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Baculovirus transfer vector pAcGP67B

PvRII Expression. Plasmid for Expression in Insect Cells. PvRII. pAcR2H. Baculovirus transfer vector pAcGP67B. {From Becton Dickenson, PharMingen, Baculovirus Expression Manual, 2001}. +. Viral DNA. pAcR2H. Sf cell. a) Reinfect for Protein prod. b) Amplify for Virus titre.

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Baculovirus transfer vector pAcGP67B

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  1. PvRII Expression Plasmid for Expression in Insect Cells PvRII pAcR2H Baculovirus transfer vector pAcGP67B {From Becton Dickenson, PharMingen, Baculovirus Expression Manual, 2001}

  2. + Viral DNA pAcR2H Sf cell a) Reinfect for Protein prod. b) Amplify for Virus titre PvRII Expression Baculovirus Expression Vector System a) Find best protein producing virus plasmid DNA only (–ve control) Experimental b) Plaque Assay and End Point Dilution Assay. • Prepare sufficient high titre virus, • (Scale-up accordingly) Recombinant Proteins Recombinant Proteins Recombinant Viruses a) Reinfect for Protein prod. b) Amplify for Virus titre

  3. Mw kDa 43 M E Mw kDa PL M FT W1 W2 E1 E2 E3 E4 R NR Mw kDa ←PvRII 9.416 6.557 4.361 2.027 1.353 1.078 9.416 6.557 4.361 2.027 1.353 1.078 79 47 33 25 Mw kDa Mw 1 2 3 4 5 6 7 8 9 Mw 1 2 3 47 0.25μg 0.5μg 1.0μg 2.0μg kb kb ←PvRII ←reduced PvRII 79 47 33 25 ←native PvRII Mobility shift of PvRII before and after reduction and alkylation ←PvRII Western blot of elution profile Silver stained elution profile of recombinant PvRII ~1 kb Mw PL M FT W1 W2 - E1 E2 E3 47 ~1 kb . kDa Silver stained SDS-PAGE gel of NiNTA purified PvRII ←PvRII BamHI and NotI RD of transformant with pAcR2H Colony PCR screening for pAcR2H RP-HPLC profile of NiNTA purified PvRII Plasmid Characterisation Protein Characterisation Agarose Gels – Colony PCR and RD Metal Affinity Chromatography Silver Stain and Mobility Shift Reverse Phase HPLC Western blot of NiNTA purified PvRII sample for RP-HPLC

  4. Add NaCl Add DBPT Protein Characterisation Erythrocyte Binding Assay (EBA) Method PvRII {after Camus and Hadley, 1985}

  5. Hu Hu ←RBC Mw c - Chy - none ←Treatment kDa 47 ←PvRII Erythrocyte binding assay with NiNTA purified PvRII Protein Characterisation EBA Results

  6. Immunogenicity Anti-PvRII Antibodies from Mice

  7. Immunogenicity ELISA Data for α- PvRII Mouse Sera

  8. Immunogenicity OD490nm Determination from ELISA     

  9. Immunogenicity α-PvRII Mouse Sera End Point Titres CLICK HERE for PART3

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