Preliminary Study Of IDH3ɑ In The Aflatoxin B₁-induced Liver Pathologicalprocess

Aflatoxin is a kind of mycotoxin produced by Aspergillus flavus and Aspergillus parasiticus.which is toxiferous to humans and animals.This toxin has a strong carcinogenic effect, aflatoxin B1 is the most damaging.Study found that many proteins are involved in the AFB₁ induced Carcinogenic process,including the TCA cycle isocitrate dehydrogenase 3É‘subunit.The protein level of isocitrate dehydrogenase 3É‘was increased in AFB₁ induced tomor formation.Idh3É‘gene was cloned,and then expressed in E.coli in our study.The protein was used to immune mice.After 2 months,the polyclonal antibody of IDH3É‘was obtained.AFB₁ was extracted from Aspergillus flavus by using methanol/water solution with ultrasonic,and then used to treat Chang Liver cells and inject it into mice.The polyclonal antibody of IDH3É‘was used to detect the IDH3É‘protein. Overexpression vector pCDNA3.1(-)-idh3É‘and RNA interference vector pGPU6/GFP/neo-shRNA were constructed,lipofectamine 2000 was used to induced these vectors to mammal cells.G418 was used to obtain stabilized cell lines,and then verified stabilized cell lines by Q-PCR and Western Blot.This study get the following results that(1)Titer of polyclonal cantibody is up to 16,000.(2)The concentration of extracted AFB₁ detected by ELISA is 100.7μg/mL.(3)IDH3É‘protein up-regulated in AFB₁ treated mice and cells.(4)The outcomes of Q-PCR,Western Blot showed that mRNA and protein levels of IDH3É‘were increased or decreased, respectively in the transfected cells.Undoubtedly, it indicates that IDH3É‘over-expression and interference cell models were successfully constructed .the moedles were benifit for the next signaling pathway research.