| Red swamp crayfish, Procambarus clarkii, is an important aquatic animal in China, and also a good model to study crustacean innate immune system. Many immune-related genes have been well studied in freshwater crayfish.C-type lectins are a protein family which can bind carbohydrates in a calcium-dependent manner. They play roles as the receptors to recognize the pathogen patterns in the immune system, and also other various functions. Through a random sequence of a cDNA library of the crayfish hemocytes, and subsequent BLAST, we identified a putative C-type lectin, and named it PcLec5. The cDNA of PcLec5 was 771 bp long, with an open reading frame of 516 bp encoding a protein of 171 amino acid residues. Besides, the 5'and 3'untanslated regions were 20 and 235 bp long, respectively. The PcLec5 protein contained a signal peptide of 17 residues, suggesting that it was a secreted protein. SMART analysis indicated PcLec5 contained a CTLD domain of 135 amino acids.Using a pair of specific primer, we performed semi-quantitative RT-PCR and found that the mRNA of PcLec5 was specifically detected in hepatopancreas at a high level. To reveal the potential role of PcLec5, we used Vibrio anguillarum and Staphylococcus aureus to challenge the crayfish, and then analyzed the expression pattern of PcLec5 post challenge by realtime RT-PCR. The expression of PcLec5 quickly increased post V. anguillarum infection and reached to a peak at 12 h post infection, and then decreased. Differently, the expression slowly increased to the top at 48 h post S. aureus infection and returned to the normal level. The expression change suggested that PcLec5 participated in the innate immunity.We then constructed recombinant expression vector PcLec5-pET30a(+), expressed the protein in Escherichia coli BL21 (DE3) cells. The purified protein could bind to bacteria, including Gram-negative and-positive bacteria, suggesting that PcLec5 might serve as a pattern recognition receptor. |
PDF Full Text Request