Cloning and expression of type 1 insulin-like growth factor in caprine mammary gland epithelial cells
Date Issued
2011
Date
2011
Author(s)
Huang, Chia-En
Abstract
Type 1 insulin-like growth factor (IGF-1), is a pluripotent growth factor and has been implicated in all phases of cell cycle dynamics, including promoting cell proliferation, differentiation and inhibiting cell death. IGF-1 applies its actions primarily through the type 1 insulin-like growth factor receptor (IGF-1R), which possesses a heterotetrameric structure with internal tyrosine kinase activity and structural homology to the insulin receptor. The bioactivity of IGF-1 is controlled by a complex system which consists of six high affinity binding proteins (IGFBP-1 to -6) and other IGFBP-interacting molecules. In mouse, IGF-1 expressed in the mammary gland nonepithelial portion in early puberty, and expressed in the epithelial cells of the terminal end buds as well as the stromal compartment during the pubertal ductal growth, and also will express in the alveolar and ductal epithelium during late pregnancy age. The spatially and temporally restricted expression of IGF-1 in the epithelium proposed that it may have the important function in the development of mouse mammary gland, but in one of the economic animals, goat, is not clear yet.
A goat IGF-1 (gIGF-1) cDNA (465 base pairs) was cloned from ear tissue. The similarity of amino acid sequences between species is from 70% to 90%. The gIGF-1 cDNA was subcloned into a mammalian expression vector and derived by a human cytomegalovirus (CMV) promoter. The gIGF-1 act as a transient expression in an immortalized caprine mammary epithelial cell line (CMC). The localization of gIGF-1 in CMC was observed by immunocytochemistry approach. The expression pattern inferred to drive endoplasmic reticulum-golgi (ER-golgi) pathway that may go through the auto-paracrine by secreting gIGF-1 out of the CMC cell. In order to overcome the question of unpredictable amount of transient expression of gIGF-1 in CMC, the pET/E. coli expression system will apply to express the mature, signal peptide fused, and gas7 gene fused recombinant gIGF-1 proteins. With all the the constructions mentioned above, the recombinant gIGF-1 protein will be induced expression by the isopropyl-β-D-1-thiogalactopyranoside (IPTG) induction. The scaled-up recombinant gIGF-1 protein purified from the pET system will be used to investigate the function of gIGF-1 in CMC in the near future.
To sum up, the immuno-staining result indicated that the constructions mentioned above could express in CMC, and according to the pattern which expressed in the cell, it is inferred that the recombinant gIGF-1 in cells transport follows the endoplasmic reticulum-golgi (ER-golgi) pathway. The confirmation whether the recombinant gIGF-1 can secrete out of cells needs further evidence.
Subjects
IGF-1
mammary epithelial cell
protein expression
mammary gland development
Type
thesis
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