1. Baculovirus mediated gene
expression and its
veterinary applications
Presented by :
Dr.Madhuvanti V. Mahajan
M.V.Sc Scholar
ABT,1246
BVC,Mumbai
2. Overview
Introduction
Various expression systems
Baculovirus
Viruses as expression systems
Baculovirus as expression systems
Advantages of BEVS
Limitations of BEVS
Applications in veterinary science
Current status of BEVS
Future Prospects
Conclusions
3. Introduction
The vector designed for the expression of i.e,
production of protein specified by the DNA insert,
is called as Expression Vector.
Expression system - A system in which a cloned
gene can be expressed.
4. Condt……
The increasing demand for production and
characterization of diverse groups of recombinant proteins
necessitates the analysis of several constructs and fusion
tags in a variety of expression systems.
For this a number of different types of vector systems have
been developed. Viz,
6. Various Expression Systems
Bacterial expression system (e.g. E.coli )
Yeast expression system (e.g S.cervesiae)
Viral expression system (e.g Baculovirus)
Mammalian cell expression system
7. Expression System Selection
– Large proteins (>100 • Eukaryote
kD)?
– Small proteins (<30 • Prokaryote
kD)?
– Glycosylation ? • Baculovirus or mammalian cell
culture
– High yields, low • E. coli
cost?
– Post-translational • Yeast or baculovirus or mammalian
modifications? cells
8.
9. Historical background
The history of the discovery of baculoviruses is
intimately related to the development of the silk
industry that occurred in China as early as 5000 years
ago.
By the 12th century it was established in Italy and
Spain and eventually spread to France and England
and to Mexico by the 1500s.
Since the 1990s they have been utilized for producing
complex eukaryotic proteins in insect cell cultures
More recently baculoviruses can transduce
mammalian cells when found that a suitable
promoter is present
10. Viral Classification
Group: Group I (dsDNA)
Family: Baculoviridae
Subfamily :
Nucleopolyhedrosis Virus (NPV)
Genera :
Alphabaculovirus
Betabaculovirus Fig. 1.1 Baculovirus occlusion
Deltabaculovirus bodies. Scanning EM by K. Hughes
Gammabaculovirus
and R.B.Addison.
11. Viral Morphology
Virions exist in two forms:
1. Polyhedra-derived Virus (PDV)
nucleocapsids packaged into
polyhedra (“occlusion bodies”)
Stable in external environment
2. Budded Virus (BV) nucleocapsids
budded from host cells envelope
Involved in secondary infection
Fig : Electron micrograph of ODV
12. Genome
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.
A diagram of the Eppo MNPV genome map
13. Host Range
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.
They are not known to replicate in mammalian or other
vertebrate animal cells.
14.
15.
16. Replication
• Ingestion
• Uncoating of polyhedra
A ) Viral • Fusion with midgut cells
infection • Viral replication in nucleus
• Budded virus is released to infect
B ) Secondary systemically
infection
17.
18. Replication
Fig : An general overview of the replication cycle of Baculoviruses.
19. Replication
Phase Description
Immediate Expression of viral transregulators and genes which do not
early require transregulators.
Delayed early Expression of genes involved in the replication of the virus
and manipulation of the host.
Late Characterised by shutdown of the host cell DNA replication
and protein synthesis.
BV is produced and disseminates
Very late Virions become occluded in the protein polyhedrin.
Viral proteases liquefy the host and degrade the chitinous
exoskeleton.
Occluded progeny virus is disseminated onto surrounding
material for horizontal spread.
21. Baculovirus Expression Vector System
Many non-essential genes
- may be replaced by gene of interest
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
22. History……
• To express recombinant genes controlled by
1983 strong insect-virus promoters in their natural
host (insect) cells
• It was first published that recombinant
1995 baculoviruses are able to deliver genes into
mammalian cells.
• Today this is a well-established and easy to
handle system for producing large quantities of
recombinant proteins for numerous purposes
• These genes are expressed provided that they are
controlled by a promoter which is active in
mammalian cells
23. Baculovirus as an Expression Vector
Autographa californica
The virus was originally isolated from the alfalfa looper.
Is multiple nuclear polyhedrosis virus (AcMNPV), which relies
on the lepidopteran species
a) Spodoptera frugiperda and
b) Trichoplusia ni
Fig :Sf9 cell line
24. Condt……
The major capsid protein VP39 together with some minor
proteins forms the nucleocapsid, that encloses the DNA
with p6.9 protein.
BV acquires its envelope from the cell membrane and
requires a glycoprotein, gp64, to be able to spread systemic
infection.
This protein forms structures called peplomers on one end
of the budded virus particle but is not found on ODV.
25. • Spodoptera frugiperda (Sf9, Sf21)
Fall armyworm
Isolated from pupal ovarian tissue
Spodoptera frugiperda
Trichoplusia ni (High Five)
Cabbage looper
Isolated from egg cell homogenates
Trichoplusia ni
28. Figure…
Tn7R p10 Gent+ Tn7L
Gene of Interest Gene construct
Gene of Interest
PpH Tn7 L
Tn7 R
pfast Bac with insert
29. Contd..
Tn7R GOI Tn7L
Transposed pfast Bac
128bp 145bp
Bacmid DNA
M 13 forward Mini att Tn7 M 13 reverse
30. 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
Helper plasmid which allows to transpose the gene of interest from pfast to
bacmid (shuttle vector)
Transposition occurs between the mini-att Tn7 target site to generate a
recombinant bacmid
31. Condt…….
PCR amplification using M-13 Forward and Reverse primers
If no transposition, then a region a bacmid alone will amplify
to gave product of 300bp
In condition of transposition then the amplified size will be
2300bp+size of insert
Recombinant bacmid is now ready to transfect to insect cell
lines
32. cells Doublin Cell appearance Medium Origin Type of
g time culture
Sf 9 72 hrs Spherical, granular, TNM-FH IPLBSF-21 cell Grow well
regular in size, firm lines of the fall as
attachment to surface army worm monolayer
spodoptera and
frugiperda suspension
Sf 21 24 hrs Spherical, granular, TNM-FH IPLBSF-21 cell Grow well
different in size, firm lines of the fall as
attachment to surface army worm monolayer
spodoptera and
frugiperda suspension
High- 18 hrs Spherical, granular, Express Ovarian cells of Grow well
five regular in size, loose five cabbage looper as
attachment to surface SFM monolayer,
also as
suspension
36. Comparison of Expression Systems
(Gene Expression Systems. Using nature for the art of expression (Fernandez, J.M. & Hoeffler, J.P., eds), Academic Press, San Diego, 1999)
Characterstics E.coli Yeast Baculovirus Mammalian cells
Cell growth Rapid Rapid Slow Slow
Complexity of growth Minimum Minimum Complex Complex
medium
Cost of growth medium Low Low High High
Expression level High Low-high Low-high Low-moderate
Extracellular Expression Secretion to Secretion to Secretion to medium Secretion to medium
periplasm medium
Post translational
modifications
Protein folding Refolding usually Refolding may Proper folding Proper folding
required be required
N-linked glycosylation None High mannose Simple, No Sialic acid Complex
O-linked glycosylation No Yes Yes Yes
Phosphorylation No Yes Yes Yes
Acetylation No Yes Yes Yes
Acylation No Yes Yes Yes
Gamma Carboxylation No No No Yes
37. Comparison of Expression Systems
Characteris Adenovirus Retrovirus Lentivirus Adeno Baculovirus
tics associated
virus
Infectivity Infects Infects Infects non Infects both Infects
dividing cells dividing cells dividing dividing and dividing cells
cells non-dividing
cells
Major Gene Therapy Gene Gene Gene Gene therapy
applications and Therapy and Delivery Therapy and vaccine
vaccination vaccination (Vectors) production
Size 7.5 kb 8kb 8kb 4.5kb >15kb
Mode of Does not provirus, Integrates in Integrates in
action integrate in remains and host genome host genome
host genome passed on to
the cell
progeny
Host Strong Moderate Moderate Mild Mild
38. Advantages
Promoter of Could be
Post Capable of
the replaced
translational producing
polyhedrin with a
modification cytotoxic
gene is very heterologou
s proteins
strong s gene
39. Condt………
Correct Has
Can express
glycosylatio chaperonins Very high
large
n & signal to help fold yields,
proteins
peptide “tough” cheap
(>50 kD)
removal prtns
40. Limitations of BEVS
Glycosylation
in insect cells large fraction
,different from RP poorly Inefficient for
Discontinuous
vertebrate cells, processed and commercial
expression
a problem for accumulates scale.
therapeutic aggregates.
proteins.
46. Condt…….
Name of vaccine Proteins Expressed Remarks
Swine Fever Vaccine Gp 55 Induce immunity and
protection against virulent
CSFV.
Influenza A/H1N1 Vaccine Heamagglutinin Alternatively Neuraminidase
and Matrix
Equine influenza strain, Hemagglutinin (HA)
A/equine/LP/93
Rota Virus Like Particle VP2, VP6, and VP7 Sf9 cell, a host of the
baculovirus.
Blue Tongue Vaccine NS2
Virus Like hemagglutinin (HA), VLPs elicit antibodies that
Particle neuraminidase (NA), and recognize a broader panel of
matrix (M1). antigenically distinct viral
isolates compared to other
vaccines in the HAI
47.
48. Gene therapy
It is applied in human medicine to a greater extent.
viz., Treatment of diabetes, hepatitis etc.
However, in veterinary sciences it is yet……..
49. Blue Tongue Diagnosis
Antigen Capture Competitive Enzyme-Linked
Immunosorbent Assays Using Baculovirus-Expressed
Antigens for Diagnosis of Bluetongue Virus and
Epizootic Hemorrhagic Disease Virus .
(J. O. Mecham et al. 2003)
The genes coding for VP7 of BTV-11 and EHDV2 are
reverse transcribed
50. FMD diagnosis
The baculovirus expressed 2C acts as a suitable antigen
for the development of a reliable diagnostic test.
The sera of convalescent animals contain antibodies to 2C,
a highly conserved non-structural protein, whereas the sera
of vaccinated animals do not.
51. Nipah virus glycoprotein production
(M. Eshaghi, et al.2004)
DNA encoding truncated G protein of NiV is cloned
into the pFastBac HT vector, and the fusion protein to
His-tag is expressed in insect cells by recombinant
baculovirus.
The resulting His-G recombinant fusion protein is
purified by affinity chromatography and used as the
coating antigen for serological testing by indirect
enzyme-linked immunosorbant assay (ELISA).
52. Economized large-scale production of high yield
of rAAV
Large-scale production of rAAV remains one of the
major challenges for continued development of pre-
clinical and clinical studies, and for its potential
commercialization.
This technology uses three different BEVs (Bac-Rep,
Bac-GFP, and Bac-VP).
53. Miscellaneous…..
The baculovirus-insect cell expression system is
widely used to produce recombinant proteins,
including glycoproteins, for various biomedical
applications.
Serological diagnosis of equine influenza using the
hemagglutinin protein produced in a baculovirus
expression system (Takeo Sugiura et al. 2001)
54. Current Status of BEVS…
Recent advances in baculovirus expression vector
technology include improvements to methods for the
selection of recombinant viruses and further
developments in virion display vectors.
Baculovirus vectors are continue to be modified to
facilitate gene expression in mammalian cells.
55. Current Status…….
The broad recognition and acceptance by the scientific
community is reflected in the determination by The
Institute of Scientific Information (ISI) that Dr.
Summers is one of the top 250 most highly cited
microbiologists in the world.
Acceptance of the BEVS by the private sector is reflected
by the commercial licenses worldwide that are held for
the BEVS technology (currently >70).
56. Conclusions
Baculovirus is one of the most important eukaryotic
expression system.
This system is capable of generating large quantities of
biologically active recombinant protein inexpensively
and quickly.
The efficiency, low cost and large-scale production of
proteins using BEVS represents breakthrough
technology that is facilitating high-throughput
proteomic studies.
57. Condt……
To date, over a thousand proteins have been expressed
using the BEVS, with 98% being biologically active.
Baculovirus has advantage to permit post translational
modifications of the protein expressed
Recombinant baculovirus have become widely used as
vectors to express heterologous genes in cultured insect
cells and insects larvae.
This gene produces up to 60% of the total protein of the
virus and can be replaced by foreign genes.
58. Condt……
Baculoviruses are noninfectious to vertebrates and
their promoter have been shown to be inactive in
most mammalian cells.
This trait gives them a real advantage in molecular
biology when the protein is destined for
diagnostic, targeted gene therapy or in medicine
59. Condt…..
The BEVS has become a core technology for:
1) The cloning and expression of genes for study of protein
structure, processing and function;
2) The production of biochemical reagents;
3) The study of regulation of gene expression;
4) The commercial exploration, development and production of
vaccines, therapeutics and diagnostics;
5) Drug discovery research;
60. Thank You for attention…..
……….Standby for discussion !!!!!
