KR20170037708A - Method for Producing and Purifying Virus-Like Particle of Rotavirus using Yeast strain - Google Patents

Abstract

The present invention relates to a method for producing and refining virus-like particles of rotavirus by using a yeast cell strain. In the present invention, major structure proteins (VP2, VP6, VP7) of rotavirus in a yeast-based system are simultaneously expressed, so triple-layered autonomous-assembled virus-like particles are produced. An expression induction condition is optimized, so an expression amount of the recombinant proteins is increased. Also, excellent refining efficiency has been confirmed according to a change in the composition of a cell pulverizing buffer solution (cell lysis buffer) capable of improving the solubility of recombinant proteins. Thus, it is expected that the method can be variously used in preventing and treating rotavirus infectious diseases.

Description

FIELD OF THE INVENTION [0001] The present invention relates to a method for producing virus-like particles of rotavirus using a yeast cell line,

The present invention relates to a method for producing and purifying virus-like particles of rotavirus using a yeast cell line.

Human Rotavirus (HRV) is a major virus causing infantile diarrhea in infants and young children. It accounts for at least 40% of the world's diarrheal diseases every year, Respectively. According to a report by the US Centers for Disease Control and Prevention (CDC), rotavirus diarrhea is estimated to cost $ 10 million a year in the US, and 70% of infants and children with acute enteritis are receiving rotavirus infection . Therefore, the World Health Organization (WHO) has been developing vaccines against rotavirus as a top priority in developing countries to reduce the incidence of rotavirus and to reduce medical costs in developed countries.

Rotavirus has no coating and consists of a triple layered protein capsid of outer capsid, inner capsid and core protein in the form of a regular icosahedral with a diameter of 75 nm. It consists of 11 segments. Each segment encodes one of six structural proteins (VP1, VP2, VP3, VP4, VP6, VP7) and six nonstructural proteins (NSP 1-6). Rotavirus is classified into 7 groups according to the antigenicity of VP6. Group A, which is the most common group in the world, is divided into G type (Glycoprotein type) by VP7, immunological protein, and P type -sensitive type). Currently, 23 types of G-type and 32 types of P-type are reported. In humans, nine serotypes such as G1-G4, G6, G8-G10 and G12 and P [3], P [4], P [6], P [8] The genotypes are infectious, and each serotype is not cross - protective.

Due to the characteristics of rotavirus that does not cross-protect all serotypes, the development of an effective rotavirus vaccine capable of protecting infections against each serotype has been required. To date, oral live attenuated vaccines and animal-human recombinant vaccines have been used but have not shown adequate protection against other serotype infections. Rotashield, developed by Wyeth-Ayerst, USA, It is a 4-valent live attenuated vaccine containing the highest G serotype (G1 to G4) and has been approved by the Food and Drug Administration (FDA). Accordingly, interest in virus-like particles (VLPs) as a new vaccine material has been increasing, and various studies have been made. At present, Escherichia coli expression system or baculovirus expression system using insect cells is mainly used. However, prokaryotic cell expression systems such as Escherichia coli have endotoxin, In case of using insect cells, it is not possible to obtain a satisfactory production effect due to the risk of contamination during the culturing process, difficulty of purification, high production cost, and low yield, and in the application to rotavirus vaccine There was a limit.

Thus, a method for stably and efficiently producing virus-like particles (VLP) has been studied (Korean Patent Laid-open Publication No. 10-2015-0045191).

Disclosure of the Invention The present invention was conceived to solve the above problems, and the present inventors produced viral-like particles simultaneously expressing virus structural proteins VP2, VP6 and VP7 of rotavirus in a yeast-based system. In addition, as a result of studying the culture and purification conditions that can increase the production yield in the virus-like particle production process of rotavirus, it was found that the increase of the protein expression amount and the purification efficiency according to the composition of the cell disruption buffer And the present invention has been completed on the basis thereof.

Accordingly, an object of the present invention is to provide a method for producing a rotavirus structural protein, comprising the steps of: a) transforming a yeast cell to express a rotavirus structural protein; b) culturing the transfected yeast cells in a medium supplemented with methanol; c) dissolving the cultured yeast cells to obtain a cell lysate; And d) separating and purifying virus-like particles of rotavirus from the cell lysate. The present invention also provides a method for producing and purifying virus-like particles (VLP) of Rotavirus.

Another object of the present invention is to provide virus-like particles of rotavirus produced and purified by the above method.

Still another object of the present invention is to provide an immunogenic composition comprising virus-like particles of the rotavirus.

However, the technical problem to be solved by the present invention is not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.

In order to accomplish the above object, the present invention provides a method for producing a rotavirus structural protein, comprising the steps of: a) transforming a yeast cell to express a rotavirus structural protein; b) culturing the transfected yeast cells in a medium supplemented with methanol; c) dissolving the cultured yeast cells to obtain a cell lysate; And d) separating and purifying virus-like particles of rotavirus from the cell lysate. The present invention also provides a method for producing and purifying virus-like particles (VLP) of Rotavirus.

In one embodiment of the present invention, the step a) comprises the steps of: a pPIC6-CAU200 VP2 vector of SEQ ID NO: 1; a pPICZ-CAU200 VP6 vector of SEQ ID NO: 2; and a pPIC3.5K-CAU200 VP7 Yeast cells can be transformed into vectors.

In another embodiment of the present invention, the yeast in step a) may be Pichia pastoris .

In another embodiment of the present invention, the step (b) may be carried out by adding methanol at a concentration of 0.5 to 1.5% for 24 to 96 hours.

In another embodiment of the present invention, the step b) may be carried out by adding methanol at a concentration of 1 to 1.5% for 24 to 48 hours.

As a further embodiment, the step c) may be dissolved in cell disruption buffer containing _{Tris-HCl, NaCl, CaCl 2} , EDTA, PMSF, glycerol, and Tween20.

The present invention provides virus-like particles of rotavirus produced and purified by the above method.

In one embodiment of the present invention, the virus-like particle can simultaneously express the rotavirus structural proteins VP2, VP6, and VP7.

The present invention provides an immunogenic composition comprising virus-like particles of said rotavirus.

The present invention provides a method for preventing or treating rotavirus infection comprising administering the virus-like particles of the rotavirus to a subject.

The present invention provides a method for preventing or treating rotavirus infection of virus-like particles of rotavirus.

According to the present invention, self-assembled virus-like particles of a triple layer were produced by simultaneously expressing the structural proteins VP2, VP6, and VP7 of the rotavirus in the yeast-based system, and the expression of the recombinant proteins In addition, the purification efficiency could be improved by using a cell lysis buffer that can increase the solubility of the recombinant proteins. Accordingly, the present invention solves the problem of low yield and high production cost, which have been pointed out in the production of virus-like particles, and is expected to be effectively utilized in the research and development of new vaccines against rotavirus .

1 schematically shows a process of transforming human rotavirus structural protein genes (VP2, VP6, VP7) isolated from patients suffering from Rotavirus infection so that they can be simultaneously expressed in Pichia pastoris to be.
FIG. 2 shows the results of colony PCR using AOX1 primer and primers specific for each gene (VP2, VP6, and VP7) for the Pichia pastoris colony transformed with all of the human rotavirus structural protein genes (VP2, VP6 and VP7) (M lane is a 1kb DNA size marker, lane 1 is a universal AOX1 primer, VP2 specific primer, lane 2 is a VP6 specific primer, and lane 3 is a VP7 specific primer).
Figure 3a shows the results of western blotting of expression of rotavirus structural proteins (VP2, VP6, VP7) in the supernatant using rat monoclonal anti-His antibodies (lane 1 shows the expression of Pichia pastoris negative In the control, the lane 2 was recombinant VP2, the lane 3 was recombinant VP6, the lane 4 was recombinant VP7, the lane 5 was expressed VP2 / VP6 / VP7 and the lane was a Thermo Scientific Spectra Multicolor Broad Range Protein Ladder ).
Figure 3b shows the results of western blotting of expression of rotavirus structural proteins (VP2, VP6, VP7) in the supernatant using polyclonal rotavirus Wa antiserum of rabbits (lane 1 is the Pichia pastoris negative control , Recombinant VP2 in lane 2, recombinant VP6 in lane 3, recombinant VP7 in lane 4, VP2 / VP6 / VP7 in lane 5 and M lane in Thermo Scientific Spectra Multicolor Broad Range Protein Ladder .).
FIG. 4 shows the result of observing virus-like particles of purified rotavirus with a transmission electron microscope.
FIG. 5 shows the result of measuring antibody titers of mouse immunity sera against rotavirus Wa antigen to confirm the immunogenicity of virus-like particles of recombinant rotavirus (() Of the recombinant antigen rotavirus VLP antibody, and (o) is the antibody of the rat serum injected only with the adjuvant not mixed with the antigen as the negative control, (5) represents the antibody value of the mouse immunized serum of the recombinant antigen rotavirus VLP, Of the mice tested).
FIG. 6 shows the results of Western blotting to determine the expression level of rotavirus structural proteins according to the concentration of methanol added and the induction time of the added methanol, and quantification of the expression level of each recombinant protein using the Image J program.
FIG. 7 shows the results of measurement of the degree of expression of each recombinant protein and quantification using an Image J program in order to compare the solubility of the protein in the supernatant protein according to the difference in the cell disruption buffer.

The present inventors produced virus-like particles expressing VP2, VP6, and VP7, which are structural proteins of rotavirus, using Pichia pastoris as an expression strain, and found that the excellent immunogenicity of the virus-like particles Respectively. In addition, the present inventors completed the present invention on the basis of the increase in the amount of rotavirus structural protein expression and the difference in solubility of the supernatant protein depending on the methanol concentration and induction time.

Accordingly, the present invention provides a method for producing and purifying virus-like particles (VLPs) of Rotavirus comprising the following steps.

a) transforming yeast cells to express a rotavirus structural protein;

b) culturing the transfected yeast cells in a medium supplemented with methanol;

c) dissolving the cultured yeast cells to obtain a cell lysate; And

d) separating and purifying virus-like particles of rotavirus from the cell lysate.

According to the method for producing and purifying virus-like particles of the present invention, virus structural proteins VP2, VP6 and VP7 of rotavirus can be simultaneously expressed in yeast cells, and the concentration of methanol, the induction time and the composition of cell- , Maximizing the production efficiency of virus-like particles, and is expected to be effectively utilized in research and development of new vaccines against rotavirus.

Hereinafter, each step of the method for producing and purifying virus-like particles according to the present invention will be described in detail.

In step a), the yeast cells are transformed to express the rotavirus structural protein.

In the present invention, 'Rotavirus' is a double helical RNA virus belonging to reoviridae having a diameter of 70 mm. The genome is composed of 11 gene segments, and the nucleus is surrounded by double capsid. To date, more than six structural proteins and four non-structural proteins have been known. There are four structural proteins (VP1, VP2, VP3, VP6) in the inner capsid and two structural proteins (VP4, VP7) in the outer capsid. Rotavirus is present in the form of rough particles with no outer capsids and smooth particles with outer capsids. Only outer particles are known to cause infection because smooth proteins are the structural proteins VP4 and VP7 that bind to specific receptors on the host cell membrane. In addition, up to now, seven serotypes have been isolated from humans, and infection by type 1 and type 3 is the most common. Because rotavirus does not cross-protect against all serotypes, it is necessary to develop an effective rotavirus vaccine that can protect against infection with each serotype. Thus, in the present invention, viral-like particles having a shape similar to that of the original virus were produced through simultaneous expression and self-assembly function of the major structural proteins of rotavirus, and gene replacement expression or simultaneous expression was easily performed in each serotype System. Specifically, it produced viral-like particles of a triple layer in which the rotavirus structural proteins VP2, VP6 and VP7 were simultaneously expressed. The virus-like particles were similar to natural infectious rotavirus, and were presented to immune cells in the body while maintaining the antigenic structure. Can induce an immune response, and has no virulence because it does not contain a virus gene necessary for virus multiplication.

As used herein, the term "transformation" refers to a process in which a DNA strand or plasmid having a heterologous gene of a different kind from that of the original cell is transfected into cells and binds to DNA originally present in the cell, It is a molecular biologic technique that changes traits. The transformation in the present invention means that the rotavirus structural protein gene is inserted into yeast cells. In general, transformation involves the insertion of a plasmid DNA capable of expressing a gene into a cell, and the insertion of a linear plasmid DNA into a genome of a host cell through a series of molecular biological reactions. In the present invention, a method of integrating the rotavirus structural protein gene into the genome of Pichia pastoris was used in consideration of the fact that it is more stable to insert into the genome than to remain in the form of plasmid DNA when differentiated into daughter cells .

In the present invention, the transformation is preferably carried out using the pPIC6-CAU200 VP2 vector represented by SEQ ID NO: 1, the pPICZ-CAU200 VP6 vector represented by SEQ ID NO: 2, and the pPIC3.5K-CAU200 VP7 vector represented by SEQ ID NO: 3, Into a cell. The rotavirus structural protein gene inserted into the yeast cells of the present invention was prepared by analyzing codons to optimize the expression in yeast cells and then changing the sequence accordingly. The VP2, VP6, and VP7 genes may preferably be SEQ ID NOS: 4 to 6, respectively, and homologues of the nucleotide sequences are included within the scope of the present invention. Specifically, the gene includes a nucleotide sequence having a sequence homology of 70% or more, preferably 80% or more, more preferably 90% or more, and most preferably 95% or more, with the nucleotide sequence of SEQ ID NO: 1 to 6 can do.

Further, in the present invention, the yeast cell may be preferably Pichia pastoris .

In step b), the transformed yeast cells in step a) are cultured in a medium supplemented with methanol. At this time, methanol is added to increase expression of rotavirus structural protein, and the culture is performed for a specific time. Specifically, the concentration of methanol may be 0.5 to 1.5%, the incubation time may be 24 to 96 hours, preferably, when the concentration of methanol is 1 to 1.5%, it may be cultured for 24 to 48 hours, But is not limited to.

In step c), the yeast cells cultured in step b) are lysed to obtain a cell lysate. At this time, a specific cell disruption buffer may be used for improving the yield of purification of virus-like particles. Specifically, a cell-destroying buffer A (Tris-HCl pH 7.5, NaCl, CaCl ₂ , EDTA (ethylenediaminetetraacetic acid), PMSF (phenylmethanesulfonyl fluoride), glycerol, Tween 20) But is not limited thereto.

In step d), virus-like particles of rotava are separated and purified from the cell lysate in step c). The separation and purification can be carried out preferably by centrifugation, but may be carried out without limitation by methods well known in the art capable of separating and purifying virus-like particles.

In one embodiment of the present invention, pPIC6-CAU200 VP2 vector, pPICZ-CAU200 VP6 vector, and pPIC3.5K-CAU200 VP7 vector were transformed by introduction into Pichia pastoris GS115 (HIS4) and transformed with the rotavirus structural protein VP2 , VP6, and VP7), and the virus-like particles were isolated and purified (see Examples 2 to 4). In another embodiment of the present invention, excellent immunogenicity of virus-like particles was confirmed in the group treated with virus-like particles of the rotavirus, and the expression level of rotavirus structural proteins was optimized by optimizing methanol concentration and induction time (Tris-HCl pH 7.5, NaCl, CaCl ₂ , EDTA, PMSF, glycerol, Tween 20) was used as a cell lysis buffer to increase the solubility of recombinant proteins It was confirmed that the purification efficiency of the virus-like particles can be increased (see Examples 5 to 7).

Accordingly, the present invention provides an immunogenic composition comprising a virus-like particle of Rotavirus.

As used herein, the term "immunogenicity" refers to the ability of a composition to elicit an immune response against a particular pathogen. The immune response is a cellular immune response that is primarily mediated by cytotoxic T-cells and cytokine-producing T-cells, or a humoral immune response that produces antibodies by activating B-cells after being mediated primarily by helper T- .

The immunogenic compositions of the present invention may further comprise a pharmaceutically acceptable carrier or adjuvant, in addition to virus-like particles of rotavirus. The carrier is used as a meaning including a diluent, a buffer, a preservative and the like. The carrier may include, but is not limited to, one or more components selected from the group consisting of excipients, disintegrants, binders and glidants known in the art. The adjuvant is a substance used for enhancing the immunogenicity of an antigen and is an adjuvant suitable for use in the present invention, and includes mineral salts such as Alum, aluminum hydroxide, aluminum phosphate and calcium phosphate; Surfactants and particulates such as nonionic block polymer surfactants (cholesterol), virosomes, saponins (Quil A, QS-21 and GPI-0100), proteosomes, immunostimulatory complexes, (Dimethyldioctadecylammonium bromide (DDA)), avidin, vitamin A, vitamin E; Bacterial products such as the RIBI Ribi Inc. cell wall skeleton of Mycobacterum phlei (Detox TM), muramyldipeptide (MDP) and tripeptide (MTP), monophosphate Bacillus Calmete-Guerin, heat-labile Escherichia coli enterotoxin, cholera toxin, trehalose dimicolate, CpG oligodeoxynucleotides; IL-12, IL-15, IL-18), granulocyte colony stimulating factor, dehydroepiandrosterone, 1,25-di Hydroxyvitamin D3; Polyanions such as dextran; Polyacrylic (e.g., polymethyl methacrylate, Carbopol 934P); Carriers such as tetanus toxide, dipteryatocide, cholera toxin B subunit, mutant heat-labile enterotoxin (rmLT) of enterotoxigenic E. coli, heat shock protein; Water-in-oil emulsions such as AMPHIGEN (R) (Hydronics, USA); And water-in-oil emulsions such as, for example, Freund's complete and incomplete adjuvants.

The immunogenic compositions of the present invention may be in any form known to those skilled in the art, for example, in the form of solutions and injections, but are not limited thereto. For liquids or injections, it may contain 10 to 40% of propylene glycol if necessary, and sodium chloride in an amount sufficient to prevent hemolysis (e.g., about 1%). The liquid or injectable solution may contain any diluents or buffers known in the art. The immunogenic compositions of the present invention may also be prepared immediately prior to use by preserving the rotavirus-like particles in a container such as a vial and adding a carrier or adjuvant, saline solution or the like necessary for the injection before use.

The immunogenic compositions of the invention may be administered by any means of administration known in the art. For example, the administration can be administered directly into an individual by intravenous, intramuscular, oral, transdermal, mucosal, intranasal, intratracheal or subcutaneous administration. The administration may be systemically or locally administered.

The immunogenic compositions of the invention may be administered in a therapeutically or prophylactically effective amount. The "therapeutically or prophylactically effective amount" means an amount required for relieving or eliminating symptoms to be treated or prevented, and may be appropriately selected in consideration of the severity of symptoms, sex, have.

In another aspect of the present invention, the present invention provides a method of preventing or treating rotavirus infection comprising administering the immunogenic composition to a subject. The term " individual "as used herein refers to a subject in need of treatment of a disease, and more specifically refers to a mammal such as a human or non-human primate, mouse, dog, cat, horse, do.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the following examples.

[Example]

Example 1. Experimental preparation

1-1. Isolation and codon optimization of rotavirus structural protein gene

Korean human rotavirus CAU200 strain (GenBank Attachment No .: KT207485, ACD50880, ACD50874) was isolated from patients with rotavirus infection. Subsequently, a gene encoding human rotavirus structural proteins (VP2, VP6, VP7) was codon optimized in GeneArt (Regensberg, Germany) to fit the expression system of the expression strain Pichia pastoris .

1-2. Preparation of yeast strains and recombinant plasmid vectors

As a strain expressing the rotavirus structural protein, Pichia pastoris GS115 (HIS4) was used. Also, in order to introduce VP2, VP6 or VP7, which is a rotavirus structural protein, into Pichia pastoris, the above structural protein genes are respectively added to pPIC6, pPICZ, or pPIC3.5K (Invitrogen, Carlsbad, CA, USA) Followed by subcloning. The pPIC6 and pPICZ plasmid vectors include Blastcidin (Invitrogen) and Zeocin (Invitrogen) as selective markers having functions in both Pichia palliatis and Escherichia coli, and the pPIC3.5K vector is Geneticin (Invitrogen) in Pichia pastoris. , And E. coli was designed to have kanamycin resistance. In addition, the gene inserted into the plasmid vector was confirmed by DNA sequencing.

Example 2. Transformation and confirmation using a recombinant plasmid vector

In this Example, the recombinant plasmid vector prepared in Example 1 was introduced into Pichia pastoris GS115 (HIS4) and transformed. The transformation process is schematically shown in FIG. Specifically, the pPIC6-CAU200 VP2 plasmid was linearized by treating with SacI (New England BioLab) restriction enzyme and transformed into Pichia pastoris GS115 strain using the Pichia EasyComp Kit (Invitrogen) according to the manufacturer's instructions. Transformed yeast cells were cultured for 3 to 4 days at 30 ° C in a YPDS agar medium containing 300 mg / ml of Blasticidin. The transformants inserted in the blasticidin-resistant colonies were subjected to colony polymerase chain reaction Polymerase chain reaction (PCR). In the second transformation, a transformant was prepared by inserting CAU 200 VP6 (pPICZ-CAU200 VP6) into Pichia pastoris GS115-cauVP2 into which VP2 was introduced, and the third transformation was carried out by adding VP2 and VP6 A transformant was prepared by inserting CAU200 VP7 (pPIC3.5K-CAU200 VP7) into the Kiapas strain GS115-cau26 (VP2 / VP6). Finally, GS115-cau267 with three rotavirus structural proteins (VP2, VP6, VP7) inserted therein was produced.

In order to confirm the expression of rotavirus structural proteins (VP2, VP6, VP7) in the transformed Pichia pastoris colonies, colony PCR was performed and electrophoresis was performed on 1.2% agarose gel. As a control group, AOX1 primer The primer sequences used in this Example are shown in Table 1 below.

As a result, as shown in Fig. 2, in the transformed Pichia pastoris colonies, VP2 of 2208 bp, VP6 of 773 bp, and VP7 structural protein of 782 bp were confirmed, and the recombinant plasmids (pPIC6-CAU200 VP2, pPICZ- (VP2, VP6, VP7) was introduced into an expression strain, Pichia pastoris GS115 (HIS4), through the expression plasmid pCIC200, CAU200 VP6 and pPIC3.5K-CAU200 VP7.

Example 3 Induction and Identification of Rotavirus Structural Protein

The positive transformant GS115-cau267 prepared in Example 2 was inoculated in 20 mL of YPD liquid medium (1% w / v yeast extract, 2% w / v peptone and 2% w / v dextrose) Under shaking culture for one day. The cultured cells were obtained by centrifugation at 5,000 rpm for 5 minutes at 4 ° C. The obtained cells were suspended in 300 mL of MGY liquid medium (1.34% w / v yeast nitrogen base, 0.05% w / v biotin and 1% v / v glycerol ), And then cultured with shaking at 30 ° C and 180 rpm at OD 600nm until reaching 2.0-6.0. The cultured cells were obtained by centrifugation and the obtained cells were suspended in 500 mL of MM liquid medium (1.34% w / v yeast nitrogen base, 0.05% w / v biotin and 0.5-1.5% v / v methanol) VP2, and VP7 proteins were expressed by shaking culturing at 30 DEG C and 180 rpm for 24-96 hours until 1.0. The cultured cells were centrifuged at 5,000 rpm for 5 minutes at 4 ° C to obtain supernatants and cell pellets. The obtained cell pellet was dissolved in cell disruption buffer A (10 mM Tris-HCl pH 7.5, 140 mM NaCl, 10 mM CaCl ₂ , 1 mM EDTA, 1 mM PMSF, 5% v / v glycerol, 0.01% v / v Tween 20) , USA) in a bead-beater (Biospec Products, USA) and shaken for 5 minutes to disrupt the cells. The lysate was centrifuged at 4,000 rpm at a rate of 12,000 xg for 30 minutes to obtain a clear supernatant.

In order to confirm the expression of rotavirus structural proteins (VP2, VP6, VP7) in viral-like particles present in the obtained supernatant, the protein in the supernatant was electrophoresed on 10% SDS-PAGE, Was transferred to an Immuno-BlotTM PVDF membrane (Bio-Rad), and the membrane was blocked with 5% skim milk. Monoclonal anti-His antibodies obtained from rats and polyclonal rotavirus Wa antiserum obtained from rabbits were used as primary antibodies and stored in 5% skim milk solution. The reaction was carried out for 1 hour using the primary antibody and reacted again with peroxidase-conjugated goat anti-rabbit IgG (1: 2000 dilution, Invitrogen) as a secondary antibody. Expressed proteins were identified by ImageQuant LAS4000 (GE Healthcare, USA) using ECL (electrochemiluminescence) technique.

As a result, as shown in Fig. 3, expression of rotavirus structural proteins VP2, VP6, and VP7 in rat monoclonal anti-His antibody (Fig. 3A) and rabbit polyclonal rotavirus Wa antiserum Respectively.

Example 4. Pure isolation and purification of virus-like particles

The cell lysate of Example 3 was subjected to ultrafast centrifugation at 12,000 rpm for 30 minutes to obtain a clear supernatant. 35% sucrose in TNC buffer (10 mM Tris-HCl, pH 7.5, 140 mM NaCl, 10 mM CaCl ₂ ) was added to the supernatant containing virus-like particles and the mixture was centrifuged at 30,000 rpm using a SW32Ti rotor (Beckman Coulter) For 120 minutes to obtain a virus-like particle precipitate. The resulting virus-like particle precipitate was suspended in 4 ml of TNC buffer, and TNC buffer containing CsCl dissolved at a concentration of 0.42 g / ml was added and centrifuged at 35,000 rpm for 18 hours using a SW41 Ti rotor (Beckman Coulter). After centrifugation, the two identified bands, the lower and upper bands, were recovered using a disposable syringe and dialysed with TNC buffer to remove CsCl.

Transmission electron microscopy analysis was performed to confirm the presence of the virus-like particles. Specifically, a purified rotavirus virus-like particle sample was placed on the surface of a 200 mesh copper grid coated with Formvar-carbon, and 30 seconds later, most of the solution was removed from the grid with filter paper. The uranyl acetate solution was placed on the grid and dyed for 30 seconds and dried. The samples were observed using a JEM1010 model (JEOL, Japan) in NICEM (National Instrumentation Center of Environmental Management, Seoul National University).

As a result, as shown in FIG. 4, virus-like particles stained with 1% uranyl acetate were confirmed through a transmission electron microscope.

Example 5. Identification of the immunity of virus-like particles

In this Example, animal models were used to determine the immunogenicity of virus-like particles of rotavirus prepared by the above method. 50 [mu] g of purified rotaviral virus-like particles were subcutaneously injected (Day 0) into 7-week-old Balb / c female rats with complete Freund's adjuvant, followed by incomplete pro- And boosted with an incomplete Freund's adjuvant. After 40 days, blood was collected from the heart, serum was separated, and antibody formation was confirmed by ELISA. As a control group, the antibody of the negative Balb / c female rat serum and the antigen Antibodies of negative Balb / c female rat sera injected with an immune response adjuvant were used. Specifically, the separated and purified rotavirus Wa antigen was prepared in a coating solution (0.1 M Na ₂ CO ₃ , 0.1 M NaHCO ₃ , pH 9.4) at a concentration of 0.1 μg per well, and then immersed in a 96-well immunoplate 0.0 > 4 C < / RTI > for one day. The coated plates were washed three times with 200 [mu] l PBS-T (1 x PBS with 0.05% Tween 20) and then blocked with 5% (w / v) skim milk. The immunized sera obtained from the rats was diluted step by step and then reacted at 37 DEG C for 1 hour. After washing, peroxidase-conjugated goat anti-rabbit IgG (diluted in PBS-T, containing 5% skim milk) was added in an amount of 100 μl per well, followed by reaction at 37 ° C for 1 hour. OPD (ortho-phenylenediamine) was added as a substrate solution, followed by reaction for 5 minutes, and 50 μl of 1M H ₂ SO ₄ was added to each well to stop the reaction. Thereafter, the absorbance was measured at 450 nm using an ELISA reader (NanoQuant) to quantify the antibody concentration (antibody value).

As a result, as shown in Fig. 5, antibody titer of significantly higher immunity sera was confirmed in the group treated with virus-like particles of rotavirus as compared with the control group. As a result, the virus-like particles of the rotavirus produced by the above method show high immunogenicity and thus can be utilized as an immunogenic composition.

Example 6. Confirmation of culture conditions for production of optimal virus-like particles

Based on the fact that the concentration of methanol per cent (v / v%) and the induction time are important factors in the production of recombinant protein in Pichia pastoris in this example, The differences in the expression levels of rotavirus structural proteins with the induction time were compared. Specifically, the final concentration was determined for each methanol content of 0.5, 0.75, 1.0, 1.5% (v / v), and cells were obtained every 24 hours and measured for 4 days. 100% methanol was added to each concentration to maintain the final concentration every 24 hours. Cell debris was removed by centrifugation at 5,000 rpm for 5 minutes at 4 ° C using buffer A. After centrifugation at 12,000xg for 10 minutes at 4 ° C, supernatants were collected and stored until use, and recombinant protein production was confirmed by Western blotting. For a precise quantitative comparison of the level of protein production, 10 ⁶ cells per condition were obtained to load the samples and the concentration of Western blot bands was compared using Image J software (Wayne Rasband, NIH, USA).

As a result, as shown in Fig. 6, the amount of expression-induced recombinant protein when cultured for 48 to 72 hours under 0.5 to 0.75% methanol addition or for 24 to 48 hours under 1 to 1.5% methanol addition conditions Respectively. Thus, it can be seen that the production of viral-like particles of rotavirus can be maximized in Pasteuris by increasing the expression amount of a recombinant protein containing rotavirus structural protein by controlling the concentration of methanol percent and induction time of expression.

Example 7. Confirmation of difference in solubility in supernatant depending on cell crush buffer difference

After the transformed cells are cultured, the resulting supernatant is used for purification, and if more recombinant proteins are dissolved in the supernatant, the production efficiency can be further increased. Therefore, in this example, based on the fact that the increase of the protein content in the supernatant is an important factor in the production of recombinant protein in the Pichia pastoris, the solubility of the protein in the supernatant protein according to the cell- Respectively. Specifically, the degree of expression of each recombinant protein was measured by Western blotting on the supernatant protein of the cell lysed with the TNC buffer commonly used and the cell lysate protein disrupted with the cell disruption buffer A, Lt; / RTI >

As a result, as shown in FIG. 7, the amount of protein expressed in the supernatant lysed with cell-disrupting buffer A was increased by more than 2 times as compared with the supernatant lysed with TNC buffer, and the amount of precipitated protein was increased from 42% to 25% As shown in Fig. Thus, it can be seen that, by using the cell disruption buffer A, the purification efficiency can be increased and the production of virus-like particles of rotavirus can be maximized in Pasteur.

It will be understood by those skilled in the art that the foregoing description of the present invention is for illustrative purposes only and that those of ordinary skill in the art can readily understand that various changes and modifications may be made without departing from the spirit or essential characteristics of the present invention. will be. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive.

<110> Chung-Ang University Industry-Academy Cooperation Foundation <120> Method for Producing and Purifying Virus-Like Particle of          Rotavirus using Yeast strain <130> CAU20151894KR_MP15-100 <160> 10 <170> KoPatentin 3.0 <210> 1 <211> 6031 <212> DNA <213> Artificial Sequence <220> <223> pPIC6-CAU200 VP2 <400> 1 agatctaaca tccaaagacg aaaggttgaa tgaaaccttt ttgccatccg acatccacag 60 gtccattctc acacataagt gccaaacgca acaggagggg atacactagc agcagaccgt 120 tgcaaacgca ggacctccac tcctcttctc ctcaacaccc acttttgcca tcgaaaaacc 180 agcccagtta ttgggcttga ttggagctcg ctcattccaa ttccttctat taggctacta 240 acaccatgac tttattagcc tgtctatcct ggcccccctg gcgaggttca tgtttgttta 300 tttccgaatg caacaagctc cgcattacac ccgaacatca ctccagatga gggctttctg 360 agtgtggggt caaatagttt catgttcccc aaatggccca aaactgacag tttaaacgct 420 gtcttggaac ctaatatgac aaaagcgtga tctcatccaa gatgaactaa gtttggttcg 480 ttgaaatgct aacggccagt tggtcaaaaa gaaacttcca aaagtcggca taccgtttgt 540 cttgtttggt attgattgac gaatgctcaa aaataatctc attaatgctt agcgcagtct 600 ctctatcgct tctgaacccc ggtgcacctg tgccgaaacg caaatgggga aacacccgct 660 ttttggatga ttatgcattg tctccacatt gtatgcttcc aagattctgg tgggaatact 720 gctgatagcc taacgttcat gatcaaaatt taactgttct aacccctact tgacagcaat 780 atataaacag aaggaagctg ccctgtctta aacctttttt tttatcatca ttattagctt 840 actttcataa ttgcgactgg ttccaattga caagcttttg attttaacga cttttaacga 900 caacttgaga agatcaaaaa acaactaatt attcgaaacg aggaattcat ggcttacaga 960 aagagaggtg ctaagagaga ggacttgcca caacagaacg agagattgca agagaaagag 1020 atcgagaaca acactgacgt tactatggaa aacaagaaca agaacaaaaa caacaataga 1080 aagcagcagt tgtccgacaa ggttttgtcc cagaaagaag agatcatcac tgacgttcag 1140 gacgacatca agatcgctga cgaagttaag aagtcctcca aagaagagtc caagcagttg 1200 ttggagatct tgaaaacaaa agaggaccac cagaaagagg ttcagtacga gatcttgcaa 1260 aagactatcc caactttcga gccaaaagag tccatcttga agaagttgga ggacatcaag 1320 ccagagcagg ctaagaaaca gatgaagttg ttcagaatct tcgagccaag acagttgcca 1380 atctacagag ctaacggtga gaaagaattg agaaacagat ggtactggaa gttgaagaaa 1440 gacactttgc cagacggtga ctacgacgtt agagagtact tcttgaactt gtacgaccag 1500 atcttgatcg agatgccaga ctacttgttg ttgaaggaca tggctgttga gaacaagaac 1560 tccagagatg ctggtaaggt tgttgactcc gagactgcta gaatctgtga cgctattttc 1620 caggacgaag agactgaggg tgttattaga agattcattg ctgacatgag acagcaggtt 1680 caggctgaca gaaacatcgt taactaccca tccatcttgc acccaatcga ccacgctttc 1740 aacgagtact ttttgaacca ccagttggtt gagccattga acaacgagat catcttcaac 1800 tacatccctg agagaatcag aaacgacgtt aattacatct tgaacatgga catgaacttg 1860 ccatccactg ctagatatat cagaccaaac ttgttgcagg acagattgaa cttgcacgac 1920 aacttcgaat ccttgtggga cactatcact acttccaact acatcttggc tagatccgtt 1980 gttccagact tgaaagagaa agagttggtt tccactgagg ctcagatcca gaagatgtcc 2040 caagacttgc aattggaggc tttgactatc cagtccgaga ctcaattctt ggctggtatt 2100 aactcccagg ctgctaacga ctgtttcaag actttgatcg ctgctatgtt gtcccagaga 2160 actatgtcct tggacttcgt tactacaaac tacatgtcct tgatctccgg tatgtggttg 2220 ttgactgtta tcccaaacga catgttcttg agagaatcct tggttgcttg tgagttggct 2280 atcatcaaca ctatcgttta cccagctttc ggtatgcaga gaatgcacta cagaaacggt 2340 gacccacaaa ctccattcca gattgctgag cagcagatcc agaacttcca agttgctaac 2400 tggttgcact tcatcaacaa caacagattc agacaggttg ttatcgacgg tgttttgaac 2460 cagactttga acgacaacat cagaaacggt caggttatca accagttgat ggaagctttg 2520 atgcagttgt ccagacagca gttcccaact atgccagttg actacaagag atccatccag 2580 agaggtatct tgttgttgtc caacagattg ggtcagttgg ttgacttgac tagacttttg 2640 tcctacaact acgagacttt gatggcttgt atcactatga acatgcagca cgttcagact 2700 ttgactactg agagattgca gttgacttcc gttacttcct tgtgtatgtt gatcggtaac 2760 actactgtta ttccatcccc acagactttg ttccactact acaacgttaa tgttaacttc 2820 cactccaact acaacgagag aatcaacgac gctgttgcta tcatcactgc tgctaataga 2880 ttgaacttgt accaaaagaa gatgaagtcc atcgttgagg atttcttgaa gagattgcag 2940 atcttcgacg ttccaagagt tccagacgac cagatgtaca gattgagaga cagattgaga 3000 cttttgccag ttgagagaag aagattggac atcttcaact tgatcttgat gaacatggaa 3060 cagatcgaga gagcttccga caagattgct cagggtgtta tcattgctta cagagatatg 3120 cagttggagc gtgatgagat gtacggtttc gttaacattg ctagaaactt ggacggttac 3180 cagcagatca acttggaaga gttgatgaga actggtgact acggtcagat cactaacatg 3240 tggttgaaca accagccagt tgctttggtt ggtgctttgc ctttcgttac tgactcctcc 3300 gttatctcct tgatcgctaa gttggacgct actgttttcg ctcagatcgt taagttgaga 3360 aagtttgaca ctttgaagcc aatcttgtac aaaatcaact ccgactccaa cgacttctac 3420 ttggttgcta actacgactg gatcccaact tccactacta aggtttacaa gcaggttcca 3480 cagccattcg acttcagagc ttctatgcac atgttgactt ccaacttgac tttcactgtt 3540 tactccgact tgttgtcctt cgtttccgct gacactgttg agcctattaa cgctatcgct 3600 ttcgacaaca tgagaatcat gaacgagttg ctcgagccgc ggcggccgcc agcttacgta 3660 gaacaaaaac tcatctcaga agaggatctg aatagcgccg tcgaccatca tcatcatcat 3720 cattgagttt gtagccttag acatgactgt tcctcagttc aagttgggca cttacgagaa 3780 gaccggtctt gctagattct aatcaagagg atgtcagaat gccatttgcc tgagagatgc 3840 aggcttcatt tttgatactt ttttatttgt aacctatata gtataggatt ttttttgtca 3900 ttttgtttct tctcgtacga gcttgctcct gatcagccta tctcgcagct gatgaatatc 3960 ttgtggtagg ggtttgggaa aatcattcga gtttgatgtt tttcttggta tttcccactc 4020 ctcttcagag tacagaagat taagtgagac cttcgtttgt gcggatcccc cacacaccat 4080 agcttcaaaa tgtttctact ccttttttac tcttccagat tttctcggac tccgcgcatc 4140 gccgtaccac ttcaaaacac ccaagcacag catactaaat tttccctctt tcttcctcta 4200 gggtgtcgtt aattacccgt actaaaggtt tggaaaagaa aaaagagacc gcctcgtttc 4260 tttttcttcg tcgaaaaagg caataaaaat ttttatcacg tttctttttc ttgaaatttt 4320 tttttttagt ttttttctct ttcagtgacc tccattgata tttaagttaa taaacggtct 4380 tcaatttctc aagtttcagt ttcatttttc ttgttctatt acaacttttt ttacttcttg 4440 ttcattagaa agaaagcata gcaatctaat ctaaggggcg gtgttgacaa ttaatcatcg 4500 gcatagtata tcggcatagt ataatacgac aaggtgagga actaaaccat ggccaagcct 4560 ttgtctcaag aagaatccac cctcattgaa agagcaacgg ctacaatcaa cagcatcccc 4620 atctctgaag actacagcgt cgccagcgca gctctctcta gcgacggccg catcttcact 4680 ggtgtcaatg tatatcattt tactggggga ccttgtgcag aactcgtggt gctgggcact 4740 gctgctgctg cggcagctgg caacctgact tgtatcgtcg cgatcggaaa tgagaacagg 4800 ggcatcttga gcccctgcgg acggtgccga caggtgcttc tcgatctgca tcctgggatc 4860 aaagccatag tgaaggacag tgatggacag ccgacggcag ttgggattcg tgaattgctg 4920 ccctctggtt atgtgtggga gggctaagca cttcgtggcc gaggagcagg actgacacgt 4980 ccgacggcgg cccacgggtc ccaggcctcg gagatccgtc ccccttttcc tttgtcgata 5040 tcatgtaatt agttatgtca cgcttacatt cacgccctcc ccccacatcc gctctaaccg 5100 aaaaggaagg agttagacaa cctgaagtct aggtccctat ttattttttt atagttatgt 5160 tagtattaag aacgttattt atatttcaaa tttttctttt ttttctgtac agacgcgtgt 5220 acgcatgtaa cattatactg aaaaccttgc ttgagaaggt tttgggacgc tcgaaggctt 5280 taatttgcaa gctggagacc aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta 5340 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa 5400 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc 5460 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt 5520 ccgcctttct cccttcggga agcgtggcgc tttctcaatg ctcacgctgt aggtatctca 5580 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg 5640 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat 5700 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta 5760 cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta tttggtatct 5820 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac 5880 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa 5940 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa 6000 actcacgtta agggattttg gtcatgagat c 6031 <210> 2 <211> 4488 <212> DNA <213> Artificial Sequence <220> <223> pPICZ-CAU200 VP6 <400> 2 agatctaaca tccaaagacg aaaggttgaa tgaaaccttt ttgccatccg acatccacag 60 gtccattctc acacataagt gccaaacgca acaggagggg atacactagc agcagaccgt 120 tgcaaacgca ggacctccac tcctcttctc ctcaacaccc acttttgcca tcgaaaaacc 180 agcccagtta ttgggcttga ttggagctcg ctcattccaa ttccttctat taggctacta 240 acaccatgac tttattagcc tgtctatcct ggcccccctg gcgaggttca tgtttgttta 300 tttccgaatg caacaagctc cgcattacac ccgaacatca ctccagatga gggctttctg 360 agtgtggggt caaatagttt catgttcccc aaatggccca aaactgacag tttaaacgct 420 gtcttggaac ctaatatgac aaaagcgtga tctcatccaa gatgaactaa gtttggttcg 480 ttgaaatgct aacggccagt tggtcaaaaa gaaacttcca aaagtcggca taccgtttgt 540 cttgtttggt attgattgac gaatgctcaa aaataatctc attaatgctt agcgcagtct 600 ctctatcgct tctgaacccc ggtgcacctg tgccgaaacg caaatgggga aacacccgct 660 ttttggatga ttatgcattg tctccacatt gtatgcttcc aagattctgg tgggaatact 720 gctgatagcc taacgttcat gatcaaaatt taactgttct aacccctact tgacagcaat 780 atataaacag aaggaagctg ccctgtctta aacctttttt tttatcatca ttattagctt 840 actttcataa ttgcgactgg ttccaattga caagcttttg attttaacga cttttaacga 900 caacttgaga agatcaaaaa acaactaatt attcgaaacg aggaattcat ggaagttttg 960 tactcattgt ccaagacttt gaaggacgct agagacaaga tcgttgaggg tactttgtac 1020 tccaacgttt ccgacttgat ccagcagttc aaccagatga tcgttactat gaacggtaac 1080 gacttccaga ctggtggtat cggtaacttg ccaatcagaa actggacttt cgacttcggt 1140 ttgttgggta ctactttgtt gaacttggac gctaactacg ttgagactgc tagaactact 1200 atcgagtact tcattgactt catcgacaac gtttgtatgg acgagatcgc tagagaatcc 1260 cagagaaacg gtgttgctcc acaatctgag gctttgagaa agttggctgg tatcaagttc 1320 aagagaatca acttcaacaa ctcctccgag tacatcgaga actggaactt gcagaacaga 1380 aggcagagaa ctggtttcgt tttccacaag ccaaacatct tcccttactc cgcttcattc 1440 actttgaaca gatctcagcc aatgcacgac aacttgatgg gtactatgtg gttgaacgct 1500 ggttccgaga ttcaggttgc tggtttcgat tactcctgtg ctttgaacgc tccagctaac 1560 attcaacagt tcgagcacat cgttcagttg cgtagagctt tgactactgc tactatcact 1620 ttgttgccag acgctgagag attctcattc ccaagagtta tcaactccgc tgacggtgct 1680 actacatggt tcttcaaccc aatcatcctt agaccaaaca acgttgaggt tgagttcttg 1740 ttgaacggtc agatcatcaa cacttaccag gctagattcg gtactattat cgctagaaac 1800 ttcgacacta tcagattgtc cttccagttg atgaggccac caaacatgac tccagctgtt 1860 aacgctttgt tcccacaagc tcaaccattc caacaccacg ctactgttgg tttgactttg 1920 agaatcgagt ccgctgtttg tgagtccgtt ttggctgatg ctaacgagac tttgttggct 1980 aacgttactg ctgttagaca agagtacgct atcccagttg gtccagtttt tccaccaggt 2040 atgaactgga ctgagttgat cactaactac tccccatcca gagaggacaa cttgcagaga 2100 gtttcactg ttgcttccat cagatccatg ttgatcaagc tcgagccgcg gcggccgcca 2160 gcttacgtag aacaaaaact catctcagaa gaggatctga atagcgccgt cgaccatcat 2220 catcatcatc attgagtttg tagccttaga catgactgtt cctcagttca agttgggcac 2280 ttacgagaag accggtcttg ctagattcta atcaagagga tgtcagaatg ccatttgcct 2340 gagagatgca ggcttcattt ttgatacttt tttatttgta acctatatag tataggattt 2400 tttttgtcat tttgtttctt ctcgtacgag cttgctcctg atcagcctat ctcgcagctg 2460 atgaatatct tgtggtaggg gtttgggaaa atcattcgag tttgatgttt ttcttggtat 2520 ttcccactcc tcttcagagt acagaagatt aagtgagacc ttcgtttgtg cggatccccc 2580 acacaccata gcttcaaaat gtttctactc cttttttact cttccagatt ttctcggact 2640 ccgcgcatcg ccgtaccact tcaaaacacc caagcacagc atactaaatt ttccctcttt 2700 cttcctctag ggtgtcgtta attacccgta ctaaaggttt ggaaaagaaa aaagagaccg 2760 cctcgtttct ttttcttcgt cgaaaaaggc aataaaaatt tttatcacgt ttctttttct 2820 tgaaattttt ttttttagtt tttttctctt tcagtgacct ccattgatat ttaagttaat 2880 aaacggtctt caatttctca agtttcagtt tcatttttct tgttctatta caactttttt 2940 tacttcttgt tcattagaaa gaaagcatag caatctaatc taaggggcgg tgttgacaat 3000 taatcatcgg catagtatat cggcatagta taatacgaca aggtgaggaa ctaaaccatg 3060 gccaagttga ccagtgccgt tccggtgctc accgcgcgcg acgtcgccgg agcggtcgag 3120 ttctggaccg accggctcgg gttctcccgg gacttcgtgg aggacgactt cgccggtgtg 3180 gtccgggacg acgtgaccct gttcatcagc gcggtccagg accaggtggt gccggacaac 3240 accctggcct gggtgtgggt gcgcggcctg gacgagctgt acgccgagtg gtcggaggtc 3300 gtgtccacga acttccggga cgcctccggg ccggccatga ccgagatcgg cgagcagccg 3360 tgggggcggg agttcgccct gcgcgacccg gccggcaact gcgtgcactt cgtggccgag 3420 gagcaggact gacacgtccg acggcggccc acgggtccca ggcctcggag atccgtcccc 3480 cttttccttt gtcgatatca tgtaattagt tatgtcacgc ttacattcac gccctccccc 3540 cacatccgct ctaaccgaaa aggaaggagt tagacaacct gaagtctagg tccctattta 3600 tttttttata gttatgttag tattaagaac gttatttata tttcaaattt ttcttttttt 3660 tctgtacaga cgcgtgtacg catgtaacat tatactgaaa accttgcttg agaaggtttt 3720 gggacgctcg aaggctttaa tttgcaagct ggagaccaac atgtgagcaa aaggccagca 3780 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc 3840 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata 3900 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc 3960 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcaatgctc 4020 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga 4080 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc 4140 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag 4200 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag 4260 gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag 4320 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca 4380 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga 4440 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagatc 4488 <210> 3 <211> 9985 <212> DNA <213> Artificial Sequence <220> <223> pPIC3.5K-CAU200 VP7 <400> 3 agatctaaca tccaaagacg aaaggttgaa tgaaaccttt ttgccatccg acatccacag 60 gtccattctc acacataagt gccaaacgca acaggagggg atacactagc agcagaccgt 120 tgcaaacgca ggacctccac tcctcttctc ctcaacaccc acttttgcca tcgaaaaacc 180 agcccagtta ttgggcttga ttggagctcg ctcattccaa ttccttctat taggctacta 240 acaccatgac tttattagcc tgtctatcct ggcccccctg gcgaggttca tgtttgttta 300 tttccgaatg caacaagctc cgcattacac ccgaacatca ctccagatga gggctttctg 360 agtgtggggt caaatagttt catgttcccc aaatggccca aaactgacag tttaaacgct 420 gtcttggaac ctaatatgac aaaagcgtga tctcatccaa gatgaactaa gtttggttcg 480 ttgaaatgct aacggccagt tggtcaaaaa gaaacttcca aaagtcgcca taccgtttgt 540 cttgtttggt attgattgac gaatgctcaa aaataatctc attaatgctt agcgcagtct 600 ctctatcgct tctgaacccc ggtgcacctg tgccgaaacg caaatgggga aacacccgct 660 ttttggatga ttatgcattg tctccacatt gtatgcttcc aagattctgg tgggaatact 720 gctgatagcc taacgttcat gatcaaaatt taactgttct aacccctact tgacagcaat 780 atataaacag aaggaagctg ccctgtctta aacctttttt tttatcatca ttattagctt 840 actttcataa ttgcgactgg ttccaattga caagcttttg attttaacga cttttaacga 900 caacttgaga agatcaaaaa acaactaatt attcgaagga tccatgtacg gtatcgagta 960 cactactatc ttgattttct tgatctccat catcttgttg aactacatct tgaagtccgt 1020 tactagaatc atggactaca tcatctacag attcttgttg atctccgttg ctttgttcgc 1080 tttgactaag gctcagaact acggtttgaa catcccaatc actggttcca tggacactgt 1140 ttactccaac tccactcaag aagaggtttt cttgacttcc actttgtgtt tgtactaccc 1200 aactgaggct tccactcaga tttctgacgg tgagtggaaa gaatccttgt cccagatgtt 1260 cttgactaag ggttggccaa ctggttccgt ttacttcaaa gagtactcca acatcgttga 1320 cttctccgtt gacccacagt tgtactgtga ctacaacttg gttttgatga agtacgacca 1380 gaacttggag ttggacatgt ctgagttggc tgacttgatc ttgaacgagt ggttgtgtaa 1440 cccaatggac atcactttgt actactacca gcaatccggt gagtccaaca agtggatttc 1500 tatgggttcc tcctgtactg ttaaggtttg tccattgaac actcagactt tgggtatcgg 1560 ttgtcagact acaaacgttg actccttcga gactgttgct gagaacgaga agttggctat 1620 cgttgacgtt gttgacggta tcaaccacaa gatcaacttg actactacta cttgtactat 1680 cagaaactgt aagaagttgg gtccaagaga gaacgttgct gttatccaag ttggtggttc 1740 caacgttttg gacatcactg ctgacccaac tacaaaccca cagatcgaga gaatgatgag 1800 agttaactgg aagagatggt ggcaggtttt ctacactatc gttgactaca tcaaccagat 1860 cgttcaggtt atgtccaaga gatccagatc cttgaactcc gctgctttct actacagagt 1920 tcatcatcat catcaccact aagcggccgc gaattaattc gccttagaca tgactgttcc 1980 tcagttcaag ttgggcactt acgagaagac cggtcttgct agattctaat caagaggatg 2040 tcagaatgcc atttgcctga gagatgcagg cttcattttt gatacttttt tatttgtaac 2100 ctatatagta taggattttt tttgtcattt tgtttcttct cgtacgagct tgctcctgat 2160 cagcctatct cgcagctgat gaatatcttg tggtaggggt ttgggaaaat cattcgagtt 2220 tgatgttttt cttggtattt cccactcctc ttcagagtac agaagattaa gtgagaagtt 2280 cgtttgtgca agcttatcga taagctttaa tgcggtagtt tatcacagtt aaattgctaa 2340 cgcagtcagg caccgtgtat gaaatctaac aatgcgctca tcgtcatcct cggcaccgtc 2400 accctggatg ctgtaggcat aggcttggtt atgccggtac tgccgggcct cttgcgggat 2460 atcgtccatt ccgacagcat cgccagtcac tatggcgtgc tgctagcgct atatgcgttg 2520 atgcaatttc tatgcgcacc cgttctcgga gcactgtccg accgctttgg ccgccgccca 2580 gtcctgctcg cttcgctact tggagccact atcgactacg cgatcatggc gaccacaccc 2640 gtcctgtgga tctatcgaat ctaaatgtaa gttaaaatct ctaaataatt aaataagtcc 2700 cagtttctcc atacgaacct taacagcatt gcggtgagca tctagacctt caacagcagc 2760 cagatccatc actgcttggc caatatgttt cagtccctca ggagttacgt cttgtgaagt 2820 gatgaacttc tggaaggttg cagtgttaac tccgctgtat tgacgggcat atccgtacgt 2880 tggcaaagtg tggttggtac cggaggagta atctccacaa ctctctggag agtaggcacc 2940 aacaaacaca gatccagcgt gttgtacttg atcaacataa gaagaagcat tctcgatttg 3000 cggatcaag tgttcaggag cgtactgatt ggacatttcc aaagcctgct cgtaggttgc 3060 aaccgatagg gttgtagagt gtgcaataca cttgcgtaca atttcaaccc ttggcaactg 3120 cacagcttgg ttgtgaacag catcttcaat tctggcaagc tccttgtctg tcatatcgac 3180 agccaacaga atcacctggg aatcaatacc atgttcagct tgagacagaa ggtctgaggc 3240 aacgaaatct ggatcagcgt atttatcagc aataactaga acttcagaag gcccagcagg 3300 catgtcaata ctacacaggg ctgatgtgtc attttgaacc atcatcttgg cagcagtaac 3360 gaactggttt cctggaccaa atattttgtc acacttagga acagtttctg ttccgtaagc 3420 catagcagct actgcctggg cgcctcctgc tagcacgata cacttagcac caaccttgtg 3480 ggcaacgtag atgacttctg gggtaagggt accatccttc ttaggtggag atgcaaaaac 3540 aatttctttg caaccagcaa ctttggcagg aacacccagc atcagggaag tggaaggcag 3600 aattgcggtt ccaccaggaa tatagaggcc aactttctca ataggtcttg caaaacgaga 3660 gcagactaca ccagggcaag tctcaacttg caacgtctcc gttagttgag cttcatggaa 3720 tttcctgacg ttatctatag agagatcaat ggctctctta acgttatctg gcaattgcat 3780 aagttcctct gggaaaggag cttctaacac aggtgtcttc aaagcgactc catcaaactt 3840 ggcagttagt tctaaaaggg ctttgtcacc attttgacga acattgtcga caattggttt 3900 gactaattcc ataatctgtt ccgttttctg gataggacga cgaagggcat cttcaatttc 3960 ttgtgaggag gccttagaaa cgtcaatttt gcacaattca atacgacctt cagaagggac 4020 ttctttaggt ttggattctt ctttaggttg ttccttggtg tatcctggct tggcatctcc 4080 tttccttcta gtgaccttta gggacttcat atccaggttt ctctccacct cgtccaacgt 4140 cacaccgtac ttggcacatc taactaatgc aaaataaaat aagtcagcac attcccaggc 4200 tatatcttcc ttggatttag cttctgcaag ttcatcagct tcctccctaa ttttagcgtt 4260 caacaaaact tcgtcgtcaa ataaccgttt ggtataagaa ccttctggag cattgctctt 4320 acgatcccac aaggtggctt ccatggctct aagacccttt gattggccaa aacaggaagt 4380 gcgttccaag tgacagaaac caacacctgt ttgttcaacc acaaatttca agcagtctcc 4440 atcacaatcc aattcgatac ccagcaactt ttgagttgct ccagatgtag cacctttata 4500 ccacaaaccg tgacgacgag attggtagac tccagtttgt gtccttatag cctccggaat 4560 agactttttg gacgagtaca ccaggcccaa cgagtaatta gaagagtcag ccaccaaagt 4620 agtgaataga ccatcggggc ggtcagtagt caaagacgcc aacaaaattt cactgacagg 4680 gaactttttg acatcttcag aaagttcgta ttcagtagtc aattgccgag catcaataat 4740 ggggattata ccagaagcaa cagtggaagt cacatctacc aactttgcgg tctcagaaaa 4800 agcataaaca gttctactac cgccattagt gaaacttttc aaatcgccca gtggagaaga 4860 aaaaggcaca gcgatactag cattagcggg caaggatgca actttatcaa ccagggtcct 4920 atagataacc ctagcgcctg ggatcatcct ttggacaact ctttctgcca aatctaggtc 4980 caaaatcact tcattgatac cattattgta caacttgagc aagttgtcga tcagctcctc 5040 aaattggtcc tctgtaacgg atgactcaac ttgcacatta acttgaagct cagtcgattg 5100 agtgaacttg atcaggttgt gcagctggtc agcagcatag ggaaacacgg cttttcctac 5160 caaactcaag gaattatcaa actctgcaac acttgcgtat gcaggtagca agggaaatgt 5220 catacttgaa gtcggacagt gagtgtagtc ttgagaaatt ctgaagccgt atttttatta 5280 tcagtgagtc agtcatcagg agatcctcta cgccggacgc atcgtggccg acctgcaggg 5340 gggggggggg cgctgaggtc tgcctcgtga agaaggtgtt gctgactcat accaggcctg 5400 aatcgcccca tcatccagcc agaaagtgag ggagccacgg ttgatgagag ctttgttgta 5460 ggtggaccag ttggtgattt tgaacttttg ctttgccacg gaacggtctg cgttgtcggg 5520 aagatgcgtg atctgatcct tcaactcagc aaaagttcga tttattcaac aaagccgccg 5580 tcccgtcaag tcagcgtaat gctctgccag tgttacaacc aattaaccaa ttctgattag 5640 aaaaactcat cgagcatcaa atgaaactgc aatttattca tatcaggatt atcaatacca 5700 tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact caccgaggca gttccatagg 5760 atggcaagat cctggtatcg gtctgcgatt ccgactcgtc caacatcaat acaacctatt 5820 aatttcccct cgtcaaaaat aaggttatca agtgagaaat caccatgagt gacgactgaa 5880 tccggtgaga atggcaaaag cttatgcatt tctttccaga cttgttcaac aggccagcca 5940 ttacgctcgt catcaaaatc actcgcatca accaaaccgt tattcattcg tgattgcgcc 6000 tgagcgagac gaaatacgcg atcgctgtta aaaggacaat tacaaacagg aatcgaatgc 6060 aaccggcgca ggaacactgc cagcgcatca acaatatttt cacctgaatc aggatattct 6120 tctaatacct ggaatgctgt tttcccgggg atcgcagtgg tgagtaacca tgcatcatca 6180 ggagtacgga taaaatgctt gatggtcgga agaggcataa attccgtcag ccagtttagt 6240 ctgaccatct catctgtaac atcattggca acgctacctt tgccatgttt cagaaacaac 6300 tctggcgcat cgggcttccc atacaatcga tagattgtcg cacctgattg cccgacatta 6360 tcgcgagccc atttataccc atataaatca gcatccatgt tggaatttaa tcgcggcctc 6420 gagcaagacg tttcccgttg aatatggctc ataacacccc ttgtattact gtttatgtaa 6480 gcagacagtt ttattgttca tgatgatata tttttatctt gtgcaatgta acatcagaga 6540 ttttgagaca caacgtggct ttcccccccc cccctgcagg tcggcatcac cggcgccaca 6600 ggtgcggttg ctggcgccta tatcgccgac atcaccgatg gggaagatcg ggctcgccac 6660 ttcgggctca tgagcgcttg tttcggcgtg ggtatggtgg caggccccgt ggccggggga 6720 ctgttgggcg ccatctcctt gcatgcacca ttccttgcgg cggcggtgct caacggcctc 6780 aacctactac tgggctgctt cctaatgcag gagtcgcata agggagagcg tcgagtatct 6840 atgattggaa gtatgggaat ggtgataccc gcattcttca gtgtcttgag gtctcctatc 6900 agattatgcc caactaaagc aaccggagga ggagatttca tggtaaattt ctctgacttt 6960 tggtcatcag tagactcgaa ctgtgagact atctcggtta tgacagcaga aatgtccttc 7020 ttggagacag taaatgaagt cccaccaata aagaaatcct tgttatcagg aacaaacttc 7080 ttgtttcgaa ctttttcggt gccttgaact ataaaatgta gagtggatat gtcgggtagg 7140 aatggagcgg gcaaatgctt accttctgga ccttcaagag gtatgtaggg tttgtagata 7200 ctgatgccaa cttcagtgac aacgttgcta tttcgttcaa accattccga atccagagaa 7260 atcaaagttg tttgtctact attgatccaa gccagtgcgg tcttgaaact gacaatagtg 7320 tgctcgtgtt ttgaggtcat ctttgtatga ataaatctag tctttgatct aaataatctt 7380 gacgagccaa ggcgataaat acccaaatct aaaactcttt taaaacgtta aaaggacaag 7440 tatgtctgcc tgtattaaac cccaaatcag ctcgtagtct gatcctcatc aacttgaggg 7500 gcactatctt gttttagaga aatttgcgga gatgcgatat cgagaaaaag gtacgctgat 7560 tttaaacgtg aaatttatct caagatctct gcctcgcgcg tttcggtgat gacggtgaaa 7620 acctctgaca catgcagctc ccggagacgg tcacagcttg tctgtaagcg gatgccggga 7680 gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc gcagccatga 7740 cccagtcacg tagcgatagc ggagtgtata ctggcttaac tatgcggcat cagagcagat 7800 tgtactgaga gtgcaccata tgcggtgtga aataccgcac agatgcgtaa ggagaaaata 7860 ccgcatcagg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct 7920 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga 7980 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc 8040 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg 8100 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg 8160 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt 8220 tctcccttcg ggaagcgtgg cgctttctca atgctcacgc tgtaggtatc tcagttcggt 8280 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg 8340 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact 8400 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt 8460 cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta tctgcgctct 8520 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac 8580 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc 8640 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg 8700 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta 8760 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca 8820 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc 8880 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc 8940 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc 9000 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat 9060 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt 9120 tgccattgct gcaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc 9180 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag 9240 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt 9300 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac 9360 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg 9420 cccggcgtca acacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat 9480 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc 9540 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc 9600 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa 9660 atgattgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg 9720 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg 9780 cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc attattatca tgacattaac 9840 ctataaaaat aggcgtatca cgaggccctt tcgtcttcaa gaattaattc tcatgtttga 9900 cagcttatca tcgataagct gactcatgtt ggtattgtga aatagacgca gatcgggaac 9960 actgaaaaat aacagttatt attcg 9985 <210> 4 <211> 2694 <212> DNA <213> Artificial Sequence <220> <223> CAU200 VP2co <400> 4 gaattcatgg cttacagaaa gagaggtgct aagagagagg acttgccaca acagaacgag 60 agattgcaag agaaagagat cgagaacaac actgacgtta ctatggaaaa caagaacaag 120 aacaaaaaca acaatagaaa gcagcagttg tccgacaagg ttttgtccca gaaagaagag 180 atcatcactg acgttcagga cgacatcaag atcgctgacg aagttaagaa gtcctccaaa 240 gaagagtcca agcagttgtt ggagatcttg aaaacaaaag aggaccacca gaaagaggtt 300 cagtacgaga tcttgcaaaa gactatccca actttcgagc caaaagagtc catcttgaag 360 aagttggagg acatcaagcc agagcaggct aagaaacaga tgaagttgtt cagaatcttc 420 gagccaagac agttgccaat ctacagagct aacggtgaga aagaattgag aaacagatgg 480 tactggaagt tgaagaaaga cactttgcca gacggtgact acgacgttag agagtacttc 540 ttgaacttgt acgaccagat cttgatcgag atgccagact acttgttgtt gaaggacatg 600 gctgttgaga acaagaactc cagagatgct ggtaaggttg ttgactccga gactgctaga 660 atctgtgacg ctattttcca ggacgaagag actgagggtg ttattagaag attcattgct 720 gacatgagac agcaggttca ggctgacaga aacatcgtta actacccatc catcttgcac 780 ccaatcgacc acgctttcaa cgagtacttt ttgaaccacc agttggttga gccattgaac 840 aacgagatca tcttcaacta catccctgag agaatcagaa acgacgttaa ttacatcttg 900 aacatggaca tgaacttgcc atccactgct agatatatca gaccaaactt gttgcaggac 960 agattgaact tgcacgacaa cttcgaatcc ttgtggga ctatcactac ttccaactac 1020 atcttggcta gatccgttgt tccagacttg aaagagaaag agttggtttc cactgaggct 1080 cagatccaga agatgtccca agacttgcaa ttggaggctt tgactatcca gtccgagact 1140 caattcttgg ctggtattaa ctcccaggct gctaacgact gtttcaagac tttgatcgct 1200 gctatgttgt cccagagaac tatgtccttg gacttcgtta ctacaaacta catgtccttg 1260 atctccggta tgtggttgtt gactgttatc ccaaacgaca tgttcttgag agaatccttg 1320 gttgcttgtg agttggctat catcaacact atcgtttacc cagctttcgg tatgcagaga 1380 atgcactaca gaaacggtga cccacaaact ccattccaga ttgctgagca gcagatccag 1440 aacttccaag ttgctaactg gttgcacttc atcaacaaca acagattcag acaggttgtt 1500 atcgacggtg ttttgaacca gactttgaac gacaacatca gaaacggtca ggttatcaac 1560 cagttgatgg aagctttgat gcagttgtcc agacagcagt tcccaactat gccagttgac 1620 tacaagagat ccatccagag aggtatcttg ttgttgtcca acagattggg tcagttggtt 1680 gacttgacta gacttttgtc ctacaactac gagactttga tggcttgtat cactatgaac 1740 atgcagcacg ttcagacttt gactactgag agattgcagt tgacttccgt tacttccttg 1800 tgtatgttga tcggtaacac tactgttatt ccatccccac agactttgtt ccactactac 1860 aacgttaatg ttaacttcca ctccaactac aacgagagaa tcaacgacgc tgttgctatc 1920 atcactgctg ctaatagatt gaacttgtac caaaagaaga tgaagtccat cgttgaggat 1980 ttcttgaaga gattgcagat cttcgacgtt ccaagagttc cagacgacca gatgtacaga 2040 ttgagagaca gattgagact tttgccagtt gagagaagaa gattggacat cttcaacttg 2100 atcttgatga acatggaaca gatcgagaga gcttccgaca agattgctca gggtgttatc 2160 attgcttaca gagatatgca gttggagcgt gatgagatgt acggtttcgt taacattgct 2220 agaaacttgg acggttacca gcagatcaac ttggaagagt tgatgagaac tggtgactac 2280 ggtcagatca ctaacatgtt gttgaacaac cagccagttg ctttggttgg tgctttgcct 2340 ttcgttactg actcctccgt tatctccttg atcgctaagt tggacgctac tgttttcgct 2400 cagatcgtta agttgagaaa ggttgacact ttgaagccaa tcttgtacaa aatcaactcc 2460 gactccaacg acttctactt ggttgctaac tacgactgga tcccaacttc cactactaag 2520 gtttacaagc aggttccaca gccattcgac ttcagagctt ctatgcacat gttgacttcc 2580 aacttgactt tcactgttta ctccgacttg ttgtccttcg tttccgctga cactgttgag 2640 cctattaacg ctatcgcttt cgacaacatg agaatcatga acgagttgct cgag 2694 <210> 5 <211> 1203 <212> DNA <213> Artificial Sequence <220> <223> CAU200 VP6co <400> 5 gaattcatgg aagttttgta ctcattgtcc aagactttga aggacgctag agacaagatc 60 gttgagggta ctttgtactc caacgtttcc gacttgatcc agcagttcaa ccagatgatc 120 gttactatga acggtaacga cttccagact ggtggtatcg gtaacttgcc aatcagaaac 180 tggactttcg acttcggttt gttggtact actttgttga acttggacgc taactacgtt 240 gagactgcta gaactactat cgagtacttc attgacttca tcgacaacgt ttgtatggac 300 gagatcgcta gagaatccca gagaaacggt gttgctccac aatctgaggc tttgagaaag 360 ttggctggta tcaagttcaa gagaatcaac ttcaacaact cctccgagta catcgagaac 420 tggaacttgc agaacagaag gcagagaact ggtttcgttt tccacaagcc aaacatcttc 480 ccttactccg cttcattcac tttgaacaga tctcagccaa tgcacgacaa cttgatgggt 540 actatgtggt tgaacgctgg ttccgagatt caggttgctg gtttcgatta ctcctgtgct 600 ttgaacgctc cagctaacat tcaacagttc gagcacatcg ttcagttgcg tagagctttg 660 actactgcta ctatcacttt gttgccagac gctgagagat tctcattccc aagagttatc 720 aactccgctg acggtgctac tacatggttc ttcaacccaa tcatccttag accaaacaac 780 gttgaggttg agttcttgtt gaacggtcag atcatcaaca cttaccaggc tagattcggt 840 actattatcg ctagaaactt cgacactatc agattgtcct tccagttgat gaggccacca 900 aacatgactc cagctgttaa cgctttgttc ccacaagctc aaccattcca acaccacgct 960 actgttggtt tgactttgag aatcgagtcc gctgtttgtg agtccgtttt ggctgatgct 1020 aacgagactt tgttggctaa cgttactgct gttagacaag agtacgctat cccagttggt 1080 ccagtttttc caccaggtat gaactggact gagttgatca ctaactactc cccatccaga 1140 gaggacaact tgcagagagt tttcactgtt gcttccatca gatccatgtt gatcaagctc 1200 gag 1203 <210> 6 <211> 990 <212> DNA <213> Artificial Sequence <220> <223> CAU200 VP7co <400> 6 gaattcatgt acggtatcga gtacactact atcttgattt tcttgatctc catcatcttg 60 ttgaactaca tcttgaagtc cgttactaga atcatggact acatcatcta cagattcttg 120 ttgatctccg ttgctttgtt cgctttgact aaggctcaga actacggttt gaacatccca 180 atcactggtt ccatggacac tgtttactcc aactccactc aagaagaggt tttcttgact 240 tccactttgt gtttgtacta cccaactgag gcttccactc agatttctga cggtgagtgg 300 aaagaatcct tgtcccagat gttcttgact aagggttggc caactggttc cgtttacttc 360 aaagagtact ccaacatcgt tgacttctcc gttgacccac agttgtactg tgactacaac 420 ttggttttga tgaagtacga ccagaacttg gagttggaca tgtctgagtt ggctgacttg 480 atcttgaacg agtggttgtg taacccaatg gacatcactt tgtactacta ccagcaatcc 540 ggtgagtcca acaagtggat ttctatgggt tcctcctgta ctgttaaggt ttgtccattg 600 aacactcaga ctttgggtat cggttgtcag actacaaacg ttgactcctt cgagactgtt 660 gctgagaacg agaagttggc tatcgttgac gttgttgacg gtatcaacca caagatcaac 720 ttgactacta ctacttgtac tatcagaaac tgtaagaagt tgggtccaag agagaacgtt 780 gctgttatcc aagttggtgg ttccaacgtt ttggacatca ctgctgaccc aactacaaac 840 ggagttaac atcgttgact acatcaacca gatcgttcag gttatgtcca agagatccag atccttgaac 960 tccgctgctt tctactacag agttctcgag 990 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> AOX1 primer <400> 7 gcaaatggca ttctgacatc c 21 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> VP2 primer <400> 8 ccaggacgaa gagactgagg 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> VP6 primer <400> 9 caacagttcg agcacatcgt 20 <210> 10 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> V7 primer <400> 10 cagttgtact gtgactacaa ctt 23

Claims (9)

A method for producing and purifying virus-like particles (VLP) of Rotavirus comprising the steps of:
a) transforming yeast cells to express a rotavirus structural protein;
b) culturing the transfected yeast cells in a medium supplemented with methanol;
c) dissolving the cultured yeast cells to obtain a cell lysate; And
d) separating and purifying virus-like particles of rotavirus from the cell lysate.
The method according to claim 1,
The above step a) is a step of transforming yeast cells with the pPIC6-CAU200 VP2 vector of SEQ ID NO: 1, the pPICZ-CAU200 VP6 vector of SEQ ID NO: 2, and the pPIC3.5K-CAU200 VP7 vector of SEQ ID NO: 3 &Lt; / RTI &gt;
The method according to claim 1,
Characterized in that the yeast in step a) is Pichia pastoris .
The method according to claim 1,
Wherein in step b), methanol is added at a concentration of 0.5 to 1.5%, and culturing is continued for 24 to 96 hours.
5. The method of claim 4,
Characterized in that in step b), methanol is added at a concentration of 1 to 1.5% and culturing is carried out for 24 to 48 hours.
The method according to claim 1,
Wherein said step c) is dissolved in a cell disruption buffer containing Tris-HCl, NaCl, CaCl ₂ , ethylenediaminetetraacetic acid (EDTA), PMSF, glycerol, and Tween 20.
A virus-like particle of Rotavirus produced and purified by the method of claim 1.
8. The method of claim 7,
Wherein said virus-like particle simultaneously expresses the rotavirus structural proteins VP2, VP6, and VP7.
An immunogenic composition comprising the virus-like particle of Rotavirus of claim 7.

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