CDs Region Cloning And Primary Function Study Of Epidermal Growth Factor Receptor Gene In Dairy Goat

Epidermal Growth Factor Receptor(EGFR)which belongs to the Receptor tyrosine kinase has an important role in regulating Growth and development process in mammals.EGFR possibly regulates the synthesis of fatty acids and cholesterol though activating mTOR signaling pathways and in turn fatty acid together with cholesterol synthesis related genes also plays an important role in regulating EGFR protein function.The research of EGFR is mainly concentrated in the field of cancer and tumor therapy instead of fatty acid metabolism.There are few reports about the interactions between EGFR and fatty acid metabolism in ruminant animals such as dairy goat and bovine.Goat EGFR gene CDs area sequence was abtained through PCR amplification in this study.Rabbit anti goat EGFR polyclonal antibody was prepared after prokaryotic expression of truncated form of EGFR protein.Pack recombinant adenovirus Ad-EGFR and synthesise specific siRNA against EGFR gene before treating goat mammary epithelial cells,we measured genes related to lipid and lactoferrin synthesis after EGFR gene overexpression and RNA interference through RT-qPCR assay.Our results provide experimental basis for regulation mechanism of goat EGFR gene on milk fat and milk protein metabolism.The main results are as follows:(1)Cloning of EGFR gene in goat mammary gland showed that the full-length of EGFR gene CDs region is 3 627 bp and encodes 1 208 amino acids.After predicting the signal peptide and hydrophobicity property,we found that the signal peptide cleavage site is between the amino acid from 24 to 25 and the region of N-terminal shows higher hydrophobicity meanwhile the C-terminal shows higher hydrophily.The expression of EGFR gene is higher in early-lactation and late-lactation and the lowest expression level is in dry-period.(2)The truncated form of Goat EGFR(ECD)protein was abtained through PCR amplification and prokaryotic expression vector pET-32a(+)-ECD was constructed successfully.The pET-32a(+)-ECD vector was transformed into E.coli BL21 and the recombinant protein was obtained with the induction condition of 0.8 mmol/L IPTG and37 ? culture for 6 h.We got High-purity protein after purification in the end.Indirect ELISAanalysis showed that the titer of the antibody reached 1:16 000.Western blot analysis showed that the specificity of antibody is capable of carrying out experiments.(3)Recombinant adenovirus Ad-EGFR was obtained through vector construction and adenovirus package.After infecting goat mammary epithelial cells with Ad-EGFR for 36 h RT-qPCR assay showed that FASN?ACC?LXR??LXR??SREBP1?SCD1?ACSS1?DGAT1?DGAT2 and SP1 gene expressions increased(P<0.05)and FABP3 gene expression decreased(P<0.05).Western blot assay showed that significant expression in EGFR protein level could be detected in goat mammary epithelial cells after 36 h Ad-EGFR infection and EGFR protein can be constitutively activated.(4)The specific siRNA against EGFR gene was synthesized successfully and then transfected into goat mammary epithelial cells.RT-qPCR assay was carried out after 24 h transfection.The result showed that ACSL1?DGAT2?PRLR and LF gene expression increased(P<0.05),meanwhile SCD1 ? FASN ? ACC ? LXR? ? LXR? and SP1 gene expression decreased(P<0.05).In summary,Goat EGFR gene CDs area sequence was abtained successfully and rabbit anti goat EGFR polyclonal antibody was prepared after purification of truncated form of Goat EGFR(ECD)protein.Genes associated with milk fat and milk protein synthesis in goat mammary epithelial cells changed after EGFR gene overexpression and interference.