Molecular Cloning, Recombinant Expression And Functional Analysis Of Two Pattern Recognition Receptor From Red Swamp Crayfish Procambarus Clarkii

Pattern recognition protein (PRR) is germ-line encoded protein which can interact with pathogen associated molecular pattern (PAMP) and activate innate immune response. In the present study, two genes encoding PRRs, PcLGBP and PcLEC, were cloned and identified from red swamp crayfish Procambarus clarkii. To investgate the function of the two PRRs, stable RNA interference (RNAi) technique was established in crustacean animals. Based on RNAi technique, the role of PcLGBP playing in defense against bacterial infection was investigated. The specificity of the recombination PcLGBP and PcLEC protein to ligand were also examined using ELISA approach.A stable RNAi technique was established by injecting dsRNA into shrimp or crayfish. Double strand RNA (dsRNA) was synthesized via in vitro transcription. The target gene can be effectively and systemically silenced by dsRNA specific for target gene. The silencing effect can last until 96 h after dsRNA injection. It is appropriate to use RNAi for investigation of gene function in crustacean.The full length cDNA of PcLGBP gene was of 1293 bp and encoded a protein of 369 amino acids which contained a glucanase-like domain and a potential recognition motif forβ-1, 3-linkage of polysaccharides. Expression analysis with RT-PCR revealed that PcLGBP gene was exclusively expressed in hepatopancreas and the expression could be up-regulated by heat-killed Listonella anguillarum. The binding specificity of PcLGBP to ligand were subsequently examined by recombinant expression of the cDNA fragment encoding its mature peptide in Escherichia coli BL21 (DE3) and ELISA approach. PcLGBP can bind Lipopolysaccharide andβ-1,3-glucan in a dose-dependent manner, but not peptidoglycan. RNAi-based silencing of PcLGBP gene resulted in significantly decreased survival rate when infected with gram-negative bacteria, but not gram-positive bacterial infection. The results collectively indicated that PcLGBP is a member of gram-negative bacterial binding protein family and mediate the defense response of red swamp crayfish against gram-negative bacterial infection.The full-length cDNA of PcLEC was of 813 bp and encoded a polypeptide of 271 amino acids. The amino acid sequence of PcLEC included a signal peptide of 17 amino acids and a glycine-riched sequence in N terminal, as well as carbohydrate recognition (CRD) of 138 amino acids in C terminal. The CRD of PcLEC was similar to C-lectin from crustacea and CTLD-containing proteins from insects. Mature peptide of PcLEC also included four conserved cysteines that likely were involved in intrachain disulfide bonds. There are twoα-helices and sevenβ-sheets in computational secondary structure of PcLEC predicted with Swiss-model software. The tissue distribution of PcLEC gene and its temporal expression in hemolymph was examined by fluorescent quantitative real-time PCR. PcLEC was expressed in all examined tissue including hemocyte, hepatopancreas, muscle, gonad, gill and heart. But the highest mRNA level was observed in hemocyte. The mRNA level of crayfish challenged by heat-killed L. anguillarum was significantly up-regulated after stimulation. The function of PcLEC was investigated by recombinant expression of the cDNA fragment encoding its mature peptide in Escherichia coli BL21 (DE3). The recombinant PcLEC agglutinated E. coli Top10, L. anguillarum, and Micrococcus luteu. The agglutinating activities were calcium-dependent and could be inhibited by D-galactose and N-Acetylgalactosamine. The binding specificity of recombinant PcLEC protein to D-galactose and N-Acetylgalactosamine were subsequently confirmed using ELISA approach. The results collectively indicated that PcLEC is a member of C-lectin family and contributed to nonself recognition as a pattern recognition receptor in the immune response of red swamp crayfish.