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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 Expression SystemsBacterial expression system (e.g. E.coli )Yeast expression system (e.g S.cervesiae)Viral expression system (e.g Baculovirus)Mammalian cell expression system
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
Historical backgroundThe history of the discovery of baculoviruses isintimately related to the development of the silkindustry that occurred in China as early as 5000 yearsago. 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 Fig. 1.1 Baculovirus occlusion Deltabaculovirus bodies. Scanning EM by K. Hughes Gammabaculovirus 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 environment2. Budded Virus (BV) nucleocapsids budded from host cells envelope Involved in secondary infection Fig : Electron micrograph of ODV
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
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.
Replication • Ingestion • Uncoating of polyhedra A ) Viral • Fusion with midgut cells infection • Viral replication in nucleus • Budded virus is released to infectB ) Secondary systemically infection
ReplicationFig : An general overview of the replication cycle of Baculoviruses.
Replication Phase DescriptionImmediate Expression of viral transregulators and genes which do notearly 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 disseminatesVery 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 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 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
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
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
Figure… Tn7R p10 Gent+ Tn7L Gene of Interest Gene construct Gene of Interest PpH Tn7 L Tn7 R pfast Bac with insert
Contd.. Tn7R GOI Tn7L Transposed pfast Bac 128bp 145bp Bacmid DNA M 13 forward Mini att Tn7 M 13 reverse
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) whichcontains a baculovirus shuttle vector bacmid having a mini-attTn7 target siteHelper 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
Condt…….PCR amplification using M-13 Forward and Reverse primersIf no transposition, then a region a bacmid alone will amplify to gave product of 300bpIn condition of transposition then the amplified size will be 2300bp+size of insertRecombinant bacmid is now ready to transfect to insect cell lines
cells Doublin Cell appearance Medium Origin Type of g time cultureSf 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 suspensionSf 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 suspensionHigh- 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
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 cellsCell growth Rapid Rapid Slow SlowComplexity of growth Minimum Minimum Complex ComplexmediumCost of growth medium Low Low High HighExpression level High Low-high Low-high Low-moderateExtracellular Expression Secretion to Secretion to Secretion to medium Secretion to medium periplasm mediumPost translationalmodificationsProtein folding Refolding usually Refolding may Proper folding Proper folding required be requiredN-linked glycosylation None High mannose Simple, No Sialic acid ComplexO-linked glycosylation No Yes Yes YesPhosphorylation No Yes Yes YesAcetylation No Yes Yes YesAcylation No Yes Yes YesGamma Carboxylation No No No Yes
Comparison of Expression SystemsCharacteris Adenovirus Retrovirus Lentivirus Adeno Baculovirustics associated virusInfectivity Infects Infects Infects non Infects both Infects dividing cells dividing cells dividing dividing and dividing cells cells non-dividing cellsMajor Gene Therapy Gene Gene Gene Gene therapyapplications and Therapy and Delivery Therapy and vaccine vaccination vaccination (Vectors) productionSize 7.5 kb 8kb 8kb 4.5kb >15kbMode of Does not provirus, Integrates in Integrates inaction integrate in remains and host genome host genome host genome passed on to the cell progenyHost Strong Moderate Moderate Mild Mild
Advantages Promoter of Could be Post Capable of the replacedtranslational producing polyhedrin with amodification cytotoxic gene is very heterologou s proteins strong s gene
Condt……… Correct HasCan express glycosylatio chaperonins Very high large n & signal to help fold yields, proteins peptide “tough” cheap (>50 kD) removal prtns
Limitations of BEVS Glycosylation in insect cells large fraction,different from RP poorly Inefficient for Discontinuousvertebrate cells, processed and commercial expression a problem for accumulates scale. therapeutic aggregates. proteins.
Condt…….Name of vaccine Proteins Expressed RemarksSwine Fever Vaccine Gp 55 Induce immunity and protection against virulent CSFV.Influenza A/H1N1 Vaccine Heamagglutinin Alternatively Neuraminidase and MatrixEquine influenza strain, Hemagglutinin (HA)A/equine/LP/93Rota Virus Like Particle VP2, VP6, and VP7 Sf9 cell, a host of the baculovirus.Blue Tongue Vaccine NS2Virus Like hemagglutinin (HA), VLPs elicit antibodies thatParticle neuraminidase (NA), and recognize a broader panel of matrix (M1). 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
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;
Thank You for attention…..……….Standby for discussion !!!!!