JP2002253239A - Genes encoding polypeptides that inhibit the activity of human immunodeficiency virus reverse transcriptase and uses thereof - Google Patents

Abstract

(57) [Problem] To provide a gene useful as a therapeutic agent for HIV infection. SOLUTION: A gene encoding Fd and VCL, which are Fab antibody genes of an anti-RT activity inhibitory antibody, capable of expressing a polypeptide having an RT inhibitory activity is obtained. A recombinant vector incorporating this gene in a vector is used as a therapeutic agent.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a human immunodeficiency virus (hereinafter referred to as HIV) reverse transcriptase (hereinafter referred to as RT).
A) a DNA molecule encoding the Fab region of a monoclonal antibody having reactivity with the antibody and its use.

[0002]

2. Description of the Related Art Hitherto, various antibodies have been used for diagnosis of HIV infection or basic research on HIV. Previous studies have shown that HIV can infect humans and human cells DN
In order for the HIV gene to be incorporated into A, it is known that RT, protease and endonuclease, which are gene products of the pol region of the HIV gene, are essential. Of these gene products, regarding RT, RT
It has been shown that the amount of polyclonal antibody that inhibits the enzyme activity (hereinafter sometimes referred to as RT activity) of E. coli decreases or disappears as the stage of HIV infection progresses [Laurence, J. et al. Saunders, A .;
and Kulkosky, J. et al. : Science, 2
56, 1501-1504 (1987); Sano,
K. et al. : J. Clin. Microbiol.
25, 2415-2417 (1987)]. Therefore, R
Producing a monoclonal antibody having high specific reactivity to T and capable of inhibiting its RT activity is important not only for understanding the pathology of HIV infection, but also for treatment and prevention. It has very important significance. From this viewpoint, Japanese Patent Application Laid-Open No. 4-75597 discloses that
Monoclonal antibodies having an activity of inhibiting RT activity (hereinafter sometimes referred to as RT inhibitory activity) have been found.

[0003] In recent years, with the progress of genetically modified vector technology, it has become possible to express specific gene products in human body cells, and gene therapy has come to the forefront. Although this technology has shown new potential for the treatment of HIV infection, the treatment of HIV infection requires a gene product that suppresses HIV growth.
For example, intracellular immunization, which is a method of gene therapy, involves introducing a DNA molecule, in which a gene encoding an antibody molecule has been genetically manipulated into a form that can be expressed in animal cells, into a body cell, and then culturing the antibody molecule in the cell. It suppresses the expression of proteins essential for HIV growth and suppresses HIV growth. Therefore, the antibody molecule used herein has an activity of inhibiting the growth of HIV (hereinafter, referred to as an activity)
HIV inhibitory activity). Monoclonal antibodies having HIV inhibitory activity in vitro (eg, anti-V3 that recognizes the V3 portion of the Env region)
Although monoclonal antibodies are also known, it is desirable to select a monoclonal antibody molecule that inhibits the activities of HIV-specific enzymes, such as RT, protease and endonuclease, from practical aspects such as effects and side effects. . When such an enzyme activity-inhibiting monoclonal antibody molecule is expressed in a cell, it is expected to inhibit the growth of HIV itself.

[0004]

Conventionally known monoclonal antibodies having HIV inhibitory activity are produced using animal cells such as mice and rats. An antibody molecule is composed of a Fab region that is an antigen recognition site and an Fc region that is a tail, and the Fc region is a peptide region derived from the cell that produced the antibody. Therefore, F of monoclonal antibodies produced using animal cells such as mice and rats
The c region can produce antibodies against these animals.
For example, when a monoclonal antibody produced using a mouse is applied to a human, the anti-mouse F
The possibility arises that c antibodies are produced. If anti-mouse F
When the c antibody is produced, the anti-mouse Fc antibody reacts with a monoclonal antibody having a mouse-derived Fc region. For this reason, a monoclonal antibody produced using a mouse cannot obtain HIV inhibitory activity in the second and subsequent applications. Indeed, if a sufficient effect can be obtained by a single application, there is no inconvenience even if an anti-mouse Fc antibody is produced in the human body, but from the viewpoint of obtaining a sufficient effect, multiple applications are realistic. . The present inventors have conducted intensive studies to solve this problem and found that
The present invention has been completed by specifying the Fab region and the Fc region of a monoclonal antibody having high specific reactivity to RT and identifying the DNA sequence encoding the Fab region.

[0005]

According to the present invention, there is provided, as a first invention, a DNA molecule encoding the amino acid sequence of SEQ ID NO: 1 or the following amino acid region (1) to (3) in the DNA molecule: A DNA molecule mutated so that any one or more of them has a homology of 50% or more. As a second invention, a DNA molecule encoding the amino acid sequence of SEQ ID NO: 2 or the following (4) Any one of the amino acid regions of (1) to (6);
DNA mutated to the extent that the above is 50% or more homology
The present invention provides, as a third invention, a recombinant vector incorporating the first and / or second DNA molecule, and as a fourth invention, a therapeutic drug for human immunodeficiency virus infection containing the recombinant vector. Is provided,
As a fifth invention, an amino acid sequence comprising a Fab region of a monoclonal antibody reactive to reverse transcriptase of human immunodeficiency virus, which is encoded by a DNA molecule of the first invention, and a DNA of the second invention A polypeptide having an amino acid sequence encoded by a molecule is provided, and as a sixth invention, a transformant expressing the polypeptide is provided. (1) 24th to 33rd amino acid region of SEQ ID NO: 1 (2) 49th to 55th amino acid region of SEQ ID NO: 1 (3) 88th to 95th amino acid region of SEQ ID NO: 1 (4 ) Amino acid region at positions 30 to 36 of SEQ ID NO: 2 (5) Amino acid region at positions 50 to 66 of SEQ ID NO: 2 (6) Amino acid region at positions 99 to 109 of SEQ ID NO: 2

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail. In the present invention, "homology" refers to the homology of amino acid sequences calculated by amino acid homology comparison software such as software DNASIS (manufactured by Hitachi Software) for comparing protein homology. In the present invention, "mutation"
The term “natural” or “artificial” means deletion, substitution, addition, omission or insertion of an amino acid sequence or base sequence.

(DNA molecule) One of the DNA molecules of the present invention is a molecule having the amino acid sequence shown in SEQ ID NO: 1 or a DNA molecule in which a specific region of this amino acid sequence is mutated (hereinafter, collectively referred to as DNA molecule 1). Is)
The other is a molecule having the amino acid sequence of SEQ ID NO: 2 and a DNA having a specific region of this amino acid sequence mutated.
Molecule (hereinafter sometimes collectively referred to as DNA molecule 2)
It is. Specific examples of the DNA sequence of DNA molecule 1 include:
The sequence of SEQ ID NO: 3 is listed, and the DN of DNA molecule 2
A specific example of the A sequence includes the sequence described in SEQ ID NO: 4.

[0008] A peptide in which the polypeptide having the amino acid sequence of SEQ ID NO: 1 and the polypeptide having the amino acid sequence of SEQ ID NO: 2 are conjugated has the activity of inhibiting the RT of HIV, which is the polypeptide of the present invention described below. Fab molecule of a monoclonal antibody (hereinafter referred to as anti-RTF
ab molecule). Here, the degree of the activity of the polypeptide to inhibit HIV RT is expressed by Ho
ffman et al. [Virology. 147 , 32
6-335 (1985)]. More specifically, according to the method described in the Examples, when the RT activity when the amount of added polypeptide is 0 is defined as 100%, if the RT activity is less than 100%, it is determined that there is an RT inhibitory activity. When a peptide is used as a therapeutic agent for HIV infection, high RT inhibitory activity is required.
RT activity when a peptide used as a therapeutic agent for V infection is added is 50% or less, preferably 30% or less, and more preferably 20% or less.

The specific region which can be mutated is the amino acid sequence of (1) to (3) for the amino acid sequence of SEQ ID NO: 1, and the amino acid sequence of (4) for the amino acid sequence of SEQ ID NO: 2. To (6) (hereinafter, the amino acid regions (1) to (6) may be collectively referred to as a hypervariable region). Even if there is a mutation in this hypervariable region, the DNA molecule can still provide a polypeptide exhibiting RT inhibitory activity, and in particular, DNA molecules with 50% or more homology, preferably 60%, A polypeptide exhibiting a high RT inhibitory activity equivalent to a polypeptide having the described amino acid sequence can be provided.

[0010] Such a DNA molecule may be synthesized based on the amino acid sequence of SEQ ID NO: 1 or 2, or may be synthesized from HIV.
CDNA may be synthesized by a predetermined method from a hybridoma producing a monoclonal antibody having an RT inhibitory activity. The obtained DNA molecule may be mutated according to a conventional method such as using a restriction enzyme site. The method for synthesizing cDNA from a hybridoma is as follows. An antibody molecule is composed of a heavy chain and a light chain, and the amino acid sequence of the variable region that binds to the antigen protein is different, but the amino acid sequence of the constant region is common. Therefore, by the PCR method using the DNA sequence of this constant region,
A DNA fragment containing the variable region can be amplified and cloned. For example, after culturing a hybridoma strain and extracting mRNA from the hybridoma cells, oligo d
A minus-strand cDNA is synthesized by an ordinary method using a T primer. Then, RA from Frohman et al.
CE method (Rapid Amplification
f cDNA ends) (Proc. Natl. Ac)
ad. Sci. USA, 85, 8998-9002 [1
988]), after adding polyA to the 3 ′ end of the minus-strand cDNA by terminal transferase, a plus-strand cDNA is synthesized with a primer having a cloning site and a poly dT sequence to obtain double-stranded DNA. Then, PCR (Polymerase Cha)
In the Reaction method, genes of the VH domain and CH1 domain of the heavy chain of the monoclonal antibody (both are collectively referred to as Fd gene) and genes of the VL domain and CL domain of the light chain (both are collectively referred to as the VCL gene) ) Can be amplified. The amplified Fd gene and VCL gene are then cloned into an E. coli vector. Note that DNA molecule 1 corresponds to the VCL gene, and DNA molecule 2 corresponds to the Fd gene.

(Recombinant vector) The recombinant vector of the present invention is a vector into which the above-mentioned DNA molecule has been incorporated. The vector into which the DNA molecule is incorporated may be any vector such as a plasmid, a phage, a cosmid or the like that is expressed in cells, or a virus vector that infects a human such as an adenovirus or a vaccinia virus.
The number of the DNA molecules of the present invention to be incorporated into the vector is not particularly limited. For example, DNA molecule 1 and DNA molecule 2 can be incorporated one by one,
Only molecules can be incorporated. In order to efficiently obtain a polypeptide corresponding to the Fab molecule of a monoclonal antibody having HIV RT inhibitory activity, it is preferable to incorporate one DNA molecule and one DNA molecule. At this time, either the DNA molecule 1 or the DNA molecule 2 may be located on the upstream side. At this time, DNA molecule 1 and DNA
Internal from encephalomyocarditis virus between molecule 2
ribosomal entry site (IRE
It is preferable to intervene S) because the protein can be efficiently synthesized. For example, the DNA molecule 1-I is contained in one mRNA.
It is preferable to link the RES-DNA molecules 2 in this order because the protein can be efficiently synthesized. For example, DNA molecule 1 is
Normally, ribosomes are bound and translated, apart from that,
From the ATG immediately after the IRES, the DN existing downstream
A molecule 2 is translated. As a result, another protein can be synthesized from one plasmid.

The recombinant vector may be constructed according to a conventional method. For example, in the case of a plasmid, a phage, a cosmid, etc., the DNA molecule of the present invention may be inserted into a restriction enzyme site existing in a cloning site, and a viral vector may be used. In this case, the DNA molecule of the present invention may be inserted by homologous recombination into a DNA region that is not essential for its growth. Usually, a promoter is arranged upstream of each DNA molecule to ensure the expression level. The promoter to be used is not particularly limited as long as it functions in a cell in which the polypeptide is expressed. Further, from the viewpoint of purification of the recombinant vector, pUC19, pBR322, pE19 having a marker gene such as the lacZ gene and a drug resistance gene such as the ampicillin resistance gene or the kanamycin resistance gene are used.
Vectors such as T series plasmids, pBKCMV and the like are preferred.

When it is necessary to isolate and purify the polypeptide of the present invention, it is preferable to insert an appropriate peptide tag gene downstream of the inserted gene in advance.
Peptides used as tags are Myc tag, His
Tags, HA tags, etc., from the widely used tags, considering the amino acid sequence of the target polypeptide,
It may be arbitrarily selected.

(Therapeutic agent for HIV infection) The above-described recombinant vector of the present invention can be applied to humans alone or with an appropriate adjuvant, and used as a therapeutic agent.
When the recombinant vector of the present invention is carried as an adjuvant by a substance (DNA transfer reagent) that can enter a cell, such as a liposome to which an oligosaccharide or a lipid described in Japanese Patent No. 2828391 is bound, the recombinant vector can enter the cell. The polypeptide of the present invention expressed by the recombinant vector contained in the cell can inhibit the RT activity of HIV in the cell and inhibit the proliferation of HIV. Therefore, gene therapy becomes possible by employing liposomes and the like as adjuvants. Of course, when the recombinant vector is a recombinant virus, the recombinant virus can be suspended and inoculated in an aqueous solution of a pharmacologically acceptable salt. If the recombinant virus gets into the cells in this way, gene therapy will be possible as well. More specifically,
The recombinant vector of the present invention can be directly or DN
A After mixing with the transfer reagent, suspend it in a buffer such as physiological saline, or mix it with a DNA transfer reagent such as liposome, and then inject it subcutaneously, intradermally, muscle, etc., or mix it with drinking water or food. Oral administration may be used. The DNA molecule of the present invention incorporated into an individual by such a method is efficiently taken up by cells, and the polypeptide of the present invention (corresponding to an anti-RT Fab molecule) is expressed in those cells. HIV present in cells
By inhibiting RT activity, it will be used as an intracellular vaccine that inhibits HIV replication.

(Polypeptide) The polypeptide of the present invention comprises Fab molecules of a monoclonal antibody reactive to HIV RT, and has an amino acid sequence encoded by two DNA molecules of the present invention. Such polypeptides include those having the amino acid sequences of SEQ ID NOS: 1 and 2. The region having the amino acid sequence encoded by the two DNA molecules forms a -S-S- structure via a sulfur atom in the molecules and connects to form a Fab molecule. The monoclonal antibody according to the present invention may be HIV (for example, HIV-1, HIV-
2, but preferably HIV-1
Is good. ) Has high specific reactivity to RT,
And a monoclonal antibody capable of inhibiting its RT activity. As such a monoclonal antibody,
7C4 acidic hybridoma (Micro-Engineering Research Strange No. 3012)
And the like. The polypeptide having the amino acid sequence of SEQ ID NOS: 1 and 2 consists of the Fab region of this monoclonal antibody. This polypeptide has high specific reactivity to both the HIV RT protein of about 66K and the protein of about 51K formed by its processing, and has its RT activity. It can inhibit, but does not show reactivity with Escherichia coli extract that does not express HIV RT (supernatant obtained by crushing E. coli by ultrasonication and centrifuging) or animal cell extract. . Further, since this polypeptide reacts with the 252nd to 335th peptide of the HIV RT protein, the "thumb subdomain (thumb su domain)"
bdomain) ”.

The monoclonal antibody according to the present invention is H
Mice can be immunized with a vaccinia virus capable of expressing IV RT as an immunogen, and lymphocytes obtained from the mice can be obtained from a hybridoma strain obtained by fusing mouse myeloma cells. As the hybridoma strain thus obtained, for example, 7C4 strain (Microtechnical Laboratory No. 3012) can be mentioned. After culturing such a hybridoma strain, extracting mRNA from the hybridoma cells, synthesizing cDNA, amplifying the antibody molecule gene by PCR, cloning into an Escherichia coli plasmid, and constructing a plasmid having a recombinant gene. Thus, it can be manufactured. Another method is to use a vaccinia virus capable of expressing HIV RT as an immunogen in a mouse, extract mRNA from lymphocytes obtained from the mouse, and perform the same operation as described above to obtain an E. coli plasmid. Can be produced by cloning into

As described above, the RT inhibitory activity of the polypeptide of the present invention can be measured by the method of Hoffman et al. Also, HIV of the polypeptide of the present invention
Has an affinity for the RT protein (affinity constant K _A ) of 1.0
0 × 10 ^{7 to} 1.00 × 10 ⁹ (Biacore
(Measured by BIACORE).

[0018]

The present invention will be described below with reference to examples and experimental examples, but the present invention is not limited to these examples.

Example 1 (1) Growth of Monoclonal Antibody-Producing Hybridoma Strain Monoclonal antibody 7C4-producing hybridoma (Microtechnical Laboratories No. 3012) was cultured in a culture flask with a MEM solution containing 10% fetal bovine serum. Culture until the cell concentration reaches 5 × 10 ⁸ and centrifuge (4 ° C., 350 G, 5
), Antibody-producing hybridomas were collected. After washing the obtained antibody-producing hybridoma once with PBS, the antibody-producing hybridoma was collected again by centrifugation,
Suspended in 10 ml of PBS. 10m to this cell suspension
1 of solution D (composition; 4M guanidine thiocyanate,
0.1 M Tris-Cl [pH 7.5], 10% 2-
Mercaptoethanol) and allowed to stand on ice for 10 minutes to melt the cells, and then 1 ml of 1 M sodium acetate (p
H4.0) was added. To this solution, 10 ml of water-saturated phenol and 2 ml of chloroform / isoamyl alcohol were added and mixed vigorously.
After centrifugation for 10 min, the supernatant was collected and 4 μl of 10 mg / m
After adding 1 g of glycogen, an equal volume of isopropanol to the supernatant was added, and the precipitate was collected by centrifugation at 4 ° C., 12,000 × G for 20 minutes. 3 ml of solution D was added to and dissolved in the precipitate, and 3 ml of isopropanol was added thereto, followed by centrifugation at 4 ° C., 12,000 × G for 20 minutes to collect the precipitate. The precipitate (RNA) was washed with 75% ethanol and dissolved in 50 μl of water.

(2) Synthesis of cDNA Of the RNA solution obtained above, 15 ng of total R
30 pmol of oligo dT [18-25] (manufactured by Life Tech) was added to NA, the whole was made 29 μl, heat-treated at 70 ° C. for 10 minutes, and then ice-cooled. In it, 1 μl of RNase inhibitor (promega) and 10 μl of 5 × RT buffer (LifeTech SuperScr)
Buffer attached to ipt Choice System. 5 × means a concentration five times the composition of the reaction solution, and similarly, a combination of a numeral and “×” is a concentration that is a numerical multiple of that. For example, if it is 10 ×, the concentration will be 10 times).
1 of 25 mM dNTP mixture, 5 μl of 0.1 M D
TT, 2 μl RT (manufactured by Life Tech Co .; SuperS
(script RT) and left at 20 ° C. for 10 minutes.
After reacting at 2 ° C. for 50 minutes, it was left still at 4 ° C. Poly (A) was added to the 5 ′ end of the single-stranded cDNA thus prepared.
First, to remove the primers from the cDNA reaction,
After removing primers and nucleotides using cDNA Spun Columns (manufactured by Pharmacia), single-stranded cDNA was recovered by ethanol precipitation. After washing the cDNA precipitate with 70% ethanol,
1 μl of sterile water and add 7 μl of 2 μl of 10 × T
dT buffer (500 mM sodium cacodylate,
5 mM CoCl ₂ ) (TaKaRa), 1.5 μl
TdT (TaKaRa), 1 μl of 10 mM dA
TP, 2 μl of 1 mM DTT and 6.5 μl of sterile water were added and reacted at 37 ° C. for 15 minutes. TdT was inactivated by heat treatment at 65 ° C. for 15 minutes, and RNaseH (TaKaRa) was used.
130 μl of TE buffer (10 mM Tris-Cl [pH 7.5], 1 mM
EDTA) was added and reacted at 37 ° C. for 10 minutes. Then
The second strand DNA was synthesized by adding 30 pm to the single-stranded cDNA (150 μl) obtained above.
ol Qt primer (sequence; CCAGTGAGCAGAGTGACGAGGA
CTCGAGCTCAAGCTTTTTTTTTTTTTTTTT), 20 μl of 10 ×
Second strand buffer (LifeTech's SuperScriptChoice System)
3 μl of 25 mM dNT
P mixture, add 5 μl of 0.1 M DTT and add 180
μl, and add 2 μl of E. coli DNA polymerase I.
After adding 00 units (20 μl) and reacting at 16 ° C. for 2.5 hours, the reaction was stopped at 4 ° C. to stop the reaction. 200 μl of phenol / chloroform / isoamyl alcohol (25
/ 24/1), and after mixing vigorously, 12,000 ×
G. After centrifugation for 10 minutes, the supernatant was collected, and the synthesized double-stranded cDNA was recovered by ethanol precipitation.
In TE buffer. From the 7C4 monoclonal antibody cDNA thus obtained, the Fd gene and the VCL gene were amplified by a two-step PCR method.

In the PCR amplification of the DNA of the Fd gene, a Qo primer (sequence: CCAGTGAGCA) was added to the first PCR reaction.
GAGTGACG) and mIgG1 (sequence; 5'-AGGCTTACTAGTACAA)
TCCCTGGGCACAAT) was used. 2 obtained by the above reaction
10 μl of main-strand cDNA, 10 μl of 10 × PCR buffer (TaKaRa), Qo and mIgG1 primer were added at 50 pmol and 10 μl of 2.5 mM, respectively.
A dNTP mixed solution was added, and the total amount was adjusted to 99 μl with water. Then, 1 μl of Taq polymerase (TaKaRa) was added. The PCR reaction conditions were as follows:
4 ° C, 10 minutes, then heat denaturation 94 ° C, 1 minute, annealing 5
Extension reaction at 8 ° C. for 2 minutes, extension reaction at 72 ° C. for 1 minute, followed by extension reaction at 72 ° C. for 10 minutes).
Transfer 10 μl of the reaction solution after completion of the first PCR to a new reaction tube, and add 10 μl of 10 × PCR buffer (T
aKaRa), Qi primer (sequence: GAGGACTCGA)
GCTCAAGC) and mGFv1 primer (sequence; TGGGGG)
TGTCGTTTTTCTAGAGGAGACGGTGACTGA) 50 pm each
ol, 10 μl of a 2.5 mM dNTP mixed solution was added, and the total volume was adjusted to 99 μl with water.
Then, 1 μl of Rase (manufactured by TaKaRa) was added, and PCR was performed under the same PCR conditions as above to obtain the Fd gene. VC
The PCR amplification of the L gene DNA is performed in the first PCR,
Qo primer and mCK1 primer (sequence; 5'-GCGC
2nd PC using CGTCTAGAATTAACACTCATTCCTGTTGAA)
R is a Qi primer and mKFv1 primer (sequence; GG
PCR using ATACAGTTGGTGCACTAGTAGCCCGTTTGATTTC)
I went in. The PCR was performed under the same conditions as in the above-mentioned FdPCR to obtain a VCL gene. From the reaction solution after the two-step PCR, take 10 μl each, and perform PCR for the Fd gene.
For the product, use restriction enzymes XhoI and XbaI. For the VCL gene PCR product, use restriction enzyme XhoI and XbaI.
After digestion with hoI and SpeI, it was digested with the same enzyme.
It was cloned into plasmid pBluescriptII KS + (Stratagene), and
-7C4H and pBS-7C4L plasmids were obtained.

(3) Analysis of DNA sequence The Fd gene of the 7C4 antibody cloned in the pBS-7C4H and pBS-7C4L plasmids, and V
The base sequence of the CL gene (hereinafter sometimes collectively referred to as Fab DNA) is converted to Thermo Sequenase.
II eterminator sequence
ing kit (Amersham-Pharmaci)
a) and 373 type sequence analyzer (ABI)
(Manufactured by the company). As a result, the DNA sequence of the VCL gene was as shown in SEQ ID NO: 3, and the DNA sequence of the Fd gene was
The sequence was as described in SEQ ID NO: 2. The amino acid sequences of polypeptides VCL and Fd, which are gene products deduced from the DNA sequences of DNA SEQ ID NOS: 3 and 4, are shown in amino acid sequence numbers 1 and 2, respectively.

(4) Expression of polypeptide The VCL and Fd proteins of 7C4 Fab were expressed by Escherichia coli. For expression, pET-22b (+) and pET-24a (+) (manufactured by NOVAGEN) were used. Downstream of the periplasmic translocation signal (pelB) DNA sequence present in pET-22b (+), pBS
To fuse the VCL and Fd genes from the -7C4L and pBS-7C4H plasmids,
A DNA fragment was prepared by CR. For the VCL gene, ETFabL5 (sequence; CTAGGATCCACAAATTGTTCTC
ACCCAG) and ETFabL3 (sequence; CTTGCGGCCGCT
CAACACTCATTCCTGTTGAA) at a final concentration of 0.5 μM,
Using 0.2 μg of the pET-7C4H plasmid,
Reaction was performed under the same PCR conditions as above, and the synthesized DNA was digested with restriction enzymes BamHI and NotI, cloned into pET-22b (+) digested with the same enzymes, and
T7C4L was obtained. Similarly, for the Fd gene, ET
FabH5 (sequence; GCAGAATTCGGAAGTGCAGCTGGTGGAG) and ETFabH3 (sequence; CTTGCGGCCGCAATATTCTTGT
CCACCTT) at a final concentration of 0.5 μM, pET-7C
Using 0.2 μg of the 4H plasmid, the same P
Reaction was performed under CR conditions, and the PCR-synthesized DNA was digested with restriction enzymes EcoRI and NotI, cloned into pET-22b (+) digested with the same enzymes, and pET-
7C4H was obtained.

PET-7C4H was treated with restriction enzymes BglII and DraIII, and treated with the same enzymes.
4a (+) plasmid and cloned into pET24-
7C4H was obtained. pET7C4L and pET24-7
C4H was introduced into BL21 (DE3) E. coli (NOVAGEN) capable of transforming, and kanamycin 15
Colonies were formed on an agar medium containing μg / ml and 50 μg / ml ampicillin. Single colonies were LB liquid medium containing the same concentration of antibiotics (10 g Bacto tryptone, 5 g per liter).
After culturing for 16 hours at 37 ° C. in Yeast extract (10 g NaCl), 20 ml thereof was added to 2 liters of the same liquid medium. After culturing at 37 ° C. for 3 hours, IPTG having a final concentration of 1 mM was added. Cultured for hours. The culture was centrifuged at 5,000 × G for 15 minutes at 4 ° C. to collect Escherichia coli bodies. Escherichia coli cells were crushed in 30 ml of a disruption buffer (30 mM Tris-Cl [pH 7.
4], 20% sucrose) and allowed to stand on ice for 1 hour. Then, 200 ml of ice-cold water is added and the cells are crushed by osmotic shock. The target anti-RTFab molecule is present in the soluble fraction from which the insoluble fraction has been removed by centrifugation of the cell disruption solution at 10,000 × G.

(5) Purification of polypeptide Anti-RT Fab present in soluble protein derived from E. coli
To purify the molecule with high purity, an affinity purification method using a His tag, in which six histidines were present in the anti-RT Fab molecule, was employed. Specifically, after adding 500 mM final concentration of NaCl to an E. coli soluble protein solution containing an anti-RT Fab molecule after centrifugation,
It is added to a Ni-NTA column (manufactured by Qiagen) to bind the anti-RT Fab molecule to the Ni-NTA resin. 9
ml of washing solution (30 mM imidazole-50 mM Tr)
After washing the column with is-Cl [pH 8.0] -500 mM NaCl), 4 ml of the eluate (100 mM imidazole-50 mM Tris-Cl [pH 8.0] -50)
Elute the bound anti-RT Fab molecules at 0 mM NaCl). The eluate was washed with 20 mM Tris-Cl [p
H8.5], and added to an anion exchange resin column (TOAEOH, DEAE-5PW).
After washing the column with 0 ml of 20 mM Tris-Cl [pH 8.5], elution is performed with a NaCl concentration gradient of 0 M to 1 M. Identification of the fraction containing the anti-RT Fab molecule
For example, it can be performed by an ELISA method coated with an RT protein. The fractions containing the anti-RT Fab molecules thus obtained were mixed and dialyzed against PBS to obtain purified anti-RT Fab molecules.

(6) Confirmation of Properties of Polypeptide The properties of the anti-RT Fab molecule were examined as follows. The specific binding of the anti-RT Fab molecule to the RT protein was examined by an ELISA method coated with the purified RT protein. 1 μg / ml RT protein (ImmunoDiag
bicarbonate)
Fifty microliters of the buffer was transferred to an ELISA plate (CORNIN
G COSTAR; No. 3690) and allowed to adsorb at room temperature for 3 hours, and then the unadsorbed portion was blocked with PBS containing 25% Block Ace (Dainippon Pharmaceutical Co., Ltd.).
The solution containing the anti-RT Fab molecule was appropriately diluted, 50 μl of the solution was added to each well, and reacted at room temperature for 1 hour.
After washing with BS / Tween 20, 50 μl of alkaline phosphatase-labeled anti-mouse IgG antibody (5,000
(Double dilution) was added. After reacting at room temperature for 1 hour, the plate was washed again, and 100 μl of pNPP (1 m
g / ml) was added for color development. By this operation, the anti-R
Since the amount of the T Fab molecule bound to the RT protein was measured as the chromogenicity of the substrate, the absorbance at 405 nm was measured, the absorbance at 630 nm was measured as the background, and the subtraction was performed to detect a specific reaction. . When the anti-RT Fab molecule was used, it was confirmed that the absorbance increased depending on the concentration of the anti-RT Fab molecule. When the color of the substrate was examined in the same manner as described above except that the RT protein was not coated, no increase in the express hall was observed. From this, the anti-RT Fab molecule is
It was found to react specifically with the protein.

Further, the RT protein was fractionated by SDS-PAGE according to the molecular weight, and then examined by Western blotting. 1 μg / ml of the above purified RT protein solution (10 μl) and sample buffer (130 mM T
ris-HCl [pH 6.8], 6% SDS, 10%
2-mercaptoethanol, 20% glycerol,
0.01% bromophenol blue) was mixed and heated at 95 ° C. for 5 minutes. This sample is called SDS-
After 10% PAGE, Polyvinylidene
Western blotting was performed on e Difluoride (PVDF) membrane (Millipore). After the completion of the Western blot, the PVDF membrane was immersed in PBS containing 25% Block Ace overnight, and the PVDF membrane containing 1 μg / ml of an anti-RT Fab molecule was prepared.
After reacting with a PBS solution containing 10% block ace for 1 hour at room temperature, washing several times with PBS / Tween20, alkaline phosphatase-labeled anti-mouse IgG F (a
b ′) ₂ (2,000-fold dilution) solution for 30 minutes.
Again, the PVDF membrane was washed several times with PBS / Tween, and then immersed in a coloring buffer. As a result, the anti-R
The T Fab antibody is 66 kilodalton (kDa) p66
And 51kDa specifically react with the p51 RT protein.

The anti-RT Fab fraction thus obtained
Hoff determines whether the offspring inhibits HIV RT activity.
Mann et al. [Virology.147, 326-
335 (1985)].
RT activity measurement solution (50 mM Tris-Cl [pH8.
0], 5 mM dethiothreitol, 5 m
M MgCl₂, 150 mM KCl, 0.3 mM g
lutathione (reduced form),
0.5 mM EDTA, 0.06% Triton X-
100, 6 μM [³H] deoxythymidine
 triphosphate (Du Pont), 3μ
M poly (rA) ・ oligo (dT) 10 (Am
ersham-Pharmacia), 0.2 mg / m
l BSA, 0.1 μg / ml RT).
Of anti-RT Fab molecules at 37 ° C. for 40 minutes
After the reaction, a part of it was passed through a DE81 filter (Wh
atman), dry and filter
SC (15 mM sodium citrate [p
H7.0], unreacted in 150 mM NaCl)
[ ³H] deoxythymidine tripho
After removing the phosphate, the synthesized cDNA contains
Tritium content ([³H]) with liquid scintillation
RT activity is measured by measuring with a counter.
You. The experiment was performed without adding an anti-RT Fab molecule.
Assuming that the amount of RT activity is 100%, an anti-RT Fab molecule
However, the antibody concentration that reduces RT activity by 50% is 0.04
5 μM, and the concentration of the antibody showing 95% inhibitory activity was 0.1 μM.
It was 4 μM.

The specificity of the thus obtained anti-RT Fab molecule against RT other than HIV RT or DNA polymerase was measured by ELISA.
Avian myeloblastosis viru
s and Moloneyline leukem
ia virus RT or E. coli DNA polymerase I, Themus aquaticus D
NA Polymerase (Taq Polymerase) protein
containing 50 μg / ml of ELISA coating solution
50 μl of an antibody diluent containing 1 μg / ml of anti-RT Fab molecule was added to the 1-plate ELISA plate, and reacted at room temperature for 1 hour. Thereafter, as a result of performing the same operation as the above-mentioned ELISA, the anti-RT Fab antibody
It did not react with T or DNA polymerase and was shown to be specific for HIV RT.

The position on the RT molecule recognized by the anti-RT Fab molecule was examined by Western blotting. RT proteins 52-98, 52-335, 145-428
, And E. coli expression plasmids containing the 155th to 250th parts were prepared.
1161, pRN2022 and pRN4891. Escherichia coli having these expression plasmids is cultured to express a part of the RT protein in the Escherichia coli. After the Escherichia coli cells were fractionated according to the molecular weight of the protein by the above-mentioned SDS-PAGE method, the protein was transferred to a PVDF membrane by Western blotting.
Reacting the PVDF membrane with an anti-RT Fab molecule,
As a result of identifying an amino acid position (epitope) on the RT protein recognized by the anti-RT Fab molecule, the anti-RT Fab molecule specifically reacted with the amino acid sequence at positions 252 to 335 of the RT protein. This region is referred to as the “thumb” subdomain of the RT protein molecule, and is one of the polypeptides of the present invention, anti-RT Fa
The b molecule was found to recognize this subdomain.

As described above, the anti-RT Fab molecule is H
It has been shown that it has an inhibitory activity on IV RT activity and specifically reacts with the amino acid sequence at positions 252 to 335 of the HIV RT protein. In addition, this relationship was investigated in detail using a competitive ELISA. First, the 7C4 and 6B9 monoclonal antibodies IgG were purchased from Ed Harlo.
w and David Lane, Antibodi
es A LABORATORY MANUAL, Co
ld Spring Harbor Laborato
ry; biotinylated by the method detailed in p340-341, 1998. The 6B9 monoclonal antibody has 250 to 155th RT with no RT inhibitory activity.
Reacts with the th amino acid and recognizes the "palm" subdomain.

After the RT protein was coated on the ELISA plate in the same manner as described above, the unadsorbed portion was reduced to 25%
Blocking was performed with PBS containing Block Ace. Anti-RT
The solution containing the Fab molecule is appropriately diluted, 50 μl is added to a well, and reacted at room temperature for 1 hour, and PBS / Tween is added.
After washing with, 50 μl of 2 μg / ml biotinylated 7C4 monoclonal antibody or biotinylated 6B9 monoclonal antibody was added, and the mixture was reacted at room temperature for 1 hour, and then the wells were washed. 50 μl of alkaline phosphatase-labeled-streptavidin (5,000-fold diluted) was added to each well, reacted at room temperature for 1 hour, washed again, and 100 μl of pNPP (1 mg / ml) was added as a substrate solution. The color was developed, and the absorbance at 405 nm was measured. 63
A specific reaction was obtained by measuring the absorbance at 0 nm as a background and subtracting the measured value. In this operation,
R of biotinylated 7C4 and 6B9 monoclonal antibodies
Prior to the reaction with the T protein, an anti-RT Fab molecule was added to react with the epitope.
If the molecule recognizes the same or very near epitope as the biotinylated monoclonal antibody added later, it inhibits the binding of the biotinylated monoclonal antibody to the RT protein, resulting in reduced absorbance I do. When monoclonal antibodies recognizing different epitopes were used, there was no competition and no change in absorbance was observed. An anti-RT Fab molecule is reacted as a competing antibody, and then is biotinylated 7C4 monoclonal antibody.
When the reaction with the RT protein was examined, the binding of the biotinylated 7C4 monoclonal antibody was inhibited. Biotinylated 7C4
The concentration of the anti-RT 7C4 Fab molecule, which inhibits the binding between the monoclonal antibody and the RT protein by 50%, is 5.1 μg.
/ Ml. In addition, it was found that the reaction between the biotinylated 6B9 monoclonal antibody and the RT protein was not inhibited by the anti-RT Fab molecule, but recognized a different epitope.

[0033]

Industrial Applicability The polypeptide of the present invention can be used for HIV RT.
And inhibits its RT activity. Therefore, the recombinant vector expressing the polypeptide of the present invention can be used for intracellular immunity gene therapy for HIV-infected patients because it is expressed in the cell into which the gene has been introduced and exhibits an RT inhibitory activity.

[0034]

[Sequence List] <110> Zeon Corp <120> Gene encoding a polypeptide that inhibits the activity of human deficiency virus reverse transcriptase and its use <130> 2000-369 <160> 4 <210> SEQ ID NO: 1 <211> 636 <212> DNA <213> Mouse <400> 1 caaattgttc tcacccagtc tccagcaatc atgtctgcat ctctggggga ggagatcacc 60 ctaacctgca gtgccaattc gagtgtgaat tacatgcact ggtaccagca gaagtcaggc 120 tcttctccca aactcttaat ttatagcaca tccaacctgg cttctggagt cccttctcgc 180 ttcagtggca gtgggtctgg gaccttttat tctctcacaa tcagcagtgt ggaggctgaa 240 gatgctgccg attattactg ccagcagtgg agtagttatc ggacgttcgg tggaggcacc 300 aaactggaaa tcaaacgggc tgatgctgca ccaactgtat ccatcttccc accatccagt 360 gagcagttaa catctggagg tgcctcagtc gtgtgcttct tgaacaactt ctaccccaaa 420 gacatcaatg tcaagtggaa gattgatggc agtgaacgac aaaatggcgt cctgaacagt 480 tggactgatc aggacagcaa agacagcacc tacagcatga gcagcaccct cacgttgacc 540 aaggacgagt atgaacgaca taacagctat acctgtgagg ccactcacaa gacatcaact 600 tcacccattg tcaagagctt caa caggaat gagtgt 636

[0035] <210> SEQ ID NO: 2 <211> 427 <212> DNA <213> Mouse <400> 2 gvagnvagsr gygygysrva gnrgyargsr yssrcysaaa asrgyhthrh srasntyraa 60 mtaatrargg nthrrgysar ggtrvaaath rsrsrgygys rtyrthrasn tyrrasgyva 120 ysgyarghth rsrargasas naagnasnth rtyrgnmtsr srargsrgas thraamttyr 180 tyrcysaaar garggtyrsr tyrastyraa mtascystrg ygngythrsr vathrvasrs 240 raaysthrth rrrsrvatyr raargysraa aagnthrasn srmtvathrg ycysvaysgy 300 tyrhrgrvat hrvathrtra snsrgysrsr srgyvahsth rhraavagns rastyrthr 420 rsrsrthr rsrsrthr rsrsrthr rsrsrthr rsrsrthr rsrsrthr rsrsrthr rsrsrthr rsrsrthr rsrsrthr rsrsrthr rsrsrthr rsrsrthr rsrsrthr rsrsrthr rsrsrthr rsrsrthr rsrsrthr rsrsrthr rsrsrthr rsrsrthr rsrsrthr rsrsrthr rsrsrthr rsrrsrr trrsrrsrrsrrsrrrsrrsrrr trr

<210> SEQ ID NO: 3 <211> 212 <212> PRT <213> Mouse <400> 3 Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Leu Gly 1 5 10 15 Glu Glu Ile Thr Leu Thr Cys Ser Ala Asn Ser Ser Val Asn Tyr Met 20 25 30 His Trp Tyr Gln Gln Lys Ser Gly Ser Ser Pro Lys Leu Leu Ile Tyr 35 40 45 Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Phe Tyr Ser Leu Thr Ile Ser Ser Val Glu Ala Glu 65 70 75 80 Asp Ala Ala Asp Tyr Tyr Cys Gln Gln Trp Ser Ser Tyr Arg Thr Phe 85 90 95 Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Asp Ala Ala Pro Thr 100 105 110 Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly Gly Ala 115 120 125 Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile Asn Val 130 135 140 Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu Asn Ser 145 150 155 160 Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser Ser Thr 165 170 175 Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr Thr Cys 180 185 190 Glu Ala Thr His L ys Thr Ser Thr Ser Pro Ile Val Lys Ser Phe Asn 195 200 205 Arg Asn Glu Cys 210

<210> SEQ ID NO: 4 <211> 217 <212> PRT <213> Mouse <400> 4 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Ala Met Ala Trp Ile Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val 35 40 45 Ala Thr Ile Ser Ser Gly Gly Ser Tyr Thr Asn Tyr Pro Asp Gly Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Gln Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Arg Glu Tyr Ser Tyr Asp Tyr Ala Met Asp Cys Trp Gly Gln 100 105 110 Gly Thr Ser Val Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val 115 120 125 Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr 130 135 140 Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr 145 150 155 160 Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Thr Val Pro Ser 180 185 190 Ser Thr Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala His Pro Ala 195 200 205 Ser Ser Thr Lys Val Asp Lys Asn Ile 210 215

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12N 1/15 C12N 1/19 4H045 1/19 1/21 1/21 C12P 21/08 5/10 C12N 15 / 00 ZNAA 15/02 5/00 A // C12P 21/08 15/00 C (72) Inventor Yasuda Yasuda 1-2-1 Yoko, Kawasaki-ku, Kawasaki-shi, Kanagawa Japan Zeon Corporation General Development Center F Term (reference) 4B024 AA01 AA14 BA51 CA04 DA06 EA04 GA11 HA01 HA17 4B064 AG27 CA02 CA19 CC24 DA01 DA15 4B065 AA26X AA97Y AB01 BA02 CA24 CA25 CA44 CA46 4C084 AA13 CA53 CA56 MA52 MA66 NA14 ZB332 ZC552 4C086 AA033 BA10 CA05 DA76 EA29 EA53 FA74

Claims (7)

[Claims] 1. A DNA molecule encoding the amino acid sequence of SEQ ID NO: 1 or at least one of the following amino acid regions (1) to (3) in the DNA molecule:
DNA molecules mutated within the range of homology of not less than%. (1) The 24th to 33rd amino acid region of SEQ ID NO: 1 (2) The 49th to 55th amino acid region of SEQ ID NO: 1 (3) The 88th to 95th amino acid region of SEQ ID NO: 1 2. A DNA molecule encoding the amino acid sequence of SEQ ID NO: 2 or at least one of the following amino acid regions (4) to (6) in the DNA molecule:
DNA molecules mutated within the range of homology of not less than%. (4) 30th to 36th amino acid region described in SEQ ID NO: 2 (5) 50th to 66th amino acid region described in SEQ ID NO: 2 (6) 99th to 109th amino acid region described in SEQ ID NO: 2 3. The DNA according to claim 1 and / or 2.
A recombinant vector incorporating a molecule. 4. A therapeutic agent for human immunodeficiency virus infection, comprising the recombinant vector according to claim 3. 5. The therapeutic agent for human immunodeficiency virus infection according to claim 4, further comprising a liposome. 6. The amino acid sequence encoded by the DNA molecule according to claim 1, which comprises the Fab region of a monoclonal antibody reactive to the reverse transcriptase of human immunodeficiency virus, and encoded by the DNA molecule according to claim 1. And a polypeptide having the amino acid sequence of the present invention. (7) A transformant which expresses the polypeptide according to (6).