JP3095248B2 - Nucleic acid carrier - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a novel carrier for introducing a nucleic acid into a cell. More specifically, the present invention relates to toxin B for efficiently and safely introducing a nucleic acid into cells.
The present invention relates to a carrier containing a chain protein and a nucleic acid binding substance.

The present invention further comprises the above carrier and a nucleic acid,
The present invention relates to a regulator for promoting the introduction of a nucleic acid into a cell.

BACKGROUND ART With the rapid development of genetic engineering, various molecular biological techniques have been developed. Accordingly, remarkable progress has been made in the analysis of genetic information and the elucidation of gene functions, and many attempts have been made to return the results obtained therefrom to actual treatment sites. Among them, one of the most remarkable fields is the field of gene therapy. With the discovery and decoding of the causative genes in various hereditary diseases, gene therapy is evolving from the stage of basic experimentation to the stage of actual clinical application. For example, in the United States, by July 1994, 54 gene therapy protocols had been approved by the NIH Recombinant DNA Committee (RAC), congenital immunodeficiency disease, familial hypercholesterolemia,
Clinical trials have already been conducted on more than 200 patients for hereditary diseases such as cystic fibrosis and various types of cancer (Experimental Medicine Vol.12-No.3 1994 pages 303-307).
page).

As described above, gene therapy is attracting attention as a method for treating diseases from the root of the cell level. One of the major technical issues in clinical application is how to efficiently and safely transfer foreign genes to target cells. There is a problem of whether to introduce.

That is, in gene therapy, there is an addition gene therapy (Augmentation Gene Therapy) in which an abnormal (pathogenic) gene is left as it is and a new (normal) gene is added, and a replacement gene therapy (Replacement) in which an abnormal gene is replaced with a normal gene.
Gene Therapy), and any therapy requires a method for efficiently and safely introducing a normal gene into target cells.

In the early 1980s, attempts were made to apply physical techniques such as microinjection.However, gene transfer efficiency was low and stable transfer was not possible. Did not connect.

Subsequently, a recombinant virus (virus vector) was developed as a carrier for efficiently introducing a foreign gene into target cells, and clinical application of gene therapy became possible for the first time. At present, there are several types of viral vectors being considered for use in gene therapy, as described below. However, these methods have not been generalized because their production methods are very complicated and, at the same time, methods for ensuring their safety have not been established.

For example, a virus vector that has received the most attention as a virus vector that can be used for gene therapy is mouse leukemia virus (MoMLV: Moloney Murine Leukemia).
Virus), which utilizes the advantages of the propagation mode of the virus. A retrovirus is an RNA virus having an envelope, and enters a cell by binding of the envelope protein to a receptor on the host cell side. After entry, single-stranded viral RNA is converted to double-stranded DNA by reverse transcriptase and integrated into infected cell genomic DNA. However, for such integration to occur, the cells must be dividing and proliferating. Therefore, the most problematic point in practice is that the gene cannot be introduced into non-dividing cells. Therefore, gene repair of nerve cells, which is a problem in many congenital metabolic disorders, cannot be performed. In addition to nerve cells, hematopoietic stem cells, hepatocytes, muscle cells, and the like, which are the target cells for gene therapy, are usually in a quiescent phase, and therefore have low gene transfer efficiency. Cells taken out of the body are treated to promote division in order to increase gene transfer efficiency, but it is considered difficult to transfer genes into these cells in vivo.

In addition, adenovirus vectors have recently received attention as being capable of introducing genes into non-dividing cells. However, since the adenovirus vector does not incorporate a foreign gene into the genomic DNA of the target cell, the effect of gene transfer is lost in several weeks to several months. Therefore, gene transfer must be repeated frequently, which causes problems such as an increase in physical and physical burden on the patient and a decrease in gene transfer efficiency due to the production of anti-adenovirus antibodies. Currently, clinical trials of transbronchially administering adenovirus vectors to the lung to treat cystic fibrosis have been initiated, but an inflammatory response that appears to be due to the immunogenicity and cytotoxicity of adenovirus particles has occurred It is said that it was done.

Herpesvirus vectors are expected to be able to introduce foreign genes into nerve cells, but they have strong cytotoxicity and the genome size of the virus itself is 150 kb.
And development is not progressing because it is very large.

The HIV vector was developed as a vector that allows specific gene transfer to CD4-positive T lymphocytes due to the host characteristics of the virus itself (Shimada T., et al., J. Clin. I
nvest. 88, 1043 (1991)). One of the biggest drawbacks of HIV vectors is the potential for contamination with wild strains.

The wild type of the AAV (Adeno-Associated Virus) vector was found to be integrated at a specific position on chromosome 19, and was noted as a vector capable of targeting the gene integration position. However, recent studies indicate that recombinant AAV vectors have lost this property and that foreign genes integrate into non-specific locations on the chromosome.
Further, the AAV vector has a drawback that the size of a foreign gene that can be introduced is limited, and that only a gene of 5 kb or less can be packaged in the vector.

Many attempts have been made to perform gene therapy using various artificial gene transfer systems other than viral vectors. For example, gene-lipid complexes with positively charged lipids have been developed as non-viral carriers for gene therapy. However, it has been pointed out that these carriers have high cytotoxicity when used in large amounts (Bioconjugate Chem. 3, 32).
3-327 (1992), Proc. Natl. Acad. Sci. USA 89, 7934-79.
38 (1992), J. Biol. Chem. 269, 12918-12924 (1994),
Japanese Patent Publication No. 6-505980, Japanese Patent Publication No. 6-507158.

Also, by utilizing the fact that nucleic acids and their derivatives have a negative charge, an electrostatic complex is formed with a positively charged synthetic polymer derivative to deliver a gene to a target cell or cell. There have been many research reports that have attempted to make this possible (Bioconjugate Chem. 3,323-327 (1992)).
2), Proc. Natl. Acad. Sci. USA 89,7934-7938 (199
2), J. Biol. Chem. 269, 12918-12924 (1994), JP-A-6-505980, JP-A-6-507158). However, it has been previously pointed out that a synthetic polymer having a positive charge has high cytotoxicity when used alone (Bio-conjuga
te Chem. 1,149-153 (1990)). At the same time, when these synthetic polymer derivatives are administered to a living body, they are recognized as foreign substances, and there is a concern about the influence on the immune system such as anaphylactic shock.

On the other hand, trials using biologically-derived nucleoproteins which are considered to be hardly recognized as foreign substances are also being studied. Since a nucleoprotein has a property of specifically binding to a nucleic acid and a derivative thereof, there is a possibility that the nucleoprotein can be a vector for gene transfer by forming an electrostatic complex. There are examples of studies using histone proteins, which are nuclear proteins, and using them as carriers for plasmid DNA (Yasufumi Kaneda, et al., SCIENCE
243,375-378 (1899), Mirjam Breeuwer and David
S. Goldfarb, Cell 60,999-1008 (1990), JIAN CHEN, et
al., Human Gene Therapy 5, 429-435 (1994)). However, even in these examples, only a method of incorporating a gene into a cell has been studied, and it is not necessarily aimed at improving the gene expression efficiency in many cell types. Therefore, there is no example designed as a vector for efficiently introducing a gene into the cytoplasm in many cell types.

As described above, in gene therapy, a highly efficient and safe method for introducing a foreign gene into cells has been required.

DISCLOSURE OF THE INVENTION The present inventors have focused on the effect of the toxin protein to introduce the toxin protein into the cytoplasm by binding to a receptor present on the cell surface, and as a result of intensive studies, completed the present invention. .

The carrier of the present invention is characterized by containing a toxin B chain protein and a nucleic acid binding substance.

The present invention provides a novel carrier for introducing a nucleic acid into a cell.

The present invention further provides a regulator for promoting the introduction of a nucleic acid into a cell, comprising the carrier and the nucleic acid.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows the incorporation of oligonucleotides into HeLa cells.

FIG. 2 shows the incorporation of plasmid DNA into HeLa cells.

FIG. 3 shows incorporation of plasmid DNA containing the β-galactosidase gene into HeLa cells and gene expression.

 FIG. 4 shows the construction of the plasmid pHSX2.

 FIG. 5 shows the construction of plasmid pUC-Neo2.

 FIG. 6 shows the construction of the plasmid pHS-Neo2.

 FIG. 7 shows the construction of the plasmid pHS-Luci.

FIG. 8 shows the expression of the luciferase gene introduced using the carrier of the present invention by thermotherapy.

BEST MODE FOR CARRYING OUT THE INVENTION The configuration and preferred embodiments of the present invention will be described in detail below.

When the toxin protein is composed of two types of parts, an A chain that actually exerts toxicity in the cytoplasm and a B chain that has an action of introducing an A chain into a cell by binding to a receptor present on the cell surface. There are many. Generally, the B chain has almost no toxicity, and the portion responsible for the toxicity of the toxin molecule can be said to be the A chain. In addition, it is known that the A chain and the B chain often form a conjugate and do not exhibit toxicity when used alone. This indicates that the A chain can be introduced into cells only by forming a conjugate between the A chain and the B chain. The carrier of the present invention utilizes such a property that the B chain of the toxin protein introduces a high molecular weight substance into the cytoplasm.

The toxin B chain protein promotes the introduction of nucleic acids into cells by binding to sugar sialic acid present on the cell surface. Glucose sialic acid is widely present in many cells in a living body. Therefore, according to the carrier of the present invention, a nucleic acid can be introduced into various cells. Therefore, it is possible to introduce a nucleic acid into non-dividing cells such as nerve cells, and further into cells taken out of a living body. Further, the toxin B chain protein has almost no toxicity, and when used as a carrier together with a nucleic acid fusion substance, there is an advantage that the nucleic acid can be safely introduced into cells. Furthermore, the carrier of the present invention containing a toxin B chain protein and a nucleic acid binding substance has the advantage that it is easier to construct and use than conventional virus vectors and the like.

The toxin B chain protein that can be used as the carrier of the present invention is not particularly limited as long as it has a property of introducing a high-molecular-weight substance into the cytoplasm, but bacterial or plant proteins are preferable. For example, ricin or a related toxin such as abrin, diphtheria toxin, exotoxin, enterotoxin, cholera toxin, pertussis toxin,
B chain proteins of attenuated strains of tetanus toxin and botulinum toxin can be used.

As the toxin B chain protein, a protein obtained by a known method from naturally occurring protein can be used. The amino acid sequences and gene sequences of these proteins are known, and they can also be produced by established genetic engineering techniques based on these sequences. The toxin B chain protein may or may not have a sugar chain bound, if desired. Toxin B chain proteins produced by genetic recombination techniques are commercially available and some are readily available. For example, ricin B chain protein is commercially available from Vector and used in the examples of the present invention.

The toxin B chain protein further introduces a high molecular weight substance into the cytoplasm even if one or more amino acid residues in the amino acid sequence of the natural protein are mutated by substitution, deletion, insertion, or the like. Any material having properties can be used as a component of the carrier of the present invention.
The mutation may be naturally occurring or may have been subjected to genetic engineering techniques. Those skilled in the art will be able to easily produce such a mutant protein by conventional methods.

The carrier of the present invention further contains a nucleic acid binding substance. As the nucleic acid binding substance, a substance that can bind to a nucleic acid to be introduced into a cell in the state of coexistence or binding with a toxin B chain protein can be used. By binding a nucleic acid to such a nucleic acid binding substance, it is possible to efficiently introduce the nucleic acid into the cytoplasm. Examples of such nucleic acid binding substances include, but are not limited to, various cationic lipids, polyamino acid derivatives, histone proteins.

Examples of the cationic lipid include lipofectin (Lipofectin), lipofectase (LipofectAce),
Lipofectamine (LipofectAmine).

Examples of the polyamino acid derivative include poly-L-lysine, poly-D-lysine, poly-L-ornithine, poly-D-ornithine, poly-L-lysine-L-serine copolymer, poly-D-lysine-D -Serine copolymer, poly-L-ornithine-L-serine copolymer,
Poly-D-ornithine-D-serine copolymer, poly-L-lysine polyethylene glycol (PEG) block copolymer, poly-D-lysine PEG block copolymer, poly-L-ornithine PEG block copolymer, poly-D-ornithine PEG block copolymer ,
Poly-L-lysine-L-serine PEG block copolymer, poly-D-lysine-D-serine PEG block copolymer, poly-L-ornithine-L-serine PEG block copolymer, poly-D-ornithine-D-serine
PEG block copolymers. These polyamino acid derivatives are ε-carbobenzoxy-L-lysine-
N-carboxylic anhydride and benzyl-L-serine-N
A carboxylic acid anhydride can be synthesized by polymerizing a primary amine such as polyethylene oxide having an amino group at one terminal (molecular weight: 200 to 250,000) as an initiator. The molecular weight of the polyamino acid moiety in the polyethylene oxide-polyamino acid block copolymer can vary from 200 to 50,000 or more.

The carrier is not particularly limited, but contains the toxin B chain protein and the nucleic acid binding substance in a molar ratio of about 1: 1 to 1:10, preferably 1: 1 to 1: 5. Further, the toxin B chain protein and the nucleic acid binding substance may exist in a free state, or may be bound by an SS bond or the like.

The size, type, and the like of the nucleic acid that can be introduced into cells by the carrier of the present invention are not particularly limited. Examples of the type of the nucleic acid include linear double-stranded DNA, circular double-stranded DNA, oligonucleotide, and RNA. For example, by using the carrier of the present invention, a structural gene encoding a useful protein can be introduced into a cell, and the gene can be expressed. When a structural gene was introduced, the ninth example described later was used.
Shows very high gene expression. In addition, the expression of a specific gene can be controlled by introducing antisense. In addition, ribozymes, triplex,
It can also be used as a carrier for aptamers and the like. Further, the nucleic acid also includes a derivative such as a phosphothioate nucleotide in which a phosphate bond is replaced with a phosphothioate bond. Also, but not limited to, about 0.1-1000 μmol of carrier is used per 1 μmol of nucleic acid.

The present invention further provides a regulator comprising the above-mentioned carrier for promoting the introduction of a nucleic acid into cells.

The modulator of the present invention can be used for autologous gene therapy (ex vivo gene therapy) in which target cells are first taken out of a patient, the target gene is introduced, and then the cells are returned to the patient. Can also be used for gene therapy in which is administered directly to patients (in vivo gene therapy). In gene therapy, the abnormal (causal) gene is left as it is, and a new (normal) gene is added (Au
gmentation Gene Therapy) and a method of replacing an abnormal gene with a normal gene (Replacement Gene Therapy).

The administration of the preparation of the invention is not limited, but
It is generally performed parenterally, and can be preferably performed, for example, by injection administration. The amount used in the present invention varies depending on the method of use, purpose of use, and the like. kg, and more preferably about 1 μg / kg daily.
1 mg / kg.

Further, by utilizing the present invention, after introducing a plasmid designed to express various marker genes and therapeutic genes by temperature stimulation, by applying a temperature stimulus such as hyperthermia, the site in gene stimulation Specific gene expression becomes possible.

The carrier containing the toxin B chain protein and the nucleic acid binding substance of the present invention forms a complex with a desired nucleic acid, and enables the safe and efficient introduction of the nucleic acid into cells. Further, when a gene is introduced by the carrier of the present invention, gene expression occurs with extremely high efficiency.

Examples Hereinafter, the present invention will be described more specifically with reference to examples. Note that the present invention is not limited by these examples.

Example 1: 2.0 g of ε-carbobenzoxy-L-lysine-N-carboxylic anhydride (manufactured by Sigma) was dissolved in 30 ml of N, N-dimethylformamide (hereinafter, DMF) (manufactured by Wako Pure Chemical), Add 15 ml of chloroform (manufactured by Wako Pure Chemical Industries). One-end methoxy One-end amino group polyethylene oxide (molecular weight 500
(0: manufactured by NOF Corporation) 4.0 g was dissolved in 15 ml of chloroform, and the solution was added to an ε-carbobenzoxy-L-lysine-N-carboxylic anhydride solution. After 26 hours, the reaction mixture is
The polymer precipitated by dropping in 0 ml of diethyl ether was collected by filtration, washed with diethyl ether (manufactured by Wako Pure Chemical Industries, Ltd.), dried in vacuo, and deprotected with a hydrogen bromide acetic acid solution (manufactured by Wako Pure Chemical Industries). A poly-L-lysine PEG block copolymer (hereinafter, referred to as “PLP”) is obtained. Yield 5.0 g.

Example 2: 1.0 g of ε-carbobenzoxy-L-lysine-N-carboxylic anhydride (Sigma) and 1.0 g of benzyl-L-serine-N-carboxylic anhydride (Sigma) were added to DMF.
Dissolve in 30 ml (manufactured by Wako Pure Chemical Industries) and add 15 ml of chloroform (manufactured by Wako Pure Chemical Industries). One end methoxy One end amino group polyethylene oxide (molecular weight 5000: manufactured by NOF Corporation) 4.0 g
Is dissolved in 15 ml of chloroform and the solution is added to the solution of ε-carbobenzoxy-L-lysine-N-carboxylic anhydride and benzyl-L-serine-N-carboxylic anhydride. After 26 hours, the reaction mixture was dropped into 330 ml of diethyl ether, and the precipitated polymer was collected by filtration, washed with diethyl ether (manufactured by Wako Pure Chemical Industries, Ltd.), dried in vacuo, and treated with a hydrogen bromide acetic acid solution (Wako Pure Chemical Industries, Ltd.). Deprotection with a drug (Pharmaceutical) to obtain a poly-L-lysine-L-serine PEG block copolymer (hereinafter, referred to as “PLSP”). Yield 4.6 g.

Example 3: Undeprotected poly-L- obtained in Example 1
Lysine PEG block copolymer 30 mg, N-succimidyl 3- (2-pyridyldithio) propionate (hereinafter SP)
DP) (Pierce) 7 mg and triethanolamine (Wako Pure Chemical Industries) 5 μl are dissolved in DMF 0.5 ml and stirred at room temperature for 16 hours. The reaction product is dropped into 10 ml of distilled water, and the precipitated polymer is recovered by filtration and freeze-dried. After that, deprotection was performed with a hydrogen bromide acetic acid solution (manufactured by Wako Pure Chemical Industries, Ltd.), and an N-terminal S-introduced poly-L-lysine PEG block copolymer (hereinafter, referred to as a
"S-PLP"). Yield 18 mg.

Example 4: Undeprotected poly-L- obtained in Example 1
Lysine-L-serine PEG block copolymer 30 mg, SPD
P7mg, triethanolamine (Wako Pure Chemical Industries) 5μl DM
Dissolve in 0.5 ml of F and stir at room temperature for 16 hours. 10 ml of reaction
The polymer precipitated by dropping into distilled water is recovered by filtration and freeze-dried. Thereafter, deprotection was performed using a hydrogen bromide acetic acid solution (manufactured by Wako Pure Chemical Industries, Ltd.) and N-terminal S-introduced poly-L-lysine-
L-serine PEG block copolymer (hereinafter referred to as “S-PLS
P ”). Yield 16 mg.

Example 5: 30 mg of polyε-carbobenzoxy-L-lysine (manufactured by Sigma), 7 mg of SPDP, and 5 μl of triethanolamine (manufactured by Wako Pure Chemical Industries) are dissolved in 0.5 ml of DMF and stirred at room temperature for 16 hours. The reaction product is dropped into 10 ml of distilled water, and the precipitated polymer is recovered by filtration and freeze-dried. Thereafter, deprotection is performed with a hydrogen bromide acetic acid solution (manufactured by Wako Pure Chemical Industries, Ltd.) to obtain N-terminal S-introduced poly-L-lysine (hereinafter, referred to as “S-PLL”).
Yield 18 mg.

Example 6: 10 mg of the N-terminal S-introduced polyamino acid obtained in Examples 3, 4 and 5 and 10 mg of lysine B chain (manufactured by Vector) are dissolved in 2 ml of Hepes buffer and stirred at 4 ° C. for 16 hours. The product was subjected to gel filtration purification using Sephadex G-75 (manufactured by Pharmacia). The desired fraction was desalted and concentrated by ultrafiltration to obtain the desired product. Each ToxB-PLP
(14 mg / ml), ToxB-PLSP (15 mg / ml), ToxB-PLL (16 m
g / ml).

Example 7: HeLa cells (Dainippon Pharmaceutical) were seeded in a 24-well cell culture dish at 10 ⁵ cells / well and cultured in DMEM (Gibco) containing 10% FCS (Gibco) for 18 hours. Was done. Then, use the DNA 5 'end labeling kit (Takara) to prepare 32P
The labeled model oligonucleotide (20 mer) was added diluted with the unlabeled model oligonucleotide to a final concentration of 10 μmol / l. At this time, the various gene transfer carriers synthesized this time were mixed with the oligonucleotide at the same ratio by weight. The oligonucleotide is
DNA synthesizer (Type 392: manufactured by Apid Biosystems)
Was synthesized using As vectors, poly-L-lysine (PLL: manufactured by Sigma), PLP, PLSP, and lipofectamine (manufactured by Gibco) were used. The oligonucleotide uptake was calculated by measuring the radiation dose of the cells in each well 2 hours later. The result is shown in FIG.
The carrier synthesized this time showed much higher oligonucleotide uptake properties than lipofectamine, a cationic liposome that was conventionally used as a carrier, and it was revealed that it was effective as a carrier for various oligonucleotides. .

Example 8: HeLa cells (Dainippon Pharmaceutical) were seeded in a 24-well cell culture dish at 10 ⁵ cells / well and cultured in DMEM (Gibco) containing 10% FCS (Gibco) for 18 hours. Was done. After that, a model plasmid DNA (PGV-C: manufactured by Toyo Ink) partially labeled with ³² P with a nick translation label kit (manufactured by Amersham) was added to a final concentration of 0.1 μm.
It was diluted with unlabeled plasmid DNA to a concentration of ol / l and added. At this time, various gene transfer carriers were mixed with the plasmid DNA at the same ratio by weight. As the carrier, poly-L-lysine (PLL: manufactured by Sigma), PL
P, PLSP and Lipofectamine (manufactured by Gibco) were used.
The amount of gene uptake was calculated by measuring the radiation dose of cells in each well 2 hours later. The result is shown in FIG. The carrier synthesized this time showed much higher gene uptake compared to lipofectamine, a cationic liposome that has been conventionally used as a carrier, and was found to be effective as a carrier.

Example 9: HeLa cells were seeded at 4 × 10 ⁵ cells / well in a 6-well cell culture dish, and 10% FCS DMEM (manufactured by Gibco)
Culture was performed for 18 hours. Then, the model plasmid DN
A (pSV β-galactosidase: manufactured by Promega) was added to a concentration of 1 μg / well, and at that time, various carriers were mixed with the plasmid DNA in the same weight ratio. 48
After a time, the gene transfer efficiency was evaluated by staining the cells with X-gal (manufactured by Gibco). The gene transfer efficiency was compared with a degree of staining of 100 when lipofectamine (manufactured by Gibco) was used. The result is shown in FIG. The carrier synthesized this time showed much higher gene expression than the lipofectamine which is a cationic liposome conventionally used as a carrier, and was proved to be effective as a carrier.

Example 10: Construction of recombinant plasmid pHSX2 heat shock promoter
The general general method used to construct the prototype plasmid, pHSX2, is shown in FIG. Plasmid pBR322 (manufactured by Pharmacia) was digested with Sal I (manufactured by Takara Shuzo) and Hind III (manufactured by Takara Shuzo) to prepare a DNA fragment of about 3.74 kb containing the ampicillin resistance gene and the replication origin. Next, plasmid p1730R (manufactured by Stress Gen) containing the promoter of heat shock protein 70B (HSP70B) was digested with Xho I (manufactured by Takara Shuzo) and Hind III to prepare a DNA fragment of about 2.57 kb containing the HSP70B promoter region. did. These two DNAs
The plasmid pHSX1 was constructed by ligating the fragments with T4 DNA ligase (Takara Shuzo). This plasmid was used for Pvu II (Takara Shuzo) and BamHI (Takara Shuzo).
And 3 'powder of the HSP70B promoter region
An approximately 2.80 kb fragment containing 0.47 kb was prepared. Mung Bean Nuclease (Takara Shuzo) and Klenow
After blunting with Fragment (Takara Shuzo), the plasmid was religated with T4 DNA ligase to construct a plasmid pHSX2.

Example 11: Construction of recombinant plasmid pUC-Neo2 Prototype plasmid pUC-N containing neomycin resistance gene
The general overall method used to construct eo2 is shown in FIG. Plasmid pUC119 (Takara Shuzo) was cut with SmaI (Takara Shuzo) and then cut with BamHI to prepare a DNA fragment of about 3.15 kb containing an ampicillin resistance gene and a replication origin. Next, a plasmid containing the neomycin resistance gene
HXN2 (provided by Nippon Medical University) was digested with XhoI to prepare a DNA fragment of about 1.20 kb containing the neomycin resistance gene. The protruding end of this DNA fragment was blunted with Klenow Fragment, and then cut with BamHI. T2 these two DNA fragments
The DNA was ligated with DNA ligase to construct a plasmid pUC-Neo. This plasmid was digested with Sph I (Takara Shuzo), and the protruding ends were treated with Mung Bean Nuclease and Klenow Frag.
After blunting with S.ment, religated with T4 DNA
Plasmid pUC-Neo2 lacking the ATG codon at the phI site
Was built.

Example 12: Construction of recombinant plasmid pHS-Neo The general procedure used to construct the prototype plasmid pHS-Neo containing a heat shock promoter and a neomycin resistance gene is shown in FIG. The plasmid pHSX2 constructed in Example 10 was digested with EcoR I (Takara Shuzo) and Hind III to prepare a DNA fragment of about 2.77 kb. Next, the plasmid pUC-Neo2 constructed in Example 11 was digested with EcoRI and HindIII to prepare a DNA fragment of about 1.25 kb. These two D
The NA fragment was ligated with T4 DNA ligase to construct pHS-Neo.

Example 13: Construction of recombinant plasmid pHS-Luci The general method used to construct plasmid pHS-Luci with the luciferase gene ligated downstream of the heat shock promoter is shown in FIG. The plasmid pHS-Neo constructed in Example 12 was digested with Hind III and Sal I, and was cut to about 4.0.
1 kb DNA was prepared. Next, the plasmid pGV-P (manufactured by Nippon Gene) containing the luciferase gene was replaced with Hind II.
The fragment was cut with I and Sal I to prepare a DNA fragment of about 2.69 kb.
These two DNA fragments were ligated with T4 DNA ligase to construct a plasmid pHS-Luci.

Example 14: HeLa cells were seeded at 4 × 10 ⁵ cells / well in a 6-well cell culture dish, and 10% FCS DMEM (manufactured by Gibco)
In the medium, cultivation was performed at 37 ° C. for 18 hours at a carbon dioxide concentration of 5%. Thereafter, pHS-Luci was added to a concentration of 1 μg / well, and in that case, various carriers were added to the plasmid DNA.
And the same amount by weight. After 24 hours, a part of the sample was cultured at 42 ° C. for 3 hours, and then returned to the original environment and further cultured for 24 hours. Thereafter, the luciferase expressed in the cells was quantified using a luciferase assay system (Picagene: manufactured by Toyo Ink Co., Ltd.). FIG. 8 shows the result. Compared to culturing at 37 ° C, when cultivated at 42 ° C, the expression level of luciferase is higher.By introducing a therapeutic gene downstream of the heat shock promoter, gene expression can be regulated by thermotherapy. It was shown to be.

────────────────────────────────────────────────── ─── Continuation of the front page (56) References JP-A-6-303987 (JP, A) JP-A-6-503552 (JP, A) (58) Fields investigated (Int. Cl. ⁷ , DB name) C12N 15/87 CA (STN) REGISTRY (STN) WPI (DIALOG) BIOSIS (DIALOG) JICST file (JOIS)

Claims (2)

(57) [Claims] (1) Toxin B chain protein and poly-L-
A carrier for promoting introduction of a nucleic acid into a cell, comprising a lysine-L-serine PEG block copolymer or a poly-D-lysine-D-serine PEG block copolymer. 2. A toxin B chain protein and poly-L-lysine-L-serine PEG block copolymer or poly-D
-Lysine-D-serine PEG block copolymer is S
The carrier according to claim 1, wherein the carrier is bonded by an -S bond.