一种新型人源化鲑鱼降钙素突变体的构建与表达

目的 降低鲑鱼降钙素（sCT）对人体的免疫原性。方法 设计并合成人源化鲑鱼降钙素（11－17）hsCT基因,用基因重组方法构建表达载体,转入大肠杆菌G1724中,经色氨酸诱导表达;用渗透压法处理菌体纯化融合蛋白;以大鼠血清钙浓度降低方法测定hsCT生物活性;用定量Western blot法测定hsCT免疫原性。结果 重组pTrxFus-hsCT质粒在大肠杆菌中获得高效可溶性融合蛋白表达,表达水平达46％以上,并得到纯度约90％的融合蛋白,rhsCT保存了鲑鱼降钙素前体活性的一半以上,免疫原性降低了55％。结论 获得一种新型人源化隆钙素（11－17）hsCT,其保存较高sCT前体的活性,免疫原性明显降低。

Construction and expression of a new humanized salmon calcitonin variant

OBJECTIVE: To study the reduction of immunogenicity of salmon calcitonin (sCT). METHODS: A new (11-17) humanized salmon calcitonin variant gene (11-17) hsCT was designed and synthesized to construct recombinant expression vector and express in E.coli G1724 with tryptophan. The fusion protein of humanized salmon calcitonin and thioredoxin (Trx-hsCT) obtained from the bacterial by means of osmotic pressure; The bioactivity of Trx-hsCT was estimated by serum calcium reduction method in rat; The quantitative Western blot analysis was used for detecting the immunogenicity of hsCT. RESULTS: Recombinant expressing vector pTrxFus-hsCT was expressed in high level and soluble in E.coli G1724, reaching 46% of total bacterial protein and obtaining the fusion protein of 90% purity; Special bioactivity of hsCT was preserved that of half pre-sCT and its immunogenicity reduced over 50% that of pre-sCT. CONCLUSION: Obtain a new humanized salmon calcitonin variant(11-17) hsCT which maintains a rather high bioactivity over pre-sCT and decreases distinctly its immunogenicity.