Construction And Expression Of Chimeric Analogy Of Human And Salmon Calcitonin

Calcitonic ( CT ) is a polypeptide with 32 amino acid residues secreted by parafollicular cells of the thyroid in mammals and ultimobranchial body in fish. It plays an important role in controlling calcium and phosphorus turnover. In various species, human calcitonin ( hCT ) appears inferior activity compared with those of bird and fish natural preparation, and salmon CT(sCT) exhibits the highest biological activity. Synthetic sCT has been used clinical for the treatment of increased bone resorption in metabolic such as Paget's disease, osteoporosis imperfecta, postmenopausal osteoporosis and bone metastases for mang years.A chimeric gene of 111bp was constructed through chemical synthesis and protease approach, which codons for analogy of 34 amino acids, including 15 amino acids of hCT and 16 amino acids, named hsCT. The antigenicity of chimeric calcitonin is lower than sCT, while the bioactivity is higher than hCT predicted by protein analysis software.The hsCT was expressed in pGEX-4T-X by recombinant form (GST-hsCT) and proved to own the epitope of hCT, but could not to obtain the target protein by enzymolysis with thrombin.Construction of a fusion gene was realized by fusing calcitonin with the C-phycocyanin β subunits of Athrospira through a flexible linker by recombinant DNA techniques. The recombinant DNA was then inserted into fusion expression vector pGEX-4T-3 and unfusion expression vector pLEX, the antigenicity and bioactivity of expressed product was analyzed, the expression of tandemly repeated chimeric calcitonin gene with cpcB at N-teminal upstream was also investigated.In the study to the antigenicity of hsCT, it was found that the expression product of fusion gene has cross-reaction with the antibody from rabbit to sCT. To obtain serials of mutants, we shifted the amino acid residues as to identify the epitope and active site of sCT.We have cloned and expressed the CPC subunits, cpcB and cpcA, and tested the activity of inhibition to the tumour cells, the monoclonal antibody of the CPC was produced at same time, which provided convenience for identification and purification of CPC.After obtaining low antigenicity and higher bioactive recombinant fusion protein BCT, we constructed transgenic Synechocystis using homologous recombinantion, theIysate of transgenic alga was proved to have hypocalcemic activity though the level of fusion protein is about 1%.