The mRNA quantitative analysis, prokaryotic expression and structure prediction of hepatocarcinogenesis related gene idh3 alpha under AFB(1) stress

By differential proteomics analysis, it was found that the expression level of IDH3 alpha (Isocitrate dehydrogenase 3 alpha) in mouse liver was significantly increased under the stress of Aflatoxin B-1. To validate the result of differential proteomics analysis, fluorescence quantitative PCR was used to detect the changing trend of idh3 alpha mRNA volume induced by Aflatoxin B-1 in mouse liver. The result showed that the expression volume of idh3 alpha mRNA showed an increasing trend with the increase of Aflatoxin B-1 concentration, which corresponded with the result of differential proteomics analysis. The protokaryon expression vector for idh3 alpha was constructed in the study with pET28a as a recipient plasmid. The expression vector (pET28a-idh3 alpha) was used to transform BL21, after which the positive expression strain (Escherichia coli BL21/pET28a-idh3 alpha) was induced to express with 100 mmol/L IPTG under 28 degrees C for 4 h, and the prokaryotic expression product of IDH3 alpha was successfully detected by SDS-PAGE electrophoresis. The molecule structure of IDH3 alpha was predicted by bioinformatics analysis, and the results showed that the total number of negatively and positively charged residues were 42 and 39, respectively. Five hydrohpobic domains were predicted in the protein, and its average hydrophobicity was -0.069. There was 43% alpha-helix and 20% beta-pleated sheet in the molecule, and the tertiary structure of IDH3 alpha was constituted by 12 alpha-helices and 12 beta-pleated sheets. Based on the results of bioinformatics analysis, the fragments of 1-17 and 112-123 residues of IDH3 alpha were selected as candidates for further effective polyclonal antibody preparation. The results of this study paved way for further exploration of the role of idh3 alpha in the process of liver carceration induced by Aflatoxin B-1.