WO2008069290A1

WO2008069290A1 - Anti-hiv drug comprising fusion protein containing multimeric actinohivin, polypeptide constituting the same, gene encoding the polypeptide and method for producing the anti-hiv drug

Info

Publication number: WO2008069290A1
Application number: PCT/JP2007/073623
Authority: WO
Inventors: Haruo Tanaka; Junji Inokoshi; Atsushi Takahashi; Satoshi Omura
Original assignee: Kiim Pharm Lab. Inc.
Priority date: 2006-11-30
Filing date: 2007-11-30
Publication date: 2008-06-12

Abstract

It is intended to provide an anti-HIV drug characterized by containing a new substance in which a fusion protein has bound to multimeric actinohivin; a polypeptide which is multimeric actinohivin; a nucleotide sequence; a transformant; and a method for producing the anti-HIV drug. The anti-HIV drug inhibits synctium formation and has an enhanced effect.

Description

Anti-HIV drug comprising fusion protein containing actinobin multimer, polypeptide constituting the same, gene encoding polypeptide and method for producing anti-HIV drug Technical Field

The present invention relates to an antiviral drug effective against human immunodeficiency virus (HIV) infection, particularly a polypeptide effective against anti-HIV drug-resistant virus, D light NA encoding this polypeptide, this DNA The present invention relates to a transformant transformed with the above-mentioned method and a method for producing the anti-HIV drug. Background art

Each year around the world, 400 million people are newly infected with HIV, but there is still no complete treatment and prevention measures. In particular, the emergence of multi-drug resistant HIV that does not work with existing anti-HIV drugs is a serious threat in both developed and developing countries. As existing therapeutic agents for HIV and AIDS, inhibitors of reverse transcriptase and protease that are involved in HIV viral replication and exist specifically only in HIV are used alone or in combination. There are nucleoside inhibitors and non-nucleoside inhibitors as reverse transcriptase inhibitors, but nucleoside inhibitors cause serious side effects by long-term administration. On the other hand, non-nucleoside inhibitors have high specificity of action, so resistant mutant viruses appear early.

Protease inhibitors alone exhibit good antiviral activity, but their effects are temporary and have side effects such as poor stability in the body and digestive disorders. In addition, when an anti-HIV drug is used alone for a long period of time, a mutant virus strain resistant to that drug frequently appears.

In recent years, multi-drug combination therapy combining multiple drugs has been recommended and effective as a treatment method that suppresses the emergence of drug-resistant HIV and enables long-term drug treatment and delays the onset of AIDS in HIV patients. However, the problems of side effects caused by the emergence of multidrug resistant mutant virus strains and long-term administration remain. Multi-drug therapy also suppresses virus growth, but it is impossible to completely eliminate virus-infected cells. Therefore, in order to prevent HIV infection and prevent the onset of AIDS, in addition to reverse transcriptase inhibitors and protease inhibitors, anti-HIV drugs with completely different mechanisms of action targeting sites other than existing therapeutic drugs Development is desired.

HIV infection is established by adhering to the host cell and entering the cell through the binding of glycoproteinl 20 (gp 120), the HIV envelope glycoprotein, and CD 4, a receptor on the host cell surface. It was thought to be. Therefore, since substances that inhibit the binding of gpl20 and CD4 are expected to inhibit HIV-target cell adhesion and prevent HIV infection, attempts have been made to inhibit the binding of gp120 and CD4. It was. As an example, attempts were made to create soluble CD4 by genetic engineering and to administer it to the human body. However, in this method, although inhibitory activity was observed in the test tube, anti-HIV activity was not observed due to the short half-life in vivo. In addition, it was clarified that no infection occurred even when human CD4 was expressed in mouse cells, suggesting the presence of a second adhesion molecule.

In recent years, there have been many reports that this second receptor is a group of chemokine receptors. HIV is roughly divided into two types: macrophage-directed strains (M-tropic HIV) and T-cell strain-directed strains (T-tropic HIV). Cell orientations seen between these virus strains It was shown that the difference in sex was due to the difference in the molecular type of the second receptor. That is, the target cell expresses either CC chemokine receptor 5 (CC R 5: M-tropic HIV receptor) or CXC chemokine receptor 4 (CXCR 4: T-tropic HIV receptor) as the second receptor. It became clear that it was decided by the difference of whether or not.

Based on these new findings, the mode of HIV adhesion / invasion to target cells is now considered as follows. First, _{§ 120} and 004 bind, followed by binding to the host cell chemokine receptor CCR5 or CXCR4. As a result, glycoprotein 41 (gp 4 1) cleaved by the protease from the precursor gp 160 is naked, and HIV membrane and host cell membrane are fused to establish HIV adhesion / invasion infection to the host cell. The chemokines also block HIV binding by competitively blocking the binding of HIV to chemokine receptors. This unique discovery not only accelerated the elucidation of the mechanism of HIV infection, but also provided a new perspective on anti-HIV drug development strategies.

From this background, the present inventors reproduced this mode of infection in an in vitro experimental system. We started construction of a test evaluation system (syncytium formation inhibitory activity test system). The HIV-1 lverb glycoprotein complex g _P 120 / gp 41 mediates the fusion (contact.invasion) between the virus and the cell membrane. This fusion also occurs between e η V expressing cells and CD 4+ cells.

These cell-cell fusions are very similar to HIV-1 cell infection. Since T-cells and M_tropic HIV-1 require the chemokine receptors CXCR4 and CCR5, respectively, as secondary receptors in addition to CD4, the inventors have developed T- and M-tropic HIV-1 In order to construct two envelope expression (env-expressing) cell lines with envelope glycoprotein genes and to measure the formed syncytia by colorimetry, en V expressing cells are transcriptionally activated. A protein (Tat) gene was introduced, and LTR (terminal repeat sequence) linked to the Lac-Z gene and a beta-galactosidase gene were introduced into the receptor-expressing cells. In this way, HeLa / T_env / Tat and HeLa / CD4 / Lac_Z (HeLa cells originally have CXCR4) for T-tropic syncytia formation. Established HeLa / Menv / Tat and HOS / CD4 / CCR5 / Lac-Z system for M-tropic This method does not use infectious virus particles Established the world's first simple and highly sensitive method without fear of biohazards (H. Chiba, S. Asamura, M. Okamoto, J. noKoshi, h. Tanaka, K. Fujita) and S. Omura, A simple screening system for anti-HIV drugs ： syncytium formation assay using Γ-cell line tropic and macrophage tropic HIV env express cell lines- Establishment and validation- J. Antibiot., 54, 818-826 (2001) )

First, we evaluated the usefulness of the test system (validation) and proved its effectiveness in screening for anti-HIV drugs. Next, He La cells expressing envelope glycoproteins (gp 120, gp 41) and He La cells expressing CD4 and CXCR 4 were obtained for the purpose of obtaining anti-HIV drugs having the above-mentioned mechanism of action. We searched for metabolites produced by microorganisms that could inhibit syncytia formation by fusion. As a result of screening over 8,000 strains of actinomycetes, fungi, and bacteria, a new actinomycete K, which is a promising syncytium formation inhibitor with anti-HIV activity named Actinohivin, was newly isolated from soil. 9 7— 0003 shares (1 February 1998 9 From the culture solution of the National Institute of Advanced Industrial Science and Technology Patent Biological Deposit Center (deposited as FERM BP-6670, Japan, 305-8566, Tsukuba Satohito 1-chome, 1-chome, Chuodai 6) (Patent Document 1: International Publication No. 0 OZ5 2 043 pamphlet).

Actinohibin is a low molecular weight protein, consisting of 1 14 amino acid residues, with a molecular weight of 12,524, as determined by its primary structure. As a result of searching the database for sequence similarity between actinohibin and translation products of existing proteins or known base sequences, the same protein and the same base sequence substance do not exist, and it may be a new compound. It was revealed.

The gene for vaccinohibin was cloned from the K97-0003 strain. This actinohibin gene was expressed in E. coli, and a recombinant actinohibin production system was established. Recombinant actinohibin showed syncytium formation inhibitory activity equivalent to natural actinohibin.

Furthermore, as a result of investigating anti-HIV activity in an in vitro experimental system, actinohibin exerted an antiviral effect against HIV-1 and HIV-2 viruses at an extremely low concentration compared to known anti-HIV drugs. .

The chemical structure of actinohibin consists of three segments that are highly homologous to each other. All of the constituent segments have affinity with gp120, but alone do not show sufficient syncytium formation inhibitory activity, and in order to show strong affinity, three or more segments are arranged in tandem It became clear that the structure was important.

Therefore, as a result of creating and examining various improved mutants by applying the production system of recombinant actinohibin, it was found that the multimer, particularly the dimer, has a superior anti-HIV effect. The reason why actinohibin multimers show excellent antiviral activity is not yet clear, but it is clear that actinobin binds to the high mannose sugar chain of the HIV envelope glycoprotein gp 120 and the binding as a cluster is It is thought to increase the HIV effect and inhibit HIV adhesion and invasion at low concentrations (Patent Document 2: International Publication No. 2006/059638 pamphlet).

Patent Document 1 International Publication No. 2000/52043 Pamphlet

Patent Document 2 International Publication No. 2006/059638 Pamphlet Disclosure of the invention

Problems to be solved by the invention

The present invention is an anti-HIV drug by inhibiting syncytium formation, and has an anti-HIV effect even more than the actinohivin dimer disclosed in Patent Document 2 (International Publication No. 2006/0596 38 Pamphlet). The purpose is to provide enhanced anti-HIV drugs.

Means for solving the problem

As a result of further investigation on the actinohibin multimer disclosed in Patent Document 2 (International Publication No. 2006/0596 38 pamphlet), the present inventors have found that a new substance in which a fusion protein is bound to the actinobin multimer has a greater anti-tumor activity. It has been found that it has an HIV effect, and the present invention has been completed.

That is, the present invention provides the following anti-HIV drugs, polypeptides, base sequences, transformants and methods for producing anti-HIV drugs.

1. an anti-HIV (human immunodeficiency virus) comprising a fusion protein comprising a lactinohibin multimer and at least one other polypeptide or protein

) Medicine.

2. The vaccinohibin multimer

(a) the polypeptide represented by SEQ ID NO: 1, and

(b) A homolog having syncytium formation inhibitory activity in which one or more amino acids in the amino acid sequence of (a) are substituted, deleted, or added.

2. The anti-HIV drug according to 1 above, wherein a plurality of polypeptides selected from (which may be the same or different) are linked by 50 or less linkers.

3. The anti-HIV drug according to 1 or 2, wherein the at least one other polypeptide or protein comprises a protease recognition sequence (polypeptide).

4. The anti-HIV drug according to any one of 1 to 3 above, wherein the at least one other polypeptide or protein comprises a recognition sequence of a His tag · TEV protease (protease derived from Tobacco Etch Virus).

5. The anti-HIV drug according to any one of 1 to 4 above, wherein the actinohibin multimer is an actinohibin dimer.

6. Any one of 1 to 5 above, wherein the linker is a peptide chain represented by SEQ ID NO: 2 Anti-HIV drugs described in 1.

7. The fusion protein consists of a His tag, a TEV protease recognition sequence, a polypeptide of SEQ ID NO: 1, a linker of SEQ ID NO: 2 and a polypeptide of SEQ ID NO: 1 in any of the following order:

(a) H i s tag—TEV protease recognition sequence—one polypeptide of SEQ ID NO: 1 one linker of SEQ ID NO: 2 polypeptide of SEQ ID NO: 1, or

(b) Polypeptide of SEQ ID NO: 1 Linker of SEQ ID NO: 2 Polypeptide of SEQ ID NO: 1 _TE V protease recognition sequence 1His tag

7. The anti-HIV drug according to any one of 1 to 6 above, which is contained in

8. A polypeptide corresponding to the fusion protein according to any one of 1 to 7 above.

9. A nucleotide sequence or complementary strand that codes for the polypeptide described in 8 above.

10. A transformant obtained by introducing the base sequence described in 9 above into a host cell.

1 1. The transformant according to 10 above, wherein the host cell is Escherichia coli, actinomycetes or yeast.

12. The transformant according to 11 above, which is Escherichia coli BL21 (DE3) plysS. 13. The above 1 to 7 comprising culturing the transformant according to 11 and 12 to produce the polypeptide according to 8 above in or in the transformant and isolating it. The manufacturing method of the anti-HIV drug in any one of.

Brief Description of Drawings

FIG. 1 shows a method for constructing a His-TEV-AH dimer / RTB-L gene according to the present invention.

FIG. 2 shows a method for constructing a His-TE V-AH dimer / RTB-L expression plasmid according to the present invention. BEST MODE FOR CARRYING OUT THE INVENTION

[Anti-HIV drug]

In a first embodiment of the present invention, an anti-feature characterized by comprising a fusion protein comprising an actinohibin multimer and at least one other polypeptide or protein (hereinafter also referred to as a fusion protein-bound actinohibin multimer). HIV drugs are provided. Where akuchinobin is

(a) the polypeptide represented by SEQ ID NO: 1, and (b) A homolog having syncytium formation inhibitory activity in which one or more amino acids in the amino acid sequence of (a) are substituted, deleted, or added.

The polypeptide in which a part of the amino acid sequence listed in (b) above is substituted, deleted, or added is an amino acid sequence described in SEQ ID NO: 1, at least 20%, preferably 30% or more, Preferably, it means a polypeptide having amino acid homology of 50% or more, more preferably 80% or more, most preferably 90% or more, particularly preferably 95% or more. As long as syncytium formation inhibitory activity is shown, amino acid substitutions, deletions, or additions can occur anywhere. Further, in the present application, “polypeptide having amino acid homology of p ° / ₀ or more” is a sequence having a length of p% or more of the original amino acid sequence length in terms of deletion. For example, in the case of the amino acid of SEQ ID NO: 1, an amino acid sequence having 50% or more homology consists of 114 X 50/100 = 57 or more amino acids.

In addition, the polypeptide represented by SEQ ID NO: 1 is composed of three homologous domains (1 to 38, 39 to 76, 77 to 114 in SEQ ID NO: 1). Actinohibin multimer (n multimer) In which 1 or 2 homologous domains are added. The number (n) of actinohibin units contained in actinohibin multimer (n-multimer) is not particularly limited as long as it exhibits syncytium formation inhibitory activity, but it is usually hexamer or less, preferably tetramer or less. For example, a dimer.

Syncytium formation inhibitory activity is measured as appropriate. For example, (i) cells expressing HIV envelope glycoprotein (gpl 20, gp41) (eg, HeLa cells) and (ii) CD4, HIV cells Contact 'Can be determined by the presence or absence of fusion with cells expressing the chemokine receptor (C XCR4 or CCR 5) gene essential for entry (eg, HeLa cells). In practice, the transcription activation protein (T at) gene is used in (i) above, and the HIV LTR (terminal repeat) and beta galactidase gene are introduced in (ii). By using such cells, the beta-galactosidase is activated by the HIV infection of the cells, so that the substrate for beta-galactosidase, X-gal (5-bromo-1,4-chloro-3-indolyl-β- The presence or absence of syncytia formation (HIV infection) can be determined by the coloration of D-galactobylanoside) or ONPG (ο—ditrophenyl J3—D-galactobylanoside). Further, in the present invention, the actinohibin multimer means the above (a) or (b) Means a mino acid sequence linked directly or via any linker sequence. The linker sequence is not particularly limited as long as it does not impair the anti-HIV action of multimers. It is a sequence comprising at most, preferably at most 20, more preferably at most 15 amino acid residues. For example, the polypeptide represented by SEQ ID NO: 2 is included. Accordingly, the main body of the actinohibin multimer of the present invention includes the linker of SEQ ID NO: 1 and the linker of SEQ ID NO: 2 and the polypeptide of SEQ ID NO: 1. This polypeptide is a dimer, but the structure of trimers and higher is the same.

Further, the fusion protein-binding actinohibin multimer of the present invention is a fusion protein of an actinohibin multimer (which may include a linker as described above) and at least one other polypeptide or protein.

The at least one other polypeptide or protein preferably includes a protease recognition sequence (polypeptide), and further preferably includes a polyhistidine tag (His-tag). Here, the protease recognition sequence is a sequence of a site where a protease acts.

These may be added to either the N-terminal side or the C non-terminal side. Proteins polyhistidine-tagged, using N i "or C o ^{+ +} etc. polyhistidine and complex purification means that carrying the metal ions to form a, for example, N i ^{+ +} Sepharose column, etc. Akuchinohibin The purification of the multimer can be performed very easily and the product can be recovered with high yield, where the polyhis tag is, for example, His 6 tag The protease in the protease recognition sequence is, for example, TEV (Tobacco Etch Virus) -derived protease), Factor Xa, etc. In general, these modified sequences reduce the anti-HIV effect, but the His tag—TEV—the polypeptide of SEQ ID NO: 1 and the linker of SEQ ID NO: 2 are consistent. The fusion protein actinohibin dimer, which is the polypeptide of SEQ ID NO: 1 (shown as SEQ ID NO: 3), is more syncytial than the original actinohibin. Growth inhibitory activity was increased to about 2 0 fold.

Therefore, the fusion protein-binding actinohibin multimer of the present invention preferably comprises a His tag, a TEV protease recognition sequence, a polypeptide of SEQ ID NO: 1, a linker of SEQ ID NO: 2, and a polypeptide of SEQ ID NO: 1 in any of the following: The order of:

(a) His tag 1 TEV protease recognition sequence—polypeptide of SEQ ID NO: 1— Each of the linkers in column number 2;

(b) Polypeptide of SEQ ID NO: 1 Linker of SEQ ID NO: 2 Polypeptide of SEQ ID NO: 1 — T E V protease recognition sequence 1 H i s tag

Including.

The fusion protein-binding actinohibin multimer contains a polypeptide between any of the elements listed above (His tag, TEV protease recognition sequence, polypeptide of SEQ ID NO: 1, linker of SEQ ID NO: 2). May be included. Examples of such polypeptides include signal peptide sequences as described below, or vector-derived V5 epitopic tags, etc., but purification and handling of other polypeptides that do not reduce the activity of fusion protein-bound actinohibin multimers. May contain useful sequences. Such a sequence may not be included.

The reason why the fusion protein-bound actinohibin dimer shows excellent activity is not clear, but the three-dimensional structure of the actinohibin dimer molecule differs depending on the type and length of the linker, and this is the reason for the binding of gp120. It is estimated that it showed different activities. In addition, the increase in activity due to the addition of Hi s * tag-TEV is an issue that needs to be investigated in order to investigate the molecular structure activity of actinohibin.

[Polypeptide]

In a second embodiment of the present invention, there is provided a polypeptide corresponding to the above fusion protein-bound actinohibin multimer characterized in that it comprises an actinohibin multimer. The details of the polypeptide are the same as those described for the anti-HIV drug.

[Base sequence]

In the third embodiment of the present invention, a nucleotide sequence encoding the polypeptide or a complementary strand thereof is provided.

For example, the base sequence provided by the present invention includes the base sequence of SEQ ID NO: 4 (including the stop codon) corresponding to the polypeptide of SEQ ID NO: 3, but more generally, (a ′) A base sequence encoding the polypeptide represented by SEQ ID NO: 1 and syncytium formation in which one or more amino acids in the amino acid sequence of (b ′) (a ′) are substituted, deleted, or added Encodes a linker sequence of actinohibin coding regions (plural) selected from nucleotide sequences encoding homologs having inhibitory activity A base sequence in which the base sequence encoding the His • tag—TEV of the fusion protein is combined with the base sequence linked by the base sequence is provided.

Here, the significance of homologue, linker sequence and syncytium formation inhibitory activity is as described above. The homologue is at least 20%, preferably 30% or more, more preferably 50% or more, even more preferably 80% or more, and most preferably, the nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 1. Has 90% or more homology. In addition, as described above, actinohibin monomer is composed of three homologous domains. However, a base sequence corresponding to one or two homologous domains may be added to a base sequence corresponding to a multimer. The number (n) of actinohibin coding regions contained in the nucleotide sequence is not particularly limited as long as the finally obtained actinohibin multimer exhibits syncytium formation inhibitory activity. Preferably, it is 3 or less, for example 2.

Furthermore, the base sequence according to the present invention may also contain a sequence useful for the production of a polypeptide. For example, if it is necessary or advantageous to form an actinohibin multimer in an organism first as a precursor polypeptide and then post-translationally modify it to obtain the actinobin multimer as a mature protein, such a nucleotide sequence May be included. Examples of such a base sequence include a signal peptide sequence region used for secretion of microorganisms outside the cells.

In the present application, the base sequence encoding the polypeptide may be encoded by any of the degenerate codons, but is preferably a base sequence suitable for the transformant described in the next section. The base sequence may be any of the above complementary strands.

[Transformant]

In a fourth embodiment of the present invention, a transformant in which the above base sequence is introduced into a host cell is provided. The transformant is not limited as long as it can stably express the actinohibin multimer, and includes E. coli, actinomycetes and other bacteria, yeast, animal cells, insect cells, plant cells, etc., cell fusion, Cells previously altered by cell engineering by means such as genetic manipulation are also included in the host of the transformant of the present invention. Particularly preferably, the host cells are Escherichia coli and actinomycetes, and an example of such a transformant is Escherichia coli BL2l (DE3) plysS strain.

Transformants can be generated by any method known in the art (for example, 1

Joseph Sambrook et al., Molecular Cloning 3 rd Ed. (Cold Spring Harbor Laboratory Press (2001) refer).

[Method for producing anti-HIV drug]

In a fifth embodiment of the present invention, the production of an actinohibin multimeric anti-HIV drug comprising culturing the transformant to produce the polypeptide in the transformant or culture and isolating the polypeptide. Law is provided.

In culturing the transformant of the present invention, a usual culture method used for culturing the transformant is applied. The medium to be used can be either a natural medium or a synthetic medium as long as it contains moderately carbon sources, nitrogen sources, inorganic substances, vitamins, growth factors and the like that can be used by transformants.

Carbon sources include carbohydrates such as glucose, fructose, mannose, maltose, sucrose and molasses, organic acids such as citrate, malic acid and acetic acid, alcohols such as methanol and ethanol, soybean oil, cottonseed oil and palm oil. Oils and fats, methane, ethane, butane, hydrocarbons such as n-paraffin or glycerol are used.

Nitrogen sources include ammonium chloride, ammonium sulfate, ammonium nitrate, ammonium salts such as ammonium phosphate, amino acids such as aspartic acid, glutamine, alanine, urea, peptone, meat extract, yeast extract, dry yeast, corn 'steam' Liquor, soy flour, solubil 'vegetable protein, cottonseed meal, casein, casamino acid, pharma media, etc. are used. As inorganic substances, monolithic phosphate, potassium dihydrogen phosphate, disodium phosphate, magnesium sulfate, ferrous sulfate, manganese sulfate, copper sulfate, cobalt sulfate, zinc sulfate, ammonium molybdate, aluminum sulfate, barium carbonate Calcium carbonate, salt, etc. are used. In addition, substances that promote the growth of bacterial cells or the production of actinohibin multimers such as trace amounts of metal salts and vitamins can be added to the medium as necessary. If a transformant requires a specific substance, it must be added as necessary. Any of these may be used as long as they are useful for the production of actinohibin multimers. For example, transformants, particularly those known as culture raw materials for Escherichia coli and actinomycetes can be used.

For mass culture of transformants, particularly microorganisms, a shaking culture method using a liquid medium, an aeration and agitation culture method and the like are preferable. At the culture temperature, transformants grow and actinohibin multimers Applicable to the extent that 2 can be produced. The culture can be appropriately selected according to the properties of the transformant. If actinohibin multimers in the culture are present in the culture solution, the culture solution containing the cells can be collected and used as is, but in general, it is cultured by filtration, centrifugation, etc. according to conventional methods. The actinohibin multimer solution after separating the actinohibin multimer and transformant in the solution is used. If actinohibin multimers are present in the transformant, the cells are collected from the obtained culture by a technique such as filtration or centrifugation, and then the cells are collected by a physical method or an enzyme such as lysozyme. Dissolve the cells using a conventional method, and if necessary, add a chelating agent such as EDTA and Z or a surfactant to solubilize the transformed cells, and separate and collect them as an aqueous solution.

The actinohibin multimer-containing solution thus obtained is subjected to, for example, vacuum concentration, membrane concentration, salt prayer treatment such as ammonium sulfate, or fractional precipitation using a hydrophilic organic solvent such as methanol, ethanol, acetone, etc. It may be precipitated by. Then

This precipitate can be dissolved in water and dialyzed with a semipermeable membrane, or a lower molecular weight impurity can be removed with a molecular sieve membrane. It can also be purified by gel filtration with an adsorbent or gel filter, adsorption chromatography, ion exchange chromatography, or reverse phase chromatography. In particular, in a production system that produces a fusion protein-bound actinohibin multimer containing a His • tag, it can be purified by a Ni ^{+ +} chelate Sepharose column that specifically adsorbs the His • tag. The fusion protein-binding actinohibin multimer obtained by these means can be obtained by purifying the containing solution by concentration under reduced pressure, lyophilization, etc. to obtain a high-purity fusion protein-binding actinohibin multimer. Example

The present invention will be described more specifically with reference to the following examples. However, these examples do not limit the present invention.

Example 1:

(1) Construction of a gene encoding a lactinohibin dimer

According to the procedure shown in FIG. 1, a gene (SEQ ID NO: 4) encoding the actinohibin dimer represented by SEQ ID NO: 3 was constructed. 3 First, a linker gene (hereinafter referred to as RTB-L gene) encoding 12 amino acid residues for linking two actinohibin genes (hereinafter referred to as AH gene) was prepared as follows.

That is, RTB-L gene is pri me r— 1 (CTG CCC AC C AAC AAC AC C CAG CCC TTC) (SEQ ID NO: 5) and primer-2 (GTT CCG GAT GGT GGT G AC GAA GGG CTG) (SEQ ID NO: 5) 6) Using the two types of primers and overlapping each of the 9 bases on the 3 'end side to form a primer dimer, PCR (reaction conditions: 30 seconds at 94 ° C, 30 seconds at 60 ° C) Second, 60 seconds at 72 ° C, and 30 cycles reaction).

Next, the plasmid pBSK +: 2A3K, which retains the AH gene cloned from the actinobine producing bacterium, is used as the AA—1 Bam (GGG AT C CGC CTC GGT GAC C) (SEQ ID NO: 7 ) And primer-3 (GTT GGT GGG CAG CCA CTT CTG GT A GTT) (SEQ ID NO: 8) were used to amplify the AH gene by PCR. As a result, an N-AH dimer / RTB-L gene was prepared by adding 12 base pairs of the RTB-L gene at the 5 'end to the 3' end of the AH gene.

Third, RTB-L gene and N-AH dimer / RTB-L gene are in a saddle type, and AA-1 Bam and primer-2 are used for SOE (Horton, RM, Hunt, HD, Ho, SN, Pullen, JK, and Pease, LR, Engineering hybrid genes without the use of restriction enzymes: PCR by gene splicing by overlap extension, Gene 77, 61-68 (1989)), R TB—L at the 3 ′ end of the AH gene L-AH dimer / RTB—L gene with the full-length gene added was prepared. On the other hand, the AH gene was amplified with PCR using Accsen (TCG GTG ACC ATC CGG AAC GCC CAG) (SEQ ID NO: 9) and TGA Hind (GA A GCT TC A GCC GGT GT AC) (SEQ ID NO: 10). C—AH dimer ZRTB—L gene was prepared.

The L-AH dimer ZRTB-L gene and the C-AH dimer ZRTB-L gene thus obtained were restricted with the restriction enzymes BamH I and A cc III and Ac c III and Hind III, respectively. Digested. Obtained two DN A The 4 fragments were mixed with E. coli cloning vector PUC19 digested with BamHI and HindIII and ligated using T4 DNA ligase. Escherichia coli J Ml 09 was transformed with this solution to prepare a plasmid pUC19: AH dimer ZRTB-L containing the AH dimer / RTB-L gene.

(2) Construction of His-TE V-AH dimer / RTB—L expression plasmid According to the procedure shown in Fig. 2, the fusion protein-bound actinohibin dimer (hereinafter, His-TEV-AH dimer / RTB— L) An expression plasmid was constructed.

p UC 1 9: AH dimer ZRT B—L as a saddle, 1 5 1 sen (C ACC GCC TCG GTG ACC) (SEQ ID NO: 1 1) and 1 5 1 asn (TT A TT A GCC GGT GTA CCA C) The AH dimer / RTB-L gene was amplified by PCR using (SEQ ID NO: 12).

Equal volume of this AH dimer ZR TB-L gene solution and E. coli expression vector p ET 1 5 1 D0 0-0 D0 solution are mixed and incubated at room temperature for 30 minutes. The Escherichia coli J Ml 09 was used to transform His-TE V -AH dimer / RTB—L expression plasmid, p ET 1 5 1: AH dimer / RTB—L.

0 00 1

(3) Expression of H i s -TEV-AH d i me r / R T B— L in E. coli

H is—TEV—AH dimer ZRTB—L expression plasmid p ET 1 5 1: Escherichia coli BL21 (DE3) plysS transformed with AH dimer ZR TB—L, ampicillin (AP) 1 0 0 μg / m 1, Chloramphenicol (CM) 34 7 in LB medium containing ί g / m 1 and 2% glucose. C. Culturing was performed with shaking. This bacterial solution was inoculated into LB medium containing AP 100 μg / m 1 and CM ₃₄ g / m 1 at an inoculum concentration of 1%, and cultured with shaking at 37 ° C. 〇. D. 600 When the value of 0 nm (growth degree of the E. coli) reaches 0.25 to 0.30, isoporyl pill] Final concentration of 3-thiogalactoviranoside (IPTG) After adding to 1 mM, the mixture was further cultured with shaking for 2 hours. The medium was quenched with ice water to stop the growth of E. coli, and the culture was centrifuged at 80,000 Xg for 10 minutes at 4 ° C to collect the cells. The obtained cells were washed twice with PBS (-). Escherichia coli expressing His-TEV-AH dimer / RTB-L in a binding buffer solution (5 mM imidazole) Five

, 0.5 M sodium chloride, 2 OmMT r i s—HCl (pH 7.9)).

Example 2:

A fusion protein-bound actinohibin dimer (hereinafter referred to as “His-TEV-AHdimerZRTB-L”) was prepared and purified by the following procedure, and tested for its anti-HIV effect.

[Preparation of E. coli extract]

According to Example 1, His-TE V-AH dimer / RTB—L was expressed in Escherichia coli. Binding buffer (5 mM imidazole, 0.5 M NaCl, 20 mM Tris · HC1 (pH 7.9)) 10 m l suspended. The suspension of Escherichia coli was subjected to an interval of 10 seconds every 10 seconds, and the cells were disrupted by ultrasonic treatment for a total of 3 minutes to obtain a bacterial extract. The extract was centrifuged at 8,000 Xg for 10 minutes at 4 ° C, and separated into a soluble fraction and an insoluble fraction. The insoluble fraction was dissolved in a binding buffer containing 6 M guanidine hydrochloride (hereinafter referred to as binding buffer DN) and used as a sample for purification by nickel chelate column chromatography.

[Affinity Chromatography using Nikkenorechelate S e p h a r o se 1]

Filled in 5 ml of Ni Sepharose High performance (GE Healthcare), equilibrated with 25 ml of binding buffer DN and dissolved in binding buffer DN. His—TE V—AH Dimer / RTB—L solution was passed through. After washing the resin with 25 ml of binding buffer DN, H s -TE V-AH dimer ZR TB-L was eluted with binding buffer DN25 ml at an imidazole concentration of 100 mM.

[OD S column chromatography]

ODS resin suspended in acetonitrile (SSC Senshiyu Kagaku Co., Ltd.) 10m 1 is packed into an open column, washed with 50% acetonitrile of 5Om 1 and 0.01% trifluoroacetic acid (TFA). The resin was equilibrated with 0.01% TFA of 1% 5% acetonitrile.

Poured H is -TE V-AH di me r / RTB one L solution was purified by nickel chelate one Tokaramu, After washing the resin with 50 m 1 of a 5% Asetonitoriru one ^{_{0. 0 1 0/0 TFA,}} 50 m The His—TE V-AH dimer ZRTB—L was eluted with 0.01% TFA of 50% acetonitrile. The resulting eluate was centrifuged and evaporated. 6 Concentrated to dryness using 1 liter and dissolved in mi 1 1 i Q water.

[High performance liquid chromatography (HP LC)]

The sample desalted with the OD S column was dissolved in a small amount of mi 1 1 i Q water and subjected to HP LC (PEGASIL 0DS 20ΦΧ250 mm (S S C Senshu Science Co., Ltd.)). As the mobile phase, 0.01% TFA of 5% acetonitrile was allowed to flow for 5 minutes, and after that, a gradient end was applied so as to reach 0.01% TFA of 70% acetonitrile after 25 minutes. The flow rate was 7 m 1 Zm in and the column temperature was set to 40 ° C. The eluate was monitored by UV with a wavelength of 21 Onm, the peak eluted at about 18 minutes was fractionated, and then freeze-drying was repeated with a centrifugal evaporator until the pH of the solution reached near neutrality.

[Measurement of syncytium formation inhibitory activity]

HeLa / T-env / Tat cells (HIV model cells) and HeLa / CD4 / LTR cells (immune system model cells) were cultured in 75 cm ² culture flasks using DME medium (Delbecco's modified Eagle medium, thigh (10 (Uig / ml), 0 · 1% carbonated water containing Natoriumu, 10% fetal bovine serum) was carried out at 5% C_〇 ₂ stream under 37 ° C.

HeLa / CD4 / LTR cells suspended in DME medium were spread in each well of a 96-well microplate at 8 × 10 ³ cells / 50 μl. Add 10 μl of the sample solution diluted with serum-free DME medium (KM 100 μg / ml), and then add 8 HeLa / T-env / Tat cells suspended in DME medium. X 10 ³ pieces / 50 β 1 were sprinkled, and the total amount was 1 10 μ 1, and the cells were cultured at 37 ° C. for 24 hours in a 5% CO ₂ stream. The culture solution was removed, 20 μ 1 of cell lysate (0.05 Triton X-100) was added, and the mixture was allowed to stand at room temperature for 15 minutes. Chromogenic substrate solution (60mM Ninatrium hydrogen phosphate, 40raM Sodium dihydrogen phosphate, 10mM potassium chloride, ImM Magnesium sulfate, 50mM] 3-Mercaptoethanol, 0.8rag / ml 0NPG (o-diphenyl 2-D-galactobiraside) Add 100 1 to each well and incubate at 37 ° C for 80 minutes, then add 50 M of reaction stop solution 2 M sodium carbonate to each well and absorb at 405 nm using a microplate reader. Was measured. Syncytium formation rate was calculated from the obtained absorbance (the following formula). Syncytium formation rate = (absorbance when inhibitor is added / absorbance when no inhibitor is added) legality of fusion protein-bound actinohibin dimer (His -TE V-AH dimer / RTB_L) thus obtained 50 ° / o inhibitory concentration on body formation (IC ₅₀ 7

) Is shown in Table 1.

—Hys-TE V-AH dimer / RTB— Combines various fusion proteins with the same procedure as in the preparation of L, with or without the addition of a His-tag, with different modified proteases, and with different linkers. Actinohibin and actinohibin dimer were prepared and the syncytium formation rate was determined. These comparison results are shown together in Table 1.

Table 1. Syncytium formation inhibitory activity of actinohibin (AH) dimer

1) Actinohibin from Actinomyces K97-0003 strain described in WO 00/52043

2) FB: Linker sequence described in WO 2006/059638 Based on the above results, there is a difference in syncytium formation inhibitory activity depending on the linker used in the dimerization of actinohibin. is -Xa (recognition sequence by protease F actor X a) 1 AH and H is— TEV— AH shows higher activity in the latter, and AH di me r and H is— TE V_AH di me r It is clear that the latter is more active, and the fusion protein-binding actinohibin dimer according to the present invention, in particular, the actinohibin dimer with SEQ ID NO: 2 as the linker is His-tag— The new derivative to which TEV was added showed syncytium formation inhibitory activity 18.6 times higher than that of actinohibin monomer.

[Measurement of anti-HIV activity (MAG I C—5 a s s a y)]

Anti-HIV activity was measured for His-TEV-AH dimer ZR TB-L, which showed the highest increase in activity. 8 Anti-HIV activity is observed in 96-well microplates, complete medium (DME medium, 10% fetal calf serum, penicillin G monosodium (100 U / m 丄 no, streptomycin (100 μg / ml)), hygromycin B (100 μ g / ml), geneticin (200 μ g / ml), blastisidin (l μ g / ml)) MAG IC one 5 cells suspended seeded by 10 ⁴ in under 5% C_〇 ₂ stream 37 ° the sample solution was added to the. cell culture medium cultured for 24 hours in C, and then the HIV solution was added, and cultured for 2 hours, were infected with HIV in the cells. subsequently, 5% C0 ₂ stream under 37 ° Cultivation was continued for 48 hours at C. After completion of the culture, the cells were fixed, stained with X_Ga1, and the number of HIV-infected cells per well was counted with a microscope. HIV activity (IC ₅ value) was calculated from the following formula (the following formula).

Number of HIV-infected cells in the sample-added medium

X 100 (%)

Number of HIV-infected cells in the test-free medium Table 2. Anti-HIV activity of AH and His-TEV-AH dimer / RTB-L

As a result, the anti-HIV activity of His-TEV-AH dimer / RTB-L was increased 2-fold to 18-fold compared to vaccinohibin (AH) (Table 2). It also shows anti-HIV activity against strain 36, which did not show anti-HIV effect with vaccinohibin. Industrial applicability

The actinohibin multimeric protein of the present invention inhibits syncytium formation, a critical pathway in HIV infection. This is thought to be due to the mechanism of action similar to the inhibition of syncytium formation by actinohibin, and is considered to prevent not only T-tropic HIV but also M_tropic HIV infection. In addition, for example, the anti-HIV effect of the actinohibin dimer protein of the present invention is nearly 20 times that of the actinohibin monomer fusion protein, showing a remarkable effect even in a very small amount. Moreover, since the molecule according to the preferred embodiment itself contains a His tag, it can be easily purified in high yield. Thus, according to the present invention, an anti-HIV drug polypeptide that can be used as a preventive or therapeutic drug for HIV and AIDS is provided. In addition, the gene encoding the actinohibin multimeric protein of the present invention can be introduced into various hosts and produced as a recombinant actinohibin multimeric protein, and can be easily produced.

Claims

The scope of the claims

1. An anti-HIV (human immunodeficiency virus) drug comprising a fusion protein comprising an actinohibin multimer and at least one other polypeptide or protein.

2. The vaccinohibin multimer

(a) the polypeptide represented by SEQ ID NO: 1, and

The anti-HIV drug according to claim 1, wherein a plurality of polypeptides selected from force (which may be the same or different) are linked by 50 or less linkers.

3. The anti-HIV drug according to claim 1, wherein the at least one other polypeptide or protein comprises a protease recognition sequence (polypeptide).

4. The anti-HIV drug according to claim 1, wherein the at least one other polypeptide or protein comprises a recognition sequence of H s tag · V protease (protease derived from Tobacco Etch Virus).

5. The anti-HIV drug according to claim 1, wherein the actinohibin multimer is an actinohibin dimer.

6. The anti-HIV drug according to claim 1, wherein the linker is a peptide chain represented by SEQ ID NO: 2.

(a) His tag 1 TEV protease recognition sequence—polypeptide of SEQ ID NO: 1— 2 Column No. 2 linker, SEQ ID No. 1 polypeptide, or

(b) Polypeptide of SEQ ID NO: 1 Linker of SEQ ID NO: 2 Polypeptide of SEQ ID NO: 1 TEV protease recognition sequence 1 H i s tag

The anti-HIV drug according to claim 1, which is contained in

8. A polypeptide corresponding to the fusion protein of claim 1.

9. A nucleotide sequence or a complementary strand for coding the polypeptide according to claim 8.

10. A transformant in which the base sequence according to claim 9 is introduced into a host cell.

11. The transformant according to claim 10, wherein the host cell is Escherichia coli, actinomycetes or yeast.

1 2. The transformant according to claim 11, which is Escherichia coli BL21 (DE3) plysS.

1 3. A method comprising culturing the transformant according to claim 1, 1, 2, producing the polypeptide according to 8 above in or in the transformant, and isolating it. A method for producing an anti-HIV drug according to any one of 1 to 7.