Construction of Eukaryotic Expression Vector of Chicken FETUB Gene and Its Expression in LMH Cells
Fetuin B(FETUB), a novel adipokine/hepatokine, is a cysteine protease inhibitor produced by the liver and released into the blood. Fetuin B plays an important role in glycolipid metabolism. The purpose of this study was to construct an eukaryotic expression vector of FETUB gene in chickens, and to study the effects of over-expression of FETUB in LMH on the expression of other lipid-related genes. Fluorescence quantitative PCR was used to detect the expression levels of FETUB gene in various tissues of chicken. The total RNA of liver tissue was used as the template to amplify the coding region of FETUB gene in chicken by RT-PCR, and the eukaryotic expression recombinant vector was constructed. Total protein and total RNA were extracted from LMH cells after transfection. The recombinant protein was detected by Western Blot, and the expression of lipid metabolism-related genes was detected by fluorescence quantitative PCR. The results show that FETUB gene was expressed in 11 kinds of chicken tissues, with high expression in liver, low expression in muscle stomach, spleen and fat, and no expression in glandular stomach. Chicken FETUB cDNA was obtained by RT-PCR, the eukaryotic expression vector pCMV-3Tag-9-ggaFETUB was constructed, and FETUB-MYC was successfully fusion expressed in LMH. Over-expression of FETUB gene significantly down-regulated the expressions of LPL and ITIH2 genes in LMH cells, but had no significant effect on the expressions of APOA4, DGAT2, FASN and ACACA genes. The indication that FETUB gene affected lipid metabolism by inhibiting LPL and ITIH2 gene expression after LMH over-expression lays a foundation for further analysis of the function of FETUB gene in chickens.
FETUB geneeukaryotic expressionglucose and lipid metabolismLPL geneITIH2 gene
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