Molecular Cloning, Expression, Functional Analysis And Polymorphism Of Insulin-like Growth Factor â…  Gene In Mink

Background and Objectives: The mink is a small precious fur-bearing animal, whose main economic value is that its fur is the expensive raw material of making leather. For mink farming, the fur quality is critical for the economic benefit of farmers, and the size of a fur is a important index in evaluation of a fur. Therefore, our unremitting target is to breed this mink that has good fur quality, big body type, strong fecundity and strong vitality. Animal growth axis, regulates animal growth, is a neuroendocrine system which is made up of a series of hormones and receptors of hypothalamus-pituitary-target organ. GH-IGFⅠis a key component of growth axis, plays an important part in growth regulation. Bioactivation of GH is implemented by IGF-1 that is a multi-function peptide has a direct impact for animal growth, development, reproduction, nutrient metabolism and histiocytic multiplication, differentiation and apoptosis .The main contents of this study are that: firstly, implement gene clone, sequence analysis, prokaryotic expression and purification of IGF-1 in mink and observe its function in mink osteoblast; secondly, investigate bioengineering methods to produce IGF-1 of mink and employ it to improve growth performance of mink; finally, analyse the gene polymorphism of IGF-1 exonls in six mink populations.Materials and Methods: Based on the lesser panda,golden monkey,giant panda,human etc,primer pairs for amplifying complete sequence of mink IGF-ⅠcDNA .The mature peptide was analyzed.It was cloned into pGEX-6p-1 plasmid through BamHI and EcoRI restriction site which was designated pGEX-6p-1-IGF-Ⅰ.The recombinant prokaryotic expression plasmid was confirmed by PCR amplification, restrictive enzymatic digestion and sequence analysis. Then the recombinant prokaryotic expression plasmid was transformed into E.coli. BL21(E3) cell. The stable transfectant, designated BL21-pGEX-6p-1-IGF-Ⅰ, was selected by the addition of Ampilin to the growth medium, followed by a series of confirmation. Bl21-pGEX-6p-1- IGF-Ⅰcells were cultured in LB medium till to OD600 value 0.6 to 0.8, then induced with IPTG to express recombinant IGF-Ⅰ, expression was detected by SDS-PAGE.The recombinant IGF-Ⅰexisted in a resoluble GST fusion protein form, its purification was employed by affinity chromatography column. The biological activity of recombinant IGF-Ⅰwas examined by mink osteoblastic cells growing tests in vitro.Otherwise, PCR-SSCP,DNA sequencing technique were applied in this study to analyze the distribution of IGF-Ⅰgene in 6 different mink populations,it is analyzed by population genetics and statistic genetics methods.Results and Conclusion:1. This study is aimed at Cloning,sequence analysis and expression,purification of IGF-Ⅰwhich playsed vital roles in growth,reproduction and milk secreting regulation.The significance and possibility of a further study towards the production of their recombinant IGF-Ⅰby recombinant DNA technique,and application in assisted growth and development.The main results were as the following:(1)By RT-PCR,liver IGF-Ⅰfrom mink,endoding 153 amino acids were isolated.,it comprised 48amino acids signal peptide ,70 amino acids mature peptide and 35 extend residues.(2)The mink IGF-Ⅰgene has a high homology with other mammal IGF-Ⅰgenes,and the homology of the sequence coding mature peptide is from 90%(cow)to 97%(golden monkey),ORF from 91%(cow)to 96%(golden monkey and lesser panda.2.The recombinant prokaryotic expressing plasmid pgex-6p-1-IGF-Ⅰwas constructed successfully.Stable transfectants BL21-pGEX-6p-1-IGF-Ⅰwere obtained and the IGF-Ⅰwas overexpressed successfully. Stable transfectants BL21-pGEX-6p-1-IGF-Ⅰwere obtained and the IGF-Ⅰwas overexpressed successfully.3. Successly cultured the osteoblast cells with enzyme digestion method and tissue adherent method in vitro.The results showed the cells obtained from mink costal bone were identified to be osteoblasts by inert microscope,ALP stain, immunochemistry stain of osteoblasts and alizarin red stain of Mineralization nodus. It indicated the cells obtained from mink costal bone showing typical osteoblasts'characteristics.4.The effect of recombinant mink IGF-Ⅰprotein on the osteoblastic cells about proliferation was studied.The results showed certain concentrations of IGF-Ⅰcould stimulate the proliferation of theosteoblasts,and the former acted in dose-dependent manner in field of 0.01～0.8mg/ml.5. PCR-SSCP,DNA sequencing technique were applied in this study to analyze the distribution of IGF-Ⅰgene in 6 different mink populations,it is analyzed by population genetics and statistic genetics methods.The results showed that one mutation site in exon1 of the mink IGF-Ⅰgene was detected,it was C→G at 185bp of the exon1.