2. AIM: IDENTIFICATION OF JEV ATTACHMENT AND RECEPTOR SYSTEM
JEV (Vellore strain P20778)
Enveloped Virus
Family : Flaviviridae
Genus : Flavivirus
Genome : 11 Kb positive sense single stranded RNA
Structural : E, C and M
E : Envelope protein is the protective antigen which folds into 3 Domains ED1, ED2 and ED3
ED3 : Receptor binding domain, which mediates attachment of virus to host cells
Non Structural : NS1, NS2a, NS2b, NS3, NS4a, NS4b, NS5
Mode of Transmission
Maintenance Host
Dead end Host
Amplifying Host
3400 a.a
Genome Organization
2
3. 14kDa
WB: Anti His
4 hours induction with 1.0 mM IPTG at 30C
EXPRESSION CHECK FOR ED3
17kDa
10kDa
M 4.55.9S Beads
pH
S:supernatant post elution
pH5.9: ED3 not eluted
pH4.9: ED3 eluted show 14kDa protein
Bead: Protein is completely eluted
Dialysis in PBSPurification of ED3 under Denaturing conditions
)
)
SDS PAGE
M MUI I UI I
17kDa
10kDa
ED3 in PBS
M
Expression of Recombinant ED3
ED3 - pet28a construct
Transformed in BL21 DE3
3
4. Generation of Antibody against ED3
Purified rED3 protein
Sent to Chromus Biotech for Ab Generation
Antibody raised in Rabbit
Polyclonal Sera
Western Blot with anti- ED3 Ab
29kDa
1 2 3 4
22kDa
1 – Purified Protein
2 – Uninfected N2a cells
3 – JEV infected N2a cells
4 - Marker
4
5. Studies on putative ED3 binding receptor molecules on PS
and N2a cells
(CELL SURFACE BIOTINYLATION)
Two cell lines were taken into consideration
PS (Porcine
kidney cells)
N2a (neuroblastoma
cells)
Biochemical Studies to find interacting partners of
JEV-ED3
Cell Surface Biotinylation and purification of biotinylated
proteins by Neutravidin beads
Elution of Biotinylated Cell Membrane Proteins via DTT and
dialysis into appropriate buffer
Preclear on Ni-NTA beads
Binding of precleared biotinylated proteins to JEV ED3
immobilized on NiNta beads
Elution, SDS-Page and Western Blotting with Strep-HRP
Western Blot:
1°Ab : S – HRP
Interaction of ED3 beads with N2a cells revealed
many high and low molecular weight fragments
especially in the range of 20-25 kDa.
Determination of putative ED3 binding receptor molecules
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6. AIM : - Expression and Purification of JEV NS5(Taiwan Strain) protein
1
2
Purification under Native conditions
Yield of NS5 protein : 15mg/lt
Induced with 1mM IPTG, for 6.5 hrs at 18⁰C M UI I Sol frac Insol
Binding Elution
NS5 - pet28c construct
Transformed in BL21 DE3
3
Expression check
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7. Cloned into pPICZαA ( 3.6Kb)
ES (secretory component of envelope) – 918 bp
ED3 (domain 3 of envelope) – 300bp
Electroporation in Pichia strains : X33, GS115, KM71H, SMD1168
Around 100 colonies for each set of transformants were checked for expression
Both ES and ED3 was not expressed in any of the strains used.
Thus, we modified the strategy as well as codon optimized the construct
for expression in Pichia system.
AIM : - To express JEV ED3 and ES in Pichia pastoris expression system
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8. Virus-like particles from Pichia pastoris
Flexible Platforms for Vaccine Development
Envelope (500 aa)ss (15 aa) prM (167 aa) 6X-H
The nucleotide sequence corresponding to JEV VLP (2060 bp, spanning nt 432–2477; coding for a 15-aa
signal peptide from the C-terminus of the C protein, together with the pre-Membrane (prM) and Envelope
(Env), codon optimized for expression in Pichia pastoris (GenScript USA Inc.)
Clone provided in pUC57
Sub-clone in pPICZ alpha vector
Transformed in P. pastoris X33 and GS115 strains
Colonies obtained screened for Zeocin resistance
Probable clones checked for expression in Buffered Methanol-Complex Medium (BMMY)
Culture media subjected to TCA precipitation and culture pellets processed via Bead breaking buffer lysis
method. Both Culture supernatant and pellet analyzed for expression; 24, 48, 72 and 96 hrs post-
induction.
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9. Expression analysis in P. pastoris X33 strain
Of the clones screened (50), only Clone 13 showed expression
The expression was visualized only in culture pellet (not in media supernatant),
48 hrs onwards, post-induction.
M Media Supernatant Culture Pellet
13/X33 V/X33 13/X33 V/X33
24 48 72 96 24 48 72 96
M Media Supernatant Culture Pellet
13/X33 V/X33 13/X33 V/X33
24 48 72 96 24 48 72 96
Expression analysis in P. pastoris GS115 strain
Of the clones screened (50), only Clone 9 showed expression
The expression was visualized both in culture pellet and in
media supernatant.
A: Culture pellet
B: Media supernatant
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10. M Media Supernatant Culture Pellet
9/GS 1/GS V/GS 9/GS 1/GS V/GS
48 hours 48 hours
Blotting with A2: the expression of Clone 9/GS115 was
confirmed in both media supernatant and culture pellet.
Maximum expression observed at 48 hrs onwards, post-
induction.
Purification of VLP from media supernatant of Clone 9/GS115
Media supernatant
Concentrated using Amicon (30 KDa)
Concentrate subjected to Ni-NTA affinity purification
1 – M
2 – W1
3 – F.T
1 2 3
95KDa
42KDa
The authenticity of protein preparation was confirmed by MS
(all three fractions correspond to envelope of JEV)
1
2
3
TEM analysis of the purified recombinant 9/GS115 protein preparation
The protein preparation contains
discrete VLPs.
Future Prospects : -
Large scale purification
Assessing the VLP preparation for its
immunogenecity in animal model of JEV.
VLP Yield: 41mg/litre
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11. General Lab Duties:-
Ordering and Maintenance of stocks of Chemicals, Glasswares, Plasticwares, Kits,
Restriction Enzymes etc.
Supervising Laboratory Technicians for various day to day activities (preparation of media, buffers, etc)
Assisted in summer training of B - Tech student.
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