Background[1-3]
Human farnesyl diphosphate farnesyl transferase 1 (FDFT1) ELISA kit is a double-antibody one-step sandwich enzyme-linked immunosorbent assay (ELISA).
Into the microwells pre-coated with human farnesyl diphosphate farnesyl transferase 1 (FDFT1) capture antibody, add specimens, standards, and HRP-labeled detection antibodies in sequence, incubate and wash thoroughly. . The substrate TMB is used to develop color. TMB is converted into blue under the catalysis of peroxidase, and converted into the final yellow under the action of acid. The depth of the color is positively correlated with the human farnesyl diphosphate farnesyl transferase 1 (FDFT1) in the sample. Use a microplate reader to measure the absorbance (OD value) at a wavelength of 450 nm and calculate the sample concentration. The human farnesyl diphosphate farnesyltransferase 1 (FDFT1) gene encodes a membrane-associated enzyme located at the branch point in the mevalonate pathway. The encoded protein is the first specific enzyme in cholesterol biosynthesis, catalyzing the dimerization of two molecules of farnesyl diphosphate to form squalene in a two-step reaction. Squalene is a polyunsaturated hydrocarbon opening agent produced during metabolic processes such as cholesterol synthesis in the human body. It contains 6 isoprene double bonds and is a terpenoid compound. Squalene is contained in many foods, including shark liver oil. The content of squalene is relatively high in a few vegetable oils such as olive oil and rice bran oil.
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Construction of shRNA and overexpression vector for FDFT1 gene and study of its effect on cholesterol metabolism of bovine fetal fibroblasts
FDFT1 gene (Farnesyl-Diphosphate Farnesyltransferase 1) is an important gene in the cholesterol metabolism pathway. In order to further study the role of the FDFT1 gene in cholesterol metabolism and lipid metabolism, this study constructed an shRNA interference vector and an overexpression vector of the FDFT1 gene with a green fluorescent label, and transfected them into Bovine Fetal Fibroblasts. BFF), fluorescence quantitative PCR and Western blot methods were used to detect the expression levels of FDFT1 gene mRNA and protein, and the interference efficiency of the shRNA interference vector and the effectiveness of the overexpression vector were verified at the cellular level.
After using the successfully constructed vector to interfere with or overexpress the FDFT1 gene, the expression levels of genes related to the upstream and downstream pathways of the FDFT1 gene, cholesterol biosynthesis, cholesterol metabolism and cholesterol transport were analyzed, revealing in a deeper level the role of the FDFT1 gene in cholesterol. Interactions among synthesis, metabolism, and transport. The role of the FDFT1 gene in lipid metabolism can also be understood by testing the cholesterol and triglyceride levels. The results showed that the FDFT1 gene mRNA expression level in the shRNA group FDFT1-Bos-520 was reduced to 40.7% of the shRNA NC group (p<0.01), and the protein expression level was reduced to 39.61% of the shRNA NC group (p<0.01); while the FDFT1 gene The expression level of FDFT1 gene mRNA in the pBI-CMV3-FDFT1 overexpression group was 137.9 times that of the pBI-CMV3 empty vector group (p<0.01), and the protein expression level increased to 2.34 times that of the empty vector group (p<0.01).
