【摘要】 目的 :将鲑鱼降钙素 (salmoncalcitonin,sCT)基因以同向串联方式连接 ,构建串联多拷贝基因的表达质粒pHis-nCT(n≤3) ,并在原核中表达 ,研究表达产物的降钙活性。方法 :采用半化学半酶促法合成sCT基因 ,利用基因的特点及特殊的酶切位点NdeI和SamI在表达载体pTrcHisC中进行sCT基因与载体基因的融合及sCT基因的串联 ,并在TOP10中表达串联多拷贝基因。表达产物形成包含体 ,对包含体变性、复性后 ,以Ni-ChelatingSepharose亲和纯化。用血清钙浓度测定法研究串联表达产物及裂解产物的降钙活性。结果 :重组菌表达的串联融合蛋白经过变性、复性及亲合层析 ,纯度达到 90 %以上。活性试验表明 ,串联融合蛋白及其裂解产物可以抑制破骨细胞 ,降低血清钙浓度 ,且呈剂量效应关系。结论 :原核表达质粒pTrcHisC可以有效表达串联的sCT基因 ,重组的串联蛋白及裂解产物均有降钙活性。更多还原

【Abstract】 Objective:To construct a tandem repeatedgene recombinant plasmid pHis-nCT（n≤3） and express recombinant His₆-3sCT in E.coli and investigate the effect of the recombinant protein on rat.Methold:The monomer sCT gene obtained by the chemical and enzyme methold was fused into the E.coli high efficient expression pTrcHisC vector resulting in pHis-CT from which a series of tandem repeated sCT gene was constructed.After the tandem repeated sCT gene was expressed in Top 10,the recombinant protein His₆-3sCT was purified by denature、renature and affinity chromatography.SDS-PAGE showed the purity is at least 90%.After the tandem protein was cleaved,the His₆-3sCT and the cleaved protein were tested on rat.The result showed both of them could suppress the osteoclast,and the effect was related with the dose.Conclusion:The tandem repeated sCT gene can be expressed in E.coli as inclusion.Both the recombinant protein His₆-3sCT and the cleaved product had the bioactivity.更多还原