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
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
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
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 ?
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
Functional genomics- Proteomic studies
To study host-tropism (molecular pathogenesis)
To develop drugs & vaccines
Why ORFeome of animalpox viruses?Why ORFeome of animalpox viruses?
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
Severe cases BPXV ZoonosisSevere cases BPXV Zoonosis
In 2011: Meerut, U.P. In 2013: BPXV Nashik, M.P.
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
attP
attB
attB
attL attR
phage
Bacterial genome
Excision
Integration
phage
Lambda phage integration & excision system Lambda phage integration & excision system
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
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
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
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
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
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
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
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
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
April 21, 2023 © VTCC - Hisar 20
Thank you for kind attention
APPLICATIONS OF RECOMBINATIONAL CLONING
Reprinted from: Dupuy et. al., Genome Research 14:2169-2175 (2004)