Download - Evaluation I.Key
Cloning, Expression, Purification and Enzymological Characterization of
NS2B/NS3 Protease / RNA Helicase protein of Japanese Encephalitis Virus.
Chakard Chalayut Advisor: Asst. Prof. Gerd Katzenmeier, Ph.D.
Laboratory of Molecular Virology Institute of Molecular Biology & Genetics
Japanese Encephalitis Virus
-Flaviviridae family-Mosquito-borne neurotropic flavivirus
•causes severe central nerve system diseases
Japanese Encephalitis Virus
Culex tritaeniorhynchus.
Source: fehd.gov.hkSource : vietnammedicalpractice.com
Japanese Encephalitis Virus
JEV causes severe central nerve system d i s e a s e s s u c h a s po l iomye l i t i s - l i ke acute flaccid paralysis, aseptic meningitis and encephalitis
Source:wonder.cdc.gov
Source: cdc.gov
30% fatality rate50,000 Cases10,000 Cases
Prevention and treatment of JEV disease
Drug No drug exist
Vaccine development
Mosquitoes control Elimination of mosquitoes breeding places
Available vaccine
Molecular biology of Japanese Encephalitis Virus
Source : molecular-virology.uni-hd.de
The NS2BHypothetical model NS2B-NS3 complex
hydrophobicity plot
51 DMWLERAADISWEMDAAITGSSRRLDVKLDDDGDFHLIDDPGVP 95
Brinkworth et al, 1999
• 130 aa• activating domain central hydrophilic region (Falgout et al, 1993)• 3 membrane spanning parts
The NS3
•Chymotrypsin-like fold2-β barrel domains •Inactive alone•Enzyme’s pocket is small
NTPase
Protease
RNA Helicase
Theoretical model from PDB 2I84
The NS3 protease
conformational change alteration of the enzyme pocket additional substrate binding site
•NS3 serine protease domain 20 kDa•catalytic residues His51, Asp75, Ser135
Complexation with NS2B cofactor
Background• NS2B(H) JE - NS3p Den did not cleaved Den
polyprotein but NS2B(H) Den - NS3p JE cleaved JEV polyprotein.
• Jan. L R et al, 1995
• The C-terminal portion of Den NS2B is required for interaction with Den NS3 to activate protease.
Background• Ser46 to Ile60 were essential region required for NS3
protease activity.
• Ala substition of Trp50, Glu55, and Arg56 in NS2B shown significantly reduced NS3 protease activity.
• Lin. C W et al,2007
Objective
• to perform cloning of the NS2B-NS3 portion of the JEV polyprotein, express in E.coli and biochemically purify to determinants of clevage activity and cofactor requirement will be analyzed and compared to dengue virus.
• The second objective is to study differences in substrate specificity and inhibitors by using peptide subst ra tes incorporated wi th fluorogenic or chromogenic reported groups.
Method & Result pLS with NS2B-NS3 JEV
NS2B(H) NS3p
SOE-PCR
NS2B(H)-NS3p
NS2B(H) NS3p NS3NS2B
NS3pNS2B(H)
1500
1000900800700
600500
400
300
200
-C
ontrol
NS2B
(H)
NS2B
(H)
1500
1000900800700600
500
400
300
200
-C
ontrol
NS3 protease
NS3 protease
NS3 protease
Figure 2 : The PCR product NS3protease JEV amplified from NS2B-NS3 JEV (Lane 3 to 5 ). The size of NS3 protease was 594 bp.
Figure 1 : The PCR product NS2B(H) JEV amplified from NS2B-NS3 JEV (Lane 3 and 4). The size of NS2B(H) was 187 bp.
1500
1000900800700600
500400
300
Figure 3 : The SOE-PCR product NS2B(H)-NS3protease JEV (Lane 2 to 5 ). The size of NS2B(H)-NS3 protease was 765 bp.
NS2B
(H)-N
S3p
-C
ontrol
1500
1000900800700600
500
400
300
200
100
Figure 4 : The NS2B(H)-NS3protease JEV (Lane 3 ) after digested with BamHI and KpnI. The size of NS2B(H)-NS3 protease was 765 bp.
-C
ontrol
NS2B
(H)-N
S3p
Method & Result pTrcHis Den with NS2B-NS3 Den
NS2B(H) NS3p
SOE-PCR
NS2B(H)-NS3p Den
NS2B
(H)
NS2B
(H)
-C
ontrol
1500
1000900800700600500
400
300
200
100
Figure5 : The PCR product NS2B(H) Den amplified from pTrc NS2B-NS3 Den (Lane 2 and 3). The size of NS2B(H) was 187 bp.
-C
ontrol
NS3 protease
NS3 protease
1500
1000900800700600500
400
300
200
100
Figure 6 : The PCR product NS3protease Den amplified from pTrc NS2B-NS3 Den (Lane 3 and 4 ). The size of NS3 protease was 594 bp.
NS2B
(H)-N
S3p
NS2B
(H)-N
S3p
NS2B
(H)-N
S3p
NS2B
(H)-N
S3p
NS2B
(H)-N
S3p
-C
ontrol
1500
1000900800700600500
400
300
200
Figure 7 : The SOE-PCR product NS2B(H)-NS3protease Den (Lane 2 and 6). The size of NS2B(H)-NS3 protease was 765 bp.
-C
ontrol
NS2B
(H)-N
S3p
NS2B
(H)-N
S3p
1500
1000900800700600500
400
300
200
Figure 8 :The NS2B(H)-NS3protease Den (Lane 3 and 4 ) after digested with BamHI and KpnI. The size of NS2B(H)-NS3 protease was 765 bp.
Method & ResultpTrcHis A
Digest with BamHI Digest with KpnI
Digest with BamHIDigest with KpnI
Gel Electrophoresis & Clean with Gel Extraction Kits
23.13 kb9.42 kb6.56 kb4.56 kb
2.32 kb2.03 kb
Figure 9 : The pTrcHis A in lane 2 without digestion
23.13 kb9.42 kb6.56 kb4.56 kb
2.32 kb2.03 kb
pTrcHis A
Digested pTrcH
is A
with Bam
HI
Digested pTrcH
is A
with K
pnI
Figure 10 : The pTrcHis A in lane digested with BamHI and KpnI in lane 3 and 4.In line 1 is pTrcHis A without digestion.
Method & Result
NS2B(H)-NS3p Den
NS2B(H)-NS3p JEVpTrcHis A
Ligation & Transformation
Site Screening & Digest with Restriction Enzyme
Dengue C
lone 1-5
Dengue C
lone 6-10
Dengue C
lone 11-15
Dengue C
lone 16-20
Dengue C
lone 21-25
JEV C
lone 1
pTrcHis A
23.13 kb
9.42 kb6.56 kb4.56 kb
2.32 kb2.03 kb
Figure 11 : The pTrcHis NS2B(H)-NS3protease JEV (lane 8) and pTrcHis NS2B(H)-NS3protease Den (lane 3 to 7) was digested with BamHI.
23.13 kb
9.42 kb6.56 kb4.56 kb
2.32 kb2.03 kb
700 bpN
S2B(H
)-NS3p
Dengue C
lone 1
Dengue C
lone 2
Dengue C
lone 3
Dengue C
lone 4
Dengue C
lone 5
Dengue C
lone 6
Dengue C
lone 7
Dengue C
lone 8
Dengue C
lone 9
JEV C
lone 1
Figure 12 : The pTrcHis NS2B(H)-NS3protease JEV (lane 12) and pTrcHis NS2B(H)-NS3protease Den clone 1-9 (lane 3 to 11) was digested with BamHI and KpnI.
Conclusion
• The NS2B(H)-NS3p JEV and NS2B(H)-NS3p Den was successfully PCR and ligated into pTrcHis A plasmid.
• The digestion of the recombinant vector shown the expect band of pTrcHis with the insert, then it need to purify and sequence the plasmid to make sure,that is the correct plasmid.
Future Work
• Check another clone to get more positive clone.
• Construct NS2B(H)Den-NS3p JE and NS2B(H) JE-NS3p Den.
• Retransform into E.coli C41.
• Purification of Protein.
• Enzyme assay.
Thank you for your attention.