文献类型：期刊文献

中文题名：斜带石斑鱼热休克蛋白基因HSP70在LPS和Poly I∶C刺激下的表达分析及重组蛋白制备

英文题名：Expression of HSP70 in spleen cells stimulated by LPS and Poly I∶C and preparation of recombinant protein in orange-spotted grouper Epinephelus coioides

作者：梁振宇[1];邹子鸿[1];汤菊芬[1];黄瑜[1];简纪常[1];蔡佳[1,2]

机构：[1]广东海洋大学水产学院,广东省水产经济动物病原生物学及流行病学重点实验室,广东省水产经济动物病害控制重点实验室,广东湛江524088;[2]广西科学院广西北部湾海洋研究中心,广西海洋天然产物与组合生物合成化学重点实验室,广西南宁530007

年份：2021

卷号：36

期号：6

起止页码：937

中文期刊名：大连海洋大学学报

外文期刊名：Journal of Dalian Ocean University

收录：CSTPCD、、CSCD2021_2022、北大核心、CSCD、北大核心2020

基金：广东海洋大学“冲一流”专项资金项目(231419013);南方海洋科学与工程广东省实验室(湛江)自主立项项目(ZJW-2019-06);广西自然科学基金(2020GXNSFAA297243)。

语种：中文

中文关键词：斜带石斑鱼;HSP70基因;表达分析;原核表达;脂多糖;聚肌胞

外文关键词：Epinephelus coioides;HSP70 gene;expression analysis;prokaryotic expression;LPS;Poly I∶C

中文摘要：为了解斜带石斑鱼Epinephelus coioides热休克蛋白(HSP70)基因(EcHSP70,GenBank登录号MW411059)在石斑鱼抗病原感染中的功能,采用荧光定量PCR检测了LPS与Poly I∶C刺激后48 h内,石斑鱼脾脏细胞(GS)中EcHSP70的表达变化;构建重组质粒EcHSP70-4T,转化到BL21(DE3)后进行诱导表达,利用SDS-PAGE电泳和Western blot检测EcHSP70重组蛋白的表达形式。结果表明:LPS与Poly I∶C刺激后,石斑鱼脾脏细胞中EcHSP70基因表达量上调;SDS-PAGE与Western blot检测表明,EcHSP70-4T重组蛋白相对分子质量约为96000,主要以包涵体形式表达。研究表明,EcHSP70蛋白可能参与了石斑鱼的免疫反应,其在大肠杆菌中能以包涵体形式高效表达,并纯化获得了EcHSP70重组蛋白,本研究结果为鉴定EcHSP70的互作蛋白及进一步了解该蛋白在免疫反应中的作用机制提供了数据参考。

外文摘要：To explore the role of HSP70 gene(EcHSP70,GenBank accession number:MW411059)from orange-spotted grouper Epinephelus coioides in immune response against pathogen infection,the expression profiles of EcHSP70 in spleen cells(GS)stimulated by lipopolysaccharide(LPS)and polyinosinic-polycytidylis acid(Poly I∶C)at a ratio of 1 mg/mL LPS to 5 mg/mL Poly I∶C for 0,4,8,12,24,and 48 h were analyzed by Real-time quantitative PCR.The prokaryotic expression vector EcHSP70-4T was constructed and transferred into Escherichia coli BL21(DE3),and the expressed EcHSP70 recombinant protein was confirmed by SDS-PAGE and western blot.The results showed that the EcHSP70 expressions were significantly up-regulated in GS exposed to the LPS and poly I∶C stimulations.SDS-PAGE and western blot indicated that the EcHSP70-4T recombinant protein had relative molecular mass of 96000 and expressed as the form of inclusion body.The EcHSP70 was shown to be involved in the immune defense of the orange-spotted grouper,expressed effectively in E.coli and the recombinant protein was purified.The findings laid a foundation to identify the interacting protein and to further clarify the mechanism of EcHSP70 during immune response.