Lectins play a crucial role in the innate immunity of invertebrates and vertebrates by recognizing and disposing of pathogens. We obtained the complete cDNA of a C-type lectin （EALecl） from Epinephelus akaara using ...Lectins play a crucial role in the innate immunity of invertebrates and vertebrates by recognizing and disposing of pathogens. We obtained the complete cDNA of a C-type lectin （EALecl） from Epinephelus akaara using RACE. The complete EALecl cDNA sequence was 827 bp. The 5-UTR and 3-UTR were 28 bp and 151 bp, respectively, in length. The sequence also contained a polyadenylation signal AATAAA and a poly（A） tail. The EALecl cDNA encodes polypeptides with 215 amino acids, including a signal peptide of 31 amino acids. The protein has a cysteine-rich region at the N terminal, a collagenous region characterized by G-X-Y repeats, a neck region, and a typical carbohydrate-recognition domain （CRD）, indicating that EALecl is a collectin. The key recognition positions of this CRD are EPD, isolated for the first time in fish. These are likely the interim types, between mannan-binding lectin and galactose-binding lectin. We evaluated the expression pattern of EALecl in 12 different tissues using RT-PCR. EALecl was expressed in all tissues, though at different levels. In addition, we inserted EALecl into an expression vector （pET-28a） for transformation into the BL21 engineering bacteria. Based on enzyme digestion and sequencing of the positive clone, we successfully constructed the EALecl recombinant expression vector.展开更多
基金Supported by the Natural Science Foundation of Fujian Province of China (No 2060203)New Century Excellent Talents supporting funding of Fujian Province
文摘Lectins play a crucial role in the innate immunity of invertebrates and vertebrates by recognizing and disposing of pathogens. We obtained the complete cDNA of a C-type lectin （EALecl） from Epinephelus akaara using RACE. The complete EALecl cDNA sequence was 827 bp. The 5-UTR and 3-UTR were 28 bp and 151 bp, respectively, in length. The sequence also contained a polyadenylation signal AATAAA and a poly（A） tail. The EALecl cDNA encodes polypeptides with 215 amino acids, including a signal peptide of 31 amino acids. The protein has a cysteine-rich region at the N terminal, a collagenous region characterized by G-X-Y repeats, a neck region, and a typical carbohydrate-recognition domain （CRD）, indicating that EALecl is a collectin. The key recognition positions of this CRD are EPD, isolated for the first time in fish. These are likely the interim types, between mannan-binding lectin and galactose-binding lectin. We evaluated the expression pattern of EALecl in 12 different tissues using RT-PCR. EALecl was expressed in all tissues, though at different levels. In addition, we inserted EALecl into an expression vector （pET-28a） for transformation into the BL21 engineering bacteria. Based on enzyme digestion and sequencing of the positive clone, we successfully constructed the EALecl recombinant expression vector.
