摘要
利用聚合酶链式反应(PCR)技术分别扩增鲑鱼降钙素的编码序列(sCT-Gly)和抗生链霉菌melCl的信号肽编码序列及其上游的调控序列,将鲑鱼降钙素编码序列融合在melCl信号肽编码序列后,定向克隆到链霉菌质粒载体pIJ680中的新霉素磷酸转移酶基因的启动子(Paph)下游,得到的表达质粒pMS680转化变铅青链霉菌(Streptomyces lividans TK54)进行分泌表达。酶联免疫测定法(EIA)显示重组菌株在YEME培养基中的分泌表达水平于96h达到最高,表达量大于100μ/L培养液。实验表明表达产物能降低大鼠血清钙的浓度。高压液相色谱(HPLC)结果表明,表达产物中主峰的保留时间与鲑鱼降钙素标准品的保留时间基本一致。以上结果提示,鲑鱼降钙素在链霉菌中获得了成功的分泌表达。
A gene coding for salmon calcitonin precursor (sCT-Gly) was amplified fromsalmon genomic DNA by Polymerase. Chain Reaction (PCR) and fused to theexpression and secretion signals of melC1 amplified by PCR. The fusion gene wascloned into the Streptomyces vector pIJ680 and expressed under the control ofaminoglycoside phosphotransferase gene (aph) promoter. Streptornyces lividans TK54transformed with the expression plasmid (pMS680) secreted biologically active sCT-Glyinto the culture medium which was confirmed by Enzyme Immunoassay (EIA) andbioassay. Production of sCT-Gly by the recombinant strain in YEME medium reacheda maximum of 100μg/L culture at about 96 h. The recombinant sCT-Gly had almostthe same HPLC retention time as the standard sCT obtained from Sigma.
基金
国家九五攻关项目(96-C02-01-08)~~
关键词
降钙素
分泌表达
链霉菌
Calcitonin
Secretory expression
Streptomyces
