摘要
大菱鲆C-型凝集素(SmLec1)是一种重要的免疫因子,本实验通过分析其活性特点以及环境胁迫对其表达调控的影响,探讨其在大菱鲆先天免疫应答过程中的潜在功能以及养殖环境中的应用。实验利用原核表达系统进行重组表达,并通过纯化获得19 ku重组目的蛋白;活性分析结果显示,SmLec1对所选6种细菌的凝集作用存在差异,其中对鳗弧菌和爱德华氏菌具有明显的凝集作用;在选取的8种糖类中,木糖、果糖、蔗糖、乳糖和甘露糖能够抑制SmLec1的凝集活性;在理化因子(pH、温度)中,pH<7和温度>37°C时对SmLec1的凝集活性产生抑制;鳗弧菌感染实验显示,感染6 h时肝组织的SmLec1表达量明显上升,在12 h时达到最高值,随后有所下降;温度胁迫时肝组织的SmLec1 m RNA表达量受到温度的影响显著,在19°C开始升高,22~27°C呈降低趋势,其他组织变化不显著。盐度胁迫对SmLec1在各盐度的表达量变化不显著。研究表明,SmLec1能够抑制鳗弧菌的感染,其表达受到温度的影响,本研究为进一步明确SmLec1蛋白的抗菌机理和探索对大菱鲆疾病的免疫学防治途径提供科学依据。
C-type lectin of Scophthalmus maximus（SmLec1） plays an important role in the non-self innate immune system. We analyzed the activity characteristics of SmLec1, and the effect of environmental stress on its expression and regulation, and explored its potential function and response in turbot innate immune system in culture environment. In this study, the molecular mass of the SmLec1 with recombinant protein was found to be 19 ku as shown by SDS-PAGE, and the target protein was obtained after purification. SmLec1 showed agglutination activity against Vibrio anguillarum and Edwardsiella sp. in the six kinds of selected bacteria to test SmLec1 bacterial agglutination activity. On this basis, eight sugars were taken for the inhibitory agglutination studies. Among the carbohydrates tested, lactose, D-xylose, D-fructose, sucrose and D-mannose effectively inhibited the agglutination activity of SmLec1. In physical and chemical factors（pH, high temperature）, the activity of SmLec1 was inhibited at pH 7, T 37 °C. QPCR was performed to examine the relative expression of the SmLec1 in V.anguillarum challenge, the expression level of liver was relatively increased after pathogenic stimulation, the highest at 6 h,then gradually began to decline and returned to its original level at 32 h. Expression of SmLec1 in high temperature stress was analyzed by q PCR, and results showed that the expression of liver m RNA starts to rise from 19 °C, and showed a decreasing trend from 22 °C to 27 °C, while expression of other tissues did not change significantly. The impact of salinity stress on expression was not significant. The study could provide a scientific basis for further study of antibacterial mechanism and immune mechanism of SmLec1.
出处
《水产学报》
CAS
CSCD
北大核心
2017年第2期161-170,共10页
Journal of Fisheries of China
基金
现代农业产业技术体系专项(CARS-50-G01)
中国博士后科学基金(2015M572096)
山东省自然科学基金(ZR2014CP001)
青岛市博士后科学基金(2014-2016)
现代农业人才支撑计划(2016-2020)~~
关键词
大菱鲆
C-型凝集素
鳗弧菌
蛋白重组
凝集活性
环境胁迫
Scophthalmus maximus
C-type lectin
Vibrio anguillarum
recombinant proteins
agglutination activity
environmental stress
