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The Construction of Recombinant Prion in Vitro and the Analysis of Recombinant Prion Infected Animal Model

Author: ZhangZhiHong
Tutor: MaJiYan
School: East China Normal University
Course: Biomedical
Keywords: Prion in vitro construction animal model
CLC: R373
Type: PhD thesis
Year: 2012
Downloads: 13
Quote: 0
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Abstract


Prion Diseases, also called Transmissible Spongiform Encephalopathies, are a group of fatal neurodegenerative disorders wildly occurred in a variety of mammals. According to the "prion" hypothesis, the agent of these disorders is Prion, which is the abnormal conformational isoform (PrPSc) of normal cellular prion protein (PrPc), PrPSc can induce more PrPc to undergo conformational change, and subsequently causes neurotoxicity and neurodegeneration.During the conformational change of PrP, cofactors other than PrP participate and promote PrP conversion, and lipid has been identified as one of the most important cofactors. Anionic lipid can interact with PrP and facilitate the conformational change, leading PrP to a PK-resistant form. In addition, RNA, with a long chain of complex polyanion structure, is also able to accelerate PrP conformational change.In this thesis, we used E.coli expressed recombinant prion protein, anionic lipid POPG and total RNA isolated from mice liver, together with the in vitro PMCA (Protein Misfolding Cyclic Amplification) technique to generate recombinant prion de novo in a brand new environment that has never been exposed to naturally occurring prions. The newly formed recombinant prion is aggregated, PK-resistant and the PK-resistant core is located at C-terminus, and biochemically indistinguishable from naturally occurring prions. Since the recombinant prion has the classic PK-resistant characteristic, we used rPrP-res to represent this form.After the construction in vitro, recombinant prion was used to infect wild type CD-1mice via i.c.(intracerebral injection) route. Recombinant prion infected mice developed neurological signs around5months post inoculation. Mice at terminal stage of the disease were sacrificed for analyses. Characterization of the mice revealed the classic neuropathology features including spongiosis, abnormal PrP deposition, astrogliosis, microgliosis and the presence of PrPSc in the brain, confirming that the de novo generated recombinant prion is capable to infect wild type CD-1mice and cause bona fide Prion disease.In summary, with lipid and RNA as cofactors, we generated a recombinant prion de novo with bacterially expressed recombinant prion protein by PMCA. Bioassy in wild type mice demonstrated that the recombinant prion are highly infectious and contain relatively high Prion infectivity. This work strongly supports the "prion" hypothesis, and set up an excellent platform to further investigate the mechanism of PrP conformational change and the molecular details of prion infectivity.

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CLC: > Medicine, health > Basic Medical > Medical Microbiology ( pathogenic bacteriology,pathogenic microbiology ) > Human Virology ( pathogenic virus)
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