BACULOVIRUS EXPRESSION SYSTEM

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

Viral Classification

•Group: Group I (dsDNA) •Family: Baculoviridae •Subfamily :

Nucleopolyhedrosis Virus (NPV) •Genera :

Alphabaculovirus Betabaculovirus Deltabaculovirus Gammabaculovirus

INTRODUCTION

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 .

•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

• 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

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

• 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.

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

Construction of recombinant AcMNPV

1. Transfer Vector MethodCo-transfect expression plasmid with second plasmid containing required viral genes, co-transfected cells produce virus, which is then amplified

2. Bac-to-bac systemTransform E. coli containing bacmid DNA and directly infect insect cells which produce virus, amplify virusThree cell types generally used : Sf9, Sf21, High-Five. Late very promoter (p10+polyhedrin) drives expression in last stages of virus cycle

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

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

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

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

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.

Culture with XGal+ IPTGKanamycinGentamicinTetracycline

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.

Transfection

9x105 Sf9 Cells per well2ml 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

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

Insect Cell Culture

• Invertebrate cells

• Insect cells grow in 25 o C to 28 o 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.

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 flaskTotal 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

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 fiveSFM

Ovarian cells of cabbage looper

Grow well as monolayer, also as suspension

• 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

Comparison

• 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

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