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[Construction and expression of a new humanized salmon calcitonin variant]
1Department of Biochemistry and Molecular Biology, Institute of Basic Medical Sciences, CAMS and PUMC, Beijing 100005, China.
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.