Akademik Sunum

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Sıra	DOSYA ADI	Format	Bağlantı
01.	Clone Cloning Genomics Recombinant	ppt	Sunumu İndir Sunuyu İndir Sunumu İndir

Transkript

[1]

Generation of Gateway clone library of virulence associated genes of zoonotic buffalopox virus: state-of-the-art resource for proteome analysis Veterinary Type Culture Collection, National Research Centre on Equines, Hisar, Haryana, IndiaB.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) repositoryObjectiveICAR created this facility for conservation of microbes of veterinary importanceAct 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 proteomesNew 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 proteinsResources for functional genomics projectsWhy ORF clones ?

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Proteins profilesORFeomeLocalizomePhenomeTranscriptomeInteractomeProteomeCloned ORFsMutational phenotypesExpression profilesProtein interactions1 2 3 4 5 nDNA Interactome Protein-DNA interactionsCellular, tissue locationORFeome: Gateway b/w Genomics & Omes

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Functional genomics- Proteomic studies To study host-tropism (molecular pathogenesis) To develop drugs & vaccinesWhy 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 7Buffalopox virus

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Severe cases BPXV ZoonosisIn 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/ligationEfficiency: 100% - only one recombinant DNA product without byproductsNo cloning step needed: no need to assay independent clonesVery precise recombination system allowing high fidelity DNA engineeringVersatile 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 systemGeneration of ORF clones by Recombinational cloning

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attPattBattBattL attRphageBacterial genomeExcisionIntegrationphageLambda phage integration & excision system

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attB2 partial attB2 partialViral DNAstart stopPartial attB1PCR1ORFattB1 partialDesigned primers ORFs of virulence associated genes of BPXVORFComplete attB2Complete attB1PCR2Gateway cloning strategy

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Genes of BPXV FunctionsVaccinia 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 defenseJanuary 19, 2020 © VTCC - Hisar 12Targeted BPXV-ORFs

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PCR amplifications of BPXV-ORFs1kb 1kbM 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 MM 1 2 3 4 5 6 7 8 9 10 11 12 13 14 M1kb 1kbM = 1kb DNA marker, L1-18 & L1-14 = amplicons of ORFs of BPXV

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PCR 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 161kb1kbM = 1kb DNA marker, L1-14 & L1-16 = amplicons of ORFs of BPXV

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Phage lambda integration:Integrase & bacterial IHFORF attB2attB1ccdB attP2attP1pDONR221ORF attL2attL1Entry clones ccdBBP reactionBy productE. Coli transformationORFattL1 attL2PCR verificationSequencing & ORF identification (BLAST)Preservation of validated clones in the repositoryConstruction of Entry clones

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Generated gateway entry clones 50 gateway entry clones of BPXV-ORFs generatedAll 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 attR2attR1Destination vector- pDEST17AmpRvORFattL2attL1Entry clones- pDONR221-vORFKanRLR reaction Phage lambda excision:Integrase, IHF & ExisionasevORF attB2attB1Destination vector- pDEST17-vORFAmpRccdB attP2attP1By productKanRE. Coli transformationSelection of Amp resistant clonesExpression of recombinant proteinRecombinational cloning into destination vector

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170 kDa130 kDa95 kDa72 kDa55 kDa43 kDa34 kDa26 kDa~32 kDa rA38LExpression of A39R protein

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ConclusionGenerated 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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January 19, 2020 © VTCC - Hisar 20Thank you for kind attention

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APPLICATIONS OF RECOMBINATIONAL CLONING Reprinted from: Dupuy et. al., Genome Research 14:2169-2175 (2004)