得基因缺失株LM△hly_(112-153),研究该缺失株对巨噬细胞的侵袭力、溶血能力并测定其LD_(50),同时评价该缺失株对小鼠的免疫保护效果。结果显示经PCR扩增及序列分析表明获得基因缺失株LM△hly_(112-153),该缺失株对巨噬细胞的侵袭能力显著下降(p<0.01);其溶血活性比野生株降低2个滴度;并且该缺失株对BALB/c小鼠的LD_(50)为4.253×10~8 cfu,高于野生株3个数量级;免疫保护试验显示,经缺失株免疫的小鼠对野生株攻毒有75%的免疫保护率。本研究构建了基因缺失株LM△hly_(112-153),该缺失株对小鼠具有较好的免疫保护效果,以上研究为减毒李斯特菌疫苗载体的研发奠定重要基础。
The Listeriolysin strain with O encoding gene(hly)-deletion was constrcted, and its biological characteristics and immunological protection were evaluated in this study. The LM △hly_(112-153) was obtained by homologous recombination technique.The biological characteristics such as the invasiveness of the deletion strain on macrophages, hemolysis ability and the 50% lethal dose(LD_(50)) were studied, and the protective immunity of LM △hly_(112-153) in mice was evaluated. The results showed that the hly gene-deletion strain LM △hly_(112-153) was constructed successfully by the identification of PCR and sequence analysis. The invasive ability of the deletion strain LM △hly_(112-153) on macrophages is significantly decreased(p<0.01). The hemolytic activity of the gene-deletion strain was reduced by 2 titers compared with the wild strain. And the LD_(50) of LM △hly_(112-153) in BALB/c mice was4.253 ×10~8 cfu, which was higher than the parent strain by 3 orders of magnitude. Furthermore, in immunological protection experiment, LM△hly_(112-153) could protect mice against wild-type strain challenge, its protection rate was as high as 75%. The data demonstrate that the hly gene deletion strain LM△hly_(112-153) constructed in this study has a good protective immunity in mice. Our study may give a basis for the research and development of attenuated Listeria vaccine carrier.
The Listeriolysin strain with O encoding gene(hly)-deletion was constrcted, and its biological characteristics and immunological protection were evaluated in this study. The LM △hly_(112-153) was obtained by homologous recombination technique.The biological characteristics such as the invasiveness of the deletion strain on macrophages, hemolysis ability and the 50% lethal dose(LD_(50)) were studied, and the protective immunity of LM △hly_(112-153) in mice was evaluated. The results showed that the hly gene-deletion strain LM △hly_(112-153) was constructed successfully by the identification of PCR and sequence analysis. The invasive ability of the deletion strain LM △hly_(112-153) on macrophages is significantly decreased(p<0.01). The hemolytic activity of the gene-deletion strain was reduced by 2 titers compared with the wild strain. And the LD_(50) of LM △hly_(112-153) in BALB/c mice was4.253 ×10~8 cfu, which was higher than the parent strain by 3 orders of magnitude. Furthermore, in immunological protection experiment, LM△hly_(112-153) could protect mice against wild-type strain challenge, its protection rate was as high as 75%. The data demonstrate that the hly gene deletion strain LM△hly_(112-153) constructed in this study has a good protective immunity in mice. Our study may give a basis for the research and development of attenuated Listeria vaccine carrier.
引文
[1]任静静,杨铭伟,王鹏雁,等.单增李斯特菌(LM90)inlA单基因及in1B/inlA双基因缺失株的构建及其生物学特性初步研究[J].中国预防兽医学报,2018, 40(4):294-300.
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[5] Seregin S S, Chen G Y, Laouar Y. Dissecting CD8+NK T cell responses to Listeria infection reveals a component of innate resistance[J]. J Immunol, 2015, 195(3):1112-1120.
[6] Lam J G T, Vadia S, Pathak-Sharma S, et al. Host cell perforation by listeriolysin O(LLO)activates a Ca2+-dependent cPKC/Racl/Arp2/3 signaling pathway that promotes Listeria monocyto-genes internalization independently of membrane resealing[J].Mol Biol Cell, 2018, 29(3):270-284.
[7] Yi R, Sasa R, Apolonija B Z, et al. Listeriolysin O membrane damaging activity involves arc formation and lineaction-implication for Listeria monocytogenes escape from phagocytic vacuole[J]. PLoS Pathog, 2016, 12(4):e1005597.
[8] Wallace N,Newton E,Abrams E,et al. Metabolic determinants in Listeria monocytogenes, anaerobic listeriolysin O production[J]. Arch Microbiol, 2017, 199(6):827-837.
[9] Lebreton A, Stavru F, Cossart P. Organelle targeting during bacterial infection:insights from Listeria[J]. Trends Cell Biol,2015, 25(6):330-338.
[10] Yin Yue-lan, Tian De-bin, Jia Ya-nan, et al. Attenuated Listeria monocytogenes', a Mycobacterium tuberculosis ESAT-6 antigen expression and delivery vector for inducing an immune response[J]. Res Microbiol, 2012, 163(8):540-549.
[11] Vdovikova S, Luhr M, Szalai P, et al. A novel role of Listeria monocytogenes'membrane vesicles in inhibition of autophagy and cell death[J]. Front Cell Infect Microbiol, 2017, 7:154.
[12] Kanki M, Naruse H, Kawatsu K. Comparison of listeriolysin O and phospholipases PlcA and PlcB activities, and initial intracellular growth capability among food and clinical strains of Listeria monocytogenes[J]. J Appl Microbiol, 2018, 124(3):899-909.
[13] Maury M M, Tsai Y H, Charlier C, et al. Uncovering Listeria monocytogenes'hypervirulence by harnessing its biodiversity[J].Nat Genet, 2016, 48(3):308-313.
[2] Tim R, Ken N, Ruaidhri C, et al. Identification of TLR10 as a key mediator of the inflammatory response to Listeria monocytogenes in intestinal epithelial cells and macrophages[J]. J Immunol, 2013, 191(12):6084-6092.
[3] Dowd G C, Bahey-El-Din M, Casey P G, et al. Listeria monocytogenes mutants defective in gallbladder replication represent safety-enhanced vaccine delivery platforms[J]. Hum Vaccine Immunother, 2016, 12(8):2059-2063.
[4] Valderrama C, Clark A, Urano F, et al. Listeria monocytogenes induces an interferon-enhanced activation of the integrated stress response that is detrimental for resolution of infection in mice[J]. Eur J Immunol, 2017, 47(5):830-840.
[5] Seregin S S, Chen G Y, Laouar Y. Dissecting CD8+NK T cell responses to Listeria infection reveals a component of innate resistance[J]. J Immunol, 2015, 195(3):1112-1120.
[6] Lam J G T, Vadia S, Pathak-Sharma S, et al. Host cell perforation by listeriolysin O(LLO)activates a Ca2+-dependent cPKC/Racl/Arp2/3 signaling pathway that promotes Listeria monocyto-genes internalization independently of membrane resealing[J].Mol Biol Cell, 2018, 29(3):270-284.
[7] Yi R, Sasa R, Apolonija B Z, et al. Listeriolysin O membrane damaging activity involves arc formation and lineaction-implication for Listeria monocytogenes escape from phagocytic vacuole[J]. PLoS Pathog, 2016, 12(4):e1005597.
[8] Wallace N,Newton E,Abrams E,et al. Metabolic determinants in Listeria monocytogenes, anaerobic listeriolysin O production[J]. Arch Microbiol, 2017, 199(6):827-837.
[9] Lebreton A, Stavru F, Cossart P. Organelle targeting during bacterial infection:insights from Listeria[J]. Trends Cell Biol,2015, 25(6):330-338.
[10] Yin Yue-lan, Tian De-bin, Jia Ya-nan, et al. Attenuated Listeria monocytogenes', a Mycobacterium tuberculosis ESAT-6 antigen expression and delivery vector for inducing an immune response[J]. Res Microbiol, 2012, 163(8):540-549.
[11] Vdovikova S, Luhr M, Szalai P, et al. A novel role of Listeria monocytogenes'membrane vesicles in inhibition of autophagy and cell death[J]. Front Cell Infect Microbiol, 2017, 7:154.
[12] Kanki M, Naruse H, Kawatsu K. Comparison of listeriolysin O and phospholipases PlcA and PlcB activities, and initial intracellular growth capability among food and clinical strains of Listeria monocytogenes[J]. J Appl Microbiol, 2018, 124(3):899-909.
[13] Maury M M, Tsai Y H, Charlier C, et al. Uncovering Listeria monocytogenes'hypervirulence by harnessing its biodiversity[J].Nat Genet, 2016, 48(3):308-313.