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...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.展开更多
[Objective] To optimize the prokaryotic expression of MCP gene of red-spotted grouper nervous necrosis virus. [Method] The MCP gene was amplified from red-spotted grouper nervous necrosis viral genome by RT-PCR. The r...[Objective] To optimize the prokaryotic expression of MCP gene of red-spotted grouper nervous necrosis virus. [Method] The MCP gene was amplified from red-spotted grouper nervous necrosis viral genome by RT-PCR. The recombinant expression vector pRSET A-MCP was constructed and transformed into BL21(DE3)plysS to express proteins with induction in different media, at different pH, or at different temperatures. [Result] The expression level of recombinant bacteria reached a peak with induction under the following condition: SOB or LB medium, pH 7.0, 37 ℃ while the fusion protein was about 44.5 kD in molecular weight. [Conclusion] This study provided a basis for the development of RGNNV-MCP vaccine.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(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.
基金Supported by Science and Technology Planning Project of Guangdong Province,China(2003B21502,2005B20301016)~~
文摘[Objective] To optimize the prokaryotic expression of MCP gene of red-spotted grouper nervous necrosis virus. [Method] The MCP gene was amplified from red-spotted grouper nervous necrosis viral genome by RT-PCR. The recombinant expression vector pRSET A-MCP was constructed and transformed into BL21(DE3)plysS to express proteins with induction in different media, at different pH, or at different temperatures. [Result] The expression level of recombinant bacteria reached a peak with induction under the following condition: SOB or LB medium, pH 7.0, 37 ℃ while the fusion protein was about 44.5 kD in molecular weight. [Conclusion] This study provided a basis for the development of RGNNV-MCP vaccine.
