Glycopolypeptide, polynucleotide encoding same, and pharmaceutical composition containing same

Abstract

To provide a modified lectin mutant (polypeptide) showing significantly high binding properties to a high-mannose sugar chain so that the same can be used as an agent for treatment and diagnosis of various virus diseases.SOLUTION: A polypeptide herein has a tandem repeat of red algae Kappaphycus alvarezii-derived lectin KAA1 (4 repeat sequences of domain consisting of 67 amino acids).SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a glycan-binding polypeptide, a polynucleotide encoding the same, and a pharmaceutical composition containing the same, and in particular to a glycan-binding polypeptide modified to have a higher glycan-binding ability than a naturally occurring lectin, a polynucleotide encoding the same, and a pharmaceutical composition containing the same. [Background technology]

[0002] Glycoconjugates such as glycoproteins and glycolipids present on cell surfaces and in body fluids function as a kind of information element and are deeply involved in important life phenomena such as development, immunity, cancer, infection, etc. Meanwhile, lectins, which are glycan-binding proteins, function as glycan-recognition molecules and play important biological roles similar to glycans.

[0003] So far, many types of lectins have been isolated from seaweed or algae (freshwater cyanobacteria), and their biochemical properties have been elucidated. Based on their binding properties, algal lectins are classified into three groups: high-mannose glycan-specific lectins, complex glycan-specific lectins, and lectins that bind to both glycans. Some lectins are known to specifically bind to viruses, such as human immunodeficiency virus (HIV) and influenza virus (Non-Patent Documents 1-12). OAA (Oscillatoria agardhii agglutinin), a lectin derived from the blue-green alga Oscillatoria agardhii, KAA (Kappaphycus alvarezii agglutinin), a lectin derived from the red alga Kappaphycus alvarezii, BCA (Boodlea coactaagglutinin), a lectin derived from the green alga Boodlea coacta, and MPL (Meristotheca papulosa lectin), a lectin derived from the red alga Meristotheca papulosa, are expected to recognize viruses that have high-mannose glycans on their surface, such as HIV and influenza viruses, due to their strong binding specificity with high-mannose glycans. In particular, Non-Patent Document 12 discloses that KAA recognizes gp120, an envelope glycoprotein of HIV, and exhibits anti-HIV activity, and is expected to be used as a pharmaceutical material. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Boyd, MR et al., Antimicrob. Agents Chemother.41, 1521-1530, 1997. [Non-Patent Document 2] O'Keefe, BRet al., Antimicrob. Agents Chemother. 47, 2518-2525, 2003. [Non-Patent Document 3] Helle, F., .et al., J. Biol. Chem.281, 25177-25183, 2006.

Non-licensed Document 4

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Non-licensed Document 6

Non-licensed Document 7

Non-licensed literature 9

Non-licensed literature 10

Non-licensed Document 11

Non-licensed Document 12

[0005] However, in order to actually utilize such lectins as pharmaceutical materials for antiviral drugs, etc., stronger binding to high-mannose glycans present on the surface of viruses such as the envelope is required. For this purpose, modification of lectins to enhance their glycan-binding ability has been considered, and although there are reports that such modification has enhanced glycan-binding ability, even stronger activity is still required.

[0006] The present invention has been made in consideration of the above-mentioned problems, and an object of the present invention is to provide a modified lectin variant (polypeptide) that exhibits significantly high binding affinity to high-mannose glycans so that it can be used as a drug for treating or diagnosing diseases caused by the above-mentioned viruses. [Means for solving the problem]

[0007] In order to achieve the above-mentioned object, the present inventors conducted intensive research and discovered that the glycan-binding ability of the lectin KAA1 derived from the red alga Kappaphycus alvarezii can be significantly enhanced by highly polyvalently modifying the glycan-binding site, thereby completing the present invention.

[0008] Specifically, the polypeptide according to the present invention is characterized by having tandem repeats of lectin KAA1 (having 4 repeat sequences of a domain consisting of 67 amino acids) derived from the red alga Kappaphycus alvarezii.

[0009] The polypeptide according to the present invention may have a linker sequence between the tandem repeats.

[0010] The polypeptide of the present invention may contain at least two amino acid sequences having the amino acid sequence of SEQ ID NO:1 or the amino acid sequence of SEQ ID NO:1 in which one or several amino acids have been substituted, deleted, inserted or added.

[0011] The polypeptide of the present invention may comprise the amino acid sequence of SEQ ID NO:2, or the amino acid sequence of SEQ ID NO:2 in which one or several amino acids have been substituted, deleted, inserted or added.

[0012] The polynucleotide according to the present invention is characterized in that it encodes the above-mentioned polypeptide according to the present invention.

[0013] The pharmaceutical composition for treating or diagnosing a viral disease according to the present invention is characterized by comprising the above-mentioned polypeptide according to the present invention, and the causative virus of the viral disease is a virus having a high-mannose type glycan on its surface. Since the pharmaceutical composition according to the present invention comprises the above-mentioned polypeptide according to the present invention in which the glycan binding ability of the polypeptide that binds to a high-mannose type glycan is significantly enhanced, it can be used for treating or diagnosing a disease caused by a virus having a high-mannose type glycan on its surface. Examples of the virus having a high-mannose type glycan on its surface include human immunodeficiency virus (HIV), influenza virus, hepatitis C virus (HCV), SARS coronavirus (SARS-CoV), and herpes simplex virus. Effect of the Invention

[0014] The polypeptide of the present invention has significantly enhanced glycan binding activity and can bind strongly to high-mannose glycans, and therefore is extremely useful as it may be used in the treatment and diagnosis of diseases caused by viruses that have high-mannose glycans on their surface. [Brief description of the drawings]

[0015] [Figure 1] FIG. 1 shows a 4-repeat sequence containing the four sugar-binding sites of KAA1. [Diagram 2] 1 is a photograph showing the results of SDS-PAGE performed on KAA1 / KAA1 in Example 2. [Diagram 3] 1 is a graph showing the results of investigating the inactivation effect of KAA1 against SARS coronavirus in Example 4. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The following preferred embodiments are merely exemplary in nature and are not intended to limit the present invention, its application method, or its uses.

[0017] One embodiment of the present invention is a polypeptide having a tandem repeat of four repeat sequences in KAA1, a lectin derived from the red alga Kappaphycus alvarezii. KAA1 shows 60.6% homology with OAA, a lectin derived from the cyanobacterium Oscillatoria agardhii, and belongs to the same OAA family. OAA consists of 132 amino acid residues that do not contain cysteine, has two repeat sequences of 67 amino acids, and approximately 75% homology is recognized between these overlapping sequences. In addition, in the tertiary structure, it has a domain swap structure in which the overlapping sequences are partially swapped, and each of the two domains formed by this has one sugar binding site. KAA1 has a four repeat sequence of approximately 67 amino acids and is composed of a structure in which two OAAs are linked together. Both OAA and KAA1 show very similar properties and functions. That is, they do not show monosaccharide binding, and the minimum sugar structure they recognize is Man(α1-3)Man(α1-6)Man(β1-4), and they specifically bind to high mannose-type sugar chains in which the (α1-3)Man residues of the D2 arm are exposed among N-type sugar chains. OAA has two sugar-binding sites and KAA1 has four sugar-binding sites per molecule, and they crosslink and aggregate cells. Therefore, they are considered to be able to recognize various viruses that have high mannose-type sugar chains on the viral surface such as the envelope. In fact, the above-mentioned Non-Patent Document 12 discloses that KAA shows strong binding to high mannose-type sugar chains possessed by the HIV envelope glycoprotein gp120, and shows anti-HIV activity. In addition to HIV, influenza virus, HCV, SARS-CoV, herpes simplex virus, etc. are known to have high mannose-type sugar chains on the viral surface.

[0018] The polypeptide according to the present embodiment is modified by repeating 4 repeat sequences corresponding to the 4 sugar-binding sites of KAA1 in tandem, thereby multiplying the sugar-binding sites. Therefore, the sugar-binding ability of the polypeptide according to the present invention is significantly increased. The 4 repeat sequence is shown in FIG. 1. In FIG. 1, amino acid positions having the same amino acids in the 4 repeat sequence are indicated by black markers, and amino acid positions having two kinds of amino acids are indicated by gray markers. As shown in FIG. 1, the 4 repeat sequence is a repeat sequence of 65 to 68 amino acids, and although the sequences are not completely identical to each other, they show high sequence identity. The polypeptide according to the present embodiment has a repeat sequence of the 4 repeat sequence, and the number of repeats of the sequence is not particularly limited, but two repeats are sufficient to obtain a significant enhancement of the sugar-binding ability. In addition, the polypeptide according to the present embodiment may contain one or several amino acid mutations in the 4 repeat sequence shown in FIG. 1, as long as the sugar-binding ability is not reduced.

[0019] In this embodiment, a specific linker sequence may be inserted between the repeated sequences. The linker sequence used is not particularly limited as long as it does not negatively affect the glycobinding ability of the polypeptide, and various well-known linker sequences can be used.

[0020] Polypeptides according to the present invention include products produced by recombinant techniques from prokaryotic or eukaryotic hosts, including, for example, bacterial cells, yeast cells, higher plant cells, insect cells and mammalian cells, as well as products of chemical synthesis procedures. Depending on the host used in a recombinant production procedure, the proteins according to the present invention may be glycosylated or non-glycosylated. Additionally, the proteins according to the present invention may contain an initial modified methionine residue as a result of host-mediated processes.

[0021] In one embodiment, the polypeptide according to the present invention preferably comprises at least two amino acid sequences of SEQ ID NO: 1, which is the amino acid sequence of the KAA1 polypeptide, or may comprise at least two amino acid sequences containing one or several amino acid mutations in the amino acid sequence of SEQ ID NO: 1. Furthermore, the polypeptide according to this embodiment may comprise the amino acid sequence of SEQ ID NO: 2, or may comprise the amino acid sequence of SEQ ID NO: 2 containing one or several amino acid mutations.

[0022] Variants include those containing deletions, insertions, inversions, repeats, and type substitutions (e.g., substitution of a hydrophilic residue for another, but usually not a strongly hydrophilic residue for a strongly hydrophobic residue). In particular, "neutral" amino acid substitutions in a polypeptide generally have little effect on the activity of that polypeptide.

[0023] 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. In addition, it is also well known that not only artificial modifications but also mutants that do not significantly change the structure or function of the protein exist in natural proteins.

[0024] Those skilled in the art can easily mutate one or several amino acids in the amino acid sequence of a polypeptide using well-known techniques. For example, any base in a polynucleotide encoding a polypeptide can be mutated according to known point mutagenesis methods. Furthermore, a primer corresponding to any site in a polynucleotide encoding a polypeptide can be designed to prepare a deletion mutant or an addition mutant. Furthermore, by using the methods described herein, it can be easily determined whether the mutant prepared is a desired mutant having glycosylation ability.

[0025] The above-mentioned mutation of "one or several amino acids" means that a number of amino acids (preferably 1 to 10, more preferably 1 to 7, even more preferably 1 to 5, and particularly preferably 1 to 3) that can be substituted, deleted, inserted, or added by known methods for producing mutant polypeptides, such as site-directed mutagenesis, have been substituted, deleted, inserted, or added.

[0026] In other embodiments, the polypeptides of the invention may be recombinantly expressed in modified forms such as fusion proteins, for example, a region of additional amino acids, particularly charged amino acids, may be added to the N-terminus of the polypeptides of the invention to improve stability and persistence within the host cell, during purification or during subsequent handling and storage.

[0027] Recombinant production can be carried out using methods well known in the art, such as using vectors and cells as described in detail below.

[0028] As described above, the present invention provides a polynucleotide encoding the polypeptide of the present invention. As used herein, the term "polynucleotide" is used interchangeably with "nucleic acid" or "nucleic acid molecule" and refers to a polymer of nucleotides. As used herein, the term "base sequence" is used interchangeably with "nucleic acid sequence" or "nucleotide sequence" and refers to a sequence of deoxyribonucleotides (abbreviated as A, G, C, and T).

[0029] The polynucleotides of the present invention may be in the form of RNA (e.g., mRNA) or in the form of DNA (e.g., cDNA or genomic DNA). The DNA may be double-stranded or single-stranded. Single-stranded DNA or RNA may be the coding strand (also known as the sense strand) or the non-coding strand (also known as the antisense strand).

[0030] As used herein, the term "oligonucleotide" refers to a compound consisting of several to several tens of nucleotides, and is used interchangeably with "polynucleotide." Oligonucleotides are expressed by the number of polymerized nucleotides, such as short ones called dinucleotides (dimers) or trinucleotides (trimers), and long ones called 30-mers or 100-mers. Oligonucleotides may be produced as fragments of longer polynucleotides, or may be chemically synthesized.

[0031] Furthermore, the polynucleotide of the present invention can be fused to a polynucleotide encoding the above-mentioned tag label (tag sequence or marker sequence) on the 5' or 3' side.

[0032] The present invention further relates to variants of the polynucleotides encoding the polypeptides of the present invention.Variants can occur naturally, such as natural allelic variants.By "allelic variant" is meant one of several interchangeable forms of a gene that occupies a given locus on the chromosome of an organism.Non-natural variants can be generated, for example, by using mutagenesis techniques well known in the art.

[0033] Such variants include variants in which one or several bases are deleted, substituted, or added in the base sequence of the polynucleotide encoding the polypeptide of the present invention. The variants may be mutated in the coding or non-coding regions, or both. Mutations in the coding regions may generate conservative or non-conservative amino acid deletions, substitutions, or additions.

[0034] The present invention further provides isolated polynucleotides comprising a polynucleotide encoding a polypeptide of the present invention or a polynucleotide that hybridizes to said polynucleotide under stringent hybridization conditions.

[0035] The above-mentioned "stringent conditions" means that hybridization occurs only when there is at least 90% identity, preferably at least 95% identity, and most preferably at least 97% identity between the sequences.

[0036] The hybridization can be carried out by a known method such as that described in Sambrook et al., Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory (1989). In general, the higher the temperature and the lower the salt concentration, the higher the stringency (the more difficult hybridization), and the more homologous polynucleotides can be obtained.

[0037] Hybridization conditions may suitably be those known in the art and are not particularly limited, but examples include 42°C, 6xSSPE, 50% formamide, 1% SDS, 100 μg / ml salmon sperm DNA, and 5xDenhardt's solution (wherein 1xSSPE is 0.18M sodium chloride, 10mM sodium phosphate, pH 7.7, 1mM EDTA; 5xDenhardt's solution is 0.1% bovine serum albumin, 0.1% Ficoll, and 0.1% polyvinylpyrrolidone).

[0038] The polynucleotide or oligonucleotide according to the present invention includes not only double-stranded DNA, but also each of the single-stranded DNAs and RNAs, such as the sense and antisense strands that constitute the double-stranded DNA. The DNA also includes cDNA and genomic DNA obtained by, for example, cloning or chemical synthesis techniques, or a combination thereof. Furthermore, the polynucleotide or oligonucleotide according to the present invention may include a sequence of an untranslated region (UTR) or a vector sequence (including an expression vector sequence).

[0039] A method for obtaining the polynucleotide or oligonucleotide of the present invention includes isolating and cloning a DNA fragment containing the polynucleotide or oligonucleotide of the present invention by a known technique. For example, a probe that specifically hybridizes with a portion of the base sequence of the polynucleotide of the present invention may be prepared and a genomic DNA library or a cDNA library may be screened. Such a probe may have any sequence and / or length as long as it specifically hybridizes with at least a portion of the base sequence of the polynucleotide of the present invention or its complementary sequence.

[0040] Alternatively, the method for obtaining the polynucleotide of the present invention may include a method using an amplification means such as PCR. For example, primers are prepared from the 5'-side and 3'-side sequences (or their complementary sequences) of the cDNA of the polynucleotide of the present invention, and PCR or the like is carried out using these primers with genomic DNA (or cDNA) or the like as a template to amplify the DNA region sandwiched between the two primers, thereby obtaining a large amount of DNA fragments containing the polynucleotide of the present invention.

[0041] The present invention also provides a pharmaceutical composition for treating or diagnosing a disease caused by a virus having a high-mannose type sugar chain on its surface, characterized by comprising the above-mentioned polypeptide.

[0042] The pharmaceutical composition according to the present invention may be either an oral preparation or a parenteral preparation. The dosage form is not particularly limited, and the composition may be formulated into tablets, granules, powders, capsules, elixirs, syrups, microcapsules, suspensions, etc., according to a conventional method.

[0043] When administered parenterally, for example, a solution containing the polypeptide of the present invention can be administered as a nasal spray or an injection, and when administered orally, it can be administered before, after, or between meals.

[0044] The pharmaceutical composition according to the present invention may contain materials such as carriers, excipients, binders, swelling agents, lubricants, sweeteners, flavoring agents, preservatives, stabilizers, coating agents and the like, as necessary.

[0045] In the pharmaceutical composition of the present invention, specific components that can be contained in, for example, tablets, capsules, etc., include binders such as tragacanth, gum arabic, corn starch, and gelatin; excipients such as microcrystalline cellulose and crystalline cellulose; bulking agents such as corn starch, pregelatinized starch, alginic acid, and dextrin; lubricants such as magnesium stearate; flow improvers such as fine silicon dioxide; glidants such as glycerin fatty acid esters; sweeteners such as sucrose, lactose, and aspartame; and flavorings such as peppermint, vanilla flavor, and cherry.

[0046] When the dosage unit is a capsule, the above-mentioned materials may further contain liquids such as oils and fats. The carrier may contain a polymeric carrier.

[0047] Various other materials may also be included as coatings or to modify the physical form of the dosage unit. Tablet coatings include, for example, shellac, sugar, or both. Syrups or elixirs may contain, for example, sucrose as a sweetener, methylparaben and propylparaben as preservatives, coloring, and cherry or orange flavor. In addition, various vitamins and various amino acids may be included.

[0048] The dosage of the pharmaceutical composition according to the present invention may be set so as to ensure the amount required by the subject of application, and the pharmaceutical composition may be used after being formulated or incorporated into food and beverages. EXAMPLES

[0049] The following examples are provided to explain in detail the polypeptides and polynucleotides encoding the polypeptides according to the present invention.

[0050] [Example 1: Preparation of a polypeptide (KAA1 / KAA1) with highly multivalent glycan-binding sites by tandem repeats of KAA1] (Construction of KAA1 / KAA1-expressing E. coli strain) pET-28a(+) (Novagen) and pColdI (Takara Bio) were used as expression vectors for constructing the KAA1 / KAA1 expression construct, and SHuffle T7 Express (Novagen) was used as the E. coli strain for expression.

[0051] First, the KAA1 / KAA1 coding region was amplified using the KAA1 expression construct as a template, high-fidelity DNA polymerase KOD Plus Neo (TOYOBO), and the primers listed in Table 1 below. As the KAA1 expression construct, pET28a-rKAA1 described in Non-Patent Document 12 above was used, which is a pET-28a(+) vector having inserted therein a sequence encoding KAA1 containing 6-His and a thrombin cleavage site at the N-terminus. The amino acid sequence of KAA1 containing 6-His and a thrombin cleavage site at the N-terminus expressed in this manner is shown in SEQ ID NO:3. In the amplification of the KAA1 / KAA1 coding region, KAA1 / KAA1(pET28a(+)) expression coding DNA was amplified using KAA-pET-1F, KAA-pET-1R, KAA-pET-2F, and KAA-pET-2R, and KAA1 / KAA1(pColdI) was amplified using KAA-pCold-1F, KAA-pET-1R, KAA-pET-2F, and KAA-pCold-2R. The PCR reaction system consisted of 5 μl of 10×PCR buffer for KOD plus NeO, 5 μl of 2 mM dNTP mix, 1.5 μl each of 10 μM forward primer and 10 μM reverse primer, 3 μl of 25 mM MgSO4, 1 μl of KAA1 expression construct, 1 μl of KOD Plus Neo, and ultrapure water to make the reaction solution 50 μl, which was then mixed thoroughly and subjected to PCR. PCR was performed using a T Gradienter 96 Thermocycler (Biometra) by repeating 35 cycles of 94°C for 2 minutes, 98°C for 10 seconds, 60°C for 30 seconds, and amplification reaction at 68°C for 15 seconds. The reaction was terminated by holding at 68°C for 5 minutes.

[0052] [Table 1] Underline: Vector homologous sequence Double underline: Linker sequence Bold: Factor Xa recognition sequence Wavy underline: Stop codon Italics: lectin gene homologous sequence Dotted underline: frameshift prevention insertion sequence

[0053] The amplified products were inserted into expression vectors pET-28a(+) (Novagen) and pColdI (Takara Bio) that had been digested with restriction enzymes NdeI and XhoI using the In-Fusion HD Cloning Kit (Takara Bio), and the resulting constructs were used to transform E. coli strains SHuffle T7 Express and SHuffle Express. + (pET-28a(+)) or LB / Amp + An appropriate amount was spread onto an agar medium (pColdI) and cultured overnight at 37° C. An insert check was performed on single colonies.

[0054] The insert check was performed by PCR using a primer pair specific to the pET-28a(+) or pColdI vector. Specifically, the pET-28a(+) vector-specific primer pair used was a T7 primer (5'-TAATACGACTCACTATAGGG-3': SEQ ID NO: 10) and a T7 terminator primer (5'-GCTAGTTATTGCTCAGCGG-3': SEQ ID NO: 11), while the pColdI vector-specific primer pair used was a pCold-F primer (5'-ACGCCATATCGCCGAAAGG-3': SEQ ID NO: 12) and a pCold-R primer (5'-GGCAGGGATCTTAGATTCTG-3': SEQ ID NO: 13). The PCR reaction system was 5μl of 10x PCR buffer for Blend Taq, 5μl of 2mM dNTP mix solution, 1μl of each 10μM primer, 0.5μL of DNA polymerase Blend Taq (TOYOBO) and ultrapure water to make a reaction solution of 50μl, mixed thoroughly, dispensed in 10μl portions, and suspended in cells partially taken from colonies for PCR. PCR was performed using a T Gradientr 96 Thermocycler (Biometra) after heat denaturation at 94℃ for 5 minutes, followed by 35 cycles of heat denaturation at 94℃ for 30 seconds, annealing at 60℃ for 30 seconds and extension reaction at 72℃ for 1 minute. The reaction was terminated by holding at 72℃ for 5 minutes.

[0055] 5 μl of the PCR product obtained was mixed with 10× loading buffer to prepare a sample for agarose electrophoresis. Electrophoresis was performed at a constant voltage of 100 V using TAE (40 mM Tris-HCl, 40 mM acetic acid, 1 mM EDTA) as the electrophoresis buffer and 1% agarose gel containing ethidium bromide as the electrophoresis gel, and bands were detected using a UV transilluminator. For positive clones in which bands of the desired size were detected, the plasmid was purified using a Hi Yield Plasmid Mini Kit according to the attached manual. The purified plasmid was used as a template and labeled with a Big Dye Terminator v3.1 Cycle Sequencing Kit using vector-specific primers, and then subjected to base sequence analysis using a Genetic Analyzer 3130xl (Applied Biosystems). The base sequence analysis was performed using Applied Biosystems Sequence Scanner Software ver.1.0. The clones whose base sequences were confirmed were subjected to LB / Kan + (pET-28a(+)) or LB / Amp + (pColdI) The cells were inoculated into liquid medium and cultured at 37°C overnight, after which an equal volume of 40% glycerol (final concentration 20%) was added to prepare glycerol stocks, which were then stored at -80°C until use.

[0056] (Expression and purification of KAA1 / KAA1) The glycerol stock of the KAA1 / KAA1 expression strain obtained as described above was cultured in LB / Kan + (pET-28a(+)) or LB / Amp + The bacteria was inoculated into 3 ml of liquid medium (pColdI) and cultured overnight at 37°C. The overnight culture was then transferred to LB / Kan + Or LB / Amp + Each was added to 250 mL of liquid medium and further cultured with shaking at 37°C until the medium reached the mid-logarithmic growth phase. 600When the pH reached 0.5, isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to a final concentration of 0.5 mM to start expression induction, and the KAA1 / KAA1 expression strain (pET-28a(+)) was cultured at 20°C for 16 hours, and the KAA1 / KAA1 expression strain (pColdI) was cultured at 15°C for 24 hours with shaking. The cells were collected by centrifugation (10,000×g, 4°C, 15 minutes), suspended in 1 / 20 volume of ultrasonic disruption buffer (20 mM phosphate buffer (pH 7.4), 500 mM NaCl, 20 mM imidazole) relative to the culture medium, and then ultrasonic disruption was performed. After disruption, the cells were centrifuged again (10,000×g, 4°C, 15 minutes), and the supernatant was collected as the soluble fraction, and the residue was collected as the insoluble fraction.

[0057] The recombinants obtained in this example were all expressed as His-tag fusions (the amino acid sequences of the recombinants obtained are shown in SEQ ID NO:2). The His-tag fusion recombinants contained in the soluble fraction were purified using a nickel chelate column (Vt=1 ml, His Gravi Trap, GE Healthcare). That is, the soluble fraction was added to a column that had been sufficiently equilibrated with an equilibration buffer (20 mM phosphate buffer (pH 7.4), 500 mM NaCl, 20 mM imidazole), and the recombinants were bound to the column. After sufficient washing with the equilibration buffer, 5 ml of an elution buffer (20 mM phosphate buffer (pH 7.4), 500 mM NaCl, 500 mM imidazole) was added to elute the recombinants. To remove the imidazole, the column was thoroughly dialyzed against ultrapure water, and the purified specimen was subjected to the following tests. The yields of the purified preparations KAA1 / KAA1(pET-28a(+)) and KAA1 / KAA1(pColdI) were 52 mg and 42 mg per liter of culture medium, respectively, and KAA1 / KAA1(pET-28a(+)), which had a higher yield, was used in subsequent tests.

[0058] [Example 2: Analysis of KAA1 / KAA1 using SDS-PAGE] (method) SDS-PAGE was performed according to the method of Schagger and Jagow (1987) by setting an acrylamide gel (12.5%) for electrophoresis in an electrophoresis apparatus (AE-6530, ATTO) at a constant voltage of 100 V. Protein molecular weight marker II (Tefco) was used as a molecular weight marker. An equal amount of 2xSDS loading buffer (50 mM Tris-HCl buffer (pH 6.8) containing 4% (w / v) SDS, 12% (w / v) glycerol, and 0.01% (w / v) bromophenol blue) was added to the above-mentioned purified product as a sample solution, and then heated at 100°C for 5 minutes to prepare a sample for electrophoresis under non-reducing conditions. In addition, a sample for electrophoresis under reducing conditions was prepared by adding the above-mentioned equal amount of 2xSDS loading buffer and 2% (w / v) mercaptoethanol. After electrophoresis of each sample, the gel was stained with Coomassie Brilliant Blue (CBB) R-250 to confirm the protein bands. The equivalent of 3 μg of the purified sample was used for electrophoresis. The results are shown in Figure 1. In Figure 1, M indicates a molecular weight marker, 1 indicates KAA1 under non-reducing conditions, 2 indicates KAA1 / KAA1 under non-reducing conditions, 3 indicates KAA1 under reducing conditions, and 4 indicates KAA1 / KAA1 under reducing conditions.

[0059] (result) As shown in Figure 1, a band of KAA1 / KAA1 was confirmed at approximately 60 kDa in both reducing and non-reducing SDS-PAGE. Since the molecular weight of wild-type KAA1 (His tag fusion) is 30.2 kDa and the theoretical molecular weight of KAA1 / KAA1 having the tandem repeat structure is 58.9 kDa, the purified preparation obtained above was confirmed to be KAA1 / KAA1.

[0060] [Example 3: Measurement of hemagglutination activity of KAA1 / KAA1] (method) Hemagglutination activity (HA) was measured using a microtiter plate. 25 μl of each 2-fold serial dilution in saline was prepared from a 16 μM solution of each sample on a microtiter plate. 25 μl of 2% trypsin-treated rabbit blood cell (TRBC) was added to each dilution, gently stirred, and allowed to stand at room temperature for 3 hours or more, after which the agglutination ability was observed. The agglutination ability was judged by the naked eye, and a positive result was obtained when 50% or more of the red blood cells were agglutinated. The strength of HA was expressed as the agglutination titer (potency), i.e., the dilution ratio of the test solution that showed a positive result. TRBC was prepared as follows. For commercially available sterile preserved rabbit blood (Hiroshima Animal Laboratory), 2 ml of red blood cells equivalent was washed three times with approximately 50 ml of saline, and then saline was added to make a measuring cup to 45 ml to prepare a 2% rabbit red blood cell suspension. Physiological saline containing 1 / 10 volume of 0.5% trypsin was added to the mixture and allowed to stand at 37°C for 60 minutes (stirred every 30 minutes). The trypsin-treated red blood cells were washed four times with 50 ml of physiological saline, and then physiological saline was added to make the total volume 45 ml to obtain 2% TRBC. The results of the hemagglutination activity test are shown in Table 2.

[0061] [Table 2]

[0062] (result) As shown in Table 2, it was confirmed that the activity of KAA1 / KAA1 dramatically increased as the concentration increased. In the 0.25 μM solution, the titer of KAA1 was 2 7 whereas KAA1 / KAA1 is 2 8 There was no significant difference, but in the 8 μM solution, the titer of KAA1 was 2 19 whereas the titer of KAA1 / KAA1 was 2 58 Furthermore, in the 16 μM solution, the titer of KAA1 was 2 20 whereas the titer of KAA1 / KAA1 was >2 144 A further dramatic increase in activity was observed.

[0063] [Example 4: Examination of SARS coronavirus inactivation effect of KAA1 / KAA1] Next, to examine whether KAA1 / KAA1 actually exhibits a virus inactivation effect, we treated SARS-CoV with KAA1 / KAA1 and evaluated the cell infectivity of SARS-CoV. The method and results are described below.

[0064] First, 10 μL of SARS-CoV-2 (2019-nCoV / Japan / AI / I-004 / 2020 strain (National Institute of Infectious Diseases): 2.0 × 10 8 TCID50 / mL) was mixed with 90 μL of KAA1 (10 μM) or KAA1 / KAA1 (10 μM). In addition, DMEM without lectin was mixed with SARS-CoV-2 as a control. After that, the reaction was allowed to proceed at room temperature for 30 minutes, and then the reaction was stopped by diluting 10-fold with cell maintenance solution DMEM. Further, 10-step serial dilution was performed to prepare virus-containing solutions of various concentrations. Then, the virus-containing solutions of each concentration were inoculated (50 μl / well, 4 wells for each concentration) into Vero / TMPRSS2 cells (JCRB1819 (JCRB Cell Bank, National Institutes of Biomedical Innovation, Health and Nutrition)) previously seeded in a 96-well plate, and after adsorption for 1 hour, the inoculation solution was aspirated and 100 μl / well of DMEM was added. After 3 days, when the cytopathic effect had spread, each well was observed under a microscope to evaluate the presence or absence of infection, and the infectivity was measured. The unit of infectious titer is 50% cell infectious concentration (TCID50) / ml. The results are shown in Figure 3.

[0065] As shown in Figure 3, SARS-CoV-2 treated with KAA1 or KAA1 / KAA1 was inactivated and the infectivity titer was reduced compared to the control (DMEM). In addition, it was revealed that the infectivity titer was lower when treated with KAA1 / KAA1 compared to when treated with KAA1. The reason why SARS-CoV-2 was inactivated and the infectivity titer was reduced by KAA1 or KAA1 / KAA1 is thought to be because KAA1 or KAA1 / KAA1 bound to the glycan on the surface of SARS-CoV-2 particles, causing steric hindrance or aggregation of viruses. The reason why KAA1 / KAA1 has a stronger inactivation ability than KAA1 is because it has more glycan binding sites (2 times) compared to KAA1, which is thought to be due to the strong aggregation of SARS-CoV-2. From these results, KAA1 / KAA1 is thought to be useful for the treatment or diagnosis of diseases caused by SARS-CoV, and is also thought to be useful for the treatment or diagnosis of viral diseases that have high-mannose type glycans on their surface like SARS-CoV.

[0066] These results suggest that KAA1 / KAA1, which is highly polyvalent due to the tandem repeat of the glycan-binding site of KAA1, has dramatically enhanced glycan-binding activity compared to wild-type KAA1. As mentioned above, KAA1 binds to the high-mannose glycan of the HIV envelope glycoprotein gp120, and therefore KAA1 / KAA1 with significantly enhanced glycan-binding activity is extremely useful because it can strongly bind to the high-mannose glycan and may be used in HIV treatment and HIV diagnosis. Furthermore, it is also thought to be useful for viral diseases other than HIV that have high-mannose glycans on their surface, such as SARS-CoV.

Claims

1. A polypeptide having tandem repeats of the 4 repeat sequence of lectin KAA1 derived from red algae Kappaphycus alvarezii, A polypeptide comprising the amino acid sequence of SEQ ID NO:1 or at least two amino acid sequences having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:1, and having glycan-binding ability.

2. The polypeptide of claim 1, further comprising a linker sequence between the tandem repeats.

3. 3. The polypeptide according to claim 1 or 2, comprising the amino acid sequence of SEQ ID NO:2 or an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:

2.

4. A polynucleotide encoding the polypeptide according to any one of claims 1 to 3.

5. A pharmaceutical composition for treating or diagnosing a viral disease caused by a virus having a high-mannose type sugar chain on its surface, comprising the polypeptide according to any one of claims 1 to 3.

6. 6. The pharmaceutical composition according to claim 5, wherein the virus is human immunodeficiency virus (HIV), influenza virus, hepatitis C virus (HCV), SARS coronavirus (SARS-CoV) or herpes simplex virus.

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