4. Transfer the supernatant into fresh centrifuge tubes and centrifuge 10 min at 3000 Â g.
5. Again, transfer the supernatant into fresh centrifuge tubes.
6. Add a protease inhibitor to the protein-containing
supernatant.
7. Store the supernatant at À80
C until further downstream
processing.
4 Notes
1. The Porcine Circovirus 2 (PCV2) infection is related to the
Post-Weaning Multisystemic Wasting Syndrome (PMWS) and
therefore a target for vaccination. The genome of this virus
comprises three open reading frames (ORF). ORF2 encodes an
approximately 30 kDa-sized capsid protein, which is the main
immunostimulating agent in infected animals.
2. The transfer vector carries the desired genetic sequences that
needs to be integrated into the baculoviral genome. For this
purpose, several commercial kits are available: (1) flashBAC
(Oxford Expression Technologies), (2) BaculoGold™
(BD Biosciences), (3) BaculoDirect™ Baculovirus Expression
System (Thermo Fisher Scientific), and (4) Bac-to-Bac
® Baculovirus Expression System (Thermo Fisher Scientific), which
was used for this chapter.
3. If a lacZα cassette is integrated in the transfer vector, X-Gal and
IPTG should be added to the agar plates to allow for an
identification of positive clones by blue–white screening. The
lacZα cassette expresses β-galactosidase, which converts the
yellow dye X-Gal into a bluish dye with the help of the inductor
IPTG, due to which the negative clones appear blue on the agar
plate. If the transduction is successful, the lacZα cassette is
interrupted and the colonies appear white.
4. The E. coli strain MAX Efficiency
® DH10-Bac™ contains the
bacmid DNA pMON14727, which carries a kanamycin resistance, and the helper plasmid, which codes for a tetracycline
resistance. Depending on which antibiotic resistances are carried by the transfer vector, it is necessary to add these antibiotics to the medium in the corresponding working
concentration. For example, if using the pFastBac-1 transfer
vector, gentamicin (working concentration 15 μg/mL) and
ampicillin (working concentration 50 μg/mL) need to be
added [6].
5. Vectors carrying the lacZα cassette allow blue–white screening.
Depending on the application, white (e.g., lacZα cassette is
replaced by gene of interest) or blue colonies (e.g., lacZα
114
Julie Harnischfeger et al.
5. Again, transfer the supernatant into fresh centrifuge tubes.
6. Add a protease inhibitor to the protein-containing
supernatant.
7. Store the supernatant at À80
C until further downstream
processing.
4 Notes
1. The Porcine Circovirus 2 (PCV2) infection is related to the
Post-Weaning Multisystemic Wasting Syndrome (PMWS) and
therefore a target for vaccination. The genome of this virus
comprises three open reading frames (ORF). ORF2 encodes an
approximately 30 kDa-sized capsid protein, which is the main
immunostimulating agent in infected animals.
2. The transfer vector carries the desired genetic sequences that
needs to be integrated into the baculoviral genome. For this
purpose, several commercial kits are available: (1) flashBAC
(Oxford Expression Technologies), (2) BaculoGold™
(BD Biosciences), (3) BaculoDirect™ Baculovirus Expression
System (Thermo Fisher Scientific), and (4) Bac-to-Bac
® Baculovirus Expression System (Thermo Fisher Scientific), which
was used for this chapter.
3. If a lacZα cassette is integrated in the transfer vector, X-Gal and
IPTG should be added to the agar plates to allow for an
identification of positive clones by blue–white screening. The
lacZα cassette expresses β-galactosidase, which converts the
yellow dye X-Gal into a bluish dye with the help of the inductor
IPTG, due to which the negative clones appear blue on the agar
plate. If the transduction is successful, the lacZα cassette is
interrupted and the colonies appear white.
4. The E. coli strain MAX Efficiency
® DH10-Bac™ contains the
bacmid DNA pMON14727, which carries a kanamycin resistance, and the helper plasmid, which codes for a tetracycline
resistance. Depending on which antibiotic resistances are carried by the transfer vector, it is necessary to add these antibiotics to the medium in the corresponding working
concentration. For example, if using the pFastBac-1 transfer
vector, gentamicin (working concentration 15 μg/mL) and
ampicillin (working concentration 50 μg/mL) need to be
added [6].
5. Vectors carrying the lacZα cassette allow blue–white screening.
Depending on the application, white (e.g., lacZα cassette is
replaced by gene of interest) or blue colonies (e.g., lacZα
114
Julie Harnischfeger et al.