摘要
【目的】过氧化氢酶(KatA)是幽门螺杆菌编码的重要毒力因子,在抵抗宿主免疫杀伤和疫苗研发等方面具有重要功能。为了鉴定幽门螺杆菌KatA的酶学特性,并分析其在幽门螺杆菌氧化耐受中的功能。【方法】首先,从幽门螺杆菌临床耐药菌株Hp_G272基因组中分离katA基因,并在大肠杆菌中克隆表达、分离纯化KatA^(G272)蛋白;然后,利用CAT检测试剂盒体外分析KatA^(G272)的过氧化氢酶活性;其次,利用同源重组技术构建katA基因工程菌株,包括敲除菌株和回补菌株;最后,通过比较野生菌株与工程菌株生长表型差异、分解过氧化氢能力差异及耐受过氧化氢能力差异,阐述katA基因的功能及其在幽门螺杆菌耐受氧化损伤中的作用。【结果】在大肠杆菌中成功克隆表达并分离纯化KatA^(G272),体外酶学分析表明,KatA^(G272)是一类嗜酸性过氧化氢酶,基因敲除分析表明,敲除该基因幽门螺杆菌丧失分解和耐受过氧化氢的能力,而回补该基因幽门螺杆菌回复分解和耐受过氧化氢的能力。【结论】katA基因是幽门螺杆菌分解和耐受过氧化氢的唯一功能基因,在幽门螺杆菌氧化耐受中具有重要功能。
【Objective】Catalase(KatA)is an important virulence factor encoded by Helicobacter pylori,and plays an important role in bacterial resistance to host immune killing and vaccine development.The objective of this study is to identify the enzymatic properties of H.pylori catalase and to analyze its function in H.pylori oxidation tolerance.【Method】Firstly,katA gene was isolated from the genome of clinical drug-resistant strain Hp_G272,G272 and KatA protein was cloned and expressed in Escherichia coli.G272 Then,the catalase activity of KatA was analyzed in vitro with CAT detection kit.Secondly,katA genetic engineering strains were constructed by homologous recombination,including knockout strains and complemented strains.Finally,the function of katA gene and its role in tolerance to oxidative damage of H.pylori were analyzed by comparing the differences in growth phenotype,decomposition and tolerance of hydrogen peroxide between wild and engineered strains.【Result】KatA^(G272) was successfully cloned,expressed and purified from E.coli.Enzymatic analysis showed that KatA^(G272) was a class of eosinophilic catalase.Gene knockout analysis showed that H.pylori lost its ability to decompose and tolerate hydrogen peroxide,while regained its ability to decompose and tolerate hydrogen peroxide after gene complement.【Conclusion】katA is the only functional gene that decomposes and tolerates hydrogen peroxide in H.pylori,and plays an important role in the oxidative tolerance of H.pylori.
作者
王鑫鑫
管玉祝
李晓苇
洪伟
吴道艳
康颖倩
刘永畅
陈峥宏
崔古贞
WANG Xin-xin;GUAN Yu-zhu;LI Xiao-wei;HONG Wei;WU Dao-yan;KANG Ying-qian;LIU Yong-chang;CHEN Zheng-hong;CUI Gu-zhen(Department of Microbiology,College of Basic Medicine,Guizhou Medical University,Guiyang 550000;Pathogenic Biology Characteristic Laboratory of Guizhou Province,Guiyang 550000;Key Laboratory of Molecular Biology of Guizhou Province,Guiyang 550000)
出处
《生物技术通报》
CAS
CSCD
北大核心
2024年第5期310-320,共11页
Biotechnology Bulletin
基金
国家自然科学基金项目(32160015,32170134)
贵州省高层次留学人才项目(2022-08)
贵州省科技计划项目([2020]1Z067)
贵州省高层次创新人才团队及人才基地项目(GCC[2022]036-1,[RCJD2018-22])
高等学校学科创新引智计划(111计划,D20009)
贵州省大学生创新训练计划项目(S202310660078)
贵州省病原生物学重点实验室(QJJ[2022]019)。
