Expression analysis and preparation of recombinant protein of T-cell immunoglobulin and mucin-domain-containing molecule 4 (TIM-4) gene from oblique grouper Epinephelus coioides
Expression analysis and preparation of recombinant protein of T-cell immunoglobulin and mucin-domain-containing molecule 4 (TIM-4) gene from oblique grouper Epinephelus coioides
1.College of Fisheries,Guangdong Provincial Key Laboratory of Aquatic Animal Disease Control and Healthy culture,Guangdong Key Laboratory of Economic Animal Disease Control of Aquatic Products,Southern Marine Science and Engineering Guangdong Laboratory (Zhanjiang),Guangdong Ocean University,Zhanjiang 524088,China;2.Guangxi Key Laboratory of Marine Natural Products and Combinatorial Biosynthesis Chemistry,Guangxi Beibu Gulf Marine Research Center,Guangxi Academy of Sciences,Nanning 530007,China
Abstract: In order to study the role of T-cell immunoglobulin and mucin-domain-containing molecule 4 (TIM-4)gene in the immune response of oblique grouper Epinepheluscoioides,protein-coding regions of the oblique grouper was obtained using polymerase chain reaction (PCR),and the structural characteristics of its protein coding region were analyzed by bioinformatics.The tissue distribution of TIM-4 gene were detected by real-time fluorescence quantitative PCR (qPCR).The expression profiles of TIM-4 gene was investigated in spleen (GS) cells exposed to lipopolysaccharide (LPS) and polyinosinic acid (Poly I:C) stimulation.The recombinant plasmid TIM-4-4T was constructed,and transformed into Escherichiacoli BL21 (DE3) for inducing prokaryotic protein expression.The results showed that the protein coding region (GenBank accession number:MZ465580) of TIM-4 was 702 bp,encoding 234 amino acids protein containing three functional domains,a V-Type(IGV)domain,a BTK domain and a PRY domain.qPCR results showed that TIM-4 was expressed in all examined 10 tissues of oblique grouper,with the maximal expression in stomach (P<0.05),and the minimal in spleen.There was significantly lower expression of TIM-4 in GS cells exposed to LPS stimulation,and significantly increase in GS cells exposed to Poly I:C stimulation (P<0.05).The SDS-PAGE analysis revealed that the TIM-4 fusion protein had relative molecular weight of about 25 100.The optimal expression TIM-4 recombinant protein were inducing with 0.4 mmol/L IPTG at 37 ℃ for 6 h.The findings indicate that TIM-4 may be involved in anti-infectious immunity of oblique grouper,which provides a theoretical basis for further study on the biological function of TIM-4.
2022, Vol. 37 
