JPH09263599A - Intracellularly intruded polypeptide and its identification - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a polypeptide having a specific amino acid sequence and derived from leishmania protozoa, playing a part in the intracellular intrusion for protozoa and having activity to induce diseases in human beings, and useful for the diagnosis of infection with protozoa, vaccines, etc. SOLUTION: This polypeptide has an amino acid sequence of the formula, being a new intracellularly intruded polypeptide for protozoa having activity to induce diseases in human beings, and playing a part in the intracellular intrusion for protozoa. This polypeptide or the gene coding this polypeptide is useful for detecting protozoa and the diagnosis of infection therewith, vaccines for treating diseases involved in protozoa and for developing such vaccines. This polypeptide is obtained by a process wherein Promastigote-type leishmania protozoa is cultured in a medium at 27 deg.C for 3 days and then collected, and a genome DNA etc., is then extracted from the protozoa by a conventional means; using the genome DNA, a DNA library is prepared and then screened to obtain a DNA coding the aimed intracellularly intruded polypeptide, which is then integrated into a vector and expressed in host cells.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an intracellular invasion polypeptide of Leishmania parasites, and more particularly to a gene sequence of an intracellular invasion polypeptide and a method for identifying the gene sequence.

[0002]

BACKGROUND OF THE INVENTION Protozoa are unicellular protozoa belonging to the eukaryote, and perform all functions necessary for survival (contact, motility, metabolism, reproduction) in single cells. Protozoa parasitizing humans cause serious diseases in humans, but they are mainly classified into the following three types.

(1) Carnivorous insects: so-called amoeba, in which dysentery ameba and large intestine ameba are included.

(2) Flagellates: Protozoa with flagella such as Trypasoma, Trichomonas, Leishmania, and Meniflagellates.

(3) Spores: having a complicated life cycle,
Often two lodging types are involved, Toxoplasma,
There are falciparum malaria, three-day fever malaria, etc.

Among these, leishmaniasis, which is a disease caused by Leishmania parasites, is a protozoal disease in which the Leishmania protozoa is mediated by the blood-sucking insect, Drosophila melanogaster. This illness is widespread, from the southern part of the United States on the new continent to the northern part of Argentina along with Asia, Africa and the Mediterranean coast of the old continent. It is estimated that there are more than 12 million patients in the world and more than 400,000 diseases occur each year, and it is one of the six major designated diseases of the World Health Organization (WHO). The clinical manifestations of this disease are extremely diverse, and in most cases, the causative species name cannot be identified only from the condition. This is because typical ulcers that occur on the skin are also detected in the mucosal type and visceral type so-called color azalea at an early stage.

[0007] Leishmania protozoa is infected with an insect as a vector. First, it invades cells of the reticuloendothelial system such as skin, mucous membrane and spleen, and then repeatedly divides and proliferates inside the cell to destroy the cell. Is known to do. However, there are still many unclear points about the mechanism of invasion, which is the most important stage as the initial stage of infection, and it is a major obstacle to the development of therapeutic methods and vaccines.

[0008]

In view of the above problems, the present inventors have expressed a molecule involved in invasion of leishmaniasis, that is, expressed on Leishmania and have the ability to invade human cells. The purpose is to clarify the intracellular invasion polypeptide and its gene structure.
Moreover, it aims at providing the new method for elucidating the intracellular invasion polypeptide regarding protozoal disease. Furthermore, it is intended to utilize the above findings for the treatment of this disease and the development of a vaccine. In addition, it is also intended to be used for the development of disease therapeutic agents and disease diagnostic agents that utilize intracellular invasion polypeptides.

[0009]

[Means for Solving the Problems] In order to achieve the above-mentioned object, the present inventor has conducted extensive studies and as a result has found that a novel polypeptide used for invading human cells from Leishmania protozoa and a DNA encoding the same are used. Succeeded in finding and identifying. That is, we succeeded in identifying a Leishmania invasion molecule capable of specifically recognizing a specific cell and invading into the cell. Furthermore, the present inventor has conducted extensive studies on a method for efficiently and stably screening a polypeptide having the above-mentioned invasive activity of protozoa, and as a result, cuts Leishmania DNA with a restriction enzyme to obtain a vector. We have succeeded in developing a new method for screening bacteria that enter into cells and invading cells, and have found a means for creating a library of the bacteria. Further, the present inventor has a method for specifically diagnosing the disease using the above-mentioned Leishmania protozoan invasion molecule and a therapeutic agent thereof,
Furthermore, they succeeded in finding a treatment method.

That is, the present invention relates to an intracellular invasion polypeptide of a protozoa which causes a disease in humans, and an intracellular invasion polypeptide having at least the amino acid sequence set forth in SEQ ID NO: 1 in Sequence Listing, SEQ ID NO: in Sequence Listing. The present invention provides an intracellular invasion polypeptide having at least the amino acid sequence set forth in 2, and an intracellular invasion polypeptide having at least the amino acid sequence set forth in SEQ ID NO: 3 in the sequence listing.

Furthermore, the present invention relates to a polynucleotide encoding the above-mentioned intracellular invasion polypeptide, and provides an intracellular invasion polynucleotide having at least the nucleotide sequence set forth in SEQ ID NO: 4 in the Sequence Listing. is there.

The present invention also relates to a polypeptide which is a mutant of the above-mentioned polypeptide and has intracellular invasion property, and further relates to a polynucleotide encoding this polypeptide.

In the present invention, the amino acid sequence of the intracellular invading polypeptide of the protozoa is (1) genomic DNA and kinetoplast DNA are simultaneously prepared from the protozoa, and (2) the DNA is cleaved with a restriction enzyme, 3) Incorporating the cleaved DNA fragment into a plasmid, (4) introducing the integrated plasmid into E. coli to prepare a gene library, and (5) infecting mammalian cells with the E. coli gene library. Escherichia coli clones that invade the cells are isolated by (6) determining the nucleotide sequence of the fragment portion of the genomic DNA of the protozoa of the plasmid of the isolated E. coli clones, (7)
The present invention provides a method for deducing and identifying from the base sequence.

Furthermore, the present invention relates to an antibody against the above-mentioned polypeptide, and to a vaccine characterized by containing at least the polypeptide. In the present invention, the term "vaccine" as used herein means one that prevents the infection of protozoa by pre-administering a polypeptide whose activity has been inactivated.

The present invention also relates to a disease therapeutic agent in which the above-described polypeptide and a therapeutic substance are fused, and also relates to a disease diagnostic agent in which a diagnostic substance is fused.

Furthermore, the present invention relates to a recombinant vector containing a polynucleotide encoding the above-mentioned intracellular entry polypeptide, or a recombinant vector in which the vector is a plasmid vector.

Furthermore, the present invention relates to a microorganism containing the above recombinant vector.

In this specification and the drawings, IUPAC-I is used when it is expressed by abbreviations such as base and amino acid.
Abbreviations from UB or abbreviations customary in the art were used. In addition, X represents an arbitrary amino acid.

Amino acids are described by three letters or one letter.

(Nucleic Acid) DNA Deoxyribonucleic Acid A Adenine C Cytosine G Guanine T Thymine (Amino Acid) Ala (A) Alanine Arg (R) Arginine Asn (N) Asparagine Asp (D) Aspartic Acid Cys (C) Cysteine Gln (Q) Glutamine Glu (E) Glutamate Gly (G) Glycine His (H) Histidine Ile (I) Isoleucine Leu (L) Leucine Lys (K) Lysine Met (M) Methionine Phe (F) Phenylalanine Pro (P) Proline Ser (S) Serine Thr (T) Threonine Trp (W) Tryptophan Tyr (Y) Tyrosine Val (V) Valine

[0021]

BEST MODE FOR CARRYING OUT THE INVENTION The Leishmania protozoa can usually be grown by selecting an appropriate culture medium in an incubator at room temperature, and the Leishmania parasite is morphologically There are two types, an amastigoto type and a promastigote type, and it is known that when these protozoa are transferred to an appropriate medium, they all become promastigoto type and proliferate. The amastigote type is a mammalian intracellular parasite type that does not have flagella, whereas the promastigote type grows in the intestinal tract of the drosophila (insect) by attaching flagella to the lumen and dividing into two.

In the present invention, the Leishmania protozoa strain, Leishmania amazonensis, is cultivated in a schneider medium (insect culture medium, manufactured by Gibco), and promastigote
Collect the protozoa into a tortoise shape. The resulting protozoa are treated with proteinase K and sarcosyl, deproteinized, and then genomic DNA and kinetoplast DNA are simultaneously purified by a conventional method.

The present invention particularly uses the following method to analyze an invading gene from the obtained DNA.

That is, a DNA library is prepared by cutting DNA with an appropriate restriction enzyme, incorporating it into a plasmid, and expressing it in E. coli.
Furthermore, by infecting human cells with the gene library,
The invading clone is separated. More specifically, as a method for preparing a library, DNA is randomly cleaved with a restriction enzyme (EcoRI, Sau3AI, etc.), inserted into a plasmid (pBluescript IISK +, etc.), and then electroporated into non-pathogenic E. coli. Introduce. Mammalian cells (VA-13, Saos2) produced by attaching and growing the Leishmania parasite DNA library thus prepared to a culture dish.
Etc.) for 3 hours. After that, Escherichia coli in the culture solution that has not entered is mechanically washed out and sterilized with an antibiotic. The remaining cells are treated with Triton X-100, and only the cell membrane is broken to take out and separate E. coli invading the cells, which is further grown in LB medium. Further, if necessary, the Escherichia coli selected by the above method can be further reacted with the cultured cells by the same method. The Escherichia coli thus obtained is cultured overnight in an LB agar medium to prepare a clone strain. in this case,
To investigate whether E. coli itself has mutated and acquired cell invasion input, a plasmid purified and separated from E. coli having cell invasion input was introduced into non-pathogenic E. coli, and the new E. coli invasion input was transformed into plasmid. It is possible to make sure that it is controlled by.

Further, the obtained insert portion of the Leishmania protozoan DNA of the plasmid having the invasion force is, for example,
ABI Prism Dye Terminator can be used to determine the DNA base sequence using a 373S autosequencer or the like. As a result, it is strongly suggested that the nucleotide sequence common to any sequenced clones is a polynucleotide encoding the above-mentioned invading polypeptide. The polypeptide deduced from the base sequence further determined is the intracellular invasion polypeptide.
In the present invention, the 614 base pairs thus determined (the sequence common to 5 clones among arbitrary 11 clones) was designated as LYM / 1.

The structure of the present invention will be described in more detail below.

(Disease Protozoa Subject to the Present Invention) The Leishmania parasite strain of the present invention can be used without particular limitation as long as it infects humans.

[0028] For example, there are reports of parasitism in humans.
The following 11 types of mania have been reported with different pathogenicity to the host: Leishmania amazonensi
s, Leishmania mexicana, Leishmania venezuelensis,
Leishmania pifanoi, Leishmania garnhami, Leishmani
a bazilliensis, Leishmania guyanensis, Leishmaniap
anamensis, Leishmania lainsoni, Leishmania peruvi
ana, Leishmania chagasi. Although it is possible to use any of these protozoan strains, in the present invention, there are many cases mainly in South America, Leishmania amazone.
Although nsis was used, it is not limited to this.

Further, the protozoa are not limited to Leishmania, and protozoa for other diseases can be used.

(Target Cell or Protein Subject to the Present Invention) In the present invention, the type of the specific target cell is not particularly limited as long as it is a cell that can be infected by a protozoan. For example, human or various animal cells can be used. Furthermore, as the origin of various animal cells,
It can be obtained from the liver, kidneys, heart, brain, lungs, thorax, pancreas, etc. Further, in the present invention, the target cell does not need to be a normal cell, and can be suitably used as a cell derived from various diseases. As the disease, for example, cells derived from cancer, AIDS, and autoimmune diseases can be used without particular limitation as long as they have the above-described properties. If there is a difference in the invasion signal between humans or various animals, it is possible to identify the Leishmania invasion signal specific to each species. In particular, identification of a human-specific invasion signal is useful because it can be applied to the treatment of various diseases.

For example, it can be used as a vaccine by appropriately modifying the cell-invading polypeptide or immunizing it with a suitable adjuvant.

Further, by using an antibody of the intracellular invasion polypeptide, it is possible to specifically prevent the invasion of cells that cause the disease of Leishmania, so that the
It is extremely efficient, and there is no fear of side effects on cells other than the cells causing the disease, and safe treatment is possible.

Further, for example, by using an antibody of the intracellular invasion polypeptide, specific detection of the intracellular invasion polypeptide secreted in the blood is possible, so that it can be applied to diagnosis and the like. is there.

(Lyshmaniella parasite DNA library) The Leishmaniella parasite-derived DNA library according to the present invention is
The DNA prepared from the Leishmaniella protozoa genome and that obtained from kinetoplast DNA were incorporated into a plasmid and expressed in an appropriate bacterium.

Protozoa belong to the order Kinetoplasts in terms of taxonomy, and are characterized by having a kinetoplast called a motility substrate or motility nucleus together with one nucleus. In the developmental stage, the body transforms into various types, and the location of kinetoplasts changes accordingly, but kinetoplasts are always present,
Its size is 1-2 μm in diameter. Kinetoplasts have a mitochondrial-like structure, and network-formed kinetoplast DNA is 10 to 20% of whole cell DNA.
It is composed of about 39 kb maxi circle (5% of kinetoplast DNA) and about 1.5 kb mini-circle DNA (95% of kinetoplast DNA). Kinetoplast DNA differs in DNA between species and protozoa species.

The present inventor considered that in identifying the invasion signal of the protozoa, the invasion signal may differ between species and strains, which may be caused by kinetoplast DNA.

Therefore, it was considered that a method of preparing DNA containing not only genomic DNA but also kinetoplast DNA was preferable, and both DNAs were prepared simultaneously. In this case, a large amount of protozoa is required to prepare DNA containing kinetoplast DNA.

Therefore, rather than collecting the amastigotes by tissue culture, it was decided to grow the protozoa in the emastigotes that can be cultured and propagated only with the artificial medium.

In this medium, 10 ¹⁰ or more of Emastigote-type protozoa obtained by culturing at 25 to 28 ° C. for 3 to 4 hours were collected. Wash the protozoa with PBS (phosphate buffer),
SE buffer (0.15M NaCl, 0.1M ED)
TA, pH 8.0) was added and suspended.

To this, proteinase K and sarcosyl were added to break the cells. Here, kinetoplast DNA and genomic DNA are not separated by centrifugation but directly extracted with phenol and precipitated with ethanol to perform kinetoplast DNA.
And genomic DNA were prepared simultaneously. As a method for incorporating these, as described above, generally, a method for expressing a protein in bacteria such as Escherichia coli, that is, first, a DNA encoding the protein is inserted into a vector such as a plasmid, and then the vector is inserted into a bacterium. It is possible to use a method in which the transformant is introduced into E. coli to produce a transformant, and the protein is expressed in the transformant.

In the present invention, there are no particular restrictions on the bacteria that can be used as the host, and include bacteria that can be selected by those skilled in the art according to the several methods described below. That is, the bacterium according to the present invention may be any bacterium that can be transformed with an appropriate vector or the like so that at least the intracellular invasion polypeptide according to the present invention can be displayed near the surface. Further, it is sufficient that the transformed bacterium can grow by a commonly known means.

Therefore, various bacteria can be preferably used in the present invention. For example, gram-positive bacteria such as staphylococci and streptococci can be used. Particularly, Gram-negative bacteria can be preferably used in the present invention. For example,
Bacteria belonging to the family Enterobacteriaceae, Vibrio or Pasteurella can be used. Among the Gram-negative bacteria, Escherichia coli of the Enterobacteriaceae family can be preferably used as disclosed in detail in the examples of the present invention.

Further, the vector incorporating the DNA encoding the intracellular invasion polypeptide described above can be used without particular limitation as long as it can be displayed on the bacterial surface and is replication-retained in the host. . For example, there are plasmid vectors and virus vectors. As the plasmid vector, for example, pBR322 (Gene, 2,95,1977), pBR
325 (Gene, 4,121,1978, derivative of pBR322), pUC12 (Gen
e, 19,259,1982), pUC13 (Gene, 19,259,1982), pUC18 (Gen
e, 33,103,1985), pUC19 (Gene, 33,103,1985), pUC118 (Me
thod in Enzymology, 153,3,1987), pUC119 (Method in E
nzymology, 153,3,1987), BluescriptII (Nucleic Acids.
Res., 17,9494, 1989) and the like.
Either can be used. Examples of viral vectors include λgt10 (Huynh, TV, Young, RA and Dav
is, RW, DNA cloning, A Practical Approach, IRL Pre
ss, Oxford, 1,49,1985), λgt11 (Proc.Natl.Acad.Sci., U.
SA, 80,1194,1983) or λZAPII (Nucleic acids.Re
s., 17,9494,1989) and the like, but other vectors may be used as long as they can be propagated in an appropriate host.

It is more preferable that the plasmid vector is stable and easy to handle. The vector used in the examples of the present invention is pBluescript.

The method for incorporating the DNA into the vector can be carried out according to a known general method.

The obtained plasmid vector or viral vector containing the DNA encoding the intracellular invasion polypeptide can be introduced into bacteria such as Escherichia coli and transformed by a known general method (molecular cloning (Molecular cloning)). Cloning, Cold Spring Harbor Laborat
ory Press, 1.3-1.72,1989)). As described above, Escherichia coli as a preferred host is not particularly limited, and examples thereof include HB101, JM109, CJ236, BL-21, DH5α, etc., which can substantially display intracellular invasion polypeptide on the surface. Anything that can be screened can be used. In this example, the case of using DH5α was disclosed in detail.

As a method for transforming a host with a plasmid vector, a generally known method, for example, molecular cloning (Molecular Cloning, Cold Spring Harb
or Laboratory Press, 1.74-1.84, 1989), the electroporation method or the calcium chloride method can be preferably used.

According to the present invention, the obtained transformant further has at least one of the nucleotide sequences of the polynucleotide encoded by the polynucleotide without changing the amino acid sequence of the polynucleotide according to the degeneracy of the genetic code.
One base can be replaced with another type of base, and thus the polynucleotide of the present invention can also contain a converted base sequence by substitution based on the degeneracy of the genetic code. In this case, no mutation occurs in the amino acid sequence deduced from the base sequence obtained by the substitution.

A part of the structure of a polynucleotide can be mutated by natural mutation or artificial mutation without changing the main activity of the polypeptide deduced from the polynucleotide. is there. Of course, the polypeptide encoded by these mutated polynucleotides may have a mutation (mutant) in its amino acid sequence as compared with the original polypeptide.

Therefore, the polynucleotide of the present invention contains a nucleotide sequence encoding a polypeptide having a structure corresponding to a variant of all the above-mentioned proteins.

(Screening Based on Intracellular Invasion) Based on the intracellular invasion property of the DNA library derived from Leishmania parasites of the present invention, only the library which reacts with the target cell and invades into the target cell Can be separated and collected. Further, by determining the amino acid sequence of the target protein of the invaded library, it is possible to identify the invasion signal into the cell.

Therefore, the screening method based on intracellular invasion according to the present invention is not limited to the well-known screening based on the recognition of various interactions on the surface of the target cell, and further penetrates into the target cell. It discloses a novel method of screening by whether or not. Furthermore, the screening method based on intracellular invasion according to the present invention also discloses a method for separating the invaded and screened clone of the library from the inside of the cell again.

Also, the change in the degree of invasiveness depending on the difference in the intracellular invasiveness condition is considered to be a property specific to the target cell, and therefore, a highly selective screening is performed based on the setting of various invasion conditions. Is possible.

In the present invention, the target cells are not particularly limited as described above, and human or various animal cells can be used.

Hereinafter, one embodiment of the screening by intracellular invasion using the library according to the present invention will be described in order.

A DNA library derived from Leishmania parasite is prepared so that the number of clones becomes an appropriate number with respect to the number of cells. The cells are allowed to react with the library in a culture medium prepared so as not to contain antibiotics or serum, and the library is allowed to enter the cells. At this time, the invasion method is not particularly limited, and known conditions can be selected and used. For example, it is desirable that the cells are reacted in a confluent state as much as possible, since there is no reaction to a non-specific culture container or the like. The number of clones of the library used in the screening is not particularly limited, but is preferably 10 ⁴ or more when using a 10 cm Petri dish, for example.

The reaction time depends on the invasion ability of each clone of the library, but is preferably about 30 minutes or more and 4 hours or less. However, a short reaction time may be used to obtain only clones that have a strong invasion ability and can invade in a short time, and conversely, a long reaction time may be used to obtain clones that do not invade unless a long time is taken. For example, in this example, when human fibroblasts were used as the cells, they were reacted for about 3 hours.
Therefore, it is possible to change the invasion conditions depending on the purpose and to control the invasion ability to the target cells depending on the degree. As a method for removing non-specific adsorption of each clone to the cell surface, it is desirable to perform sufficient washing with, for example, a phosphate buffer.

The clone that has entered the cell can be extracted again from the cell. Various known means can be used. For example, it can be extracted from cells with a surfactant or the like. For example, according to an embodiment of the present invention, 1%
Triton-X100 can isolate clones invading cells without damaging E. coli. Therefore, if necessary, the above screening can be repeated. For example, after culturing the clone group in an LB medium containing an antibiotic, it is desirable to repeat the cell invasion test twice or more under the same conditions as above. This is because the number of clones having high invasion ability can be increased by repeating the process. However, if it is desired to obtain a clone that invades cells without being biased, it need not be repeated.

Further, it is possible to isolate a single clone expressing a peptide having an invasion signal of interest on the surface from a DNA library derived from Leishmania protozoa by an ordinary method, for example, the clone. It is possible to inoculate the cells invaded by the above method to an LB plate containing an antibiotic such as ampicillin so as to form a single clone, culture the cells, and then separate the colonies.

In order to identify the signal that has invaded into the cell, plasmid DNA is prepared from the invaded bacterium, and after synthesizing a primer of an appropriate length capable of sequencing the part encoding the target protein, for example, , Maxiam-Gilbert method (Methods in Enzymolo
gy, 65, 499-560, 1980), dideoxy method (Messing, J.et
al., Nuclear Acids Research, 9, 309, 1981), Taq cycle sequencing method using fluorescent dye (Biotechni
ques, 7, 494-499, 1989) etc. to determine the nucleotide sequence. Based on this base sequence, the amino acid sequence of the target protein can be determined.

(Disease Diagnosis Method, Disease Treatment Agent, and Disease Treatment Method Using Protozoa-Derived DNA Library or Intracellular Invasion Polypeptide) As described above, the protozoa-derived DN according to the present invention
Screening with the A library allows the identification of invasion signals specific to particular cells. In this case, by repeating the screening with the library according to the present invention, it becomes possible to identify an entry signal with extremely high selectivity. Therefore, the obtained intracellular invasion polypeptide of the present invention having the invasion signal can highly specifically invade the specific cell. In this case, the bacterium itself is not required, but at least the intracellular invading polypeptide derived from the protozoa is required. These intracellular invasion polypeptides have very specific properties in that they specifically invade only certain preselected cells.

Therefore, by applying the intracellular invasion polypeptide to a particular diseased cell, the disease is diagnosed, a therapeutic agent with extremely few side effects to the disease is developed, and a therapeutic method for the disease is further developed. It becomes possible.

Specifically, when the intracellular invasion polypeptide specifically invades the diseased cell, an appropriate therapeutic substance (therapeutic agent, therapeutic agent, etc.) is additionally introduced to induce the disease. It has a therapeutic effect in cells. For example, when a specific therapeutic substance is present near the cell surface of diseased cells, the therapeutic effect on the diseased cells is low, but when present within the cells, the therapeutic effect is effective. When it is exerted, the therapeutic substance is brought into the diseased cells by adding the therapeutic substance to the intracellular invasion polypeptide, and the therapeutic effect can be exerted on the diseased cells.

In the present invention, the therapeutic substance is not particularly limited, and various drugs, a composition containing the therapeutic substance, or a cytotoxic substance can be used depending on the type of disease. For example, those based on proteins such as physiologically active protein substances, cytokines, growth factors, or DNAs such as antisenses that suppress the symptoms of diseases are possible. Furthermore, it is presumed that immunosuppressive therapeutic agents (immunological diseases, immunosuppressive therapeutic agents at the time of organ transplantation), for example, epidermal growth factor receptor (EFG) plays an important role in breast cancer or ovarian cancer. Tumor-specific receptors for existing cancer cells can be used.

As described above, the diseased cells are not particularly limited, and in the case where the normal cell and the diseased cell of a particular cell have different intracellular invasion signals, the intracellular cell specific to the diseased cell is different. By using an invasion signal, it becomes possible to invade only the diseased cells. For example, when the disease is cancer (malignant tumor, lung cancer, leukemia, colon cancer, ovarian cancer, breast cancer, pancreatic cancer, etc.), it is possible to identify a target protein (invasion signal) that invades only cancerous cells. This is possible by using the disclosed library according to the present invention, and thus enables specific invasion into cancer cells only.
Furthermore, autoimmune diseases (rheumatism, multiple sclerosis,
It is also applicable to systemic lupus erythematosus), allergies, AIDS, hepatitis (B type, C type), and other organ transplant cells.

The means for fusing the desired disease cell therapeutic substance to the intracellular invading polypeptide derived from protozoa is not particularly limited. For example, in advance, a DNA encoding the specific therapeutic agent for disease cells or a derivative thereof is added to the DNA encoding the intracellular invasion polypeptide,
It can be prepared by appropriately cleaving and separating from the bacterium expressing the intracellular invasion polypeptide (after growth if necessary). Alternatively, as one of preferred embodiments, after the screening, if necessary, the intracellular invasion polypeptide or the protein containing the intracellular invasion polypeptide is isolated after the intracellular invasion polypeptide is separated (after the proliferation if necessary). It can be prepared by fusing the required therapeutic substance by various fusing methods. At this time, means used in various conventional drug delivery systems can be suitably selected.

Therefore, a disease which enables the treatment at the cellular level with less side effects by using the above-obtained intracellular invasion polypeptide having an appropriately selected and bound therapeutic substance as a therapeutic agent Treatments are disclosed.

Furthermore, as described above, the screening method according to the present invention based on the specific binding to the specific production substances (proteins etc.) derived from the various diseases mentioned above (in the case of using the protozoan-derived DNA library) And both with the intracellular entry polypeptide)
The development of a simple and highly specific diagnostic method for the disease is disclosed. As an actual diagnostic method, an assay method based on various known methods, for example, an immunoassay method or the like that can quantitatively determine a protein or an antibody specifically produced by a disease according to the method described above is selected. It is possible.

[0069]

EXAMPLES The present invention will be specifically described below based on examples, but the present invention is not limited to the following examples as long as the gist thereof is not exceeded.

(Example 1) Leishmania DNA Library- Promasteigototype Leishmania protozoa were cultured in Schneider medium (insect cell medium, Gibco) at 27 ° C for 3 days, and 10 ¹⁰ protozoa were collected. Add this to phosphate buffer (PB
After washing twice with S), the cells are suspended in an SE buffer solution (0.15M NaOH, 0.1M EDTA pH 8.0) in approximately the same amount as the collected protozoa and stored. Treated with sarcosin (Sodium sarkosinate) and proteinase K at 60 ℃ for 3 hours,
-One extraction with chloroform and one extraction with chloroform were performed to remove protein components. By performing ethanol precipitation, the genome
DNA and kinetoplast DNA were prepared simultaneously.

The obtained Leishmania DNA was digested with the restriction enzyme Ec.
It was cleaved with oR I and Sau 3AI to obtain a DNA fragment.

Plasmid vector-, pBluescripr IISK +
(Stratagene) was cut with EcoR I and BamHI, and NuSi
It was recovered with eve gel (Takara).

The vector after digestion with the enzyme and the DNA fragment were ligated with a DNA ligation kit (Takara). This was introduced into Escherichia coli (DH5α) by the electroporation method and cultured at 37 ° C. for 3 hours to prepare a Leishmania DNA library. This library was plated on an LB plate containing ampicillin (100 μg / ml).
I started 30 clones were randomly extracted from each clone, and DNA was purified from each clone and electrophoresed on an agar plate to confirm that the plasmid was introduced into E. coli.

(Example 2) Mammalian cell invasion experiment of non-pathogenic E. coli In order to identify signals invading human cells,
The following intracellular invasion experiments were carried out by culturing the Schumania DNA library with cells.

(1) Preparation of cells VA13 (human fibroblasts) was cultured in a DME culture medium containing 10% fetal calf serum until confluent in a 10 cm Petri dish. Then, the cells were treated with phosphate buffer (PB
Washed 3 times with S).

(2) Intracellular invasion experiment DME culture medium containing no serum or antibiotics (DME
(−)) After adding the Leishmania DNA library prepared in Example 1 to 10 ml, the culture solution was added to the petri dish in which the cells were cultured in (1), and the mixture was added for 3 hours at 37 ° C. in an incubator. By culturing, E. coli was allowed to enter the cells. This was washed 8 times with PBS to remove intracellular non-invasive clones.

(3) Death of intracellular non-invading clone Gentamicin (50 μg / m 2) was added to DME (-) culture medium.
After 1) was added, the culture solution was added to the dish prepared in (2) and incubated for 2 hours to kill cells to which Escherichia coli had not entered. After that, the cells invading the clone were washed 10 times with PBS.

(4) Extraction of invading clones 2 ml of 1% Triton-X100 was added to the petri dish prepared in (3).
Aqueous solution was added to lyse the cells for 10 minutes at 37 ° C.
To this, 10 ml of PBS was added and washed. Then 60
The invading E. coli was recovered by centrifugation at 00 rpm for 5 minutes. 5 ml of LB medium containing ampicillin (LB / Amp) was added to the pellets of the obtained ESPEL clones to give 1
Incubated for 2 hours.

(5) Extraction of plasmid Plasmids can be extracted by using a commercially available plasmid extraction kit (Q
IAGEN) according to the procedure described.

(6) Electroporation 10 ⁴ electrocompetent cells DH5α / 1 ml
The extracted plasmid (10 ng) was added to the culture solution containing the concentration of 0.2 c with BIO-RAD Gene Pulser.
m cuvette, 2500 V, 40 μF, 200 ohm. Immediately after the end of the pulse, 1 ml of S
The OC solution was added. After culturing for 1 hour, add 9 LB / Amp
ml was added and the cells were cultured for 12 hours.

(7) Intracellular invasion experiment 500 μl of Escherichia coli obtained in (6) was invaded into VA13 (human fibroblast) in the same manner as in (2), and (3)
From the above, an intracellular invading clone was obtained according to the method described in (4).

(8) Plasmid extraction and DNA sequencing The clones obtained in (7) above were plated on LB / Amp plates, 30 clones were randomly selected, and the plasmids were prepared in the same manner as in (5) above. Extracted. This is used as a primer for the primer M13-20 (5'-GTAAAACGACGGCCAG) near the vicinity of the target protein.
T-3 ') or reverse primer (5'-AACAG
It was determined by the Taq cycle sequencing method using a DNA sequencer 373A manufactured by Perkin Elmer Co., Ltd.

As a result of analyzing the obtained DNA sequence, as shown in FIG. 1, invasion clones having different lengths but having a common DNA sequence were obtained. Nos. 6, 11, and 13 were the same clones of 614 bp. Also, No. 19, No. 15, and No. 20 are
It was found to be a clone with a region that partially overlaps with No. 6.

Therefore, 614 bp common to Nos. 6, 11, and 13
It can be seen that the clone is required for effective intrusion. Here, this clone was named LYM / 1 and deposited on March 27, 1996 at the Institute of Biotechnology, Institute of Biotechnology (FERM P-15530).

The polypeptide encoded by this DNA is shown in FIG. The first line in FIG. 4 is the nucleotide sequence of the DNA, and the number above it indicates the position from the 5'end of the DNA. The second line shows the amino acid sequence of the polypeptide encoded by the DNA when read from the 5'end. Line 3
The fourth line shows the amino acid sequence of the polypeptide when read from the second G and the third A of the DNA, respectively. The asterisk (*) corresponds to the stop codon.

[0086] (Example 3) deli - Shonmyu - 614Bp black Tanto creation Figure 2, wherein - down deli - Shonmyu - tanto kilo sequence for deletion kit (Takara Co.)
Was prepared by using the above, and an invasion test against VA13 was performed for each clone. As a result, the 5'-N terminal side 61-bp deletion significantly reduced the invasion input, and the 3'-C terminal side had a significantly reduced invasion input due to the 283bp deletion. It was presumed to be present (Fig. 2). From these results, it was found that among the polypeptides encoded by the 614 bp DNA, there are three that can be considered as intracellular invasion polypeptides. The amino acid sequences thereof are shown in SEQ ID NOs: 1, 2, and 3 of the sequence listing.

(Example 4) Northern of mRNA reacting with LYM / 1 DNA invading Leishmania
Analysis by Blot (1) Northern blot analysis of mRNA expression level in Leishmania was performed using a polynucleotide of LYM / 1 DNA as a probe.

As the probe, (4) of (Example 1)
Using the recombinant obtained in step 1, plasmid DNA was prepared from E. coli DH-5α by QIAwell Plasmid P
Urification System (QIAGEN
Co., Ltd.). Use this plasmid with the restriction enzyme BamH 1
After treatment with EcoR I (Takara), separation was performed by 2% agarose gel electrophoresis, and a 0.64 kbp probe was attached to QIA Qui.
ck GEL Extraction Kit (QIA
GEN).

This was labeled with ³² P using a multiprime DNA labeling system (Amersham) and used as a probe.

MRNA (1) extracted from Leishmania
μg and 2 μg) were blotted on a positively charged nylon filter (Amersham).

As a prehybridization solution,
5xSSC (NaCl 0.15M, sodium citrate (pH 7.0)
0.015M), 50% formamide, 1 x denhardt
(Bovine serum albumin (Fraction V) 0.2%, polyvinylpyrrolidone 0.2%, Ficoll400 0.2%) solution, 0.1% SD
S, 200 μg / ml salmon spar DNA was used.

The filter was incubated with the prehybridization solution at 42 ° C. for 3 hours, followed by the hybridization solution (10%) to which the labeled probe was added.
(Pre-hybridization solution containing dextran sulfate) at 42 ° C. for 16 hours to carry out hybridization.

The filter is 2 × SSC, 0.1% SDS
Wash 4 times at 42 ° C for 15 minutes, then 1xSSC,
Washed once with 0.1% SDS for 30 minutes.

Autoradiography is based on Kodal X
Performed at -70 ° C using AR-5 film.

As a result of autoradiography, as shown in FIG. 3, LYM / 1 DNA showed mRN of about 0.5 kb and 1.2 kb.
Reacted with A, the expression level was dependent on the applied sample amount.

[0096]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 71 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Gly Lys Asn Glu Cys Arg Asn Pro Val His Asn Phe Pro Glu Asn 5 10 15 Gly Glu Phe Arg Leu Glu Arg Trp Lys Leu Gly Val Gly Val Lys 20 25 30 Tyr Ala Val Val Gly Thr Cys Gly Phe Gly Leu Leu Ile Tyr Val 35 40 45 Gly Leu Gly Arg Cys Phe Tyr Met Leu Ile Arg Phe Leu Phe Tyr 50 55 60 Phe Ile Ser Phe Leu Tyr Phe Gly Phe Thr Phe 65 70 SEQ ID NO: 2 Sequence length: 28 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Gly Lys Met Ser Ala Glu Thr Pro Phe Ile Ile Ser Arg Lys Met 5 10 15 Gly Asn Phe Gly Ser Asn Gly Gly Asn Trp Gly Leu Val 20 25 SEQ ID NO: 3 Sequence Length: 100 Sequence Type: Amino Acid Topology: Linear Sequence Type: Peptide Sequence Phe Pro Gly Lys Trp Gly Ile Ser Ala Arg Thr Val Glu Thr Gly 5 10 15 Gly Trp Cys Lys Ile Arg Gly Gly Gly Tyr Val Trp Val Trp Ser 20 25 30 Phe Asp Leu Cys Gly Ser Arg Ala Leu Phe Leu Tyr Val Asn Ser 35 40 45 Phe Ser Val Leu Phe Tyr Phe Phe Phe Ile Phe Trp Ile Tyr Phe 50 55 60 Leu Val Tyr Leu Ala Val Asp Arg Tyr Val Gly Arg Leu Tyr Asp 65 70 75 Leu Ala Leu Leu Cys Ala Phe Val Ile Ser Cys Leu Cys Gly Leu 80 85 90 Ile Leu Leu Asp Val Val Val Ala Leu Met 95 100 SEQ ID NO: 4 Sequence length: 614 Sequence type: Number of nucleic acid chains : 2-stranded topology: linear sequence type: DNA sequence GGGAAAAATGAGTGCAGAAACCCCGTTCATAATTTCCCGGAAAAT 45 GGGGAATTTCGGCTCGAACGGTGGAAACTGGGGGTTGGTGTAAAA 90 TACGCGGTGGTGGGTACGTGTGGGTTTGGTCTTTTGATTTATGTG 135 GGTCTCGGGCGCTGTTTTTATATGTTAATTCGTTTTCTGTTTTAT 180 TTTATTTCTTTTTTATATTTTGGATTTACTTTTTAGTTTACTTGG 225 CTGTTGATCGATATGTGGGTAGGTTATATGATTTGGCGTTACTGT 270 GCGCTTTTGTTATTAGTTGTCTGTGTGGGTTAATTTTATTGGATG 315 TGGTCGTGGCGCTTATGTGATGTTCCTTTTGGATATGATATTTCT 360 AATATAAAAGGTAATTGATTAGGTAATAACGGTGGTGGGTAATAG 405 GCGTTGTTGTAATGTGCCGATAACGTTAAGTTTTTGTAAATAATT 450 GATGTTACTTATACGCAATTAATATTATGTTTAACGTTT GTATGG 495 GCACGTGATTTTGTACGGTGCTTGGTTAGTGTGTTACTATATTGA 540 TGGGTTTAGATTTCTTTTTGATGTTAATGATTGTTGGTAGTGGGT 585 AGTAACGTGGATGTAGTTTGTGTAGGCTG 614

[Brief description of drawings]

FIG. 1 is a schematic diagram of cell-invading clones obtained from a Leishmania DNA library according to the present invention. Numbers are clone names and parentheses are the number of DNA bases.

FIG. 2 shows the delivery of LYM / 1 cell entry clone according to the present invention.
FIG. 6 is a diagram showing the length of DNA of Schomutant and the cell invasion input to VA13 cells by the number of cell invasion colonies.

FIG. 3 is a diagram showing the analysis results of Northern blot for Ryushimania mRNA using DNA of LYM / 1 cell-invading clone according to the present invention. .

[Fig. 4] LY, which is a Leishmania protozoa DNA according to the present invention
FIG. 3 is a diagram showing the nucleotide sequence of M / 1 and the polypeptide encoded by the DNA.

Continuation of front page (51) Int.Cl. ⁶ Identification code Office reference number FI Technical display location C12Q 1/02 9282-4B C12N 15/00 ZNAA

Claims (15)

[Claims] 1. A protozoan intracellular invasion polypeptide that causes disease in humans. 2. An intracellular invasion polypeptide having at least the amino acid sequence set forth in SEQ ID NO: 1 in the Sequence Listing. 3. An intracellular invasion polypeptide having at least the amino acid sequence set forth in SEQ ID NO: 2 in the Sequence Listing. 4. An intracellular invasion polypeptide having at least the amino acid sequence set forth in SEQ ID NO: 3 in the Sequence Listing. 5. A polynucleotide encoding the intracellular invasion polypeptide according to any one of claims 1 to 4. 6. An intracellular invasion polynucleotide having at least the nucleotide sequence set forth in SEQ ID NO: 4 in Sequence Listing. 7. A polypeptide which is a variant of the polypeptide according to any one of claims 2 to 4 and has intracellular invasion property. 8. A polynucleotide encoding the polypeptide according to claim 7. 9. The amino acid sequence of the intracellular invading polypeptide of the protozoa is (1) genomic DNA from the protozoa and kinetoplast DN.
A is prepared at the same time, (2) cut the DNA with a restriction enzyme,
(3) Incorporating the cleaved DNA fragment into a plasmid, (4) Introducing the integrated plasmid into Escherichia coli to prepare a gene library, and (5) Infecting mammalian cells with the E. coli gene library. (6) the genomic DNA of the protozoa of the plasmid of the isolated E. coli clones.
(7) A method of determining the base sequence of the fragment portion, and (7) deducing and specifying from the base sequence. 10. An antibody against the polypeptide according to any one of claims 1 to 4. 11. A vaccine comprising at least the polypeptide according to any one of claims 1 to 4. 12. A disease therapeutic agent comprising the polypeptide according to any one of claims 1 to 4 and 7 and a therapeutic substance fused together. 13. A disease diagnostic agent comprising the polypeptide according to any one of claims 1 to 4 and 7 fused with a diagnostic substance. 14. A recombinant vector comprising the polynucleotide according to claim 8. 15. A microorganism containing the recombinant vector according to claim 14.