Expression of HSP70 in spleen cells stimulated by LPS and Poly I∶C and preparation of recombinant protein in orange-spotted grouper Epinephelus coioides
LIANG Zhenyu， ZOU Zihong， TANG Jufen， HUANG Yu， JIAN Jichang， CAI Jia
1.College of Fisheries, Guangdong Provincial Key Laboratory of Pathogenic Biology and Epidemiology for Aquatic Economic Animals, Key Laboratory of Control for Diseases of Aquatic Economic Animals of Guangdong Higher Education Institutes, Guangdong Ocean University, Zhanjiang 524088, China; 2.Guangxi Beibu Gulf Marine Research Center, Guangxi Key Laboratory of Marine Natural Products and Combinatorial Biosynthesis Chemistry, Guangxi Academy of Sciences, Nanning 530007, China
Abstract: To explore the role of HSP70 gene (EcHSP70, GenBank accession number: MW411059) from orange-spotted grouper Epinepheluscoioides 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 Escherichiacoli 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 96 000 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.
梁振宇, 邹子鸿, 汤菊芬, 黄瑜, 简纪常, 蔡佳. 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[J]. 大连海洋大学学报, 2021, 36(6): 937-942.
LIANG Zhenyu, ZOU Zihong, TANG Jufen, HUANG Yu, JIAN Jichang, CAI Jia. Expression of HSP70 in spleen cells stimulated by LPS and Poly I∶C and preparation of recombinant protein in orange-spotted grouper Epinephelus coioides. Journal of Dalian Ocean University, 2021, 36(6): 937-942.
2021, Vol. 36 
