cDNA cloning and expression of a collectin from red-spotted grouper(Epinephelus akaara)

【摘要】 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(EALec1) from Epinephelus akaara using RACE.The complete EALec1 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 EALec1 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 EALec1 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 EALec1 in 12 different tissues using RT-PCR.EALec1 was expressed in all tissues,though at different levels.In addition,we inserted EALec1 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 EALec1 recombinant expression vector.更多还原

【Abstract】 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(EALec1) from Epinephelus akaara using RACE.The complete EALec1 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 EALec1 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 EALec1 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 EALec1 in 12 different tissues using RT-PCR.EALec1 was expressed in all tissues,though at different levels.In addition,we inserted EALec1 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 EALec1 recombinant expression vector.更多还原