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Biologicals xxx (2015) 1e7

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Biologicals

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Construction and characterization of yeast two-hybrid cDNA libraryderived from LFBK cell line

Sonalika Mahajan, Gaurav Kumar Sharma, Rakesh Matura, Saravanan Subramaniam,Jajati Keshari Mohapatra, Bramhadev Pattnaik*

Project Directorate on Foot and Mouth Disease, Indian Council of Agricultural Research, IVRI Campus, Mukteswar, Uttarakhand 263138, India

a r t i c l e i n f o

Article history:Received 7 November 2014Accepted 28 January 2015Available online xxx

Keywords:FMDYeast two-hybridcDNA librarySMART

* Corresponding author. Tel.: þ91 5942286004; faxE-mail address: pattnaikb@gmail.com (B. Pattnaik

http://dx.doi.org/10.1016/j.biologicals.2015.01.0031045-1056/© 2015 The International Alliance for Biol

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a b s t r a c t

The cDNA libraries are indispensable and critical tools for performing proteineprotein interactionstudies. In this study, a high quality yeast two-hybrid cDNA library from the LFBK cell line was con-structed and characterized. LFBK cell line was originally derived from the swine kidney cells and is highlysusceptible to foot-and-mouth disease virus (FMDV) infection. The total RNA was extracted from theLFBK cells and the switching mechanism at the 50 end of RNA template (SMART) technique was employedfor the cDNA synthesis. Subsequently, double stranded cDNA was amplified by long-distance PCR,purified and co-transformed with pGADT7-rec vector in yeast strain Y187. The quality parameters of theconstructed library were evaluated to qualify the constructed library. Nucleotide sequencing of therandomly selected clones from the library confirmed the swine genotype of LFBK cell line. The LFBKcDNA library was mated with the 2C protein of FMDV in yeast two-hybrid (YTH) system and severalputative interaction partners were identified in the preliminary screening. The LFBK library was observedto be of high quality and could potentially be applied to protein interaction studies between FMDV andthe host cells using YTH system.

© 2015 The International Alliance for Biological Standardization. Published by Elsevier Ltd. All rightsreserved.

1. Introduction

Foot-and-mouth disease (FMD) is a highly contagious viraldisease of domestic livestock and several wild life species and iscaused by foot-and-mouth disease virus (FMDV), a prototype of thegenus Aphthoviruswithin the family Picornaviridae [1]. FMDV existsas seven immunologically distinct serotypes (A, O, C, Asia 1, SAT1,SAT2, and SAT3) without any cross-protection between them [2].Even though the FMDV is the first animal virus to be discovered, themolecular intricacies of viral pathogenesis are yet to be completelyunderstood [3]. Despite the small genome size of ~8.2 kb, FMDVproteins play plethora of roles during viral replication [4e6]. FMDVproteins interact with the host factors and exploit host cell ma-chinery for the virus survival and replication. Thus, it is of para-mount significance to study the host-virus interactions for furtherinsights into the virus replication and pathogenesis.

: þ91 5942286307.).

ogical Standardization. Published b

, et al., Construction and char1016/j.biologicals.2015.01.003

The yeast two-hybrid (YTH) screens have been used widely tostudy interactions between host and viral proteins [7]. A wellcharacterized, high quality cDNA library is a prerequisite and crit-ical tool for studying host-virus proteineprotein interactions usingYTH system. To this end, many workers have previously reportedthe construction of cDNA libraries from the pathogen susceptiblecell lines and tissues [8e12]. For FMDV, several susceptiblecontinuous cell lines have been described which include BHK-21[13], IB-RS-2 [14], IB-RS-11 [15], MVPK-1 [16] and LFBK [17].Amongst these, LFBK cell line is a continuous cell line derived fromthe kidney cells of FMDV susceptible host species that survivedinfection with temperature sensitive mutant of FMDV C3 [17].Previously, LFBK cell line was thought to be derived from the calfkidney cells but the recent studies indicated the swine genotype ofcell line (Personal communication, Luis L. Rodriguez, PIADC, ARS,USDA). Besides, LFBK cells have been found to be equally suscep-tible to all seven serotypes of FMDV and have been routinely usedfor various assays like plaque assay, immunofluorescent assay orplaque neutralization assay in different laboratories worldwide.

Here, we describe the construction and characterization of cDNAlibrary derived from LFBK cells employing the switching

y Elsevier Ltd. All rights reserved.

acterization of yeast two-hybrid cDNA library derived from LFBK cell

S. Mahajan et al. / Biologicals xxx (2015) 1e72

mechanism at the 50-end of the RNA transcript (SMART) technol-ogy. Using SMART technique, almost all of the mRNAs can bereverse-transcribed, even the low copy genes, thus facilitating thestudy of even less abundant proteins in protein interaction studies[18]. The developed cDNA library was evaluated for various pa-rameters to qualify its application as host prey library to study in-teractions between FMDV and host cells using YTH system.

2. Materials and methods

2.1. Cell line and virus

LFBK cells were maintained as monolayers in Glasgow mini-mum essential medium (GMEM, Sigma, USA) supplemented with10% fetal bovine serum (Hyclone, Thermo Scientific, USA). Thecultured cells served as the source of RNA for LFBK- cDNA library.

2.2. Isolation of total RNA and mRNA

The LFBK monolayers were trypsinized at 37 �C for 10 min toobtain individual cells. The cells were suspended in culture me-dium and centrifuged followed by washing of the cell pellet in PBS.The washed cell pellet thus obtained was used for the total RNAextraction using MN-Nucleospin RNA kit (Machery-Nagel, Ger-many) as per the manufacturer's instructions. The concentrationand purity of the total RNA was determined using nanodrop spec-trophotometer (Thermo Fisher Scientific, USA). The integrity of thetotal RNAs was analyzed by 1% TAE-agarose gel electrophoresis.Isolation of poly(A) mRNA from total RNA was carried out usingMN-NucleoTrap® mRNA kit (Machery-Nagel, Germany) accordingto the manufacturer's instructions. The isolated mRNA was furthervacuum concentrated using Concentrator plus™ (Eppendorf, Ger-many). The quantity and integrity of the isolated mRNA wasdetected by nanodrop spectrophotometer and agarose gel elec-trophoresis, respectively.

2.3. cDNA synthesis

Single-stranded (ss) cDNA was synthesized using Make YourOwn “Mate&Plate” Library System(Clontech,USA). ThemRNA (2ml)was reverse transcribed using SMART III oligo (50- AAGCAGTGG-TATCAACGCAGAGTGGCCATTATGGCCGGG-30), CDS III primer (50-ATTCTAGAGGCCGAGGCGGCCGACATG-d(T)30VN-30) and SMARTMMLV Reverse Transcriptase to synthesize ss cDNA. The double-stranded (ds) cDNA was amplified by long distance PCR (LD-PCR)using 2 ml of ss cDNA product and Advantage 2 PCR kit (Clontech,USA) according to the manufacturer's protocol. The PCR ampliconswere analyzed using 1% agarose/EtdBr gel. Thereafter, size-fractionation of ds cDNA was carried out using CHROMA-SPIN TE-400 columns (Clontech, USA). The ds cDNA was precipitated usingsodium acetate and ethanol. The quantity and quality of the purifiedds cDNA was analyzed with nanodrop spectrophotometer and 1%agarose/EtdBr gel, respectively.

2.4. Construction of cDNA library

The purified ds cDNA (3.9 mg) and 3 mg of SmaI-linearizedpGADT7-rec vector were co-transformed into Y187 yeast strainusing lithium acetate transformation method [19] and cultured onSD/-Leu agar plates at 30 �C for 4 days. The resultant transformantswere harvested in freezing medium (YPDA þ 25% glycerol) andstored at �80 �C in 1 ml aliquots. The transformed cells (1 ml) werediluted in YPDAmedium and 100 ml of 10�1 and 10�2 dilutions weregrown on SD/-Leu plates at 30 �C for 4 days to calculate the variousparameters of transformed libraries.

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2.5. Evaluation of the cDNA library

The constructed cDNA library was evaluated for number ofindependent clones and transformation efficiency from 10�2

dilution SD/-Leu plates [11]. The harvested and pooled librarieswere also plated on SD/-Leu plates at 10�2, 10�3, 10�4 and 10�5

dilutions to calculate cell density, library titer and library quan-tity [20]. The average insert size and recombination rate of thelibraries was estimated from the randomly selected 60 coloniesfrom LFBK cDNA library. The selected colonies were subjected toPCR using Matchmaker InsertCheck PCR Mix 2 (Clontech, USA)and the products were analyzed on 1% agarose gel. Plasmids wereextracted from the selected colonies using Easy Yeast PlasmidIsolation kit (Clontech, USA) and re-transformed in competentEscherichia coli (E. coli) DH5a cells by chemical transformationmethod [21] and plated on Luria Bertani-ampicillin (LB-Amp)agar plates. Plasmids were extracted from the positive trans-formants using PureLink™ Quick Plasmid MiniPrep kit (Invi-trogen, USA). The extracted plasmids were subjected tonucleotide sequencing.

2.6. Nucleotide sequencing and analysis

Sixty clones were randomly selected from the LFBK cDNA libraryand were nucleotide sequenced with a single pass reading from the50end on an ABI 3130 Genetic Analyzer using Big dye terminatorv3.1 (Applied Biosystems) and forward primer GBK-ADF (50-GTAATACGACTCACTATAGGGCGA-30). Raw sequences were manu-ally trimmed to remove the vector and low-quality sequences. Theassembled cDNA sequences were subjected to BLAST search againstthe nonredundant (nr) GenBank database to compare them withthe currently available ESTs and genes. The BLASTn results with bitscores >80 and e-values less than 10�10 were considered as sig-nificant matches. The ESTs homologous to known proteins werefurther annotated for gene ontology (GO) terms and the GO analysiswas carried out using WEB-based GEne SeT AnaLysiS Toolkit(WebGestalt) [22,23].

3. Results

3.1. Isolation and analysis of total RNA and mRNA

The A260/280 ratio and the average concentration of the isolatedLFBK total RNA was found to be 2.11and 2081 ng/ml, respectively.The gel electrophoresis revealed bands of 28S and 18S rRNAs andthe intensity of 28S band was twice as that of the 18S rRNA (Fig. 1)suggesting that the isolated RNA was of high quality and purity tobe used for mRNA isolation. The concentration of mRNAwas foundto be 114.8 ng/ml (40 ml volume) and the A260/280 ratio was 2.26. ThemRNA was concentrated to ~350 ng/ml using the Concentratorplus™ (Eppendorf, Germany). The gel electrophoresis revealed asmear up to ~6 kb (Fig. 2). Although some rRNA bands were faintlyvisible but the good length of smear reflected the quality of theextracted mRNA.

3.2. cDNA synthesis

Approximately 700 ng of mRNA (2 ml) was used for the ss cDNAsynthesis using oligo-d(T) primer. The ss cDNA was further ampli-fied by LD-PCR for ds cDNA synthesis. The optimal number of PCRcycles for ds cDNA synthesis was found to be 24. The ds cDNA smearranged from <100 bp to 4 kb (Fig. 3a) and ~500 bp to 4 kb (Fig. 3b),before and after purification, respectively.

acterization of yeast two-hybrid cDNA library derived from LFBK cell

Fig. 1. Agarose gel electrophoresis of total RNA from LFBK cells. Lane 1, total RNA; LaneM, RiboRuler High Range RNA Ladder (ThermoScientific, USA).

Fig. 3. Analysis of ds cDNAs of LFBK cell line on 1% agarose gel. (a) unpurified ds cDNAand (b) purified ds cDNA; Lane M, GeneRuler 1 kb DNA Ladder (#SM0313, Thermo-Scientific, USA); Lane 1, LFBK ds cDNA; Lane 2, positive control ds cDNA.

S. Mahajan et al. / Biologicals xxx (2015) 1e7 3

3.3. Construction and evaluation of DNA library

The YTH compatible LFBK was generated using pGADT7-recvector in Y187 strain. The resultant cDNA library represented2.41�106 independent clones. The transformation efficiency of theconstructed library was found to be 8.0 � 105/mg of GADT7-recvector. Other parameters of library qualification are described inTable 1. The insert size of the constructed library varied from 500 bpto 2000 bp with an average insert size of 1124 bp (Fig. 4aed). Of the60 colonies screened, 59 were found positive and the recombina-tion rate was estimated to be 98.33%.

3.4. Nucleotide sequencing analysis

Sixty ESTs from the cDNA library were sequenced and depositedin EST database of Genbank (dBEST; Table 2). After assembly, 53ESTs revealed top hits from the pig sequences on BLASTanalysis andtwo clones were found homologous to functionally characterizedproteins of horse (DLN42 and DLN 43). Clone DLN81 revealed

Fig. 2. Agarose gel electrophoresis of LFBK mRNA. Lane M, RiboRuler High Range RNALadder (#SM1821, ThermoScientific, USA); Lane 1, mRNA.

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similarity to NS5A protein of bovine viral diarrhea virus (BVDV).However, no significant similarity was observed for five clones(DLI016, DLI019, DLI020, and DLN051). 45 ESTs (out of 60) whichwere found identical to the known protein coding genes weresubjected to the GO analysis and their ontology in terms of bio-logical processes, molecular function and cellular compartmentwas determined (Fig. 5aec).

4. Discussion

FMDV has developed complex mechanisms to evade host im-mune responses and facilitates its own replication even in vacci-nated animals by subverting the cellular pathways. And whiledoing so, viral proteins distract several host proteins from theirnatural pathway to their advantage. Thus, study of virus-host pro-tein interactions is of utmost significance for better understandingof the molecular mechanisms of viral replication and pathogenesisand to identify the checkpoints for development of therapeutics infuture. The techniques available for studying interactions between

Table 1Parameters for qualification of LFBK cDNA library.

Parameter LFBK cDNA library

Independent clones 2.41 � 106

Transformation efficiency 8.0 � 105/mg of GADT7-recCell density (pre-freezing) 6.5 � 108 cells/mlLibrary titre (pre-freezing) 2.88 � 108 cfu/mlCell density (post-freezing) 5.1 � 108 cells/mlLibrary titre (post-freezing) 2.6 � 108 cfu/mlRecombination % 98.33Insert size 1124 bp (500e2000 bp)

acterization of yeast two-hybrid cDNA library derived from LFBK cell

Fig. 4. Insert-check PCR of randomly selected colonies from LFBK cDNA library. Lane 1-60, recombinant individual colonies; Lane M, GeneRuler 1 kb DNA Ladder (#SM0313,ThermoScientific, USA).

S. Mahajan et al. / Biologicals xxx (2015) 1e74

the host and viral proteins include YTH, siRNA screens and affinity-purificationmass spectrophotometry [24]. However, YTH screeningis the most widely used, high-throughput and low-cost approach[25] for studying proteineprotein interactions. It is an in-vivoscreening approach with the ability to identify even low affinity,transient interactions as well [26]. A high quality cDNA library (preylibrary) expressing host proteins is indispensable for screening ofinteractions between virus and host using YTH method. Previously,Gladue et al. [27,28] have used the cDNA library derived from

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bovine tissue susceptible to FMDV while Wang et al. [3] and Li et al.[29] have used swine spleen cDNA library for studying FMDV-hostinteractions. But, till now to the best of our knowledge, cDNA li-brary of any of the FMDV susceptible cell lines is not available.

In this study, cDNA library of the FMDV-susceptible LFBK cellline (porcine origin) was constructed and characterized. This cellline was chosen since it has been shown to be highly susceptible toall the serotypes of FMDV [17]. The LFBK cDNA library was con-structed using SMART technique and less than 1 mg of the high

acterization of yeast two-hybrid cDNA library derived from LFBK cell

Table 2Putative genes identified in cDNA clones of YTH LFBK library on their sequences comparison through BLASTn search engine of NCBI database.

Clone no. GenBankaccession

Best hit in the NCBI database

mRNA Accession Organism e-value Identity

DLI02 JZ761152 Synovial sarcoma translocation, chromosome 18 (SS18), mRNA NM_001244804.1 Sus scrofa 2E-78 98%DLI04 JZ761153 RNA binding motif protein, X-linked (RBMX) NM_001244096.1 Sus scrofa 0 100%DLI07 JZ761154 Alpha-kinase 2 (ALPK2), XM_001925970.5 Sus scrofa 0 93%DLI010 JZ761155 Cell division cycle associated 3 (CDCA3) XM_001928347.2 Sus scrofa 2.00E-102 100%DLI011 JZ761156 Aconitase 1 XM_003357729.2 Sus scrofa 1.00E-19 98%DLI013 JZ761157 NFKB inhibitor interacting Ras-like 1 (NKIRAS1),

transcript variant X2XM_005669299.1 Sus scrofa 0 99%

DLI014 JZ761158 mRNA, clone:BKFL10074E06, expressed in back fat AK344159.1 Sus scrofa 7.00E-71 93%DLI015 JZ761159 mRNA, clone:OVRM10094E10, expressed in ovary AK235577.1 Sus scrofa 1.00E-18 98%DLI016 JZ761160 No similiarityDLI017 JZ761161 Glutaredoxin 3 (GLRX3) NM_001243896.1 Sus scrofa 9.00E-132 98%DLI019 JZ761162 No similiarityDLI020 JZ761163 No similiarityDLI022 JZ761164 Ribosomal protein S26 (RPS26) NM_001097481.2 Sus scrofa 2.00E-32 99%DLI026 JZ761165 Adipogenesis regulatory factor-like (LOC100517243) XM_003133100.3 Sus scrofa 1.00E-13 100%DLI028 JZ761166 Fibronectin 1 (FN1), transcript variant X13 XM_005672174.1 Sus scrofa 8.00E-82 95%DLI030 JZ761167 Guanine nucleotide binding protein (G protein),

beta polypeptide 2-like 1 (GNB2L1)NM_214332.1 Sus scrofa 0 99%

DLI032 JZ761168 Vacuolar protein sorting 29 homolog(S. cerevisiae) (VPS29), transcript variant 1

XM_003483426.2 Sus scrofa 0 99%

DLI034 JZ761169 Histone H3.3-like (LOC100521680) XM_003131194.2 Sus scrofa 0 98%DLI039 JZ761170 Dual specificity phosphatase 6 (DUSP6) NM_001267842.1 Sus scrofa 6.00E-99 96%DLI040 JZ761171 Lysosomal protein transmembrane 4 beta (LAPTM4B) XM_005655356.1 Sus scrofa 0 94%DLN041 JZ761172 Angiotensinogen (serpin peptidase inhibitor, clade A,

member 8) (AGT)XM_001924659.5 Sus scrofa 0 99%

DLN042 JZ761173 Kelch-like family member 14 (KLHL14) XM_005614848.1 Equus caballus 0 91%DLN043 JZ761174 Colony stimulating factor 1 (macrophage) (CSF1) XM_005610548.1 Equus caballus 0 84%DLN044 JZ761175 mRNA, clone:TES010041B06, expressed in testis AK238492.1 Sus scrofa 0 97%DLN045 JZ761176 Cytochrome c oxidase subunit 8A, mitochondrial-like

(LOC100522725), transcript variant 1 COX8aXM_003122583.3 Sus scrofa 0 99%

DLN046 JZ761177 Glutathione S-transferase alpha 1 (GSTA1), transcript variant X4 XM_005666467.1 Sus scrofa 0 97%DLN047 JZ761178 Nuclear autoantigenic sperm protein

(histone-binding) (NASP), transcript variant X2XM_005658787.1 Sus scrofa 2.00E-160 95%

DLN049 JZ761179 Signal peptidase complex subunit 3 homolog (S. cerevisiae) (SPCS3) XM_003133320.2 Sus scrofa 6.00E-104 86%DLN050 JZ761180 Transformer 2 alpha homolog (Drosophila)

(TRA2A), transcript variant X1XM_005656690.1 Sus scrofa 0 99%

DLN051 JZ761181 no similiarityDLN052 JZ761182 Syndecan 4 (SDC4) NM_214284.1 Sus scrofa 7.00E-70 99%DLN053 JZ761183 Pim-2 oncogene (PIM2) XM_003360311.2 Sus scrofa 6.00E-149 100%DLN054 JZ761184 Clone CH242e311B2 on chromosome X CU915480.5 Sus scrofa 1.00E-24 83%DLN055 JZ761185 THUMP domain containing 1 (THUMPD1), transcript variant X1 XM_005662086.1 Sus scrofa 0 95%DLN056 JZ761186 Glycyl-tRNA synthetase (GARS), transcript variant X2 XM_005673280.1 Sus scrofa 0 91%DLN057 JZ761187 Sec23 homolog B (S. cerevisiae) (SEC23B), transcript variant X1 XM_005672715.1 Sus scrofa 0 95%DLN058 JZ761188 Trafficking protein particle complex 6B

(TRAPPC6B), transcript variant 2XM_003121779.2 Sus scrofa 3.00E-137 99%

DLN060 JZ761189 Ribosomal protein L23 (RPL23) NM_001129955.1 Sus scrofa 1.00E-161 98%DLN061 JZ761190 RNA binding motif (RNP1, RRM) protein 3 (RBM3),

transcript variant X2XM_005657796.1 Sus scrofa 2.00E-141 94%

DLN062 JZ761191 no similiarityDLN063 JZ761192 Hematological and neurological expressed 1 (HN1) NM_001048069.1 Sus scrofa 0 97%DLN064 JZ761193 G protein-coupled receptor, family C, group 5, member C (GPRC5C) XM_005674260.1 Sus scrofa 5.00E-166 98%DLN065 JZ766042 Ancient ubiquitous protein 1 (AUP1) XM_005662443.1 Sus scrofa 3.00E-142 93%DLN066 JZ766043 Eukaryotic translation elongation factor 1 alpha 1 (EEF1A1) NM_001097418.2 Sus scrofa 0 97%DLN067 JZ761194 Delta(4)-desaturase, sphingolipid 1 (DEGS1) NM_001244121.1 Sus scrofa 0 98%DLN069 JZ761195 Clone CH242e116P23 on chromosome X FP015872.10 Sus scrofa 5.00E-105 94%DLN071 JZ761196 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 8, 19 kDa

(NDUFA8), transcript variant X1XM_005654528.1 Sus scrofa 0 96%

DLN072 JZ761197 H2A histone family, member Z (H2AFZ) NM_001123122.1 Sus scrofa 0 99%DLN073 JZ761198 NOP10 ribonucleoprotein (NOP10) XM_001925450.3 Sus scrofa 0 98%DLN074 JZ761199 Methionyl-tRNA synthetase (MARS) XM_005674389.1 Sus scrofa 0 99%DLN075 JZ761200 Histone H1.0-like (LOC100737738) XM_005663789.1 Sus scrofa 1.00E-155 99%DLN076 JZ761201 Malignant T-cell-amplified sequence 1-like (LOC100738684) XM_003482845.2 Sus scrofa 5.00E-135 98%DLN077 JZ761202 Ferritin, light polypeptide (FTL) NM_001244131.1 Sus scrofa 0 96%DLN078 JZ761203 Adenylate kinase 3-like 1 (AK3L1) EF488234.1 Sus scrofa 2.00E-116 93%DLN079 JZ761204 Keratin 10 (KRT10), transcript variant X2 XM_005668847.1 Sus scrofa 1.00E-99 98%DLN081 JZ761205 Bovine viral diarrhea virus type 1a strain 6010 polyprotein gene JN380080.1 Bovine viral

diarrhea virus0 91%

DLN082 JZ761206 Chromosome 10 open reading frame 58 ortholog (C14H10orf58),transcript variant X6 FAM213A

XM_005671142.1 Sus scrofa 0 95%

DLN083 JZ761207 Succinate dehydrogenase complex, subunit B, iron sulfur (Ip) (SDHB) NM_001104953.1 Sus scrofa 0 99%DLN084 JZ761208 NADH dehydrogenase (ubiquinone) complex I,

assembly factor 4 (NDUFAF4)XM_001925477.3 Sus scrofa 0 99%

DLN085 JZ761209 Small nuclear ribonucleoprotein 70 kDa (U1) (SNRNP70) XM_005664707.1 Sus scrofa 9.00E-90 88%

S. Mahajan et al. / Biologicals xxx (2015) 1e7 5

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a

b

c

Fig. 5. Functional categorization of the ESTs with Gene Ontology (GO) terms under three categories (a) biological process, (b) molecular function and (c) cellular compartment withrespective GO Slim terms.

S. Mahajan et al. / Biologicals xxx (2015) 1e76

quality mRNA (derived from LFBK total RNA; Figs. 1 and 2) was usedfor first strand cDNA synthesis. The full length ss cDNAs wereamplified by LD-PCR with the optimal number of cycles (24) tominimize the PCR induced mutations and the selection bias forsmaller size cDNA. The ds cDNAs were further purified usingCHROMA-SPIN TE-400 columns (Fig. 3a and b) to limit the

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preponderance of the small inserts (lowmolecular weight ds cDNA,adapters and un-incorporated nucleotides). The purified ds cDNAsalong with the pGADT7-rec vector were directly transformed intothe prey yeast strain Y187, thus making it very convenient for YTHscreening. The constructed cDNA library was qualified by esti-mating the number of independent clones in the library to be more

acterization of yeast two-hybrid cDNA library derived from LFBK cell

S. Mahajan et al. / Biologicals xxx (2015) 1e7 7

than 2 million with the cell density greater than 107 cells/ml andthe titre of the generated library was more than 1 � 107 cfu/ml(Table 1). The transformation efficiency, pre- and post-freezing li-brary titre were also calculated to evaluate the quality of the con-structed library. The titre and the cell density of the library weremaintained above the standard after storage at �80 �C (Table 1).The LFBK cDNA library had a high recombination rate of 98.33%with an average insert size of 1124 bp (Fig. 4). All these attributesindicated that the constructed cDNA library is of high quality and issuitable for use as the prey library in YTH system for identifyingFMDV-interacting host partners.

The nucleotide sequence analysis of the randomly picked clonesfrom the cDNA library indicated that 88.33% of the ESTs were ho-mologous to the characterized or hypothetical proteins or themRNAs of the pig (Table 2). Initially, LFBK cell line was published tobe of calf kidney origin [17] but the recent Isoenzyme and real timeRT-PCR studies confirmed that the LFBK cells are of porcine geno-type (Personal communication, Luis L. Rodriguez, PIADC, ARS,USDA). The sequence analysis of the LFBK cDNA library corrobo-rated the findings. Besides, the clone DLN81 revealed similarity tothe NS5A protein of the BVDV (Table 2) suggesting possible pesti-virus contamination in the cell line. The presence of transmissiblenon-cytopathic BVDV in LFBK cells has also been established bymonoclonal antibody and microarray studies (Personal communi-cation, Luis L. Rodriguez, PIADC, ARS, USDA). The GO analysis of thesequenced ESTs suggested the diversity of the transcripts present inthe LFBK library without much redundancy (Fig. 5aec). Besides, weperformed a proof-of-concept YTH screen using the LFBK cDNAlibrary as prey and FMDV 2C protein as bait. Several host proteinshave been identified as the putative 2C-interacting proteins onpreliminary screening (unpublished data). Further confirmation ofthe identified interactions using the in-vitro methods is underway.The constructed cDNA library would be one of the useful genomicsources to study the FMDV-host interactions. As it has been re-ported that LFBK cell line is susceptible to bluetongue virus and torinderpest virus as well [17], the constructed library could be usefultools for studying host-virus protein interactions for these impor-tant animal pathogens as well.

In conclusion, we reported the construction and characterisationof a YTH system compatible cDNA library from FMDV susceptibleLFBKcell line. The librarywasgenerated fromhighqualityLFBKmRNAusing SMART technique. The sequencing of library clones determinedthe swine genotype of the cell line. The library was evaluated forvarious parameters and found suitable to be used as prey for studyingFMDV-host protein interactions. The LFBK cDNA library constructedin this study will be available for other researchers.

Acknowledgements

This work was funded by the Indian Council of AgriculturalResearch under the project IXX10801. We sincerely thank Dr. Luis L.Rodriguez, Research Leader, PIADC, USA, for kindly sharing the LFBKcell line.

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acterization of yeast two-hybrid cDNA library derived from LFBK cell