摘要
以变铅青链霉菌为宿主研究了人TNFβ(hTNFβ)的异源表达。应用链霉菌S .venezuelaeCBS76 2 70分泌产生的枯草杆菌蛋白酶抑制剂vsi基因的启动子、表达调控序列和分泌信号肽序列 ,分别对hTNFβ进行了直接分泌表达、分泌融合表达和胞内表达。将hTNFβ的cDNA分别直接融合于vsi信号肽序列下游 2个氨基酸处、vsi全长基因之后以及vsi起始密码子ATG的下游 ,获得的表达盒分别克隆至链霉菌高拷贝质粒pIJ486 ,转化StreptomyceslividansTK2 4,获得了重组菌株S .lividans(pIJ486 hTNFβ)、S .lividans(pIJ486 vsi hTNFβ)和S .lividans(pIVPA hTNFβ)。分别对不同的重组菌株进行摇瓶培养 ,对其培养的上清液和细胞裂解液进行SDS PAGE和Western杂交 ,结果表明 :hTNFβ在重组菌株中均获得了表达 ,且直接分泌产物和胞内表达产物均具有生物学活性。hTNFβ直接分泌表达产物的分子量约为 16kDa,NB培养基中培养 48h时表达水平约为 0 7mg L。胞内表达产物分子量与对照重组hTNFβ一致(18 7kDa) ,但随培养时间的延长逐步降解为 16kDa ,NB培养基中培养 48h时的表达水平 (2 5 1mg L)远高于其直接分泌表达水平。
The production of human tumor necrosis factor β(hTNFβ) in Streptomyces lividans was studied.The expression of hTNFβ uses the transcription,translation and secretion signals of subtilisin inhibitor VSI which is naturally produced by Streptomyces venezuelae CBS762.70.In direct secretory expression cassette,hTNFβ cDNA was fused 2 amino acids after the signal peptidase cleavage site.In fusion expression cassette,hTNFβ cDNA was fused after the total vsi gene.In intracellular expression cassette,hTNFβ cDNA was fused after initiation codon ATG.The expression cassettes were subcloned into Streptomyces multi-copy plasmid pIJ486 respectively and transformed into S.lividans TK24.The recombinant strains were designated as S.lividans (pIJ486-hTNFβ),S.lividans (pIJ486-vsi-hTNFβ) and S.lividans (pIVPA-hTNFβ).The analysis of expressed proteins by Western blotting and biological activity measurements revealed hTNFβ was expressed by the recombinant strains with bioactivity.The molecular weight of directly secreted product is around 16 kDa,and the expression level at 48 hours in NB medium was 0.7 mg/L.Intact product could be obtained when hTNFβ was expressed intracellularly,although it may be degraded to lower molecular weight product when cultivation was prolonged.The intracellular expression level at 48 hours in NB medium was 25.1 mg/L.
基金
国家自然科学基金 ( 30 0 70 0 0 9)
中国与比利时国际合作项目 (BIL97 4 2 )资助~~
