WO2019117700A1 - Process for the expression of recombinant proteins in microalgae using viral vectors - Google Patents
Process for the expression of recombinant proteins in microalgae using viral vectors Download PDFInfo
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- WO2019117700A1 WO2019117700A1 PCT/MX2018/050030 MX2018050030W WO2019117700A1 WO 2019117700 A1 WO2019117700 A1 WO 2019117700A1 MX 2018050030 W MX2018050030 W MX 2018050030W WO 2019117700 A1 WO2019117700 A1 WO 2019117700A1
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- C—CHEMISTRY; METALLURGY
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/12—Unicellular algae; Culture media therefor
- C12N1/125—Unicellular algae isolates
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/89—Algae ; Processes using algae
Definitions
- the present invention relates to the technical field of Biotechnology, as it relates to a process for the expression of recombinant proteins, which comprises the use of a viral DNA vector to induce expression and protocols to achieve transient expression said microalgae
- the process is characterized by including the use of Schizochytrium sp. strain ATCC 20888 and Chlamydomonas reinhardtii as expression hosts, Agrobacterium tumefaciens GV3101 as vector for delivery of viral DNA by inclusion in a binary vector. It also includes the construction and use of a viral vector that includes the Ori origin of the geminivirus Ageratum enantion virus (AgEV).
- AgEV geminivirus Ageratum enantion virus
- the process of the present invention is used for the production of recombinant proteins of biopharmaceutical interest useful in the preparation of vaccines for use in living beings that need them and / or in bioprocesses that require it.
- recombinant proteins that can be obtained by the process of the present invention, those coming from Zaire ebolavirus and / or Escherichia coli stand out, as application examples, without limiting its application in other recombinant proteins useful in the industry.
- microalgae for the production of proteins of interest is gaining relevance in the field of biotechnology.
- transplastomic clones require a laborious construction of species-specific vectors and the system is unable to carry out complex post-translational modifications (eg, glycosylation) or secrete the recombinant protein.
- complex post-translational modifications eg, glycosylation
- secrete the recombinant protein secrete the recombinant protein.
- genetic engineering faces two challenges: the improvement of yields and the genetic stability of transformed clones (Rosales-Mendoza 2016b).
- Viral vectors have been widely applied in the expression of heterologous proteins in plants (Salazar-González et al., 2015, Dugdale et al., 2013; Chen, et al; 201 1; Lico et al., 2008; Gleba et al. ., 2007).
- the Magnifection system described ten years ago became a powerful tool for the transient expression of recombinant proteins in higher plants (Gleba et al., 2013, Marillonnet et al., 2005).
- some viral elements have been used to innovate approaches to genetic engineering.
- the picornaviral 2A sequence has allowed the expression of policistrons (Rasala 2012) and the use of synthetic promoters has led to improved expression (Scranton 2016).
- TW201224145 integrates an expression vector that includes a protein expression cassette and at least two recombinant DNA homologous fragments flanking the protein expression cassette, which comprises a first marker of selection, promoters, a multiple cloning site adjacent to backflow of the promoter and a second selection marker, wherein the promoter comprises a 356S promoter of Cauliflower mosaic virus (CAMV). It contemplates a promoter of Ribulose bisphosphate carboxylase, an adenine methyltransferase (AMT) promoter, a nitrate reductase promoter or a combination thereof.
- AMT adenine methyltransferase
- WO2013182484 refers to a method for the expression and secretion of a fully assembled and functional protein complex having medical or biotechnological utility in microalgae, wherein the protein complex is an antibody, an enzyme, hormone or vaccine and said complex it is secreted in a medium.
- the micro alga used is Phaeodactylum tricornutum.
- Document KR20130006178 claims a method for preparing a target protein using a recombinant vector for transforming fungi, provided to express and obtain various proteins in fungi, especially Pleurotus eryngii.
- Constitution A recombinant vector for transforming fungi comprising: CaMV 35S (cauliflower mosaic virus 35S) promoter; CaMV 35S 5'- untranslated sequence (UTS); CaMV 35S 3'-UTS; NOS (nopaline synthase) terminator; and a nucleotide sequence that encodes a target protein.
- CaMV 35S cauliflower mosaic virus 35S
- UTS CaMV 35S 5'- untranslated sequence
- NOS nopaline synthase
- the method for preparing the fungal target protein comprises: a step to transform a fungus conidium with a recombinant vector to transform the fungi; and a step to grow mushroom conidia and collect mushroom mycelia.
- the white protein is bovine growth hormone and the method further comprises the step of growing the mycelium.
- US2014127254 claims a system for releasing a biologically active protein to a host animal comprising an algal cell transformed by an expression vector and the expression vector comprising a nucleotide sequence encoding the biologically active protein, operably linked to a promoter.
- the algae is suspended in water for immersion in the host animal, the release system is selected from hormones and antimicrobial peptides.
- the algae is mixed with a sample or food for the animal host
- the animal is selected from mammals, fish, birds and crustaceans.
- the protein is a peptide derived from bactericidal proteins, insecticidal proteins, growth hormones and antigens.
- the antigen delivery system comprises an algal cell transformed by an expression vector, and the expression vector comprises a nucleotide sequence encoding a determinant antigen and the expression vector further comprises a promoter operably linked to the nucleotide sequence encoding for the determinant antigen.
- the algal cell expresses the determinant antigen in an area selected from the group consisting of nucleus, chloroplast, mitochondria, periplasmic space, cell membrane or cell wall.
- the algal cell is Chlamydomonas reinhardtii.
- US2015164965 claims a transgenic eukaryotic microalgae comprising an expression cassette comprising at least one transcriptable polynucleotide encoding an exogenous biologically active protein expressed within a subcellular compartment of the microalgae cell.
- the compartment is selected from cellular vacuole, endoplasmic reticulum, Golgi system, Lisosome and peroxisome.
- the cassette further comprises a polynucleotide which encodes a peptide directed to vacuoles, which improves the consumption of the exogenous protein expressed by a xenogenetic cell or tissue.
- the microalga used is of marine origin and is selected from: Phaeodactylum tricornutum, Dunaliella spp.,
- Nannochloropsis spp. Nannochloris spp., Tetraselmis spp., Isochrysis galbana; Pavlova spp .; Amphiprora hyaline; Chaetoceros muellerr, and Neochloris oleoabundans.
- the document MX201001 151 claims a method for generating genotypic variation in a genome of a plant, the method characterized in that it comprises introducing into the plant at least one viral expression vector encoding at least one chimeric nuclease comprising a DNA domain. DNA binding, a nuclease and a localization signal to an organelle containing DNA, where the DNA binding domain mediates the specific nuclease direction to the plant genome, in order to generate genotypic variation in the genome of plant.
- the method for treating a plant infection by a pathogen comprises introducing into the plant at least one viral expression vector encoding at least one chimeric nuclease comprising a DNA binding domain and a nuclease, in where the DNA binding domain mediates the nuclease's address to the pathogen's genome, in order to prevent or treat an infection of the plant by a pathogen. It also claims a plant viral expression vector, comprising a nucleic acid sequence encoding at least one chimeric nuclease comprising a DNA binding domain, a nuclease and a location signal to an organelle containing DNA.
- An expression vector based on pTRV comprising a nucleic acid sequence encoding at least two heterologous polypeptide sequences.
- a transgenic plant characterized in that it comprises the aforementioned plant viral expression vector, the generation of genotypic variation is transient.
- the viral expression vector comprises a Tobacco Rattle virus (TRV) expression vector.
- US20130295607 claims a method of producing a protein, comprising: cultivating a recombinant microorganism of the order Thraustochytrols in a medium, comprising a molecule isolated from nucleic acid and a gene that encodes the protein to produce the protein and the recovery of the protein.
- the microorganism is of the selected species of the group: Schizochytrium sp., Schizochytrium aggregatum, Schizochytrium iimacinum, Schizochytrium minutum, Thraustochytrium sp., Thraustochytrium striatum, Thraustochytrium aureum, Thraustochytrium roseum, and Japonochytrium sp.
- the document MX304364 claims a process to produce one or more proteins of interest, characterized in that it comprises
- a first heterologous nucleotide sequence comprising: a nucleotide sequence encoding an RNA replicon, and a first inducible promoter operably linked to the nucleotide sequence which encodes said RNA replicon; the RNA does not code for a protein that provides cell-to-cell movement of the RNA replicon in the plant; the RNA replicon codes for a polymerase to replicate the RNA replicon and the one or more of a protein of interest; Y
- a second heterologous nucleotide sequence comprising a sequence encoding a protein that enables cell-to-cell movement of the RNA replicon, wherein the second heterologous nucleotide sequence comprises a second inducible promoter operably linked to the sequence encoding the protein that enables the cell to cell movement of the DNA replicon;
- step (a) Inducing, in the plant or plant cell of step (a), the first and second inducible promoters thereby producing one or more of a protein of interest in the plant or in the cell of the plant, respectively.
- the RNA replicon is derived from a positive strand single-stranded RNA virus, belonging to the tobacco mosaic virus or the potato X virus.
- Figure 1 It shows the map of the viral vector of the present invention (pAlgevir).
- Figure 2 Shows the detection of replicons by inverse PCR.
- Figure 3 Shows the immunodetection by means of Western blot of the recombinant proteins GP1 (A) and LTB (B) produced in Shizochrytrium sp.
- Figure 4 Shows yields of the recombinant proteins produced in Shizochrytrium sp. with the process of the present invention (Algevir).
- the present invention relates to a process for the expression of recombinant proteins in microalgae through viral DNA vectors, to induce the expression that is delivered through Agrobaterium tumefaciens; as well as protocols to achieve transient expression in microalgae.
- the invention comprises the use of viral vectors that, through an efficient transient transformation, lead to an accumulation of the recombinant protein at high levels (up to 1.25 mg / g in fresh weight), at the same time, the expression process is inducible by ethanol , which allows to phase out the moment of transformation with that of the beginning of the expression.
- the process has been validated both in photosynthetic freshwater microalgae and in non-photosynthetic marine microalgae.
- the process for the expression of recombinant proteins of the present invention in microalgae comprises the use of:
- At least one photosynthetic freshwater microalgae and / or a non-photosynthetic marine microalgae as an expression host is selected from the species: Schizochytrium sp. and / or Chlamydomonas reinhardtir, ii) The use of Agrobacterium tumefaciens as a vector for the delivery of viral DNA through its inclusion in a binary vector; iii) At least one recombinant protein (z) of biopharmaceutical interest related to a disease that affects a living being, or in bioprocesses that require it, as a molecule of interest, such as antigens for the preparation of vaccines, hormones or enzymes .
- VMC Cauliflower Mosaic Virus
- the elements of (1) to (7) are included in the context of the binary vector pB1121 which allows the transfer of the expression cassettes by Agrobacterium tumefaciens.
- the process for the expression of recombinant proteins in microalgae through viral vectors of the present invention comprising the steps of: a) Construct the vector of Figure 1, which has as a base structure a functional binary vector in Agrobacterium tumefaciens; b) Transiently transform a microalga host expression by inoculating Agrobacterium tumefaciens, in a medium supplemented with 100 pM Acetosyringone and a subsequent addition of 250 mg / L cefotaxime.
- Chlamydomonas reinhardtii and / or Schizochytrium sp. Indistinctly; c) Express the gene of interest (may be GP1, from Zaire ebolavirus or LTB from E. coli, or a recombinant protein (z) in which antigens may be included for the preparation of vaccines, hormones or enzymes, (to cite some examples) in the transiently transformed microalga of step b) by the addition of a solution of an alcohol.
- the gene of interest may be GP1, from Zaire ebolavirus or LTB from E. coli, or a recombinant protein (z) in which antigens may be included for the preparation of vaccines, hormones or enzymes, (to cite some examples) in the transiently transformed microalga of step b) by the addition of a solution of an alcohol.
- strain Schizochytrium sp. refers to strain ATCC 20888
- Chlamydomonas reinhardtii refers to the strain available at: http://www.chlamycollection.org/product/cc-125-wild-type-mt-137c
- Agrobacterium tumefaciens refers to strain GV3101 available for consultation at: http://www.mpipz.mpg.de/koncz/publications.
- the process for the expression of recombinant proteins in microalgae through viral vectors of the present invention shows an expression yield of recombinant proteins of interest of at least 1.25 mg / g in fresh weight in some embodiments, however , yields of at least 0.12 mg / g in fresh weight can be obtained, depending on the nature and / or origin of the recombinant protein to be obtained.
- the expression process of the present invention is functional and leads to the efficient expression of recombinant proteins.
- AlcA promoter from Aspergillus nidulans with restriction sites Smal (5 ' ) and BamHI and Pstl (3 ' ) in which the gene of interest can be introduced.
- This DNA cassette was synthesized by GenScript Inc. (New Jersey, USA) and subcloned into the binary vector pBI121 at the Xbal-Sacl sites through standard digestion-ligation procedures.
- a positive clone identified by the restriction profile was used and sequencing was subsequently performed to confirm the integrity of the sequence.
- the construction was subsequently transferred to Agrobacterium tumefaciens (strain GV3101) by electroporation (Cangelosi et al., 1991).
- a clone carrying the expression vector was confirmed by polymerase chain reaction (PCR) and subsequently cultured overnight in Luria Bertani medium at 25 ° C and 200 rpm to perform expression assays.
- the marine microalga Schizochytrium sp. strain 20888 was obtained from ATCC (USA), cultured at 25 ° C in modified seawater medium (5 g / L of peptone, 1 g / L of yeast extract, 0.2 g / L of FeS04, g / L of agar and 35 g / L of NaCl).
- modified seawater medium 5 g / L of peptone, 1 g / L of yeast extract, 0.2 g / L of FeS04, g / L of agar and 35 g / L of NaCl.
- 679BY medium (1 g / L of yeast extract, 1 g / L of peptone, 5 g / L of dextrose and 35 g / L of NaCl
- the freshwater microalgae Chlamydomonas reinhardtii was cultured in 100 ml of the TAP medium (tris-acetate-phosphates), at 25 ° C with
- the expression of the gene of interest in the transiently transformed microalgae was induced 20 h after inoculation by the addition of 1 mL of absolute ethanol (1% of the final concentration). Culture samples were collected before induction and 12, 24 and 48 h after induction. Samples were stored at -80 ° C until later use.
- Circular DNA replicons were detected by inverse PCR using the following sets of primers:
- forward SEQ ID NO: 1 5 ' CATCACCAAGATACCGGAGAAG and reverse (SEQ ID NO: 2) 5TTTAGTTTCCCAGAAGGCCC 3 ' ;
- forward (SEQ ID NO: 3) 5 ' CATTTCCCTCTTTCCAGCCA and reverse (SEQ ID NO: 4) 5TTATGGAGAAACTCGAGCTTGT 3 ' .
- the total DNA was isolated from cultures of Schizochytrium sp. According to Dellaporta et al. (1983).
- the PCR reaction mixtures contained 1 c PCR buffer, 100 ng DNA, 1.5 mM magnesium chloride, 2.5 U Taq DNA polymerase, 1 mM dNTPs, and 1 pM of the corresponding set of primers. Cyclic conditions were: 94 ° C for 5 min (initial denaturation); 35 cycles at 94 ° C for 30 s, 55 ° C for 30 s, 72 ° C for 120 s, and a final extension at 72 ° C for 10 min. The PCR products were detected by electrophoresis in 1% agarose gels. The negative control consisted of 100 ng of culture DNA of Schizochytrium sp. Protein analysis
- the integrity of the proteins obtained from Schizochytrium were evaluated by Western blot analysis.
- the cultures for sample were collected in different induction times (0, 12, 24, and 48 h), and the protein extracts were obtained as follows: 20 mg of fresh biomass was resuspended in 200 pL of an extraction buffer (750 mM Tris-Hydrochloric acid, pH 8, 15% sucrose, 100 mM b-mercaptoethanol, and 1 mM PMSF) and sonicated (4 pulses of 4 s with 4 s of delay each other) using an ultrasonic processor equipment with 24% amplitude (model GEX130PB).
- an extraction buffer 750 mM Tris-Hydrochloric acid, pH 8, 15% sucrose, 100 mM b-mercaptoethanol, and 1 mM PMSF
- sonicated 4 pulses of 4 s with 4 s of delay each other
- the samples were subsequently centrifuged at 8000 rpm for 15 min and the supernatants were transferred to new microtubes.
- the total concentration of soluble proteins was determined in the extracts by Lowry method and a volume corresponding to 80 pg of TSP (approximately 100 pL) was mixed with the same volume of 2x reducing buffer, and denatured by boiling for 5 min at 95 ° C , and subsequently subject to SDS-PAGE analysis.
- the gels were used to transfer the proteins to Bio-Rad nitrocellulose membranes of 0.45 L m (http://www.bio-rad.com).
- Protein transfer was performed using a TV100-EBK Electroblotter (AlphaMetrix Biotech, GER) for 1 h at 150 V in a methanol-based buffer. After blocking with 5% of fat-free milk (Carnation, Nestle) dissolved in PBS-0.1% Tween, the membranes were incubated with anti-mouse serum (1: 200 dilution) against both, LTB or GP1; which were produced in mice as previously described (Orellana-Escobedo et al., 2015). The membranes were washed and incubated with a secondary mouse anti-IgG antibody conjugated with goat radish peroxidase (1: 2,000 Sigma dilution) for 2 h at room temperature.
- a secondary mouse anti-IgG antibody conjugated with goat radish peroxidase (1: 2,000 Sigma dilution
- Antigen detection was revealed by incubation of the membranes with SuperSignal West Dura solution, following the supplier's instructions (Thermo Scientific, http://www.thermoscientific.com) and exposing the film to standard procedures.
- the purified proteins LTB and GP1 (250 ng) produced in recombinant E. coli were used as positive controls.
- coli were expressed, inserted in the Sma I site at the 5 'end and any of the Bam Hl or Pst I sites at the 3' end, whose expression is under the control of the AlcA inducible promoter.
- the transcription factor AlcR is activated leading to the expression of the gene of interest and the Rep protein.
- the latter acts on the Ori elements and through the generation and replication of circular DNA molecules that carry the cassette. expression for the recombinant protein. (See figure 5).
- Algevir allows the efficient expression of viral and bacterial antigens.
- Figure 2 shows the detection of replicons by inverse PCR in DNA samples of Schizochrytrium sp. Transiently transformed with the vectors pAlgevir-GP1 (A) and pAlgevir-LTB (B); using oligonucleotides to detect circular replicons generated by the Rep protein.
- the lanes show the following: M, 1 Kb molecular weight marker (New England, biolabs); 1-5, Schizochrytrium DNA samples sp. transiently transformed with A. tumefaciens carrying the vector pAlgevir, taken at 0, 12, 24, 48 and 72 h post-induction, respectively; 6: DNA sample from Schizochrytrium sp. untransformed (WT); 7, negative control (water).
- the amplicons expected for the detection of the GP1 and LTB replicons are 1800 and 918 bp in length, respectively.
- FIG. 3 shows the immunodetection by means of Western blot of the recombinant proteins GP1 (A) and LTB (B) produced in Schizochrytrium sp .; as well as the GP1 protein in Chlamydomonas reinhardtii (C), through the Algevir system. Positive controls consisted of 250 ng of GP1 or LTB.
- the recombinant protein is produced 48 h after induction and reached values of up to 1.25 mg / g of fresh weight (FW) for GP1 (6 mg / L of culture) and 0.12 mg / g FW (0.6 mg / L culture) for LTB (Figure 4).
- Figure 4 shows the yields of the recombinant proteins produced in Schizochrytrium sp. with the process of the present invention (Algevir).
- a quantitative enzyme-linked immunosorbent assay (ELISA) was performed to determine the accumulation levels of Schizochrytrium sp proteins. at 48 h postinduction. Pure proteins were used to construct standard curves for GP1 (A) and LTB (B).
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Abstract
The invention relates to a process for the expression of recombinant proteins, comprising the use of a viral DNA vector in order to induce expression and protocols in order to obtain transient expression in said microalgae. The process is characterised in that it comprises the use of Schizochytrium sp. strain ATCC 20888 and Chlamydomonas reinhardtii as expression hosts, and Agrobacterium tumefaciens GV3101 as a viral DNA delivery vector, by means of its inclusion in a binary vector. The invention also relates to the construction and use of a viral vector that includes the replication origin Ori of the geminivirus Ageratum enation virus (AgEV). The process of the invention is used to produce recombinant proteins of biopharmaceutical interest, including those from Zaire ebolavirus and Escherichia coli, which can be used to develop vaccines for use by living beings that need same and/or in bioprocesses in which they are required.
Description
PROCESO PARA LA EXPRESIÓN DE PROTEÍNAS RECOMBINANTES EN MICROALGAS A TRAVÉS DE VECTORES VIRALES PROCESS FOR THE EXPRESSION OF RECOMBINANT PROTEINS IN MICROALOGS THROUGH VIRAL VECTORS
CAMPO TÉCNICO TECHNICAL FIELD
La presente invención se relaciona con el campo técnico de la Biotecnología, ya que se refiere a un proceso para la expresión de proteínas recombinantes, que comprende el uso de un vector de ADN viral para inducir la expresión y los protocolos para lograr la expresión transitoria dichas microalgas. El proceso se caracteriza por incluir el uso de Schizochytrium sp. cepa ATCC 20888 y Chlamydomonas reinhardtii como hospederos de expresión, Agrobacterium tumefaciens GV3101 como vector para la entrega de ADN viral mediante su inclusión en un vector binario. Así mismo, incluye la construcción y el uso de un vector viral que incluye el origen de replicación Orí del geminivirus Ageratum enantion virus (AgEV). El proceso de la presente invención es utilizado para la producción de proteínas recombinantes de interés biofarmacéutico útiles en la elaboración de vacunas para uso en seres vivos que las necesiten y/o en bioprocesos que lo requieran. Entre las proteínas recombinantes que pueden ser obtenidas mediante el proceso de la presente invención, se destacan las provenientes de Zaire ebolavirus y/o Escherichia coli, como ejemplos de aplicación, sin que ello limite su aplicación en otras proteínas recombinantes útiles en la industria. The present invention relates to the technical field of Biotechnology, as it relates to a process for the expression of recombinant proteins, which comprises the use of a viral DNA vector to induce expression and protocols to achieve transient expression said microalgae The process is characterized by including the use of Schizochytrium sp. strain ATCC 20888 and Chlamydomonas reinhardtii as expression hosts, Agrobacterium tumefaciens GV3101 as vector for delivery of viral DNA by inclusion in a binary vector. It also includes the construction and use of a viral vector that includes the Ori origin of the geminivirus Ageratum enantion virus (AgEV). The process of the present invention is used for the production of recombinant proteins of biopharmaceutical interest useful in the preparation of vaccines for use in living beings that need them and / or in bioprocesses that require it. Among the recombinant proteins that can be obtained by the process of the present invention, those coming from Zaire ebolavirus and / or Escherichia coli stand out, as application examples, without limiting its application in other recombinant proteins useful in the industry.
ANTECEDENTES BACKGROUND
El uso de microalgas para la producción de proteínas de interés está adquiriendo relevancia en el campo de la biotecnología. Varios antígenos vacunales funcionales, anticuerpos y otras proteínas terapéuticas; así como enzimas para uso industrial, se han producido en microalgas (Scranton 2015; Rosales-Mendoza 2016a). Los rendimientos más altos, observados hasta el momento, se consiguieron en microalgas fotosintéticas transformadas en el genoma del cloroplasto; con rendimientos de hasta 3 mg/L de cultivo (1 ,86 % de la proteína total soluble (TSP), Gimpel et al., 2015). No
obstante, el desarrollo de clonas transplastómicas requiere una construcción laboriosa de vectores específicos de especie y el sistema es incapaz de llevar a cabo modificaciones postraduccionales complejas (por ejemplo, glicosilación) o secretar la proteína recombinante. En el caso de la expresión nuclear, la ingeniería genética se enfrenta a dos desafíos: la mejora de los rendimientos y la estabilidad genética de las clonas transformadas (Rosales-Mendoza 2016b). En un esfuerzo por superar estos obstáculos, en el estado de la técnica, se ha utilizado la mutagénesis inducida por luz UV para generar cepas de Chlamydomonas reinhardtii que presentan una mejora en la expresión (con rendimientos de hasta el 0,2% de TSP) (Neupert et al., 2009; Kurniasih et al., 2016). También se han aplicado estrategias basadas en la secreción de la proteína recombinante al medio de cultivo, logrando rendimientos de hasta 10 mg de proteína secretada por litro de cultivo (Lauersen et al., 2013). The use of microalgae for the production of proteins of interest is gaining relevance in the field of biotechnology. Several functional vaccine antigens, antibodies and other therapeutic proteins; as well as enzymes for industrial use, have been produced in microalgae (Scranton 2015, Rosales-Mendoza 2016a). The highest yields, observed so far, were achieved in photosynthetic microalgae transformed in the chloroplast genome; with yields of up to 3 mg / L of culture (1, 86% of total soluble protein (TSP), Gimpel et al., 2015). Do not However, the development of transplastomic clones requires a laborious construction of species-specific vectors and the system is unable to carry out complex post-translational modifications (eg, glycosylation) or secrete the recombinant protein. In the case of nuclear expression, genetic engineering faces two challenges: the improvement of yields and the genetic stability of transformed clones (Rosales-Mendoza 2016b). In an effort to overcome these obstacles, in the state of the art, the mutagenesis induced by UV light has been used to generate strains of Chlamydomonas reinhardtii that present an improvement in the expression (with yields of up to 0.2% of TSP) (Neupert et al., 2009; Kurniasih et al., 2016). Strategies based on the secretion of the recombinant protein have also been applied to the culture medium, achieving yields of up to 10 mg of secreted protein per liter of culture (Lauersen et al., 2013).
Los vectores virales se han aplicado ampliamente en la expresión de proteínas heterólogas en plantas (Salazar-González et al., 2015; Dugdale et al., 2013; Chen, et al; 201 1 ; Lico et al., 2008; Gleba et al., 2007). Por ejemplo, el sistema Magnifection descrito hace diez años se convirtió en una poderosa herramienta para la expresión transitoria de proteínas recombinantes en plantas superiores (Gleba et al., 2013, Marillonnet et al., 2005). En el caso de las microalgas, algunos elementos virales se han utilizado para innovar los enfoques de la ingeniería genética. Por ejemplo, la secuencia picornaviral 2A, ha permitido la expresión de policistrones (Rasala 2012) y el uso de promotores sintéticos ha conducido a una expresión mejorada (Scranton 2016). Viral vectors have been widely applied in the expression of heterologous proteins in plants (Salazar-González et al., 2015, Dugdale et al., 2013; Chen, et al; 201 1; Lico et al., 2008; Gleba et al. ., 2007). For example, the Magnifection system described ten years ago became a powerful tool for the transient expression of recombinant proteins in higher plants (Gleba et al., 2013, Marillonnet et al., 2005). In the case of microalgae, some viral elements have been used to innovate approaches to genetic engineering. For example, the picornaviral 2A sequence has allowed the expression of policistrons (Rasala 2012) and the use of synthetic promoters has led to improved expression (Scranton 2016).
Además de lo antes mencionado, en el estado de la técnica se han identificado referencias sobre diversos procesos para la expresión de proteínas recombinantes, como lo son los documentos TW201224145, WO2013182484, KR20130006178, US2014127254, US2015164965, MX/a/2010/01 151 1 , US20130295607 y el MX304364.In addition to the aforementioned, references have been identified in the state of the art on various processes for the expression of recombinant proteins, such as documents TW201224145, WO2013182484, KR20130006178, US2014127254, US2015164965, MX / a / 2010/01 151 1 , US20130295607 and the MX304364.
El documento TW201224145 integra un vector de expresión que incluye un casete de expresión de proteína y al menos dos fragmentos homólogos de ADN recombinantes flanqueando el casete de expresión de proteínas, el cual comprende un primer marcador
de selección, promotores, un sitio de clonación múltiple adyacente a contraflujo del promotor y un segundo marcador de selección, en donde el promotor comprende un promotor 356S de virus mosaico de Coliflor (CAMV). Contempla un promotor de Ribulosa bifosfato carboxilasa, un promotor de adenina metiltransferasa (AMT), un promotor nitrato reductasa o una combinación de los mismos. TW201224145 integrates an expression vector that includes a protein expression cassette and at least two recombinant DNA homologous fragments flanking the protein expression cassette, which comprises a first marker of selection, promoters, a multiple cloning site adjacent to backflow of the promoter and a second selection marker, wherein the promoter comprises a 356S promoter of Cauliflower mosaic virus (CAMV). It contemplates a promoter of Ribulose bisphosphate carboxylase, an adenine methyltransferase (AMT) promoter, a nitrate reductase promoter or a combination thereof.
El WO2013182484, se refiere a un método para la expresión y secreción de un complejo de proteínas completamente ensamblado y funcional que tiene utilidad médica o biotecnológica en microalgas, en donde el complejo de proteínas es un anticuerpo, una enzima, hormona o vacuna y dicho complejo es secretado en un medio. La micro alga utilizada es Phaeodactylum tricornutum. WO2013182484, refers to a method for the expression and secretion of a fully assembled and functional protein complex having medical or biotechnological utility in microalgae, wherein the protein complex is an antibody, an enzyme, hormone or vaccine and said complex it is secreted in a medium. The micro alga used is Phaeodactylum tricornutum.
El documento KR20130006178 reivindica un método para preparar una proteína blanco usando un vector recombinante para transformar hongos, provista para expresar y obtener varias proteínas en hongos, especialmente Pleurotus eryngii. Constitución: Un vector recombinante para transformar hongos que comprende: CaMV 35S(virus del mosaico de coliflor 35S) promotor; CaMV 35S 5'- secuencia sin traducir (UTS); CaMV 35S 3'-UTS; NOS (nopalina sintasa) terminador; y una secuencia de nucleótido que codifica una proteína blanco. El método para preparar la proteína blanco de hongos comprende: un paso para transformar un conidio de hongo con un vector recombinante para transformar los hongos; y un paso para cultivar los conidios de hongos y colectar micelios de hongo. La proteína blanco es hormona de crecimiento bovina y el método comprende además el paso de cultivo del micelio. Document KR20130006178 claims a method for preparing a target protein using a recombinant vector for transforming fungi, provided to express and obtain various proteins in fungi, especially Pleurotus eryngii. Constitution: A recombinant vector for transforming fungi comprising: CaMV 35S (cauliflower mosaic virus 35S) promoter; CaMV 35S 5'- untranslated sequence (UTS); CaMV 35S 3'-UTS; NOS (nopaline synthase) terminator; and a nucleotide sequence that encodes a target protein. The method for preparing the fungal target protein comprises: a step to transform a fungus conidium with a recombinant vector to transform the fungi; and a step to grow mushroom conidia and collect mushroom mycelia. The white protein is bovine growth hormone and the method further comprises the step of growing the mycelium.
El documento US2014127254 reivindica un sistema de liberación una proteína biológicamente activa a un animal hospedero que comprende una célula de alga transformada por un vector de expresión y el vector de expresión comprende una secuencia de nucleótidos que codifica para la proteína biológicamente activa, operablemente ligada a un promotor. El alga es suspendida en agua para inmersión en el animal hospedero, el sistema de liberación es seleccionado de hormonas y péptidos antimicrobianos. El alga es mezclada con una muestra o alimento para el animal
hospedero. El animal se selecciona de mamíferos, peces, aves y crustáceos. La proteína es un péptido derivado de proteínas bactericidas, proteínas insecticidas, hormonas de crecimiento y antígenos. El sistema de liberación de antígenos comprende una célula algal transformada por un vector de expresión, y el vector de expresión comprende una secuencia nucleotídica que codifica para un antígeno determinante y el vector de expresión además comprende un promotor operablemente ligado a la secuencia de nucleótidos que codifica para el antígeno determinante. La célula algal expresa el antígeno determinante en un área seleccionada del grupo que consiste de núcleo, cloroplasto, mitocondrias, espacio periplásmico, membrana celular o pared celular. La célula algal es Chlamydomonas reinhardtii. US2014127254 claims a system for releasing a biologically active protein to a host animal comprising an algal cell transformed by an expression vector and the expression vector comprising a nucleotide sequence encoding the biologically active protein, operably linked to a promoter. The algae is suspended in water for immersion in the host animal, the release system is selected from hormones and antimicrobial peptides. The algae is mixed with a sample or food for the animal host The animal is selected from mammals, fish, birds and crustaceans. The protein is a peptide derived from bactericidal proteins, insecticidal proteins, growth hormones and antigens. The antigen delivery system comprises an algal cell transformed by an expression vector, and the expression vector comprises a nucleotide sequence encoding a determinant antigen and the expression vector further comprises a promoter operably linked to the nucleotide sequence encoding for the determinant antigen. The algal cell expresses the determinant antigen in an area selected from the group consisting of nucleus, chloroplast, mitochondria, periplasmic space, cell membrane or cell wall. The algal cell is Chlamydomonas reinhardtii.
El documento US2015164965, por su parte, reivindica una microalga eucariótica transgénica que comprende un casete de expresión que comprende al menos un polinucleótido transcriptible que codifica una proteína exógena biológicamente activa expresada dentro de un compartimento sub celular de la célula de microalga. El compartimento es seleccionado de vacuola celular, retículo endoplásmico, sistema de Golgi, Lisosoma y peroxisoma. El casete además comprende un polinucleótico que codifica un péptido dirigido a vacuolas, que mejora el consumo de la proteína exógena expresada por una célula xenogenéica o tejido. La microalga utilizada es de origen marino y se selecciona de: Phaeodactylum tricornutum, Dunaliella spp., US2015164965, for its part, claims a transgenic eukaryotic microalgae comprising an expression cassette comprising at least one transcriptable polynucleotide encoding an exogenous biologically active protein expressed within a subcellular compartment of the microalgae cell. The compartment is selected from cellular vacuole, endoplasmic reticulum, Golgi system, Lisosome and peroxisome. The cassette further comprises a polynucleotide which encodes a peptide directed to vacuoles, which improves the consumption of the exogenous protein expressed by a xenogenetic cell or tissue. The microalga used is of marine origin and is selected from: Phaeodactylum tricornutum, Dunaliella spp.,
Nannochloropsis spp., Nannochloris spp., Tetraselmis spp., Isochrysis galbana; Pavlova spp.; Amphiprora hyaline; Chaetoceros muellerr, y Neochloris oleoabundans. Nannochloropsis spp., Nannochloris spp., Tetraselmis spp., Isochrysis galbana; Pavlova spp .; Amphiprora hyaline; Chaetoceros muellerr, and Neochloris oleoabundans.
El documento MX201001 151 1 , reivindica un método para generar variación genotípica en un genoma de una planta, el método caracterizado porque comprende introducir en la planta por lo menos un vector de expresión viral que codifica por lo menos una nucleasa quimérica que comprende un dominio de enlace de ADN, una nucleasa y una señal de localización a un organelo que contiene ADN, en donde el dominio de enlace de ADN media la dirección específica de la nucleasa al genoma de la planta, para de esta manera generar variación genotípica en el genoma de la planta.
El método para tratar una infección de planta por un patógeno, el método caracterizado porque comprende introducir en la planta por lo menos un vector de expresión viral que codifica por lo menos una nucleasa quimérica que comprende un dominio de enlace de ADN y una nucleasa, en donde el domino de enlace de ADN media la dirección de la nucleasa al genoma del patógeno, para de esta manera prevenir o tratar una infección de la planta por un patógeno. Así mismo, reivindica un vector de expresión viral de planta, que comprende una secuencia de ácido nucléico que codifica por lo menos una nucleasa quimérica que comprende un dominio de enlace de ADN, una nucleasa y una señal de localización a una organelo que contiene ADN. Un vector de expresión basado en pTRV, que comprende una secuencia de ácido nucléico que codifica por lo menos dos secuencias de poliéptido heterólogas. Una planta transgénica, caracterizada porque comprende el vector de expresión viral de planta antes mencionado, la generación de variación genotípica es transitoria. El vector de expresión viral comprende un vector de expresión de virus Rattle de Tabaco (TRV). The document MX201001 151 1, claims a method for generating genotypic variation in a genome of a plant, the method characterized in that it comprises introducing into the plant at least one viral expression vector encoding at least one chimeric nuclease comprising a DNA domain. DNA binding, a nuclease and a localization signal to an organelle containing DNA, where the DNA binding domain mediates the specific nuclease direction to the plant genome, in order to generate genotypic variation in the genome of plant. The method for treating a plant infection by a pathogen, the method characterized in that it comprises introducing into the plant at least one viral expression vector encoding at least one chimeric nuclease comprising a DNA binding domain and a nuclease, in where the DNA binding domain mediates the nuclease's address to the pathogen's genome, in order to prevent or treat an infection of the plant by a pathogen. It also claims a plant viral expression vector, comprising a nucleic acid sequence encoding at least one chimeric nuclease comprising a DNA binding domain, a nuclease and a location signal to an organelle containing DNA. An expression vector based on pTRV, comprising a nucleic acid sequence encoding at least two heterologous polypeptide sequences. A transgenic plant, characterized in that it comprises the aforementioned plant viral expression vector, the generation of genotypic variation is transient. The viral expression vector comprises a Tobacco Rattle virus (TRV) expression vector.
El documento US20130295607, reivindica un método de producción de una proteína, que comprende: cultivar un microorganismo recombinante del orden Thraustochytríales en un medio, que comprende una molécula aislada de ácido nucléico y un gen que codifica la proteína para producir la proteína y la recuperación de la proteína. El microorganismo es de las especies seleccionadas del grupo: Schizochytrium sp., Schizochytrium aggregatum, Schizochytrium iimacinum, Schizochytrium minutum, Thraustochytrium sp., Thraustochytrium striatum, Thraustochytrium aureum, Thraustochytrium roseum, y Japonochytrium sp. US20130295607, claims a method of producing a protein, comprising: cultivating a recombinant microorganism of the order Thraustochytrols in a medium, comprising a molecule isolated from nucleic acid and a gene that encodes the protein to produce the protein and the recovery of the protein. The microorganism is of the selected species of the group: Schizochytrium sp., Schizochytrium aggregatum, Schizochytrium iimacinum, Schizochytrium minutum, Thraustochytrium sp., Thraustochytrium striatum, Thraustochytrium aureum, Thraustochytrium roseum, and Japonochytrium sp.
El documento MX304364 reivindica un proceso para producir una o más proteínas de interés, caracterizado porque comprende The document MX304364 claims a process to produce one or more proteins of interest, characterized in that it comprises
a) Proporcionar una planta o célula de planta que comprende: a) Provide a plant or plant cell comprising:
i. En un cromosoma nuclear una primera secuencia nucleotídica heteróloga que comprende: una secuencia nucleotídica que codifica para un replicón de ARN, y un primer promotor inducible ligado operativamente a la secuencia nucleotídica que
codifica para dicho replicón de ARN; el ARN no codifica para una proteína que proporciona movimiento célula a célula del replicón de ARN en la planta; el replicón de ARN codifica para una polimerasa para replicar el replicón de ARN y la una o más de una proteína de interés; y i. In a nuclear chromosome a first heterologous nucleotide sequence comprising: a nucleotide sequence encoding an RNA replicon, and a first inducible promoter operably linked to the nucleotide sequence which encodes said RNA replicon; the RNA does not code for a protein that provides cell-to-cell movement of the RNA replicon in the plant; the RNA replicon codes for a polymerase to replicate the RNA replicon and the one or more of a protein of interest; Y
ii. Una segunda secuencia nucleotídica heteróloga que comprende una secuencia que codifica para una proteína que habilita el movimiento célula a célula del replicón de ARN, donde la segunda secuencia nucleotídica heteróloga comprende un segundo promotor inducible ligado operativamente a la secuencia que codifica para la proteína que habilita el movimiento célula a célula del replicón de ADN; e ii. A second heterologous nucleotide sequence comprising a sequence encoding a protein that enables cell-to-cell movement of the RNA replicon, wherein the second heterologous nucleotide sequence comprises a second inducible promoter operably linked to the sequence encoding the protein that enables the cell to cell movement of the DNA replicon; and
a) Inducir, en la planta o célula de la planta del paso (a), el primer y segundo promotores inducibles produciendo por lo tanto, una o más de una proteína de interés en la planta o en la célula de la planta, respectivamente. a) Inducing, in the plant or plant cell of step (a), the first and second inducible promoters thereby producing one or more of a protein of interest in the plant or in the cell of the plant, respectively.
El replicón de ARN es derivado de un ARN virus de una sola hebra de sentido positivo, perteneciente al virus del mosaico del tabaco o el virus X de la papa. The RNA replicon is derived from a positive strand single-stranded RNA virus, belonging to the tobacco mosaic virus or the potato X virus.
Como se puede observar, en ninguno de los documentos anteriormente mencionados, se describe, evidencia o sugiere un método para expresión de proteínas recombinantes en microalgas a través de vectores virales con las características de la presente invención. Es por ello que, se determinó que el uso de vectores virales no ha sido explorado en la biotecnología de microalgas hasta el momento. En este sentido, en la presente invención se muestra la expresión de proteínas recombinantes utilizando un vector viral en microalgas, con un rendimiento por encima de lo descrito en el estado de la técnica, lo cual, se presenta como una opción mejorada para la producción de proteínas recombinantes. Si bien las microalgas se han empleado para producir proteínas recombinantes en distintas especies y con distintos elementos genéticos, el uso de vectores de expresión basados en replicones virales es algo novedoso en el estado de la técnica referida.
BREVE DESCRIPCION DE LAS FIGURAS As can be seen, in none of the aforementioned documents, a method for expression of recombinant proteins in microalgae through viral vectors with the characteristics of the present invention is described, evidenced or suggested. That is why, it was determined that the use of viral vectors has not been explored in microalgae biotechnology until now. In this sense, the expression of recombinant proteins using a viral vector in microalgae is shown in the present invention, with a performance above that described in the state of the art, which is presented as an improved option for the production of recombinant proteins. Although microalgae have been used to produce recombinant proteins in different species and with different genetic elements, the use of expression vectors based on viral replicons is something new in the referred state of the art. BRIEF DESCRIPTION OF THE FIGURES
Figura 1 . Muestra el mapa del vector viral de la presente invención (pAlgevir). Figure 1 . It shows the map of the viral vector of the present invention (pAlgevir).
Figura 2. Muestra la detección de replicones por PCR inversa. Figure 2. Shows the detection of replicons by inverse PCR.
Figura 3. Muestra la inmunodetección por medio de Western blot de las proteínas recombinantes GP1 (A) y LTB (B) producidas en Shizochrytrium sp. Figure 3. Shows the immunodetection by means of Western blot of the recombinant proteins GP1 (A) and LTB (B) produced in Shizochrytrium sp.
Figura 4. Muestra Rendimientos de las proteínas recombinantes producidas en Shizochrytrium sp. con el proceso de la presente invención (Algevir). Figure 4. Shows yields of the recombinant proteins produced in Shizochrytrium sp. with the process of the present invention (Algevir).
Figura 5. Perfil de restricción del vector Algevir. Figure 5. Restriction profile of the Algevir vector.
DESCRIPCIÓN DETALLADA DE LA INVENCIÓN DETAILED DESCRIPTION OF THE INVENTION
La presente invención se refiere a un proceso para la expresión de proteínas recombinantes en microalgas a través de vectores de ADN viral, para inducir la expresión que es entregada a través de Agrobaterium tumefaciens ; así como los protocolos para lograr la expresión transitoria en microalgas. The present invention relates to a process for the expression of recombinant proteins in microalgae through viral DNA vectors, to induce the expression that is delivered through Agrobaterium tumefaciens; as well as protocols to achieve transient expression in microalgae.
La invención comprende el uso de vectores virales que mediante una transformación transitoria eficiente conducen a una acumulación de la proteína recombinante a altos niveles (hasta 1 .25 mg/g en peso fresco), a la par, el proceso de expresión es inducible por etanol, lo que permite desfasar el momento de la transformación con el del inicio de la expresión. El proceso se ha validado tanto en microalgas de agua dulce fotosintéticas, como en microalgas marinas no fotosintéticas. The invention comprises the use of viral vectors that, through an efficient transient transformation, lead to an accumulation of the recombinant protein at high levels (up to 1.25 mg / g in fresh weight), at the same time, the expression process is inducible by ethanol , which allows to phase out the moment of transformation with that of the beginning of the expression. The process has been validated both in photosynthetic freshwater microalgae and in non-photosynthetic marine microalgae.
El proceso para la expresión de proteínas recombinantes de la presente invención en microalgas, comprende el uso de: The process for the expression of recombinant proteins of the present invention in microalgae, comprises the use of:
i) Al menos una microalga de agua dulce fotosintéticas y/o una microalga de origen marino no fotosintética como hospedero de expresión. Como uso preferente, se selecciona de las especies: Schizochytrium sp. y/o Chlamydomonas reinhardtir,
ii) El uso de Agrobacterium tumefaciens como vector para la entrega del ADN viral mediante su inclusión en un vector binario; iii) Al menos una proteína recombinante (z) de interés biofarmacéutico relacionada a una enfermedad que afecta a un ser vivo, o bien en bioprocesos que lo requieran, como molécula de interés, como por ejemplo antígenos para la elaboración de vacunas, hormonas o enzimas. i) At least one photosynthetic freshwater microalgae and / or a non-photosynthetic marine microalgae as an expression host. As a preferred use, it is selected from the species: Schizochytrium sp. and / or Chlamydomonas reinhardtir, ii) The use of Agrobacterium tumefaciens as a vector for the delivery of viral DNA through its inclusion in a binary vector; iii) At least one recombinant protein (z) of biopharmaceutical interest related to a disease that affects a living being, or in bioprocesses that require it, as a molecule of interest, such as antigens for the preparation of vaccines, hormones or enzymes .
Así como la construcción y uso de un vector viral, denominado en lo subsecuente pAlgevir, referido e ilustrado en la Figura 1 , el cual, cuenta con un mapa físico que comprende en orden de mención, los elementos genéticos: As well as the construction and use of a viral vector, referred to in the subsequent pAlgevir, referred to and illustrated in Figure 1, which has a physical map that includes, in order of mention, the genetic elements:
(1) Un Promotor 35S del Virus del Mosaico de la Coliflor (VMC), (1) A 35S Promoter of the Cauliflower Mosaic Virus (VMC),
(2) Un gen AlcR de Aspergillus nidulans. (2) An AlcR gene from Aspergillus nidulans.
(3) Un Terminador de la Nopalina Sintasa (nos ter) (3) A Terminator of the Nopalina Sintasa (nos ter)
(4) Un origen de replicación "Orí" del geminivirus Ageratum enation virus (AgEV) (4) An "Ori" origin of replication of the geminivirus Ageratum enation virus (AgEV)
(5) Un promotor AlcA de Aspergillus nidulans con sitios de restricción Smal (5') y(5) An AlcA promoter from Aspergillus nidulans with Smal (5 ' ) restriction sites and
BamHI y Pstl (3') en los cuales se puede introducir un gen de interés, BamHI and Pstl (3 ' ) in which a gene of interest can be introduced,
(6) Un Terminador 35S, (6) A 35S Terminator,
(7) Un origen de replicación Orí (repetido), un Promotor AlcA, y un gen de proteína Rep de AgEV; y un Terminador Nos, (7) An Ori origin of replication (repeated), an AlcA promoter, and an AgEV Rep protein gene; and a Terminator Nos,
Los elementos del (1 ) al (7), se incluyen en el contexto del vector binario pB1121 que permite la transferencia de los casetes de expresión mediante Agrobacterium tumefaciens. The elements of (1) to (7) are included in the context of the binary vector pB1121 which allows the transfer of the expression cassettes by Agrobacterium tumefaciens.
El proceso para la expresión de proteínas recombinantes en microalgas a través de vectores virales de la presente invención, que comprende los pasos de:
a) Construir el vector de la Figura 1 , el cual tiene como estructura base un vector binario funcional en Agrobacterium tumefaciens ; b) Transformar transitoriamente una microalga hospedero de expresión mediante la inoculación de Agrobacterium tumefaciens, en un medio suplementado con 100 pM de Acetosiringona y una adición posterior de 250 mg/L de cefotaxima. Se sugiere, pero no se limita al uso de Chlamydomonas reinhardtii y/o Schizochytrium sp., indistintamente); c) Expresar el gen de interés (puede ser GP1 , de Zaire ebolavirus o LTB de E. coli, o bien una proteína recombinante (z) en las que se pueden incluir antígenos para la elaboración de vacunas, hormonas o enzimas, (por citar algunos ejemplos) en la microalga transitoriamente transformada del paso b) mediante la adición de una solución de un alcohol. Se prefiere el uso de etanol absoluto, o bien en una solución acuosa al 1 %; y, d) Obtener la proteína recombinante de interés que se expresa eficientemente gracias a la localización del gen codificante (puede ser de origen viral o bacteriano, por ejemplo, de Zaire ebolavirus o de Escherichia coli), entre los elementos Orí del vector de la Figura 1 , y mediante la generación y replicación de moléculas de ADN circulares que contienen llevan el casete de expresión para el gen de interés del vector iv). The process for the expression of recombinant proteins in microalgae through viral vectors of the present invention, comprising the steps of: a) Construct the vector of Figure 1, which has as a base structure a functional binary vector in Agrobacterium tumefaciens; b) Transiently transform a microalga host expression by inoculating Agrobacterium tumefaciens, in a medium supplemented with 100 pM Acetosyringone and a subsequent addition of 250 mg / L cefotaxime. It is suggested, but not limited to the use of Chlamydomonas reinhardtii and / or Schizochytrium sp., Indistinctly); c) Express the gene of interest (may be GP1, from Zaire ebolavirus or LTB from E. coli, or a recombinant protein (z) in which antigens may be included for the preparation of vaccines, hormones or enzymes, (to cite some examples) in the transiently transformed microalga of step b) by the addition of a solution of an alcohol. The use of absolute ethanol is preferred, or in a 1% aqueous solution; and, d) Obtain the recombinant protein of interest that is efficiently expressed thanks to the location of the coding gene (it may be of viral or bacterial origin, for example, of Zaire ebolavirus or of Escherichia coli), between the Ori elements of the vector of the Figure 1, and by generating and replicating circular DNA molecules that contain the expression cassette for the gene of interest of vector iv).
La cepa Schizochytrium sp. se refiere a la cepa ATCC 20888, Chlamydomonas reinhardtii se refiere a la cepa disponible en: http://www.chlamycollection.org/product/cc-125-wild-type-mt-137c , y en lo que respecta a Agrobacterium tumefaciens se refiere a la cepa GV3101 disponible para su consulta en: http://www.mpipz.mpg.de/koncz/publications.
El proceso para la expresión de proteínas recombinantes en microalgas a través de vectores virales de la presente invención, muestra un rendimiento de expresión de proteínas recombinantes de interés de al menos 1 .25 mg/g en peso fresco en algunas formas de realización, sin embargo, se pueden obtener rendimientos de al menos 0.12 mg/g en peso fresco, dependiendo de la naturaleza y/u origen de la proteína recombinante a obtener. The strain Schizochytrium sp. refers to strain ATCC 20888, Chlamydomonas reinhardtii refers to the strain available at: http://www.chlamycollection.org/product/cc-125-wild-type-mt-137c, and with respect to Agrobacterium tumefaciens refers to strain GV3101 available for consultation at: http://www.mpipz.mpg.de/koncz/publications. The process for the expression of recombinant proteins in microalgae through viral vectors of the present invention shows an expression yield of recombinant proteins of interest of at least 1.25 mg / g in fresh weight in some embodiments, however , yields of at least 0.12 mg / g in fresh weight can be obtained, depending on the nature and / or origin of the recombinant protein to be obtained.
A continuación, se muestra un ejemplo de realización de la presente invención, en donde para ilustrar la eficiencia del proceso, se utilizan dos antígenos modelos relacionados a enfermedades virales, (glicoproteína 1 de Zaire ebolavirus, GP1) y bacterianas (la subunidad B de la enterotoxina termolábil de Escheríchia coli, LTB) relevantes en vacunación. In the following, an embodiment of the present invention is shown, where to illustrate the efficiency of the process, two model antigens related to viral diseases (Zaire ebolavirus glycoprotein, GP1) and bacterial (the B subunit of the thermolabile enterotoxin of Escherichia coli, LTB) relevant in vaccination.
El proceso de expresión de la presente invención es funcional y conduce a la expresión eficiente de proteínas recombinantes. The expression process of the present invention is functional and leads to the efficient expression of recombinant proteins.
Lo que a continuación se ilustra, de ninguna manera se considera como limitante de aplicación de la presente invención. What is illustrated below is in no way considered as limiting the application of the present invention.
Ejemplo 1 : Example 1 :
1. Diseño del vector pAlgevir 1. Design of the pAlgevir vector
Se diseñó un vector inducible utilizando los siguientes elementos en el siguiente orden (Ver Figura 1): An inducible vector was designed using the following elements in the following order (See Figure 1):
(1) Promotor 35S del virus del mosaico de la coliflor, (1) 35S promoter of cauliflower mosaic virus,
(2) El gen AlcR (2) The AlcR gene
(3) Terminador de la nopalina sintasa (nos), (3) Terminator of nopaline synthase (nos),
(4) El origen de replicación "Orí" del geminivirus Ageratum enation virus (AgEV).
(5) Promotor AlcA de Aspergillus nidulans con sitios de restricción Smal (5') y BamHI y Pstl (3') en los cuales se puede introducir el gen de interés. (4) The "Orí" origin of replication of the geminivirus Ageratum enation virus (AgEV). (5) AlcA promoter from Aspergillus nidulans with restriction sites Smal (5 ' ) and BamHI and Pstl (3 ' ) in which the gene of interest can be introduced.
(6) Terminador 35S (6) Terminator 35S
(7) Orí (repetido), Promotor AlcA, y ORF de proteína Rep de AgEV; (7) Orí (repeated), Promoter AlcA, and Rep protein ORF of AgEV;
(8) Terminador nos. (8) Terminator nos.
Este casete de ADN fue sintetizado por GenScript Inc. (New Jersey, EE.UU.) y subclonado en el vector binario pBI121 en los sitios Xbal-Sacl a través de procedimientos estándar de digestión-ligación. This DNA cassette was synthesized by GenScript Inc. (New Jersey, USA) and subcloned into the binary vector pBI121 at the Xbal-Sacl sites through standard digestion-ligation procedures.
Se usó una clona positiva identificada por el perfil de restricción y posteriormente se realizó secuenciación para confirmar la integridad de la secuencia. La construcción se transfirió posteriormente a Agrobacteríum tumefaciens (cepa GV3101) por electroporación (Cangelosi et al., 1991). Una clona que portaba el vector de expresión se confirmó por reacción en cadena de la polimerasa (PCR) y posteriormente se cultivó durante la noche en medio Luria Bertani a 25 °C y 200 rpm para realizar ensayos de expresión. A positive clone identified by the restriction profile was used and sequencing was subsequently performed to confirm the integrity of the sequence. The construction was subsequently transferred to Agrobacterium tumefaciens (strain GV3101) by electroporation (Cangelosi et al., 1991). A clone carrying the expression vector was confirmed by polymerase chain reaction (PCR) and subsequently cultured overnight in Luria Bertani medium at 25 ° C and 200 rpm to perform expression assays.
2. Transformación transitoria de la microalga 2. Transient transformation of the microalgae
La microalga marina Schizochytrium sp. cepa 20888 se obtuvo de ATCC (USA), se cultivó a 25 °C en medio de agua de mar modificado (5 g/L de peptona, 1 g/L de extracto de levadura, 0,2 g/L de FeS04, 15 g/L de agar y 35 g/L de NaCI). Para cultivos líquidos, se usó el medio 679BY (1 g/L de extracto de levadura, 1 g/L de peptona, 5 g/L de dextrosa y 35 g/L de NaCI).
La microalga de agua dulce Chlamydomonas reinhardtii se cultivó en 100 mi del medio TAP (tris-acetato-fosfatos), a 25 °C con agitación de 150 rpm. The marine microalga Schizochytrium sp. strain 20888 was obtained from ATCC (USA), cultured at 25 ° C in modified seawater medium (5 g / L of peptone, 1 g / L of yeast extract, 0.2 g / L of FeS04, g / L of agar and 35 g / L of NaCl). For liquid cultures, the 679BY medium (1 g / L of yeast extract, 1 g / L of peptone, 5 g / L of dextrose and 35 g / L of NaCl) was used. The freshwater microalgae Chlamydomonas reinhardtii was cultured in 100 ml of the TAP medium (tris-acetate-phosphates), at 25 ° C with 150 rpm stirring.
La transformación se llevó a cabo mediante la inoculación de Schizochytrium sp. (100 mi, Densidad óptica (OD) 650nm = 0,7) o Chlamydomonas reinhardtii (100ml, OD 600595 nm = 0.7) con 1 mL de cultivo de Agrobacterium tumefaciens (OD660 nm = 1 ,0). El medio se suplemento con acetosiringona 100 mM. 16 h después de la inoculación se añadió cefotaxima a una concentración final de 250 mg/L. La expresión del gen de interés en las microalgas transitoriamente transformadas se indujo 20 h después de la inoculación mediante la adición de 1 mL de etanol absoluto (1 % de la concentración final). Las muestras de cultivo se recogieron antes de la inducción y 12, 24 y 48 h después de la inducción. Las muestras se almacenaron a -80 °C hasta su uso posterior. The transformation was carried out by inoculating Schizochytrium sp. (100 ml, Optical density (OD) 650nm = 0.7) or Chlamydomonas reinhardtii (100ml, OD 600595 nm = 0.7) with 1 mL of Agrobacterium tumefaciens culture (OD660 nm = 1.0). The medium is supplemented with 100 mM acetosyringone. Cefotaxime was added 16 h after the inoculation to a final concentration of 250 mg / L. The expression of the gene of interest in the transiently transformed microalgae was induced 20 h after inoculation by the addition of 1 mL of absolute ethanol (1% of the final concentration). Culture samples were collected before induction and 12, 24 and 48 h after induction. Samples were stored at -80 ° C until later use.
PCR Inverso Inverse PCR
Los replicones circulares de ADN fueron detectados por PCR inverso usando los siguientes conjuntos de cebadores: Circular DNA replicons were detected by inverse PCR using the following sets of primers:
Para GP1 , delantero SEQ ID NO:1 5'CATCACCAAGATACCGGAGAAG y reversa (SEQ ID NO:2) 5TTTAGTTTCCCAGAAGGCCC 3'; Para LTB, delantero (SEQ ID NO:3) 5'CATTTCCCTCTTTCCAGCCA y reversa (SEQ ID NO:4) 5TTATGGAGAAACTCGAGCTTGT 3'. El ADN total fue aislado de cultivos de Schizochytrium sp. De acuerdo a Dellaporta et al. (1983). Las mezclas de reacción PCR (25pL) contenidos 1 c búfer PCR, 100 ng ADN, 1 .5 mM cloruro de magnesio, 2.5 U Taq ADN polimerasa, 1 mM dNTPs, y 1 pM del correspondiente conjunto de cebadores. Condiciones cíclicas fueron: 94°C por 5 min (desnaturalización inicial); 35 ciclos a 94°C por 30 s, 55°C por 30 s, 72°C por 120 s, y una extensión final a 72°C por 10 min. Los productos de PCR fueron detectados por electroforesis en geles agarosa al 1 %. El control negativo consistió en 100 ng de ADN de cultivo de Schizochytrium sp.
Análisis de proteínas For GP1, forward SEQ ID NO: 1 5 ' CATCACCAAGATACCGGAGAAG and reverse (SEQ ID NO: 2) 5TTTAGTTTCCCAGAAGGCCC 3 ' ; For LTB, forward (SEQ ID NO: 3) 5 ' CATTTCCCTCTTTCCAGCCA and reverse (SEQ ID NO: 4) 5TTATGGAGAAACTCGAGCTTGT 3 ' . The total DNA was isolated from cultures of Schizochytrium sp. According to Dellaporta et al. (1983). The PCR reaction mixtures (25pL) contained 1 c PCR buffer, 100 ng DNA, 1.5 mM magnesium chloride, 2.5 U Taq DNA polymerase, 1 mM dNTPs, and 1 pM of the corresponding set of primers. Cyclic conditions were: 94 ° C for 5 min (initial denaturation); 35 cycles at 94 ° C for 30 s, 55 ° C for 30 s, 72 ° C for 120 s, and a final extension at 72 ° C for 10 min. The PCR products were detected by electrophoresis in 1% agarose gels. The negative control consisted of 100 ng of culture DNA of Schizochytrium sp. Protein analysis
La integridad de las proteínas obtenidas de Schizochytrium fueron evaluadas por análisis Western blot. Los cultivos para muestra fueron recolectados en diferentes tiempos de inducción (0, 12, 24, y 48 h), y los extractos de proteínas fueron obtenidos como sigue: 20 mg de biomasa fresca fue resuspendida en 200 pL de un búffer de extracción (750 mM Tris-Ácido Clorhídrico, pH 8, 15% sucrosa, 100 mM b- mercaptoetanol, y 1 mM PMSF) y sonificado (4 pulsos de 4 s con 4 s de retraso entre sí) usando un equipo de procesador ultrasónico con 24% de amplitud (modelo GEX130PB). The integrity of the proteins obtained from Schizochytrium were evaluated by Western blot analysis. The cultures for sample were collected in different induction times (0, 12, 24, and 48 h), and the protein extracts were obtained as follows: 20 mg of fresh biomass was resuspended in 200 pL of an extraction buffer (750 mM Tris-Hydrochloric acid, pH 8, 15% sucrose, 100 mM b-mercaptoethanol, and 1 mM PMSF) and sonicated (4 pulses of 4 s with 4 s of delay each other) using an ultrasonic processor equipment with 24% amplitude (model GEX130PB).
Muestras: Samples:
Las muestras fueron subsecuentemente centrifugadas a 8000 rpm por 15 min y los sobrenadantes fueron transferidos a nuevos microtubos. La concentración total de proteínas solubles fue determinada en los extractos por método Lowry y un volumen correspondiente a 80 pg de TSP (aproximadamente 100 pL) fue mezclado con el mismo volumen de 2x buffer reductor, y desnaturalizado por ebullición por 5 min a 95°C, y subsecuentemente sujeto a análisis SDS-PAGE. Los geles fueron utlizados para transferir las proteínas a membranas de nitrocelulosa Bio-Rad de 0.45 L m (http://www.bio-rad.com). La transferencia de proteínas fue realizada utilizando un TV100-EBK Electroblotter (AlphaMetrix Biotech, GER) por 1 h a 150 V en un buffer de base metanol. Después de bloquear con 5% de leche libre de grasa (Carnation, Nestle) disuelto en PBS-0.1 % Tween, las membranas fueron incubadas con anti-suero de ratón (1 :200 dilución) contra ambos, LTB o GP1 ; los cuales fueron producidos en ratones como previamente fue descrito (Orellana-Escobedo et al. 2015). Las membranas fueron lavadas e incubadas con un anticuerpo anti-lgG de ratón secundario conjugado con peroxidasa de rábano de cabra (1 :2,000 dilución Sigma) por 2 h a temperatura ambiente. La detección del antígeno fue revelada por incubación de las membranas con
solución SuperSignal West Dura, siguiendo las instrucciones del proveedor (Thermo Scientific, http://www.thermoscientific.com) y exponiendo la película a procedimientos estándar. Las proteínas purificadas LTB y GP1 (250 ng) producidas en E.coli recombinante fueron usadas como controles positivos. The samples were subsequently centrifuged at 8000 rpm for 15 min and the supernatants were transferred to new microtubes. The total concentration of soluble proteins was determined in the extracts by Lowry method and a volume corresponding to 80 pg of TSP (approximately 100 pL) was mixed with the same volume of 2x reducing buffer, and denatured by boiling for 5 min at 95 ° C , and subsequently subject to SDS-PAGE analysis. The gels were used to transfer the proteins to Bio-Rad nitrocellulose membranes of 0.45 L m (http://www.bio-rad.com). Protein transfer was performed using a TV100-EBK Electroblotter (AlphaMetrix Biotech, GER) for 1 h at 150 V in a methanol-based buffer. After blocking with 5% of fat-free milk (Carnation, Nestle) dissolved in PBS-0.1% Tween, the membranes were incubated with anti-mouse serum (1: 200 dilution) against both, LTB or GP1; which were produced in mice as previously described (Orellana-Escobedo et al., 2015). The membranes were washed and incubated with a secondary mouse anti-IgG antibody conjugated with goat radish peroxidase (1: 2,000 Sigma dilution) for 2 h at room temperature. Antigen detection was revealed by incubation of the membranes with SuperSignal West Dura solution, following the supplier's instructions (Thermo Scientific, http://www.thermoscientific.com) and exposing the film to standard procedures. The purified proteins LTB and GP1 (250 ng) produced in recombinant E. coli were used as positive controls.
Para la cuantificación ELISA, un protocolo descrito fue aplicado (Ríos-Huerta et al. 2017). Brevemente, los extractos de proteínas fueron obtenidos como se describe enseguida y diluidos con búfer de carbonato 0.2 M (pH = 9.6). La mezcla diluida fue agregada a una placa ELISA para adsorción de proteínas a 4°C. Después de bloquear con solución al 5% de leche seca libre de grasa por 2 h a temperatura ambiente, las placas fueron incubadas con suero de ratón anti-LTB (1 :500 dilución) o anti-GP1 (1 : 1000 dilución) durante la noche a 4 °C. Las placas fueron subsecuentemente incubadas con IgG anti-ratón conjugada con peroxidasa de rábano picante (1 :2000 dilución) por 2 h a 25 °C. Una reacción colorimétrica fue inducida por adición de solución substrato ABTS (0.6 mM ácido 2,2'-azino-bis(3-etilbenzotiazolin-6-sulfonico), 0.1 M ácido cítrico, pH= 4.35, y 1 mM Agua Oxigenada), después de 30 min de incubación a 25 °C los valores OD fueron medidos a 405 nm. Curvas estándar fueron construidas usando LTB o GP1 puras para estimar los niveles de expresión. For the ELISA quantification, a described protocol was applied (Ríos-Huerta et al., 2017). Briefly, the protein extracts were obtained as described below and diluted with 0.2 M carbonate buffer (pH = 9.6). The diluted mixture was added to an ELISA plate for protein adsorption at 4 ° C. After blocking with 5% solution of fat-free dry milk for 2 h at room temperature, the plates were incubated with mouse anti-LTB serum (1: 500 dilution) or anti-GP1 (1: 1000 dilution) overnight at 4 ° C. The plates were subsequently incubated with anti-mouse IgG conjugated with horseradish peroxidase (1: 2000 dilution) for 2 h at 25 ° C. A colorimetric reaction was induced by addition of ABTS substrate solution (0.6 mM 2,2'-azino-bis (3-ethyl-6-sulfonic acid), 0.1 M citric acid, pH = 4.35, and 1 mM Peroxide), then After 30 min of incubation at 25 ° C, OD values were measured at 405 nm. Standard curves were constructed using pure LTB or GP1 to estimate expression levels.
Resultados: Results:
1) El vector pAlgevir es entregado mediante Agrobacterium y replicado en microalgas. 1) The vector pAlgevir is delivered by Agrobacterium and replicated in microalgae.
La síntesis génica y las técnicas de clonación molecular permitieron la construcción del vector pAlgevir, cuyo mapa físico se muestra en la Figura 1 , el perfil de restricción se muestra en la figura 5 y la secuencia expresada por los casetes de expresión SEQ ID NO:5. El gen AlcR de Aspergillus nidulans se encuentra bajo control del promotor 35s de virus de mosaico de Coliflor y del terminador nos, mientras que la proteína Rep está
bajo control del promotor inducible AlcA de Aspergillus nidulans y el terminador nos. Se expresaron los genes GP1 , de Zaire ebolavirus o LTB de E. coli, insertados en el sitio Sma I en el extremo 5 'y cualquiera de los sitios Bam Hl o Pst I en el extremo 3', cuya expresión está bajo el control del promotor inducible AlcA. Tras la inducción del etanol, el factor de transcripción AlcR se activa conduciendo a la expresión del gen de interés y de la proteína Rep. Este último actúa sobre los elementos Orí y mediante la generación y replicación de moléculas de ADN circulares que llevan el casete de expresión para la proteína recombinante. (Ver figura 5). Gene synthesis and molecular cloning techniques allowed the construction of the vector pAlgevir, whose physical map is shown in Figure 1, the restriction profile is shown in Figure 5 and the sequence expressed by the expression cassettes SEQ ID NO: 5 . The AlcR gene of Aspergillus nidulans is under the control of the 35s promoter of Cauliflower mosaic virus and the nos terminator, while the Rep protein is under control of the AlcA inducible promoter of Aspergillus nidulans and the nos terminator. The GP1 genes of Zaire ebolavirus or LTB of E. coli were expressed, inserted in the Sma I site at the 5 'end and any of the Bam Hl or Pst I sites at the 3' end, whose expression is under the control of the AlcA inducible promoter. After the induction of ethanol, the transcription factor AlcR is activated leading to the expression of the gene of interest and the Rep protein. The latter acts on the Ori elements and through the generation and replication of circular DNA molecules that carry the cassette. expression for the recombinant protein. (See figure 5).
2) Algevir permite la expresión eficiente de antígenos virales y bacterianos. 2) Algevir allows the efficient expression of viral and bacterial antigens.
Se realizaron estudios de expresión para evaluar la eficiencia del proceso Algevir. Los cultivos de Schizochytrium sp. se inocularon con agrobacterias recombinantes y se indujeron con etanol 20 h después de la inoculación, con el subsiguiente análisis para determinar la replicación del vector y los rendimientos de la proteína recombinante en diferentes tiempos posteriores a la inducción. En primer lugar, se investigó la presencia de replicones de ADN a través de análisis de PCR inversa para probar la expresión de Rep funcional que conduce a la replicación del casete de expresión. En este enfoque, los oligonucleótidos de PCR que alinean en los flancos del casete de expresión en una posición divergente permiten la amplificación específica de moléculas circulares que se generan por replicación de círculo rodante. Los cultivos transformados e inducidos de Schizochytrium sp. mostraron un resultado positivo en todos los tiempos experimentales post-inducción, lo que indica que el vector pAlgevir media la generación de replicones mediante replicación del círculo rodante (Figura 2). Expression studies were conducted to evaluate the efficiency of the Algevir process. The cultures of Schizochytrium sp. they were inoculated with recombinant agrobacteria and induced with ethanol 20 h after inoculation, with the subsequent analysis to determine the replication of the vector and the yields of the recombinant protein at different times after induction. First, the presence of DNA replicons was investigated through inverse PCR analysis to test the expression of functional Rep that leads to replication of the expression cassette. In this approach, the PCR oligonucleotides that line the flanks of the expression cassette in a divergent position allow the specific amplification of circular molecules that are generated by rolling circle replication. Transformed and induced cultures of Schizochytrium sp. showed a positive result at all post-induction experimental times, indicating that the pAlgevir vector mediates the generation of replicons by replication of the rolling circle (Figure 2).
En la figura 2 se muestra la detección de replicones por PCR inversa en muestras de ADN de Schizochrytrium sp. transformada transitoriamente con los vectores pAlgevir- GP1 (A) y pAlgevir-LTB (B); usando oligonucleótidos para detectar repliconas circulares generados por la proteína Rep. Los carriles muestran lo siguiente: M, marcador de peso molecular de 1 Kb (New England, biolabs); 1 -5, muestras de ADN de Schizochrytrium
sp. transformado transitoriamente con A. tumefaciens portando el vector pAlgevir, tomadas a las 0, 12, 24, 48 y 72 h post-inducción, respectivamente; 6: muestra de ADN de Schizochrytrium sp. sin transformar (WT); 7, control negativo (agua). Los amplicones esperados para la detección de los replicones GP1 y LTB son de 1800 y 918 pb de longitud, respectivamente. Figure 2 shows the detection of replicons by inverse PCR in DNA samples of Schizochrytrium sp. Transiently transformed with the vectors pAlgevir-GP1 (A) and pAlgevir-LTB (B); using oligonucleotides to detect circular replicons generated by the Rep protein. The lanes show the following: M, 1 Kb molecular weight marker (New England, biolabs); 1-5, Schizochrytrium DNA samples sp. transiently transformed with A. tumefaciens carrying the vector pAlgevir, taken at 0, 12, 24, 48 and 72 h post-induction, respectively; 6: DNA sample from Schizochrytrium sp. untransformed (WT); 7, negative control (water). The amplicons expected for the detection of the GP1 and LTB replicons are 1800 and 918 bp in length, respectively.
La amplificación también se detectó en la fase de pre-inducción, lo que sugirió una transcripción basal de la proteína Rep. En términos de producción de proteínas recombinantes, Schizochytrium sp. mostró un nivel mínimo de proteína recombinante en la fase de pre-inducción. Posteriormente los niveles de proteína recombinante aumentaron espectacularmente después de la inducción con etanol, alcanzando los niveles más altos 48 h después de la inducción (Figura 3). En la figura 3 se muestra la inmunodetección por medio de Western blot de las proteínas recombinantes GP1 (A) y LTB (B) producidas en Schizochrytrium sp.; así como la proteína GP1 en Chlamydomonas reinhardtii (C), a través del sistema Algevir. Los controles positivos consistieron en 250 ng de GP1 o LTB. Amplification was also detected in the pre-induction phase, which suggested a basal transcription of the Rep protein. In terms of production of recombinant proteins, Schizochytrium sp. showed a minimum level of recombinant protein in the pre-induction phase. Subsequently, the levels of recombinant protein increased dramatically after induction with ethanol, reaching the highest levels 48 h after induction (Figure 3). Figure 3 shows the immunodetection by means of Western blot of the recombinant proteins GP1 (A) and LTB (B) produced in Schizochrytrium sp .; as well as the GP1 protein in Chlamydomonas reinhardtii (C), through the Algevir system. Positive controls consisted of 250 ng of GP1 or LTB.
De acuerdo con los resultados de ELISA, la proteína recombinante se produce 48 h después de la inducción y alcanzó valores de hasta 1 ,25 mg/g de peso fresco (FW) para GP1 (6 mg/L de cultivo) y 0,12 mg/g de FW (0,6 mg/L de cultivo) para LTB (Figura 4). La figura 4 muestra los rendimientos de las proteínas recombinantes producidas en Schizochrytrium sp. con el proceso de la presente invención (Algevir). Se realizó un ensayo por inmunoabsorción ligado a enzimas (ELISA) cuantitativo para determinar los niveles de acumulación de las proteínas de Schizochrytrium sp. a las 48 h postinducción. Se usaron proteínas puras para construir curvas estándar para GP1 (A) y LTB (B). Los niveles de acumulación se expresan en miligramos de proteína recombinante por gramo de biomasa de microalgas frescas o miligramos de proteína recombinante por litro de cultivo de microalgas (C). Los valores presentados son la media de los triplicados ± desviación estándar. (Ver. Figura 4).
El análisis ELISA cuantitativo confirmó que 48 h después de la inducción era el punto de tiempo con rendimientos máximos para la especie Schizochytrium sp., mientras que en la microalga Chlamydomonas reinhardtii la mayor concentración se obtuvo a las 24 horas post inducción. En los tiempos de post-inducción más cortos o extendidos mostraron rendimientos de proteína significativamente más bajos. En el caso de Chlamydomonas los rendimientos estimados para la proteína la proteína GP1 fueron de hasta 0.125 mg/g de peso fresco.
According to the results of ELISA, the recombinant protein is produced 48 h after induction and reached values of up to 1.25 mg / g of fresh weight (FW) for GP1 (6 mg / L of culture) and 0.12 mg / g FW (0.6 mg / L culture) for LTB (Figure 4). Figure 4 shows the yields of the recombinant proteins produced in Schizochrytrium sp. with the process of the present invention (Algevir). A quantitative enzyme-linked immunosorbent assay (ELISA) was performed to determine the accumulation levels of Schizochrytrium sp proteins. at 48 h postinduction. Pure proteins were used to construct standard curves for GP1 (A) and LTB (B). Accumulation levels are expressed in milligrams of recombinant protein per gram of fresh microalgae biomass or milligrams of recombinant protein per liter of microalgae culture (C). The values presented are the mean of the triplicates ± standard deviation. (See. Figure 4). The quantitative ELISA analysis confirmed that 48 h after induction was the time point with maximum yields for the species Schizochytrium sp., Whereas in the microalga Chlamydomonas reinhardtii the highest concentration was obtained 24 hours after induction. In shorter or extended post-induction times they showed significantly lower protein yields. In the case of Chlamydomonas, the estimated yields for protein GP1 were up to 0.125 mg / g of fresh weight.
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Claims
1 . Un proceso para la expresión de proteínas recombinantes en microalgas, caracterizado porque comprende: one . A process for the expression of recombinant proteins in microalgae, characterized in that it comprises:
i) Al menos una microalga de agua dulce fotosintética y/o una microalga de origen marino no fotosintética como hospedero de expresión; i) At least one photosynthetic freshwater microalgae and / or a non-photosynthetic marine microalgae as an expression host;
ii) Al menos una bacteria vector para la entrega de ADN viral mediante su inclusión en un vector binario; ii) At least one vector bacterium for the delivery of viral DNA by its inclusion in a binary vector;
iii) Al menos una proteína recombinante (z) de interés biofarmacéutico relacionada a una enfermedad que afecta a un ser vivo, y/o un bioproceso que lo requiera, como molécula de interés; y iii) At least one recombinant protein (z) of biopharmaceutical interest related to a disease that affects a living being, and / or a bioprocess that requires it, as a molecule of interest; Y
iv) Un vector viral que comprende en orden de mención, los elementos genéticos: Un promotor 35S del virus del mosaico de la Coliflor (1 ), un gen AlcR de Aspergillus nidulans. (2), un terminador de la nopalina sintasa (nos ter) (3), el origen de replicación "Orí" del geminivirus Ageratum enation virus (AgEV) (4), un promotor AlcA de Aspergillus nidulans con sitios de restricción Smal (5') y BamHI y Pstl (3') en los cuales se puede introducir un gen de interés (5), un Terminador 35S (6), un origen de replicación Orí (repetido), AlcA Promotor, y el gen de la proteína Rep de AgEV (7), y un terminador Nos ter. iv) A viral vector comprising, in order of mention, the genetic elements: A 35S promoter of the Cauliflower mosaic virus (1), an AlcR gene of Aspergillus nidulans. (2), a terminator of nopaline synthase (nos ter) (3), the "Ori" origin of the geminivirus Ageratum enation virus (AgEV) (4), an AlcA promoter from Aspergillus nidulans with Smal restriction sites (5) ' ) and BamHI and Pstl (3 ' ) in which a gene of interest (5), a 35S Terminator (6), an Ori origin of replication (repeated), AlcA Promoter, and the Rep protein gene can be introduced. of AgEV (7), and a terminator Nos ter.
2. El proceso para la expresión de proteínas recombinantes en microalgas de la reivindicación 1 , caracterizado porque la microalga utilizada como hospedero de expresión i) es preferentemente seleccionada de: Schizochytrium sp., Chlamydomonas reinhardtii. 2. The process for the expression of recombinant proteins in microalgae of claim 1, characterized in that the microalga used as host for expression i) is preferably selected from: Schizochytrium sp., Chlamydomonas reinhardtii.
3. El proceso para la expresión de proteínas recombinantes en microalgas de la reivindicación 1 , caracterizado porque la bacteria vector para la entrega de ADN viral es Agrobacterium tumefaciens.
3. The process for the expression of recombinant proteins in microalgae of claim 1, characterized in that the vector bacterium for the delivery of viral DNA is Agrobacterium tumefaciens.
4. El proceso para la expresión de proteínas recombinantes en microalgas de la reivindicación 1 , caracterizado porque la proteína recombinante (z) de interés iii) se selecciona de: antígenos y/u hormonas y/o enzimas. 4. The process for the expression of recombinant proteins in microalgae of claim 1, characterized in that the recombinant protein (z) of interest iii) is selected from: antigens and / or hormones and / or enzymes.
5. El proceso para la expresión de proteínas recombinantes en microalgas de la reivindicación 1 , caracterizado porque la proteína recombinante (z) de interés iii) se selecciona de: Glicoproteína 1 de Zaire ebolavirus (GP1) y/o la subunidad B de la enterotoxina termolábil de Escheríchia coli (LTB). 5. The process for the expression of recombinant proteins in microalgae of claim 1, characterized in that the recombinant protein (z) of interest iii) is selected from: Zaire ebolavirus glycoprotein 1 (GP1) and / or the B subunit of the enterotoxin thermolabile of Escherichia coli (LTB).
6. El proceso para la expresión de proteínas recombinantes en microalgas de la reivindicación 1 , caracterizado porque el vector viral iv), tiene como estructura base un vector binario funcional en Agrobacterium tumefaciens. 6. The process for the expression of recombinant proteins in microalgae of claim 1, characterized in that the viral vector iv) has as a base structure a functional binary vector in Agrobacterium tumefaciens.
7. El proceso para la expresión de proteínas recombinantes en microalgas de las reivindicaciones 1 a 6, caracterizado porque comprende los pasos: 7. The process for the expression of recombinant proteins in microalgae of claims 1 to 6, characterized in that it comprises the steps:
a) Construir el vector iv); a) Construct the vector iv);
b) Transformar transitoriamente una microalga hospedero de expresión mediante la inoculación de Agrobacterium tumefaciens, en un medio suplementado con Acetosiringona y Cefotaxima; b) Transiently transform a microalga host expression by inoculating Agrobacterium tumefaciens, in a medium supplemented with Acetosyringone and Cefotaxime;
c) Expresar el gen de interés en la microalga transitoriamente transformada del paso b) mediante la adición de una solución de un alcohol; y, c) Expressing the gene of interest in the transiently transformed microalga of step b) by adding a solution of an alcohol; Y,
d) Obtener la proteína recombinante de interés mediante la introducción de la proteína de interés, sobre los elementos Orí del vector iv), y mediante la generación y replicación de moléculas de ADN circulares que llevan el casete de expresión del vector iv). d) Obtaining the recombinant protein of interest by introducing the protein of interest, on the Ori elements of vector iv), and by generating and replicating circular DNA molecules carrying the vector expression cassette iv).
8. El proceso para la expresión de proteínas recombinantes en microalgas de la reivindicación 7, caracterizado porque el alga transitoriamente transformada y utilizada en el paso b) se selecciona de: Chlamydomonas reinhardtii y/o Schizochytrium sp. 8. The process for the expression of recombinant proteins in microalgae of claim 7, characterized in that the alga transiently transformed and used in step b) is selected from: Chlamydomonas reinhardtii and / or Schizochytrium sp.
9. El proceso para la expresión de proteínas recombinantes en microalgas de la reivindicación 7, caracterizado porque el gen de interés en la microalga transitoriamente transformada del paso c), se selecciona de: antígenos y/u hormonas y/o enzimas.
9. The process for the expression of recombinant proteins in microalgae of claim 7, characterized in that the gene of interest in the transiently transformed microalga of step c) is selected from: antigens and / or hormones and / or enzymes.
10. El proceso para la expresión de proteínas recombinantes en microalgas de la reivindicación 9, caracterizado porque el gen de interés en la microalga transitoriamente transformada del paso c), se selecciona de: Zaire ebolavirus y/o Escheríchia coli.10. The process for the expression of recombinant proteins in microalgae of claim 9, characterized in that the gene of interest in the transiently transformed microalga of step c) is selected from: Zaire ebolavirus and / or Escherichia coli.
1 1 . El proceso para la expresión de proteínas recombinantes en microalgas de la reivindicación 7, caracterizado porque la solución de alcohol del paso c) es en grado absoluto, y/o en solución al 1 %. eleven . The process for the expression of recombinant proteins in microalgae of claim 7, characterized in that the alcohol solution of step c) is in absolute degree, and / or in 1% solution.
12. El proceso para la expresión de proteínas recombinantes en microalgas de la reivindicación 1 1 , caracterizado porque el alcohol es etanol. 12. The process for the expression of recombinant proteins in microalgae of claim 1, characterized in that the alcohol is ethanol.
13. El proceso para la expresión de proteínas recombinantes en microalgas de la reivindicación 7, caracterizado porque la proteína recombinante de interés d) es de interés biofarmacéutico relacionada a una enfermedad que afecta a un ser vivo, y/o un bioproceso que lo requiera como molécula de interés. 13. The process for the expression of recombinant proteins in microalgae of claim 7, characterized in that the recombinant protein of interest d) is of biopharmaceutical interest related to a disease that affects a living being, and / or a bioprocess that requires it as molecule of interest.
14. El proceso para la expresión de proteínas recombinantes en microalgas de la reivindicación 13, caracterizada porque la proteína recombinante de interés biofarmacéutico relacionada a una enfermedad que afecta a un ser vivo puede ser de Zaire ebolavirus y/o de Escheríchia coli. 14. The process for the expression of recombinant proteins in microalgae of claim 13, characterized in that the recombinant protein of biopharmaceutical interest related to a disease that affects a living being can be of Zaire ebolavirus and / or of Escherichia coli.
15. El proceso para la expresión de proteínas recombinantes en microalgas de las reivindicaciones 1 a 14, caracterizado porque la replicación se realiza por medio de círculo rodante. 15. The process for the expression of recombinant proteins in microalgae of claims 1 to 14, characterized in that the replication is carried out by means of a rolling circle.
16. El proceso para la expresión de proteínas recombinantes en microalgas de las reivindicaciones 1 a 14, caracterizado porque la inducción en el sitio Orí, se realiza con soluciones de un alcohol al 1 % o en grado absoluto. 16. The process for the expression of recombinant proteins in microalgae of claims 1 to 14, characterized in that the induction in the Ori site is carried out with solutions of a 1% alcohol or absolute grade.
17. El proceso para la expresión de proteínas recombinantes en microalgas de la reivindicación 16, caracterizado porque el alcohol es etanol. 17. The process for the expression of recombinant proteins in microalgae of claim 16, characterized in that the alcohol is ethanol.
18. El proceso para la expresión de proteínas recombinantes en microalgas de las reivindicaciones 1 a 17, caracterizado porque muestra un rendimiento de expresión de proteínas comprendido entre 0.12 a 1 .25 mg/g en peso fresco, dependiendo de la naturaleza y/u origen de la proteína recombinante de interés a obtener.
18. The process for the expression of recombinant proteins in microalgae of claims 1 to 17, characterized in that it shows a protein expression yield between 0.12 to 1.25 mg / g in fresh weight, depending on the nature and / or origin of the recombinant protein of interest to obtain.
19. El proceso para la expresión de proteínas recombinantes en microalgas de las reivindicaciones 1 a 18, caracterizado porque muestra un rendimiento de expresión de proteínas comprendido de al menos 0.12 mg/g cuando se usa para obtener proteínas recombinates de Escheríchia coli. 19. The process for the expression of recombinant proteins in microalgae of claims 1 to 18, characterized in that it shows a protein expression yield of at least 0.12 mg / g when used to obtain recombinant Escherichia coli proteins.
20. El proceso para la expresión de proteínas recombinantes en microalgas de las reivindicaciones 1 a 18, caracterizado porque muestra un rendimiento de expresión de proteínas comprendido de al menos 1.25 mg/g en peso fresco para obtener proteínas recombinates de Zaire ebolavirus. 20. The process for the expression of recombinant proteins in microalgae of claims 1 to 18, characterized in that it shows a protein expression yield of at least 1.25 mg / g fresh weight to obtain recombinant proteins of Zaire ebolavirus.
21. El uso de una proteína recombinante obtenida mediante el proceso de las reividicaciones 1 a 20 en la elaboración de una vacuna para tratar una enfermedad que afecta a un ser vivo. 21. The use of a recombinant protein obtained through the process of subdivisions 1 to 20 in the preparation of a vaccine to treat a disease that affects a living being.
22. El uso de una proteína recombinante obtenida mediante el proceso de la reivindicación 21 en la elaboración de una vacuna para tratar la enfermedad causada por Zaire ebolavirus. 22. The use of a recombinant protein obtained by the process of claim 21 in the manufacture of a vaccine to treat the disease caused by Zaire ebolavirus.
23. El uso de una proteína recombinante obtenida mediante el proceso de la reivindicación21 en la elaboración de una vacuna para tratar la enfermedad causada por la enterotoxina de Escheríchia coli.
23. The use of a recombinant protein obtained by the process of claim 21 in the manufacture of a vaccine to treat the disease caused by the enterotoxin of Escherichia coli.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
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MX2018000828A MX2018000828A (en) | 2017-12-15 | 2017-12-15 | Process for the expression of recombinant proteins in microalgae using viral vectors. |
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CN116479027A (en) * | 2023-06-15 | 2023-07-25 | 中国农业科学院北京畜牧兽医研究所 | Recombinant expression vector for expressing bovine lactoferrin as well as construction method and application thereof |
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WO2015168733A1 (en) * | 2014-05-05 | 2015-11-12 | Queensland University Of Technology | Decoy molecules |
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CN116479027A (en) * | 2023-06-15 | 2023-07-25 | 中国农业科学院北京畜牧兽医研究所 | Recombinant expression vector for expressing bovine lactoferrin as well as construction method and application thereof |
CN116479027B (en) * | 2023-06-15 | 2023-08-18 | 中国农业科学院北京畜牧兽医研究所 | Recombinant expression vector for expressing bovine lactoferrin as well as construction method and application thereof |
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