人白介素-3是一种造血系统和免疫系统调节剂,在治疗造血系统疾病、肿瘤、先天或获得性免疫功能缺陷等方面发挥着重要作用.应用牛乳腺生物反应器生产人白介素-3的研究具有重要的临床应用和经济价值.研究选用具有红色荧光蛋白报告基因(R e...人白介素-3是一种造血系统和免疫系统调节剂,在治疗造血系统疾病、肿瘤、先天或获得性免疫功能缺陷等方面发挥着重要作用.应用牛乳腺生物反应器生产人白介素-3的研究具有重要的临床应用和经济价值.研究选用具有红色荧光蛋白报告基因(R ed2)和新霉素抗性基因(neor)表达框架的pD sR ed2-1质粒为骨架构建人白介素-3乳腺表达载体.通过PCR方法分别扩增牛β-酪蛋白基因5′端上游调控序列、人IL-3基因以及CM V启动子序列,将它们按先后顺序分别定向克隆于质粒pD sR ed2-1的多克隆位点内,使牛β-酪蛋白基因调控序列位于人IL-3基因的上游,指导人IL-3基因在乳腺组织中特异性表达,而CM V启动子位于红荧光蛋白基因的上游,指导红荧光蛋白基因在所有的组织中非特异性表达.限制性酶切片段分析及部分DNA序列鉴定结果表明,所构建载体结构正确.展开更多
Background Nowadays it is now a focus topic in schistosomiasis research to find ideal vaccine candidates and new drug targets for developing anti-schistosomiasis vaccine. We cloned a new gene, casein kinase Ⅱ beta s...Background Nowadays it is now a focus topic in schistosomiasis research to find ideal vaccine candidates and new drug targets for developing anti-schistosomiasis vaccine. We cloned a new gene, casein kinase Ⅱ beta subunit, of Schistosoma japonicum (S. japonicum) and express it in Escherichia coli (E.coli).Methods The ESTs obtained in our laboratory were analyzed by homologous searching, and a new gene was recognized. The full-length cDNA of the new gene was obtained by joining the 3’RACE PCR fragment and the EST clone. To express the new gene, the cDNA was cloned into pGEX-4T-1 vector and then transformed into E.coli JM109. The recombinant protein was analyzed by SDS-PAGE and Western-blot. Results A 908 bp cDNA was isolated from S. japonicum and identified to be casein kinase Ⅱ beta subunit gene by sequence analysis. The open reading frame of the gene encodes a protein of 217 amino acids exhibiting 75.8%, 75.8%, 73.9%, 68.2%, 51.6% identity to the amino acids sequence of the corresponding genes of Homo sapiens (H. sapiens), Xenopus laevi (X. laevi), Drosophila melanogaster (D. melanogaster), Caenorhabditis elegan (C. elegan), and Schizosaccharomyces pombe (S. promber) respectively. The predicted molecular weight of the protein was 24.921 kDa. The new cDNA sequence had been submitted to GenBank, and its accession number is AY241391. This cDNA was subcloned into the pGEX-4T-1 vector and expressed in E.coli JM109.The recombinant protein could be recognized by the S. japonicum infected rabbit serum. Conclusion The full-length cDNA sequences encoding S. japonicum casein kinase Ⅱ beta subunit were firstly sequenced, cloned, and expressed in E.coli.展开更多
文摘人白介素-3是一种造血系统和免疫系统调节剂,在治疗造血系统疾病、肿瘤、先天或获得性免疫功能缺陷等方面发挥着重要作用.应用牛乳腺生物反应器生产人白介素-3的研究具有重要的临床应用和经济价值.研究选用具有红色荧光蛋白报告基因(R ed2)和新霉素抗性基因(neor)表达框架的pD sR ed2-1质粒为骨架构建人白介素-3乳腺表达载体.通过PCR方法分别扩增牛β-酪蛋白基因5′端上游调控序列、人IL-3基因以及CM V启动子序列,将它们按先后顺序分别定向克隆于质粒pD sR ed2-1的多克隆位点内,使牛β-酪蛋白基因调控序列位于人IL-3基因的上游,指导人IL-3基因在乳腺组织中特异性表达,而CM V启动子位于红荧光蛋白基因的上游,指导红荧光蛋白基因在所有的组织中非特异性表达.限制性酶切片段分析及部分DNA序列鉴定结果表明,所构建载体结构正确.
基金ThisprojectwassupportedbytheNationalNaturalScienceFoundationofChina (No 3 0 0 70 683 )andDoctorFoundationofNationalMinistryofEducation (No 2 0 0 0 45 )
文摘Background Nowadays it is now a focus topic in schistosomiasis research to find ideal vaccine candidates and new drug targets for developing anti-schistosomiasis vaccine. We cloned a new gene, casein kinase Ⅱ beta subunit, of Schistosoma japonicum (S. japonicum) and express it in Escherichia coli (E.coli).Methods The ESTs obtained in our laboratory were analyzed by homologous searching, and a new gene was recognized. The full-length cDNA of the new gene was obtained by joining the 3’RACE PCR fragment and the EST clone. To express the new gene, the cDNA was cloned into pGEX-4T-1 vector and then transformed into E.coli JM109. The recombinant protein was analyzed by SDS-PAGE and Western-blot. Results A 908 bp cDNA was isolated from S. japonicum and identified to be casein kinase Ⅱ beta subunit gene by sequence analysis. The open reading frame of the gene encodes a protein of 217 amino acids exhibiting 75.8%, 75.8%, 73.9%, 68.2%, 51.6% identity to the amino acids sequence of the corresponding genes of Homo sapiens (H. sapiens), Xenopus laevi (X. laevi), Drosophila melanogaster (D. melanogaster), Caenorhabditis elegan (C. elegan), and Schizosaccharomyces pombe (S. promber) respectively. The predicted molecular weight of the protein was 24.921 kDa. The new cDNA sequence had been submitted to GenBank, and its accession number is AY241391. This cDNA was subcloned into the pGEX-4T-1 vector and expressed in E.coli JM109.The recombinant protein could be recognized by the S. japonicum infected rabbit serum. Conclusion The full-length cDNA sequences encoding S. japonicum casein kinase Ⅱ beta subunit were firstly sequenced, cloned, and expressed in E.coli.
