2 Materials
2.1 Transfer Vector
Cloning
In this part of the chapter, the required materials for the cloning of
the transfer vector are described. In addition to the genetic
sequence of interest, which codes for the target protein, it is
assumed that a lacZα cassette for blue–white screening is
integrated.
1. Transfer cloning vector (e.g., pFastBac-1, Thermo Fisher Scientific, Waltham, MA, USA).
2. Genetic sequence of interest (e.g., ORF2 of PCV2).
3. Other sequences of genetic elements (a reporter gene, a secretion signal, etc.).
2.2 Generation
of Recombinant
Bacmid
(Baculoviral DNA)
This part of the chapter describes the materials for the generation
and analysis of recombinant bacmid DNA, based on the Bac-toBac™ Baculovirus Expression Vector System using MAX Efficiency
® DH10-Bac™ (see Note 2).
2.2.1 Materials
for Transformation
of Competent MAX
Efficiency
® DH10-Bac™
1. Baculovirus expression vector system kit (e.g., Bac-to-Bac™
Baculovirus Expression System, Thermo Fisher Scientific, Waltham, MA, USA).
2. Cloned transfer vector (see Subheading 2.1).
3. Crushed ice for thawing the MAX Efficiency
® DH10-Bac™.
4. S.O.C. medium (e.g., New England Biolabs, Ipswich,
Massachusetts, USA).
5. LB medium for casting agar plates (e.g., granulated LB
medium, Carl Roth, Karlsruhe, Germany, prepare according
to the manufacturer’s instructions).
6. Liquid LB medium for the dilution of the bacterial cell suspension (e.g., granulated LB medium, Carl Roth, Karlsruhe,
Germany).
7. Agar plates containing 5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside (X-Gal) and isopropyl-β-D-thiogalactopyranoside
(IPTG) (see Note 3) and also kanamycin (working concentration 25 μg/mL), tetracycline (working concentration 15 μg/
mL). Depending on the transfer vector, further or other antibiotics may be required (see Note 4).
8. Sterile single-use pipette tips (e.g., pipette tips, Sarstedt, Nu ¨mbrecht, Germany).
9. Pipette holder/rubber bulb (e.g., pipetus, Hirschmann Laborger€ ate, Eberstadt, Germany).
10. 1.5 mL centrifuge tubes (e.g., Sarstedt, Nu ¨mbrecht,
Germany).
Antigen Production in Insect Cells
97
2.1 Transfer Vector
Cloning
In this part of the chapter, the required materials for the cloning of
the transfer vector are described. In addition to the genetic
sequence of interest, which codes for the target protein, it is
assumed that a lacZα cassette for blue–white screening is
integrated.
1. Transfer cloning vector (e.g., pFastBac-1, Thermo Fisher Scientific, Waltham, MA, USA).
2. Genetic sequence of interest (e.g., ORF2 of PCV2).
3. Other sequences of genetic elements (a reporter gene, a secretion signal, etc.).
2.2 Generation
of Recombinant
Bacmid
(Baculoviral DNA)
This part of the chapter describes the materials for the generation
and analysis of recombinant bacmid DNA, based on the Bac-toBac™ Baculovirus Expression Vector System using MAX Efficiency
® DH10-Bac™ (see Note 2).
2.2.1 Materials
for Transformation
of Competent MAX
Efficiency
® DH10-Bac™
1. Baculovirus expression vector system kit (e.g., Bac-to-Bac™
Baculovirus Expression System, Thermo Fisher Scientific, Waltham, MA, USA).
2. Cloned transfer vector (see Subheading 2.1).
3. Crushed ice for thawing the MAX Efficiency
® DH10-Bac™.
4. S.O.C. medium (e.g., New England Biolabs, Ipswich,
Massachusetts, USA).
5. LB medium for casting agar plates (e.g., granulated LB
medium, Carl Roth, Karlsruhe, Germany, prepare according
to the manufacturer’s instructions).
6. Liquid LB medium for the dilution of the bacterial cell suspension (e.g., granulated LB medium, Carl Roth, Karlsruhe,
Germany).
7. Agar plates containing 5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside (X-Gal) and isopropyl-β-D-thiogalactopyranoside
(IPTG) (see Note 3) and also kanamycin (working concentration 25 μg/mL), tetracycline (working concentration 15 μg/
mL). Depending on the transfer vector, further or other antibiotics may be required (see Note 4).
8. Sterile single-use pipette tips (e.g., pipette tips, Sarstedt, Nu ¨mbrecht, Germany).
9. Pipette holder/rubber bulb (e.g., pipetus, Hirschmann Laborger€ ate, Eberstadt, Germany).
10. 1.5 mL centrifuge tubes (e.g., Sarstedt, Nu ¨mbrecht,
Germany).
Antigen Production in Insect Cells
97