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Screenning and Identification of Potential Host Interactors of Hepatitis B Virus Polymerase

Author: ChenMengZuo
Tutor: ZhouTianHong
School: Jinan University
Course: Genetics
Keywords: Yeast two-hybrid Hepatitis B virus polymerase PSMB5 Protein interactions
CLC: R373
Type: Master's thesis
Year: 2011
Downloads: 12
Quote: 0
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Abstract


The hepatitis B virus polymerase (Hepatitis B Virus Polymerase, HBV Pol) is an important protein of the viral life cycle , and to play a key role in the viral genome replication and assembly of core particles . However, less functional studies of Pol , Pol play the activity of specific molecular mechanism remains unclear . Protein interactions important ways , this experiment by the host protein interaction screening Pol , to reveal the molecular mechanisms of Pol functioning further understanding of the mechanism of HBV replication in cells provide the basis of protein function . First , from the blood sample of HBV carriers Balloting proposed RC -DNA of the HBV genome , amplified by PCR after built into the pcDNA3.1 ( ) vector , sequenced important structural domains of the HBV genotypes and Pol mutation analysis results to determine the HBV gene is type B , sub- genotype B2 type , Pol RT domain without mutations . Then the pCDNA3.1-HBV as a template , PCR amplification Pol gene sequences , to construct the recombinant plasmid pGBKT7 - Pol ; bait plasmid pGBKT7-Pol , using the yeast two-hybrid system to screen a human liver cDNA library , selected 290 clones for p- galactosidase activity detection , screening positive clones plasmid library insert fragment PCR and restriction map analysis , remove duplicate library plasmid , 39 non - repeated cloning and sequencing . Sequencing results in Genbank sequence alignment analysis , one part of the fragment encoding the proteasome subunit beta type 5 (PSMB5) protein . From HepG2 cDNA PCR amplification PSMB5 gene sequences , construct the recombinant plasmid pGADT7-PSMB5 yeast Reply hybridization to verify Pol with PSMB5 of interaction ; build of pCDNA3.1-Pol-Flag and the pCDNA3.1-PSMB5-Myc recombinant plasmid , turn the dyeing He la cells co-immunoprecipitation experiments verify the interaction of Pol and PSMB5 , the results showed that the Pol and PSMB5 the existence of a direct and specific interaction . In summary , this topic screened by yeast two-hybrid system the host protein PSMB5 with HBVPol interaction , and by co-immunoprecipitation experiments confirmed the interaction . The results provide clues to further research the Pol function in the viral life cycle .

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