Molecular cloning and functional study of mannose receptor gene in Epinephelus coioides

Mannose receptor (MR), also known as CD206, is an endocytosis receptor involved in pathogen recognition and antigen presentation. As a link between innate immunity and adaptive immunity, MR plays an important role in immune response. Viral nerve necrosis (VNN) caused by nerve necrosis virus (NNV) is a highly contagious neuropathological disease, which has caused the death of more than 50 important farmed juvenile fishes worldwide. At the same time, NNV is also one of the main viral pathogens causing huge economic losses to the Epinephelus coioides culture industry. Innate immunity can recognize pathogens by combining pattern recognition receptor (PRR) with pathogen-associated molecular pattern (PAMP). C-type lectins belong to PRR. MR as a member of C-type lectins family, is of great significance to study whether MR has immune function in NNV. Most of the studies on MR in fish are focused on the antibacterial activity of MR, but the relationship between MR and antivirus in fish is unknown. In order to study the immune function of EcMR gene against red grouper nerve necrosis virus (Red-spotted grouper nervous necrosis virus, RGNNV), EcMR gene was cloned and expressed. The results showed that the EcMR cDNA full length sequence was 4 877 bp, and its open reading frame encoded 1446 amino acids. The protein domain of EcMR was composed of a signal peptide, an extracellular ricin-like β-type clover domain (RICIN), a fibronectin type Ⅱ domain (FN Ⅱ), eight tandem C-type lectin-like domains (CLECTs) and a transmembrane domain. The results of real-time quantitative PCR (qRT-PCR) showed that EcMR gene widely existed in 8 examined tissues and the relative expression of mRNA was in the following order of gill > head kidney > brain > spleen > liver > peripheral blood > heart > muscle. The results showed that RGNNV could proliferate rapidly in GF-1 cells and significantly activate the expression of EcMR gene at 6 h and 24 h after infection. In order to further explore the effect of RGNNV on GF-1 cells, the apoptosis of GF-1 cells infected by RGNNV was detected by the Alexa Fluor^® 488 annexin V/Dead Cell Apoptosis Kit. The results showed that RGNNV infection could promote the apoptosis of GF-1 cells, and the apoptosis rate increased with the prolongation of infection time. At the same time, the results of qRT-PCR and enzyme activity assay showed that RGNNV infection could significantly promote the transcription level of apoptosis-related genes, and the expression of inflammatory cytokines and the enzyme activity levels of Caspase-3, Caspase-8 and Caspase-9. To sum up, this study successfully cloned the EcMR gene and revealed its correlation in the process of RGNNV invasion. The results of this study will provide some basic theoretical reference for the prevention and control of grouper viral diseases.