WO2015037979A1

WO2015037979A1 - A lactococcal bicistronic vector system for gene delivery and method of preparation thereof

Info

Publication number: WO2015037979A1
Application number: PCT/MY2014/000232
Authority: WO
Inventors: Nurulfiza MAT ISA; Raha Abdul Rahim; Noorjahan Banu MOHAMED ALITHEEN; Nur Elina ABDUL MUTALIB
Original assignee: Universiti Putra Malaysia (UPM)
Current assignee: Universiti Putra Malaysia (UPM)
Priority date: 2013-09-11
Filing date: 2014-09-10
Publication date: 2015-03-19
Anticipated expiration: 2016-03-11
Also published as: MY178094A

Abstract

The present invention provides a bicistronic vector system based on Lactococcus lactis plasmid DNA into which an eukaryotic promoter, a first gene to be transcribed by the promoter, internal ribosome entry (IRES), a second gene to be transcribed by IRES and transcription terminator signal are incorporated in said order downstream of each other. The invention also provides a method of preparation of such vector.

Description

A lactococcal bicistronic vector system for gene delivery and method of preparation thereof

Field of Art

The present invention relates to a novel lactococcal bicistronic vector system for delivery of genes into eukaryotic cells.

Background Art

IRES-containing bicistronic vectors allow the simultaneous expression of two proteins separately but from the same RNA transcript. The IRES of the encephalomyocarditis virus (ECMV) permits the translation of two open reading frames from one messenger RNA. Although translation initiation of eukaryotic mRNAs occurs almost exclusively at the 5' cap, the IRES allows ribosomes to bind and initiate translation at a second, internal location. Thus, two proteins are expressed simultaneously from the same bicistronic mRNA transcript. Fluorescent protein-containing IRES vectors allow coexpression of a target protein and a fluorescent protein. Successfully transfected target cells are easily identified by fluorescence microscopy or flow cytometry.

(http://www.clontech.com US Products Fluorescent_Proteins_and_Reporters/Coexpressio n IRES_Bicistronic)

There was disclosed a bi-cistronic vector construction incorporating two different eukaryotic promoters (SV40 and CMV promoter) to express multiple genes in eukaryotic cells (WO 2011/ 113841 Al).

Further, IRES incorporation in bicistronic vector utilizing DNA vectors specific for certain hosts (retrovirus, yeast and Escherichia coli) enabling either replication or expression or both for vaccine development and cancer treatment was disclosed (WO 2004/067746 Al; EP1985709A1 ; Martin, P., Albagli, O., Poggi, M.C., Boulukos, K.E., Pognonec, P., 2006. Development of a new bicistronic retroviral vector with strong IRES activity. BMC Biotechnology 6, 4.; Morgan, R. A, Couture, L., Elroy-Stein, O., Ragheb, J., Moss, B., Anderson, W.F., 1992. Retroviral vectors containing putative internal ribosome entry sites: development of a polycistronic gene transfer system and applications to human gene therapy. Nucleic acids research 20, 1293-9.; Douin, V., Bornes, S., Creancier, L., Rochaix, P., Favre, G., Prats, A.-C, Couderc, B., 2004. Use and comparison of different internal ribosomal entry sites (IRES) in tricistronic retroviral vectors. BMC Biotechnology 4, 16.). IRES elements eliminate the requirement of two promoters and transcription terminator signals to enable multiple genes expression. It only requires several initiation of translation factors for translation thus, the host machinery translation system do not have to compete for resources to obtain the production of target proteins. Retroviral vectors are efficient to transfect mammalian cells, but downstream applications and manufacture retroviral-based vaccines are still doubted by consumers. Lactic acid bacteria (LAB) have been granted with the status of being generally regarded as safe (GRAS) and its potential in many applications have yet to be fully explored, therefore creating many possible lactococcal vector and host applications for vaccine, nutrients and beneficial genes expression and delivery.

IRES-based bicistronic vector with lactococcal DNA backbone which would solve the above problems was not designed up to date. Disclosure of the invention

The present invention provides a bicistronic vector system based on Lactococcus lactis plasmid DNA into which an eukaryotic promoter, a first gene to be transcribed by the promoter, internal ribosome entry (IRES), a second gene to be transcribed by IRES and transcription terminator signal are incorporated in said order downstream of each other,

In one preferred embodiment, the eukaryotic promoter is Pcmv.

In another preferred embodiment, the IRES is the IRES of encephalomyocarditis virus (EMCV IRES) (US4937190).

In another preferred embodiment, the terminator signal is PolyA. In yet another preferred embodiment, a first gene or a second gene to be transcribed is a gene suitable for signaling of successful transfection, such as a gene encoding a

fluorescent protein, e.g., GFP gene.

The transcription initiated by the eukaryotic promoter promptly transcribes the first gene, the IRES element and the second gene. The translation initiation will both occur in the cap region of the first gene and in the IRES structure. To maintain the ribosome binding site of the IRES element, the IRES element and the second gene is preferably amplified as a single DNA fragment as studied by Bochkov, Y.A., Palmenberg, A.C., 2006. Translational efficiency of EMCV IRES in bicistronic vectors is dependent upon IRES sequence and gene location. BioTechniques 41, 283-292.

The present invention also includes a method of preparation of the bicistromc vector system, comprising the steps of: ligating a fragment containing IRES and a second gene downstream of IRES to a first gene so that the first gene is upstream of IRES, and cloning the resulting fragment into lactococcal plasmid harboring eukaryotic promoter and transcription termination signal. The method that requires the presence of eukaryotic promoter and transcription termination signal were the ones disclosed and reviewed in Schirmbeck, R., Von Kampen, J, Metzger, K, Wild, J., Griiner, B., Schleef, M., Kroger, A., Hauser, H., Reimann, J, Kampen, J Von, 2000. DNA-Based Vaccination with Polycistronic Expression Plasmids. Methods in Molecular Medicine 29, 313-322 and Williams, J. a, Carnes, A.E., Hodgson, CP., 2009. Plasmid DNA vaccine vector design: impact on efficacy, safety and upstream production. Biotechnology Advances 27, 353-70 as a process on how to construct expression vectors for eukaryotic cells.

The resulting plasmids can be used for research purposes as well as in vaccine products, cancer research, pharmaceutical and nutraceutical industry. Polycistronic expression of proteins in mammalian cells will be possible by the introduction of GRAS status- lactococcal DNA vector into them. In particular, the designated vector has the potential to be utilized as multivalent vaccine carrying more than one or more epitope of the same or different infectious agents. This system also will be useful in a drug delivery. Brief description of Figures

Figure 1. Schematic representation of the arrangement of the bi-cistronic construct via addition of Pcmv promoter (upstream of the first gene, VP2) and IRES (upstream of reporter gene, GFP).

Figure 2. Schematic representation of the arrangement of proteins of interest, VP2 gene and GFP for the bicistronic construct. The fragment are inserted downstream of the promoter and upstream of the terminator transcription signal.

Figure 3. Schematic representation of the addition of cytomegalovirus promoter(Pcmv) and polyA, terminator transcription signal, (a) The RE digestion profile of ligated Pcmv in pNZ8048 when RE-digested with Bglll while (b) the gel pictograph indicates the successful insertion of polyA signal to the construct firstly via PCR verification of ligation.

Figure 4. The RE digestion profile of possible putative recombinant plasmids from the colonies formed from the selective agar, SGM17 supplemented with chloramphenicol. From the RE digestion profile, sample in lane 4 contain the correct band size indicating the presence of cassette VP2-IRES-GFP while the rest are incorrect ligation fragment of vp2 or IRES-GFP into pNZ:CA.

Figure 5. Western blot profile of expression of vp2 gene and gfp gene with different concentration of pNZ:vig in in-vitro translation and transcription system with antibody against His-Tag. M: Pageruler Plus Prestained Protein Ladder; l :pNZ:vig (^g) and 2: pNZ:vig ^g).

Figure 6. Western blot profile of expressed vp2 gene of vector, pNZ:vig (at different DNA concentration) and pNZ: vp2 against antibody anti-vp2 (Genetex, 1 :5000 dilution) when subjected to the in vitro transcription- translation system. M: PageRuler Plus Prestained Protein Ladder (Fermentas); lactococcal plasmid at different concentration, 1 : pNZ: vig (1 μg DNA), 2: pNZ:vig (2 μg DNA) and 3: pNZ:vp2. The expected size of expressed VP2 gene is 49 kDa.

Figure 7. Western blot profile of gfp gene of pNZ:gfp and pNZ:vig in in-vitro translation and transcription system against antibody against reporter gene, GFP; -ve: pNZ8048 as negative control, M: Pageruler Plus Prestained Protein Ladder; 1-2: Protein samples from pNZ: gfp, 3-4: Protein samples from pNZ: gfp. Both vectors when subjected to the cell- free system were at the concentration of 2 g. The expected size of expressed GFP gene is 29 kDa. Detailed Description of the Invention

The foreign DNA inserts are introduced into lactococcal vector in this manner; eukaryotic promoter, followed by the VP2 gene (coding for VP2 protein from infectious bursal disease virus, the first gene to be transcribed by the promoter), IRES, gfp gene (gene encoding for green fluorescent protein, the second gene to be transcribed by IRES) and transcription terminator signal polyA (Figure 1). The IRES element must be placed between 2 cistronic regions as shown in Figure 2. The transcription initiated by eukaryotic promoter will promptly transcribe the VP2 gene, IRES element and the gfp gene. The translation initiation will both occur in the cap region of the VP2 gene and IRES structure. To maintain the ribosome binding site of IRES element, the IRES element and the GFP gene was amplified as a single DNA fragment.

The IRES-GFP fragment was ligated to the VP2 gene of IBDV, then cloned into lactococcal plasmid harboring eukaryotic promoter (Pcmv) and transcription termination signal (polyA) (Figure 3). The verification of the possible positive recombinant plasmids carrying the functional bicistronic fragment is shown in Figure 4. It will be noted that the transcribed mRNA of the IRES and the VP2 gene will be in the polycistronic manner, similar to bacterial operon; it will be highly dependent on the activation of the eukaryotic promoter to initiate transcription of DNA.

The expression of the VP2 gene is mediated by the eukaryotic promoter (in this example, cytomegalovirus promoter) while the expression of the GFP gene is mediated by the IRES element (in this example, EMCV IRES). The Western blot profile in Figure 5 indicated the co-expression of both VP2 and gfp gene when the bicistronic vector was subjected to in vitro transcription and translation system.

To further verify the expressed genes in the in vitro system, the proteins were detected on Western blot with specific antibodies, anti-GFP and anti-VP2 as shown in Figure 6 and Figure 7 respectively. The expression of the gfp gene is regarded as cap-independent translation as the translation of the GFP gene did not undergo the interaction between capped mRNA and initiation complex for the ribosome to bind to mRNA, but the interaction of the initiation complex factor will bind to the secondary structure of the IRES element. The expression of the VP2 gene will be regarded as cap-dependent translation as the translation of the VP2 gene has undergone the recognition of ribosomal binding site via the interaction of the eukaryotic initiation complex of the capped structure of the transcribed mRNA at 5' end.

• Bacterial strains, plasmids and media

Plasmidless L. lactis NZ9000 was grown in Ml 7 media supplemented with 0.5% glucose while L.lactis NZ9000 harboring plasmids was grown in Ml 7 media (Merck) with 0.5% lactose supplemented with chloramphenicol. For transformation purposes, Ml 7 media with 0.5% glucose and 0.5% sucrose (SGM17) supplemented with chloramphenicol were used. Lactococcal cultures were incubated as a standing culture at 30°C. TOP10 E. coli cells harboring plasmids which encode vp2 gene and IRES-GFP fragments [pUCIDT VP2 UPM 04190 and pRetroX IRES ZsGreenl (Clontech)] respectively was grown in LB media (Merck) supplemented with ampicillin. The eukaryotic elements (promoter P_cmv and polyadenylation signal) were isolated from pCDNA 3.1 His A (Invitrogen). E. coli cells grown in liquid media was subjected to shaking at 250 rpm at 37°C. Antibiotic chloramphenicol and ampicillin were used when necessary at the final concentration of 7.5 μg/ml and 100 μg/ml respectively.

• Construction of recombinant plasmids Encephalomyocarditis virus' internal ribosome entry site (EMCV IRES) is the most common and versatile IRES for internal initiation of translation utilized in IRES- incorporated vectors. In this study, EMCV IRES and reporter gene, green fluorescent protein (Zoanthus sp. gfp) was fished out from pRetroX IRES ZsGreenl (Clontech). To maintain the functional internal initiation codon for IRES-mediated translation, both DNA fragments were isolated as a single cassette. VP2 gene of infectious bursal disease virus (wIBDV) from UPM isolates (UPM 04190) was synthetically produced based on accession AY791998.1 (Nurulfiza, I., Hair-Bejo, M., Omar, A.R., Aini, I., 2006. Molecular characterization of recent infectious bursal disease virus isolates from Malaysia. Acta Virologica 50, 45-51.) from the NCBI database into pUCIDT VP2 vector (IDT Technologies). The VP2 gene was isolated by PCR amplification. Primers were designed as F-vp2 (5 'ACCCCATGGAGCCGACC-ATGACAAACCTGCAAGAT3 ', SEQ ID NO. 1) and R-vp2 (5'TAGCCATGGTCTAGAGAATTCG-GCCCGGATTATGTCTTTG 3', SEQ ID NO. 2). For EMCV IRES and gfp gene, the fragments were fished out from pRetroX IRES ZsGreenl (Clontech) with using these designated primers; F-IRES (5' AAGGAATTCGCCCCTCTCCCTCCCCCCCCCCTAA 3', SEQ ID NO. 3) and R-GFP (5'AACGAGCTCTCAATCATCATCATCATCATGGGGCAA-GGCGGAGCCGGA 3', SEQ ID NO. 4). As for amplification of eukaryotic elements, P_cmv was amplified using F- Pcmv (5' AACCAG-ATCTTAGTTATTAATAGTAATCAATTACGGGGTC 3', SEQ ID NO. 5) and R-Pcmv (5' AGCAG-ATCTCCCTATAGTGAGTCGTATTAA 3', SEQ ID NO. 6) whereas polyadenylation signal was amplified using primers F-PA (5' GTAGAGCTCTGCCTTCTAGTTGCCAGCCATCTGTTG 3', SEQ ID NO. 7) and R-PA (5' CATGAGCTCCAG-AAGCCATAGAGCCCACCGCATCC 3', SEQ ID NO. 8), respectively from plasmid pCDNA 3.1. PCR amplification was carried out with the reaction mixture of lx Pfu buffer with MgSo₄ (Fermentas), 0.2mM dNTP mix (Invitrogen), 5 μΜ of forward and reverse primers, 150 ng of template DNA and 1.25U/50 μΐ Pfu polymerase. PCR reaction was set up as follows: initial denaturation at 95°C for 5 min, 25 cycles of denaturation at 95°C for 1 min, annealing at 64°C for 1 min and extension at 68°C for 1 min/kb and lastly final extension at 72°C for 10 min.

Firstly, both vp2 and IRES-GFP fragments were cut with EcoRl and ligated with T4 DNA ligase (Fermentas) forming 2691 bp DNA cassette. The ligated fragments were amplified again, RE-digested with Ncol and Sad at 37°C for 2 h and ligated into similarly RE- digested lactococcal plasmid, pNZ8048 (Mobitec).

As for inclusion of P_cmv fragment upstream of multiple cloning site of pNZ8048, the vector was RE-digested with BgRl and Ncol in 2x Tango buffer (Fermentas). The polyadenylation signal was RE-digested with Sacl and ligated downstream of gfp gene. All the eukaryotic elements are ligated to the positive transformants of pNZ8048 with the insertion of vp2-ires-gfp.

The ligation mix was electroporated into competent L. lactis NZ9000 using the methods from Holo, H, Nes, IF., 1989. High-Frequency Transformation, by Electroporation, of Lactococcus lactis subsp. cremoris Grown with Glycine in Osmotically Stabilized Media. Applied and Environmental Microbiology 55, 3119-23. The transformants were selected on SGM17 supplemented with chloramphenicol. To verify the possible putative recombinant plasmids, the clones were subjected to verification via RE digestion and sequencing.

• In-vitro expression via coupled transcription/ translation system and rabbit reticulocyte lysate

To check the functionality of the recombinant lactococcal IRES-incorporated vectors for transient transfections, the plasmids were subjected to coupled transcription-translation system via TNT® Quick Coupled Transcription/Translation System (Promega). 1.0 μg of plasmid DNA was mixed together with 40.0 μΐ of TNT Quick Mix and labeled with FluorotectGreen_LystRNA (Promega) according to the manfacturer's protocol for detection of proteins. The mixture was incubated at 30°C for 90 min.

The supernatants were loaded on a 12% SDS-PAGE/ Tris-glycine gels and the separated proteins was semi-wet blotted onto polyvinylidene fluoride (PVDF) membrane under constant current 65 mA for 1 hour. The Western blot analysis was performed using Western Max™ Horseradish Peroxidase Chromogenic Detection Kit (Amresco, USA) following the manufacturer's protocol. The membranes were blocked with 5% skimmed milk (Merck) powder for 1 h followed by incubation with mouse anti-his monoclonal antibody 1 : 5000 (Genetex) at room temperature for 1 h. Next, the membranes were incubated with secondary anti-mouse horseradish peroxidase-IgG 1 : 2000 (Merck) antibody for lh.

To further verify the presence of functional proteins both expressed via PCMV and EMCV IRES respectively, the samples were immunoblotted with anti-VP2 and anti-GFP (Clontech) via Western blotting. >pNZ:vig sequence (SEQ ID NO. 9) is:

AGATTGCAGATCTCGATGTACGGGCCAGATATACGCGTTGACATTGATTA TTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCC ATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCT GACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCC ATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTT ACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTA CGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCC CAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATT AGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGC GTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGAC GTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATG TCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGT GGGAGGTCTATATAAGCAGAGCTCTCTGGCTAACTAGAGAACCCACTGCT TACTGGCTTATCGAAATTAATACGACTCACTATAGGGAGATCTGCTCATG GAGCCGACCATGACAAACCTGCAAGATCAAACCCAACAGATTGTTCCGTT CATACGGAGCCTTCTGATGCCAACAACCGGACCGGCGTCCATTCCGGACG ACACCCTAGAGAAGCACACTCTCAGGTC AGAGACCTCGACCTACAATTTG ACTGTGGGGGACACAGGGTCAGGGCTAATTGTCTTTTTCCCTGGCTTCCC TGGCTCAATTGTGGGTGCTCACTACACACTGCAGAGCAATGGGAGCTACA AGTTCGATCAGATGCTCCTGACTGCCCAGAACCTACCGGCCAGCTACAAC TACTGCAGGCTAGTGAGTCGGAGTCTCACAGTGAGGTCAAGCACACTCCC TGGTGGCGTATATGC ACTAAATGGCACCATAAACGCCGTGACCTTCC AAG GAAGCCTGAGCGAACTGACAGATGTTAGCTACAACGGGTTGATGTCTGCA ACAGCCAACATCAACGACAAAATCGGGAACGTCCTAGTAGGGGAAGGGGT GACCGTCCTCAGCCTACCCACATCATACGACCTTGGGTATGTTAGACTCG GTGACCCCATTCCCGCTATAGGGCTCGACCCAAAAATGGTAGCAACATGT GACAGCAGTGATAGGCCCAGAGTCTACACCATAACTGCAGCCAATGATTA TCAGTTCTCATCACAGTACCAAGCAGGTGGGGTAACAATCACACTGTTCT CAGCTAATATCGATGCCATCACAAGCTTCAGCATCGGGGGAGAACTTGTG TTTGAAACAAGCGTCCAAGGCCTTATACTGGGTGCCACCATCTACCTTAT GGGCTTTGATGGGACTGCTGTAATCACCAGAGCTGTGGCCGCAGACAATG GGCTAACGGCCGGCACTGACAACCTTATGCCATTCAATATTGTGATCCCA ACCAGCGAGATAACCCAGCCAATCACATCCATCAAACTGGAAATAGTGAC CTCCAAGAGTGGTGGACAGGCGGGGGATCAGATGTCATGGTCAGCTAGTG GGAGCCTAGCAGTGACGATCCACGGTGGCAACTATCCAGGGGCCCTCCGT CCTGTCACACTAGTGGCCTACGAAAGAGTGGCAACAGGGTCTGTCGTCAC AGTTGCCGGGGTGAGCAACTTCGAGCTGATCCCAAATCCTGAACTGGCAA AGAACCTGGTCACAGAATACGGCCGATTTGACCCAGGGGCCATGAACTAC ACAAAATTGATACTGAGTGAGAGGGACCGTCTTGGCATCAAGACCGTATG GCCAACAAGGGAGTACACTGACTTTCGCGAGTACTTCATGGAGGTGGCCG ACCTCAACTCTCCCCTGAAGATTGCAGGAGCATTTGGCTTCAAAGACATA ATCCGGGCCATCATCATCATCATCATTGAGAATTCGCCCCTCTCCCTCCC CCCCCCCTAACGTTACTGGCCGAAGCCGCTTGGAATAAGGCCGGTGTGCG TTTGTCTATATGTTATTTTCCACCATATTGCCGTCTTTTGGCAATGTGAG GGCCCGGAAACCTGGCCCTGTCTTCTTGACGAGCATTCCTAGGGGTCTTT CCCCTCTCGCCAAAGGAATGCAAGGTCTGTTGAATGTCGTGAAGGAAGCA GTTCCTCTGGAAGCTTCTTGAAGACAAACAACGTCTGTAGCGACCCTTTG CAGGCAGCGGAACCCCCCACCTGGCGACAGGTGCCTCTGCGGCCAAAAGC CACGTGTATAAGATACACCTGCAAAGGCGGCACAACCCCAGTGCCACGTT GTGAGTTGGATAGTTGTGGAAAGAGTC AAATGGCTC ACCTC AAGCGTATT CAACAAGGGGCTGAAGGATGCCCAGAAGGTACCCCATTGTATGGGATCTG ATCTGGGGCCTCGGTGCACATGCTTTACATGTGTTTAGTCGAGGTTAAAA AACGTCTAGGCCCCCCGAACCACGGGGACGTGGTTTTCCTTTGAAAAACA CGATGATAATATGGCCCAGTCCAAGCACGGCCTGACCAAGGAGATGACCA TGAAGTACCGCATGGAGGGCTGCGTGGACGGCC ACAAGTTCGTGATCACC GGCGAGGGCATCGGCTACCCCTTCAAGGGCAAGCAGGCCATCAACCTGTG CGTGGTGGAGGGCGGCCCCTTGCCCTTCGCCGAGGACATCTTGTCCGCCG CCTTCATGTACGGCAACCGCGTGTTCACCGAGTACCCCCAGGACATCGTC GACTACTTCAAGAACTCCTGCCCCGCCGGCTACACCTGGGACCGCTCCTT CCTGTTCGAGGACGGCGCCGTGTGCATCTGCAACGCCGACATCACCGTGA GCGTGGAGGAGAACTGCATGTACCACGAGTCCAAGTTCTACGGCGTGAAC TTCCCCGCCGACGGCCCCGTGATGAAGAAGATGACCGACAACTGGGAGCC CTCCTGCGAGAAGATCATCCCCGTGCCCAAGCAGGGCATCTTGAAGGGCG ACGTGAGCATGTACCTGCTGCTGAAGGACGGTGGCCGCTTGCGCTGCCAG TTCGACACCGTGTACAAGGCCAAGTCCGTGCCCCGCAAGATGCCCGACTG GCACTTCATCCAGCACAAGCTGACCCGCGAGGACCGCAGCGACGCCAAGA ACCAGAAGTGGCACCTGACCGAGCACGCCATCGCCTCCGGCTCCGCCTTG CCCCATCATCATCATCATCATTCAGAGCTCCTGCCTTCTAGTTGCCAGCC ATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCA CTCCC ACTGTCCTTTCCTAATAAAATGAGGAAATTGC ATCGC ATTGTCTG AGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGG GGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTA TGGCTTCTGGAGCTAAGCTTTCTTTGAACCAAAATTAGAAAACCAAGGCT TGAAACGTTCAATTGAAATGGCAATTAAACAAATTACAGCACGTGTTGCT TTGATTGATAGCCAAAAAGCAGCAGTTGATAAAGCAATTACTGATATTGC TGAAAAATTGTAATTTATAAATAAAAATCACCTTTTAGAGGTGGTTTTTT TATTTATAAATTATTCGTTTGATTTCGCTTTCGATAGAACAATCAAAGCG AGAATAAGGAAGATAAATCCCATAAGGGCGGGAGCAGAATGTCCGAGACT AATGTAAATTTGTCCACCAATTAAAGGACCGATAACGCGAGCTTCTCGAG TGCATATTTTCGGCAATCTTCTCAATGAGATGCTCTTCAGCATGTTCAAT GATGTCGATTTTTTATTAAAACGTCTCAAAATCGTTTCTGAGACGTTTTA GCGTTTATTTCGTTTAGTTATCGGCATAATCGTTAAAACAGGCGTTATCG TAGCGTAAAAGCCCTTGAGCGTAGCGTGCTTTGCAGCGAAGATGTTGTCT GTTAGATTATGAAAGCCGATGACTGAATGAAATAATAAGCGCAGCGTCCT TCTATTTCGGTTGGAGGAGGCTCAAGGGAGTTTGAGGGAATGAAATTCCC TCATGGGTTTGATTTTAAAAATTGCTTGCAATTTTGCCGAGCGGTAGCGC TGGAAAATTTTTGAAAAAAATTTGGAATTTGGAAAAAAATGGGGGGAAAG GAAGCGAATTTTGCTTCCGTACTACGACCCCCCATTAAGTGCCGAGTGCC AATTTTTGTGCCAAAAACGCTCTATCCCAACTGGCTCAAGGGTTTGAGGG GTTTTTCAATCGCCAACGAATCGCCAACGTTTTCGCCAACGTTTTTTATA AATCTATATTTAAGTAGCTTTATTGTTGTTTTTATGATTACAAAGTGATA CACTAATTTTATAAAATTATTTGATTGGAGTTTTTTAAATGGTGATTTCA GAATCGAAAAAAAGAGTTATGATTTCTCTGACAAAAGAGCAAGATAAAAA ATTAACAGATATGGCGAAACAAAAAGGTTTTTCAAAATCTGCGGTTGCGG CGTTAGCTATAGAAGAATATGCAAGAAAGGAATCAGAACAAAAAAAATAA GCGAAAGCTCGCGTTTTTAGAAGGATACGAGTTTTCGCTACTTGTTTTTG ATAAGGTAATATATCATGGCTATTAAAAATACTAAAGCTAGAAATTTTGG ATTTTTATTATATCCTGACTCAATTCCTAATGATTGGAAAGAAAAATTAG AGAGTTTGGGCGTATCTATGGCTGTCAGTCCTTTACACGATATGGACGAA AAAAAAGATAAAGATACATGGAATAGTAGTGATGTTATACGAAATGGAAA GCACTATAAAAAACCACACTATCACGTTATATATATTGCACGAAATCCTG TAACAATAGAAAGCGTTAGGAACAAGATTAAGCGAAAATTGGGGAATAGT TCAGTTGCTCATGTTGAGATACTTGATTATATCAAAGGTTCATATGAATA TTTGACTCATGAATCAAAGGACGCTATTGCTAAGAATAAACATATATACG ACAAAAAAGATATTTTGAACATTAATGATTTTGATATTGACCGCTATATA ACACTTGATGAAAGCCAAAAAAGAGAATTGAAGAATTTACTTTTAGATAT AGTGGATGACTATAATTTGGTAAATACAAAAGATTTAATGGCTTTTATTC GCCTTAGGGGAGCGGAGTTTGGAATTTTAAATACGAATGATGTAAAAGAT ATTGTTTCAACAAACTCTAGCGCCTTTAGATTATGGTTTGAGGGCAATTA TCAGTGTGGATATAGAGCAAGTTATGCAAAGGTTCTTGATGCTGAAACGG GGGAAATAAAATGACAAACAAAGAAAAAGAGTTATTTGCTGAAAATGAGG AATTAAAAAAAGAAATTAAGGACTTAAAAGAGCGTATTGAAAGATACAGA GAAATGGAAGTTGAATTAAGTACAACAATAGATTTATTGAGAGGAGGGAT TATTGAATAAATAAAAGCCCCCCTGACGAAAGTCGACGGCAATAGTTACC CTTATTATCAAGATAAGAAAGAAAAGGATTTTTCGCTACGCTCAAATCCT TTAAAAAAACACAAAAGACCACATTTTTTAATGTGGTCTTTATTCTTCAA CTAAAGCACCCATTAGTTCAACAAACGAAAATTGGATAAAGTGGGATATT TTTAAAATATATATTTATGTTACAGTAATATTGACTTTTAAAAAAGGATT GATTCTAATGAAGAAAGCAGACAAGTAAGCCTCCTAAATTCACTTTAGAT AAAAATTTAGGAGGCATATCAAATGAACTTTAATAAAATTGATTTAGACA ATTGGAAGAGAAAAGAGATATTTAATCATTATTTGAACCAACAAACGACT TTTAGTATAACCACAGAAATTGATATTAGTGTTTTATACCGAAACATAAA ACAAGAAGGATATAAATTTTACCCTGCATTTATTTTCTTAGTGACAAGGG TGATAAACTCAAATACAGCTTTTAGAACTGGTTACAATAGCGACGGAGAG TTAGGTTATTGGGATAAGTTAGAGCCACTTTATACAATTTTTGATGGTGT ATCTAAAACATTCTCTGGTATTTGGACTCCTGTAAAGAATGACTTCAAAG AGTTTTATGATTTATACCTTTCTGATGTAGAGAAATATAATGGTTCGGGG AAATTGTTTCCCAAAACACCTATACCTGAAAATGCTTTTTCTCTTTCTAT TATTCCTTGGACTTCATTTACTGGGTTTAACTTAAATATCAATAATAATA GTAATTACCTTCTACCCATTATTACAGCAGGAAAATTCATTAATAAAGGT AATTCAATATATTTACCGCTATCTTTACAGGTACATCATTCTGTTTGTGA TGGTTATCATGCAGGATTGTTTATGAACTCTATTCAGGAATTGTCAGATA GGCCTAATGACTGGCTTTTATAATATGAGATAATGCCGACTGTACTTTTT ACAGTCGGTTTTCTAATGTCACTAACCTGCCCCGTTAGTTGAAGAAGGTT TTT ATATT AC AGCTC C A

Claims

1. A bicistronic vector system based on Lactococcus lactis plasmid DNA into which an eukaryotic promoter, a first gene to be transcribed by the promoter, internal ribosome entry (IRES), a second gene to be transcribed by IRES and transcription terminator signal are incorporated in said order downstream of each other.

2. The bicistronic vector system according to claim 1, wherein the eukaryotic promoter is Pcmv.

3. The bicistronic vector system according to claim 1, wherein the IRES is the IRES of encephalomyocarditis virus (EMCV IRES).

4. The bicistronic vector system according to claim 1, wherein the terminator signal is PolyA.

5. The bicistronic vector system according to claim 1, wherein the first gene or the second gene to be transcribed is a gene suitable for signaling of successful transfection, such as a gene encoding a fluorescent protein.

6. A method of preparation of the bicistronic vector system, comprising the steps of:

ligating a fragment containing IRES and a second gene downstream of IRES to a first gene so that the first gene is upstream of IRES, and

cloning the resulting fragment into lactococcal plasmid harboring eukaryotic promoter and transcription termination signal.

7. Use of the bicistronic vector system according to claim 1 for research purposes, in vaccine products, in cancer research, in pharmaceutical and/or nutraceutical industry.

8. Use of IRES-integrated Lactococcus lactis plasmid backbone for producing a bicistronic vector in eukaryotic system.