construction of an expression system for hbv pseudo-viral particles candidate no: candidate no:
TRANSCRIPT
Construction of an expression Construction of an expression system for HBV pseudo-viral system for HBV pseudo-viral
particlesparticles
Candidate No:Candidate No:
Introduction: Hepatitis B Virus
• Liver Specific Hepadnavirus• Vaccine available• > 2 billion people infected worldwide with > 350 million
chronically infected patients at high risk of liver cirrhosis and hepatocellular cancer
• No specific treatment for patients with acute infection• Need for new anti-HBV drugs• Many possible liver specific viral receptors but no direct
evidence
Aims
• Construct heterologous expression system for HBV based on HIV1 minimal three plasmid transfection (TPT) systemi.e. Pseudo-type HIV1 TPT system
• Infect heterologous cell line expressing human liver cDNA to identify putative receptors
• Unambiguously link receptor viral interactions to uptake and infection
HIV1 Minimal Vector
HIV1 based minimal vector
HIV1
Deletion of certain regions & separation of protein coding regions
Genome componentVector Gag-pol component
Vector
Envelope componentVector
CMV CMVR-U5- -Gal
pGM
Gag-polCMV
pGP
EnvCMV
pEnv
Three plasmid transfection (TPT) system
HIV1 Minimal Vector
HIV1 based minimal vector
HIV1
Deletion of certain regions & separation of protein coding regions
Genome componentVector Gag-pol component
Vector
Envelope componentVector
CMV CMVR-U5- CD8 - GFP
pGM
“δHC”Gag-polCMV
pGP
HBV EnvCMV
pEnv
Three plasmid transfection (TPT) system
Pseudo-typing
Strategy• Construct heterologous expression system for HBV based on HIV1 minimal
three plasmid transfection (TPT) system• Pseudo-type HIV1 TPT system:
Replace MA with truncated HBV core “δHC”• Straight forward replacement not possible due to
Lack of suitable sites for primes (PCR mutagenesis approach)Lack of convenient unique restriction enzyme sites (partial digest approach failed)
• HBV assembles via the ER whereas HIV buds from the plasma membrane. Need to redirect protein synthesis to the ER
HIV MA HBV truncated core protein “δHC”
signal target for the plasma membrane
signals transport to the ER
interacts with HBV envelope
RNA binding region deleted (amino acids 150-183)
gagNucleocapsid NC
p7
HIV1 Minimal Vector Pseudo-typed with HBV Envelope
HBV truncated core
HBV envelope protein
Methods & Controls
• All HIV vectors supplied by Oxford Biomedica• Transformations:
Heat shock, XL1blue and SURE cells (E.coli)• Plasmid preparation:
– SS-phenol/chloroform extraction, QIAGEN and SIGMA kits
RE digest at each stage to verify plasmid integritySpectrophotometry:
A260 [DNA]A260/280 - purity
• In-vitro transcription/translation followed by SDS-PAGE and western blotting
pGP 11kb
pBSdGAG
5.5Kb
XhoI
EcoRV
NotI δHC
EcoRV
NotI
*Double digest
EcoRV NotI
Linearised vector
LIGATION
δHC
EcoRV
NotI
pBSm1GP 8.5 kb
XhoI
LIGATION
Difficult…*small scale ligations unsuccessful transformation of SURE cells*large scale approach1:6 vector: insert De-phosphorylated insert rather than vector
Markers Insert vector ligation mix
3kb unligated insert
8.5 kb5.5kb unligated vector
11kb vector-vector ligation
8.5 kb ligation product Extracted and purified for transformation
Difficult to extract, transformations unsuccessful
Alternative approach…
PCR approach on synthetic, humanised plasmids
• psynGP and pHBΔC - Humanised plasmids – same protein coding regions but very different from the viral plasmid sequences
1.2kb
pHBΔC
psynGP
primer 1
primer 2
primer 3
primer 4
500bp
700bp
primer 4
primer 1
PCR 1
98°C 5 min slow cool to anneal
PCR 3
Gag-Pol genesCapsid
δHC
δHC Gag-Pol genes
1.2kb δHC Gag-Pol genes
Gag-Pol genesCapsidpsynGP
Mlu1 EcoRI double digest
Purification
Ligation
Quantitation of 500bp and 700bp PCR fragments for annealing reaction
Separation of products of PCR annealing reaction
MluI EcoRI digest of purified 1.2kb PCR fragment
1kb 1kb
1kb
Nested PCR approach
Obtain pure insert in greater quantity
Primer design
Optimisation of reactions (36 trials)
pHBΔC
psynGP
primer 1primer 1
primer 2
primer 3
Gag-Pol genesCapsid
δHC
primer 5
primer 4 primer 6
Conclusions
• Closer to construction of part of HBV pseudo-typed HIV1 TPT system
• PCR based method adopted in favour of large scale RE digest approach.
• Optimisation of nested PCR achieved.
Acknowledgements
• Dr D Patil
• Dr N Ramamurthy
• Dr N Zitzmann
• All of the Virus Group
• With thanks to Prof. R A Dwek
Thank you for your constant patience, advice and support.