WO2002006305A1

WO2002006305A1 - Method of collecting viral envelope from germinating baculovirus

Info

Publication number: WO2002006305A1
Application number: PCT/JP2001/006109
Authority: WO
Inventors: Takao Hamakubo; Tatsuhiko Kodama; Hiroko Iwanari; Yukio Itoh
Original assignee: Institute Of Immunology Co., Ltd.; Center For Advanced Science And Technology Incubation, Ltd.
Priority date: 2000-07-17
Filing date: 2001-07-16
Publication date: 2002-01-24
Also published as: JP2002030098A; AU2001271050A1

Abstract

It is intended to provide a method of collecting the enucleated virion envelope (EVE) in which a target protein is expressed by separating the viral envelope from the nucleocapsid of a germinating virus. Namely, a method of collecting the viral envelope comprising treating a germinating baculovirus, in which a target protein is expressed, by a physicochemical treatment and thus separating the viral envelope from the nucleocapsid of a germinating virus.

Description

Specification

Method for recovering virus envelope from baculovirus budding virus

Technical field

The present invention relates to a method for recovering a virus envelope from a baculovirus germination virus. More specifically, the present invention relates to a method for recovering a virus envelope from a germinated virus, which comprises treating the budding virus of the Baculovirus expressing the protein of interest with a physicochemical technique. The present invention also relates to a method for screening a chemical substance, a method for producing an antibody, and a method for purifying a target protein using the above-mentioned recovery method. Background art

A protein expression system using baculovirus and insect cells is widely used as a membrane protein expression system. Compared to expression systems using Escherichia coli or yeast, such an expression system has the advantage that it does not easily form aggregates and contains post-translational modifications necessary for protein functions such as sugar chain addition and metal ion coordination. There are many.

The baculovirus expression system is a system for expressing a target gene in large amounts in insect cells by using a promoter of a viral gene such as a baculovirus polyhedrin protein.

Normally, the expressed protein is recovered from insect cells, but the seven transmembrane receptors (Loisel TP, Ansanay H, St-Onge S, Gay B, Boulanger P, Strosberg AD, Marullo S, Bouvier M., Nat Biotechnol 1997 Nov; 15 (12): 1300-4., Recovery of homogeneous and functional beta 2-adrenergic receptors from extracellular baculovirus particles.) ゃ Japanese Patent Application No. 2000-0- 15 8 2 9 4 Various membrane proteins have been reported to be expressed in the viral envelope.

Membrane proteins expressed in the viral envelope are proteins expressed in insect cells It has been reported that the proportion of proteins that retain their functions is higher than that of proteins. In other words, in addition to using proteins expressed in insect cells such as Sf9, a new method using membrane proteins expressed in the envelope of the virus has been considered. It has come to be. In addition, a monoclonal antibody against the target protein was prepared using the method of fusing the target protein to gp64, the only viral membrane protein present in the viral envelope, and displaying it on the viral envelope (Novagen, pBACsurf-1 expression system). Reported how to

(Lindley KM, Su JL., Hodges PK, Wisely B., Bledsoe RK, Condreay J.P., Winegar DA, Hutchins JT, Kost TA Production of monoclonal antibodies using recombinant baculovirus displaying gp64-fusion proteins.Journal of Immunological Methods 234 , 2000, 123-135).

As described above, in the baculovirus expression system, a protein expressed in a budding virus in addition to a system using a protein expressed in insect cells such as Sf9 and a system using a secreted protein as in the past. The system to be used is beginning to be used for the production of monoclonal antibodies.

Baculoviruses have two life cycles: occlusion derived virus (OCD) and budded virus (BV), which are adapted to the process of infection between insects and between cells in individuals. are doing. Both consist of a viral DNA and a viral membrane called the virion envelope, which surrounds a part called nucleocapsid, which is made of structural proteins. Only gp64 is known as a virus-derived membrane protein in the virus envelope. The membrane protein expressed in the budding virus is considered to be present in the virus envelope.

Membrane proteins include receptors for hormones and chemicals, channel proteins, proteins involved in mass transport, adhesion factors, membrane enzymes, enzyme substrate proteins, enzyme activators, proteins involved in antigen presentation, higher order It has important functions related to the physiological functions of cells, such as proteins involved in structure formation. Express these membrane proteins and use their functions Building a system that can be used is an important technology that will lead to the development of pharmaceuticals, biosensors, and monoclonal antibodies.

However, no simple method has been reported so far to separate the nucleoside peptide of the budding virus from the virus envelope and recover the viral membrane fraction (enucleated virion envelope, EVE) in which the target membrane protein is expressed. Disclosure of the invention

That is, an object of the present invention is to provide a method for separating a nucleocabside of a budding virus from a virus envelope and recovering a virus membrane fraction (enucleated virion envelope, EVE) in which a target membrane protein is expressed. did.

Furthermore, the present invention solves the problem of providing a method for screening a chemical substance, a method for producing an antibody, and a method for purifying a target protein using the above-mentioned method for recovering a viral membrane fraction in which a target membrane protein is expressed. It should be a task to be done.

The present inventors have conducted intensive studies in order to solve the above-mentioned problems. As a result, the baculovirus budding virus (Budded Virus) was subjected to a physicochemical method (specifically, a combination of surfactant treatment and density gradient centrifugation). Thus, the present invention succeeded in separating the nucleoside peptide of the budding virus from the virus envelope, and recovering the virus membrane fraction (enucleated virion envelope, EVE) in which the target membrane protein was expressed. It was completed.

That is, according to the present invention, the following inventions are provided.

(1) A method for recovering a virus envelope by separating a nucleoforce of the budding virus and a virus envelope, comprising treating the baculovirus germinating virus expressing the target protein by a physicochemical technique.

(2) The method according to (1), wherein the treatment by a physicochemical technique is a treatment with a surfactant or a treatment by freeze-thawing and a separation operation by density gradient centrifugation.

(3) The method according to (1) or (2), wherein tween20 is used as a surfactant.

(4) The method according to any one of (1) to (3), wherein the target protein is a membrane protein. (5) A virus envelope in which a target protein is expressed, obtained by the method according to any one of (1) to (4).

(6) Interaction between the target protein expressed on the viral envelope and the test substance using the viral envelope expressing the target protein obtained by the method according to any one of (1) to (4). A method of screening for chemicals, including measuring effects.

(7) Using a virus envelope in which a target protein obtained by the method according to any one of (1) to (4) has been expressed, a polyclonal antibody against the target protein expressed on the virus envelope or A method for producing a monoclonal antibody.

(8) A polyclonal antibody or a monoclonal antibody obtained by the method according to (7).

(9) A method for treating a viral envelope in which a target protein obtained by the method according to any one of (1) to (4) is expressed with a lysing agent, solubilizing and purifying the target protein. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the measurement results of sucrose concentration and protein concentration of each fraction obtained by sucrose density gradient ultracentrifugation.

FIG. 2 shows the results of silver staining of the 3rd to 18th fractions obtained by sucrose density gradient ultracentrifugation, after 12% polyacrylamide SDS electrophoresis (SDS-PAGE). FIG. 3 is an electron micrograph of the ninth fraction obtained by sucrose density gradient ultracentrifugation. FIG. 4 is an electron microscope image of the tenth fraction obtained by sucrose density gradient ultracentrifugation.

FIG. 5 is an electron microscope image of the eleventh fraction obtained by sucrose density gradient ultracentrifugation.

Figure 6 shows an electron microscope image of the 12th fraction obtained by sucrose gradient ultracentrifugation. It is. '

FIG. 7 shows the results of Western blotting using each fraction obtained by sucrose density gradient ultracentrifugation and a specific antibody against SREBP2. BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, an embodiment and an implementation method of the present invention will be described in detail.

The method of the present invention relates to a method for separating a nucleoside of a budding virus and a virus envelope, which comprises treating a baculovirus budding virus expressing a target protein by a physical-chemical method.

The target protein referred to in the present specification is preferably a membrane protein, and more preferably a membrane-bound enzyme, a substrate of the membrane-bound enzyme, a membrane-bound enzyme activator, a membrane-bound transport protein, a channel protein, It is a protein selected from membrane structural proteins, proteins involved in adhesion, proteins involved in antigen presentation, and proteins involved in the formation of higher-order structures of proteins. Hereinafter, the membrane protein will be described in more detail.

As used herein, the term “membrane-bound” broadly means that the protein is present in the plasma membrane of cell membranes and intracellular organelles (eg, endoplasmic reticulum, Golgi apparatus, etc.). Not limited. Preferably, a membrane-bound receptor, a membrane-bound enzyme, a substrate of the membrane-bound enzyme, a membrane-bound enzyme activator or a membrane-bound transport protein S, a channel protein, a structural protein of a membrane, an adhesion-related protein A protein, a protein involved in antigen presentation, or a protein involved in formation of a higher-order structure of a protein is a membrane-bound protein of an intracellular organelle, for example, a protein bound to the membrane of the endoplasmic reticulum or the Golgi apparatus.

Membrane-bound receptors include seven transmembrane receptors for hormones, odors, taste, light, etc., one transmembrane receptors for LDL receptor ゃ scavenger-one receptor, growth hormone dinsulin, TNF, glutamate, etc. Receptors, ion channel receptors such as GABA, acetylcholine, and ryanodine, and those forming complexes with T cell receptors, Fc receptors, and the like.

Examples of membrane-bound enzymes include HMG-CoA reductase involved in cholesterol metabolism and ACAT -: such as _{(acyl coenzyme A cholesterol acyltransferase) s} 7 a- hydroxylase , and the like. Examples include cytochrome P450 related to detoxification, electron transfer enzymes such as ATP synthase ゃ cytochrome oxidase and reductase, and NADH-Q reductase present in mitochondria. In addition, a group of processing proteases related to the processing of hormones, regulatory factors, and nutritional factors, etc .: SIP (site 1 protease), furin, PC (proprotein convertase, S2P (site 2 protease)) _s endothelin converting enzyme, Angiotensin converting enzyme The ADAMS (a disintegrin and metal loprotease) family involved in the signal transduction system such as _N neprilysin and notch signal, and the matrix metal loprotease group involved in the degradation of extracellular matrix. Examples include membrane lipid metabolizing enzymes such as synthase, phosphatidic acid phosphatase, and phosphatidylserine synthase, and enzymes involved in signal transmission such as adenylate cyclase.

Examples of membrane-bound enzyme substrate proteins include sterol regulatory protein (SREBP), Notch, Irel, and ATF6 as proteins involved in signal transduction and transcriptional regulation, as well as other amyloid precursor proteins, Tu (tumor necrosis factor) precursors Stem cell factors M-CSF (monocyte colony stimulating factor) precursor ^ Klotho.

The membrane-bound enzyme activators, Purese diphosphate (presenil lin), SCAP (SREBP cleavage activating protein), and the like ₀

Membrane-bound transport proteins include NPC (Niemann-Pick type c) 1, which transports lipids such as cholesterol, ABC (ATP-binding cassette), transposon, caveolin, and fatty acid transport protein. And sugar transports including glucose transport such as GLUT-4 and amino acid transposers such as glutamate tanspoter and serotonin transporter. In addition, Secl2 and the like can be mentioned as a membrane protein involved in substance transport between intracellular vesicles. In addition, there are channel proteins that selectively pass molecules that do not pass through the membrane under certain conditions. Among them, aquaporin famili, which is a selective channel of water, and potassium, calcium, and sodium ions For example, an ion channel, which is a selective channel for such a channel, may be used.

Other membrane structural proteins and proteins involved in adhesion include NCAM (Neural cell adhesion molecu 丄 e), ICAM (mterecellular adhesion molecule), Dherin family, integrin, desmocholine, desmoglein, L-selectin, connexin, glycoprotein, etc. Is raised. Major histocompatibility complex (MHC) involved in antigen presentation in immune cells, calnexin, PDI (protein disulfide isomerase), and CFTR (cystic nbrosis transmembrane conductance regulator), which are thought to be involved in the formation of higher-order structures of proteins ) And major prion protein precursors (prions).

In the present invention, at least one type of recombinant baculovirus containing a gene encoding a target protein as described above is used.

Baculovirus, a virus that infects insects and causes disease, is an enveloped virus that has a circular double-stranded DNA as a gene and is susceptible to insects such as Lepidoptera, Hymenoptera, and Diptera. Among the baculoviruses, the nuclear polyhedrosis virus (NPV) is a group of viruses that produce large amounts of inclusion bodies called polyhydra in the nucleus of infected cells. Polyhedra are composed of a polyhedrin protein with a molecular weight of 31 kDa and are produced in large quantities at the late stage of infection, in which numerous virus particles are embedded. Polyhedra are essential for the virus to survive in nature, but are not necessary for the growth of the virus itself, so even if a foreign gene that you want to express in place of the polyhedron gene is inserted, the virus will not infect at all. And proliferate.

Examples of the baculovirus used in the present invention include Autographa californica NPV (AcNP V) of NPV subfamily and Bombyxmori NPV (BmNP V) of silkworm. Of viruses can be used as vectors.

AcNPV hosts (infected, passaged cells) include Spodoptera frugiperda cells (Sf cells), etc. BmNPV hosts (infected, passaged cells) include BmN4 cells, etc. . AcNPV vectors are preferred because Sf cells have a higher growth rate than BmN4 cells and the like, and AcNPV also has the ability to infect human hepatocytes and human fetal kidney cells.

As hosts, Spodoptera Frugiperda cell lines Sf9 and Sf21 have been established from ovarian tissues of S. frugiperda larvae, and are available from Invitrogen, Pharmingen (San Diego, CA), ATCC, and the like. In addition, live insect larvae can also be used as host cell systems.

The method of constructing the recombinant virus used in the present invention may be performed according to a conventional method, and for example, can be performed by the following procedure.

First, the gene of the protein to be expressed is inserted into a transfer vector to construct a recombinant transfer vector.

The overall size of the transfer protein is generally about several kb to 10 kb, of which about 3 kb is a plasmid-derived skeleton, which is resistant to antibiotic resistance genes such as ampicillin and bacteria. Contains the signal for initiation of DNA replication. In addition to this backbone, a normal transfer vector contains 5 'and 3' regions of the polyhedron gene, each of which is several kb, and when transfection is performed as described below, the target Homologous recombination occurs between the gene and the polyhedron gene. It is preferable that the transfer vector contains a plasmid for expressing the protein gene. Examples of the promotion include the promotion of the polyhedron gene, the promotion of the P10 gene, and the promotion of the capsid gene.

The type of transfer vector is not particularly limited. A specific example of the transfer vector is pEVmX as an AcNPV-based transfer vector. IV2, pAc SGl, VL 1392/1393, pAcMP2 / 3, p Ac JPI s pAcUW21, pAcDZl, pB lueBac III, pAcUW 51, pAcAB3, p Ac 360 s pB lueBacHi s _s p VT -B ac 33, pAcUWls pAcUW42 / 43 etc. Examples of BmNPV transfer vectors include ρΒΚ283, ρΒΚ5, ρΒΒ30, ρΒΕ1, ρΒΕ2, ρΒΚ3, ρΒΚ52, pBKblue, ρΒKb 1 ue2, pBF series (Funakoshi Corporation, (Available from Fujisawa Pharmaceutical Co., Ltd.).

Next, in order to produce a recombinant virus, the above-mentioned recombinant transfer vector is mixed with the virus and then transferred to a cultured cell used as a host, or the above-described vector is added to a cultured cell used as a host previously infected with the virus. Then, homologous recombination occurs between the recombinant transfer vector and the viral genomic DNA to construct a recombinant virus.

Here, the cultured cells used as a host include the above-mentioned hosts, and are usually insect cultured cells (such as Sf9 cells and BmN cells). Culture conditions are appropriately determined by those skilled in the art. Specifically, when Sf9 cells are used, culture is preferably performed at about 28 ° C. in a medium containing 10% fetal bovine serum. The recombinant virus thus constructed can be purified by a conventional method, for example, plaque assay. In addition, the recombinant virus thus produced cannot transform into a polyhedron because foreign DNA has been substituted or inserted into the gene region of the polyhedrin protein of the nuclear polyhedrosis virus. It can be easily distinguished from a replacement virus.

In the present invention, the above-mentioned recombinant baculovirus is infected to an appropriate host (cultured cells such as Spodoptera Frugiperda cell lines Sf9 and Sf21, or insect larvae), and after a certain period of time (for example, after 72 hours) In addition, the target protein can be recovered by collecting extracellular budding virus (BV) from the culture supernatant by a separation operation such as centrifugation. Only one type of recombinant baculovirus The infection may be carried out, or two or more recombinant baculoviruses may be combined and co-infected.

The extracellular budding baculovirus can be collected, for example, as follows.

First, the culture of the infected cells is centrifuged at 500 to 1,000 g, and the supernatant containing the extracellular budding baculovirus is recovered. The supernatant is centrifuged at about 30,000 to 500,000 g to obtain a precipitate containing extracellular budding baculovirus. The virus (BV) fraction containing the extracellular budding baculovirus can be obtained by suspending the precipitate in an appropriate buffer.

In the present invention, a baculovirus budding virus having a target protein obtained as described above is treated by a physicochemical technique to separate a nucleoforce peptide from a virus envelope.

The physicochemical method used in the present invention is not particularly limited as long as it can separate the nucleocabside and the virus envelope from the budding virus of baculovirus, and examples thereof include a treatment with a surfactant and a separation operation by density gradient centrifugation. Alternatively, treatment by freeze-thawing and separation by density gradient centrifugation may be mentioned. The type of surfactant that can be used in the present invention is not particularly limited, and examples thereof include tween 20, triton X 305, and the like, and particularly preferably tween 20.

For treatment with a detergent, mix the fraction containing the budding virus with a detergent solution (for example, a 0.5% tween20 / PBS solution) in a suitable buffer such as PBS, and allow to stand at room temperature for an appropriate time. Can be performed.

In general, freeze-thaw treatment involves suspending the virus (BV) in PBS and cooling to 120 ° C. After 30 minutes, the procedure of returning to room temperature and melting may be repeated, for example, about three times. A method of combining with a hypotonic treatment such as phosphate buffer or water instead of or together with PBS, a method of lowering the temperature further (at 180 ° C or in liquid nitrogen, etc.), and a higher melting temperature. Yes (for example, It is also possible to adopt a more rapid freeze-thaw method, such as a method at 37 ° C. For the process by freeze-thaw, optimal conditions can be set by appropriately combining such conditions.

Examples of other physicochemical methods used in the present invention include ultrasonic treatment and a method in which pressure is applied and pressure is rapidly reduced.

As an example of sonication, there is a method of suspending the virus in PBS and treating it on ice three times for 20 seconds on a machine such as a Branson Sonifier 250, but it is better to perform the treatment under milder conditions. It may be preferable.

Further, the method of rapidly vacuum keep over pressure, specifically, N ₂ by using an apparatus such as Nitrogen cavitation apparatus, under reduced pressure once and allowed to dissolve N ₂ over pressure This method is based on the principle that cells are destroyed by evaporating them. Alternatively, a cell destruction method called French press is also available. Specifically, it is a method in which a cell suspension is broken by applying pressure and passing through a small hole.

As described above, the means of the physicochemical technique used in the present invention is not particularly limited, but particularly preferably includes a treatment with a surfactant and a separation operation by density gradient centrifugation.

In the present invention, the baculovirus budding virus is treated with a surfactant or freeze-thawing as described above, and then the separation operation is performed by density gradient centrifugation.

As the density gradient centrifugation, for example, sucrose density gradient ultracentrifugation can be performed. Specifically, a sucrose solution is overlaid with a suitable concentration gradient (for example, 66.5%, 45%, and 30%), and a germinated virus-containing solution treated with a surfactant or freeze-thaw on the upper layer. After centrifugation at 100,000 g to 500,000 g (for example, 350,000 g) for a certain time, fractionate from the bottom layer.

Measure the sucrose concentration (refractometer) and protein concentration (Bio-Rad protein assay system, BSA standard) of each fraction, perform polyacrylamide SDS electrophoresis (SDS-PAGE), and then perform silver staining Confirm protein by Can be. As a result, it is possible to identify the fraction in which the virus protein is considered to be present. Further, by observing the fractions in which the viral proteins are thought to be present in detail with an electron microscope, it is possible to isolate the fractions containing the viral envelope without the nucleoside peptide.

The virus envelope obtained by the above method and expressing the target protein is also within the scope of the present invention.

The present invention further includes measuring the interaction between the target protein expressed on the viral envelope and the test substance using the virus envelope in which the target protein obtained by the above method is expressed. It relates to screening methods for chemical substances.

Chemicals to be screened include, for example, peptides, polypeptides, synthetic compounds, fermented microorganisms, extracts from organisms (including plant or animal tissues, microorganisms, cells, etc.), and their libraries. Is mentioned. Libraries include synthetic compound libraries (such as combinatorial libraries) and peptide libraries (such as combinatorial libraries). The chemicals to be screened may be natural or synthetic, and even if a single candidate chemical is tested independently, a mixture of several candidate chemicals (including ) May be tested. Further, it is also possible to screen a fractionated mixture such as a cell extract, and to repeat the fractionation to isolate a substance having a desired activity.

These chemicals include a target protein (preferably a membrane protein, particularly preferably a membrane-bound receptor, a membrane-bound enzyme, a substrate of the membrane-bound enzyme, a membrane-bound enzyme, expressed on the virus envelope). Interacting with activators, membrane-bound transport proteins, channel proteins, membrane structural proteins, proteins involved in adhesion, proteins involved in antigen presentation, or proteins involved in the formation of higher-order structures of proteins) And more preferably an inhibitor or an activating drug for the above protein. The present invention further provides a method for producing a polyclonal antibody or a monoclonal antibody against the target protein expressed on the virus envelope using the viral envelope in which the target protein obtained by the above method is expressed, and It relates to a polyclonal antibody or a monoclonal antibody obtained by the method. In the method for producing an antibody of the present invention, a virus envelope in which a target protein obtained by the above method is expressed is used as an immunogen.

Antibodies can be prepared by a conventional method. When producing a polyclonal antibody, a mammal is immunized with the viral envelope in which the target protein is expressed as an antigen, blood is collected from the mammal, and the antibody is separated and purified from the collected blood. Can be obtained. For example, mammals such as mice, hamsters, guinea pigs, chickens, rats, rabbits, dogs, goats, sheep, and mice can be immunized. Immunization can be performed according to the usual immunization method, for example, by administering the antigen one or more times.

For antigen administration, for example, it is preferable to administer the antigen twice or three times at intervals of 7 to 30 days, particularly 12 to 16 days, and the dose can be appropriately selected. The route of administration of the antigen is also not particularly limited, and subcutaneous administration, intradermal administration, intraperitoneal administration, intravenous administration, intramuscular administration, etc. can be appropriately selected, but injection is performed intravenously, intraperitoneally or subcutaneously. It is preferable to administer by the following method. The antigen may be an appropriate buffer, for example, complete Freund's adjuvant, RAS CMPL (Monophosphoryl Lipid A) + TDM (Synthetic Trehalose Dicorynomy co 1 ate) + CWS (Ce 11 Wall Skeleton) adjuvant system), aluminum hydroxide Can be used after dissolving it in an appropriate buffer containing a commonly used adjuvant, but the above adjuvant may not be used depending on the administration route and conditions.

After the immunized mammal is bred for 0.5 to 4 months, for example, a small amount of serum from the mammal is sampled from an ear vein or the like, and the antibody titer is measured. If the antibody titer rises, administer the antigen an appropriate number of times according to the situation. For example, booster immunization is performed using an antigen of 100 / g to 100000. 1 to 2 months after last dose Blood is collected from the sensitized mammal by a conventional method, and the blood is collected, for example, by centrifugation, precipitation using ammonium sulfate or polyethylene glycol, gel filtration chromatography, ion exchange chromatography, and affinity chromatography. A desired polyclonal antibody can be obtained as a polyclonal antiserum by separating and purifying by ordinary methods such as chromatography such as two-take mouth chromatography.

When a monoclonal antibody is produced, a desired monoclonal antibody can be obtained, for example, by producing a hybridoma by cell fusion between an antibody-producing cell and a myeloma cell line. A hybridoma producing a monoclonal antibody can be obtained by the following cell fusion method.

As antibody-producing cells, spleen cells, lymph node cells, B lymphocytes and the like from immunized animals are used. As the antigen, a virus envelope expressing the target protein is used. Mice, rats, and the like are used as animals to be immunized, and administration of the antigen to these animals is performed according to a conventional method. For example, a suspension or emulsion of an adjuvant, such as complete Freund's adjuvant or incomplete Freund's adjuvant, and a germinated baculovirus as an antigen is prepared and administered several times subcutaneously, intradermally, intraperitoneally, etc., to an animal. Immunize the animal. By obtaining, for example, spleen cells as antibody-producing cells from the immunized animal and fusing them with myeloma cells by a method known per se (G. Kohler et al. ₃ Nature, 256 495 (1975)). High pre-doma can be produced. Examples of myeloma cell lines used for cell fusion include P3X63Ag8, P3U1 strain, and Sp2 / 0 strain in mice. When performing cell fusion, a fusion promoter such as polyethylene glycol or Sendai virus is used. For selection of hybridomas after cell fusion, use of a hypoxanthine 'aminopterin' thymidine (HAT) medium in a conventional manner. Can be.

Hybridomas obtained by cell fusion are cloned by limiting dilution, etc., and screened to specifically recognize the desired protein. Thus, a cell line producing a monoclonal antibody can be obtained.

To produce the desired monoclonal antibody from the thus obtained hybridoma, the hybridoma is cultured by a usual cell culture method or ascites formation method, and the monoclonal antibody is isolated from the culture supernatant or ascites. It may be purified. Purification of the monoclonal antibody from the culture supernatant or ascites can be performed by a conventional method. For example, ammonium sulfate fractionation, gel filtration, ion exchange chromatography, affinity chromatography, and the like can be used in appropriate combination.

The present invention further provides a method of solubilizing and purifying the target protein, which comprises treating the viral envelope expressing the target protein obtained by the above-described method with a solubilizing agent. Specifically, the virus envelope in which the target protein is expressed is suspended in an appropriate buffer, treated with a lysing agent such as lyso-phosphatidylcholin, and further centrifuged (for example, at 300 rpm). This allows separation into a supernatant and a precipitate. The solubilized autologous protein is recovered in the supernatant.

All the contents described in Japanese Patent Application No. 2000-0-215 54 16 which is the basis of the priority claim of the present application are incorporated herein by reference.

The present invention will be specifically described by the following examples, but the present invention is not limited to the examples. Example

Example 1: Extracellular baculovirus expressing sterol-regulated protein (SREBP-2) treated with surfactant and separation of envelope by ultracentrifugation

(1) Sterol regulatory protein (SREBP-2)

SREBP2 is a transcription factor that regulates cholesterol-dependent transcriptional regulation of intracellular cholesterol regulation enzymes and transport proteins such as LDL receptor and HMG-CoA reductase (Brown MS, Goldstein J., Proc Natl Acad Sci USA 1999 Sep 28; 96 (20): 11041-8, A proteolytic pathway that controls the cholesterol content of membranes, cells, and blood.). SREBP 2 is 1 2 at steady state It is present in the endoplasmic reticulum membrane as a 5 kd twice transmembrane precursor protein. Lack of cholesterol in the details causes a two-step cleavage by the protease near the transmembrane site of the SREBP2 precursor protein, which cuts off the amino terminus, including the SREBP2 DNA binding site, from the membrane to the cytoplasm. Released and translocated to the nucleus. It activates transcription by binding to sre sequences on the promoter regions of various cholesterol regulatory genes.

(2) Preparation of recombinant paculovirus and culture of Sf9 cells

Human SREBP2 full-length gene (Hua X, Yokoyama C, Wu J, Briggs MR, Brown MS, Goldstein JL, Wang X, Proc Natl Acad Sci USA 1993 Dec 15; 90 (24): 11603-7., SREBP-2, a second basic-helix-loop-helix- leucine zipper protein that stimulates transcription by binding to a sterol regulatory element.) was incorporated into pBlueBacTM vector (Invitrogen, Carlsbad, CA). Sf9 cells (Invitrogen) is a 1 Ocm dish at 27 ° C with Grace's supplemented media (GIBCO BRL) containing 10% fetal serum (Sigma), penicillin 100 units / ml, streptmycin 100 jug / ml. Was subcultured. The recombinant baculovirus was prepared according to the instruction manual (Bac-N-BlueTM Transfection Kit, Invitrogen), and Sf9 cells were co-infected with Bac-N-Blue DNA (from ApMNPV) and 4 μg of pBlueBac-SREBP2 to form SREBP2. A recombinant virus was created.

(3) Expression of SREBP 2 in virus and recovery of virus

For large-scale expression, Sf9 cells were cultured on eight 15 cm dishes at a density of 2 x 10 ⁷ cells per dish, and the SREBP2 recombinant virus was infected with M0I (multiplicity of infection) 5 and 48 hours later. The culture supernatant was collected. The collected culture supernatant was centrifuged at 800 g for 10 minutes to remove cells, the supernatant was ultracentrifuged at 40,000 g for 20 minutes, and the precipitate was suspended in 4 ml of phosphate buffered saline (PBS). Virus (BV) fraction (LoiselTP, et al., Nat Biotechnol. 1997

(4) EVE fractionation by virus detergent treatment and sucrose density gradient ultracentrifugation SREBP2 expression BV fraction 250〃1 and 0.5% tween20 in PBS solution 250 1 and 1:

1 was mixed and left standing at room temperature for 10 minutes.

66.5% (W / V) sucrose / PBS solution 0.5 ml, 45% (W / V) sucrose / PBS solution 1.0 ml, 30% (W / V) sucrose PBS solution 1.0 ml 0.5 ml of the above-mentioned SREBP2-expressing BV fraction in tween20 treatment was overlaid on the upper layer, centrifuged at 350,000 g for 2 hours, and fractionated 100/1 from the bottom layer.

The sucrose concentration (refractometer) and protein concentration (Bio-Rad Protein Atsie System, BSA standard) of each fraction were measured (Figure 1). 3; For 1 8 fraction, sample 1 0 1 to 5 xsds Roh source off § one (2 M Tris-HCl, pH6.8 5 15% 3 - mercaptoethano丄, 15% SDS, 50¾ glycerol, 1.5% bromophenolblue ) Add 2.5 ju 1 and heat-treat at 95 ° C for 10 minutes. Perform 12% polyacrylamide SDS electrophoresis (SDS-PAGE) and stain the protein with silver staining (Daiichi Kagaku Kit). There were protein bands considered to be viral proteins such as gp64 and VP39 (Fig. 2). Therefore, 8 to 13 fractions were observed with an electron microscope (AKASHI EM002A) (Figures 3 to 6 show the results of electron microscope observation of 9 to 12 fractions).

(5) Electron microscopy and western western printing

Electron microscope observation was performed as follows. A collodion film was applied to a 400 grid mesh for sample observation, and carbon deposition (carbon coating) was performed. In order to easily adsorb the sample, the 400 grid mesh was pre-etched by Ionco overnight. The sample ιο〃ι was placed on a 400 g mesh and dried, and then negatively stained with a phosphoric acid solution of ginstenic acid (ΡΤΑ) and observed.

In the fractions from the sucrose gradient ultracentrifugation, BV was recovered at a sucrose concentration of 44.5 to 34.5% (8 to 13 fractions) in accordance with the evening protein staining, and the nucleoforce pseudo (Fig. 3), the virus envelope (EVE) was concentrated in 12 fractions with a sucrose concentration of 37.0% (Fig. 6), and in the 10 and 11 fractions, a mixture of nucleocabushid and envelope was observed. (Figures 4 and 5). VP 39 on SDS-PAGE Molecular weight 3 9 kDa protein distributed in agreement with the nucleosid image of the electron microscope, and molecular weight 6 known as the only viral protein present in the virus envelope 6 The density of the protein band in SDS-PAGE of gp64 of 4 kilodaltons and the distribution of the envelope image of the electron microscope are in agreement (Fig. 2 and Figs. 3 to 6). In addition, to perform immunostaining (Western plot) using a specific antibody against SREBP2, these samples were subjected to gel electrophoresis by 8% SDS-PAGE, and then subjected to nitrocellulose membrane (Hybond ECL, Amersham) at 38 V for 20 hours. Transferred to After blocking the transfer membrane with Proceed Ace for 30 minutes, a salting-out purified antibody (10 zg / ml) of the monoclonal antibody 1C6 (ATCC No CRL-2224) recognizing the carboxyl terminus of SREBP 2 was added at room temperature. Incubate for 1 hour, wash 4 times with TBS (2 OmM Tris-buffered saline, pH 7.4), react with peroxidase conjugated anti-mouse IgG antibody (CAPPEL) for 1 hour, wash with TBS in the same manner, and use ECL reagent (Amersham Pharmacia ), And exposed to an x-ray film.

As a result, a band corresponding to the 126 kd SREBP2 precursor protein was found in agreement with the distribution of EVE (Fig. 7), suggesting that the membrane protein SREBP2 expressed in the virus envelope was concentrated in EVE. . Industrial applicability

According to the present invention, a simple method for separating a nucleoside peptide of a budding virus and a virus envelope and recovering a virus membrane fraction (enucleated virion envelope, EVE) in which a target membrane protein is expressed is provided. Was.

By using the method of the present invention, the target membrane protein can be concentrated, and the interference of the Atsushi system with the DNA / nucleoside protein of the virus can be avoided. In addition, membrane proteins that have an active site inside the membrane (nucleoside side), proteins that interact with other proteins or chemicals, etc., have a part of the membrane destroyed, and substrates or chemicals added from outside cannot be used. Can reach active sites and interaction sites Need to be. In such cases, EVE technology is better than using the virus itself.

Claims

The scope of the claims

1. A method of recovering a virus envelope by separating a nucleoside of a budding virus and a virus envelope, comprising treating a baculovirus budding virus in which a target protein is expressed by a physicochemical technique.

2. The method according to claim 1, wherein the treatment by a physicochemical method is a treatment with a surfactant or a treatment by freeze-thawing and a separation operation by density gradient centrifugation.

3. The method according to claim 1, wherein tween20 is used as a surfactant.

4. The method according to any one of claims 1 to 3, wherein the target protein is a membrane protein.

5. A virus envelope in which a target protein is obtained, which is obtained by the method according to any one of claims 1 to 4.

6. Using a virus envelope expressing the target protein obtained by the method according to any one of claims 1 to 4, measuring the interaction between the target protein expressed on the virus envelope and the test substance. Screening method for chemical substances, including:

7. A polyclonal antibody or a monoclonal antibody against the target protein expressed on the viral envelope is prepared using the viral envelope expressing the target protein obtained by the method according to any one of claims 1 to 4. how to.

8. A polyclonal antibody or a monoclonal antibody obtained by the method according to claim Ί.

9. A method for solubilizing and purifying the target protein by treating the viral envelope expressing the target protein obtained by the method according to any one of claims 1 to 4 with a lysing agent.