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鱼类致病嗜水气单胞菌外膜蛋白P5的原核表达、免疫保护及抗血浆杀菌作用研究
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引用本文:荣 娜,简思杰,孙 薇,康 超,伍娜娜,刘 祥.鱼类致病嗜水气单胞菌外膜蛋白P5的原核表达、免疫保护及抗血浆杀菌作用研究[J].西北农业学报,2021,(3):333~342
DOI:10.7606/j.issn.1004-1389.2021.03.002
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作者单位
荣 娜,简思杰,孙 薇,康 超,伍娜娜,刘 祥 (陕西理工大学 生物科学与工程学院中德天然产物研究所陕西汉中 723001) 
基金项目:国家外国专家局项目(GDT20186100426);陕西省科技厅重点研发计划项目(2020LSFP2-38);陕西理工大学人才项目(SLGQD1803)。
中文摘要:研究鱼类致病嗜水气单胞菌外膜蛋白P5的免疫学功能,为相关疫苗开发奠定理论基础。采用生物信息学方法分析P5的理化性质;分子克隆构建P5表达菌株并确定其最佳诱导表达条件,包涵体洗涤和SDS-PAGE电泳切胶纯化P5蛋白,并免疫小鼠制备P5抗血清;Western blotting检测抗血清特异性与效价,免疫酶联法模拟P5抗血清对嗜水气单胞菌的体外识别;P5抗血清被动免疫红鲫,攻毒以评价P5蛋白的免疫保护功能;Western blotting分析P5蛋白抵抗鱼血浆的杀菌作用。结果表明:P5蛋白在气单胞菌属间同源性较好,进化保守。成功克隆表达、纯化P5蛋白,其最佳表达条件为:诱导时菌液浓度OD600=1.0,IPTG浓度0.5 mmol/L,32 ℃诱导8 h。Western blotting验证P5抗血清特异性较好,效价达到1∶1 600,ELISA显示P5抗血清与嗜水气单胞菌存在识别作用,表明P5蛋白具有较好的免疫原性与抗原性。被动免疫显示P5抗血清对红鲫的免疫保护率为显著性的56%,具有良好的免疫保护作用。Western blotting发现P5蛋白可通过表达下调有效抵抗鱼血浆的杀菌作用,表明其可能与细菌抗血浆杀菌有关。综上,嗜水气单胞菌外膜蛋白P5是一种保护性抗原,有望作为防治嗜水气单胞菌感染的疫苗候选成分。
中文关键词:嗜水气单胞菌  外膜蛋白P5  多克隆抗血清  血浆杀菌作用  被动免疫
 
Study on Prokaryotic Expression, Immunoprotective Effect and Resistance to Plasma Sterilization of Outer Membrane Protein P5 of Fish Pathogenic Aeromonas hydrophila
Abstract:To evaluate the immunological function of outer membrane protein P5 of fish pathogenic Aeromonas hydrophila(A.hydrophila) and provide a theoretical basis for vaccine development.The bioinformatics method was used to analyze the physicochemical properties of P5 protein.The molecule a cloning method was used to develop recombinant strain expressing P5 protein,which was purified by inclusion body washing and SDS-PAGE, and immunized mice to prepare P5 antiserum.Western blotting was used to detect the specificity and titer of the antiserum, and enzyme-linked immunosorbent assay (ELISA) was used to simulate the recognition effect between P5 antiserum and A.hydrophila.P5 antiserum was passively immunized to Carassius auratus and challenged with A.hydrophila to evaluate the immune protective function.Western blotting was used to detect the P5 protein effect for bacteria against fish plasma sterilization.The results showed that P5 had high homology and conservative evolution among Aeromonas.The P5 gene was cloned, expressed and purified successfully, and the optimal expression conditions for P5 protein included a strain OD600 value of 1.0, final isopropyl-β-d-thiogalactoside (IPTG) concentration of 0.5 mmol/L, an inducing temperature of 32 ℃ and an inducing time of 8 hours.Western blotting and ELISA analysis showed that the antiserum of P5 held high specificity and titer(1∶1 600).The P5 antiserum could recognize with A.hydrophilain vitro, indicating that P5 had high immunogenicity and antigenicity.The results of passive immunity showed that P5 protein demonstrated a significant immune protection function (56%) against A.hydrophila infection.P5 protein could protect effectively bacteria against fish plasma sterilization by down regulating expression, and P5 may be related to plasma sterilization.In summary, the outer membrane protein P5 of A.hydrophila is a protective antigen and expected to be a potential vaccine against A.hydrophila in fection.
keywords:Aeromonas hydrophila  Outer membrane protein P5  Polyclonal antiserum  Plasma sterilization  Passive immunity
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