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)