JP2011177135A - Method for producing n36-binding peptide by yeast - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a DNA encoding an N36-binding peptide fused with a polypeptide, and exhibiting a new effect in an yeast, and the DNA for highly producing a complete length N36-binding peptide by using the yeast. <P>SOLUTION: This DNA obtained by fusing a DNA consisting of an amino acid sequence having ≥70% homology with a specific amino acid sequence originated from Aspergillus and encoding a protein having an endoglucanase activity with a DNA encoding a thrombin-recognition sequence and a DNA encoding the N36-binding peptide binding with the N36 protein of a retrovirus, and the DNA obtained by fusing a DNA encoding a polypeptide consisting of an amino acid sequence having ≥70% homology with a specific amino acid sequence originated from the yeast and functioning as a signal sequence with a DNA encoding His tag and a DNA encoding the N36-binding peptide binding with the N36 protein of a retrovirus causing immunodeficiency to mammalian animals are provided. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a DNA for producing an N36-binding peptide in yeast, a yeast transformant having the DNA, and a method for producing an N36-binding peptide using the transformant.

  AIDS (Acquired Immuno Deficiency Syndrome) is an abbreviation for acquired immune deficiency syndrome. When infected with a retrovirus that causes immunodeficiency (eg, HIV, SIV), the immune system against pathogens does not function normally. It is a general term for various diseases that develop.

  It is estimated that HIV uses a Type-I type fusion fusion protein for membrane fusion between a virus and a target cell during infection of the target cell. From the functional analysis of proteins such as HIV-1 having Type-I type fusion protein, the following is estimated as the function of HIV gp41 during membrane fusion. (1) gp41 has two regions with high α-helix properties (often called HR1 and HR2 from the N-terminal side). (2) The N-terminal side of gp41 is first anchored to the target cell membrane, and then three N36s (HR1) interact to form a helix bundle. (3) To this three-stranded α-helical coiled-coil, three C34s (corresponding to the HR2 region) are bound in antiparallel so as to surround from the outside to form a 6-helix bundle. (4) As a result, it is a dynamic supramolecular mechanism in which HIV and the target cell membrane come close to each other and membrane fusion is established. Based on this mechanism, it is believed that the C34-derived peptide of HIV gp41 inhibits 6-helix bundle formation and exhibits antiviral activity. From the sequence analysis of gp41, search and identification of sequences corresponding to N36 and C34, as well as evaluation of anti-HIV activity of these peptide sequences, were conducted by several research groups including our group. (For example, Patent Documents 1 to 3). In addition, an anti-HIV agent T-20 (trade name Fuzeon) already on the market is a peptide that inhibits this process (Non-patent Document 1). On the other hand, recent studies have revealed that drug-resistant strains emerge due to long-term administration of Fuzeon, and there is a concern that the effect of preventing the onset of infected individuals may be diminished. ing.

  We have X-EE-XX-KK scan, ie, a 7-residue sequence of X-EE-XX-KK (X: amino acids required for interaction with HR1; E: Glu; K: Lys) It was discovered that peptides designed by a unique molecular conversion strategy using the repetition of the above were further highly active (Patent Documents 4 and 5, Non-Patent Documents 2 and 3). In Fuzeon resistant strains, amino acid substitutions based on single nucleotide mutations in the viral genome occur in the N36 region to which Fuzeon binds, and in the C34 region that is thought to be in contact with the N36 region when forming a 6-helical bundle structure. Similar amino acid substitutions have occurred. The inventor conducted comprehensive synthesis and evaluation of antiviral activity of derivatives of the N36 peptide in which the amino acid substitution position generated at the contact site of the C34 region with the N36 region was changed, including wild strains and Fuzeon resistant strains. Peptides showing strong anti-HIV activity against drug-resistant strains have been identified (Patent Document 5, Non-Patent Document 4).

  The supply of these anti-HIV active peptides has heretofore been performed by chemical synthesis combining a solid phase synthesis method and a liquid phase method (Non-patent Document 5). However, when considering stable supply to developing countries and other regions where inexpensive anti-HIV drugs are needed, it is necessary to supply cheap pharmaceutical raw materials using fermentation technology.

  Patent Documents 6 and 7 describe a method for producing this N36-binding peptide inexpensively and in large quantities by using at least one selected from the group consisting of Escherichia coli, yeast and filamentous fungi as a host. Further, in Patent Documents 6 and 7, N36-binding peptides are expressed as N36-binding peptides when expressed in a host alone, because the yield of the target peptide is low even if various high-expressing substance production systems are used. It is described that it is preferable to link with a polypeptide that can be expressed in a host or to express an N36-binding peptide as a multimer (for example, 3 to 7 mer).

In addition, it is considered preferable to use yeast from the following points as a host for producing an N36-binding peptide.
-In Escherichia coli, the produced target protein remains in the microbial cells, but yeast can be secreted and expressed. By secreting the yeast, the subsequent purification becomes very simple, leading to a significant cost reduction.
・ It is easier to control the culture compared to filamentous fungal yeast in terms of culture, and the fungus body is easier to control. Therefore, high-density culture is possible, and the substance after production is relatively stable (not susceptible to degradation). Yield is expected to improve. In addition, the culture time is short and the handling is easy. On the other hand, since filamentous fungi are rich in degrading enzymes, there is an aspect that, when secreted and produced in the same way, the product is susceptible to degradation during culture, and it is difficult to stably produce the target product. .

  Patent Documents 6 and 7 include alpha factor, invertase, acid phosphatase and the like as a polypeptide preferably fused with an N36-binding peptide when the host is yeast. In Example 2, an N36-binding peptide is produced by fusing and expressing an alpha-factor signal sequence and an N36-binding peptide using yeast.

International Publication No. 2005/067960 International Publication No. 01/51673 International Publication No. 96/19495 International Publication No. 03/029284 International Publication No. 2008/050830 International Publication No. 2008/013242 JP 2008-29239 A

Nature Reviews Drug Discovery, Volume 3, 215-225 (2004) Angewante Chemie International Edition in English, 41, 2937-2940 (2002) Journal of Medicinal Chemistry, 51, 388-391 (2008) Journal of Biological Chemistry, 284, 4914-4920 (2009) Nature Reviews Drug Discovery, Volume 2, pages 587-593 (2003)

  However, in Patent Documents 6 and 7, the only polypeptide used for expression by fusing with an N36-binding peptide in yeast is the alpha-factor signal sequence, and the effects when other polypeptides are used. Has not been considered.

  Furthermore, in Example 2 of Patent Documents 6 and 7, the expression of the N36-binding peptide in yeast was confirmed only by Western blotting, and the expression level of the N36-binding peptide was very small. In addition, the N36-binding peptide obtained by the production method using koji molds of Patent Documents 6 and 7 sometimes lacks or is degraded, and there is a problem that it is difficult to obtain a complete N36-binding peptide.

  The present invention has been made to solve the above-mentioned problems, and DNA encoding an N36-binding peptide fused with a polypeptide having a new effect in yeast, a yeast transformant having the DNA, and the transformation An object of the present invention is to provide a method for producing an N36-binding peptide using the body. The present invention also provides DNA for high production of full-length N36-binding peptide using yeast, a yeast transformant having the DNA, and a method for producing N36-binding peptide using the transformant. The purpose is to do.

  The present inventors have obtained the knowledge that cleavage occurs at the thrombin recognition sequence by fusing and expressing CelA, thrombin recognition sequence and N36 binding peptide using yeast. Furthermore, the inventors have found that an N36-binding peptide can be produced at high yield by fusing and expressing an α-factor prepro sequence, a His tag and an N36-binding peptide in yeast.

  The present invention has been completed based on these findings, and has been completed. The present invention provides the following DNA, yeast transformant, N36-binding peptide production method, and the like.

  Item 1. DNA encoding a protein comprising an amino acid sequence having 70% or more identity with the amino acid sequence represented by SEQ ID NO: 1 and having endoglucanase activity, DNA encoding a thrombin recognition sequence, and a mammal A DNA fused with a DNA encoding an N36-binding peptide that binds to the N36 protein of a retrovirus that causes immunodeficiency.

  Item 2. A DNA obtained by fusing the DNA of Item 1 with a DNA encoding an amino acid sequence for purification.

  Item 3. A transformant of yeast into which a gene expression cassette containing the DNA according to Item 1 or 2 is introduced.

  Item 4. Item 4. The transformant according to Item 3, wherein the yeast is Saccharomyces cerevisiae.

Item 5. Item 5. A method for producing an N36-binding peptide, comprising culturing the transformant according to Item 3 or 4 to produce and accumulate an N36-binding peptide in the culture, and collecting the N36-binding peptide from the culture.

  Item 6. DNA encoding a polypeptide consisting of an amino acid sequence represented by SEQ ID NO: 2 and having an identity of 70% or more and functioning as a signal sequence, DNA encoding a His tag, and immunodeficiency in mammals A DNA fused with a DNA encoding an N36-binding peptide that binds to the N36 protein of the retrovirus that occurs.

  Item 7. A gene expression cassette comprising the DNA of Item 6, wherein the promoter comprises a base sequence having 70% or more identity with the base sequence represented by SEQ ID NO: 3, and comprises a DNA that functions as a promoter in yeast Gene expression cassette.

  Item 8. A transformant of yeast into which the gene expression cassette comprising the DNA of item 6 or the gene expression cassette of claim 7 has been introduced.

Item 9. Item 9. A method for producing an N36-binding peptide, comprising culturing the transformant according to Item 8 to produce and accumulate an N36-binding peptide in the culture, and collecting the N36-binding peptide from the culture.

  Item 10. Item 9 is characterized in that the culture is performed in a medium containing 0.01-10% (w / v) yeast extract, 0.01-10% (w / v) peptone, and 0.1-20% (w / v) galactose. The method described in 1.

  Item 11. Item 15. The method according to Item 9, 9 or 10, further comprising the step of allowing cyanogen bromide to act on the N36-binding peptide, wherein the N-terminal pyroglutamic acid residue and the C-terminal homoserine lactone group are introduced. Production method.

  Item 12. Item 11. A prophylactic or therapeutic agent for a retroviral infection that causes immunodeficiency in a mammal comprising an N36-binding peptide obtained by the method according to Item 5, 9 or 10 as an active ingredient.

  By culturing a yeast transformant having DNA fused with DNA encoding the thrombin recognition sequence of the present invention, a full-length N36-binding peptide cleaved with the thrombin recognition sequence can be secreted and produced outside the cell. it can. In addition, by culturing yeast transformants having DNA fused with the DNA encoding the His tag of the present invention, a full-length N36-binding peptide can be produced at high yield, and the N36-binding peptide after secretory production The stability of the can also be improved.

It is a figure which shows the construction method of plasmid pYES2- (alpha) F and pYES2-CelA. Plasmids pYES2-αF-MQ-SC35EK-MQ, pYES2-αF-M- (Q-SC35EK-M) ₃ -Q, pYES2-CelA-MQ-SC35EK-MQ and pYES2-CelA-M- (Q-SC35EK-M ) It is a figure which shows the construction method of _3- Q. pYES2-αF, pYES2-αF-MQ-SC35EK-MQ and pYES2-αF-M- (Q-SC35EK-M) ₃ -Q were used to induce expression using transformants of YNN27, BY2777 and INVSc1 strains. It is a figure which shows the result of SDS-PAGE at the time. Use pYES2-αF-MQ-SC35EK-MQ and pYES2-αF-M- (Q-SC35EK-M) ₃ -Q transformants of YNN27 to induce expression in uracil-requiring galactose medium or YPG medium. FIG. 3 is a diagram showing the results of confirming the increase in the number of cells over time and the expression level by SDS-PAGE. pYES2-CelA, pYES2-CelA-MQ-SC35EK-MQ, pYES2-CelA-M- (Q-SC35EK-M) ₃ -Q of YNN27, BY2777 and INVSc1 transformants were used to induce expression. It is a figure which shows the result of SDS-PAGE at the time. 6 is a diagram showing the principle of ELISA performed in Test Example 2. FIG. 6 is a diagram showing the results of ELISA of Test Example 2. FIG. It is a figure which shows the result of the western blotting using the anti- His tag quality antibody which uses the microbial cell obtained by the test example 1 of the CelA fusion protein, and a culture supernatant. Each of the culture supernatants expressing CelA-His-MQ-SC35EK-MQ and CelA-His-M- (Q-SC35EK-M) ₃ -Q when purified using N36 peptide-binding Ni-NTA agarose It is a figure which shows the result of having analyzed the step by SDS-PAGE. It is a figure which shows the cutting | disconnection position assumed when CelA-His-MQ-SC35EK-MQ and CelA-His-M- (Q-SC35EK-M) _3- Q are secreted out of a microbial cell. It is a figure which shows the mass contained in the chromatogram of HPLC of the target protein refine | purified, and each peak. It is a figure which shows the arrangement | sequence considered to correspond to each peak of FIG. It is a figure which shows the LC-MS analysis result of the both terminal cyclization peptide cut out from the expressed protein. It is a figure which shows the measurement result of the circular dichroism (CD) spectrum about modified | denatured SC35EK and both ends unmodified SC35EK. It is a figure which shows the thermal stability about SC35EK modified and SC35EK without both terminal modification by circular dichroism spectrum analysis.

  Hereinafter, the DNA of the present invention, yeast transformant, N36-binding peptide production method and the like will be described in detail.

  Specific examples of the method for producing DNA, yeast transformant, and N36-binding peptide in the present invention include those shown in the following Embodiments 1 and 2.

<Embodiment 1>
DNA
The DNA of Embodiment 1 of the present invention is a DNA encoding a protein comprising an amino acid sequence having 70% or more identity with the amino acid sequence represented by SEQ ID NO: 1 and having endoglucanase activity, thrombin recognition It is characterized by being a DNA fused with a sequence-encoding DNA and a DNA encoding an N36-binding peptide that binds to an N36 protein of a retrovirus that causes immunodeficiency in mammals.

  A protein comprising an amino acid sequence having 70% or more identity with the amino acid sequence represented by SEQ ID NO: 1 and having endoglucanase activity (hereinafter also referred to as protein A) is the identity in the amino acid sequence. Is 80% or more, preferably 85% or more, more preferably 90% or more, and still more preferably 95% or more. For example, CelA derived from Aspergillus oryzae (AO090026000102) can be mentioned. The nucleotide sequence and amino acid sequence of CelA are shown as SEQ ID NO: 4 and SEQ ID NO: 1, respectively, in the sequence listing. The endoglucanase activity means the activity of hydrolyzing the β1 → 4 glucoside bond of cellulose into an endo type. By fusing the N36-binding peptide with the protein A and expressing it in yeast, it becomes possible to produce a full-length N36-binding peptide.

Endoglucanase activity can be measured by the following method. Mix 200 μl of 2% AZO-CM-Cellulose (Megazyme) (pH 5.0) and 200 μl of enzyme solution (diluted with 50 mM acetic acid-sodium acetate buffer (pH 5.0)) and react at 37 ° C. for 15 minutes. Add 1 ml of precipitant, vortex, let stand for a few minutes, then centrifuge. Measure the absorbance of the supernatant at 590 nm. Enzyme activity that increases the absorbance at 590 nm by 1 per minute is defined as 1 unit.
(Precipitant) Dissolve CH ₃ COONa 3H ₂ O 10 g and Zn (CH ₃ COO) ₂ 1 g in deionized water, adjust to pH 5.0, make up to 50 ml, and then add 200 ml EtOH And make.

In the present invention, DNA encoding protein A is synthesized by synthesizing an appropriate oligonucleotide primer pair based on a known gene sequence and extracted from the host cell or tissue of the gene or poly A ⁽⁺⁾ RNA or Cloning can be performed by RT-PCR or PCR using chromosomal DNA as a template. At this time, in order to facilitate subsequent cloning into a plasmid, an appropriate restriction enzyme recognition sequence can be added to the end of the oligo primer to be used.

  As a thrombin recognition sequence, a well-known amino acid sequence: LVPRGS can be mentioned, but the thrombin recognition sequence is not limited to this as long as it is cleaved by thrombin and exhibits the effects of the present invention. By fusing protein A (leader sequence) and thrombin recognition sequence to N36-binding protein and expressing it in yeast, it is cleaved by thrombin recognition sequence when secreted and produced outside the cell, and N36-binding protein is secreted without leader sequence Can be produced. Therefore, the work of removing the leader sequence after expression (enzyme treatment, chemical treatment) becomes unnecessary.

  In the present invention, the retrovirus causing immune deficiency in mammals includes human immunodeficiency virus (HIV), simian immunodeficiency virus (SIV), bovine immunodeficiency virus (BIV), etc. Can be mentioned.

  In the present specification, the “N36-binding peptide unit” means the following.

(Definition of N36-binding peptide unit)
It means a polypeptide having binding ability to N36 of a retrovirus that causes immunodeficiency in mammals, including at least one of the following module structures 1 to 3. The modular structure is a polypeptide consisting of 6 or 7 amino acids, and the following three types are exemplified.
(Y ′) _m1 −X _1a X _1b −A ₁ −X _1c X _1d X _1e −A ₂ − (X ″) _n1 : Module structure 1 (MS1)
(Y ′) _m2 −X _2a −A ₁ A ₁ −X _2b X _2c −A ₂ A ₂ − (X ″) _n2 : Module structure 2 (MS2)
_{(Y ') m3 -X 3a -A} 1 -X 3b X 3c X 3d -A 2 -X': Module structure 3 (MS3)
In each module in the polypeptide, A ₁ represents an acidic amino acid, preferably glutamic acid, aspartic acid or cysteic acid, more preferably glutamic acid or aspartic acid. A ₂ represents a basic amino acid, preferably lysine, arginine, ornithine or histidine, more preferably lysine, arginine or ornithine, and further preferably lysine or ornithine.

A ₁ and A ₂ have a relationship of i-th position and i + 4-th position in each module, respectively. By making the amino acids at the i-position and i + 4-position into a combination of an acidic amino acid and a basic amino acid (or vice versa), a salt bridge is formed between them, and α- of the polypeptide of the present invention A helix is easily formed. In addition, since the dipole direction of the salt bridge is opposite to the dipole direction formed by the peptide main chain, the thermodynamic stability of the molecule when the helix is formed is increased.

  Among the combinations of the acidic amino acid and the basic amino acid, the combination of glutamic acid (E) -lysine (K) is more preferable.

  One or two acidic amino acid and basic amino acid combinations are preferably present in each module.

  Further, the N36-binding peptide unit is not limited to only within a module, and may have a combination of the i-position and the i + 4-position between modules (a range exceeding the module structure). By having this combination between modules, the N36-binding peptide unit can further stabilize the α-helix.

Amino acids X _{1a to} X _3d (X _1a , X _1b , X _1c , X _1d , X _1e , X _2a , X _2b , X _2c , X _3a , X _3b , X _3c and X _3d ) in the N36-binding peptide unit are Represents any amino acid that is the same or different.

  Examples of the arbitrary amino acids include glycine, alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, tryptophan, serine, threonine, cysteine, cysteic acid, methionine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine. , Proline, ornithine, sarcosine, β-alanine, norleucine (Nle), naphthylalanine (Nal) and the like.

  Note that proline should not be used when there is a possibility of destroying the α-helix structure by use. May be used when there is no possibility of destroying the α-helix structure.

For example, the amino acid X _1a , X _2a or X _3a is preferably tryptophan, leucine, isoleucine, glutamine, serine, threonine, asparagine, valine, phenylalanine, tyrosine, methionine, glycine, alanine, naphthylalanine and the like.

As the amino acid at the position of X _1b or `` c '', that is, the amino acid of X _3b , for example, glycine, alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, tryptophan, serine, threonine, cysteine, cysteic acid, methionine, asparagine, Examples include glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, proline, ornithine, sarcosine, β-alanine, norleucine, naphthylalanine and the like.

  Proline should not be used when there is a possibility of destroying the α-helix structure by use. However, when proline is located at the N-terminal or C-terminal and there is no possibility of destroying the α-helix structure. May be used.

Examples of the amino acids X _1c , X _2b , X _3c , X _1d , X _2c or X _3d include tryptophan, leucine, isoleucine, asparagine, glutamine, aspartic acid, glutamic acid, serine, threonine, valine, phenylalanine, tyrosine, methionine. Glycine, alanine and the like are preferable.

Examples of the amino acid of X _1e include glycine, alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, tryptophan, serine, threonine, cysteine, cysteic acid, methionine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine. , Proline, ornithine, sarcosine, β-alanine, norleucine, naphthylalanine and the like.

  Proline should not be used when there is a possibility of destroying the α-helix structure by use, but it is not possible to destroy the α-helix structure at the N-terminal or C-terminal. May be used.

X ′ represents any amino acid optionally having a protecting group, —OR ¹ or —NR ² R ³ , and X ″ represents —OR ⁴ or —NR ⁵ R ⁶ .

  Examples of amino acids include glycine, alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, tryptophan, serine, threonine, cysteine, cysteic acid, methionine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, proline, Examples include ornithine, sarcosine, β-alanine, norleucine, naphthylalanine and the like.

  Proline should not be used when there is a possibility of destroying the α-helix structure by use, but it is not possible to destroy the α-helix structure at the N-terminal or C-terminal. May be used.

  When the N36-binding peptide unit has a protected amino acid, examples of the protecting group include the following. Ethoxycarbonyl group, methoxycarbonyl group, 9-fluorenylmethoxycarbonyl group, benzyloxycarbonyl group, 4-methoxybenzyloxycarbonyl group, 2,2,2-trichloroethyloxycarbonyl group, formyl group, acetyl group, propionyl group And a butyryl group.

R ^{1 to} R ⁶ (R ¹ , R ² , R ³ , R ⁴ , R ⁵ and R ⁶ ) are the same or different and represent H, an alkyl group, an aryl group or an aralkyl group.

Examples of the alkyl group include, for example, a linear or branched alkyl group of C ₁ -C _4, preferably, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert- butyl, and the like. Examples of the aryl group include a phenyl group and a phenyl group having a substituent. Examples of the substituent include the alkyl group, halogen atom, cyano, carboxylic acid, nitro, amino, acetylamino, alkoxy group and hydroxyl group exemplified above. The number of substituents is not particularly limited, and is 1 to 5, preferably 1 to 3.

Y ′ represents H, R ⁷ CO—, a toluenesulfonyl group or a methanesulfonyl group. R ⁷ represents an alkyl group, a phenyl group which may have a substituent, or a benzyl group which may have a substituent. As the alkyl group, those described above can be used. Examples and numbers of substituents are also as exemplified above.

  n1 is 1 when the module structure 1 is C-terminal, and is 0 when the module structure 1 is not C-terminal. n2 is 1 when the module structure 2 is C-terminal, and is 0 when the module structure 2 is not C-terminal.

  m1 is 1 when the module structure 1 is N-terminal, and is 0 except when the module structure 1 is N-terminal. m2 is 1 when the module structure 2 is N-terminal, and is 0 except when the module structure 2 is N-terminal. m3 is 1 when the module structure 3 is N-terminal, and is 0 except when the module structure 3 is N-terminal.

  In the module structures 1 to 3, the amino acid means -NH-CH (R ')-CO- (R' represents a side chain of the amino acid).

  The polypeptide of the present invention is a polypeptide comprising at least one of the module structures 1 to 3, preferably at least one of the module structures 1 to 3. The polypeptide of the present invention may consist only of the same module structure as the module structure.

  The polypeptide of the present invention can contain, for example, 2 to 15, preferably 3 to 12, more preferably 4 to 10, and particularly preferably 5 to 7, the above-mentioned module structure. The binding order of each module structure is not limited as long as the N36-binding peptide unit has an α-helix structure and binds to N36.

  For example, one of preferred embodiments when an N36-binding peptide unit is used as an anti-HIV agent is a polypeptide having a module structure 1 at the amino terminus (hereinafter referred to as “N-terminus”). Among them, the module structure 1 located at the N-terminus is particularly preferably “Trp-Z-Glu-Trp-Asp-Arg-Lys” (“Z” represents norleucine, methionine, or glutamic acid).

Further, as another preferred embodiment, a polypeptide in which module structure 3 is located at the C-terminus can be exemplified, and among them, X ′ is more preferably —NH ₂ .

  In the N36-binding peptide unit, it is particularly preferred that no amino acid other than the amino acid forming the module structure is present between the modules. However, as long as the polypeptide of the present invention can form an α-helix and bind to N36, a 7 amino acid insertion sequence consisting of seven arbitrary amino acids may be included between each module structure. If it is N terminal or C terminal, you may add 1-6 arbitrary amino acids.

  As an optional amino acid, for example, glycine, alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, tryptophan, serine, threonine, cysteine, cysteic acid, methionine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, Examples include proline, ornithine, sarcosine, β-alanine, norleucine, naphthylalanine and the like. These amino acids may have a protecting group as described above.

  Proline should not be used when there is a possibility of destroying the α-helix structure by use, but it is not possible to destroy the α-helix structure at the N-terminal or C-terminal. May be used.

Examples of 7 amino acid insertion sequences that may be included between modules include “X _2a -A ₂ A ₂ -X _2b X _2c -A ₁ A ₁ ” (X _2a , X _2b , X _2c , A ₁ and A ₂ Is as defined above.) Is a preferred embodiment.

  This 7-amino acid insertion sequence can be included in the polypeptide of the present invention, for example, if it is between module structures, 1 to 3 if it is N-terminal or C-terminal.

  Further, the N36-binding peptide unit can have a compound capable of binding to N36 at its N-terminal and / or C-terminal.

  The N36-binding peptide unit has, for example, 13 or 14 to 104 or 105 amino acids, preferably 13 or 14 to 83 or 84, more preferably 13 or 14 to 48 or 49, particularly preferably 34 or 35. be able to.

  The amino acid used in the N36-binding peptide unit is preferably L-form (L amino acid), but D-form may be used. When using D-form, it is necessary to make all optically active amino acids D-form.

  Furthermore, a peptide bond (—NH—CO—⇔—N═C (OH) —) in the polypeptide of the present invention is replaced with a biologically equivalent bond such as an alkene (—CH═CH—) or fluoro Alkenes (-CF = CH-) can also be used. Regarding alkenes, for example, S. Oishi, T. Kamano, A. Niida, Y. Odagaki, N. Hamanaka, M. Yamamoto, K. Ajito, H. Tamamura, A. Otaka, and N. Fujii. Diastereoselective Synthesis of New Ψ [(E) -CH = CMe]-and Ψ [(Z) -CH = CMe] -type Alkene Dipeptide Isosteres by Organocopper Reagents and Application to Conformationally Restricted Cyclic RGD Peptidomimetics. J. Org. Chem. 2002, 67, For example, A. Otaka, H. Watanabe, A. Yukimasa, S. Oishi, H. Tamamura, and N. Fujii. New Access to a-Substituted ( Z) -Fluoroalkene Dipeptide Isosteres Utilizing Organocopper Reagents under “Reduction-Oxidative Alkylation (R-OA)” Conditions. Tetrahedron Lett. 2001, 42, 5443-5446.

As a preferred embodiment of the N36-binding peptide unit, for example, a polypeptide represented by the following formula can be exemplified (MS1 indicates module structure 1, MS2 indicates module structure 2, MS3 indicates module structure 3, and AA indicates 7 Shows an insertion sequence consisting of two amino acids.);
MS1-MS1-MS1-MS1-MS1, MS2-MS2-MS2-MS2-MS2, MS3-MS3-MS3-MS3-MS3, AA-MS1-MS1-MS1-MS1, MS1-AA-MS1-MS1-MS1, MS1-MS1-AA-MS1-MS1, MS1-MS1-MS1-AA-MS1, MS1-MS1-MS1-MS1-AA, AA-MS2-MS2-MS2-MS2, MS2-AA-MS2-MS2-MS2, MS2-MS2-AA-MS2-MS2, MS2-MS2-MS2-AA-MS2, MS2-MS2-MS2-MS2-AA, MS3-AA-MS3-MS3-MS3, MS3-MS3-AA-MS3-MS3, MS3-MS3-MS3-AA-MS3, MS3-MS3-MS3-MS3-AA, AA-MS3-MS3-MS3-MS3, MS1-MS2-AA-MS1-MS3, MS1-MS2-MS2-MS1-MS3, MS1-AA-MS2-MS1-MS3, MS1-MS2-MS1-AA-MS3, MS1-MS2-MS2-MS1-MS3, MS1-MS1-MS2-MS1-MS3, MS1-MS1-MS2-MS2-MS3, MS1-MS1-MS2-MS3-MS3, MS1-MS1-MS1-MS1-MS3, MS1-MS1-MS1-MS2-MS3, MS1-MS1-MS1-MS3-MS3, MS1-MS1-MS3-MS1-MS3, MS1-MS1-MS3-MS2-MS3, MS1-MS1-MS3-MS3-MS3, MS1-MS2-MS1-MS1-MS3, MS1-MS2-MS1-MS2-MS3 MS1-MS2-MS1-MS3-MS3, MS1-MS2-MS2-MS2-MS3, MS1-MS2-MS2-MS3-MS3, MS1-MS2-MS3-MS1-MS3, MS1-MS2-MS3-MS2-MS3, MS1-MS2-MS3-MS3-MS3, MS1-MS3-MS1-MS1-MS3, MS1-MS3-MS1-MS2-MS3, MS1-MS3-MS1-MS3-MS3, MS1-MS3-MS2-MS1-MS3, MS1-MS3-MS2-MS2-MS3, MS1-MS3-MS2-MS3-MS3, MS1-MS3-MS3-MS1-MS3, MS1-MS3-MS3-MS2-MS3, MS1-MS3-MS3-MS3-MS3.

More preferable specific examples of N36 protein include, for example, “Ac-Trp-Nle-Glu-Trp-Asp-Arg-Lys-Ile-Glu-Glu-Tyr-Thr-Lys-Lys-Ile-Lys-Lys-Leu -Ile-Glu-Glu-Ser-Gln-Glu-Gln-Gln-Glu-Lys-Asn-Glu-Lys-Glu-Leu-Lys-NH ₂ (SC34-a), Ac-Trp-Met-Glu -Trp-Asp-Arg-Lys-Ile-Glu-Glu-Tyr-Thr-Lys-Lys-Ile-Lys-Lys-Leu-Ile-Glu-Glu-Ser-Gln-Glu-Gln-Gln-Glu-Lys -Asn-Glu-Lys-Glu- Leu-Lys-NH 2 "(SC34-b)," Ac-Trp-Nle-Glu- Trp-Asp-Arg-Lys-Ile-Glu-Glu-Tyr-Thr-Lys -Lys-Ile-Glu-Glu-Leu-Ile-Lys-Lys-Ser-Gln-Glu-Gln-Gln-Glu-Lys-Asn-Glu-Lys-Glu-Leu-Lys-NH ₂ '' (SC34 (EK ) -A), `` Ac-Trp-Met-Glu-Trp-Asp-Arg-Lys-Ile-Glu-Glu-Tyr-Thr-Lys-Lys-Ile-Glu-Glu-Leu-Ile-Lys-Lys- Ser-Gln-Glu-Gln-Gln-Glu-Lys-Asn-Glu-Lys-Glu-Leu-Lys-NH ₂ (SC34 (EK) -b), Ac-Trp-Glu-Glu-Trp-Asp -Lys-Lys-Ile-Glu-Glu-Tyr-Thr-Lys-Lys-Ile-Glu-Glu-Leu-Ile-Lys-Lys-Ser-Glu-Glu-Gln-Gln-Lys-Lys-Asn-Glu -Glu-Glu-Leu-Lys- Lys-NH 2 "(SC35EK) (" Ac ". an acetyl group) can be exemplified, and the like.

  The above N36 protein is exemplified mainly for sequences effective against HIV, but the amino acid sequence of N36 protein against simian immunodeficiency virus (SIV) and bovine immunodeficiency virus (BIV) also causes the corresponding immunodeficiency. It can be similarly designed according to the retroviral C34 peptide sequence.

  N36 protein is a protein having an α-helix structure to which a trimer of C34 having an α-helix structure in a gp41 protein of a retrovirus that causes immunodeficiency in mammals is bound. And the N36 trimer combine to form a hexamer.

  Examples of the N36-binding peptide unit include C34 or a derivative thereof. Examples of the C34 derivative having binding ability to N36 include derivatives obtained by substituting or extending / shortening amino acid residues for the purpose of improving the water solubility and stability of C34. For example, as described in JP-A-2003-176298, it has a plurality of modular structures consisting of 6 or 7 amino acids, and the i-position and i + 4-position amino acids are referred to as acidic amino acids and basic amino acids, respectively. By combining them (or vice versa), a salt bridge is formed between them, and an α-helix of the polypeptide of the present invention is easily formed. In this specification, the amino acids at positions i and i + 4 of the C34 protein are acidic amino acids (Glu (E) is represented as “E” as a representative example) and basic amino acids (Lys (K) as a representative example). Derivatives containing one module (X-EE-XX-KK) substituted with “K”) may be labeled as “SC34” derivatives and derivatives containing two or more as “SC34EK” derivatives .

  N36-binding peptide units include those whose peptide ends are carboxyl or amino groups, or derivatives that are substituted with other chemical structures (amide groups, lactam rings, etc.) when cleaving the N36-binding peptide unit from the fusion protein. It is. Moreover, the peptide end of the obtained fusion protein / peptide may be in a state where it is substituted with another chemical structure by a conventional method. Furthermore, this peptide can be used not only in the peptide state but also in the form of a derivative in which the peptide bond is substituted with a structure such as a carbon bond such as alkene, fluoroalkene or alkane or an ether bond. By replacing the peptide bond, it is difficult to be degraded by the enzyme, and an improvement in in vivo stability can be expected.

  Furthermore, in the present invention, “N36-binding peptide” refers to “N36-binding peptide unit”, “N36-binding peptide unit” linked directly or via any amino acid sequence (hereinafter also referred to as a conjugate), and The term “N36-binding peptide unit” and “conjugate” means that one or more amino acids are further added before and after.

  In the present invention, the DNA encoding the thrombin recognition sequence and the N36-binding peptide is determined based on the amino acid sequence in consideration of codon usage in the host, and the DNA / RNA automatic synthesizer is used. , By synthesizing the partial sequence of the sense strand and the partial sequence of the antisense strand so as to partially overlap, and obtaining a longer partial sequence as a double-stranded DNA by PCR method. .

  In the present invention, DNA encoding an amino acid sequence for purification may be further fused to the above three types of DNA. Examples of the amino acid sequence for purification include His tag, GST, MBP (maltose binding protein) and the like, and preferably His tag. By such an amino acid sequence for purification being bound to the N36-binding peptide unit, it becomes possible to purify the N36-binding peptide by affinity chromatography.

  Further, in the present invention, it is preferable that the three or four kinds of DNAs are fused via a recognition site capable of cleaving the peptide bond so that the target N36-binding peptide can be easily recovered after expression. The recognition sites include amino acid sequences that can be cleaved by proteases such as processing enzymes and digestive enzymes (consisting of two or more amino acids), chemical modifiers such as cysteine and methionine, and physical cleavage such as ultrasound, laser, and heat. Examples include cleavable amino acids. The digestive enzyme (processing enzyme) capable of cleaving the recognition site preferably includes factor Xa, renin, thrombin, trypsin, V8 protease, Pseudomonas endoprotease, Arthrobacter lysyl endopeptidase, etc. Examples include Ile-Glu-Gly-Arg which is a factor Xa recognition sequence. Examples of the chemical modifier include CNBr (Cyanogen bromide) that recognizes Met, dilute hydrochloric acid that recognizes Asn, Asp, and Glu, and DMAP-CN that recognizes Cys.

  When N36-binding peptide is expressed by fusion with protein A, thrombin recognition sequence and amino acid sequence for purification (hereinafter referred to as N36-binding peptide, protein A and thrombin recognition sequence (further amino acid sequence for purification) if necessary) N36 binding that is an anti-HIV activator from the fusion protein by inserting a Met-Gln sequence into both N and C ends of this N36-binding peptide, using a chemical reagent such as CNBr. The peptide can be cleaved. At the N-terminus, the Met-Gln bond is cleaved, the Met residue is released, and Gln is automatically cyclized under acidic conditions to become a pyroglutamic acid residue. In addition, the C-terminus is cleaved from the Met-Gln bond and cyclized to a homoserine lactone group by automatic modification of the Met residue. Thus, by cyclizing both ends, resistance to peptidase degradation in blood is improved, and maintenance of in vitro bioactivity and biophysical properties can be expected (Bioorganic and medicinal Chemistry, 17 (2009), p.7964-7970). In addition, when expressing N36-binding protein units in tandem, this Met-Gln sequence is interposed between N36-binding peptide units, so that N36-binding peptide units can be easily expressed by chemical reagents after expression in tandem. Obtainable.

  Specific methods of cleavage and cyclization include the following methods. Under acidic conditions (70% formic acid, 0.1N hydrochloric acid solution or 0.1N trifluoroacetic acid solution), 10 equivalents of TCEP (Tris (2-carboxyethyl) phosphine) per 1 methionine residue in the fusion protein sequence The target compound can be obtained by reacting 100 equivalents of CNBr at 60 ° C. for 2 hours in the presence of Hydrochloride).

  The order of the three or four types of fused DNA may be any order as long as the effect of the present invention can be obtained, but preferably protein A → thrombin recognition sequence → N36 binding from the 5 ′ end. Peptide or protein A → thrombin recognition sequence → purification amino acid sequence → N36 binding peptide.

Yeast Transformant A yeast transformant according to Embodiment 1 of the present invention is a yeast transformant into which a gene expression cassette containing the DNA according to Embodiment 1 is introduced.

The gene expression cassette of the present invention includes a promoter, DNA encoding protein A, DNA encoding thrombin recognition sequence, and DNA encoding N36 binding peptide (if necessary, DNA encoding further amino acid sequence for purification) In which DNA is fused (hereinafter sometimes referred to as fused DNA) , and a terminator.

  The promoter is not particularly limited as long as it can function in yeast serving as a host, and examples thereof include promoters such as PHO5 promoter, PGK promoter, GAP promoter, ADH1 promoter, GAL promoter, and GAL1 promoter. In addition, improved promoters obtained by adding sequence modifications to these promoters may be used. As the terminator, a commonly used natural or synthetic terminator can be used, and there is no particular limitation as long as it is a terminator that can function in a yeast serving as a host.

  The gene expression cassette of the present invention may contain a marker gene. When co-transformation is performed, the marker gene may be present in a fragment or plasmid different from the gene expression cassette.

  The marker gene is not particularly limited as long as it can be expressed in yeast as a host. Examples thereof include genes that are resistant to geneticin, genes that complement gene mutations involved in auxotrophy, LEU2, URA3, TRP1, and HIS3. .

  Examples of the yeast used as a host in the present invention include yeasts belonging to the genus Saccharomyces, Hansenula, Pichia, Schizosaccharomyces, Krivellomyces, and specifically include Saccharomyces cerevisiae, Pichia pastoris, Pichia Examples include methanolica, Schizosaccharomyces pombe, Hansenula anomala, Krivellomyces lactis. Among them, Saccharomyces cerevisiae is preferable, and YNN27 strain is most preferable. Since a transformed strain can be easily selected, a mutant strain in which the marker gene to be introduced is mutated or a strain in which the marker gene to be introduced is disrupted is preferably used.

  As a method for introducing the gene expression cassette into yeast, a known method can be adopted, and examples thereof include PEG-calcium method, calcium phosphate method, DEAE dextran method, electroporation method, lipofection method, microinjection method and the like. .

Method for Producing N36-Binding Peptide The method for producing an N36-binding peptide according to Embodiment 1 of the present invention comprises the steps of culturing the transformant of Embodiment 1 above to produce and accumulate N36-binding peptide in the culture, and the culture. And collecting N36-binding peptide from

The step of generating and accumulating N36-binding peptide in the culture In this step, the transformant produces N36-binding peptide in the culture, and the N36-binding peptide is accumulated in the culture. A thing means a culture medium and a transformant. In addition, by culturing the transformant in a liquid medium and secreting the N36-binding peptide outside the transformant, it is cleaved by the thrombin recognition sequence during secretory production outside the cell, and the N36-binding protein is removed without the leader sequence. It can be secreted.

  The medium used for culturing the transformant of the present invention contains a carbohydrate such as glucose, fructose, glycerol or starch as a carbon source. Further, it may contain an inorganic or organic nitrogen source (for example, ammonium sulfate, ammonium chloride, casein hydrolyzate, yeast extract, polypeptone, bactotryptone, beef extract, etc.). These carbon sources and nitrogen sources do not need to be used in pure form, and those having low purity are advantageous because they are rich in trace amounts of growth factors and inorganic nutrients. Further, if desired, other nutrient sources [eg, inorganic salts (eg, sodium diphosphate, potassium diphosphate, dipotassium hydrogen phosphate, magnesium chloride, magnesium sulfate, calcium chloride), vitamins (eg, vitamin B1) Antibiotics (for example, ampicillin, kanamycin, etc.) may be added to the medium. In particular, culturing in a medium containing 0.01 to 10% (w / v) yeast extract, 0.01 to 10% (w / v) peptone, and 0.1 to 20% (w / v) galactose increases the N36-binding peptide. It is preferable because it is produced.

  The culture temperature of the transformant is usually 25 to 35 ° C., preferably about 28 to 32 ° C., and the culture time is usually about 24 to 72 hours. These can be appropriately changed depending on the culture conditions and culture scale.

-Step of collecting the protein and N36-binding peptide from the culture This step is characterized in that the N36-binding peptide is recovered by purifying the N36-binding peptide produced by the transformant from the culture.

  The N36-binding peptide can be purified by affinity chromatography when the N36-binding peptide is bound to a purification amino acid sequence (preferably a His tag). If the N36-binding peptide does not have an amino acid sequence for purification, it can be purified by various chromatographic operations such as ion exchange, hydrophobicity, gel filtration, etc. In this case, purification using the N36 peptide It is also possible to do.

  In the production method of the present invention, in addition to the above two steps, cyanogen bromide was further allowed to act on the N36-binding peptide recovered from the culture, and a pyroglutamic acid residue was introduced at the N-terminus and a homoserine lactone group was introduced at the C-terminus. N36-binding peptide units may be obtained.

<Embodiment 2>
DNA
The DNA of Embodiment 2 of the present invention comprises a DNA encoding a polypeptide consisting of an amino acid sequence having 70% or more identity with the amino acid sequence represented by SEQ ID NO: 2 and functioning as a signal sequence, encoding a His tag And DNA encoding an N36-binding peptide that binds to the N36 protein of a retrovirus that causes immunodeficiency in mammals.

  A polypeptide (hereinafter, also referred to as polypeptide B) comprising an amino acid sequence having 70% or more identity with the amino acid sequence represented by SEQ ID NO: 2 and functioning as a signal sequence has identity in the amino acid sequence. 80% or more, preferably 85% or more, more preferably 90% or more, and still more preferably 95% or more is desirable, and examples include α-factor prepro sequences derived from Saccharomyces cerevisiae. The base sequence and amino acid sequence of the α-factor prepro sequence are shown as SEQ ID NO: 5 and SEQ ID NO: 2, respectively. By allowing polypeptide B to function as a signal sequence, it is possible to secrete an N36-binding peptide outside the cell.

  In the present invention, the His tag is an amino acid sequence containing 6 histidine residues, and the His tag is linked to an N36-binding peptide, so that high production of N36-binding protein is possible, and N36 after secretory production is produced. The stability of the bound peptide can also be improved. Furthermore, the N36-binding peptide can be purified by affinity chromatography.

  In the present invention, it is preferable to fuse the above three types of DNA with a recognition site capable of cleaving the peptide bond interposed, so that the target N36-binding peptide can be easily recovered after expression. The recognition sites include amino acid sequences that can be cleaved by proteases such as processing enzymes and digestive enzymes (consisting of two or more amino acids), chemical modifiers such as cysteine and methionine, and physical cleavage such as ultrasound, laser, and heat. Examples include cleavable amino acids. Preferred digestive enzymes (processing enzymes) capable of cleaving the recognition site include Factor Xa, renin, trypsin, V8 protease, Pseudomonas endoprotease, Arthrobacter lysyl endopeptidase, etc. Examples of the recognition site include Factor Xa The recognition sequence Ile-Glu-Gly-Arg can be mentioned. Examples of the chemical modifier include CNBr (Cyanogen bromide) that recognizes Met, dilute hydrochloric acid that recognizes Asn, Asp, and Glu, and DMAP-CN that recognizes Cys.

  When N36-binding peptide is expressed by fusion with polypeptide B and His tag, anti-HIV can be expressed by chemical reagents such as CNBr by inserting Met-Gln sequences at both N and C ends of this N36-binding peptide. The active N36 binding peptide can be cleaved.

  The order of the three types of fused DNAs may be any order as long as the effects of the present invention can be obtained. Preferably, the order of polypeptide B → His tag → N36-binding peptide from the 5 ′ end is used. In order.

  The N36-binding peptide and the N36-binding peptide unit are the same as described above.

Gene Expression Cassette The gene expression cassette of Embodiment 2 of the present invention includes a promoter, the DNA of Embodiment 2 above, and a terminator.

  The promoter is not particularly limited as long as it functions in the yeast serving as a host and can achieve the effects of the present invention, but the nucleotide sequence having 70% or more identity with the nucleotide sequence represented by SEQ ID NO: 3 And those containing DNA that functions as a promoter in yeast. The identity of the DNA is more preferably 80% or more, further preferably 85% or more, particularly preferably 90% or more, and most preferably 95% or more. An example of such a promoter is the GAL1 promoter derived from Saccharomyces cerevisiae. The base sequence of the GAL1 promoter is shown as SEQ ID NO: 3 in the sequence listing. As the terminator, a commonly used natural or synthetic terminator can be used, and there is no particular limitation as long as it is a terminator that can function in a yeast serving as a host.

  The gene expression cassette of the present invention may contain a marker gene. When co-transformation is performed, the marker gene may be present in a fragment or plasmid different from the gene expression cassette.

  The marker gene is not particularly limited as long as it can be expressed in yeast as a host. Examples thereof include genes that are resistant to geneticin, genes that complement gene mutations involved in auxotrophy, LEU2, URA3, TRP1, and HIS3. .

Yeast Transformant A yeast transformant according to Embodiment 2 of the present invention is a yeast transformant into which the gene expression cassette according to Embodiment 2 is introduced.

  The method for introducing yeast and gene expression cassette is the same as described above.

Method for Producing N36-Binding Peptide A method for producing an N36-binding peptide according to Embodiment 2 of the present invention includes the step of culturing the transformant of Embodiment 2 to produce and accumulate N36-binding peptide in the culture, and the culture. And collecting N36-binding peptide from

The step of generating and accumulating N36-binding peptide in the culture In this step, the transformant produces N36-binding peptide in the culture, and the N36-binding peptide is accumulated in the culture. A thing means a culture medium and a transformant. Preferably, the culture is performed in a medium containing yeast extract 0.01 to 10% (w / v), peptone 0.01 to 10% (w / v), and galactose 0.1 to 20% (w / v). . Further, by culturing the transformant in a liquid medium, the N36-binding peptide can be secreted outside the transformant, and can be produced and accumulated in the medium.

  The medium used for culturing the transformant of the present invention is 0.01-10% (w / v) of yeast extract, preferably 0.5-5% (w / v), more preferably 1-2% ( w / v), 0.01-10% (w / v) peptone, preferably 0.5-5% (w / v), more preferably 1-2% (w / v), 0.1-20% galactose (w / v), preferably 1-10% (w / v), more preferably 2-5% (w / v). In addition to these, as a medium component, the above-described components may be added as long as the effects of the present invention can be obtained. By using such a medium, the N36-binding peptide can be produced at a high level, and the stability of the N36-binding peptide after secretory production can also be improved.

  The culture temperature of the transformant is usually 25 to 35 ° C., preferably about 28 to 32 ° C., and the culture time is usually about 24 to 72 hours. These can be appropriately changed depending on the culture conditions and culture scale.

-Step of collecting the protein and N36-binding peptide from the culture This step is characterized in that the N36-binding peptide is recovered by purifying the N36-binding peptide produced by the transformant from the culture.

  The N36-binding peptide can be purified by affinity chromatography when the N36-binding peptide is bound to a His tag. If the N36-binding peptide does not have an amino acid sequence for purification, it can be purified by various chromatographic operations such as ion exchange, hydrophobicity, gel filtration, etc. In this case, purification using the N36 peptide It is also possible to do.

  In the production method of the present invention, in addition to the above two steps, cyanogen bromide was further allowed to act on the N36-binding peptide recovered from the culture, and a pyroglutamic acid residue was introduced at the N-terminus and a homoserine lactone group was introduced at the C-terminus. N36-binding peptide units may be obtained.

Preventive or therapeutic agent for retrovirus infection (AIDS) causing immune deficiency in mammals The preventive or therapeutic agent for AIDS in mammals of the present invention comprises the N36-binding peptide obtained by the production method of Embodiments 1 and 2 above. It is characterized by being an active ingredient. The retrovirus that causes immunodeficiency in the mammal is preferably human immunodeficiency virus (HIV). The N36-binding peptide obtained by the above production method according to Test Example 2 described later inhibits the binding of HR1 and C34, so that it is effective as a preventive or therapeutic agent for retroviral infection that causes immune deficiency in mammals. Can be inferred.

  In addition, the AIDS preventive or therapeutic agent for the mammal of the present invention can optionally contain biologically acceptable carriers, excipients, and the like depending on the form of use. The preventive or therapeutic agent for AIDS in the mammal of the present invention can be produced according to conventional means. For example, orally as tablets, capsules, elixirs, microcapsules, etc. with sugar coating or enteric coating as needed, or as a topical agent such as an ointment or plaster, as a spray or inhalant, It can be used parenterally, nasally or tracheally, or in the form of injections such as sterile solutions or suspensions with water or other pharmaceutically acceptable fluids.

  The dose of the agent for preventing or treating AIDS of the present invention varies depending on symptoms, but in the case of oral administration, generally for an adult (with a body weight of 60 kg), about 100 mg to 4000 mg, preferably about 200 mg per day. ~ 3000 mg, more preferably about 300 mg to 2000 mg. When administered parenterally, the single dose varies depending on the administration subject, symptom, administration method, etc. For example, in the form of an injection, it is about 1 per day for administration to an adult (with a body weight of 60 kg). It is convenient to administer 0.00001 g to 1 g, preferably about 0.01 mg to 30 mg, more preferably about 0.1 mg to 20 mg, more preferably about 0.1 mg to 10 mg by intravenous injection. This dose is the same for other animals.

  Hereinafter, production examples and test examples are given to describe the present invention in more detail. However, the present invention is not limited to these production examples.

Production example: Creating an expression vector for yeast
(1) Preparation of expression vector containing leader sequence A vector for secreting and expressing SC35EK represented by SEQ ID NO: 6 was prepared using yeast. As a leader sequence for selecting and secreting a multi-copy type pYES2 vector having a Gal1 promoter, α-factor prepro sequence (SEQ ID NO: 2, 5) or CelA protein derived from Aspergillus (SEQ ID NO: 1, 4) and downstream thereof A His tag for purification and a thrombin recognition sequence (SEQ ID NOs: 7 and 8) were inserted into the DNA. A leader sequence was inserted into the KpnI / BamHI site of the pYES2 / CT vector, and a His tag / thrombin site was inserted into the SacI / BamI site to create pYES2-αF and pYES2-CelA shown in FIG.

・ Experimental method YEpFLAG1 vector (Sigma) containing the prepro sequence cDNA sequence of α-factor or pYCUE vector containing the CelA cDNA sequence as a template using PCR method and KpnI (GGATCC) at the 3 ′ end The α-factor prepro sequence or the CelA cDNA to which a restriction enzyme recognition site of SacI (GAGCTC) was added was amplified. Furthermore, a SacI at the 5 'end and a cDNA sequence encoding a thrombin recognition sequence downstream of the His tag with a BamHI (GGATCC) restriction enzyme recognition site at the 3' end, and a Klenow fragment (TOYOBO) using primers as templates. ) Was used for the extension reaction.

  PBlueScriptII KS (+) vector (Takara) restricted with KpnI / BamHI and α-factor prepro sequence or CelA PCR product restricted with KpnI / SacI, His tag / thrombin treated with SacI / BamHI Each site was ligated using DNA Ligation Kit Ver2.1 (Takara). Transform Escherichia coli JM109 with the ligation solution, select and culture transformants containing the target vector using LB agar medium and LB medium containing antibiotic ampicillin, and then use Plasmid Mini Kit (QIAGEN) from E. coli Was used to purify the plasmid. The nucleotide sequence of the obtained plasmid was confirmed, and the target plasmids, pBSII KS-αF and pBSII KS-CelA, were obtained.

  The obtained pBSII KS-αF and pBSII KS-CelA were each cut at the KpnI / BamHI site and developed by 1.2% agarose gel electrophoresis, and then the DNA fragment was extracted from the gel and purified. The purified DNA fragment was incorporated into a pYES2 / CT vector (Invitrogen) treated with KpnI / BamHI restriction enzyme, and E. coli JM109 was transformed. Using LB agar medium and LB medium containing ampicillin, a transformant containing the target vector was selected and cultured to obtain the target plus pYES2-αF and pYES2-CelA.

(2) Preparation of vector containing SC35EK peptide sequence Next, as shown in FIG. 2, the cDNA sequence of the target peptide SC35EK (SEQ ID NO: 9 or 10) was inserted downstream of pYES2-αF and pYES2-CelA. In SC35EK, amino acid sequences of 2 residues were arranged at both ends of SC35EK, respectively, so that methionine and glutamine were in this order from the N-terminal side. By designing in this way, SC35EK is expected to be cut out by reacting with cyanogen bromide under acidic conditions. In addition, a trimer was synthesized in the hope of improving the yield of the cyclized peptide.

·experimental method
Nucleic acids having cDNA base sequences represented by MQ-SC35EK-MQ and M- (Q-SC35EK-M) ₃ -Q were synthesized using an extension reaction using a Klenow fragment and a PCR method, respectively. These have restriction enzyme recognition sites of BamHI at the 5 ′ end and EcoRI (GAATTC) or XhoI (CTCGAG) at the 3 ′ end, respectively.

PBlueScriptII SK (+) vector (Takara) treated with restriction enzyme with BamHI / EcoRI or BamHI / XhoI and MQ-SC35EK-MQ PCR product treated with restriction enzyme with BamHI / EcoRI, M- treated with restriction enzyme with BamHI / XhoI (Q-SC35EK-M) ₃ -Q PCR products were ligated using DNA Ligation Kit Ver2.1. Transform E. coli strain JM109 with the ligation solution, select and culture transformants containing the target vector using LB agar medium and LB medium containing the antibiotic ampicillin, and then use Escherichia coli with the Plasmid Mini Kit. The plasmid was purified. The nucleotide sequence of the obtained plasmid was confirmed, and the desired plasmid pBSII SK-MQ-SC35EK-MQ and pBSII SK-M- (Q-SC35EK-M) ₃ -Q were obtained.

The obtained pBSII SK-MQ-SC35EK-MQ and pBSII SK-M- (Q-SC35EK-M) ₃ -Q were cleaved at the BamHI / EcoRI site or BamHI / XhoI site, respectively, and developed by 1.2% agarose gel electrophoresis. Thereafter, the DNA fragment was extracted from the gel and purified. The purified DNA fragment was incorporated into pYES2-αF and pYES2-CelA vectors treated with BamHI / EcoRI or BamHI / XhoI, and E. coli JM109 was transformed. Using LB agar medium and LB medium containing ampicillin, select and culture the transformant containing the target vector, and pYES2-αF-MQ-SC35EK-MQ, pYES2-αF-M- (Q-SC35EK-M) ₃ -Q, pYES2-CelA-MQ-SC35EK-MQ, pYES2-CelA-M- (Q-SC35EK-M) ₃ -Q) were prepared.

Test Example 1: Examination of secretory expression conditions in yeast Secretory leader sequence (α prepro, CelA), host (YNN27 strain, BY2777 strain, INVSc1 strain), medium composition (expression induction medium), culture time (0-30 hours) ) And the conditions for secretory expression in yeast were examined.

・ Experimental Method Using the plasmids obtained in the production examples, laboratory yeast strain YNN27 (MATα, ura3, trp1), protease deficient strain BY2777 (MATa, prb1-1122, prc1-407, pep4-3, ura3- 52, leu2, trp1) and INVSc1 strain (Invitrogen) (MATa, his3D1, leu1, trp1-289, ura3-52) were transformed, respectively. Each isolated colony was picked up and placed in uracil-requiring medium (0.67% (w / v) Yeast nitrogenbase, 0.37% (w / v) amino acid mix (-Ura) (Clontech), 2% (w / v) v) galactose), and cultured overnight at 30 ° C., so that the culture OD ₆₀₀ of 0.4, the expression-inducing medium (uracil auxotrophy galactose medium (0.67% (w / v) Yeast nitrogenbase, 0.37% (w / v) amino acid mix (−Ura), 2% (w / v) galactose), or YPG medium (2% (w / v) peptone, 1% (w / v) yeast extract, 2% (w / v) ) Galactose)), diluted with culturing at 30 ° C. to induce protein expression. After expression induction, sampling was performed over time, and the culture supernatant was collected by centrifugation at 3,000 rpm for 20 minutes. Ni-NTA agarose (QIAGEN) was added to the culture supernatant and stirred for 2 hours. Ni-NTA agarose was recovered and washed with a washing buffer (20 mM phosphate buffer, pH 6.0, 0.5 M NaCl). The expression of the target protein at each time was analyzed using SDS-PAGE (15-20% or 10-20% gradient gel).

・ Results Fig. 3 shows the transformants of pYES2-αF, pYES2-αF-MQ-SC35EK-MQ, pYES2-αF-M- (Q-SC35EK-M) ₃ -Q, YNN27 strain, BY2777 strain, INVSc1 strain. The results are shown when expression induction is applied. When expression induction was performed using a uracil-requiring galactose medium, the target protein could not be confirmed in the culture supernatant. However, when the expression induction was performed using the YPG medium, as shown in FIG. 3, a band could be confirmed near the theoretical value of the target protein in the medium 6 to 24 hours after the expression induction. From the above results, the expression was confirmed in any yeast strain by changing the uracil-requiring medium to YPG. In addition, the YNN27 strain was more stable than the product obtained after the other strains were used, and the yield was increased by increasing the culture time.

FIG. 4 shows that uracil-requiring galactose medium or YPG medium using transformants of pYES2-αF-MQ-SC35EK-MQ and pYES2-αF-M- (Q-SC35EK-M) ₃ -Q strain YNN27. The results of confirming the increase in the number of cells over time and the expression level by SDS-PAGE are shown. When uracil-requiring galactose medium was used, the number of cells became almost steady after 36 hours. In addition, expression of MQ-SC35EK-MQ could not be confirmed at any time, but expression of tandem type M- (Q-SC35EK-M) ₃ -Q could be confirmed after 36 and 48 hours. However, the 48-hour band was thinner than 36 hours and was considered to have been degraded in the culture supernatant. On the other hand, when YPG medium is used, the number of cells is almost steady after 30 hours, and MQ-SC35EK-MQ is tandem type M- (Q-SC35EK-M) ₃ -Q after 24 hours. Expression was confirmed after 12 hours, and the expression level increased up to 36 hours. That is, by using the YPG medium, the expression amount of the target protein was increased and the stability in the culture supernatant after the expression was improved as compared with the case of using the uracil-requiring galactose medium.

FIG. 5 shows pYES2-CelA, pYES2-CelA-MQ-SC35EK-MQ, and pYES2-CelA-M- (Q-SC35EK-M) ₃ -Q transformants of YNN27, BY2777, and INVSc1 strains. The result when expression induction is applied is shown. In the YNN27 strain, the target protein could not be confirmed in the culture supernatant when expression induction was performed using uracil-requiring galactose medium, as in the case of using αF as the leader sequence. As shown in FIG. 5, protein expression could be confirmed in the medium 24 hours after the induction of expression. However, the band size did not match the theoretical value of the target protein. Similar results were obtained with the BY2777 and INVSc1 strains.

Test Example 2: ELISA using culture supernatant
In order to confirm that the target peptide is contained in the culture supernatant obtained in Test Example 1, using the culture supernatant when the expression of the transformant of the YNN27 strain was induced in the YPG medium, The ELISA experiment shown in FIG.

·experimental method
Maltose binding protein (MBP) fusion HIV-HR1 was diluted to 50 nM with 50 mM sodium carbonate buffer (pH 9.4), added 50 μl on an ELISA plate, and reacted at 4 ° C. overnight. After washing three times with 200 μl of 0.25% Tween 20 in PBS (T-PBS), 200 μl of 0.1% BSA in T-PBS was added and blocking was performed at room temperature for 2 hours. After blocking, the plate was washed 3 times with T-PBS, 100 μl each of the yeast transformant culture solution was added, incubated at 37 ° C. for 2 hours, and then washed 3 times with T-PBS. Thioredoxin (TRX) -His tag fused alkaliphosphatase (ALP) -C34 / S138A was diluted to 50 nM with PBS, 100 μl was added to each well, and incubated at 37 ° C. for 2 hours. After washing three times with T-PBS, 100 μl of BluePhos Microwell (manufactured by KPL), which is an ALP substrate, was added and reacted at room temperature for 2 hours. As a control, wells to which only medium was added were also reacted at the same time.

Results The well image after 2 hours of reaction is shown in FIG. When any culture solution was used, the reaction was inhibited as compared with the control, suggesting that the target SC35EK peptide was contained in the culture solution.

Test Example 3: Expression of CelA fusion protein in cells and culture supernatant In Example 1 , the expression of CelA fusion protein in the culture supernatant was confirmed, but the band size did not match the theoretical value. On the other hand, the result of the ELISA in Test Example 2 suggested that the target peptide may be contained in the culture supernatant. From the above results, it was considered that the CelA fusion protein was expressed in the microbial cells and was degraded in the process of secretion into the culture supernatant. Therefore, in order to confirm the expression of the CelA fusion protein in the microbial cells and in the culture supernatant, Western blotting using an anti-His tag antibody was performed using the microbial cells obtained in Test Example 1 and the culture supernatant. It was.

Experimental Method Experiments were performed using the culture supernatant and cells collected 6, 12, and 24 hours after induction of expression in Test Example 1. An equal volume of 2 × sample buffer was added to the culture supernatant. After washing the cells twice with PBS, add 3 times the amount of Y-PER Plus, Yeast Protein Extraction Reagent (PIERCE) and 1 mM dithiothreitol (nacalai), and react for 20 minutes at room temperature. An equal volume of 2 × sample buffer was added. Each sample was developed on SDS-PAGE (10 to 20% gradient gel) and then transferred to a PVDF membrane using a Western Blotting apparatus. After the transfer, the PVDF membrane was washed with TBS (20 mM Tris, 200 mM NaCl, pH 7.5) and then blocked in 3% gelatin at 4 ° C. overnight. After blocking, wash 3 times with TTBS (20 mM Tris, 200 mM NaCl, 0.05% (w / v) Tween-20, pH 7.5) and 2000 with antibody dilution solution (1% (w / v) gelatin / TTBS). The mixture was reacted for 2 hours at room temperature in an anti-His tag antibody (Sigma, Cat. H1029) solution diluted twice. After the reaction, it was washed 3 times with TTBS and reacted in an alkaline phosphatase-labeled anti-mouse IgG antibody (Bio-Rad, Cat. 170-6461) solution diluted 3000 times with an antibody dilution solution at room temperature for 2 hours. After the reaction, it was washed twice with TTBS and once with TBS, and visualized by adding a substrate solution (Bio-Rad, Cat. 170-6461).

-Results As shown in FIG. 8, the CelA fusion protein showed a band near the target molecular weight in the cells, and the band became deeper as the culture time passed. On the other hand, in the samples collected from the supernatant, bands were confirmed in the culture supernatant after 12 and 24 hours. CelA broadly detects a band with a size larger than the target molecular weight, and CelA-MQ-SC35EK-MQ and CelA-M- (Q-SC35EK-M) ₃ -Q show similar band patterns. Compared to the band confirmed in, the size was down. From the above results, it was suggested that the CelA fusion protein may have undergone some degradation during the secretion process from the microbial cells to the outside of the microbial cells.

Test Example 4: Recovery of C-terminal peptide As shown in Test Examples 2 and 3, CelA-His-MQ-SC35EK-MQ and CelA-His-M- (Q-SC35EK-M) ₃ -Q expressed in yeast This suggests that the protein may be cleaved behind the His tag after secretion from the microbial cells. Moreover, since the cleaved peptide does not contain a tag, it was impossible to recover it using Ni-NTA agarose. Then, the refinement | purification using the N peptide (sequence number 11) which has high affinity with SC35EK peptide from the culture supernatant again, and the analysis of the obtained peptide were tried.

Experimental section
Shake transformants of pYES2-CelA-MQ-SC35EK-MQ and pYES2-CelA-M- (Q-SC35EK-M) ₃ -Q strain YNN27 overnight at 30 ° C in 10 ml of uracil-requiring medium Cultured. This culture solution was transferred to 100 ml of uracil-requiring medium and further cultured with shaking at 30 ° C. The obtained culture broth was diluted with 1 L of YPG medium so that OD ₆₀₀ was 0.4, and cultured with shaking at 30 ° C. After 24 hours of expression induction, the culture supernatant was collected by centrifuging the culture at 4,000 rpm for 20 minutes.

  N peptide N36 was synthesized by Fmoc solid phase synthesis as His tag N36 with a His tag added to the N-terminal side. The peptide was dissolved in DMSO to 1 mM and further diluted to 10 μM with PBS. In advance, Ni-NTA agarose and His-tagged N36 peptide were reacted for 1 hour to prepare His-tagged N36 peptide-bound Ni-NTA agarose.

  His-tagged N36 peptide-bonded Ni-NTA agarose was added to the culture supernatant and stirred for 2 hours. Ni-NTA agarose was recovered, washed with PBS, and eluted with 0.1N TFA. After confirming each step by SDS-PAGE, the sample after elution was subjected to LC-MS, and the molecular weight of the obtained sample was confirmed.

result
Each of the culture supernatants expressing CelA-His-MQ-SC35EK-MQ and CelA-His-M- (Q-SC35EK-M) ₃ -Q when purified using N36 peptide-binding Ni-NTA agarose The results of analyzing the steps by SDS-PAGE are shown in FIG. In both cases, bands other than N36 peptide were confirmed in the elution fraction after purification with N36 peptide-bound Ni-NTA agarose (lanes 4, 9). As a result of analyzing the molecular weight of the sample after elution using LC / MS, it was 5,300 and 14,775, respectively. As shown in FIG. 10, assuming that it was cleaved at the thrombin site (LVPR ↓ GS) behind the CelA-His tag. The molecular weight was consistent.

Test Example 5: Purification of secretory expressed protein YNN27 of pYES2-αF-MQ-SC35EK-MQ and pYES2-αF-M- (Q-SC35EK-M) ₃ -Q obtained the best results in Test Example 1. Using the transformant of the strain, further detailed examination was performed and the target protein was purified.

·experimental method
Shake the transformants of pYES2-αF-MQ-SC35EK-MQ and pYES2-αF-M- (Q-SC35EK-M) ₃ -Q strain YNN27 overnight at 30 ° C in 10 ml of uracil-requiring medium. Cultured. This culture solution was transferred to 100 ml of uracil-requiring medium and further cultured with shaking at 30 ° C. The obtained culture broth was diluted with 1 L of YPG medium so that OD ₆₀₀ was 0.4, and cultured with shaking at 30 ° C. After 24 hours of expression induction, the culture supernatant was collected by centrifuging the culture at 4,000 rpm for 20 minutes. Ni-NTA agarose was added to the culture supernatant, and the mixture was stirred for 2 hours. Ni-NTA agarose was recovered, washed with a washing buffer, and then the protein was eluted using an acid as a cutting reaction solvent. The sample after elution was subjected to LC-MS, and the molecular weight of the obtained sample was confirmed. Furthermore, each peak was fractionated and analyzed for the N-terminal amino acid. The yield of the fusion protein was measured using Protein Assay Kit (BIO-RAD Laboratories, Hercules, CA).

result
The chromatogram of HPLC and the mass contained in each peak are shown in FIG. From this result, the culture supernatant of the transformant of pYES2-αF-MQ-SC35EK-MQ contained peak 1 and peak 2, and the respective masses were 1,998 and 7,437. In addition, the culture supernatant of the transformant of pYES2-αF-MQ-SC35EK-MQ_tandem contained peak 1 and peak 3, and the respective masses were 1,998, 16,908, and 14,771. The masses contained in the peaks 1 to 3 almost coincided with the molecular weights of the sequences 1 to 3 shown in FIG. Analysis of the N-terminal amino acid of each peak gave ELGSG, which was consistent with this result. In addition, the mass of 14,771 contained in peak 3 is almost the same as the molecular weight of sequence 4, and it is considered that the molecule of sequence 3 was decomposed. The sample concentrations after elution were 0.7 mg / L and 0.7 mg / L.

Test Example 6: Excision and Purification of Both End Cyclized Peptides from the Expressed Protein The fusion protein of Test Example 5 was prepared using 100 equivalents of cyanogen bromide and 10 equivalents of tris (2-carboxyethyl) phosphine [methionine residue [ Tris (2-carboxyethyl) phosphine (TCEP); source SIGMA, product number C4706], in the presence of acid (70% formic acid or 0.1N hydrochloric acid or 0.1N trifluoroacetic acid) at 60 ° C for 2 hours, both ends SC35EK was cut out in a cyclic structure (N-terminal side pyroglutamic acid, C-terminal side homoserine lactone group). As shown in FIG. 13, the product was analyzed by LC-MS, and the target pGlu-SC35EK-Hsl was purified by HPLC fractionation. As a result, 50 ig of pGlu-SC35EK-Hsl could be obtained from 1 L of the culture solution.

Test Example 7: Evaluation of the modified nuclear peptide (i) Anti-HIV activity and (ii) physicochemical properties of the modified SC35EK were evaluated as follows. For control, the activity was similarly evaluated for SC35EK (N-terminal acetylation, C-terminal amide) and unmodified form of both ends of SC35EK (N-terminal unprotected and C-terminal carboxylic acid).

First, each of these modified SC35EK was evaluated for anti-HIV activity. Evaluation was performed by the following MAGI assay (Kodama, EI et al., Antimicrob. Agents. Chemother. 2001, 45, 1529-1546; Maeda Y. et al., J. Infec. Dis., 1998 , 177, 1207-1213). Target cells (Hela-CD4-LTR-β-gal) 10 ⁴ cells / well were seeded in a 96-well flat microtiter culture plate. The next day, HIV-1 clone NL4-3 was seeded so that 60 MAGI U / well, 60 blue cells appeared after 48 hours. Thereafter, the specimen was added and cultured. After 48 hours, the number of cells stained with X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside) was counted. The activity of the analytes was evaluated at a concentration that prevents 50% replication of HIV-1 (EC ₅₀ ). As a result, as shown in Table 1, it was revealed that these modified SC35EK showed the same activity as SC35EK. The introduction of a cyclic structure was expected to impair the activity, but this result was contrary.

  For these modified SC35EK, it was also verified whether the physicochemical characteristics based on the structural modification were impaired. This physiological feature is an important feature associated with anti-HIV activity. Analysis was performed as follows from a circular dichroism (CD) spectrum. 10 μM of peptide was placed in 5 mM HEPES buffer (pH 7.2) and treated at 37 ° C. for 30 minutes. The CD spectrum was measured at 25 ° C. with an average of 8 scans using a circular dichroism spectrophotometer (J-710 manufactured by JASCO Corporation). After 0.25 minutes of parallelization, after an integration time of 1.0 second, thermal denaturation was measured every 0.5 ° C.

  As a result, as shown in FIG. 14, it was revealed that the physiological characteristics based on the α-helix structure were not impaired.

In addition, these modified SC35EKs were also verified for thermal stability. A midpoint of thermal denaturation (melting point Tm) was calculated based on [θ] ₂₂₂ value by circular dichroism spectrum analysis.

  As a result, as shown in FIG. 15, it became clear that the thermal stability was not impaired.

Claims (12)

  DNA encoding a protein comprising an amino acid sequence having 70% or more identity with the amino acid sequence represented by SEQ ID NO: 1 and having endoglucanase activity, DNA encoding a thrombin recognition sequence, and a mammal A DNA fused with a DNA encoding an N36-binding peptide that binds to the N36 protein of a retrovirus that causes immunodeficiency.   A DNA obtained by fusing the DNA of claim 1 with a DNA encoding an amino acid sequence for purification.   A yeast transformant into which the gene expression cassette comprising the DNA according to claim 1 or 2 is introduced.   The transformant of Claim 3 whose said yeast is Saccharomyces cerevisiae (Saccharomyces cerevisiae). A method for producing an N36-binding peptide comprising the steps of culturing the transformant according to claim 3 or 4 to produce and accumulate an N36-binding peptide in the culture, and collecting the N36-binding peptide from the culture.   DNA encoding a polypeptide consisting of an amino acid sequence represented by SEQ ID NO: 2 and having an identity of 70% or more and functioning as a signal sequence, DNA encoding a His tag, and immunodeficiency in mammals A DNA fused with a DNA encoding an N36-binding peptide that binds to the N36 protein of the retrovirus that occurs.   A gene expression cassette comprising the DNA according to claim 6, wherein the promoter comprises a nucleotide sequence having 70% or more identity with the nucleotide sequence represented by SEQ ID NO: 3, and a DNA that functions as a promoter in yeast. Gene expression cassette containing.   A transformant of yeast into which the gene expression cassette containing the DNA according to claim 6 or the gene expression cassette according to claim 7 has been introduced. A method for producing an N36-binding peptide, comprising culturing the transformant according to claim 8 to produce and accumulate an N36-binding peptide in the culture, and collecting the N36-binding peptide from the culture.   The culture is performed in a medium containing yeast extract 0.01-10% (w / v), peptone 0.01-10% (w / v), and galactose 0.1-20% (w / v). 9. The method according to 9.   11. The method according to claim 5, 9 or 10, further comprising a step of allowing cyanogen bromide to act on an N36-binding peptide, wherein a pyroglutamic acid residue is introduced at the N-terminus and a homoserine lactone group is introduced at the C-terminus. Manufacturing method.   A preventive or therapeutic agent for a retroviral infection that causes immunodeficiency in a mammal, comprising the N36-binding peptide obtained by the method according to claim 5, 9 or 10 as an active ingredient.

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