6. Elastin: Elastin from human aorta is dissolved in Tris buffer to a
concentration of 1 mg/mL and stored at 2–8
C.
7. Fibronectin: The solution of fibronectin from human fibroblasts is kept at À20
C (see Note 4).
2.2 Cloning,
Expression,
and Purification
of Recombinant
Proteins
1. Genomic DNA of L. interrogans serovar Copenhageni strain
FIOCRUZ L1-130 (see Note 5).
2. Specific primers for sequence amplification.
3. Thermocycler.
4. Microtubes, 0.2 mL volume.
5. DNA and Gel Band Purification Kits.
6. Vectors for cloning.
7. Restriction enzymes.
8. Tris-acetate-EDTA (TAE) buffer: 40 mM Tris, 20 mM acetic
acid, 1 mM EDTA. Store at room temperature.
9. 1% agarose gel: Dissolve 1 g of agarose in 100 mL TAE buffer.
10. Vectors for expression.
11. E. coli BL21.
12. Luria-Bertani (LB) medium: 1% NaCl, 0.5% yeast extract, 1%
tryptone. For solid medium, use 1.5% bacteriological agar.
Adjust the pH to 7.0 and sterilize by autoclaving for 20 min
at 121
C.
13. Isopropyl-β-D-thiogalactopyranoside (IPTG): 1 M solution in
water. Sterilize with a 0.2 μm filter and store in 1 mL aliquots at
À20
C.
14. Ampicillin: 100 mg/mL solution in water. Sterilize by filtration, aliquot, and store at À20
C.
15. 5-Bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-Gal):
20 mg/mL solution in dimethylformamide. Sterilize with a
0.2 μm filter, and store in the dark in aliquots of 0.5 mL at
À20
C.
16. Chloramphenicol: 30 mg/mL solution in ethanol. Sterilize by
filtration, aliquot, and store at À20
C.
17. Phenylmethylsulfonyl fluoride (PMSF): 0.1 M solution in isopropanol (see Note 6). PMSF is unstable in aqueous solution.
Store at 20–25
C.
18. Lysozyme: 10 mg/mL solution in water. Aliquot and store at
À20
C.
19. Lysis buffer: 10 mM Tris–HCl, pH 8.0, 150 mM NaCl,
100 μg/mL lysozyme, 1% Triton X-100, 2 mM PMSF.
20. Cell disruptor.
Cell Adhesion Assay for Leptospiral Proteins
173
concentration of 1 mg/mL and stored at 2–8
C.
7. Fibronectin: The solution of fibronectin from human fibroblasts is kept at À20
C (see Note 4).
2.2 Cloning,
Expression,
and Purification
of Recombinant
Proteins
1. Genomic DNA of L. interrogans serovar Copenhageni strain
FIOCRUZ L1-130 (see Note 5).
2. Specific primers for sequence amplification.
3. Thermocycler.
4. Microtubes, 0.2 mL volume.
5. DNA and Gel Band Purification Kits.
6. Vectors for cloning.
7. Restriction enzymes.
8. Tris-acetate-EDTA (TAE) buffer: 40 mM Tris, 20 mM acetic
acid, 1 mM EDTA. Store at room temperature.
9. 1% agarose gel: Dissolve 1 g of agarose in 100 mL TAE buffer.
10. Vectors for expression.
11. E. coli BL21.
12. Luria-Bertani (LB) medium: 1% NaCl, 0.5% yeast extract, 1%
tryptone. For solid medium, use 1.5% bacteriological agar.
Adjust the pH to 7.0 and sterilize by autoclaving for 20 min
at 121
C.
13. Isopropyl-β-D-thiogalactopyranoside (IPTG): 1 M solution in
water. Sterilize with a 0.2 μm filter and store in 1 mL aliquots at
À20
C.
14. Ampicillin: 100 mg/mL solution in water. Sterilize by filtration, aliquot, and store at À20
C.
15. 5-Bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-Gal):
20 mg/mL solution in dimethylformamide. Sterilize with a
0.2 μm filter, and store in the dark in aliquots of 0.5 mL at
À20
C.
16. Chloramphenicol: 30 mg/mL solution in ethanol. Sterilize by
filtration, aliquot, and store at À20
C.
17. Phenylmethylsulfonyl fluoride (PMSF): 0.1 M solution in isopropanol (see Note 6). PMSF is unstable in aqueous solution.
Store at 20–25
C.
18. Lysozyme: 10 mg/mL solution in water. Aliquot and store at
À20
C.
19. Lysis buffer: 10 mM Tris–HCl, pH 8.0, 150 mM NaCl,
100 μg/mL lysozyme, 1% Triton X-100, 2 mM PMSF.
20. Cell disruptor.
Cell Adhesion Assay for Leptospiral Proteins
173