Abstract
Aim. To construct a recombinant eukaryotic expression plasmid containing human calcitonin (hCT) gene and express the gene in murine fibroblast NIH3T3 cells. Materials and Methods. A murine Igκ-chain leader sequence and hCT gene were synthesized and cloned into pCDNA3.0 to form the pCDNA3.0-Igκ-hCT eukaryotic expression vector, which was transfected into NIH3T3 cells. The mRNA and protein expressions and secretion of hCT were detected. Primarily cultured osteoclasts were incubated with the supernatant of pCDNA3.0-Igk-hCT-transfected NIH3T3 cells, and their numbers were counted and morphology observed. Results. The expression and secretion of hCT were successfully detected in pCDNA3.0-Igk-hCT-transfected NIH3T3 cells. The number of osteoclasts was decreased and the cells became crumpled when they were incubated with the supernatant of pCDNA3.0-Igk-hCT-transfected NIH3T3 cells. Conclusion. A recombinant eukaryotic expression vector containing hCT gene was successfully constructed and expressed in NIH3T3 cells. The secreted recombinant hCT inhibited the growth and morphology of osteoclasts.
Highlights
Calcitonin is a 32-residue peptide hormone produced by specialized C-parafollicular cells of the thyroid glands in mammals or by cells of the ultimobranchial glands in reptiles and fish
The recombinant plasmid was transferred into cells of murine fibroblast cell line, NIH3T3, and the expression and secretion of recombinant human calcitonin (hCT) in NIH3T3 cells were observed in this study
To demonstrate the mRNA expression of Igκ-hCT mediated by pCDNA3.0-Igk-hCT vector in NIH3T3 cells, totally cellular RNA was extracted from the NIH3T3 cells by TRIzol method
Summary
Calcitonin is a 32-residue peptide hormone produced by specialized C-parafollicular cells of the thyroid glands in mammals or by cells of the ultimobranchial glands in reptiles and fish. Calcitonins that are used in clinical practice are mainly extracted from the gills of salmon and pig thyroid glands [10]. These heterologous products are short of resources and expensive. There have been some studies on the synthesis of calcitonin [14, 15], there is little information in literature on its application in gene therapy. For this purpose, we constructed a recombinant eukaryotic expression plasmid, pCDNA3.0-Igκ-hCT, which contains human calcitonin gene and murine Igκ-chain leader sequence. The recombinant plasmid was transferred into cells of murine fibroblast cell line, NIH3T3, and the expression and secretion of recombinant hCT in NIH3T3 cells were observed in this study
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