Evaluation I.Key

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Evaluation I.Key

  1. 1. 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
  2. 2. Japanese Encephalitis Virus -Flaviviridae family -Mosquito-borne neurotropic flavivirus •causes severe central nerve system diseases
  3. 3. Japanese Encephalitis Virus Culex tritaeniorhynchus. Source : vietnammedicalpractice.com Source: fehd.gov.hk
  4. 4. Japanese Encephalitis Virus JEV causes severe central nerve system diseases such as 50,000 Cases 10,000 p o l i o mye l i t i s - l i ke 30% fatality rate acute flaccid paralysis, aseptic meningitis and encephalitis Source:wonder.cdc.gov Source: cdc.gov
  5. 5. Prevention and treatment of JEV disease Drug No drug exist Vaccine development Available vaccine Elimination of mosquitoes Mosquitoes control breeding places
  6. 6. Molecular biology of Japanese Encephalitis Virus Source : molecular-virology.uni-hd.de
  7. 7. The NS2B • 130 aa Hypothetical model • activating domain NS2B-NS3 complex central hydrophilic region (Falgout et al, 1993) • 3 membrane spanning parts hydrophobicity plot ฺBrinkworth et al, 1999 51 DMWLERAADISWEMDAAITGSSRRLDVKLDDDGDFHLIDDPGVP 95
  8. 8. The NS3 Protease NTPase •Chymotrypsin-like fold 2-β barrel Helicase RNA domains •Inactive alone Theoretical model from PDB •Enzyme’s pocket is small 2I84
  9. 9. The NS3 protease Complexation with NS2B cofactor • conformational change serine protease NS3 alteration of the enzyme pocket additional substrate binding 20 kDa domain site •catalytic residues His51, Asp75, Ser135
  10. 10. Background • NS2B(H) JE - NS3p Den did not cleaved Den polyprotein but NS2B(H) Den - NS3p JE cleaved JEV polyprotein. • The C-terminal portion of Den NS2B is required for interaction with Den NS3 to activate protease. • Jan. L R et al, 1995
  11. 11. Background • Ser to Ile60 were essential region required for NS3 46 protease activity. • Ala substition of Trp 50, Glu55, and Arg56 in NS2B shown significantly reduced NS3 protease activity. • Lin. C W et al,2007
  12. 12. 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 substrates incorporated with fluorogenic or chromogenic reported groups.
  13. 13. Method & Result pLS with NS2B-NS3 JEV NS2B(H) NS3p SOE-PCR NS2B(H)-NS3p
  14. 14. NS2B(H) NS2B NS3p NS3 NS2B(H) NS3p
  15. 15. - Control NS2B(H) NS2B(H) NS3 protease NS3 protease NS3 protease - Control 1500 1500 1000 900 800 1000 700 900 800 600 700 500 600 400 500 400 300 300 200 200 Figure 1 : The PCR product NS2B(H) JEV amplified Figure 2 : The PCR product NS3protease JEV from NS2B-NS3 JEV (Lane 3 and 4). The size of amplified from NS2B-NS3 JEV (Lane 3 to 5 ). The size NS2B(H) was 187 bp. of NS3 protease was 594 bp.
  16. 16. NS2B(H)-NS3p - Control NS2B(H)-NS3p - Control 1500 1000 900 1500 800 700 600 1000 900 500 800 400 700 600 300 500 400 200 300 100 Figure 3 : The SOE-PCR product NS2B(H)- Figure 4 : The NS2B(H)-NS3protease JEV NS3protease JEV (Lane 2 to 5 ). The size of (Lane 3 ) after digested with BamHI and KpnI. NS2B(H)-NS3 protease was 765 bp. The size of NS2B(H)-NS3 protease was 765 bp.
  17. 17. Method & Result pTrcHis Den with NS2B-NS3 Den NS2B(H) NS3p SOE-PCR NS2B(H)-NS3p Den
  18. 18. NS3 protease NS3 protease - Control - Control NS2B(H) NS2B(H) 1500 1500 1000 1000 900 900 800 800 700 700 600 600 500 500 400 400 300 300 200 200 100 100 Figure5 : The PCR product NS2B(H) Den amplified Figure 6 : The PCR product NS3protease Den amplified from pTrc NS2B-NS3 Den (Lane 2 and 3). The size of from pTrc NS2B-NS3 Den (Lane 3 and 4 ). The size of NS2B(H) was 187 bp. NS3 protease was 594 bp.
  19. 19. NS2B(H)-NS3p NS2B(H)-NS3p NS2B(H)-NS3p NS2B(H)-NS3p NS2B(H)-NS3p NS2B(H)-NS3p NS2B(H)-NS3p - Control - Control 1500 1000 900 1500 800 700 1000 600 900 500 800 700 400 600 500 300 400 200 300 200 Figure 7 : The SOE-PCR product NS2B(H)-NS3protease Figure 8 :The NS2B(H)-NS3protease Den (Lane 3 and Den (Lane 2 and 6). The size of NS2B(H)-NS3 protease 4 ) after digested with BamHI and KpnI. The size of was 765 bp. NS2B(H)-NS3 protease was 765 bp.
  20. 20. Method & Result pTrcHis A Digest with BamHI Digest with KpnI Digest with KpnI Digest with BamHI Gel Electrophoresis & Clean with Gel Extraction Kits
  21. 21. with BamHI Digested pTrcHis A with KpnI Digested pTrcHis A pTrcHis A 23.13 kb 9.42 kb 23.13 kb 6.56 kb 9.42 kb 4.56 kb 6.56 kb 4.56 kb 2.32 kb 2.03 kb 2.32 kb 2.03 kb Figure 9 : The pTrcHis A in lane 2 without digestion 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.
  22. 22. Method & Result NS2B(H)-NS3p JEV pTrcHis A NS2B(H)-NS3p Den Ligation & Transformation Site Screening & Digest with Restriction Enzyme
  23. 23. Dengue Clone 1-5 Dengue Clone 11-15 Dengue Clone 16-20 Dengue Clone 21-25 Dengue Clone 6-10 Dengue Clone 4 Dengue Clone 5 Dengue Clone 6 Dengue Clone 8 Dengue Clone 9 Dengue Clone 1 Dengue Clone 2 Dengue Clone 3 Dengue Clone 7 NS2B(H)-NS3p JEV Clone 1 JEV Clone 1 pTrcHis A 23.13 kb 23.13 kb 9.42 kb 9.42 kb 6.56 kb 6.56 kb 4.56 kb 4.56 kb 2.32 kb 2.32 kb 2.03 kb 2.03 kb 700 bp Figure 12 : The pTrcHis NS2B(H)-NS3protease JEV Figure 11 : The pTrcHis NS2B(H)-NS3protease JEV (lane 12) and pTrcHis NS2B(H)-NS3protease Den (lane 8) and pTrcHis NS2B(H)-NS3protease Den (lane clone 1-9 (lane 3 to 11) was digested with BamHI and 3 to 7) was digested with BamHI. KpnI.
  24. 24. 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.
  25. 25. 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.
  26. 26. Thank you for your attention.

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