Enzymes - Identification and characterization of specific amino acid residues in the NS1 protei

2.1 Materials

2.1.5 Enzymes

AccuPrime Pfx DNA Polymerase Invitrogen

GoTaq flexi DNA polymerase Promega

Platinum^R Pfx DNA polymerase Invitrogen

SuperScript ^TM III reverse transcriptase Invitrogen

T4 Ligase NEB

T4 polynuleotide kinase (10,000 units/ml) NEB

2.1.6 Kits

AEC staining kit Sigma

Bradford Protein Assay Kit

ECL (enhanced chemiluminescence) solution Kit Amersham/GE High pure PCR product purification Kit Roche

In situ cell death detection Kit, fluorescein Roche

NucleoPond Xtra Maxi Kit for DNA plasmids prep Macherey-Nagel pcDNA^TM 3.1 directional TOPO expression Kit Invitrogen

QIAprep Spin Miniprep Kit (250) Qiagen

QIAamp Viral RNA Mini Kit (250) Qiagen

Qiagen plasmid Maxi Kit Qiagen

Nucleotide removal Kit TOPO XL PCR cloning Kit Qiagen

Caspase-3 activity Abcam

Rapid DNA ligation Kit Roche

QuikChange® Site-Directed Mutagenesis Kit Stratagene

Human Interferon beta Kit PBL Assay Science

2.1.7 E. coli strain, recombinant viruses and cell lines E. coli strain

XL1-Blue Stratagene

TOP 10 Invitrogen

Materials and Methods

35 Recombinant influenza viruses

A/FPV/Rostock/34 (H7N1) (FPV-WT) Rescued at the Institute of Medical Virology, Giessen

FPV-NS^GD Rescued at the Institute

of Medical Virology, Giessen

FPV-NS^MA Rescued at the Institute

of Medical Virology, Giessen

FPV-NS^MA_P3S Rescued at the Institute

of Medical Virology, Giessen

FPV-NS^MA_R41K Rescued at the Institute

of Medical Virology, Giessen

FPV-NS^MA_D74N Rescued at the Institute

of Medical Virology, Giessen

FPV-NS^MA_I98M Rescued at the Institute

of Medical Virology, Giessen

FPV-NS^MA_D153S Rescued at the Institute

of Medical Virology, Giessen

FPV-NS^MA_M166V Rescued at the Institute

of Medical Virology, Giessen

FPV-NS^MA_P213S Rescued at the Institute

of Medical Virology, Giessen

FPV-NS^MA_R224K Rescued at the Institute

of Medical Virology, Giessen

Materials and Methods

FPV-NSMA_P3S+R41K Rescued at the Institute

of Medical Virology, Giessen

FPV-NSMA_P3S+R41K+D74N Rescued at the Institute

of Medical Virology, Giessen

FPV-NSMA_P3S+R41K+D74N+I98M Rescued at the Institute

of Medical Virology, Giessen

FPV-NSMA_P3S+R41K+D74N+I98M+D153S Rescued at the Institute of Medical Virology, Giessen

FPV-NSMA_P3S+R41K+D74N+I98M+D153S+M166V Rescued at the Institute of Medical Virology, Giessen

FPV-NSMA_P3S+R41K+D74N+I98M+D153S+M166V+P213S Rescued at the Institute of Medical Virology, Giessen

FPV-NSMA_P3S+R41K+D74N+I98M+D153S+M166V+P213S+R224K Rescued at the Institute of Medical Virology, Giessen

FPV-NS^GD/MA Rescued at the Institute

of Medical Virology, Giessen

FPV-NS^MA/GD Rescued at the Institute

of Medical Virology, Giessen

Materials and Methods

37 Cell lines

A549 (Human Alveoar Epithelial cells) ATCC

MDCK-II (Madin-Darby canine kidney cells) Cell-collection of the Institute of Medical Virology, Giessen 293T (Human embryonic kidney cells) Cell-collection of the

Institute of Medical Virology, Giessen

QT6 (quail fibroblast cells) Kind gift from the

Institute for Virology, Marburg

2.1.8 Plasmids

pPolI-FPV-PB1 (A/FPV/Rostock/34 (H7N1)) Kind gift from the Institute for Virology, Marburg

pPolI-FPV-PB2 (A/FPV/Rostock/34 (H7N1)) Kind gift from the Institute for Virology, Marburg

pPolI-FPV-PA (A/FPV/Rostock/34 (H7N1)) Kind gift from the Institute for Virology, Marburg

pPolI-NP (A/FPV/Rostock/34 (H7N1)) Kind gift from the Institute for Virology, Marburg

pPolI-FPV-HA (A/FPV/Rostock/34 (H7N1)) Kind gift from the Institute for Virology, Marburg

Materials and Methods

38 pPolI-FPV-NA (A/FPV/Rostock/34 (H7N1)) Kind gift from the

Institute for Virology, Marburg

pPolI-FPV-M (A/FPV/Rostock/34 (H7N1)) Kind gift from the Institute for Virology, Marburg

pPolI-FPV-NS (A/FPV/Rostock/34 (H7N1)) Kind gift from the Institute for Virology, Marburg

pCI-GD-NS (A/Goose/Guangdong/1/96 (H5N1)) Kind gift from the Institute for Virology, Marburg

pPolI-MA-NS (A/Mallard/NL/12/2000 (H7N3)) Kind gift from the Institute for Virology, Helsinki

pPolI-Ma-NS^GD/MA (A/Mallard/NL/12/2000 (H7N3)) Kind gift from the Institute for Virology, Helsinki

pPolI-Ma-NS^MA/GD (A/Mallard/NL/12/2000 (H7N3)) Kind gift from the Institute for Virology, Helsinki

pHMG-PA (A/Puerto Rico/8/34(H1N1)) Kind gift from the Institute for Virology, Zurich

pHMG-PB1 (A/Puerto Rico/8/34(H1N1)) Kind gift from the Institute for Virology, Zurich

pHMG-PB2 (A/Puerto Rico/8/34(H1N1)) Kind gift from the Institute for Virology, Zurich

pHMG-NP (A/Puerto Rico/8/34(H1N1)) Kind gift from the Institute for Virology, Zurich

Materials and Methods

39 pPolI-CAT-RT (Human Pol promoter) Kind gift from the

department of microbiology, Mount Sinai School of Medicine, New York pcDNA3.0-GD-NS1(A/Goose/Guangdong/1/96 (H5N1)) Constructed at the

Institute of Medical Virology, Giessen pcDNA3.0-MA-NS1(A/Mallard/NL/12/2000 (H7N3)) Constructed at the

Institute of Medical Virology, Giessen pcDNA3.0-MA-NS1^D74N(A/Mallard/NL/12/2000 (H7N3)) Constructed at the

Institute of Medical Virology, Giessen pcDNA3.0-MA-NS1P3S/R41K/D74N

(A/Mallard/NL/12/2000 (H7N3)) Constructed at the

Institute of Medical Virology, Giessen pcDNA3.1-FPV-NP (A/FPV/Rostock/34 (H7N1)) Constructed at the

Institute of Medical Virology, Giessen pcDNA3.0-FPV-PB1(A/FPV/Rostock/34 (H7N1)) Constructed at the

Institute of Medical Virology, Giessen pcDNA3.0-FPV-PB2(A/FPV/Rostock/34 (H7N1)) Constructed at the

Institute of Medical Virology, Giessen pcDNA3.0-FPV-PA(A/FPV/Rostock/34 (H7N1)) Constructed at the

Institute of Medical Virology, Giessen

Materials and Methods

40 2.1.9 Medias, buffers and solutions

Avicel Medium (100 ml)

10 ml 10X MEM

33 ml ddH2O

1 ml Penicillin/Strepromycin liquid (100X) 1 ml BSA solution (35%)

50 ml Avicell Stock (2.5%)

1 ml DEAE-Dextran (1%)

4 ml NaHCO3 (7.5%)

Avicel Stock (2.5%)

5 g Avicel-Powder

200 ml ddH2O

Cell culture medium for QT6 cells (500 ml) 425 ml HAM's-F10 media

5 ml Penicillin/Strepromycin liquid (100X)

5 ml L-Glutamine

50 ml Fetal Calf Serum (FCS)

5 ml Chicken Serum

10 ml Tryptose Phosphate Broth Cell fixing buffer (100 ml)

95 ml PBS++

4 ml Formaldehyde (PFA) (37%)

1 ml Triton X-100 (t-Octylphenoxypolyethoxyethanol) DMEM/BA (500 ml)

492 ml DMEM

5 ml Penicillin/streptomycin (100X) 3 ml Bovine Albumin (BA) (30%)

Materials and Methods

41 2X DMEM/BA for Plaque-Assay (500 ml)

100 ml MEM (10X)

10 ml Penicillin/Streptomycin (100X) 20 ml NaHCO3 (7.5%)

6 ml Bovine Albumin (BA) (30%) 354 ml ddH2O

Luria-Broth (LB) medium

Extract of yeast powder (0.5%) (w/v)

Peptone from meat trypsin-digested (1%) (w/v)

adjust pH to 7.5 with NaOH and total volume to 1 L with ddH2O, autoclaved for plates, add agar high-gel strength (1.5%) (w/v), autoclaved

Infection media (for MDCK, A549 and 293T) (100 ml) 98.5 ml DMEM (1X)

1 ml Penicillin/Strepromycin liquid (100X) 500 ul BSA (30%)

Infection media (QT6) (100 ml) 95.5 ml HAM's-F10 media

1 ml Penicillin/Strepromycin liquid (100x)

1 ml L-Glutamine

2 ml Tryptose Phosphate Broth 500 µl BSA (30%)

Mowiol mounting medium Mowiol 4-88

Glycerol

Tris-HCl (0.2 M, pH 8.5)

DABCO (1,4-diazabicyclo-[2,2,2]-octane)

Materials and Methods

42 SDS-PAGE stacking gel (1 gel)

2.9 ml ddH2O

1.25 ml Tris-HCl, pH 6.8 (0.5 M)

50 μl SDS (10%)

750 μl Polyacrylamide (PAA) (30%)

50 μl APS (10%)

4 μl TEMED

SDS-PAGE resolving gel (10%) (1 gel)

4 ml ddH2O

2.5 ml Tris-HCl, pH 8.8 (1.5 M)

100 μl SDS (10%)

3.3 ml Polyacrylamide (PAA) (30%)

SM medium Gelatin (0.01%) NaCl 250 mM MgSO4 8.5 mM

Tris–HCl 50 mM, pH 7.5

SOC Medium

Tryptone (2%) (w/v) Yeast extract (0.5%) (w/v) NaCl 10 mM

MaCl2 10 mM Glucose 20 mM

Prepare a solution containing the first four reagents, sterilize at 121°C, then add sterile MgCl2 and Glucose

Materials and Methods

43 6%, 7M urea polyacrylamide gel

21 g Urea

5 ml 10X TBE

7.5 ml Acrylamide (40%) Fill up the tube to 50ml with sterile water 0.5 ml APS (10%)

0.05 ml TEMED

Blocking buffer and antibody diluting solution Non-fat dry milk (5%) (w/v) in 1XTBS-T (see below) Lysis buffer

10 ml TLB buffer (see TLB buffer) 10 µl Pefablock (200 mM)

10 µl Aprotinin (5 mg/ml) 10 µl Leupeptin (5 mg/ml)

100 µl Sodium orthovanadate (100 mM) 50 µl Benzamidine (1 M)

1XPBS⁺⁺ buffer (500 ml)

495 ml 1XPBS (autoclaved) 5 ml Ca²⁺/Mg²⁺ solution (100X) PBS/Ca²⁺/Mg²⁺/BA/antibiotic (200ml)

20 ml 10X PBS (see above) 174.8 ml ddH2O (sterile)

2 ml Penicillin/Streptomycin (100X) 1.2 ml BSA (30%)

2 ml Ca²⁺/Mg²⁺ (100X)

Materials and Methods

44 SDS-PAGE loading buffer (2X) (Laemmli Buffer)

100 mM Tris-HCl, pH 6.8 200 mM DTT (dithiothreitol)

SDS (4%) Glycerol (20%)

Bromophenol blue (0.2%) SDS-PAGE running buffer (5X) (1000 ml)

1 g SDS

6 g Tris-HCl

28.8 g Glycin

adjust total volume to 1 L with ddH2O SDS-PAGE transfer buffer (1000 ml) 5.8 g Tris-HCl

2.9 g Glycin

0.17 g SDS

200 ml Methanol

adjust to total volume to 1 L with ddH2O TAE buffer (10X) (1000 ml)

48.5 g Tris

11.4 ml Glacial acetic acid 20 ml 0.5 M EDTA (pH 8.0) TAE buffer (1X)

Dilute stock solution 10X (10:1) in ddH2O 1XTAE buffer contain

40 mM Tris

20 mM Acetic acid

1 mM EDTA

Materials and Methods

45 TLB buffer

20 mM Tris-HCl, pH 7.4 137 mM NaCl

Glycerol (10%) (v/v) Triton X-100 (1%) (v/v)

2 mM EDTA

50 mM Sodium-β-glycerophosphate 20 mM Sodiumpyrophosphate

Tris-Buffered Saline (10X TBS) (1000 ml) 24.2 g Tris-HCl

80 g NaCl

dissolve by adding 900 ml ddH2O

adjust pH to 7.6 and total volume to 1 L with ddH2O TBS/Tween (0.05%) (1XTBS-T) (1000 ml)

100 ml 10X TBS

900 ml ddH2O 0.5 ml Tween 20

Im Dokument Identification and characterization of specific amino acid residues in the NS1 protein that facilitated viral replication of avian influenza viruses in mammalian host (Seite 49-61)