王一帆　陈子茵　贾新蕾　黄增朝　吕静　黄郁葱

摘 要：根据GenBank上登录的红笛鲷SR-BI基因设计引物，采用RT-PCR方法扩增该基因胞外段序列，然后将该基因胞外段序列定向克隆到原核表达载体pET-28a（+）中，将重组质粒转入大肠杆菌BL21进行IPTG诱导表达，并对重组表达菌株的表达条件进行优化。结果显示，成功构建了原核表达载体pET28a-SR-BI，并能在大肠杆菌BL21中表达，表达的融合蛋白分子量为50.0ku。融合蛋白的最佳诱导表达温度、IPTG浓度和时间分别为16℃、0.05mmol/L和8h。研究结果为进一步研究SR-BI的功能奠定了基础。

关键词：红笛鲷;SR-BI基因;原核表达;条件优化

中图分类号 S942.1文献标识码 A文章编号 1007-7731（2021）03-0001-06

Prokaryotic Expression and Condition Optimization of SR-BI Gene in Lutjanus sanguineus

WANG Yifan et al

（Fisheries College of Guangdong Ocean University/Provincial Key Laboratory of Pathogenic Biology and Epidemiology for Aquatic Economic Animals of Guangdong & Key Laboratory of Control for Diseases of Aquatic Economic Animals of Guangdong Higher Education Institute， Zhanjiang 524088， China）

Abstract：A pair of primers were designed based on Scavenger receptor class B type I（SR-BI） gene sequence published in GenBank. The extracellular region sequence of SR-BI gene was amplified by RT-PCR and then inserted into the pET-28a（+） vector to construct prokaryotic expression plasmid. The recombinant plasmid was transformed into E.coli BL21 to overexpressed in the presence of isopropyl-β-D-thiogalato pyranos ide （IPTG）， and the expression conditions of the recombinant strain were optimized. The results showed that the prokaryotic expression plasmid pET28a-SR-BI was successfully constructed and expressed in E. coli BL21. The molecular weight of the expressed recombinant fusion protein was 50.0 ku. The optimal induction expression temperature， IPTG concentration and induction time of the recombinant fusion protein was 16℃， 0.05mmol/L and 8h， respectively. The research results laid the foundation for the further research on the functions of SR-BI.

Key words： Lutjanus sanguineus; SR-BI gene; Prokaryotic Expression; Condition optimization

清道夫受體（Scavenger Recptors，SRs）是一类与膜相关或可溶性的、能结合修饰后的LDL（Low-Density Lipoprotein）或其他多聚阴离子配体的跨膜糖蛋白[1]，首次发现于小鼠动脉粥样硬化斑块的巨噬细胞上存在摄取和降解乙酰化低密度脂蛋白（ac-LDL）的结合位点，后来被称为巨噬细胞清道夫受体1[2]。根据分子结构、表达特征及与配体识别机制，目前已发现的清道夫受体至少包含8种不同类别（A、B、C、D、E、F、G、H）[1]。清道夫受体具有广泛的受体识别谱，能够识别修饰低密度脂蛋白、革兰氏阴性菌细胞壁成分的脂多糖（LPS）和革兰氏阳性菌细胞壁成分磷壁酸（LTA）多种微生物结构成分，还可识别由细胞膜内转运到膜外的磷脂酰丝氨酸，并能够结合多种蛋白质、核苷酸、多糖以及脂类等，参与机体一系列的生理和病理过程，包括动脉粥样硬化形成或抑制、阿尔茨海默氏病、宿主免疫防御、细胞粘附、凋亡细胞清除、组织保护和维护机体平衡等[3-5]，在动物生命代谢过程中起着重要作用。……