JP2016146809A - Viral isolation device - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a viral isolation device that is excellent in hemocompatibility and is capable of efficiently isolating viruses contained in samples, even if the samples are human whole blood.SOLUTION: A viral isolation device capable of isolating a virus and the like contained in a sample from the sample comprises a given polypeptide specifically bound to a high mannose type sugar chain, and a given immobilization support that immobilizes the above polypeptide, wherein the above polypeptide is immobilized on the above immobilization support.SELECTED DRAWING: Figure 4

Description

  The present invention relates to a virus separation device. More specifically, the present invention relates to a virus separation device that has excellent blood compatibility and can separate viruses contained in a specimen and / or cells infected with the virus from the specimen even when whole blood is used as the specimen.

  Human immunodeficiency virus (hereinafter “HIV” or “AIDS virus”), influenza virus, hepatitis C virus (hereinafter “HCV”), Ebola virus, human herpes virus, severe acute respiratory syndrome (hereinafter “SARS”) virus, etc. Viruses often cause serious symptoms on the human body, and the spread of infection has become a social problem.

  These viruses have a glycoprotein having a high mannose type sugar chain on the surface, and it is known that the glycoprotein plays an important role in infecting a host (for example, Non-Patent Document 1). . In addition to the above, West Nile virus, dengue virus, Marburg virus, yellow fever virus, and cytomegalovirus have been reported as viruses having a glycoprotein having a high mannose sugar chain on the surface. In addition, various hemorrhagic viruses such as severe febrile thrombocytopenia syndrome (SFTS) virus are infected by dendritic cells via the DC-SIGN receptor, and thus glycoproteins having high mannose-type sugar chains on the surface. It is considered to have.

  In addition to viruses, protozoan Leishmania, mold Candida albicans, other mycobacteria, Schistosoma mansoni, etc. also have glycoproteins with high mannose sugar chains on their surfaces. It has been reported that

  So far, many types of lectins have been isolated from seaweeds or algae (freshwater cyanobacteria) and their biochemical properties have been elucidated. It is known that a part of the lectin specifically binds to a high mannose sugar chain and specifically binds to the above-described viruses such as HIV and influenza virus (Non-patent Documents 2 to 12). ). In addition, an anti-virus targeting a lectin (MPL) derived from Meristotheca papulosa (Montagne) J. Agardh that targets a virus having a high mannose sugar chain (a virus in which a high mannose sugar chain is present on the surface of the virus) It is also known that it can be used as an agent (Patent Document 5).

  Regarding the separation of HIV and the like using a lectin, as an invention relating to the binding of lectin and HIV, for example, HIV or a part thereof is supported on a fine particle-immobilized carrier comprising a hydrophobic substrate to which a lectin such as concanavalin A (ConA) is bound. Virus in the blood of an individual infected with a virus by passing blood through a porous hollow fiber membrane fixed with a lectin such as GNA, which contains microparticles obtained by capturing the protein or peptide (Patent Document 1) And a method for reducing lectin-binding fragments thereof (Patent Document 2), and a composition containing a mannan-binding lectin oligomer containing a mannan-binding lectin subunit and the like in the manufacture of a medicament for the prevention and / or treatment of infectious diseases in cancer patients (Patent Document 3), succinyl-concanava Using lectin such emissions A as a binding component, such as a method of increasing the viral titer of the sample comprising viral particles (Patent Document 4) is known.

  Further, as a protein array for quantifying the amount of protein on the protein array, a protein array is known in which proteins are aligned on a monolith gel substrate and immobilized at a high density (Patent Document 6).

  The lectin is derived from the Latin lectus (meaning selection), and was first discovered in 1888 as a hemagglutinin from castor bean extract by Hermann Stillmark, a Russian researcher. is there. And many lectins discovered so far have been discovered by the hemagglutination activity and the inhibition test (nonpatent literature 13).

JP 2001-335510 A (published on December 4, 2001) Special table 2007-525232 gazette (announced September 6, 2007) Special table 2008-535872 publication (announced September 4, 2008) Special Table 2004-500831 Publication (announced on January 15, 2004) JP 2012-213382 A (published November 8, 2012) JP 2010-127853 A (released on June 10, 2010)

Kilby, J. M. and Eron, J. J., N. Engl. J. Med. 348, 2228-2238, 2003. Boyd, M. R. et al., Antimicrob. Agents Chemother. 41, 1521-1530, 1997. O’Keefe, B. R. et al., Antimicrob. Agents Chemother. 47, 2518-2525, 2003. Helle, F., .et al., J. Biol. Chem. 281, 25177-25183, 2006. Barrientos, L. G., et al., Antiviral. Res. 58, 47-56, 2003. Dey, B., et al., J. Virol. 74, 4562-4569, 2000. O’Keefe, B. R. et al., J. Virol. 84, 2511-2521, 2010. Hori, K. et al., Glycobiology, 17, 479-491, 2007. Sato, Y. et al., J. Biol. Chem. 282, 11021-11029, 2007. Sato, Y. et al., Biochem. Biophys. Res. Commun. 405, 291-296, 2011. Yuichiro Sato, Makoto Hirayama, Yoshifumi Fujiwara, Kinjiro Morimoto, Kanji Hori (2010) Abstract of the 13th Annual Meeting of the Marine Biotechnology Society (2010. 5.29 presentation) Sato, Y. et al., J. Biol. Chem. 286, 19446-19458, 2011. Abstracts of the 2009 Annual Meeting of the Japan Society for Agricultural Chemistry 2009 Date: July 4, 2009 Venue: Nippon Veterinary and Life Science University Presentation: 2009 Agricultural Chemistry Society Encouragement Award Lecture R1-01 Structure of lectins Functional analysis and application to glycobiology, Hiroaki Kanno (National Institute of Advanced Industrial Science and Technology, Glycomedical Engineering Research Center)

  However, the lectins used in Patent Documents 1 to 4 have a hemagglutination effect. For this reason, the lectin cannot be used directly in a device for separating blood virus from whole blood, and it has been necessary to make contact after plasma separation.

  On the other hand, seaweeds or algae-derived lectins are known to exhibit binding properties to viruses as described above, and are known to aggregate rabbit and sheep erythrocytes treated with trypsin or pronase. ing. However, the compatibility with human blood was not known, and there was no knowledge as to whether it was possible to isolate viruses contained in blood without affecting human blood. . Further, there has been no knowledge as to what kind of immobilization carrier the lectin is immobilized on, so that viruses in blood can be efficiently separated even when whole blood is treated.

  The present invention has been made in view of such problems, and the object thereof is excellent in blood compatibility, and even if the specimen is human whole blood, the virus contained in the specimen is efficiently separated. It is to provide a possible virus isolation device.

  In order to solve the above-mentioned problems, the present inventors have intensively studied and produced a device in which a polypeptide that specifically binds to a high mannose sugar chain is immobilized on an immobilization carrier. And by using the device, even when the specimen is human whole blood, it has been found that the virus contained in the specimen and / or cells infected with the virus can be efficiently separated, and the present invention has been completed. It came to do. That is, the present invention includes the following inventions.

  [1] A virus separation device capable of separating a virus contained in a sample and / or a virus-infected cell from the sample, and immobilizing the polypeptide that specifically binds to a high-mannose sugar chain and the polypeptide The polypeptide is immobilized on the immobilization carrier, the immobilization carrier is a monolith gel, and the polypeptide includes type I lectin, type II lectin, type III lectin and type A device comprising one or more lectins selected from the group consisting of IV lectins.

  [2] The specimen is human whole blood, and the virus contained in the whole blood and / or cells infected with the virus can be separated from the whole blood by contacting the whole blood with the polypeptide. [1] The device according to [1].

  [3] The device according to [1] or [2], wherein the monolith gel has a macropore diameter of 1 μm to 200 μm and a mesopore diameter of 40 nm to 200 nm.

  [4] The device according to [3], wherein the monolith gel has a macropore diameter of 20 μm to 200 μm.

  [5] The device according to any one of [1] to [4], wherein the monolith gel is a silica monolith.

  [6] The device according to any one of [1] to [5], wherein 2 μg or more of the polypeptide is immobilized per 1 ml of the immobilization carrier.

  [7] The device according to any one of [1] to [6], wherein the polypeptide is immobilized on the immobilization carrier at a carboxy terminus of a peptide chain.

  [8] The device according to [7], wherein the naturally occurring amino acid sequence of the polypeptide has 1 or less cysteine residues.

  [9] The device according to [8], wherein the naturally occurring amino acid sequence of the polypeptide does not contain a cysteine residue.

  [10] The device according to [9], wherein the naturally occurring amino acid sequence of the polypeptide does not contain a cysteine residue or a lysine residue.

  [11] The device according to any one of [1] to [10], wherein the virus is a virus having a high mannose sugar chain.

  [12] The virus is one or more viruses selected from the group consisting of influenza virus, hepatitis C virus, human herpes virus, dengue virus, AIDS virus, Ebola virus and severe acute respiratory syndrome virus. The device according to any one of [1] to [11], which is characterized.

  [13] The device according to any one of [1] to [12], wherein the virus and / or a cell infected with the virus is an AIDS virus and / or an AIDS virus-infected T cell.

  In the virus separation device according to the present invention, a predetermined polypeptide that specifically binds to a high-mannose sugar chain is immobilized on a monolith gel that is an immobilization carrier, so that it does not affect human blood, The virus and / or virus-infected cells can be efficiently separated from the specimen.

It is a schematic diagram which shows the outline of the structure of polypeptide for carrier fixation | immobilization which is a recombinant. It is a graph which shows the grade of the hemolysis when the contact time to a lectin is 5 minutes when 1% hemolysis (Positive Control) is 1.0. It is a graph which shows the grade of the hemolysis in which the contact time to a lectin is 30 minutes when 1% hemolysis (Positive Control) is 1.0. It is a photograph which shows the result of having dye | stained P24 protein which is an antigen of HIV-1 by the fluorescent antibody method about the column passage fraction of a virus separation device. It is a figure which shows an example of the structure of a high mannose type sugar chain.

  Hereinafter, the virus separation device according to the present invention will be described in detail, but the scope of the present invention is not limited to these explanations, and other than the following examples, as appropriate, within the scope not impairing the spirit of the present invention, Can be implemented.

  In the present specification, “A to B” indicating a range represents A or more and B or less. Moreover, the patent document and nonpatent literature described in this specification are used as reference in this specification.

[1. Virus isolation device
The virus separation device according to the present invention is a virus separation device capable of separating a virus contained in a specimen and / or a virus-infected cell from the specimen, and a polypeptide that specifically binds to a high-mannose sugar chain An immobilization carrier that immobilizes the polypeptide, the polypeptide is immobilized on the immobilization carrier, the immobilization carrier is a monolith gel, and the polypeptide includes type I lectin and type II lectin. , One or more lectins selected from the group consisting of type III lectins and type IV lectins.

(1) Polypeptide that specifically binds to high mannose-type sugar chains High-mannose-type sugar chains are called “trimannosyl cores” common to N-type sugar chains [Manα1-6 (Manα1-3) Manβ1-4GlcNAcβ1 -4GlcNAc] in addition to the α-mannose residue in the branched structure, [Manα1-6 (Manα1-3) Manα1-6 (Manα1-3) Manβ1-4GlcNAcβ1-4GlcNAc] This sugar chain contains the heptasaccharide as a common mother nucleus.

  The phrase “specifically binds to a high mannose type sugar chain” means having a binding property to a high mannose type sugar chain and not having a binding property to a complex type sugar chain or a mixed sugar chain.

  As described above, viruses such as HIV virus and influenza virus have glycoproteins having high mannose type sugar chains on the surface.

  On the other hand, it is considered that high mannose-type sugar chains and the above-mentioned glycoprotein hardly exist on the surface of human somatic cells. Since the virus separation device according to the present invention includes a polypeptide that specifically binds to a high mannose sugar chain, for example, when it comes into contact with a biological sample such as human whole blood, the virus having the glycoprotein Is contained in the sample, it specifically binds to the high mannose type sugar chain of the virus, and therefore the influence on other components in the sample is very small.

  In addition, the virus separation device according to the present invention is in a state in which the polypeptide is easily contacted with the virus because the polypeptide is immobilized on a monolith gel that is an immobilization carrier, and the virus is very efficiently produced. And / or cells infected with the virus (hereinafter, the virus and cells infected with the virus may be simply referred to as “virus”) can be separated from the specimen.

  From the above, the virus separation device according to the present invention can very efficiently separate the virus in the specimen from the specimen without affecting the specimen such as human whole blood.

  Here, the “sugar chain” of the high mannose type sugar chain means a linear or branched oligosaccharide or polysaccharide. An oligosaccharide refers to a product obtained by dehydrating 2 to 10 monosaccharides or substituted derivatives of monosaccharides. Furthermore, a saccharide to which a large number of monosaccharides are bonded is called a polysaccharide.

In the present invention, polypeptides that specifically bind to high mannose-type sugar chains are classified into the following four types based on the recognition site of branched oligomannoside and the difference in primary structure (Hirukiru Osamu, Bioscience and Industry vol.71 No.2 (2013) 129-133), that is, one or more lectins selected from the group consisting of type I lectin, type II lectin, type III lectin and type IV lectin are used. FIG. 5 is a diagram showing an example of the structure of a high mannose sugar chain. In the figure, D1 to D3 represent D1 arm to D3 arm, respectively.
(A) Type I: Strongly bound to an α (1-3) Man residue at the non-reducing end of the D2 arm of a high mannose sugar chain, and α (1-2) Man was added to the residue In the case of bonding, the bonding force is significantly reduced. In addition, a Manα1-6 (Manα1-3) Manα1-6 (Manα1-3) -Man structure is used as a recognition site.
(B) Type II: α (1-2) Man residue at the non-reducing end of the D1 arm, α (1-2) Man residue at the non-reducing end of the D2 arm, and α ( 1-2) Uses a Man residue as a recognition site and binds more strongly to a high-mannose sugar chain having a large number of α (1-2) Man residues. It does not bind to a high mannose sugar chain that does not have an α (1-2) Man residue at the non-reducing end.
(C) Type III: Recognizing the difference in structure of the branched sugar chain, all high mannose sugar chains and free trimannosyl core structure ((Manα1-6 (Manα1-3) Manβ1-4GlcNAcβ1-4GlcNAc-PA) The binding to a high mannose sugar chain is higher than the binding to a free trimannosyl core structure.
(D) Type IV: binds only to those having an α (1-2) Man residue at the non-reducing end of the D3 arm.

  Examples of the lectin belonging to any one of the above (a) to (d) include algae-derived lectins which are lectins that can be isolated from seaweeds or cyanobacteria. “Lectin” is a generic term for proteins having a sugar-binding domain in the molecule, excluding antibodies.

  Examples of the algae-derived lectin belonging to any of the above (a) to (d) include, for example, type I lectin, OAA (UniProtKB / Swiss-Prot Accession No .: P84330) derived from the freshwater cyanobacteria Oscillatoria agardhii; No. 5), ESA derived from seaweed Eucheuma serra (ESA-1, ESA-2 (GenBank Accession No .: P84331; amino acid sequence shown in SEQ ID NO: 29)), KAA derived from seaweed Kappaphycus alvarezii ( KAA-1 (GenBank Accession No: LC007080; amino acid sequence shown in SEQ ID NO: 6), KAA-2 (GenBank Accession No: LC007081; amino acid sequence shown in SEQ ID NO: 30), KAA-3), derived from the seaweed Kappaphycus striatum KSA (KSA-1, KSA-2), EAA derived from seaweed Eucheuma amakusaensis (EAA-1, EAA-2, EAA-3), seaweed Eucheum EDA derived from a denticulatum (EDA-1, EDA-2 (GenBank Accession No .: LC007085; amino acid sequence shown in SEQ ID NO: 33), EDA-3), Solin derived from seaweed mirin (Solieria pacifica) (Solnin A, Solnin) B, Solnin C), MPA derived from the seaweed Meristotheca papulosa (MPA-1 (GenBank Accession No: LC008514; amino acid sequence shown in SEQ ID NO: 34)), MPA-2 (GenBank Accession No: LC008515; amino acid sequence No. 35)), Granin-BP derived from the seaweed Shiramo (Gracilaria bursa-pastoris), ASL derived from the seaweed Agardhiella subulata (ASL-1 (GenBank Accession No: LC007083; amino acid sequence shown in SEQ ID NO: 31)), ASL- 2 (GenBank Accession No: LC007084; amino acid sequence shown in SEQ ID NO: 32)), PF derived from the bacterium Pseudomonas fluorescens L (GenBank Accession No: ABA72252; amino acid sequence is shown in SEQ ID NO: 37), MBHA derived from bacteria Myxococcus xanthus (GenBank Accession No: M13831; amino acid sequence is shown in SEQ ID NO: 38), BOA derived from bacteria Burkholderia oklahomensis (GenBank Accession No: AIO69853; the amino acid sequence is shown in SEQ ID NO: 39) and the like.

  In addition, the present inventors have found from the database (GenBank) search that theoretical proteins having a sequence in common with the amino acid sequence of type I lectin are also present in other species (bacteria, cyanobacteria). theoretical proteins can also be used. Examples of the theoretical proteins include, for example, a protein having the deduced amino acid sequence shown in SEQ ID NO: 40 of Lyngbya sp. And a protein having a deduced amino acid sequence shown in SEQ ID NO: 42 of Stigmatella aurantiaca DW4 / 3-1 (Accession No. ZP — 01464390).

  As the type II lectin, for example, BCA (GenBank Accession No: BAK23238; amino acid sequence is shown in SEQ ID NO: 7) derived from seaweed Boodlea coacta can be used.

  Examples of type III lectins include BPL-17 (GenBank Accession No: BAI43482; amino acid sequence shown in SEQ ID NO: 8) derived from seaweed honey (Bryopsis plumosa), BML-17 (GenBank Accession No: BAI94585; the amino acid sequence is shown in SEQ ID NO: 36) and the like.

  Examples of type IV lectins include MPL-1 derived from the seaweed Meristhotheca papulosa (GenBank Accession No: LC007082; amino acid sequence shown in SEQ ID NO: 9), MPL-P2, MPL-2 and MPL-P4. In the amino acid sequence shown in SEQ ID NO: 9, the N-terminus is pyroglutamic acid.

  As mentioned above, although the type I to type IV lectins are exemplified, the polypeptide that specifically binds to the high mannose type sugar chain used in the present invention is not limited to the lectins exemplified above. When using 2 or more types of said lectins, ratio of the usage-amount may be arbitrary.

  As used herein, the term “polypeptide” is used interchangeably with “peptide” or “protein”. “Polypeptides that specifically bind to high mannose-type sugar chains” including the algal-derived lectins may be isolated from natural sources or chemically synthesized.

  The algae-derived lectin can be isolated from seaweeds or cyanobacteria by a conventionally known method. For example, OAA is a method disclosed in literature (Sato, Y. et al., Comp. Biochem. Physiol. B Biochem. Mol. Biol., 125, 169-177, 2000); ESA-1 and ESA-2 are literature (Kawakubo, A. et al., J. Appl. Phycol. 9, 331-338, 1997); EAA-1, EAA-2 and EAA-3 are described in the literature (Kawakubo, A. et al., J. Appl. Phycol. 11, 149-156, 1999); EDA-1, EDA-2 and EDA-3 are published in the literature (Hung, LD et al., J. Appl. Phycol. In press (DOI 10.1007 / s10811-014-0441-0)); KAA-1, KAA-2 and KAA-3 are described in the literature (Hung, LD et al., Fish. Sci. 75, 723-730, 2009). Disclosed methods; KSA-1 and KSA-2 are disclosed in literature (Hung, LD et al., Phytochemistry 72, 855-861, 2011); Solnin A, Solnin B and Solnin C are published in literature (Hori, K. et al., Phytochemistry 27, 2063-2067,1988) Granin-BP is the method disclosed in the literature (Okamoto, T. et al., Experientia. 46, 975-977, 1990); MBHA is the literature (Cumsky, MG, Zusman, DR, J. Biol. Chem. 256, 12581-12588, 1981), respectively.

  BOA is disclosed in the literature (Whitley, MJ et al., FEBS J. 280, 2056-2067, 2013), and PFL is published in the literature (Sato, Y. et al., PLoS One 7, e45922, 2012). Each can be isolated using the E. coli expression system by the disclosed method.

  MPA-1 and MPA-2 were prepared by pulverizing the seaweed Meristotheca papulosa fresh algal bodies under liquid nitrogen, followed by extraction with 20 mM phosphate buffer (pH 7.0) containing 0.85% NaCl. It can be isolated by subjecting it to ammonium sulfate salting out and ion exchange chromatography.

  ASL-1 and ASL-2 were prepared by pulverizing seaweed Agaldhiella subulata raw algal bodies under liquid nitrogen, extraction with 20 mM phosphate buffer (pH 7.0) containing 0.85% NaCl, It can be isolated by subjecting it to ammonium sulfate salting out, ion exchange chromatography and gel filtration.

  BCA can be isolated by the method disclosed in the literature (Sato, Y. et al., J. Biol. Chem. 286, 19446-19458, 2011). BML-17 can be isolated from the method disclosed in Japanese Patent No. 4876258, and BPL-17 can be isolated from the seaweed Bryopsis plumosa by a method similar to the method for isolating BML-17 disclosed in the above publication.

  The term “isolated” polypeptide or protein is intended to be a polypeptide or protein that has been removed from its natural environment. For example, recombinantly produced polypeptides and proteins expressed in a host cell can be isolated in the same manner as natural or recombinant polypeptides and proteins that have been substantially purified by any suitable technique. It is thought that there is.

  Synthetic peptides can be synthesized using known methods of chemical synthesis. For example, Houghten is prepared for the synthesis of multiple peptides such as 10-20 mg of 248 different 13 residue peptides that represent a single amino acid variant of the HA1 polypeptide segment prepared and characterized in less than 4 weeks. A simple method is described (Houghten, RA, Proc. Natl. Acad. Sci. USA 82: 5131-5135 (1985)). This “Simultaneous Multiple Peptide Synthesis (SMPS)” process is further described in US Pat. No. 4,631,211 to Houghten et al. (1986). In this procedure, individual resins for solid phase synthesis of various peptides are contained in separate solvent permeable packets, allowing optimal use of many identical iteration steps associated with solid phase methods. Complete manual procedures allow 500-1000 or more syntheses to be performed simultaneously (Houghten et al., Supra, 5134). These documents are incorporated herein by reference.

  Polypeptides that specifically bind to the high mannose-type sugar chains include natural purified products, products of chemical synthesis procedures, and prokaryotic or eukaryotic hosts (eg, bacterial cells, yeast cells, higher plant cells, insects). Cells, and products produced by recombinant techniques from mammalian cells). Depending on the host used in the recombinant production procedure, the polypeptide may be glycosylated or non-glycosylated. In addition, the polypeptide may also include an initiating modified methionine residue in some cases as a result of host-mediated processes.

  The amino acid sequence and base sequence of the algae-derived lectin belonging to any one of the above-described (a) to (d) are known. In one embodiment, the algal-derived lectin may be a polypeptide consisting of each published amino acid sequence, or may be a variant of the polypeptide.

  Variants include deletions, insertions, inversions, repeats, and type substitutions (eg, replacement of a hydrophilic residue with another residue, but usually a strong hydrophilic residue with a strong hydrophobic residue Are not substituted). In particular, “neutral” amino acid substitutions in a polypeptide generally have little effect on the activity of the polypeptide.

  It is well known in the art that some amino acids in the amino acid sequence of a polypeptide can be easily modified without significantly affecting the structure or function of the polypeptide. Furthermore, it is also well known that there are variants in natural proteins that do not significantly alter the structure or function of the protein, not only artificially modified.

  One skilled in the art can readily mutate one or several amino acids in the amino acid sequence of a polypeptide using well-known techniques. For example, according to a known point mutation introduction method, an arbitrary base of a polynucleotide encoding a polypeptide can be mutated. In addition, a deletion mutant or an addition mutant can be prepared by designing a primer corresponding to an arbitrary site of a polynucleotide encoding a polypeptide.

  Preferred variants have conservative or non-conservative amino acid substitutions, deletions, or insertions. Silent substitution, insertion and deletion are preferred, and conservative substitution is particularly preferred. These do not alter the polypeptide activity according to the invention.

  Representative conservative substitutions are substitutions of one amino acid for another in the aliphatic amino acids Ala, Val, Leu, and Ile; exchange of hydroxyl residues Ser and Thr, acidic residues Asp and Glu exchange, substitution between amide residues Asn and Gln, exchange of basic residues Lys and Arg, and substitution between aromatic residues Phe, Tyr.

  As detailed above, further guidance on which amino acid changes are likely to be phenotypically silent (ie, not likely to have a significant adverse effect on function) can be found in Bowie, J. . U. “Deciphering the Message in Protein Sequences: Tolerance to Amino Acid Substations”, Science 247: 1306-1310 (1990), which is incorporated herein by reference.

  The algae-derived lectin is a polypeptide comprising each published amino acid sequence; or a polypeptide comprising an amino acid sequence in which one or several amino acids are substituted, deleted, inserted, or added in the amino acid sequence. A peptide is preferred.

  The above “one or more amino acids are substituted, deleted, inserted, or added” means substitution, deletion, insertion, or the like by a known mutant polypeptide production method such as site-directed mutagenesis. It means that as many amino acids as can be added (preferably 10 or less, more preferably 7 or less, most preferably 5 or less) are substituted, deleted, inserted or added. As described above, such a mutant polypeptide is not limited to a polypeptide having a mutation artificially introduced by a known mutant polypeptide production method, and a naturally occurring polypeptide is isolated and purified. It may be what you did.

  On the other hand, the “polypeptide that specifically binds to a high-mannose sugar chain” used in the virus separation device according to the present invention is preferably immobilized on an immobilization carrier by controlling the orientation. In other words, the polypeptide is preferably immobilized on the immobilization carrier at one end of the peptide chain. As will be described later, in order to control the orientation and immobilize it on the immobilization carrier, (1) a naturally occurring amino acid sequence of a polypeptide that specifically binds to a high mannose-type sugar chain has a cysteine residue. (2) the naturally occurring amino acid sequence of a polypeptide that specifically binds to a high mannose sugar chain does not contain a cysteine residue; or (3) the high mannose sugar chain contains More preferably, the naturally occurring amino acid sequence of the polypeptide that specifically binds does not contain cysteine and lysine residues. Therefore, although it is possible to perform the amino acid substitution or amino acid addition described above, it is more preferable not to substitute a certain amino acid with cysteine or lysine.

  The “polypeptide that specifically binds to high mannose-type sugar chains” used in the virus separation device according to the present invention may be any polypeptide in which amino acids are peptide-bonded, but is not limited thereto. Alternatively, it may be a complex polypeptide containing a structure other than the polypeptide. As used herein, examples of the “structure other than the polypeptide” include sugar chains and isoprenoid groups, but are not particularly limited.

  In addition, the “polypeptide that specifically binds to a high mannose-type sugar chain” may include an additional polypeptide. Examples of the additional polypeptide include epitope-tagged polypeptides such as His, Myc, and Flag.

  The “polypeptide that specifically binds to a high-mannose sugar chain” can be expressed as a recombinant in a modified form such as a fusion protein. As will be described later, when a monolith gel such as silica monolith is used as an immobilization carrier, it is preferably expressed as a recombinant in which a linker sequence, an immobilization reaction sequence, and a purification tag sequence are added to the carboxy terminus of the polypeptide. . The purification tag sequence can contribute to convenient purification of the fusion protein and can be removed prior to final preparation of the polypeptide.

  Purification tag sequences include, for example, hexahistidine peptides (eg, tags provided in the pQE vector (Qiagen, Inc.)), “HA useful for purification corresponding to epitopes derived from influenza hemagglutinin (HA) protein. ”Tag or the like.

(2) Immobilization carrier for immobilizing a polypeptide A virus separation device according to the present invention includes an immobilization carrier for immobilizing the above-mentioned “polypeptide that specifically binds to a high-mannose sugar chain”. The immobilization carrier includes macropores (macropores) that serve as solution flow paths and mesopores (mesopores) that serve as separation sites, which facilitates immobilization of the polypeptide and also has whole blood. A monolith gel is used as the immobilization carrier because it can be processed and the high-mannose sugar chain contained in the virus contained in the specimen can be efficiently bound.

  The monolith gel refers to a gel made of a monolith material (monolith type polymer). The monolith material is composed of a single continuous structure and has pores that are continuous channels through the structure. The monolith gel can be produced by a solation step for producing an aqueous solution sol, a gelation step for heating the obtained sol to form a gel, and a firing step for firing the obtained gel.

  The monolith gel is obtained, for example, by causing a reaction solution containing silica as a main component to cause a sol-gel transition accompanied by phase separation. Examples of precursors of network components that cause gel formation used in sol-gel reactions include metal alkoxides, complexes, metal salts, organically modified metal alkoxides, organic cross-linked metal alkoxides, and partial hydrolysis products and partial polymerization products thereof. Can be used. Sol-gel transition by changing the pH of water glass and other silicate aqueous solutions can be used as well.

  More specifically, the monolith gel is obtained by dissolving a water-soluble polymer and a thermally decomposable compound in an acidic aqueous solution, adding a metal compound having a hydrolyzable functional group to the hydrolyzed reaction, and solidifying the product. Then, it is preferable to heat the gel in a wet state, thereby thermally decomposing the low-molecular compound previously dissolved at the time of gel preparation, and then drying and heating.

  Here, the water-soluble polymer is a water-soluble organic polymer that can be theoretically formed into an aqueous solution having an appropriate concentration, and includes a reaction system including an alcohol generated by a metal compound having a hydrolyzable functional group. Any material that can be uniformly dissolved therein may be used. Specifically, sodium or potassium salt of polystyrene sulfonic acid, which is a polymer metal salt, polyacrylic acid which is a polymer acid and dissociates to become a polyanion, a polymer base which produces a polycation in an aqueous solution. Polyallylamine and polyethyleneimine, or a neutral polymer, polyethylene oxide having an ether bond in the main chain, polyvinylpyrrolidone having a carbonyl group in the side chain, and the like are preferable. In addition, formamide, polyhydric alcohol, or surfactant may be used in place of the organic polymer. In this case, glycerin is optimal as the polyhydric alcohol, and polyoxyethylene alkyl ethers are optimal as the surfactant.

  As the metal compound having a hydrolyzable functional group, a metal alkoxide or an oligomer thereof can be used, and those having a small number of carbon atoms such as a methoxy group, an ethoxy group, and a propoxy group are preferable. Further, as the metal, an oxide metal finally formed, for example, Si, Ti, Zr, or Al is used. This metal may be one type or two or more types. On the other hand, any oligomer can be used as long as it can be uniformly dissolved and dispersed in alcohol. Specifically, it can be used up to about 10-mer.

  Moreover, as said acidic aqueous solution, the thing of 0.001 mol concentration or more of mineral acids, such as hydrochloric acid and nitric acid, or organic acid 0.01 mol concentration or more of acetic acid, formic acid, etc. is preferable normally.

  Phase separation and gelation can be achieved by storing the solution at room temperature of 40 to 80 ° C. for 0.5 to 5 hours. Phase separation / gelation is a process in which an initially transparent solution becomes cloudy, causing phase separation between a silica phase and an aqueous phase and finally gelling. By this phase separation / gelation, the water-soluble polymers are in a dispersed state and their precipitation does not substantially occur.

  Specific examples of the thermally decomposable compound to coexist in advance include urea or hexamethylenetetramine, formamide, N-methylformamide, N, N-dimethylformamide, acetamide, N-methylacetamide, N, N-dimethyl. Although organic amides such as acetamide can be used, since the pH value of the solvent after heating is an important condition, there is no particular limitation as long as it is a compound that makes the solvent basic after thermal decomposition.

  Although the said thermally decomposable compound to coexist is based also on the kind of compound, in the case of urea, for example, it is 0.05-0.8g with respect to 10g of reaction solutions, Preferably it is 0.1-0.7g. The heating temperature is preferably 40 to 200 ° C. in the case of urea, for example, and the pH value of the solvent after heating is preferably 6.0 to 12.0.

  Moreover, what produces the compound with the property to melt | dissolve a silica like hydrofluoric acid by thermal decomposition can be utilized similarly.

  In the above method, when a water-soluble polymer is dissolved in an acidic aqueous solution, and a hydrolysis reaction is performed by adding a metal compound having a hydrolyzable functional group thereto, a gel separated into a solvent-rich phase and a skeleton phase is generated. . After the product (gel) has solidified, an appropriate aging time has passed, and then the wet gel is heated to thermally decompose the amide compound dissolved in the reaction solution in advance. The pH of the solvent in contact with increases. The solvent erodes the inner wall surface, and gradually changes the pore size by changing the uneven state of the inner wall surface.

  In the case of gels containing silica as the main component, the degree of change is very small in the acidic or neutral region, but as thermal decomposition becomes more vigorous and the basicity of the aqueous solution increases, the part constituting the pore dissolves. Then, by reprecipitation in a flatter portion, a reaction in which the average pore diameter becomes large occurs remarkably.

  In gels that have only three-dimensionally confined pores without huge pores, the elution substance cannot be diffused to the external solution even if it can be dissolved as an equilibrium condition. Remains in a considerable proportion. On the other hand, in a gel having a solvent-rich phase that becomes giant pores, there are many pores that are restricted only two-dimensionally, and exchange of substances with an external aqueous solution occurs frequently enough. In parallel with the development, small pores disappear and the entire pore size distribution does not spread significantly.

  In the heating process, it is effective to place the gel in a hermetically sealed condition so that the vapor pressure of the pyrolysis product is saturated and the pH of the solvent quickly takes a steady value.

  The heat treatment time required for the dissolution / reprecipitation reaction to reach a steady state and obtain the corresponding pore structure varies depending on the size of the huge pores and the volume of the sample. It is necessary to determine the shortest processing time at which the pore structure does not change.

  The gel after the heat treatment becomes a dry gel that is in close contact with the tube wall in the groove by vaporizing the solvent. Since the coexisting substances in the starting solution may remain in this dry gel, the target inorganic porous material can be obtained by heat-treating at an appropriate temperature and thermally decomposing organic matter. it can. In addition, drying is performed by leaving at 30-80 degreeC for several hours-several dozen hours, and heat processing is heated at about 200-800 degreeC.

  As the monolith gel, for example, an organic monolith such as an acrylamide type, a methacrylic acid ester type, a styrene-divinylbenzene type, a silica monolith, or the like can be used, and a silica monolith is particularly suitable because it easily introduces a primary amino group described later. However, the present invention is not limited to this.

  The monolith gel preferably has a macropore diameter of 1 to 200 μm and a mesopore diameter of 40 to 200 nm. According to the above configuration, the macropore diameter is a size that allows blood cells to pass through, and the mesopore diameter is about twice or more the size of the virus to be separated. The amount of separable virus per volume can be kept high.

  Therefore, by fixing the “polypeptide that specifically binds to a high mannose sugar chain” to the monolith gel, the high mannose sugar chain of the virus can be specifically bound to the polypeptide. Therefore, the virus can be efficiently separated from the specimen.

  In addition, it is not preferable that the mesopore diameter exceeds 200 nm because the gel strength is remarkably lowered and the gel cannot endure actual use.

  More preferably, the monolith gel has a macropore diameter of 20 μm or more and 200 μm or less, and a mesopore diameter of 40 nm or more and 200 nm or less. According to the above configuration, it is possible to pass blood cells and maintain high resolution while reducing the load pressure on the macropore. Therefore, the virus can be more efficiently separated from the specimen, and the whole blood can be easily processed.

  Monolith gels can be prepared by the sol-gel method as described above. A monolith gel having a macropore diameter of a desired size such as 1 μm or more and 200 μm or less, 20 μm or more and 200 μm or less is a block copolymer that affects the molecular weight and addition amount of a water-soluble polymer added to induce phase separation, and the phase separation timing It can be obtained by adjusting the addition amount of the sol and adjusting the viscosity of the sol.

  The mesopore diameter of the monolith gel can be controlled by the aging conditions after gelation. For example, by adjusting the solvent composition, the heating temperature, and the heating time in aging, a monolith gel having a mesopore diameter of a desired size such as 10 nm to 200 nm or 40 nm to 200 nm can be obtained.

  The macropore diameter and the mesopore diameter can be confirmed, for example, by measuring the pore size distribution by a conventionally known method such as a mercury intrusion method or by observing the monolith gel with a microscope.

  Moreover, a monolith gel may use a commercial item. As a commercially available product, for example, MonoBis column, low pressure type, unmodified product number: 3250L30SI, macropore diameter 1.4 μm, mesopore diameter 30 nm (manufactured by Kyoto Monotech Co., Ltd.), etc. can be used.

  The “polypeptide that specifically binds to a high mannose sugar chain” is preferably immobilized at 2 μg or more, more preferably at least 10 μg, more preferably at least 100 μg, per ml of the immobilized carrier. Preferably, 1 mg or more is fixed. According to the said structure, since the said polypeptide is fix | immobilized with high density per unit volume of the fixed support | carrier, it is preferable when isolate | separating the virus in a test substance from a test substance efficiently.

  The upper limit of the amount of the polypeptide immobilized on 1 ml of the carrier is preferably 10 mg or less.

  Whether the polypeptide is immobilized in a desired amount on the immobilization carrier, for example, the absorbance of the polypeptide in the reaction solution before and after the reaction is measured at 280 nm, and the reaction is performed based on the measurement result. This can be confirmed by determining the amount of the polypeptide consumed and setting this amount as the immobilized amount. Details will be described later in Example 4.

(3) Method of immobilizing the polypeptide on the immobilization carrier The method of immobilizing the polypeptide on the immobilization carrier (monolith gel) is not particularly limited, but in order to immobilize at a high density as described above, It is preferable to immobilize the polypeptide on an immobilization carrier by controlling the orientation. The immobilization of a polypeptide whose orientation is controlled means that the polypeptide is immobilized on the above-mentioned immobilization carrier at one end of the peptide chain, for example, it is immobilized at the carboxy terminus of the peptide chain. Examples of the method for immobilizing the polypeptide by controlling the orientation include the methods described in Japanese Patent No. 2517861, Japanese Patent Application Laid-Open No. 2000-119300, and Japanese Patent Application Laid-Open No. 2003-344396.

  Since the immobilization carrier is a monolith gel, primary amino groups are introduced into the monolith gel at high density by introducing an epoxy group into the monolith gel and then introducing an amino group-containing polymer in order to immobilize the polypeptide. It is preferable to do. As a result, the polypeptide can be immobilized on a monolith gel with efficient orientation control.

  Introduction of an epoxy group into the monolith gel can be performed, for example, by immersing the monolith gel in a solution of epoxy silane. The introduction of primary amino groups may be achieved by adding an amino group-containing polymer to the surface of the monolith gel and allowing it to penetrate inside. By these operations, an epoxy group can be introduced into the monolith gel, and a primary amino group can be further covalently bonded to the epoxy group.

  As an example of the immobilization of the polypeptide to the primary amino group, a method based on the method disclosed in JP 2000-119300 A will be described.

The “polypeptide that specifically binds to a high mannose-type sugar chain” is represented by the general formula (2).
NH ₂ —R ₁ —COOH (2)
(Wherein R ₁ represents any amino acid residue)
Next, a fusion polypeptide represented by the following general formula (4) in which a peptide represented by the following general formula (3) is bound to the polypeptide represented by the general formula (2) is prepared, By cyanating the SH group of this fusion polypeptide, it is converted into a cyano group-containing polypeptide represented by the following general formula (5), and this is converted into an immobilization carrier represented by the following general formula (6) ( By binding to a primary amino group introduced into the monolith gel), it can be immobilized on an immobilization carrier as a polypeptide represented by the following general formula (1).

NH ₂ —R ₂ —CO—NH—CH (CH ₂ —SH) —CO—X (3)
(Wherein R ₂ represents any amino acid residue, and X represents OH or any amino acid residue)
NH ₂ —R ₁ —CO—NH—R ₂ —CO—NH—CH (CH ₂ —SH) —CO—X (4)
(Wherein R ₁ and R ₂ are arbitrary amino acid residues, and X is OH or any amino acid residue)
NH ₂ —R ₁ —CO—NH—R ₂ —CO—NH—CH (CH ₂ —SCN) —CO—X (5)
(Wherein R ₁ and R ₂ are arbitrary amino acid residues, and X is OH or any amino acid residue)
NH ₂ -Y (6)
(In the formula, Y represents a monolith gel which is an immobilization carrier)
NH ₂ —R ₁ —CO—NH—R ₂ —CO—NH—Y (1)
(In the formula, R ₁ and R ₂ are arbitrary amino acid residues, Y represents a monolith gel which is an immobilization carrier)
As shown in the general formula (1), R ₂ becomes a linker peptide (linker sequence) between the polypeptide represented by the general formula (2) to be immobilized and the primary amino group. R ₂ is an arbitrary amino acid residue, and the type and number of amino acids are not particularly limited. For example, a sequence consisting of 5 residues of glycine shown in SEQ ID NO: 1, a sequence consisting of 6 residues of glycine shown in SEQ ID NO: 2, etc. Can be used. R _{1 is} also an arbitrary amino acid residue, and the type and number of amino acids are not particularly limited.

In the general formula (3), a moiety represented by —NH—CH (CH ₂ —SH) —CO—X is referred to as an immobilization reaction sequence in the present specification, and as shown in the general formula (5) By being cyanated to produce a polypeptide having a cyano group, the reaction with the immobilized carrier represented by the general formula (6) is enabled.

  The immobilization reaction sequence needs to contain one residue of cysteine as shown in the general formula (3). X is OH or any amino acid residue (the type and number of amino acids are not particularly limited), and is not particularly limited, but the isoelectric point of the substance represented by the general formula (4) is preferably 4-5. Since it is easy to adjust the isoelectric point to 4 to 5, a sequence containing a large amount of aspartic acid and glutamic acid is preferable as X. For example, an aspartic acid 6-residue sequence or alanyl-polyaspartic acid can be suitably used. Alanyl-polyaspartic acid has an amino acid side chain that easily causes an amide bond formation reaction via a cyanocysteine residue by changing the amino acid next to cyanocysteine represented by the general formula (5) to alanine. This is because the carboxyl group of aspartic acid is the most acidic among them, and the isoelectric point is easily adjusted to 4-5.

  The immobilization reaction sequence is not particularly limited as long as it contains one residue of cysteine. For example, the sequence consisting of 8 residues of amino acid including 1 residue of cysteine shown in SEQ ID NO: 3 (CADDDDD ) Etc. can be used. As X, it is preferable that the purification tag sequence described above is further added to the C-terminal side of the immobilized reaction sequence.

  Conversion of the polypeptide represented by the general formula (2) into the fusion polypeptide represented by the general formula (4) can be performed by using a conventionally known recombinant DNA technique. That is, the fusion represented by the general formula (4) is obtained by binding the polynucleotide encoding the polypeptide represented by the general formula (2) and the polynucleotide encoding the peptide sequence represented by the general formula (3). By producing a polynucleotide encoding the polypeptide, expressing it in a host organism such as Escherichia coli, and then separating and purifying the expressed polypeptide, the desired fusion polypeptide can be prepared.

  Conversion from the fusion polypeptide represented by the general formula (4) to the fusion polypeptide represented by the general formula (5), so-called cyanation reaction, can be performed using a cyanation reagent. As a cyanating reagent, 2-nitro-5-thiocyanobenzoic acid (NTCB) (Y. Degani, A. Pthornnik, Biochemistry, 13, 1-11 (1974) is usually used. Or a method using 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) or the like.

Commercially available NTCB and CDAP can be used as they are. Cyanation using NTCB can be carried out efficiently between pH 7-9, and the reaction efficiency is increased by increasing the absorbance of 412 nm of liberated thionitrobenzoic acid (molecular extinction coefficient = 13,600 M ⁻¹ cm ⁻¹ ). Can be examined. The cyanation of the SH group can also be carried out, for example, according to the method described in the literature (J. Wood & Catsipolas, J. Biol. Chem. 233, 2887 (1963)).

  The reaction between the cyanated fusion polypeptide represented by the general formula (5) and the immobilized carrier (monolith gel) represented by the general formula (6) is performed at room temperature under weak alkaline conditions (pH 8 to 10). be able to.

  As a solvent for the immobilization reaction, any solvent that can dissolve the cyanated fusion polypeptide represented by the general formula (5) and that can adjust pH can be used. For example, various buffers such as a phosphate buffer and a borate buffer, alcohols such as methanol and ethanol, dimethylformamide, dimethylsulfoxide, and the like can be used. The reaction temperature is high as long as the reaction efficiency can be obtained at room temperature. However, if the solvent used does not freeze or boil, and the temperature range in which the cyanated fusion polypeptide represented by the general formula (5) does not aggregate as a result of denaturation is problematic. Can be used.

  Thus, the polypeptide is immobilized on the immobilization carrier at the carboxy terminus of the peptide chain by immobilizing the polypeptide that specifically binds to the high mannose sugar chain to the immobilization carrier. That is, since the orientation is controlled and immobilized on the immobilization carrier, the immobilization is performed on the carrier with high density. Therefore, since the binding efficiency of the high mannose type sugar chain can be improved, the virus contained in the sample can be more efficiently separated from the sample. In addition, since the immobilized polypeptides cannot collide with each other, denaturation of the polypeptides can be made reversible.

(4) Structure of a polypeptide that specifically binds to a high mannose-type sugar chain As described above, a polypeptide that specifically binds to a high-mannose-type sugar chain is immobilized on the above-mentioned immobilization carrier at the carboxy terminus of the peptide chain. It is preferable that

  In the present specification, a polypeptide isolated from nature is referred to as a “naturally-derived polypeptide”. A recombinant having the same amino acid sequence as a naturally-occurring polypeptide is referred to as a “wild-type polypeptide”, and one or several amino acids other than cysteine and / or lysine in the amino acid sequence of the naturally-occurring polypeptide A polypeptide in which is substituted with another amino acid is also referred to as “wild-type polypeptide”. A polypeptide having an amino acid sequence obtained by substituting one or more cysteine and / or lysine of a naturally-derived polypeptide or wild-type polypeptide with another amino acid is referred to as a “modified polypeptide”. The above “one or several” has already been described.

  As shown in Example 2 described later, naturally-occurring KAA-1 contains 1 residue of cysteine and 11 residues of lysine, but does not contain cysteine but contains 11 residues of lysine, Both KAA-1 ("wild-type KAA-1" in Example 2) in which cysteine residues and lysine residues are left as they are are linked to "polypeptides that specifically bind to high mannose-type sugar chains". A “carrier immobilization polypeptide”, which is a polypeptide to which a sequence, an immobilization reaction sequence and a purification tag sequence are added, has been successfully prepared. The carrier immobilization polypeptide corresponds to the polypeptide represented by the general formula (4) described above.

  Naturally-derived BCA contains 1 residue of cysteine and 10 residues of lysine, but both modified BCA containing 9 residues of lysine and no cysteine, and wild-type BCA, The peptide has been successfully prepared.

  Furthermore, naturally-occurring OAA does not contain cysteine and contains one residue of lysine, but modified OAA that does not contain cysteine and lysine has also been successfully prepared as a carrier immobilization polypeptide.

  From the above, in order to prepare a polypeptide for carrier immobilization, (1) the naturally occurring amino acid sequence of a polypeptide that specifically binds to a high mannose sugar chain has 1 or less cysteine residues. (2) the naturally occurring amino acid sequence of a polypeptide that specifically binds to a high mannose sugar chain does not contain a cysteine residue; or (3) specifically binds to a high mannose sugar chain More preferably, the naturally occurring amino acid sequence of the polypeptide satisfies one of the conditions that it does not contain a cysteine residue or a lysine residue.

  The “naturally-occurring amino acid sequence of a polypeptide that specifically binds to a high mannose sugar chain” refers to the same amino acid sequence as that of the polypeptide isolated from nature.

When R _{1 of} the naturally-derived “polypeptide that specifically binds to a high mannose-type sugar chain” represented by the above general formula (2) does not contain a cysteine residue, the carrier immobilization represented by the general formula (4) R ₁ in the polypeptide for use in the present invention can be prevented from being cyanated and hydrolyzed during the immobilization reaction to the immobilization carrier, so that R ₁ does not contain a cysteine residue ( The above conditions (2) and (3) are most preferable.

When R ₁ contains one cysteine residue, the cysteine residue may be present without being exposed to the outside, and thus the immobilization reaction can be suitably performed even in the case of the above condition (1). When R ₁ contains two or more cysteine residues, there is a possibility that an SS bond exists and R ₁ is likely to be cyanated, which is not preferable.

  It should be noted that a wild-type polypeptide and a modified polypeptide, which are polypeptides in which a part of the naturally-occurring amino acid sequence of “polypeptide that specifically binds to a high-mannose sugar chain” is modified, If the amino acid sequence of the naturally-occurring polypeptide corresponding to the wild-type polypeptide and the modified polypeptide does not satisfy the above conditions even if the above conditions such as not containing a cysteine residue are satisfied, the carrier is immobilized. This is not preferable in preparing a polypeptide for crystallization. For example, in Example 2, which will be described later, the results were that the expression of modified BPL-17, wild-type BPL-17, modified MPL-1 and wild-type MPL-1 was unsuccessful.

  As described above, in order to immobilize a polypeptide that specifically binds to a high mannose-type sugar chain on an immobilization carrier by controlling its orientation, first, as described above, the carrier immobilization polypeptide ( It is preferable to prepare (corresponding to the polypeptide represented by the general formula (4)). Therefore, when a polypeptide that specifically binds to a high mannose sugar chain satisfies any of the above conditions, it is considered suitable for performing the above orientation control.

(5) Separation of specimen and virus In the virus separation device according to the present invention, since a polypeptide that specifically binds to a high mannose-type sugar chain is immobilized on an immobilization carrier, no virus is contained in the specimen. Thus, it can be efficiently separated from the specimen, and cells infected with the virus can also be separated.

  The specimen is not particularly limited as long as the polypeptide is not aggregated and can be brought into contact with the polypeptide immobilized on an immobilization carrier. Examples thereof include human whole blood, plasma, serum, saliva, nasal mucosa, urine, and other body fluids. These may be used as a specimen per se, or may be used as a liquid prepared by adding to a MEM medium, physiological saline, or the like.

  In the virus separation device according to the present invention, the virus contained in the whole blood and / or a cell infected with the virus can be obtained by contacting the whole blood with the polypeptide even when the specimen is human whole blood. Can be separated from the whole blood.

  The lectins used in Patent Documents 1 to 4 have a hemagglutination action as described above. For this reason, the lectin cannot be used directly in a device for separating blood viruses from human whole blood, and it has been necessary to make contact after plasma separation. In other words, there has not been a device that can directly contact human whole blood with a polypeptide to separate virus and / or virus-infected cells from whole blood.

  For example, in Patent Document 2, a lectin selected from the group consisting of GNA, NPA, ConA and cyanobilin is fixed to the porous outer portion of a porous hollow fiber membrane, and blood is allowed to pass through the porous hollow fiber. Is disclosed (claims 7, 9, etc. of Patent Document 2).

  However, the above GNA, NPA, and ConA are based on literatures and analytical data, and other than high mannose type sugar chains, monosaccharide mannose, trimannosyl core structure (core pentasaccharide of N-glycan common structure), complex type sugar chains And GNA / NPA (Lectin Frontier Database (http://jcggdb.jp/rcmg/glycodb/LectinSearch) for ConA, Mega, T. et al., J. Biochem., 111, 396-400, (1992)). Cyanovirin-N (CV-N) binds to free mannobiose and high mannose-type sugar chains, but α (1-2) at the non-reducing end of D1 and D3 arms of branched mannosides in high-mannose sugar chains. The Man residue is a recognition site, and the α (1-2) Man residue at the non-reducing end of the D2 arm is not a recognition site.

  On the other hand, as described above, the “polypeptide that specifically binds to a high mannose-type sugar chain” used in the present invention has a binding property to a high-mannose-type sugar chain, and is a complex-type sugar chain and a hybrid sugar. A polypeptide that does not have binding properties to the chain. Further, as described above, the polypeptide is one or more lectins selected from the group consisting of type I lectin, type II lectin, type III lectin and type IV lectin, and the branched mannoside of the lectin. The recognition site is as described in (a) to (d) of (1) above.

  That is, the polypeptide used in the present invention is different from CV-N used in Patent Document 2 in the recognition site of branched mannosides of high mannose sugar chains. Moreover, unlike GNA, NPA, and ConA, complex sugar chains and mixed sugar chains are not recognized.

  And in patent document 2, the lectin is being fixed to the porous outer part of the porous hollow fiber membrane. That is, the lectin is prevented from contacting blood cells. From this, it is surmised that the blood compatibility of the lectin is not high.

  On the other hand, in the virus separation device according to the present invention, a polypeptide that specifically binds to a high mannose-type sugar chain is immobilized on an immobilization carrier (monolith gel). Very high compatibility. That is, the hemagglutination action is very low and the influence on complement activity is very small. Therefore, even if the polypeptide directly contacts human whole blood, there is no influence on the whole blood.

  Therefore, human whole blood can be used as a specimen, and viruses and / or cells infected with viruses contained in human whole blood can be separated from the whole blood. For example, in the examples described below, even when OAA-1 is directly contacted with human whole blood by using a virus separation device in which OAA-1 is immobilized on silica monolith, thrombus formation, hemolysis, blood cell adhesion It has been demonstrated that HIV can be efficiently separated from whole blood without causing aggregation, complement activation, and the like.

  Thus, since the virus separation device according to the present invention can use human whole blood as a sample, for example, whole blood of a patient is collected and introduced into the device using a peristaltic pump or the like. In the extracorporeal circulation, the virus in the whole blood and / or virus-infected cells are separated from the whole blood by contacting with a polypeptide that specifically binds to a sugar chain, and the separated whole blood is returned to the patient's body again. The process can isolate the virus and / or virus-infected cells contained in the patient's whole blood. The isolated virus and / or cells infected with the virus may be discarded without returning to the patient.

  Of course, the mode of separation is not limited to the extracorporeal circulation of whole blood. For example, after introducing a diluted solution of saliva into the device and separating the virus, the virus bound to the polypeptide is analyzed, and the patient is analyzed. It can also be used for purposes such as confirming what kind of virus the virus is infected with.

  The immobilization carrier (monolith gel) on which the above polypeptide is immobilized should be packed in a column or the like so that the sample can be easily introduced and the high mannose sugar chain can be smoothly immobilized on the above polypeptide. Is preferred. As the column, for example, a conventionally known spin column, a stainless steel column for liquid chromatography, or the like can be used.

  The target of separation from the specimen may be a cell infected with a virus having a high mannose sugar chain and / or a virus having a high mannose sugar chain. Examples of the virus include one or more viruses selected from the group consisting of influenza virus, hepatitis C virus, human herpes virus, dengue virus, AIDS virus, Ebola virus, and severe acute respiratory syndrome virus. Can do. As the virus and / or cells infected with the virus, AIDS virus and / or AIDS virus-infected T cells can be used more preferably.

The separation of virus and / or virus-infected cells from the specimen by the above device can be confirmed by methods such as TCID ₅₀ method and fluorescent antibody method shown in the examples described later.

  The present invention is not limited to the above-described embodiments, and various modifications are possible within the scope shown in the claims, and embodiments obtained by appropriately combining technical means disclosed in different embodiments. Is also included in the technical scope of the present invention.

  EXAMPLES Hereinafter, although this invention is demonstrated according to an Example, this invention is limited to an Example and is not interpreted.

[Example 1: Confirmation of binding property between HIV and algae-derived lectin]
In order to use as an index of the ability of the polypeptide to bind to HIV, the ability to neutralize HIV infectivity by algae-derived lectins (polypeptides that specifically bind to high-mannose sugar chains) was measured.

  Algae-derived lectins were diluted 2-fold in a flat-bottom 96-well plate for cell culture so that the final concentration of lectin during virus mixing was in the range of 1 μM to 1 nM (4 or 8 wells per dilution).

About 100 HIV-1 LAV strains (derived from pNL4-3) were added to each well and incubated for 10 minutes. Thereafter, 5 × 10 ⁴ Jurkat cells were added per well and cultured for about one week. Jurkat cells are immortalized T lymphocyte cells. Using the appearance of membrane fusion in Jurkat cells as an index, the dilution rate at which membrane fusion expression was inhibited by 50% by the lectin was evaluated by the Behrens-Karber method, and the 50% inhibition concentration (IC ₅₀ ) was calculated. At the same time, the amount of virus added was quantified and corrected for a concentration that would block 100 viruses. The experiment was performed at least three times at different times, and the average value was obtained.

  The algae-derived lectins subjected to the test and their 50% inhibitory concentration (nM) are summarized in Table 1. All algae-derived lectins suppressed HIV infection at the nM level and showed high binding to HIV. In particular, MPL showed an extremely high infection neutralizing property of 1.8 to 6.3 nM. On the other hand, the core α1-6 fucose-specific polypeptide, HypninA-1, used as a comparative example, had a 50% inhibitory concentration of 163.1 nM and weak binding to HIV.

[Example 2: Design of polypeptide for carrier immobilization, gene expression and purification of the polypeptide]
When immobilizing algal-derived lectins (polypeptides that specifically bind to high mannose-type sugar chains) on silica monoliths as immobilization carriers, a recombinant of the above lectins is used using the method disclosed in JP-A-2000-119300. A carrier immobilization polypeptide was prepared.

  As the algae-derived lectin, type I OAA, type I KAA-1, type II BCA, type III BPL-17, and type IV MPL-1 subunit were used. The amino acid sequences of these lectins are known. The amino acid sequences of naturally-derived OAA, KAA-1, BCA, BPL-17, and MPL-1 subunits are shown in SEQ ID NOs: 5 to 9, respectively.

  Naturally-derived OAA does not contain a cysteine residue in the amino acid sequence, and contains a lysine 1 residue at the 128th position from the N-terminus. First, a modified OAA in which the lysine at position 128 was substituted with arginine and the proline at position 82 was substituted with phenylalanine was prepared. In the present specification, the preparation of a polypeptide in which a part of the amino acid of a naturally derived polypeptide is substituted was performed according to a conventional method.

  Naturally-derived KAA-1 contains a cysteine residue at position 252 from the N-terminal in the amino acid sequence and an lysine 11 residue. Here, a modified KAA-1 in which the 252nd cysteine is substituted with alanine and the first glycine is substituted with alanine, and a lectin (wild type KAA) in which only the first glycine of naturally-derived KAA-1 is substituted with alanine. -1)).

  Naturally-derived BCA contains 1 cysteine residue at the 90th position from the N-terminal in the amino acid sequence, and 10 lysine residues in the sequence. Here, a modified BCA in which the 90th cysteine was replaced with alanine and the 124th lysine was replaced with leucine was prepared and used for preparation of a recombinant described later together with the wild type BCA.

  Naturally derived BPL-17 contains 6 cysteine residues and 11 lysine residues. Here, modified BPL-17 in which the 27th, 89th, and 130th cysteines from the N-terminus are substituted with alanine, and the 43rd, 113th, and 151st cysteines are substituted with valine; A lectin (referred to as wild-type BPL-17) in which the first (N-terminal) valine was substituted with alanine was prepared.

  The naturally derived MPL-1 subunit contains 3 cysteine residues and no lysine. Here, the modified MPL-1 in which the fifth and 27th cysteines from the N-terminus are substituted with alanine, the 121st cysteine with valine, and the first pyroglutamic acid with alanine, respectively, and a naturally derived MPL-1 subunit A lectin (referred to as wild-type MPL-1) in which the first pyroglutamic acid was substituted with alanine was prepared.

  Next, using these modified lectins and wild-type lectins, recombinants were prepared based on the method disclosed in JP-A-2000-119300. The recombinant is obtained by adding a linker sequence, an immobilization reaction sequence, and a purification tag sequence to the carboxy terminus of a lectin.

  As the linker sequence, a sequence consisting of 5 residues of glycine shown in SEQ ID NO: 1 or a sequence consisting of 6 residues of glycine shown in SEQ ID NO: 2 was used. As the immobilization reaction sequence, the sequence consisting of 8-residue amino acids including 1 residue of cysteine shown in SEQ ID NO: 3 was used. As the purification tag sequence, a sequence consisting of 6 histidine residues shown in SEQ ID NO: 4 was used.

  FIG. 1 is a schematic diagram showing an outline of the structure of a carrier immobilization polypeptide which is a recombinant. The algae-derived lectin in the figure corresponds to the modified lectin or the wild-type lectin. As shown in the figure, a linker sequence, an immobilization reaction sequence and a purification tag sequence are added to the carboxy terminal side of the algal-derived lectin.

  The carrier immobilization polypeptide is prepared by introducing a predetermined 80 base pair sequence upstream of the start codon (ATG) in the base sequence encoding the modified lectin polypeptide or the wild-type lectin polypeptide. Using a recombinant plasmid obtained by inserting a synthetic DNA (TAAGAATTC) prepared by introducing an EcoRI sequence (6 base pairs) downstream of the stop codon (TAA) into the BamHI / EcoRI site of pUC18 went. The above 80 base pair sequence and initiation codon are shown in SEQ ID NO: 28.

  The amino acid sequences of the prepared polypeptide for immobilizing a carrier are shown in SEQ ID NOs: 10 to 18. SEQ ID NO: 10 is a modified OAA, SEQ ID NO: 11 is a modified KAA-1, SEQ ID NO: 12 is a wild type KAA-1, SEQ ID NO: 13 is a modified BCA, SEQ ID NO: 14 is a wild type BCA, SEQ ID NO: 15 is a modified BPL-17, SEQ ID NO: 16 is a wild-type BPL-17, SEQ ID NO: 17 is a modified MPL-1, and SEQ ID NO: 18 is a wild-type MPL-1 amino acid sequence added with a linker sequence, an immobilization reaction sequence and a purification tag sequence, respectively. It represents the amino acid sequence of the polypeptide for immobilization.

  The nucleotide sequences of the polynucleotides encoding the carrier immobilization polypeptides shown in SEQ ID NOs: 10 to 18 (start codon and start codon are not shown) are shown in SEQ ID NOs: 19 to 27, respectively.

  The polynucleotide encoding the carrier immobilization polypeptide is amplified, the resulting polynucleotide is cleaved with the restriction enzyme BamHI, then combined with the cloning vector pUC19 cleaved with BamHI, and the resulting recombinant plasmid is introduced into Escherichia coli. Thus, it was confirmed whether or not the carrier immobilization polypeptide could be expressed in Escherichia coli cells.

  As a result, the modified BPL-17, wild-type BPL-17, modified MPL-1 and wild-type MPL-1 could not be expressed. Could be expressed.

  As described above, naturally-occurring OAA does not contain a cysteine residue in the amino acid sequence, but contains one lysine residue. Naturally derived KAA-1 contains 1 cysteine residue in the amino acid sequence and 11 lysine residues. Naturally-derived BCA contains 1 cysteine residue in the amino acid sequence and 10 lysine residues.

  From the result of expression of the above carrier immobilization polypeptide in E. coli, as described above, in order to prepare a carrier immobilization polypeptide, (1) a polypeptide that specifically binds to a high mannose sugar chain The naturally-occurring amino acid sequence has 1 or less cysteine residues; (2) the naturally-occurring amino acid sequence of a polypeptide that specifically binds to a high-mannose sugar chain does not contain a cysteine residue; or (3) It is found that it is preferable that the naturally-occurring amino acid sequence of a polypeptide that specifically binds to a high mannose-type sugar chain does not contain any cysteine residue or lysine residue. It was.

  After the obtained cultured cells were crushed, the purification method from the supernatant from which insoluble substances were removed by centrifugation was examined. That is, the conditions for removing nucleic acid, the separation conditions for chromatography using a nickel chelate column, and the separation conditions for chromatography using an ion exchange column were examined.

  In the chromatography operation using the column, the purified protein with a purity of 80% or more was obtained by the purification with the nickel chelate column alone using the independently developed continuous chromatography. Furthermore, the purity could be increased to 95% or more by purifying with an anion exchange column. As a result, about 600 mg of purified protein could be obtained from 8 L of the culture solution.

  The continuous chromatography is a method in which two columns are used and purification is performed while performing different operations. In this method, first, a sample is applied to the first column, and washing, elution, and column preparation are performed. However, while washing, elution, and column preparation are performed, the sample is applied to the second column. Apply. The first column is ready during this time, and the second sample is applied while the second column is being washed, eluted and prepared. In the same way, purification will proceed while performing different operations.

  The purification using the anion exchange column was performed by continuous chromatography in the same manner as the purification using the nickel chelate column using two anion exchange columns.

[Example 3: Immobilization of purified protein with controlled orientation on immobilization support and measurement of immobilization efficiency]
The above-mentioned linker sequence, the above-mentioned immobilization reaction sequence and the above-mentioned purification tag sequence are added to the carboxy terminus of the modified OAA, and the carrier immobilization polypeptide obtained as the purified protein in Example 2 is hereinafter referred to as “OAA-1”. (SEQ ID NO: 10). In this example, in order to immobilize OAA-1 on the immobilization support by controlling the orientation, that is, OAA-1 is a carboxyl group at the carboxy terminal and covalently bonded to the amino group of the immobilization support. In order to be immobilized on the immobilization carrier (OAA-1 was bonded to the immobilization carrier at one position of the carboxy terminal and via the main chain), a cyanation reaction of a cysteine residue was performed.

In this example, 0.1 mL of aminocellulofine (trade name: sold by Seikagaku Corporation; amine content: about 10 ⁻⁵ moles NH ₂ / ml gel) immobilized carrier was used as the amino group-immobilized carrier, and the reaction efficiency was improved. Measurements were made.

  Using 20 mM sodium phosphate buffer (pH 7.0) as a solvent, 1 mL of an OAA-1 protein solution having a concentration of about 10 μM was prepared. The solution was mixed with 0.1 mL of the aminocellulose fine immobilization carrier, and the supernatant solution was separated from the immobilization carrier by centrifugation (1000 rpm, 20 seconds) to obtain the supernatant solution (A). The protein concentration of the supernatant solution (A) was estimated by measuring the absorbance at 280 nm of the supernatant solution (A). Under these conditions, the OAA-1 protein was adsorbed quantitatively on the immobilized carrier.

  Next, 1 mL of 2-nitro-5-thiocyanobenzoic acid (NTCB) solution as a cyanation reagent is added to the above-mentioned aminocellulose fine immobilization carrier to perform a cyanation reaction, and then the reaction is performed by centrifugation (1000 rpm, 20 seconds). The solution and the immobilization carrier were separated, and the supernatant was designated as (B). The protein concentration in the supernatant solution (B) was measured. No OAA-1 protein was detected in this supernatant.

  Next, 1 mL of 20 mM borate buffer solution (pH 9.5) is added to the above-mentioned immobilized carrier to cause an immobilization reaction. After the reaction, the buffer solution and the immobilized carrier are separated by centrifugation (1000 rpm, 20 seconds). After separation, the supernatant solution was designated as (C). In this supernatant solution, no OAA-1 protein was detected.

  Next, 1 mL of KCL solution was added to the immobilized carrier, and the protein bound to the immobilized carrier was released by ion adsorption. The solution and the immobilized carrier were separated by centrifugation (1000 rpm, 20 seconds), and the supernatant solution was defined as (D). The protein concentration in the supernatant solution (D) was measured. As a result, about 2 μM protein was detected from the supernatant solution (D).

  This result indicates that a protein corresponding to about 8 μM is retained in the immobilization carrier, that is, immobilization occurs at a high rate of about 80%. When the same experiment was repeated 5 times, a value of about 75% ± 5% was obtained as the immobilization efficiency value.

  When the amount of OAA-1 protein used for immobilization is increased, the protein is detected in the supernatant solution (A). When an amount of OAA-1 exceeding about 100 nmol per 1 mL of the above-mentioned immobilization carrier is used, the efficiency in the adsorption stage of OAA-1 to the immobilization carrier is reduced, but the protein adsorbed on the immobilization carrier is immobilized. The conversion reaction is considered to be almost constant (about 75% ± 5%, ie 70% or more).

[Example 4: Immobilization of lectin on silica monolith]
In immobilizing the carrier immobilization polypeptide on the silica monolith, a primary amino group was introduced as a surface functional group on the surface of the solid phase serving as an immobilization reaction field. When introducing a primary amino group, an unmodified silica monolith (referred to as unmodified silica monolith) is reacted with a silane coupling agent having an epoxy group, and a silica monolith having an epoxy group introduced on the solid surface (epoxidation) (Referred to as silica monolith). This is reacted with poly-L-lysine, which is an amino polymer having a primary amino group in the side chain, to produce a silica monolith with a primary amino group introduced on the solid surface (referred to as an aminated silica monolith). did.

  Poly-L-lysine can be polymerized with the epoxy group introduced on the solid surface to form a uniform film and cover the solid surface. This also has the effect of reducing silica exposed on the solid phase surface and preventing non-specific adsorption with biological materials. The aminated silica monolith thus prepared was reacted with a modified lectin protein that had been converted to cyanocysteine to effect immobilization. After the reaction, the unreacted amino group was treated with acetic anhydride. Details of the preparation of the aminated silica monolith, the preparation of the polypeptide for immobilizing the carrier, and the immobilization were as follows.

  Introduction of an epoxy group into an unmodified silica monolith (ie, preparation of an epoxidized silica monolith) is achieved by placing an unmodified silica monolith in an epoxy silane (3-glycidyloxypropyltrimethoxysilane) solution diluted to 20% with toluene. Dipped and left overnight at 80 ° C.

  Next, poly-L-lysine (molecular weight 4,000 to 15,000: purchased from Sigma), which is an amino group-containing polymer, is dissolved in 20 mM borate buffer (pH 9.5) so as to be 0.2% (w / v). An amount 6 times the volume of the silica monolith column was prepared. The total amount of this solution was added to the epoxidized silica monolith prepared above and allowed to react for 16 hours with gentle stirring. That is, poly-L-lysine was immersed in an epoxidized silica monolith, covalently bonded to an epoxy group, and an amino group was introduced into the epoxidized silica monolith (ie, preparation of an aminated silica monolith).

  Thereafter, the aminated silica monolith was washed with 20 mM sodium phosphate buffer (pH 7.0) containing 0.5 M sodium chloride, followed by 0.5 M monoethanolamine solution (pH = 8. The whole aminated silica monolith was immersed in 5) (adjusted with hydrochloric acid) and gently stirred for 12 hours to block residual epoxy groups and remove unreacted poly-L-lysine. After that, by washing several times with ultrapure water 5 times the volume of the silica monolith column, monoethanolamine and unreacted poly-L-lysine are removed, and the above-mentioned aminated silica monolith column is prepared. This was used for the immobilization reaction of the polypeptide for immobilizing the carrier.

  Based on the method described in Example 2, a recombinant (polypeptide for carrier immobilization) in which a linker sequence, an immobilization reaction sequence, and a purification tag sequence are added to the carboxy terminus of the lectin is described in Example 3. In order to efficiently perform the cyanation reaction, it was subjected to pretreatment. Specifically, 12 mg of the carrier immobilization polypeptide and 4.5 mg of dithiothreitol per 1 mL volume of the silica monolith column used for the immobilization reaction, 10 mM sodium phosphate buffer (pH 7.0) containing 2.5 mM EDTA. The sample solution having the same volume as the column volume was prepared. This was reacted in a thermostat at 30 ° C. for 1 hour to reduce the disulfide bond due to cysteine contained in the carrier immobilization polypeptide.

  Thereafter, the buffer was exchanged using a desalting column (manufactured by GE Healthcare, HiPrep 26/10 Desalting) to cyanate the polypeptide. Specifically, the desalting column was equilibrated in advance with 20 mM sodium phosphate buffer (pH 7.0) containing 1 mM of 2-nitro-5-thiocyanobenzoic acid (NTCB), which is a cyanating reagent, By exchanging the buffer by adding the sample solution containing dithiothreitol, excess dithiothreitol contained in the sample solution is quickly removed, and at the same time, the solvent of the polypeptide is quickly added to the NTCB solution. Replaced with

  The cyanation reaction of the carrier immobilization polypeptide was carried out in this way, and the cyanated sample was again subjected to buffer exchange using the desalting column, and NTCB and reaction byproducts were removed to remove 20 mM sodium phosphate. Replaced with buffer (pH 7.0). The cyanated polypeptide thus obtained was added to the aminated silica monolith column into which the primary amino group had been introduced, and adsorbed for 30 minutes while gently shaking. Thereafter, 0.5M borate buffer (pH 9.5) was added 1/4 times the amount of the above-mentioned cyanated polypeptide to adjust the pH of the reaction system to 9.2, and gently shaken at room temperature. However, the immobilization reaction of the carrier immobilization polypeptide to the aminated silica monolith was started. The immobilization reaction was completed after 20 hours.

  After completion of the reaction, the reaction solution was removed, and the remaining polypeptide that was not immobilized was washed with 20 mM sodium phosphate buffer (pH 7.0) containing 0.5 M sodium chloride. Specifically, an operation of adding and washing a buffer containing sodium chloride in an amount of 5 times the volume of the silica monolith column to the silica monolith column after immobilization was repeated several times to remove unreacted residual polypeptide. Removed (washing step). Subsequently, a solution prepared by dissolving acetic anhydride in 1.5M sodium acetate buffer (pH 5.2) to a concentration of 1% (v / v) was prepared in an amount 5 times the volume of the silica monolith column. A series of operations of adding to a silica monolith column and reacting for 10 minutes and then removing the solution is repeated five times to acetylate the primary amino group remaining on the unreacted solid phase surface, thereby remaining residual primary amino The group was protected.

  The amount of immobilization of the polypeptide for immobilizing the carrier is calculated from the absorbance at the wavelength based on the extinction coefficient at 280 nm estimated from the amino acid sequence of the polypeptide, and the reaction before and after the immobilization reaction is started. The amount of the carrier immobilization polypeptide contained in the solution and the recovery solution in the washing step was determined, respectively, and the balance was defined as the amount of immobilization of the carrier immobilization polypeptide. That is, [immobilization amount] = [content in liquid before reaction] − ([content in liquid after reaction] + [content in liquid of washing liquid]).

  As a result of immobilizing the carrier-immobilizing polypeptide OAA-1 on the silica monolith column as described above, the silica monolith column (φ4.8 mm × 1.5 mm, macropore diameter 2 μm, mesopore diameter 200 nm) was obtained. 37 μg (1.36 mg / mL carrier) of OAA-1 could be immobilized.

[Example 5: Blood compatibility test (hemolysis test)]
Using a high mannose-type sugar chain-specific polypeptide OAA (naturally-derived OAA), histidine-tagged OAA (His-rOAA), OAA-1 immobilized on silica monolith, and commercially available lectin ConA derived from red bean, Sex tests and complement activation tests were performed. Each test was conducted according to ISO10993-part4 (DRAFT). The hemolysis test was evaluated by comparing the absorbance of the sample with 1% hemolysis. The His-rOAA is a fusion of OAA having the amino acid sequence shown in SEQ ID NO: 5 and a purified tag sequence consisting of 6 histidine residues shown in SEQ ID NO: 4.

  Immobilization of OAA-1 on silica monolith was performed according to the method described in Example 4. The silica monolith used was prepared as follows. That is, 0.80 g of polyethylene oxide (product number 85,645-2 manufactured by Aldrich) and 0.90 g of urea, which are water-soluble polymers, are dissolved in 10 g of 0.01 N acetic acid aqueous solution, and 4 ml of tetramethoxysilane is stirred into this solution. In addition, a hydrolysis reaction was performed. After stirring for several minutes, the obtained transparent solution was poured into a glass tube having an inner diameter of 6 millimeters and kept in a constant temperature bath at 40 ° C., and solidified after about 30 minutes.

  The solidified sample was further aged for several hours and kept at 240 ° C. for 1 hour under sealed conditions. After this treatment, the gel was dried at 40 ° C. for 3 days and heated to 800 ° C. at a heating rate of 100 ° C./h to obtain a rod-shaped inorganic porous body (ie, silica monolith) having a diameter of 4.8 mm. It was.

  In the obtained porous body, it was confirmed that through-holes having a center hole diameter of about 3.5 μm (= 3500 nm) were present in a structure entangled in a three-dimensional network. And it was confirmed by nitrogen adsorption measurement that many pores with a diameter of about 200 nm exist on the inner wall of the through-hole.

  The amount of OAA-1 immobilized on the silica monolith subjected to the test was 7 mg per mL of the silica monolith.

  Next, 100 μg / mL solution and 1 mg / mL solution of each of the above lectins were prepared using physiological saline as a solvent. Next, the solution and human whole blood of type A, type B, and type O collected from heparin were mixed and contacted at a ratio of lectin solution: whole blood = 1: 20. The contact time was 5 minutes and 30 minutes and the reaction temperature was 37 ° C.

  The results are shown in FIGS. FIGS. 2 and 3 are graphs showing the degree of hemolysis when the contact time with the lectin is 5 minutes or 30 minutes when 1% hemolysis (Positive Control) is 1.0. In the figure, “negative control (saline)” is the result when physiological saline was used. “Commercially available device” indicates the result when a cellulose gel (liposorber) having dextran sulfate fixed by Kaneka Corporation is used at a final concentration of 100 mg / mL. “100 μg / mL OAAHistag” shows the results when 100 μg / mL of OAA with a histidine tag (His-rOAA described above) was used.

  As shown in FIGS. 2 and 3, hemolysis did not occur in naturally occurring OAA, histidine-tagged OAA (His-rOAA), and OAA-1 immobilized on silica monolith, but the contact time was 5 minutes and 30 minutes. In both cases, hemolysis was observed when ConA was supplied at 1 mg / mL. Moreover, activation of complement of C3a, C5a, and SC5b-9 was not observed for all samples.

  From the above results, all of OAA, naturally-derived OAA, His-rOAA and OAA-1 immobilized on silica monolith have excellent blood compatibility. It became clear that there was no.

[Example 6: Human whole blood passage test]
Using a virus separation device in which OAA-1 was immobilized on silica monolith, a blood compatibility test was performed according to ISO10993-part4. Immobilization of OAA-1 on silica monolith was performed according to the method described in Example 4. The silica monolith is a cylindrical one with a macropore diameter of 100 μm, a mesopore diameter of 161 nm, a diameter of 4 mm, and a thickness of 2 mm. After fixing OAA-1, it is packed into a spin column (self work) to obtain a virus separation device. .

  Using a peristaltic pump, 15 mL of human whole blood was injected into the spin column at a flow rate of 0.5 mL / min (linear velocity: 4.0 cm / min), and the passing fraction was collected. ACD-A was used as an anticoagulant. In addition, as a comparative example, silica monolith on which OAA-1 was not immobilized was used. Table 2 shows the results of subjecting the passage fraction to a blood compatibility test.

  As a result, in the virus separation device in which OAA-1 was immobilized on silica monolith, formation of thrombus, hemolysis, adhesion of blood cells, aggregation, activation of complement, etc. were not observed, and blood was not affected. Since bradykinin production was reduced by immobilization of OAA-1, it was speculated that OAA-1 was immobilized at a high density on the silica monolith surface.

  In the table, “pre-device pressure” means the pressure at the device inlet when whole blood is flowed at a flow rate of 0.5 mL / min with a peristaltic pump. For each item described in the table, thrombus formation is performed by observing the surface and cut surface of the silica monolith using an optical microscope and observing whether thrombus is formed; The blood after the passage was separated into blood cells, and the hemolysis of plasma was measured by a method of comparing with 1% hemolysis (Positive Control). The white blood cell count, red blood cell count, and platelet count were measured using a blood cell counter. Complement activation and bradykinin production were measured by ELISA using a commercially available kit.

[Example 7: Separation of HIV using virus separation device]
OAA-1 was used as the carrier immobilization polypeptide, and this was immobilized on a silica monolith and used. As a silica monolith, a cylindrical one having a macropore diameter of 2 μm, a mesopore diameter of 60 nm, a diameter of 4 mm, and a thickness of 2 mm is used. After fixing OAA-1, a spin column (self-made) is packed into a virus separation device. did.

In the separation test, 1 mL of HIV-1 solution having an infectivity value of 1.3 × 10 ⁴ (TCID ₅₀ / mL) was loaded at a flow rate of 0.1 mL, and the infectivity titer before loading and the infectivity titer of the flow-through after loading were loaded. Was measured. The passing liquid was collected in 10 fractions, and the infectious titer of each fraction was measured. The infectious titer was measured based on the TCID ₅₀ method using changes in cytopathogenicity caused by viruses as indices. All the fractions had an infectious titer of 1.3 × 10 ² or less, which is the detection limit, and could remove at least 99% of HIV-1.

[Example 8: Infected lymphocyte removal test]
OAA-1 was used as the carrier immobilization polypeptide, and this was immobilized on a silica monolith and used. As the silica monolith, a cylindrical one having a macropore diameter of 100 μm, a mesopore diameter of 160 nm, a diameter of 4.5 mm, and a thickness of 100 mm was used. A silica monolith with OAA-1 immobilized and covered with a heat-shrinkable fluororesin is inserted into a stainless steel empty column with an inner diameter of 5 mm, and the gap is hardened with an epoxy resin to produce a column. A device.

The HIV-1 infected lymphocyte removal test was performed using Jurkat cells, which were immortalized T lymphocytes, as a model of infected cells. The day before, approximately 10 ⁶ Jurkat cells were infected with 10 or more HIV-1 cells. About 5 × 10 ⁵ Jurkat cells infected with HIV-1 were dispersed in 4 mL of cell culture medium RPMI 1640, and negatively added to the stainless steel column with a peristaltic pump. As a control, Jurkat cells not infected with HIV-1 (uninfected Jurkat cells) were also used. The flow rate was 0.1 mL / min, and 500 μL of the fraction passed through the column was collected from the time when the loaded cell solution began to be discharged.

  Next, for each fraction, the cells were collected by low speed centrifugation and subjected to the fluorescent antibody method. In the fluorescent antibody method, cells are adhered to a poly-L-lysine-coated slide glass (AGC Techno Glass), fixed with 3% paraformamide, and permeabilized with a surfactant. Rabbit antiserum (Bio Academia) and Dylight488 labeled anti-rabbit IgG antibody.

  FIG. 4 is a photograph showing the result of staining the P24 protein, which is an antigen of HIV-1, by the fluorescent antibody method for the fraction passing through the column of the virus separation device. FIG. 4A is a dispersion of HIV-1 infected Jurkat cells before being applied to the column, and fluorescence is confirmed. However, no fluorescence was observed in any of the column passage fractions (b) to (h) in FIG. That is, all Jurkat cells infected with HIV-1 were adsorbed to the virus separation device. Since uninfected Jurkat cells and sheep erythrocytes passed through the column (results not shown), it was considered that HIV-1 infected Jurkat cells could be specifically adsorbed and removed by OAA-1 on the surface of the virus separation device.

  The present invention has high blood compatibility and is excellent in the ability to separate a virus in a specimen. Therefore, the present invention can be used very suitably in the field of medical equipment and the like.

Claims (13)

A virus separation device capable of separating a virus contained in a specimen and / or a virus-infected cell from the specimen, and a polypeptide that specifically binds to a high-mannose sugar chain and an immobilization that immobilizes the polypeptide A carrier, wherein the polypeptide is immobilized on the immobilization carrier, the immobilization carrier is a monolith gel,
The device is characterized in that the polypeptide is one or more lectins selected from the group consisting of type I lectin, type II lectin, type III lectin and type IV lectin.   The specimen is human whole blood, and the virus contained in the whole blood and / or cells infected with the virus can be separated from the whole blood by contacting the whole blood with the polypeptide. The device of claim 1, characterized in that:   The device according to claim 1, wherein the monolith gel has a macropore diameter of 1 μm to 200 μm and a mesopore diameter of 40 nm to 200 nm.   The device according to claim 3, wherein the monolith gel has a macropore diameter of 20 μm to 200 μm.   The device according to claim 1, wherein the monolith gel is a silica monolith.   The device according to any one of claims 1 to 5, wherein the polypeptide is immobilized at 2 µg or more per ml of the immobilization carrier.   The device according to any one of claims 1 to 6, wherein the polypeptide is immobilized on the immobilization carrier at a carboxy terminus of a peptide chain.   The device according to claim 7, wherein the naturally-occurring amino acid sequence of the polypeptide has 1 or less cysteine residues.   The device according to claim 8, wherein the naturally occurring amino acid sequence of the polypeptide does not contain a cysteine residue.   The device according to claim 9, wherein the naturally occurring amino acid sequence of the polypeptide does not contain a cysteine residue and a lysine residue.   The device according to any one of claims 1 to 10, wherein the virus is a virus having a high mannose sugar chain.   The virus is one or more viruses selected from the group consisting of influenza virus, hepatitis C virus, human herpes virus, dengue virus, AIDS virus, Ebola virus and severe acute respiratory syndrome virus. The device according to claim 1.   The device according to any one of claims 1 to 12, wherein the virus and / or the virus-infected cell is an AIDS virus and / or an AIDS virus-infected T cell.