This document summarizes the baculovirus expression system. Baculoviruses can be used as expression vectors by replacing a non-essential viral gene with a gene of interest. The recombinant baculovirus is produced through homologous recombination or using the Bac-to-Bac system. Insect cells are infected with the recombinant baculovirus, which drives high-level expression of the foreign gene. The baculovirus expression system allows safe, scalable production of recombinant proteins for research applications.

1. BACULOVIRUS EXPRESSION
SYSTEM
Thilina Herath (Bsc. University of Peradeniya)
Faculty of veterinary Medicine,
Chungnam National University,
South Korea.

2. Viral Classification
•Group: Group I (dsDNA)
•Family: Baculoviridae
•Subfamily :
Nucleopolyhedrosis Virus (NPV)
•Genera :
Alphabaculovirus
Betabaculovirus
Deltabaculovirus
Gammabaculovirus
INTRODUCTION

3. Baculovirus structure
•Virions exist in two forms:
1. Budded Virus (BV) nucleocapsids budded from host cells envelope.
Involved in secondary infection
2. Polyhedra-derived Virus (PDV)
nucleocapsids packaged into polyhedra (“occlusion bodies”)
Stable in external environment .

4. •Circular, double stranded DNA genome.
•A number of small repeated sequences
•known as homologous regions (hrs)
interspersed in the genome.
•Hrs enhance early gene transcription
and also to act as origins of replication.
Autographa californica multiple nuclear polyhedrosis virus (AcMNPV)
Genome

5. • Species-specific tropisms among the invertebrates with over 600 host
species.
• Immature (larval) forms of moth species are the most common hosts, but
these viruses have also been found infecting sawflies, mosquitoes, and
shrimp.
• Not replicate in mammalian or other vertebrate animal cells.
Host Range

6. B)Secondary infection
•Ingestion
•Uncoating of polyhedra
•Fusion with midgut cells
•Viral replication in nucleus
•Budded virus is released to infect
systemically
Replication
A ) Viral infection

7. • Immediate early
• Delayed early
• Late
• Very late
Expression of viral transregulators and genes
which do not require transregulators.
Expression of genes involved in the replication
of the virus and manipulation of the host.
Characterised by shutdown of the host cell
DNA replication and protein synthesis. BV is
produced and disseminates
Virions become occluded in the protein
polyhedrin.
Viral proteases liquefy the host and
degrade the chitinous exoskeleton.
Occluded progeny virus is released
onto surrounding material for horizontal
spread.

9. Baculovirus as an Expression Vector………..
• Many non-essential genes
- may be replaced by gene of interest
Baculovirus Expression Vector System
• The resulting recombinant Baculovirus lacks one of
nonessential gene (polh, v-cath, chiA etc.) replaced with foreign
gene
• Powerful viral promoters
- particularly for late (L) and very late (VL) phase genes

10. Construction of recombinant AcMNPV
1. Transfer Vector Method
Co-transfect expression plasmid with second plasmid containing
required viral genes,
co-transfected cells produce virus, which is then amplified
2. Bac-to-bac system
Transform E. coli containing bacmid DNA and directly infect insect cells which
produce virus, amplify virus
Three cell types generally used : Sf9, Sf21, High-Five. Late very promoter
(p10+polyhedrin) drives expression in last stages of virus cycle

11. Homologous Recombination
•Transfer vector – pVL 1393, pVL1392 (9.8kb)
Features
•Multiple cloning sites
•Recombination sequences for insetion in to the Baculovirus genome
•Ployhedrin enhancer- promoter
•Ampiciline resistance gene for selection

13. Generating a recombinant Virus by Site Specific Transposition
(Bac-to-Bac expression system)

14. Steps in recombinant baculovirus production
• Clone the gene of interest in pfast Bac donor plasmid
• Expression cassette in pfast Bac is flanked by left and right arms of Tn7
• Cloned pfast Bac is transformed in E.coli host strain (DH10Bac) which contains a
baculovirus shuttle vector bacmid having a mini-attTn7 target site
• Transposition occurs between the mini-att Tn7 target site to generate a recombinant
bacmid
• PCR amplification using M-13 Forward and Reverse primers

15. Transposition
Gene of Interest
Tn7R p10 Gent+ Tn7L
Gene
construct
Gene of Interest
Tn7 R
PpH Tn7 L
pfast Bac with insert
•pFASTBACTM vector
•Site specific transposition
with Tn7

16. Transform recombinant plasmid in to DH10BACTM competent cells which contain the Bacmid
•Mini-Tn7 element on the pFASTBAC plasmid can transpose to the mini-attTn7 target site on
theBacmid
•Helper plasmid facilitate transposition by transposition proteins
•Transformation of the recombinant plasmid
•Identification by Antibiotic selection and blue white screening.

17. Culture with XGal+ IPTG
Kanamycin
Gentamicin
Tetracycline
white colonies, where X-gal is not hydrolyzed,
presence of an insert in lacZα
•Isolation of Bacmid DNA from DH10BAC with the CONCERT high purity plasmid miniprep
system according to producer protocol.

18. Transfection
9x105 Sf9 Cells per well
2ml of Sf-900 II SFM
1hour incubation at 28C
A. Bacmid DNA (5μl)
Sf-900 II SFM (100 μl)
B. CELLFECTIN Reagent(1.5-9 μl)
Sf-900 II SFM (100 μl)
Mix B in to A (keep15min @
room T.)
(Allow to form Lipid/ DNA
complex)
Dialute with Sf-900 II SFM (0.8 ml)
Over lay Lipid/ DNA complexes on to washed cells
Incubate 5hr in 27C
Remove transfection mixture. Add 2ml Sf-900 II SFM per well
& incubate at 27C for 72 h.
Harvest the virus at 72 h post transfection

19. Virus plaque Assay
• The infectious potency of stock of Baculovirus is determined
– In an immobilized monolayer culture (using agarose overlay)
• Identifying the plaques
– Wild-type
• Decreased cell density , and enlarged nuclei.
• Many large, dark, angular occlusion bodies in the nuclei.
– Recombinant
• The milky gray plaques (Small and low contrast)
• By staining with neutral red solution
• By recombinant expressing chromotogenic markers
(luciferace/B galactocidase/ Bluo-gal & X-gal

20. Insect Cell Culture
• Invertebrate cells
• Insect cells grow in 25 o C to 28o C
• pH of the growth medium, 6-6.4
• Non-CO2 equilibration and open capped culture system
• Osmolality within 345-380 mOsm/kg
Insect Species Cell Line
Spodoptera frugiperda Sf9
Spodoptera frugiperda Sf-21
Trichoplusia ni Tn-368
Trichoplusia ni High-Five™ BTI-TN-5B1-4
Table: Insect cell lines commonly used in BEVS applications.

21. Cell density >2x106 viable cells/mL >80% confluency
Culture vessel 125 or 250 mL Erlenmeyer
flask containing 35-50mL
or 75-100mL cell
suspension respectively
T-75 cm2 or T-162 cm2 T
flask
Total working volume of
15-20mL or 40-50mL
Seeding density 3 to 5 x106 Viable cells/ mL 2 to 5 x104 Viable cells/cm2
Incubation condition 28oC non-humidified,
ambient air-regulated
incubator or warm room
on an orbital shaker (125-
150rpm)
28oC non-humidified,
ambient air-regulated
incubator
Table: Recommended conditions

22. Types of Insect cell lines
cells Doubling
time
Cell appearance Medium Origin Type of
culture
Sf 9 72 hrs Spherical, granular,
regular in size, firm
attachment to
surface
TNM-FH IPLBSF-21 cell
lines of the fall
army worm
spodoptera
frugiperda
Grow well as
monolayer
and
suspension
Sf 21 24 hrs Spherical, granular,
different in size,
firm attachment to
surface
TNM-FH IPLBSF-21 cell
lines of the fall
army worm
spodoptera
frugiperda
Grow well as
monolayer
and
suspension
High-five 18 hrs Spherical, granular,
regular in size, loose
attachment to
surface
Express five
SFM
Ovarian cells
of cabbage
looper
Grow well as
monolayer,
also as
suspension

23. • One-Step purification and Amplification
• Higher purity of the isolated recombinant viral DNA
– Not mixed with parental or nonrecombinant viruses
• Rapid and simultaneous isolation of multiple recombinant viruses
Advantages of Bac-to-Bac expression system over Homologous
recombination

24. Comparison

25. • Safety
– baculoviruses are essentially nonpand plantsathogenic to mammals
• Ease of scale up
– Reproducibly scaled up for the large scale production of recombinant
products
• High level of recombinant gene expression
• Accuracy
• Ideal for suspension culture
Advantages of BAVS Technology

26. THANK YOU