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Preliminary Study of IDH3ɑ in the Aflatoxin B1-induced Liver Pathologicalprocess

Author: TangGuangBo
Tutor: WangShiHua
School: Fujian Agriculture and Forestry University
Course: Microbiology
Keywords: aflatoxin B1 isocitric dehydrogenase 3ɑsubunit RNA interference
CLC: R363
Type: Master's thesis
Year: 2011
Downloads: 22
Quote: 0
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Abstract


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 3ɑsubunit.The protein level of isocitrate dehydrogenase 3ɑwas increased in AFB1 induced tomor formation.Idh3ɑgene 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 IDH3ɑwas 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 IDH3ɑwas used to detect the IDH3ɑprotein. Overexpression vector pCDNA3.1（-）-idh3ɑand 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）IDH3ɑprotein up-regulated in AFB1 treated mice and cells.（4）The outcomes of Q-PCR,Western Blot showed that mRNA and protein levels of IDH3ɑwere increased or decreased, respectively in the transfected cells.Undoubtedly, it indicates that IDH3ɑover-expression and interference cell models were successfully constructed .the moedles were benifit for the next signaling pathway research.

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