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Baculovirus mediated gene expression and its
veterinary applications
Presented by : Dr.Madhuvanti V. Mahajan
M.V.Sc Scholar ABT,1246
BVC,Mumbai
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
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.
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,
Various Vector 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
Various Expression Systems
Expression System Selection • Eukaryote – Large proteins (>100
kD)?
• Prokaryote – Small proteins (<30 kD)?
• Baculovirus or mammalian cell culture
– Glycosylation ?
• E. coli – High yields, low cost?
• Yeast or baculovirus or mammalian cells
– Post-translational modifications?
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
Viral Classification
Group: Group I (dsDNA)
Family: Baculoviridae
Subfamily : Nucleopolyhedrosis Virus (NPV)
Genera : Alphabaculovirus
Betabaculovirus
Deltabaculovirus Gammabaculovirus
Fig. 1.1 Baculovirus occlusion bodies. Scanning EM by K. Hughes
and R.B.Addison.
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
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.
Genome
A diagram of the Eppo MNPV genome map
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.
Host Range
Replication
A ) Viral infection
• Ingestion
• Uncoating of polyhedra
• Fusion with midgut cells
• Viral replication in nucleus
B ) Secondary infection
• Budded virus is released to infect systemically
Replication
Fig : An general overview of the replication cycle of Baculoviruses.
Replication
Phase
Description
Immediate early
Expression of viral transregulators and genes which do not 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.
Baculovirus as an Expression Vector………..
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
History…… • To express recombinant genes controlled by
strong insect-virus promoters in their natural host (insect) cells 1983
• It was first published that recombinant baculoviruses are able to deliver genes into mammalian cells. 1995
• 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
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
Baculovirus as an Expression Vector
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.
• 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
The
baculovirus
expression
vector
system
(BEVS)
Steps in recombinant baculovirus production
Figure…
Gene of Interest
Tn7R p10 Gent+ Tn7L
Gene construct
Gene of Interest
Tn7 R
PpH Tn7 L
pfast Bac with insert
Contd..
128bp 145bp
Mini att Tn7 M 13 forward M 13 reverse
Tn7R GOI Tn7L
Bacmid DNA
Transposed pfast Bac
Steps in recombinant baculovirus production
Transposition occurs between the mini-att Tn7 target site to generate a recombinant bacmid
Helper plasmid which allows to transpose the gene of interest from pfast to bacmid (shuttle vector)
Cloned pfast Bac is transformed in E.coli host strain (DH10Bac) which contains a baculovirus shuttle vector bacmid having a mini-attTn7 target site
Expression cassette in pfast Bac is flanked by left and right arms of Tn7
Clone the gene of interest in pfast Bac donor plasmid
Condt…….
Recombinant bacmid is now ready to transfect to insect cell lines
In condition of transposition then the amplified size will be 2300bp+size of insert
If no transposition, then a region a bacmid alone will amplify to gave product of 300bp
PCR amplification using M-13 Forward and Reverse primers
cells Doublin
g 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
Why BEVS ?????????
Comparison
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 medium
Minimum Minimum Complex Complex
Cost of growth medium Low Low High High
Expression level High Low-high Low-high Low-moderate
Extracellular Expression Secretion to periplasm
Secretion to medium
Secretion to medium Secretion to medium
Post translational modifications
Protein folding Refolding usually required
Refolding may be required
Proper folding Proper folding
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
Comparison of Expression Systems Characteristics
Adenovirus Retrovirus Lentivirus Adeno associated virus
Baculovirus
Infectivity Infects dividing cells
Infects dividing cells
Infects non dividing cells
Infects both dividing and non-dividing cells
Infects dividing cells
Major applications
Gene Therapy and vaccination
Gene Therapy and vaccination
Gene Delivery (Vectors)
Gene Therapy
Gene therapy and vaccine production
Size 7.5 kb 8kb 8kb 4.5kb >15kb
Mode of action
Does not integrate in host genome
provirus, remains and passed on to the cell progeny
Integrates in host genome
Integrates in host genome
Host Immune Response
Strong Moderate Moderate Mild Mild
Advantages
Post translational modification
s
Promoter of the
polyhedrin gene is very
strong
Could be replaced
with a heterologou
s gene
Capable of producing cytotoxic proteins
Can express large
proteins (>50 kD)
Correct glycosylation & signal
peptide removal
Has chaperonins to help fold
“tough” prtns
Very high yields, cheap
Condt………
Glycosylation in insect cells
,different from vertebrate cells,
a problem for therapeutic
proteins.
large fraction RP poorly
processed and accumulates aggregates.
Discontinuous expression
Inefficient for commercial
scale.
Limitations of BEVS
Baculovirus Successes (National Library of Medicine (US), NCBI;2008)
• Alpha and beta interferon
• Adenosine deaminase
• Erythropoietin
• Interleukin 2
• Poliovirus proteins
• Tissue plamsinogen activator (TPA)
SSX2 tumor antigen (R.G. Kyyamova et. al. 2006)
Vaccines produced……. Blue Tongue disease
Porcine parvovirus
African equine fever
Avian Influenza
H1N1 Vaccine
Rota Virus Vaccine
Swine fever vaccine
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, A/equine/LP/93
Hemagglutinin (HA)
Rota Virus Like Particle VP2, VP6, and VP7 Sf9 cell, a host of the baculovirus.
Blue Tongue Vaccine NS2
Virus Like Particle
hemagglutinin (HA), neuraminidase (NA), and matrix (M1).
VLPs elicit antibodies that recognize a broader panel of antigenically distinct viral isolates compared to other vaccines in the HAI
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……..
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
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.
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).
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).
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)
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.
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).
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.
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.
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
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;
Condt…..
Thank You for attention…..
……….Standby for discussion !!!!!