幽门螺杆菌katA基因功能及其在耐受氧化损伤中的作用分析

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国家科技期刊平台
NETL
NSTL
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中文摘要：[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KatAG272 蛋白;然后,利用 CAT 检测试剂盒体外分析KatAG272 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 KatAG272,体外酶学分析表明,KatAG272 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.

外文标题：Function of katA in Helicobacter pylori and Its Role in the Tolerance to Oxidative Damage

外文摘要：[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,and KatAG272 protein was cloned and expressed in Escherichia coli.Then,the catalase activity of KatAG272 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]KatAG272 was successfully cloned,expressed and purified from E.coli.Enzymatic analysis showed that KatAG272 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.

外文关键词：

Helicobacter pyloricatalasekatAtoleranceclone and expressiongene knockout

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

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作者单位：

贵州医科大学基础医学院微生物学教研室,贵阳 550000

贵州省病原生物学特色重点实验室,贵阳 550000

贵州省分子生物学重点实验室,贵阳 550000

关键词：

幽门螺杆菌过氧化氢酶 katA 耐受性克隆表达基因敲除

基金：

国家自然科学基金国家自然科学基金贵州省高层次留学人才项目贵州省科技计划贵州省高层次创新人才团队及人才基地项目贵州省高层次创新人才团队及人才基地项目高等学校学科创新引智计划(111计划)贵州省大学生创新训练计划贵州省病原生物学重点实验室项目

项目编号：

32160015321701342022-08[2020]1Z067GCC[2022]036-1[RCJD2018-22]D20009S202310660078QJJ[2022]019

出版年：

2024

DOI：

10.13560/j.cnki.biotech.bull.1985.2023-1114

生物技术通报

中国农业科学院农业信息研究所

生物技术通报

CSTPCD北大核心

影响因子：0.505

ISSN：1002-5464

年,卷(期)：2024.40(5)