ISSN 2736-1756
Advanced Journal of Microbiology Research ISSN 2241-9837 Vol. 13 (3), pp. 001-008, March, 2019. © International Scholars Journals
Full Length Research Paper
The mRNA quantitative analysis, prokaryotic expression and structure prediction of hepatocarcinogenesis related gene idh3 under AFB1 stress
Zhuang Zhenhong, Zhang Feng, Li Yanyun, Yuan Jun, Yang Yanling, Lin Ling and Wang Shihua*
Key Laboratory of Biopesticide and Chemical Biology, Ministry of Education, and College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002, China.
Accepted 19 February, 2019
Abstract
By differential proteomics analysis, it was found that the expression level of IDH3 (Isocitrate dehydrogenase 3 ) in mouse liver was significantly increased under the stress of Aflatoxin B1. To validate the result of differential proteomics analysis, fluorescence quantitative PCR was used to detect the changing trend of idh3 mRNA volume induced by Aflatoxin B1 in mouse liver. The result showed that the expression volume of idh3 mRNA showed an increasing trend with the increase of Aflatoxin B1 concentration, which corresponded with the result of differential proteomics analysis. The protokaryon expression vector for idh3 was constructed in the study with pET28a as a recipient plasmid. The expression vector (pET28a-idh3 ) was used to transform BL21, after which the positive expression strain (Escherichia coli BL21/pET28a-idh3 ) was induced to express with 100 mmol/L IPTG under 28°C for 4 h, and the prokaryotic expression product of IDH3 was successfully detected by SDS-PAGE electrophoresis. The molecule structure of IDH3 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% - helix and 20% -pleated sheet in the molecule, and the tertiary structure of IDH3 was constituted by 12 - helices and 12 -pleated sheets. Based on the results of bioinformatics analysis, the fragments of 1- 17 and 112-123 residues of IDH3 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 in the process of liver carceration induced by Aflatoxin B1.
Key words: IDH3 , aflatoxin B1, liver, canceration, bioinformatics analysis.