generation of gateway clone library of virulence associated genes of zoonotic buffalopox virus:...
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Generation of Gateway clone library of virulence Generation of Gateway clone library of virulence associated genes of zoonotic buffalopox virus: associated genes of zoonotic buffalopox virus: state-of-the-art resource for proteome analysis state-of-the-art resource for proteome analysis
Veterinary Type Culture Collection, National Research Centre on Equines, Hisar, Haryana, India
B.C. Bera, Taruna Anand, Sanjay Barua, R. K. Vaid, Nitin Virmani, Riyesh T. & Praveen Malik
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Generation of repository of Open Reading Frames (ORFs) clones – ORFeome of zoonotic buffalopox virus in Veterinary Type Culture Collection (VTCC) repository
ObjectiveObjective
ICAR created this facility for conservation of microbes of veterinary importance
Act as biological resource bank for R&D
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Refers to the libraries of complete set of clones of protein-coding open reading frames (ORFs)
Collection of plasmids containing ORFs of a genome
Flexible & versatile library allows transfer of ORFs into different destination vectors
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Advent of systems biology necessitates the cloning of nearly entire sets of ORFs to allow functional studies of proteomes
New challenges in post genomic era:to understand the function of the many genes predictedAnalysis of all genes at a timehigh-throughput preparation of versatile resource for the
functional and structural studies of proteins
Resources for functional genomics projects
Why ORF clones ?Why ORF clones ?
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Proteins profiles
ORFeome
Localizome
Phenome
Transcriptome
Interactome
Proteome
Cloned ORFs
Mutational phenotypes
Expression profiles
Protein interactions
1 2 3 4 5 n
DNA Interactome Protein-DNA interactions
Cellular, tissue location
ORFeome: Gateway b/w Genomics & OmesORFeome: Gateway b/w Genomics & Omes
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Functional genomics- Proteomic studies
To study host-tropism (molecular pathogenesis)
To develop drugs & vaccines
Why ORFeome of animalpox viruses?Why ORFeome of animalpox viruses?
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Zoonotic infections Reduction of cohort- immunity against poxviruses in humans
Discontinuation of vaccination against smallpox since 1980
Change of host tropism (inter-species jumping)
BPXV – Human & Cow
© VTCC - Hisar 7
Buffalopox virus
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Severe cases BPXV ZoonosisSevere cases BPXV Zoonosis
In 2011: Meerut, U.P. In 2013: BPXV Nashik, M.P.
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One step site specific recombination based cloning technology
Not dependent on restriction/ligation
Efficiency: 100% - only one recombinant DNA product without byproducts
No cloning step needed: no need to assay independent clones
Very precise recombination system allowing high fidelity DNA engineering
Versatile cloning technology: Genes can be easily transferred into a range of vector systems Expression, Gene fusion, RNAi…
GATEWAY Recombinational Cloning Based on the bacteriophage lambda integration & excision system
Generation of ORF clones by Recombinational Generation of ORF clones by Recombinational cloning cloning
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attP
attB
attB
attL attR
phage
Bacterial genome
Excision
Integration
phage
Lambda phage integration & excision system Lambda phage integration & excision system
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attB2 partial
attB2 partialViral DNAstart stopPartial attB1
PCR1
ORF
attB1 partial
Designed primers ORFs of virulence associated genes of BPXV
ORF
Complete attB2Complete attB1
PCR2
Gateway cloning strategyGateway cloning strategy
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Genes of BPXV Functions
Vaccinia virus homologue genes:
CrmB, CKBP, INFA, IL-18, C7L, C3L, ZFA, N1L, K1L, K2L, K3L, B29R, K7R, A39R, A46R, B5R, VACWR208, L5R, H1L, H2R, H3L, VACWR217, A9L, A17L, A21L, VACWR207, A28L, B1R, N2L, F3L, F10L, F34L, A36L, A38L, A40R, A43R, A44L, A46R, A55R, B4R, B6R, B8R, B12R, B13R, B19R, B25R, C12L, M1L, A56R, B18R
Modulation of host immune defense
April 21, 2023 © VTCC - Hisar 12
Targeted BPXV-ORFs Targeted BPXV-ORFs
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PCR amplifications of BPXV-ORFsPCR amplifications of BPXV-ORFs
1kb 1kb
M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 M
M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 M
1kb 1kb
M = 1kb DNA marker, L1-18 & L1-14 = amplicons of ORFs of BPXV
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PCR amplifications of BPXV-ORFsPCR amplifications of BPXV-ORFs
M 1 2 3 4 5 6 7 8 9 10 11 12 13 14
M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
1kb
1kb
M = 1kb DNA marker, L1-14 & L1-16 = amplicons of ORFs of BPXV
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Phage lambda integration:Integrase & bacterial IHF
ORF attB2attB1ccdB attP2attP1
pDONR221
ORF attL2attL1
Entry clones ccdB
BP reaction
By product
E. Coli transformation
ORFattL1 attL2
PCR verification
Sequencing & ORF identification (BLAST)
Preservation of validated clones in the repository
Construction of Entry clonesConstruction of Entry clones
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Generated gateway entry clonesGenerated gateway entry clones
50 gateway entry clones of BPXV-ORFs generated
All clones validated by sequencing and BLAST analysis
Clones preserved in the VTCC repository: 5 clones (recombinant E.coli) of each ORF stored as
glycerol stock at -80oC
Purified recombinant plasmids stored at -80oC as ethanol precipitate
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ccdB attR2attR1
Destination vector- pDEST17
AmpR
vORF
attL2
attL1
Entry clones- pDONR221-vORF
KanR
LR reaction Phage lambda excision:Integrase, IHF & Exisionase
vORF attB2attB1
Destination vector- pDEST17-vORF
AmpR
ccdB attP2
attP1
By productKanR
E. Coli transformation
Selection of Amp resistant clones
Expression of recombinant protein
Recombinational cloning into destination vectorRecombinational cloning into destination vector
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170 kDa
130 kDa
95 kDa
72 kDa
55 kDa
43 kDa
34 kDa
26 kDa
~32 kDa rA38L
Expression of A39R protein Expression of A39R protein
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ConclusionConclusion
Generated entry clone resource of 50 ORFs of virulence associated genes of buffalopox virus -
Platform for functional genomics
Basic biology: molecular networks, structural &
functional analysis
Understanding pathogenesis: virus−host interaction
Identifying vaccine candidates : reverse vaccinology
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April 21, 2023 © VTCC - Hisar 20
Thank you for kind attention
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APPLICATIONS OF RECOMBINATIONAL CLONING
Reprinted from: Dupuy et. al., Genome Research 14:2169-2175 (2004)