JPH11322794A - Peptide, nucleic acid, recombinant vector, and transformant, production of peptide, and agent for antiviral disease - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new peptide capable of suppressing the activity of a trans activation factor for transcribing human immunodeficient virus in cells/in a body, and useful as an anti-AIDS agent, an antitumor agent or the like by introducing a specific amino acid sequence. SOLUTION: This peptide has an amino acid sequence of formula I, or an amino acid sequence where one to several amino acids are deleted, substituted or added in the amino acid sequence of formula I. In addition, the peptide has an activity for suppressing transcription activation reaction of HIV-LTR by a trans activation factor, Tat, of transcription of human immunodeficient virus, HIV, in cells. Further, the peptide is preferably produced by culturing a transformant containing a recombinant vector which binds a nucleic acid encoding peptide having an amino acid sequence of formula I and having a base sequence of formula II in such a state that the genetic information can be expressed.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a peptide, a nucleic acid, a recombinant vector, a transformant, a method for producing a peptide, and an antiviral disease agent.

More specifically, the present invention relates to a method for producing human immunodeficiency virus HIV (Human Immunodeficient Virus) in a cell / in vivo.
Tat, a transcriptional transactivator of iency Virus)
HIV-LTR (Trans-activator of transcription)
(Long Terminal Repeat), a novel peptide having the effect of suppressing the transcription activation reaction, a nucleic acid encoding such a peptide, a recombinant vector in which such nucleic acid is linked so that its genetic information can be expressed, and a microorganism containing such a recombinant vector Or a transformant that is a cell, a method for producing the peptide using the transformant, and an antiviral agent (eg, an anti-AIDS agent or an antitumor agent) containing the peptide or the recombinant vector as an active ingredient. About.

[0003]

2. Description of the Related Art Tat is a protein encoded and expressed by an HIV gene.
Transcription reaction from R (introduced into chromosome in host cell
RNA polymerase from host cells from HIV provirus
Reaction read from the transcription start site of HIV-LTR by II)
In particular, by activating the elongation reaction of transcription, the proliferation of HIV is activated about 100 times.

[0004] The mechanism of action is that Tat
Binds to kinases such as (cyclin-dependent kinase 9) and CDK9 (cyclin-dependent kinase 9),
It is believed that by enhancing their phosphorylation ability, they phosphorylate the C-terminal domain (CTD) of RNA polymerase II and activate the elongation reaction of transcription.

Accordingly, Tat, a transcriptional activator of HIV,
If the function of Tat can be suppressed, the proliferation of HIV can be markedly suppressed and, consequently, an effective antiviral activity such as an anti-AIDS action or an antitumor action can be expected. Regarding drugs that inhibit the function, for example, Ming-Chu Hsu et al., “Inhibition of type 1 hum
an immunodeficiency virus Replication by a TatAnta
gonist to which the virus remains sensitive after
prolonged exposure in vitro ”Proc. Natl. Acad. Sc
i. USA, Vol. 90, pp. 6395-6399, July 1993 Cell Biolo
There are reports such as gy.

[0006]

However, the inhibitors disclosed in these reports are not necessarily related to the fact that the mechanism of action of Tat as described above was not sufficiently elucidated. It is presumed that it is not a specific and effective inhibitor, and it is feared that it is not practical due to problems such as cytotoxicity. Also, it is a so-called “in vitro” report.

In recent years, the mechanism of action of Tat has been elucidated and CDK
Under the circumstances that it is clear that kinases such as CD7 and CDK9 mediate their functions, peptides that suppress CDK7 as shown in SEQ ID NO: 1 have been reported (Cujec., TP, Okamoto). , H., et al., “The HIV tr
ans-activator Tat binds to the CDK-activating Kina
se polymeraseII ”Genes & Dev. 11, 2645-2657, 1997
).

However, although this peptide has an inhibitory effect in a cell-free experiment in a test tube, it has been found that it does not function in an experimental system using cells and is not effective in vivo.

Accordingly, the present invention provides a novel peptide which effectively suppresses the action of Tat in a cell / in vivo, provides a means for producing the same by genetic engineering, and further provides the peptide or recombinant vector. It is an object of the present invention to provide an antiviral disease agent utilizing the same.

[0010]

Means for Solving the Problems The structure of the first invention of the present application (the invention of claim 1) for solving the above-mentioned problems is the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing. Is a peptide having

(Structure of the Second Invention) The structure of the second invention of the present application (the invention according to claim 2) for solving the above problems is as follows.
A transactivator of human immunodeficiency virus HIV transcription in a cell having an amino acid sequence in which one to several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing; HIV-LT by Tat
It is a peptide that has the effect of suppressing the transcription activation reaction of R.

(Structure of Third Invention) The structure of the third invention (the invention according to claim 3) for solving the above-mentioned problem is as follows.
It is a nucleic acid encoding a peptide having the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing.

(Structure of Fourth Invention) The structure of the fourth invention (the invention described in claim 4) for solving the above-mentioned problem is as follows.
It is a nucleic acid having the base sequence shown in SEQ ID NO: 3 in the sequence listing.

(Structure of Fifth Invention) The structure of the fifth invention (the invention according to claim 5) for solving the above-mentioned problem is as follows.
HIV-LTR transcriptional activity by the transactivator Tat of human immunodeficiency virus HIV in cells hybridizes with a nucleic acid having the nucleotide sequence shown in SEQ ID NO: 3 in the sequence listing under stringent conditions. It is a nucleic acid that encodes a peptide that has an action to inhibit the chemical reaction.

(Structure of the Sixth Invention) The structure of the sixth invention (the invention according to claim 6) for solving the above problems is as follows.
A recombinant vector comprising a nucleic acid having the nucleotide sequence according to any one of the third to fifth aspects of the present invention ligated so that its genetic information can be expressed.

(Structure of Seventh Invention) The structure of the seventh invention of the present application (the invention according to claim 7) for solving the above problems is as follows.
A transformant which is a microorganism or a cell containing the recombinant vector according to the sixth invention.

(Structure of Eighth Invention) The structure of the eighth invention (the invention according to claim 8) for solving the above problems is as follows.
This is a method for producing the peptide according to any of the first or second invention using the transformant according to the seventh invention.

(Structure of the ninth invention) The structure of the ninth invention (the invention according to claim 9) for solving the above problems is as follows.
An antiviral disease agent comprising, as an active ingredient, the peptide according to the first or second invention or the recombinant vector according to the sixth invention.

(Structure of the tenth invention) In the structure of the tenth invention (the invention according to the tenth invention) for solving the above-mentioned problem, the antiviral agent in the ninth invention is an anti-AIDS agent. , An antiviral agent.

(Structure of the eleventh invention) In the structure of the eleventh invention (the invention according to the eleventh invention) for solving the above-mentioned problems, the antiviral agent in the ninth invention is an antitumor agent. , An antiviral agent.

[0021]

The peptide having the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing according to the first invention is a SV40 (Simian Vir) consisting of amino acids 1 to 7 shown in the same sequence.
us 40) (amino acid sequence present in SV40, present in the nucleus, and translocating) and amino acid number 8
And has an action of effectively suppressing the HIV-LTR transcriptional activation reaction by Tat, which is a transactivator of HIV transcription, even in cells. confirmed.

This peptide has a Tat of CD as described above.
By binding to kinases such as K7 and CDK9, the phosphorylation ability is enhanced to phosphorylate the CTD of RNA polymerase II, thereby activating the elongation reaction of transcription. -It is thought that the function of Tat is suppressed.

Therefore, the growth of HIV can be remarkably suppressed in cells infected with HIV or in a living body, and excellent anti-AIDS action (or anti-HIV action) can be expected. Furthermore, as in the case of HIV-Tat, it is considered that there is an excellent inhibitory effect also on various tumors including other viral diseases and malignant tumors in which activation of transcription elongation reaction is considered to be involved in the pathological condition. In addition, not only HIV, but also retroviruses (SIV) that exist and infect mammals other than humans
Etc.) can be expected to fulfill the same function.

The reason why the peptide according to the first invention acquired the function of effectively acting in cells as described above by binding the SV40 nuclear localization signal to the CDK7 inhibitory peptide is as follows. It is presumed to be efficiently accumulated in the nucleus, which is a field, and to stabilize the peptide itself in cells.

The peptide according to the second invention can be expected to have the same effect as the peptide according to the first invention.

The nucleic acid according to the third invention to the fifth invention, the recombinant vector according to the sixth invention, the transformant according to the seventh invention, and the method for producing the peptide according to the eighth invention can be used to genetically engineer the peptide. An efficient and inexpensive manufacturing method using means is provided. Further, the recombinant vector according to the sixth invention can be used as an effective therapeutic means for an antiviral disease by introducing itself into cells by an appropriate method.

The ninth to eleventh inventions relate to the use of the peptide of the first or second invention or the recombinant vector of the sixth invention as an antiviral disease agent, particularly preferably as an anti-AIDS agent or an antitumor agent. Which gives
Specific treatments are provided for various viral diseases, especially AIDS and various tumors / malignancies.

[0028]

Next, embodiments of the first invention to the eleventh invention will be described. In the following, the term "the present invention" simply refers to the first to eleventh inventions collectively.

[Usage of the Present Invention] In the present invention, it is possible to prepare a plasmid for expressing a peptide by binding a nucleic acid encoding the peptide to any known vector, and to amplify the plasmid in, for example, Escherichia coli for use. it can. Then, cells of a human or animal to be administered are taken out and cultured, and after introducing the vector into a form in which the vector is encapsulated in liposome or polyethylene glycol (PEG) -liposome or by a method such as electroporation, The cells can be returned to the human or animal body again. Therefore, it can be used as a means of gene therapy.

The nucleic acid can be administered intravenously using a retrovirus vector, or the vector can be encapsulated in the PEG-liposome and administered intravenously.

In constructing a recombinant vector,
According to a conventional method, the following means a) to d) can be applied alone or in combination. B) The nucleic acid (gene) is introduced downstream of any promoter such as CMV (cytomegalovirus) and SV40.
In particular, the efficiency can be enhanced by introducing the gene downstream of a promoter (CD2, Thy1, etc.) specifically expressed in HIV-infected T cells or macrophages. B) An expression vector having a polyA sequence downstream of the gene sequence is introduced. C) Immediately upstream of the gene sequence, a Kozak sequence for efficiently expressing (translating) the peptide of the present invention is introduced. D) GFP (Green Fluorescence P) downstream of the gene sequence
(Rotein) and the like, and the expression is monitored.

The recombinant vector to which the nucleic acid encoding the peptide of the present invention is linked is used not only as a kind of antiviral agent for therapeutic means as described above, but also as a means for producing the peptide of the present invention. At that time, selection of microorganisms or cells to be used for the transformant, production of the peptide by culturing the transformant, and recovery / purification of the peptide from the culture medium, etc., are performed. And a method may be appropriately selected and used.

In order to use the peptide mass-produced efficiently as an antiviral agent, the solubilized peptide itself is used in the form of a solution or encapsulated in PEG-liposome for the human or animal to be administered. It can be administered in the form of a so-called PEG-immunoliposome in which PEG-liposome is modified with an anti-CD4 antibody, an anti-chemokine receptor antibody, or the like.

[Application object of the present invention] The present invention is preferably applied to various viral diseases such as HIV infection and neoplastic growth in any organs of all mammals including humans, particularly malignant tumors. can do. That is, HIV
In addition to infection, activation of the transcription elongation reaction is thought to be involved in its pathology, similar to HIV-Tat. Other viral diseases and various tumors, and retroviruses (SIV, etc.) that exist and infect mammals other than humans ) Can also be expected to have an excellent inhibitory effect. Here, “suppression” includes suppression of virus growth, suppression of tumor cell growth, and suppression of metastasis to other organs.

[Peptide according to the present invention] The peptide according to the first invention has the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing.
A nuclear localization signal of SV40 consisting of
~ 29 CDK7 inhibitory peptides.

The peptide according to the second invention has an amino acid sequence in which one to several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing,
In addition, it includes, without limitation, peptides having an action of suppressing the transcription activation reaction of HIV-LTR in cells.

The peptide according to the present invention further comprises the first
The peptides of the invention or the second invention include those in which other amino acid sequences are added for purposes related to the effects of the present invention or for various purposes not related to the effects of the present invention. The “amino acid” in these peptides includes derivatives and chemically modified amino acids.

[Nucleic acid according to the present invention] The nucleic acid according to the present invention includes at least SEQ ID NO: 2 in the sequence listing according to the third invention.
A nucleic acid encoding a peptide having the amino acid sequence shown in SEQ ID NO: 3, a nucleic acid having the nucleotide sequence shown in SEQ ID NO: 3 of the Sequence Listing according to the fourth invention, and a nucleotide sequence shown in SEQ ID NO: 3 in the Sequence Listing according to the fifth invention Hybridizes under stringent conditions with a nucleic acid having
Nucleic acids encoding peptides having the effect of inhibiting the transcription activation reaction of V-LTR are included.

The term "nucleic acid" used herein is a concept including, without limitation, sense / antisense DNA or RNA, hybridization probes, PCR primers and the like.

[0040]

EXAMPLES ( Example 1 ) In this example, a reporter gene assay [CAT (chloramphenicol acetyltransferase) Assay] using COS cells (monkey kidney fibroblast cell line) was carried out, and the present invention was carried out. H
The function-suppressing action of IV-Tat was evaluated.

Using a liposome, COS cells were prepared by using HIV-CAT as a so-called reporter plasmid and pSVTat (a Tat gene introduced downstream of the SV40 promoter) as a so-called effector plasmid. Introduced.

Each of the plurality of sample cells was used as a Dulb
100 per mL in ecco's modified Eagle's medium
U penicillin, 100 μg streptomycin, 2
The cells were cultured in a medium supplemented with 92 μg of L-glutamine, and a predetermined amount of the peptide of the first invention (referred to as “MC2”) was mixed with 10 μL of a commercially available lipofectamine, lipofectamine (manufactured by Gibco BRL, USA). By adding to the culture solution, each dose shown in FIG.
Incubation was performed at 7 ° C. in a 5% carbon dioxide atmosphere for 48 hours.

Thereafter, the sample cells are ground with NP40, the total protein in the cells is mixed with ³ H-labeled chloramphenicol and acetyl coenzyme A, and the acetylated chloramphenicol is measured by a scintillation counter. The enzyme activity of CAT contained in the cells was evaluated.

The evaluation results are shown in FIG. 1 in terms of the ratio of the activity of each case to the CAT enzyme activity in the absence of pSVTat. As is clear from FIG. 1, depending on the dose of MC2, H
The transcriptional activation of HIV-LTR by IV-Tat was suppressed.

On the other hand, as a control, a peptide consisting of a SV40 nuclear localization signal and a sequence of 22 random amino acids (referred to as "XC2") was prepared in a predetermined amount under the same procedure and conditions as described above. After administration, evaluation was made. As shown in FIG. 1, no significant effect was observed regardless of the dose of XC2.

( Embodiment 2 ) In this embodiment, the COS
Using the cells, the proliferation of HIV was examined by HIV p24 antigen assay, and the inhibitory effect of the present invention on HIV proliferation was evaluated.

Expression of all HIV genes in COS cells,
The replicating plasmid clone pHXB2 or pNL4.3
Was introduced using liposomes.

Each of the plurality of sample cells was used as a Dulb
100 per mL in ecco's modified Eagle's medium
U penicillin, 100 μg streptomycin, 2
The cells were cultured in a medium to which 92 μg of L-glutamine had been added.
FIG. 2 by mixing with
And incubated at 37 ° C. in a 5% carbon dioxide atmosphere for 48 hours.

Thereafter, an HIV p24 antigen assay (RETRO-TEK HIV-1 p24 Antigen ELISA kit, Cellular
Products Inc., NY, USA). FIG. 2 shows the evaluation results. As is clear from FIG. 2, the proliferation of HIV was suppressed depending on the dose of MC2.

On the other hand, as a control, a predetermined amount of XC2 was administered under the same procedure and under the same conditions as above, and evaluation was performed. As shown in FIG. 2, regardless of the dose of XC2, No significant effect was observed.

Example 3 An experiment similar to that of Example 1 was performed by using the above-mentioned CD having no SV40 nuclear localization signal.
When the K7 inhibitory peptide (peptide shown in SEQ ID NO: 1 in the sequence listing) was also tested, HIV-Lat by HIV-Tat was used.
Did not suppress transcriptional activation. FIG. 3 shows the evaluation results. FIG. 3 also shows a graph according to the first embodiment of FIG. 1 again for convenience of comparison.

[0052]

[Sequence Listing] SEQ ID NO: 1 Sequence length: 22 Sequence type: amino acid Sequence characteristics Other information: The following sequence shows the amino acid sequence of the CDK7 inhibitory peptide. Sequence: Ala Arg Ala Phe Gly Val Pro Val Arg Thr Try Ala His Glu Val Val 1 5 10 15 Thr Leu Trp Tyr Arg Ala 20 22 SEQ ID NO: 2 Sequence length: 29 Sequence type: Amino acid Sequence characteristics Other Information: The sequence below consists of a SV40 nuclear localization signal consisting of amino acids 1-7 and a CDK7 inhibitory peptide consisting of amino acids 8-29. Sequence: Pro Lys Lys Lys Arg Lys Val Ala Arg Ala Phe Gly Val Pro Val Arg 1 7 8 10 15 Thr Try Ala His Glu Val Val Thr Leu Trp Tyr Arg Ala 20 25 29 SEQ ID NO: 3 Sequence length: 87 Sequence Type: Nucleic acid Sequence: CCA AAA AAG AAG CGA AAG GTA GCC CGA GCT TTT GGA GTC CCT GTT CGT 48 ACT TAC GCC CAT GAG GTG GTG ACC CTG TGG TAC CGA GCT 87

[Brief description of the drawings]

FIG. 1 is a graph showing evaluation results of Example 1.

FIG. 2 is a graph showing evaluation results of Example 2.

FIG. 3 is a graph showing evaluation results of Example 3.

Claims (11)

[Claims] 1. A peptide having an amino acid sequence represented by SEQ ID NO: 2 in the sequence listing. 2. The human immunodeficiency virus HIV has an amino acid sequence in which one to several amino acids have been deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2 of the sequence listing, and has been transcribed intracellularly. Transactivator of Tat
A peptide having an effect of inhibiting the transcriptional activation reaction of HIV-LTR by lipase. 3. A nucleic acid encoding a peptide having the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing. 4. A nucleic acid having the nucleotide sequence shown in SEQ ID NO: 3 in the sequence listing. 5. A human immunodeficiency virus HIV that hybridizes with a nucleic acid having the nucleotide sequence shown in SEQ ID NO: 3 in the sequence listing under stringent conditions and in a cell.
Nucleic acid encoding a peptide having an action of suppressing the transcriptional activation reaction of HIV-LTR by the transactivator of transcription of Tat. 6. A recombinant vector comprising a nucleic acid having the nucleotide sequence according to any one of claims 3 to 5 ligated so that its genetic information can be expressed. [7] A transformant which is a microorganism or cell containing the recombinant vector according to [6]. (8) A method for producing the peptide according to (1) or (2) using the transformant according to (7). 9. An antiviral disease agent comprising the peptide of claim 1 or the recombinant vector of claim 6 as an active ingredient. AIDS agent. 10. The antiviral disease agent according to claim 9, wherein the antiviral disease agent is an anti-AIDS agent. 11. The antiviral agent according to claim 9, wherein the antiviral agent is an antitumor agent.