Molecular cloning and characterization in immune response of a novel C-type lectin gene (LvLc2) from Litopenaeus vannamei

Litopenaeus vannamei is the largest economic shrimp breed in the world at present. However, with the expansion of shrimp culture, diseases and other problems are exposed gradually. Shrimp culture industry was affected seriously by diseases all over the world. Shrimp belongs to invertebrate, and has no typical acquired immune system. They mainly rely on the innate immune system for defense and protection when pathogens invade. In innate immune response, crustaceans recognize non-self components through pattern recognition receptors (PRRs). The components recognized by PRRs are mainly constitutive and conserved polysaccharide molecules on the surface of microorganisms, which are not produced by eukaryotic cell hosts and are necessary for the survival of pathogenic microorganisms. They are called pathogen associated molecular patterns (PAMPs). The recognition and binding of PRRs to PAMPs is the beginning of humoral immunity. The recognition of PRRs to pathogens can directly or indirectly activate proteases, hemocytes and intracellular signal pathways related to humoral immunity. A variety of immune defense reactions were triggered. It produces antibacterial substances and effective molecules to inhibit and eliminate pathogens. As a pattern recognition receptor and terminal effector, C-type lectin participates in the process of “non-self” recognition and elimination of invasive pathogens, which is crucial in the innate immune defense mechanism of crustaceans. In this study, a novel C-type lectin gene (LvLc2, GenBank accession number: KR020738) was cloned from hemocytes of L. vannamei according to our previous transcriptome results. Bioinformatics analysis showed that the ORF region of LvLc2 gene was 465 bp encoding 154 aa with 11 bp 5'-UTR and 126 bp 3'-UTR. The predicted molecular weight was 17.16 ku and the theoretical isoelectric point was 4.54. The deduced amino acid sequence contained a signal peptide sequence with 17 amino acids at the amino end and a conserved carbohydrate recognition domain (CRD) at the carboxyl end, in which four conserved cysteines formed two disulfide bonds. The motif associated with PAMP binding specificity is EPS (Glu¹¹³-Pro¹¹⁴-Ser¹¹⁵). Blast homology analysis showed that LvLc2 had high similarity with mannose receptor C-type lectin from different species, and 38% similarity with the mannose receptor of Rimicaris exoculata. LvLc2 gene was expressed in different tissues of L. vannamei. The highest expression was in intestine, followed by stomach, hepatopancrease, hemocytes, and the lowest expression level was in muscle. After shrimps were challenged by LPS and PGN, the expression profiles of LvLc2 gene in hemocytes were analyzed by qPCR. Compared with the control group, the expression of LvLc2 showed a slight down-regulated expression profile at the initial stage of LPS injection (0.5 h), and then reached the highest value at 12 h after LPS injection, which was about 2.2 times of the initial expression level. At 24-72 h after injection, the expression of LvLc2 basically returned to the initial level, and there was no significant change. At the early stage after PGN injection, the expression of LvLc2 fluctuated slightly. At the 48th hour, the expression of LvLc2 was significantly up-regulated, which was about 4.5 times of that in the control group and then fell back to slightly lower than the initial level. Different pathogen-associated molecular patterns (PAMP) could induce response changes of LvLc2 gene after stimulation, which indicated that it had a potential broad-spectrum response pattern. At the same time, it had different expression profiles for different pathogens, which showed the differences of recognition, combination and indirect immune effects. The research of its characterization and function are helpful to better understand the mechanism of shrimp C-type lectin during the process of pathogen infection.