ires-incorporated lactococcal bicistronic vector for target gene expression in a eukaryotic system

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Plasmid xxx (2014) xxx–xxx

YPLAS 2202 No. of Pages 8, Model 3G

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Contents lists available at ScienceDirect

Plasmid

journal homepage: www.elsevier .com/ locate/yplas

IRES-incorporated lactococcal bicistronic vector for target geneexpression in a eukaryotic system

http://dx.doi.org/10.1016/j.plasmid.2014.04.0030147-619X/� 2014 Published by Elsevier Inc.

⇑ Corresponding author. Fax: +60 3 89467510.E-mail address: [email protected] (N. Mat Isa).

Please cite this article in press as: Mutalib, N.E.A., et al. IRES-incorporated lactococcal bicistronic vector for target gene expressieukaryotic system. Plasmid (2014), http://dx.doi.org/10.1016/j.plasmid.2014.04.003

Nur Elina Abdul Mutalib a, Nurulfiza Mat Isa a,⇑, Noorjahan Banu Alitheen a,Adelene Ai-Lian Song a, Raha Abdul Rahim a,b

a Department of Cell and Molecular Biology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 UPM Serdang,Selangor Darul Ehsan, Malaysiab Institute of Bioscience, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor Darul Ehsan, Malaysia

a r t i c l e i n f o a b s t r a c t

2829303132333435363738

Article history:Received 29 November 2013Accepted 18 April 2014Available online xxxxCommunicated by Dhruba K. Chattoraj

Keywords:Bicistronic vectorInternal ribosome entry site (IRES)Lactococcus lactisInfectious bursal disease virus (IBDV)

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Plasmid DNAs isolated from lactic acid bacteria (LAB) such as Lactococcus lactis (L. lactis)has been gaining more interests for its positive prospects in genetic engineering-relatedapplications. In this study, the lactococcal plasmid, pNZ8048 was modified so as to be ableto express multiple genes in the eukaryotic system. Therefore, a cassette containing aninternal ribosome entry site (IRES) was cloned between VP2 gene of a very virulent infec-tious bursal disease (vvIBDV) UPM 04190 of Malaysian local isolates and the reporter gene,green fluorescent protein (GFP) into pNZ:CA, a newly constructed derivative of pNZ8048harboring the cytomegalovirus promoter (Pcmv) and polyadenylation signal. The new bicis-tronic vector, denoted as pNZ:vig was subjected to in vitro transcription/translation systemfollowed by SDS–PAGE and Western blot analysis to rapidly verify its functionality. Immu-noblotting profiles showed the presence of 49 and 29 kDa bands that corresponds to thesizes of the VP2 and GFP proteins respectively. This preliminary result shows that thenewly constructed lactococcal bicistronic vector can co-express multiple genes in a eukary-otic system via the IRES element thus suggesting its feasibility to be used for transfection ofin vitro cell cultures and vaccine delivery.

� 2014 Published by Elsevier Inc.

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1 Introduction

Lactococcus lactis is fast becoming an ideal cell factoryfor producing heterologous proteins. Many previousstudies have shown the effectiveness of these lactic acidbacteria (LAB) in genetic engineering and DNA manipula-tions (Chatel et al., 2001; Noreen et al., 2011; Song et al.,2012). Despite that, there have been few lactococcalvectors to accommodate the same versatility shown byEscherichia coli-derived plasmids where a vast array of vec-tors with diverse functions such as shuttle vectors, vectorswith tags, vectors for secretion, vectors for recombination

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and many others are available. In comparison to E. coli, L.lactis and its vectors have not yet been fully utilized formolecular biology approaches. For example, lactococcalvectors have yet to be developed as eukaroytic DNA deliv-ery systems for targeted gene expression to match the fea-sibility of E. coli/pUC-derived vectors. The vectors in thesestudies were modified to confer the eukaryotic transcrip-tion and translation machinery regulatory system such asa eukaryotic promoter to ensure the genes encoding theproteins of interest it harbors will be delivered to theintended cells (Guimarães et al., 2009, 2006; Innocentinet al., 2009).

There are a few strategies that can be employed toco-express two or more genes such as the use of two ormore promoters, the fusion of genes translated in-frame

on in a

http://dx.doi.org/10.1016/j.plasmid.2014.04.003

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2 N.E.A. Mutalib et al. / Plasmid xxx (2014) xxx–xxx


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to produce a chimeric protein or the use of bicistronic/mul-ticistronic vectors containing viral 2A peptide or IRES ele-ments (Goedhart et al., 2011; Wong et al., 2002). IRES arewidely known to mediate internal initiation of translation.Upon transcription, IRES RNA forms secondary structuresthat allows the initiation factors–ribosome complexes tobind (Borman et al., 1995; Douin et al., 2004; Jang andWimmer, 1990). Binding of initiation factors–ribosomecomplexes at these regions allow translation of the down-stream gene(s) thus enabling multiple proteins to be trans-lated at the same time (Fernández-Miragall et al., 2009;Jang and Wimmer, 1990; Martínez-Salas et al., 2001). Thisfeature of IRESs has garnered the interest in producingIRES-incorporated vectors to simultaneously co-translatetwo or more proteins (Clarke et al., 1997; Fitzgerald andSemler, 2009; Havenga et al., 1998; Kang et al., 2009). Mostdelivery of DNA vectors into mammalian cells utilize co-transfection of two different plasmids; one carrying thegene encoding a protein of interest and the other harboringthe gene encoding a reporter protein (Sun et al., 2005).Upon successful transfection of the reporter gene, the othergene of interest is usually presumed to be successfullytransfected as well. With the incorporation of IRES elementinto a single vector, multiple genes including a reportergene can be cloned together into a single vector, thus elim-inating uncertainty in transfection of target genes.

Infectious bursal disease virus (IBDV) outbreaks in poul-tries are the most dreaded viral infections in the industry.The immunosuppression and susceptibility to secondaryinfection caused by this virus could result in chicken mor-tality that could wipe out the whole farm population (Saif,1991). There are many researches to alleviate the problemsthat cause the outbreaks, but the best prevention so far isthrough vaccination. IBDV DNA vaccine has been devel-oped extensively in many previous studies, but the currenttrend is to co-express the VP2 gene of the viral protein (thecausative virulence protein) together with other accessoryproteins such as VP3/VP4 gene, avian-related cytokines orimmunostimulatory sequences (ISS) to induce chickenimmune response towards the VP2 antigen (Chang et al.,2002; Sun et al., 2005; Wang et al., 2003).

In this study, the nisin-inducible lactococcal vector,pNZ8048 was modified in such a way as to accommodateboth promoter- (cap-dependent) and IRES- (cap-indepen-dent) translation. For this purpose, the eukaryotic pro-moter (Pcmv), including the ribosome binding site (Kozaksequence) and a polyadenylation signal from an E. coli-mammalian shuttle vector were cloned into the lactoccocalplasmid together with a modified IRES element. To checkthe functionality of the modified vector, gene for the VP2protein of IBDV and a reporter gene, gfp were clonedupstream and downstream of IRES region, respectively.The ability of the newly constructed vector to properlyexpress both VP2 and GFP was analyzed.

2. Materials and methods

2.1. Bacterial strains, plasmids and media

Bacterial strains and plasmid vectors used in this studyare tabulated in Table 1. Plasmidless L. lactis NZ9000 was

Please cite this article in press as: Mutalib, N.E.A., et al. IRES-incorporaeukaryotic system. Plasmid (2014), http://dx.doi.org/10.1016/j.plasmid.

grown in M17 media (Merck, Germany) supplementedwith 0.5% glucose while L. lactis NZ9000 harboring plas-mids was grown in M17 media (Merck) with 0.5% glucosesupplemented with 7.5 lg/ml chloramphenicol. For trans-formation purposes, M17 media with 0.5% glucose and0.5% sucrose (SGM17) also supplemented with 7.5 lg/mlchloramphenicol were used. Lactococcal cultures wereincubated as standing cultures at 30 �C. TOP10 E. coli cellsharboring plasmids which encode the VP2 gene and IRES-GFP fragments [pUCIDT VP2 UPM 04190 (IDT Technolo-gies) and pRetroX IRES ZsGreen1 (Clontech, USA)], respec-tively, were grown in LB media (Merck, Germany)supplemented with 100 lg/ml ampicillin. Meanwhile, theeukaryotic elements (promoter Pcmv and polyadenylationsignal) were isolated from pCDNA 3.1 His A (Invitrogen,USA). E. coli cells grown in liquid media were grown byshaking vigorously at 250 rpm at 37 �C.

2.2. Construction of recombinant plasmids

2.2.1. Insertion of the eukaryotic promoter and terminationsignal

The cytomegalovirus promoter (Pcmv) and terminatortranscription signal, bovine growth hormone polyA wasamplified from pCDNA 3.1 His A. These two combinationsof promoter and polyA signal have been studied previouslyto show high level of transcription in eukaryotic cells. Asfor amplification of eukaryotic elements, Pcmv was ampli-fied using F-Pcmv and R-Pcmv whereas polyadenylationsignal was amplified using primers F-PA and R-PA, respec-tively, from plasmid pCDNA 3.1, as depicted in Table 2.

PCR amplification was carried out with the reactionmixture of 1� Pfu buffer with MgSO4 (Fermentas, USA),0.2 mM dNTP mix (Invitrogen, USA), 5 lM of forward andreverse primers, 150 ng of template DNA and 2.5 units/llPfu polymerase (Fermentas, USA). PCR reaction was setup as follows: initial denaturation at 95 �C for 5 min,followed by 25 cycles of denaturation at 95 �C for 1 min,annealing at 64 �C for 1 min and extension at 68 �C for1 min/kb of PCR product size and lastly final extension at68 �C for 10 min.

The original PnisA promoter from pNZ8048 was firstremoved from the vector by digesting the plasmid with BglIIand NcoI (Fermentas, USA) resulting in two fragments ofPnisA and pNZ8048-PnisA (pNZ8048 minus the PnisA pro-moter). The overhangs of pNZ8048-PnisA were filled inusing Pfu polymerase by incubating the mixtures at 72 �Cfor 10 min. Then, the blunt-ended fragment of pNZ8048-PnisA was recircularized back with T4 DNA ligase.

As for inclusion of the Pcmv fragment upstream of themultiple cloning site of pNZ8048-PnisA, Pcmv was insertedinto the vector at the BglII and NcoI site by RE digestion in2� Tango buffer (Fermentas) while the polyadenylationsignal was inserted in the SacI site. The ligated plasmid,denoted pNZ:CA, was electroporated into competentL. lactis NZ9000 using the methods from Holo and Nes(1989). The transformants were selected on SGM17 agarsupplemented with chloramphenicol. To verify the possi-ble putative recombinant plasmids, the clones weresubjected to verification via RE digestion and sequencing.

ted lactococcal bicistronic vector for target gene expression in a2014.04.003


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Table 1The bacterial strains and plasmids used in this study and its relevant features.

Bacterial strains/plasmids

Relevant genotype(s) and/or genotype(s) References

L. lactis NZ9000 Plasmid-less strain, with the nisR and nisK gene integrated in its genome for nisin induction, host fornisin-inducible vectors

Mobitec

E. coli TOP 10 Plasmid-less strain, host for pCDNA 3.1 His A and pUCIDT AMP VP2-UPM 04190 in this study InvitrogenF� mcrA D(mrr-hsdRMS-mcrBC) U80lacZD M15 DlacX74 recA1 araD139 D(araleu)7697 galU galKrpsL (StrR) endA1 nupG

pNZ8048 3.3 kb, PnisA promoter for inducible expression upon nisin introduction, chloramphenicol resistant MobitecpUCIDT AMP VP2-

UPM 041904.19 kb, VP2 gene from vvIBDV Malaysian isolates, UPM 04190, ampicillin-resistant Genbank accession no:

AY791998.1pCDNA 3.1 His A 5.5 kb, derivatives of pCDNA 3.1, human immediate early cytomegalovirus promoter (Pcmv) and

bovine growth hormone polyadenylation signal (polyA), ampicillin-resistantInvitrogen

pRetroX-IRES-ZsGreen1

5.9 kb, bicistronic, fluorescent, retroviral vector harboring encephalomyocarditis virus IRES (EMCVIRES) and gene encoding Zoanthus sp. green fluorescent protein

Clontech

pNZ:CA 3.5 kb; modified pNZ8048 with cytomegalovirus promoter (Pcmv) and polyA signal upstream anddownstream of the multiple cloning site (MCS); chloramphenicol resistant

In this study

pNZ:VIG 6.4 kb; ChlR containing VP2-IRES-GFP with cytomegalovirus promoter (Pcmv) and polyA signal;chloramphenicol resistant

In this study

Table 2Primers design for PCR amplification and DNA sequencing purposes.

Primer Sequence (50?30) RE

F-IRES2 AAGGAATTCGCCCCTCTCCCTCCCCCCCCCCTAA EcoRIR-GFP2 AACGAGCTCTGAATCATCATCATCATCATGGGGCAAGGCGGAGCCGGA SacI

F-VP2 ACCCCATGGAGCCGACCATGACAAACCTGCAAGAT NcoIR-VP2 CAGGAATTCTCAATCATCATCATCATCATGGCCCGGATTATGTCTTTG EcoRI

F-Pcmv AACCAGATCTTAGTTATTAATAGTAATCAATTACGGGGTC BglIIR-Pcmv AGCAGATCTCCCTATAGTGAGTCGTATTAA BglIIF-BghPA GTAGAGCTCTGCCTTCTAGTTGCCAGCCATCTGTTG SacIR-BghPA CATGAGCTCCAGAAGCCATAGAGCCCACCGCATCC SacI

Restriction enzyme sites are italicized while His tags sequences are underlined.

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2.2.2. Insertion of VP2 and gfp geneEncephalomyocarditis virus’ internal ribosome entry

site (EMCV IRES) is the most common and versatile IRESelement for internal initiation of translation utilized inIRES-incorporated vectors. In this study, EMCV IRES andreporter gene, green fluorescent protein (Zoanthus sp.GFP) was amplified from pRetroX IRES ZsGreen1 (Clon-tech). To maintain the functional internal initiation codonto facilitate IRES-mediated translation, both DNA frag-ments were isolated as a single cassette. The VP2 gene ofvvIBDV from UPM isolates (UPM 04190) was syntheticallyproduced based on Genbank accession no. AY791998.1(Nurulfiza et al., 2006) and was provided in the pUCIDTVP2 vector (IDT Technologies). The VP2 gene was isolatedby PCR amplification. Primers were designed as F-VP2and R-VP2 to amplify the 1350 bp PCR product. For EMCVIRES and green fluorescent protein, the fragments wereamplified from pRetroX IRES ZsGreen1 (Clontech) usingthe designated primers; F-IRES and R-GFP.

Both VP2 and IRES-GFP fragments were first digestedwith EcoRI and ligated together with T4 DNA ligase (Fer-mentas), forming a 2691 bp DNA cassette. The ligated frag-ments were amplified again using F-VP2 and R-GFP2 toincrease the copy number, then linearized with NcoI (1 U/ll) and SacI (0.25 U/ll) at 37 �C for 2 h and ligated intosimilarly RE-digested lactococcal plasmid, pNZ:CA. Then,the ligation mix was cloned into L. lactis NZ9000 in thesame manner as stated in Section 2.2.1.


2.3. In vitro expression via cell-free coupled transcription/translation system

To check the functionality of the recombinant lactococ-cal IRES-incorporated vectors prior to transient transfec-tions, the plasmids were subjected to coupledtranscription–translation system via TNT� Quick CoupledTranscription/Translation System (Promega). 1.0–3.0 lgof plasmid DNA was mixed together with 40.0 ll of TNTQuick Mix and labeled with Flourotect GreenLys tRNA (Pro-mega) according to the manufacturer’s protocol for detec-tion of proteins. The mixture was incubated at 30 �C for90 min. The lysates were diluted with PBS when requiredand were loaded on a 12% SDS–PAGE/Tris–glycine gel andthe separated proteins was transferred onto polyvinyli-dene fluoride (PVDF) membrane under constant current65 mA for 1 h. Western blot analysis was performed usingthe Western Max™ Horseradish Peroxidase ChromogenicDetection Kit (Amresco, USA) according to the manufac-turer’s protocol with some modifications. The membranewas blocked with 5% skimmed milk (Merck, USA) for 1 hfollowed by incubation overnight with mouse anti HisTag� Monoclonal IgG diluted at 1:2000 (Novagen, USA) toverify the presence of functional proteins both expressedby the regulation of Pcmv and EMCV IRES respectively. Next,the membrane was incubated with anti-mouse goat IgGconjugated with horseradish peroxidase as the secondaryantibody at 1:2000 dilution for 1 h. To further verify the



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presence of proteins expressed via Pcmv, samples wereimmunoblotted with mouse infectious bursal disease virus(IBDV) VP2 monoclonal antibody, 1:10,000 (GTX41331,GeneTex, USA) at room temperature for 1 h in a separateWestern blot. The samples were electrophoresed togetherwith a positive control, VP2 gene expressed from pNZ:vp2.Similarly, the presence of functional GFP expressed viaEMCV IRES-regulated translation was verified by immuno-blotting with Living Colors� Full-Length ZsGreen Poly-clonal Antibody (Clontech, USA). As mentioned before, acontrol was electrophoresed together with the samples,but using GFP expressed from pNZ:gfp. The interaction ofthe described antibodies to detect the presence ofexpressed genes of VP2 and GFP was observed as browncolored bands developed on the membrane when exposedto DAB substrate.

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3. Results and discussion

3.1. Construction of recombinant bicistronic plasmid

The newly constructed bicistronic vector, pNZ:vig hasthe backbone of the lactococcal vector, pNZ8048 (Fig. 1).This particular vector was chosen as a DNA backbone dueto its high copy number. The plasmid pNZ:CA was first con-structed by replacing the original PnisA promoter withPcmv promoter to allow transcription in an eukaryotic sys-tem. pNZ:CA also contains the terminator signal, polyA.Both were amplified from pCDNA 3.1 (Fig. 2(b)). After con-struction of pNZ:CA, pNZ:vig which is the final bicistronic

Fig. 1. Schematic representation of the arrangement of the bicistronic construct(upstream of reporter gene, gfp) to yield pNZ:vig.


vector developed in this study was constructed by inser-tion of the VP2 gene and IRES-GFP fragment, amplifiedfrom their respective template plasmids, (Fig. 2(a)) intopNZ:CA. This yielded pNZ:vig which was verified by doubledigestion with NcoI/SacI (Fig. 3) and sequencing (data notshown).

The IRES element and reporter gene was amplified as asingle cassette as described in Fig. 1 to ensure correct rec-ognition of the start codon of GFP by IRES-mediated trans-lation. Previous studies have emphasized on thecomposition and arrangement of genes to enhance thetranscription level of a bicistronic mRNA. Depending onthe type of IRES used to initiate internal translation, theplacement of the first and second gene divided by an IRESis very important (Hennecke et al., 2001; Kaminski et al.,1990). In this study, the IRES incorporated was amplifiedfrom Clontech’s optimized EMCV IRES fragment where itmediates equivalent amount of translated products fromthe first and second cistron regulated by cap-dependentand IRES-mediated translation, respectively (Bochkov andPalmenberg, 2006). This will ease the quantification andanalysis of the expression of target proteins when thebicistronic vector is delivered to cell culture for in vitrostudy.

3.2. In vitro expression via cell-free coupled transcription/translation system mediated by bicistronic vector

The efficiency of the final construct, pNZ:vig, to mediatebicistronic translation was tested using a cell-free in vitrocoupled transcription and translation system. Made up

via addition of Pcmv promoter (upstream of the first gene, VP2) and IRES



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Fig. 2. (a) PCR amplification of VP2 gene (1390 bp; lane 1 and 2) and EMCV IRES-GFP (�1300 bp; lane 3 and 4) from their respective templates, pUCIDT VP2and pRetroX IRES ZsGreen1. (b) PCR amplification of Pcmv (i) and bovine polyadenylation signal (ii) from pCDNA 3.1. M: GeneRuler DNA Ladder (Fermentas).

Fig. 3. The RE digestion profile of putative positive recombinant plasmids from the colonies formed from the selective agar, SGM17 supplemented withchloramphenicol. Lane 1,2, 3, 4, 5 and 6 corresponds to the undigested possible recombinant clones. These clones were digested with NcoI (labeled a) andNcoI/SacI (labeled b) to verify the presence of insert. Lane 7 corresponds to the empty vector of pNZ8048. From the RE digestion profile, sample in lane 2bcontains the correct band size indicating the presence of insert cassette of VP2-IRES-GFP (2700 bp) when linearized with NcoI and SacI while the rest areincorrect ligation fragment of VP2 or IRES-GFP into pNZ:CA.



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from lysates of rabbit reticulocyte, the cell-free coupledtranscription–translation and its predecessor cell-freein vitro translation system have been utilized for eukary-


otic in vitro translation. Apart from mediating transcriptionand translation of normal eukaryotic mRNA (cap-dependent translation), it can also mediate initiation of



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Fig. 4. Western blot profile of expression of VP2 gene and gfp gene ofpNZ:vig in in vitro translation and transcription system with antibodyagainst His-Tag with different DNA concentrations. M: Pageruler PlusPrestained Protein Ladder (Fermentas, USA); 1: pNZ:vig (1 lg DNA) and 2:pNZ:vig (2 lg DNA). The expected sizes of the expressed genes are 49 kDaand 29 kDa respectively.

M 1 2

40 kDa

50 kDa

Fig. 5. Western blot profile of expressed VP2 gene of vector, pNZ:vp2 andpNZ;vig against antibody anti-VP2 (Genetex,1:5000 dilution) whensubjected to the in vitro transcription–translation system. M: SpectraMulticolor Broad Range Protein Ladder (Thermoscientific, USA); 1:pNZ:vp2; 2: pNZ:vig. The expected size of expressed VP2 gene is 49 kDa.

Fig. 6. Western blot profile of expressed reporter gene, GFP of vector,pNZ:gfp and pNZ:vig against antibody anti-GFP (Clontech, 1:2000 dilu-tion) when subjected to the in vitro transcription–translation system. M:Pageruler Plus Prestained Protein Ladder (Fermentas, USA); 1: pNZ:gfp; 2:pNZ:vig. The expected size of expressed gfp gene is 29 kDa.



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translation from IRES elements (Jang et al., 1988;Niepmann, 2009; Pelham, 1978).

Fig. 4 shows the Western blot profile of the VP2 proteinand the GFP reporter when blotted with anti-histidineantibodies which bind to the His tags of the recombinantproteins incorporated into the primers during amplifica-tion. The expressed, VP2 and gfp gene was detected as49 kDa and 29 kDa, respectively, showing that pNZ:vigwas able to mediate both cap-dependent and cap-


independent translation. To further verify this, Westernblot was also performed using specific antibodies againstinfectious bursal disease virus (IBDV) VP2 antibody (Gene-tex) and GFP as shown in Figs. 5 and 6 respectively. Thedetected bands confirmed that genes for both VP2 andgfp could be transcribed and translated for pNZ:vig, dueto the addition of IRES element. With these results, pNZ:vigis expected to successfully transfect in vitro avian cell cul-ture or even be used for delivery of VP2 to chickens viaintramuscular administration.

Construction of bicistronic DNA vectors that can co-express multiple proteins has been performed extensivelysince the discovery of IRES elements in picornaviruses(Jang et al., 1989, 1988). IRES element from poliovirusand encephalomyocarditis viruses (EMCV) are often usedto construct bi- or oligocistronic expression vectors to co-express various genes from one mRNA (Alexander et al.,1994; Balvay et al., 2009; Hennecke et al., 2001). The cur-rent trend for bicistronic vector utilizes retroviral vectors(Adam et al., 1991; Morgan et al., 1992), plant-based vec-tors (Ali et al., 2010) and yeast-based vectors(Mäkeläinen and Mäkinen, 2007; Seino et al., 2005). Thecommon trait between all these vectors are the utilizationof E. coli/pUC derived backbone for cloning and large scalereplication of the plasmids. To further expand the feasibil-ity of L. lactis vectors, this study focuses on the incorpora-tion of IRES and related eukaryotic elements asprerequisites to accommodate the use of plasmid DNAsfor transcription and translation of proteins of interest in



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a eukaryotic host (See Donnelly et al. (1997) andSchirmbeck et al. (2000) for reviews). Insertion of eukary-otic elements (such as eukaryotic promoters and its termi-nation signals) into lactococcal host has been previouslyexplored and has been shown to be able to transfect targeteukaryotic cells (Guimarães et al., 2009). However, thereare no reported studies that include IRES elements toaccommodate co-translation of multiple proteins in lacto-coccal vectors.

Plasmid DNA construction normally utilizes E. colimainly as the cell host for replication and/or protein pro-duction. E. coli possesses many ideal parameters for DNAmanipulations (i.e. high cell density, high copy number ofplasmid replication, etc.) but like other bacterial hostchosen for heterologous protein cell factory and replica-tion of recombinant plasmids, this organism also hassome limitations (Douillard et al., 2011). E. coli are cate-gorized under Gram-negative bacteria and accountablygenerate a lot of endotoxins. The presence of endotoxinsmay affect plasmid preparations and the efficiency ofDNA-based delivery for clinical purposes such as vaccina-tion, for instance. Therefore, extra steps for purification ofplasmids or purchasing endotoxin-free kits are requiredbefore the plasmid DNAs can be delivered to eukaryoticcells (Ferreira et al., 2000).

Nisin expression system (NICE) is a widely known host-vector system for cloning and expression in L. lactis. In thisstudy, one of its vectors, pNZ8048 was utilized as a DNAbackbone for lactococcal bicistronic vector construction.The pNZ8048 vector and its derivatives are well-estab-lished and extensively used in many applications such asoverexpression of eukaryotic membrane proteins, proteinsecretion and metabolic engineering (Mierau andKleerebezem, 2005). Gram et al. (2007) have describedthe differences of the antibody titer stimulated by antigensharbored by E. coli-derived vector compared to L. lactis-based DNA vectors when administered in vivo. From theirstudy, L. lactis vector harboring viral antigen can conferprotection in the host animal during the vaccination trialsalthough the E. coli-derived vectors showed more pro-nounced effect in increasing the specific cell-mediatedresponse (Gram et al., 2007). This is mainly due to theDNA composition of L. lactis vectors that consist predomi-nantly of AT nucleotides than GC compared to E. coli vec-tors. Owing to the DNA properties of L. lactis vectors, itsDNA can be manipulated not only to act as a potent adju-vant in chicken immunization against bacterial and viralinfections, but also to mediate specific immune responsethat correlates to the antigens that was introduced to thecells (Hung et al., 2011; Wang et al., 2003).

It has been demonstrated that lactoccal vectors cantransfect mammalian cells (Bermúdez-Humarán et al.,2011; Guimarães et al., 2005). However, the researcheswere limited to evaluate the functionality of reporter geneslike green flourescent protein downstream of the eukary-otic promoter and the transfection efficiency. By incorpo-rating IRES element in the vector, the ability tosimultaneously validate these parameters can be per-formed as IRES-mediated translation more or less occurwhen the cap-dependent translation is initiated (Attalet al., 1999; Hennecke et al., 2001) since not all initiation


factors are required for IRES-mediated translation.(Pisarev et al., 2005).

For future studies, pNZ:vig will be used to transientlytransfect an avian cell line and/or intramuscular injectionin chickens for a vaccination development study againstIBDV. The gene encoding VP2 protein from its RNA genomehas been identified as the major host-protective immuno-gen of IBDV and contains major epitopes responsible foreliciting neutralizing antibodies (Kim et al., 2000; Sharmaet al., 2000). In view of that, delivery of pNZ:vig into chick-ens is expected to effectively confer immunity againstIBDV.

4. Conclusion

In this study, IRES-incorporated lactococcal vector wassuccessfully constructed together with the appropriateeukaryotic promoter and transcription terminator signal.This lactococcal vector has the ability to perform cap-dependent and cap-independent translation in eukaryoticcell-free system based on the expression of viral proteinof IBDV, VP2, and of a reporter gene, gfp. The results sug-gest that the modified lactococcal plasmid can be deliveredto avian cells for the expression of VP2 antigen to alleviateIBDV outbreaks among poultries.

Acknowledgments

This work was supported by the Sciencefund researchgrant number 02-01-04-SF1046 from the Ministry of Sci-ence, Technology and Innovation of Malaysia (MOSTI).The first author was financially supported throughout herstudy by Ministry of Higher Education, Malaysia (MOHE)scholarship, MyBrain15 MyPhd. The authors would alsolike to thank Prof Kees Leenhouts for the kind gifts of thelactococcal vector and host used in this study.

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ires-incorporated lactococcal bicistronic vector for target gene expression in a eukaryotic system

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