KR20010082143A - Production method of recombinant rotavirus structural proteins and vaccine composition - Google Patents

Abstract

PURPOSE: A method for producing capside proteins of recombinant rotavirus is provided. And vaccine compositions produced thereby is also provided. Therefore, the rotavirus can be cheaply and effectively produced in higher yield and low contamination possibility. CONSTITUTION: The method for producing capside proteins of recombinant rotavirus comprises the steps of: preparing an expression plasmid containing cDNA fragment encoding the capside protein of rotavirus to be controlled by a promotor that regulates plant specific expression and a selection mark gene; inserting the expression plasmid into a plant cell; inducing the formation of calluses from the plasmid containing plant cell; incubating the calluses; recovering proteins expressed from the calluses. Wherein, cDNA encoding VP2 of human rotavirus is represented by SEQ ID NO: 1 and rotavirus is derived from humans, bovines, monkeys, pigs, dogs, cats, birds or mice. The vaccine composition useful for the treatment of rotavirus related diseases contains the capside proteins of recombinant rotavirus as an active ingredient.

Description

Production method of recombinant rotavirus structural proteins and vaccine composition

The present invention relates to a method for producing a recombinant rotavirus construct protein and a vaccine composition comprising the proteins as an active ingredient by culturing transformed plant cells, and more specifically, to a gene encoding a construct protein of rotavirus. The present invention relates to a method for constructing a plant expression vector comprising the same, transforming plant cells using the same, and preparing a vaccine composition comprising a rotavirus antigen from the culture.

Rotavirus, first discovered in 1973 by Bishop in Australia, is a type of double-stranded RNA virus belonging to the Reoviridae family that causes acute gastroenteritis that commonly occurs in infants. It spreads from person to person after an incubation period of about 1 to 3 days. It appears to be a serious disease in children 6 to 24 months of age, but mild or asymptomatic in newborns or most adults. Rotavirus-induced acute infectious diarrheal disease is the leading cause of death in many parts of the world, and in developing countries, it is estimated that about 1 million patients die each year from rotavirus-induced diarrhea. , NR & Greenburg, HB, (1991) Viral gastroentertitis N. Engl. J. Med ., 325: 152-164, 1991]. Therefore, the World Health Organization (WHO) has prioritized research to more effectively suppress and prevent rotavirus infection [Ref. Glass, R. I et al. , (1994) Rotavirus vaccines: success by reassortment Science 265: 1389-1391.

Rotaviruses are generally spherical and their names are derived from the unique outer and inner or double-shell capsid structures. The outer capsid is about 70 nm in diameter and the inner capsid is about 55 nm in diameter. Typically, the double-shell capsid structure of rotaviruses surrounds the core containing the inner protein shell or genome. The genome of rotavirus consists of segments of double stranded RNA encoding at least 11 unique viral proteins. The inner capsid of rotavirus consists of proteins named VP6 and VP2, which are outside the double-shell capsid structure and form the outer capsid. VP6 is a group-specific antigen. Rotavirus is divided into seven groups of A-G according to the antigenicity of VP6. VP2 protein is involved in RNA binding. Group A rotaviruses are further divided into glycoproteins (G7) and glycoproteins (Glycoprotein) and VP4 (Ptease-cleaved proteins). Estes MK,et al., (1987) Synthesis and immunogenicity of the rotavirus major capsid antigen using a baculovirus expression system,J. Virol. 61: 1488-1494; Estes M. K & Cohen J., (1989) Rotavirus gene structure and function.Microbiol. Rev.53: 410-419; Desselberger U & McCrae M. A., (1994) The rotavirus genome.Curr. Microbiol. Immuno.185: 31-66.

Therapies for acute diarrheal diseases caused by rotavirus have been limited to the administration of nonspecific fitness aids, such as replenishment of water and electrolytes, and the development of effective rotavirus vaccines that provide complete protection against all serotypes of human rotavirus. Has been required. This approach uses a live vaccine of attenuated human rotavirus, or a combination of rotavirus strains of animals such as cattle, or different serotypes of human rotavirus and RNA segments of animal rotavirus. Has been. As a result of these studies, in 1998, US vaccine company Wyeth Laboratories released RotaShield, the first rotavirus vaccine approved by the US Food and Drug Administration (FDA), which is based on strains derived from rhesus monkeys. A vaccine produced by reassortant three human rotavirus serotypes. However, the vaccine was temporarily revoked by the US Food and Drug Administration (FDA) in 1999 due to side effects such as intestinal overlap [Ref .: MMWR Morb. Mortal Wkly. Rep., (1999) 5; 48 (43): 1007.

Separately, studies are being conducted to mass-produce a part of rotavirus constructs using genetic recombination technology and use it as a subunit vaccine. In view of this, many researchers have studied the expression system of rotavirus capsid proteins using E. coli, baculovirus, and mammalian expression systems [Ref . Smith RE et al. , (1989) Cloning and expression of the major inner capsid protein of SA-11 simian rotavirus in E. coli . Gene 79: 239-248; Tosser G. et al. , (1992) Expression of the major capsid protein VP6 of group C rotavirus and synthesis of chimeric single-shelled particles by using recombinant baculoviruses. J. Virol . 66: 5825-5831; Ito H. et al. , (1997) Expression of the major inner capsid protein, VP6, of avian rotavirus in mammalian cells. Vet. Microbiol . 49: 257-265.

However, rotaviruses infected through mucosal cells are difficult to mass-produce through cell culture. That is, prokaryotic expression systems such as Escherichia coli are difficult to produce viral protein particles similar to those of natural forms, and thus do not exhibit antigenicity properly.In case of using animal cells, the risk of contamination and difficulty in purification, high production cost, and low yield are obtained. As a result, satisfactory production effects were not obtained.

In addition, most of the diseases caused by rotavirus are developed in developing countries where sanitary facilities and vaccines are difficult to spread. However, even if the technical success of the virus-constituted proteins and vaccine production methods using other cells is possible, Considering the high selling price, the actual commercialization requires considerable time and research. On the other hand, if the rotavirus component protein can be produced and used as a vaccine through plants, particularly edible plants, it can be economically effective in terms of reducing production costs, eliminating the need for purification, transportation and storage.

Accordingly, the present inventors transformed plant cells with plant expression vectors comprising a capsid gene of rotavirus, and when cultured under appropriate conditions, rotavirus construct proteins capable of inducing a desirable mucosal and systemic immune response from the plant cells, and The present invention has been completed by discovering that a vaccine composition using the same can be produced.

Accordingly, it is an object of the present invention to provide a method for the treatment of rotavirus that can induce a desirable immune response by culturing transgenic plant cells. Construct proteins It is to provide a method for mass production. It is another object of the present invention to provide a plant expression vector for producing the transgenic plant.

It is also an object of the present invention to provide a vaccine composition for the treatment or prophylaxis of enteritis comprising the Rotavirus construct protein recovered from the culture medium of the transformed plant cells as an active ingredient. In particular, an object of the present invention is to provide a use for use as an edible vaccine or an oral vaccine by producing the virus like particles from edible plant cells.

Furthermore, an object of the present invention is a vaccine for the treatment or prevention of rotavirus-related diseases, which is low in production cost, mass-produced, requires little purification, and can induce desirable mucosal and systemic immune responses. It is an object to provide a vaccine for use.

1A to 1D are cleavage maps showing the structures of binary vector for plant transformation, pILTAB357-VP6, pILTAB357-VP2, pILTAB357-VP4, and pILTAB357-VP7, respectively.

CsVMV promoter: cassava lobe mosaic virus promoter

NOS promoter: promoter of nopalin synthase

NOS 3 ': transcription termination site of noparin synthase

NPTII: gene of neomycin phosphotransferase II

2A to 2D show the results of Western blot analysis of normal and transformed tomato cells for recombinant rotavirus proteins VP6, VP2, VP4 and VP7, respectively.

M: molecular weight marker,

1: intracellular fraction of normal cells,

2: medium fraction of normal cells,

3: intracellular fraction of transformed tomato cells

4: Media fraction of transformed tomato cells, wherein arrows indicate recombinant rotavirus proteins VP6, VP2, VP4 and VP7, respectively.

3A is a graph showing changes over time in cell concentration and recombinant Rotavirus protein VP6 expression in shake flask cultures of transformed cells,

1st operation 2nd operation

Figure 3b shows the results of Western blot analysis on the expression of recombinant rotavirus protein VP6 at 12, 15, 18, 21 days after incubation in the first and second operations, respectively. Herein, "C" in lane 5 represents purified fusion VP6 protein obtained from 0.2 µg E. coli.

4A is a graph showing the change over time of recombinant Rotavirus protein VP7 expression with cell density and G1 serotype in shake flask culture of transformed cells,

1st operation 2nd operation

Figure 4b shows the results of Western blot analysis on the 2nd, 3rd, 4th, 5th, 6th, and 7th day after incubation in primary and secondary operations to examine the expression of recombinant rotavirus protein VP7 with G1 serotype. will be.

In order to achieve the above object, in the present invention, the gene for producing a constitutive protein of rotavirus is inserted into a plant cell and transformed, and the cultivated by culturing the rotavirus constitutive protein and a vaccine composition containing the protein as an active ingredient. Provide a way to.

Method for producing rotavirus construct protein using the plant cell of the present invention,

1) preparing an expression plasmid comprising cDNA fragments encoding rotavirus construct proteins regulated by a promoter regulating plant specific expression and additionally comprising a selection marker gene;

2) introducing said expression plasmid into plant cells;

3) inducing callus formation from the transformed plant cells;

4) culturing the callus; And

5) recovering the recombinant rotavirus construct protein expressed from the cDNA encoding the rotavirus construct protein in the culture.

As the gene encoding the rotavirus constituent protein of step 1, any gene encoding the rotavirus capsid protein can be used, as well as genes derived from human rotaviruses, as well as genes derived from animal rotaviruses such as cows. It is preferable to use a gene encoding a protein, a gene encoding a VP2 inner capsid protein, a gene encoding a VP4 outer capsid protein, a gene encoding a VP7 outer capsid protein, and the like. The desired rotavirus gene can be obtained by amplification from known excretion of a subject suffering from rotavirus-related disease through a known polymerase chain reaction (PCR). In a preferred embodiment of the present invention, the plasmid pGEM-VP6 (KCTC 8984P) containing the gene fragment encoding the VP6 protein was digested with a restriction enzyme, and in another preferred embodiment of the present invention, a rotavirus-related disease The cDNA fragments encoding VP2, VP4 and VP7 proteins having the serotypes of G1, G2, G3, and G4 were isolated and amplified from human feces.

In addition, recombinant plasmids expressing cDNA fragments encoding the desired rotavirus construct protein of step 1 can be prepared using known plant expression vectors as a base vector, and include a general binary vector and a cointegration vector. vector) or a general vector which does not contain the T-DNA site but is designed to be expressed in plants can be used.

As an example of a preferred binary vector, the present invention includes a left border and a right border involved in the infection of a foreign gene in T-DNA for transformation of plant cells, with a cassava leaf vein mosaic in between. Encoding a rotavirus construct protein VP6, VP2, VP4 or VP7 in a binary vector containing a viral promoter, a promoter of noparin synthase, a transcription termination site of noparin synthase, and a label gene for transformant selection The final binary vectors constructed by inserting the genes respectively, such as pILTAB357-VP6, pILTAB357-VP2, pILTAB357-VP4, pILTAB357-VP7 and the like, were used.

In addition to the cassava leaf vein mosaic virus promoter described above, promoters for controlling plant-specific expression include the ubiquitin promoter, the cabbage mosaic virus 35S promoter, the actin promoter, the PG promoter, or the endosperm specific promoter. All known promoters for maximizing expression of recombinant proteins in plant cells, such as -specific promoters, can be used.

In the transformed plant of step 2 cells, when using a binary vector or co-integration vectors are available for transposition transformed plant cells mediated by Agrobacterium (Agrobacterium), Agrobacterium tube Mapo when Enschede (Agrobacterium tumefaciens ) or Agrobacterium rhizogenes .

In a preferred embodiment of the present invention, the constructed binary vector was introduced into Agrobacterium tumefaciens through known triparental mating. Swallowed hybridization method includes E. coli strain containing a helper plasmid having the function necessary for conjugation, E. coli strain containing a recombinant plasmid containing the target gene, and Agrobacterium strain for plant cell transformation to which the target gene is to be introduced. It is a method of introducing a plasmid having a target gene into Agrobacterium by co-culture. Specifically, E. coli strains having pILTAB357-VP6 were used as parent strains used in the parenting hybridization method, and cotyledon cut from tomato seedlings was transformed with the Agrobacterium strain transformed by the parental hybridization method. .

In addition, when transforming monocotyledonous plants using vectors containing no T-DNA site, polyethylene glycol-mediated uptake, electroporation or microparticle bombardment using polyethylene glycol is used. ) May be used, and a transformation using a single DNA and a simultaneous transformation using multiple DNAs may be used.

Transformed cells are re-differentiated into plants using standard techniques known in the art. As plants that can be transformed by the above method, both dicotyledonous plants such as lettuce, cabbage, radish, potatoes, tomatoes, and monocotyledonous plants such as rice, barley, and banana can be used. In particular, in the case of transforming edible plants to produce rotavirus structural proteins and analogous particles, the immune response can be induced by directly ingesting the plants themselves, and thus can be effectively used as an edible vaccine.

Antibiotic resistance genes are mainly used as marker genes for selecting transgenic plants, but are not limited thereto. Herbicide resistance genes, metabolic genes, luminescent genes, GFP (green fluorescence protein), GUS (β-glucuronidase) gene, GAL (β-galactosidase) gene and the like can be used. Specifically, neomycin phosphotransferase II (NPT II) gene, hygromycin phosphotransferase gene, phosphinothricin acetyltransferase gene or dihydrofolate reductase gene and the like can be used.

On the other hand, the culture of the transformed plant cells of the present invention can be used in addition to the general suspension culture microgravity culture method using a high-phase rotary wall reactor. When using the high-phase rotary wall reactor, the growth rate of the cells was found to be very slow compared to the general suspension culture because the lag phase of the cell growth is long to adapt to the microgravity simulation environment. However, the production of each rotavirus protein per cell weight was found to be similar to that of shaking culture.

In addition, the present invention is a rotavirus prepared as described above Construct proteins VP2, VP6, VP4 and VP7 are folded to each other to provide rotavirus analogs that can be prepared by binding. As used herein, 'rotavirus like particle' refers to a combination of one or more capsid proteins or particles that are antigenic as being self-assembly in a cell.

Furthermore, the present invention is a rotavirus prepared as described above Construct proteins It provides a vaccine composition for the treatment or prevention of rotavirus-related diseases as an active ingredient. Specifically, the vaccine composition may comprise any one or more selected from the group consisting of VP4 with VP4 and G1 serotypes, VP7 with G2 serotypes, VP7 with G3 serotypes and VP7 with G4 serotypes. More preferably, it is preferable to include VP2 and VP6.

Rotavirus construct protein produced by plant cells for the production of the vaccine composition may be used by pure separation from the cell culture by a known method, but when transformed using an edible plant without separation and purification It is preferable in terms of cost and process to prepare the plant cell itself, a part of the transformed plant re-differentiated therefrom, or an extract thereof.

The vaccine for the treatment or prophylaxis of rotavirus-related diseases of the present invention can be administered orally or parenterally during clinical administration and can be used in the form of general pharmaceutical preparations.

That is, the rotavirus vaccine composition produced by the plant cell of the present invention When formulated, it can be formulated using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc., which are commonly used pharmaceutically. Solid form preparations for oral administration include tablets, pills, powders, granules, capsules, and the like. At least one excipient such as starch, calcium carbonate, sucrose or lactose, gelatin and the like is mixed and prepared. In addition to simple excipients, lubricants such as magnesium styrate talc are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As a suppository base, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

However, since most of the vaccines for rotavirus are intended for infants and are preferably given before the first exposure to rotavirus, especially in infants aged 2-24 months, the formulation of the vaccine is intended for infants. More preferred are liquid oral, injectable or suppository forms that are easy to inoculate.

Hereinafter, the present invention will be described in more detail with an example of an expression system for expressing complementary DNA (cDNA) encoding a recombinant rotavirus construct protein from a suspension culture of transformed tomato cells, and expressing the recombinant rotavirus construct protein. An expression system using a High Aspect Rotating-Wall Vessel designed by NASA as a system for optimally culturing transformed tomato cells is described. However, since these methods can also be carried out by modifying the methods taught in the documents cited herein or the methods described in these documents, the present invention is not limited to these specific embodiments.

Construction of a vector comprising a cDNA fragment encoding a rotavirus construct protein

Gene cloning methods used in the present invention can be easily carried out by employing a methodology known in the art. Such methodologies are described, for example, in Sambrook et al., 1989, Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory, N.Y .; And Ausubel et al., 1989, Current Protocols in Molecular Biology, Greene Publishing Associate and Wiley Interscience, N.Y.

There are a wide variety of binary vectors that are widely used in the transformation of dicotyledonous plants, especially tomatoes, and the majority of binary vectors are international organizations such as CAMBIA (Center for the Application of Molecular Biology to International Agriculture, GPO Box 3200, Canberra ACT2601, Australia, etc., and the skeleton of the basic binary vector is used by variously transforming transformant selection marker genes, promoters, transcriptional termination genes, etc. in the left and right boundary regions where the genes are delivered.

In the embodiment of the present invention the human body for the configuration of the binary vector PGEM-VP6 (KCTC 0944BP) containing the cDNA fragment encoding the rotavirus VP6 protein was used, and also the cDNA fragment encoding human rotavirus VP2 represented by SEQ ID NO: 1, human rotavirus VP4 represented by SEQ ID NO: 2 A plasmid comprising a cDNA fragment encoding human body, a cDNA fragment encoding human rotavirus VP6 represented by SEQ ID NO: 3, and a cDNA fragment encoding human rotavirus VP7 having a different serotype represented by SEQ ID NOs: 4 to 7. Although used, the vectors suitable for the practice of the present invention are not limited thereto, and any vector can be used without particular limitation as long as it can efficiently express the gene using any cloning vector capable of expressing the proteins. In the present invention, the cDNA fragment is used in the known plasmid pILTAB357 (Scripps Research Institute, USA; Verdaguer et al., Isolation and expression in transgenic tobacco and rice plants, of the cassava vein mosaic virus promoter,Plant Molecular Biology31: 1129-1139, 1996) A vector was constructed by inserting it into the cloning site, and a cassava leaf vein mosaic virus (CsVMV) promoter was used as the promoter for the T-DNA site of the binary vector pILTAB357 for plant transformation, and neomycin phosphotransferase II (NPTII) as a selection marker. ) Genes were inserted to construct plasmids for plant cell expression (see FIGS. 1A-1D).

Transformation Procedure

Transformation can be carried out using conventional methods known in the art. Transformation to plants may utilize Agrobacterium-mediated transformation and the methods illustrated in Paszkowsky et al., EMBO J 3: 2717-2722 (1984). For example, Agrobacterium-mediated transformation methods for tomatoes are known in the art (An et al., EMBO J 4: 227-288 (1985)) and the like. For the transformation of monocotyledonous plants, it is possible to use a method of grafting genes directly into protoplasts using PEG or electroshock method or particle bombardment into callus tissue. Transformed cells are re-differentiated into whole plants using standard techniques known in the art. Agrobacterium tumefaciens is mainly used as a host for transformation, and Agrobacterium tumafaciens strain LBA4404, which is widely known as a preferred example, was used in the present invention.

In the present invention, the constructed binary vector is known as Agrobacterium tumafaciens through known triparental mating.Agrobacterium tumefaciens) Was introduced. For example, in this case, the parent strain contains a human rotavirus VP6 protein cDNA fragment. E. coli MM294 strain with pILTAB357-VP6 was used. Then, using recombinant Agrobacterium tumafaciens LBA 4404 tomato (Lycopersicon esclentumCotyledon cut from the seedling of Mill) was transformed.

Transformation Process of Transformant

In culturing the transgenic tomato cells, they were cultured using suspension culture and high-phase rotary wall reactor. To this end, the seeds of Lycopersicon esclentum Mill are sterilized, inoculated onto MS (Murashige and Skoog) medium, and then germinated to co-culture with suspension culture of Agrobacterium tumafaciens LBA 4404 with recombinant plasmids. Passage. In addition, as a culture system for optimally producing recombinant rotavirus construct proteins VP6, VP2, VP4 and VP7 proteins, transformed tomato cells were cultured using a High Aspect Rotating-Wall Vessel designed by NASA. Was carried out to simulate the microgravity.

Expression of Recombinant Rotavirus Construct Protein

Suspension culture of transformed tomato cells from selected callus in individual kanamycin resistant callus was established and maintained for 3 months in liquid medium added with kanamycin 200 mg / L. After culture, the expression levels of genes were analyzed using transformed cells and normal tomato cells. As shown in Figures 2a to 2d, recombinant rotavirus construct proteins VP6, VP2, VP4 and VP7 could be detected in the transformed tomato cells by Western analysis.

For example, the recombinant rotavirus protein VP6 is mainly in the intracellular fraction and has a molecular weight of about 44 kDa, VP2 has a molecular weight of 94 kDa, VP4 has a molecular weight of 87 kDa, and VP7 has a molecular weight of 34 kDa. The molecular weight was almost identical to the one inferred from the cDNA base sequence. Recombinant rotavirus proteins VP6, VP2, VP4 and VP7 were not detected in untransformed cell or media fractions (lanes 1-2 in FIGS. 2A-2D, respectively). This indicates that the expression of VP6, VP2, VP4 and VP7 proteins is due to pILTAB357-VP6, pILTAB357-VP2, pILTAB357-VP4 and pILTAB357-VP7 integrated into transformed tomato cells, respectively.

Hourly Expression

Cell concentration and recombinant rotavirus protein during shake flask culture of transformed tomato cells The change in the expression of VP6 over time is shown in Figure 3a. The highest cell concentration showed a fresh weight of 192 g / L at 15 days post culture when inoculated with 40 g / L fresh weight. Western analysis using a fused VP6 protein having a molecular weight of 45.5 kDa produced from Escherichia coli as a control, The production of rotavirus protein VP6 was 0.14, 0.21 and 0.19 mg / L at 15, 18 and 21 days after incubation, respectively, when the fresh weight of 14 g / L was inoculated. Similarly, when inoculated with a fresh weight of 40 g / L, the recombinant rotavirus protein The production of VP6 was 0.29, 0.33 and 0.28 mg / L at 15, 18 and 21 days after incubation, respectively. The maximum yield of recombinant rotavirus protein VP6 was 0.33 mg / L fresh weight on day 18 of culture. The recombinant protein yield of 0.33 mg / L obtained from the transformed tomato cells according to the present invention is reported by LaCount et al. [LaCount W.et al., (1997) The effect of polyvinylpyrrolidone on the heavy chain monoclonal antibody production from plant suspension culture.Biotechnol. Lett. 19: 93-96] higher than 0.19 mg / L, the production of the heavy chain of recombinant monoclonal antibodies from transformed tobacco cells cultured without the addition of polyvinylpyrrolidone.

Hereinafter, the present invention will be described in more detail with reference to Examples.

However, the following examples are illustrative of the present invention, and the content of the present invention is not limited thereto.

Example 1 Preparation of Binary Vector for Transformation

anatomy A fragment of pGEM-VP6 (KCTC 0944BP), which encodes a rotavirus VP6 protein, digested with restriction enzyme EcoR I was inserted into the EcoR I cleavage site of the pILTAB357 plasmid (Scripps Research Institute, USA) to produce a 13.94 kb recombinant plasmid pILTAB357-VP6. Was prepared. 1A shows a cleavage map showing the structure of pILTAB357-VP6. In this construct, a promoter for transcription regulation of the VP6 gene is a cassava leaf vein mosaic virus (CsVMV) promoter inserted between the left and right borders of the T-DNA of the plant vector, pILTAB357, for plant transformation. References: Verdaguer B., et al., (1996) Isolation and expression in transgenic tobacco and rice plants, of the cassava vein mosaic virus (CsVMV) promoter.Plant Mol. Biol.31: 1129-1139], a neomycin phosphotransferase II (NPTII) gene was inserted to select a transformant in a growth medium containing kanamycin (see FIG. 1A). The alignment and translation frame of the VP6 gene was correctly inserted into the recombinant plasmid pILTAB357-VP6 by mapping and DNA sequencing using restriction enzymes. The binary vector constructed in this way is known as Agrobacterium tumafaciens through known triparental mating.Agrobacterium tumefaciens) LBA 4404 (Cat. No. 18313-015, Gibco BRL, USA) [Ainsworth C. et al., (1996)Techniques in Plant Molecular Biology,Wye, UK: Wye College]. As the parent strain in this swallowed hybridization method, Escherichia coli MM294 (KCCM 70079) containing pILTAB357-VP6 was used. Tomato using recombinant Agrobacterium tumafaciens LBA 4404Lycopersicon esclentumThe cotyledons cut from the seedlings of Mill were transformed according to the known Agrobacterium-mediated leaf disc transformation, Horsch R.B. et al., Science 227: 1229-1231, 1985.

Example 2 Transformation Procedure and Establishment of Suspension Culture of Transformed Cells

Lycopersicon esculentum Mill seeds are sterilized with 70% ethanol solution for 1 minute, surface sterilized with 1.2% sodium hypochlorite (NaOCl) solution for 30 minutes, washed several times with sterile distilled water, and then agar 8g / L On the added MS (Murashige and Skoog) medium (sucrose 30g / l, 2,4-D (2,4-dichlorophenoxyacetic acid) 2mg / l, kinetin 0.2mg / l, thimentin 150mg / l, pH 5.7) Sprinkled Seeds sown in magenta boxes were germinated in a 27 ° C. incubator for a 12 hour light cycle. Aseptic young seedlings germinated about 2 weeks later were used in the transformation experiments. After cutting the cotyledons of the seedlings, they were immersed in the suspension culture of recombinant Agrobacterium tumafaciens LBA 4404 containing pILTAB357-VP6 plasmid for about 3 minutes, and the excess microorganisms were removed by filter paper. The prepared explants were then co-cultured with Agrobacterium tumafaciens LBA4404 in solid MS medium to which 8 g of agar was added. After co-culture explants were transferred to the medium added 200 mg / L of kanamycin B and 150 mg / L of thimentin. After that, the explants were incubated for 4 weeks in a culture chamber at 27 ° C. with a photoperiod of 12 hours to induce callus. The induced callus was cut out and propagated while maintaining subculture at 4 week intervals again. Then, the callus having kanamycin resistance was selected and transferred to a liquid medium to which kanamycin 200 mg / L was added without suspension, followed by suspension culture.

Suspension culture of the transformed plant cells was maintained and propagated while subcultured at 100 rpm for 3 weeks using a rotary shaker at 27 ° C. using a liquid medium to which kanamycin B 200 mg / L was added. As a control, all the procedures except for the Agrobacterium transformation process were conducted in the same manner to induce callus of normal tomato cells which were not transformed, and the suspension culture was established and maintained under the same conditions.

Example 3 Suspension Culture and Gene Expression Analysis

1) Suspension Culture of Transgenic Plant Cells and Isolation of VP6

Transformed tomato cells were suspended and cultured in a shake flask containing 50 mg / L of MS liquid medium added with 200 mg / L of kanamycin at 27 ° C. and 100 rpm. Cells were collected at regular intervals during the incubation period to analyze cell growth and expression of recombinant proteins.

After separating the cells by centrifuging the culture solution at 3,000 rpm for 5 minutes, the supernatant was used to analyze the extracellular recombinant protein. After freezing the cell fractions with liquid nitrogen, the protein extraction solution (Tris-HCl, pH 7.5) 50 mM, ethylenediaminetetraacetic acid (EDTA) 2 mM, ethylenebis (oxyethylenenitrilo) tetraacetic acid (EGTA) 0.5 mM, phenylmethylsul Phenyl fluoride (PMSF) 1 mM, 1% Triton X-100) was added to the pestle and induction, and then transferred to a centrifuge tube and sonicated three times for 5 minutes at 4 ° C. Cell extracts were then centrifuged at 14,000 rpm for 15 minutes at 4 ° C. to remove cell debris and supernatants were used to analyze intracellular recombinant proteins. Unless otherwise specified, the analysis of total protein production used a mixture of intracellular and extracellular fractions.

2) Western blot analysis

The yield of recombinant rotavirus VP6 protein was measured using Western analysis using 44.5 kDa fused VP6 protein expressed in Escherichia coli as a control. The fusion VP6 protein was expressed in Escherichia coli using a pET-15b plasmid from Novagen, USA, which was designed to express VP6 protein in E. coli with a molecular weight of 44.5 kDa. The base sequence was fused and purified using His-Bind kit (Novagen).

Protein samples were subjected to SDS-PAGE electrophoresis according to Laemmli method [Laemmli UK (1970) Cleavge of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685. After electrophoresis, the proteins were transferred to nitrocellulose membranes, which were then combined with guinea pig anti-rotavirus polyclonal antibodies (Seoul National University of Korea) and rabbit anti-guinea pig immunoglobulin G (Kirkegaard & Perry) bound to alkaline phosphatase. Laboratories Co.). After washing the membrane with the buffer solution, color development was performed using BM Purple AP substrate solution (Boehringer Mannheim), and the color reaction was stopped by adding distilled water.

As a result, as shown in Figure 2a, recombinant rotavirus VP6 protein was mainly present in the transformed plant cell fraction and has a molecular weight of about 44.5 kDa. This is consistent with the molecular weight of the VP6 protein expressed in Escherichia coli, indicating that the transformed plant cells of the present invention successfully express the Rotavirus VP6 protein.

3) Analysis of Expression of VP6 Protein in Shake Cultures

3A and 3B show changes in cell concentration and expression time of recombinant Rotavirus VP6 protein in shaking cultures of transformed plant cells.

As shown in Figures 3a and 3b, the highest cell concentrations were inoculated with 40 g / L fresh weight (primary operation, ), The fresh weight of 192 g / L was shown at 15 days after the culture. Western analysis using the fusion VP6 protein of 45.5 kDa molecular weight produced from Escherichia coli as a control showed that the production of recombinant Rotavirus VP6 protein was inoculated at a fresh weight of 14 g / L (secondary operation, ) Showed VP6 production of 0.14, 0.21 and 0.19 mg / L at 15, 18 and 21 days after incubation, respectively. The maximum yield of recombinant VP6 was 0.33 mg / L at 18 days of culture. Converting this figure into non-productivity on the basis of cell weight corresponds to 0.0018 mg / g, which is known in 1997 when the recombinant protein is produced in plant cells, for example, the trait reported by LaCount et al. In 1997. Significantly higher than heavy chain production (0.19 mg / L) of recombinant monoclonal antibodies from the converting tobacco cells.

Example 4 Microgravity Culture

Meanwhile, in order to simulate the microgravity, the transformed tomato cell culture medium was completely filled in a 10 ml high-level rotary wall reactor (HARV, Systhecon, Houston, TX) to produce recombinant Rotavirus VP6 protein, and then cultured for 18 days. . The reactor was rotated at 27 rpm at 10 rpm. The results are shown in Table 1 below.

Cell growth of transformed cells in the high phase rotary wall reactor (HARV) increased to 73 g / L and 76 g / L fresh weight. Compared to the culture results in the shake flasks shown in FIGS. 3A and 3B, it can be seen that the high-phase rotary wall reactor has a slow growth rate due to the lag phase for adapting to the microgravity simulation environment. It is considered to be. The yield of recombinant rotavirus VP6 protein in the high phase rotary wall reactor was 0.13 mg / L and 0.15 mg / L, respectively. However, the specific productivity of VP6 protein on a cell basis was similar to that of shake flask.

Further scientific studies will be needed to further understand the intracellular and molecular mechanisms involved in culturing plant cells in a high-phase rotary wall reactor, but the results obtained by the present inventors have shown that It is shown that the growth of converted plant cells and the production of recombinant protein can be maintained.

Example 5

PGEM comprising a cDNA fragment having the cDNA sequence represented by SEQ ID NO: 1 encoding a VP2 protein derived from human rotavirus, instead of pGEM-VP6 (KCTC 0944BP) containing a cDNA fragment encoding human rotavirus VP6 protein PILTAB357- was prepared in the same manner as in Examples 1 to 3, except that 15.0 kb of pILTAB357-VP2 recombinant plasmid was prepared using -VP2 (KCTC 0947BP). The VP2 binary vector was introduced and the cotyledons cut from tomato seedlings were transformed. Thereafter, the transformed tomato cells were suspended in culture, the recombinant rotavirus protein VP2 was isolated, and Western blot analysis confirmed that the transformed tomato cells successfully expressed the rotavirus VP2 protein (FIG. 2B). . The maximum yield of recombinant VP2 protein was 0.2 mg at 18 days of culture.

PGEM-VP2, which contains cDNA fragments encoding VP2 from human rotavirus, was deposited on February 8, 2001 with the Korea Biotechnology Research Institute Gene Bank (KCTC), an international depository under the Budapest Treaty on International Approval of Microbial Deposits. No. was deposited with KCTC 0947BP. The cleavage map of the pILTAB357-VP2 recombinant plasmid is shown in FIG.

<Example 6>

PGEM comprising a DNA fragment having the cDNA sequence represented by SEQ ID NO: 2 encoding a VP4 protein derived from human rotavirus, instead of pGEM-VP6 (KCTC 0944BP) containing a cDNA fragment encoding human rotavirus VP6 protein PILTAB357- was carried out in the same manner as in Examples 1 to 3 except that 14.8 kb of pILTAB357-VP4 recombinant plasmid was prepared using -VP4 (KCTC 0945BP). The VP4 binary vector was introduced and the cotyledons cut from the tomato seedlings were transformed. Thereafter, the transformed tomato cells were suspended and cultured, and the recombinant rotavirus VP4 protein was isolated, and then Western blot analysis was used to confirm that the transformed tomato cells successfully expressed the rotavirus VP4 protein (FIG. 2C). . The maximum yield of recombinant VP4 protein was 0.2 mg on day 18 of culture, as in Example 5 above.

PGEM-VP4, which contains cDNA fragments encoding VP4 derived from human rotavirus, was deposited on February 8, 2001 with the Korea Biotechnology Research Institute Gene Bank (KCTC), an international depository organization under the Budapest Treaty on International Approval of Microbial Deposits. Deposited with the number KCTC 0945BP. The cleavage map of the pILTAB357-VP4 recombinant plasmid is shown in FIG. 1C.

<Example 7>

CDNA having a cDNA sequence represented by SEQ ID NO: 4 encoding a VP7 protein with a G1 serotype derived from human rotavirus, instead of pGEM-VP6 (KCTC 0944BP) containing a cDNA fragment encoding human rotavirus VP6 protein Agrobacterium tumafaci was prepared according to the same method as Examples 1 to 3, except that 13.7 kb of pILTAB357-VP7 recombinant plasmid was prepared using pGEM-VP7 (KCTC 0946BP) containing the fragment. The pILTAB357-VP7 binary vector was introduced into the end, and cotyledons cut from tomato seedlings were transformed. Thereafter, the transformed tomato cells were suspended in culture, the recombinant rotavirus VP7 protein having G1 serotype was isolated, and then, using Western blot analysis, the transformed tomato cells were successfully rotavirus VP7 protein having G1 serotype. It was confirmed that the expression (Fig. 2d). The maximum yield of recombinant VP7 protein with G1 serotype was 0.2 mg on day 18 of culture, as in Example 5 above.

PGEM-VP7, which contains a cDNA fragment encoding a VP7 protein with a G1 serotype derived from human rotavirus, was assigned to the Korea Biotechnology Research Institute, an international depository institution under the Budapest Convention on International Approval of Microbial Deposits, dated February 8, 2001. Deposited with Bank No. KCTC 0946BP. The cleavage map of the pILTAB357-VP7 recombinant plasmid is shown in FIG. 1D.

<Example 8>

Instead of pGEM-VP7 (KCTC 0946BP) comprising a cDNA fragment encoding a VP7 protein with a G1 serotype derived from human rotavirus in Example 7, the VP7 protein with a G2 serotype derived from human rotavirus was encoded. Except for using pCR2.1-VP7 (KCTC 0948BP) comprising a cDNA fragment having a cDNA sequence represented by SEQ ID NO: 5, Rota having a G2 serotype according to the same method as in Example 7 above It can be seen that the viral VP7 protein is expressed.

PCR2.1-VP7, which contains a cDNA fragment encoding a VP7 protein with a G2 serotype derived from human rotavirus, is an international depository institution under the Budapest Convention on International Approval of Microbial Deposits, dated February 8, 2001. It was deposited with Researcher Gene Bank (KCTC) under accession number KCTC 0948BP.

Example 9

Instead of pGEM-VP7 (KCTC 0946BP) comprising a cDNA fragment encoding a VP7 protein with a G1 serotype derived from human rotavirus in Example 7, the VP7 protein with a G3 serotype derived from human rotavirus was encoded. Except for using pCR2.1-VP7 (KCTC 0949BP) containing a cDNA fragment having a cDNA sequence represented by SEQ ID NO: 6, Rota having a G3 serotype was carried out in the same manner as in Example 7. It can be seen that the viral VP7 protein is expressed.

PCR2.1-VP7, which contains a cDNA fragment encoding a VP7 protein with a G3 serotype derived from the human rotavirus, is an international depository organization under the Budapest Convention on International Approval of Microbial Deposits, dated February 8, 2001. It was deposited with Researcher Gene Bank (KCTC) under accession number KCTC 0949BP.

<Example 10>

Instead of pGEM-VP7 (KCTC 0946BP) comprising a cDNA fragment encoding a VP7 protein with a G1 serotype derived from human rotavirus in Example 7, the VP7 protein with a G4 serotype derived from human rotavirus was encoded. Except for using pCR2.1-VP7 (KCTC 0950BP) containing a cDNA fragment having a cDNA nucleotide sequence represented by SEQ ID NO: 7, Rota having a G4 serotype in the same manner as in Example 7 It can be seen that the viral VP7 protein is expressed.

PCR2.1-VP7, which contains a cDNA fragment encoding a VP7 protein with a G4 serotype derived from human rotavirus, is an international depository institution under the Budapest Convention on International Approval of Microbial Deposits, dated February 8, 2001. It was deposited with Researcher Gene Bank (KCTC) under accession number KCTC 0950BP.

The method for mass production of rotavirus construct protein and vaccine composition containing the protein as an active ingredient in plant cells using the genetic recombination technique of the present invention is low in production cost, less necessity of purification, and from edible plants As it is produced, the viral component proteins can be isolated and made into vaccines with known additives or used as food vaccines or oral vaccines by ingesting the plant itself. In particular, when induction of mucosal immune response is important, such as rotavirus, oral vaccines can induce desirable mucosal and systemic immune responses, which is useful for the manufacture of vaccines for the treatment or prevention of rotavirus-related diseases. Can be used. In addition, the vaccine is easier to produce than the production using other culture systems and is effective in terms of efficiency of transportation and storage.

Claims (25)

1) preparing an expression plasmid comprising cDNA fragments encoding rotavirus construct proteins regulated by a promoter regulating plant specific expression and additionally comprising a selection marker gene; 2) introducing the expression plasmid of step 1 into plant cells; 3) inducing callus formation from the plant cell of step 2; 4) culturing the callus of step 3; And 5) A method for producing a recombinant rotavirus construct protein having antigenicity from plant cells, comprising recovering the expressed protein from the cDNA encoding the rotavirus construct protein in the culture of step 4. The rotavirus of claim 1, wherein the rotavirus of step 1 is a rotavirus derived from a human body or a rotavirus derived from an animal including a cow, a rhesus monkey, a pig, a dog, a cat, a bird, a mouse, and the like. A method of producing recombinant rotavirus construct protein having antigenicity from plant cells. The method of claim 1, wherein the rotavirus construct protein of step 1 is VP2, VP4, VP6 or VP7. According to claim 1, wherein the cDNA fragment encoding the rotavirus constituent protein has a cDNA base sequence represented by SEQ ID NO: 1 encoding VP2 derived from human rotavirus, having antigenicity from plant cells Method of producing recombinant rotavirus construct protein. According to claim 1, wherein the cDNA fragment encoding the rotavirus constituent protein has a cDNA sequence represented by SEQ ID NO: 2 encoding VP4 derived from human rotavirus, having antigenicity from plant cells Method of producing recombinant rotavirus construct protein. The cDNA fragment encoding the rotavirus constituent protein has a cDNA sequence represented by SEQ ID NO: 3 encoding VP6 derived from human rotavirus, and has antigenicity from plant cells. Method of producing recombinant rotavirus construct protein. The plant cell according to claim 1, wherein the cDNA fragment encoding the rotavirus construct protein has a cDNA sequence represented by SEQ ID NO: 4 encoding VP7 having a G1 serotype derived from human rotavirus. A method for producing a recombinant rotavirus construct protein having antigenicity. The plant cell according to claim 1, wherein the cDNA fragment encoding the rotavirus construct protein has a cDNA sequence represented by SEQ ID NO: 5 encoding VP7 having a G2 serotype derived from human rotavirus. A method for producing a recombinant rotavirus construct protein having antigenicity. The plant cell according to claim 1, wherein the cDNA fragment encoding the rotavirus construct protein has a cDNA sequence represented by SEQ ID NO: 6 encoding VP7 having a G3 serotype derived from human rotavirus. A method for producing a recombinant rotavirus construct protein having antigenicity. The plant cell according to claim 1, wherein the cDNA fragment encoding the rotavirus construct protein has a cDNA sequence represented by SEQ ID NO: 7 encoding VP7 having a G4 serotype derived from human rotavirus. A method for producing a recombinant rotavirus construct protein having antigenicity. The method of claim 1, wherein the promoter regulating the plant specific expression of step 1 is a cassava leaf vein mosaic virus promoter, ubiquitin promoter, CaMV 35S promoter, actin promoter, PG promoter or endosperm-specific promoter A method for producing a recombinant rotavirus construct protein having antigenicity from plant cells. The method of claim 1, wherein the selection marker gene of step 1 is a neomycin phosphotransferase II (NPT II) gene, hygromycin phosphotransferase gene, phosphinothricin acetyltransferase gene or dihydrofolate reductase A method for producing a recombinant rotavirus construct protein having antigenicity from plant cells, characterized in that it is a gene. The expression plasmid of claim 1, wherein the expression plasmid of step 1 comprises the right border of T-DNA, noparin synthase promoter, neomycin phosphotransferase II (NPT II) gene, noparin transcription termination site, cassava lobe mosaic virus promoter, A method of producing a recombinant rotavirus construct protein having antigenicity from plant cells, characterized in that it is a plasmid comprising a cDNA fragment encoding a rotavirus construct protein, a noparin transcription termination site, and a left boundary of T-DNA in that order. The plasmid pILTAB357-VP6 represented by the cleavage map of FIG. 1A, the plasmid pILTAB357-VP2 represented by the cleavage map of FIG. 1B, and the plasmid pILTAB357-VP4 represented by the cleavage map of FIG. 1C. Or plasmid pILTAB357-VP7 represented by the cleavage map of FIG. 1D, wherein the recombinant rotavirus construct protein having antigenicity is derived from plant cells. The method of claim 1, wherein the plant cell introduction of the expression plasmid of step 2 is characterized by transforming Agrobacterium with a recombinant binary vector or coin integration vector, and co-culturing the transformed Agrobacterium with plant cells. , A method for producing a recombinant rotavirus construct protein having antigenicity from plant cells. 16. The method of claim 15, wherein Agrobacterium is Agrobacterium tumafaciens or Agrobacterium lyzogenes. The method of claim 1, wherein the plant cell introduction of the expression plasmid of step 2 is characterized by polyethylene glycol precipitation (PEG-mediated uptake), microparticle bombardment or electroporation (electroporation). A method for producing a recombinant rotavirus construct protein having antigenicity from plant cells. 2. The recombinant rotavirus having antigenicity from plant cells according to claim 1, wherein the plant cells of step 2 are edible plant cells comprising tomatoes, lettuce, cabbage, bananas, potatoes, radish, rice, and the like. Method of producing construct protein. 19. A recombinant rotavirus construct protein produced by the method of any one of claims 1-18. A rotavirus like particle prepared by folding and binding the recombinant rotavirus construct proteins VP2, VP6, VP4 and VP7 produced by the method of any one of claims 1 to 18. A rotavirus like particle prepared by folding and binding the recombinant rotavirus construct proteins VP2, VP4 and VP7 produced by the method of any one of claims 1-18. A vaccine composition for the treatment or prophylaxis of rotavirus-related diseases comprising a recombinant rotavirus construct protein produced by the method of any one of claims 1 to 18 as an active ingredient. The method of claim 22, wherein the recombinant rotavirus construct protein is in a group consisting of VP2, VP4, VP6, VP7 with G1 serotype, VP7 with G2 serotype, VP7 with G3 serotype and VP7 with G4 serotype A vaccine composition for treating or preventing rotavirus-related diseases, characterized in that any one or more selected. The vaccine composition of claim 22, wherein the recombinant rotavirus construct protein is present in the form of a transgenic plant or extract thereof re-differentiated from the plant cell itself or from the plant cell. 23. The vaccine composition according to claim 22, which is ingested orally or for food for immunity.