JP2004350502A - Partial histone tail type immunoporter - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a system having high nucleic acid transport efficiency in a recombination immunogene method. <P>SOLUTION: A fused protein for transporting nucleic acid into a cell comprises a single strand antibody for binding to the nucleic acid and a partial histone polypeptide for binding to the nucleic acid. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

【図面の簡単な説明】
【図１】本発明の一実施例におけるヒト型一本鎖抗体ＣａＣをコードする組換えＤＮＡの塩基配列（配列番号９２）とＣａＣのアミノ酸配列（配列番号９３）を示す図である。
【図２】本発明の一実施例における部分ヒストンＨ１テイル型イムノポーターをコードする組換えＤＮＡの塩基配列（配列番号９４）とイムノポーターのアミノ酸配列（配列番号９５）を示す図である。
【図３】本発明の一実施例における２３アミノ酸残基ヒストンＨ１テイル型イムノポーター遺伝子をコードする組換えＤＮＡの塩基配列（配列番号９６）とイムノポーターのアミノ酸配列（配列番号９７）を示す図である。
【図４】本発明の一実施例における２８アミノ酸残基ヒストンＨ１テイル型イムノポーター遺伝子をコードする組換えＤＮＡの塩基配列（配列番号９８）とイムノポーターのアミノ酸配列（配列番号９９）を示す図である。
【図５】本発明の一実施例における部分ヒストンＨ１テイル型イムノポーターを用いた遺伝子導入実験の結果を示すグラフである。ｙ軸は、単位量あたりのルシフェラーゼ活性測定値を示す。をＨ３Ｌ−の後の数字はゲル濾過におけるピーク分画を表し、第１の分画では遺伝子導入が観察されていないが、この分画は処理中に幾分か分解された分画であると思われる。
【図６】本発明の一実施例における２３アミノ酸残基及び２８アミノ酸残基ヒストンＨ１テイル型イムノポーターを用いた遺伝子導入実験の結果を示すグラフである。ｙ軸は、図５と同じ単位量あたりのルシフェラーゼ活性測定値を示す。本図でも、図５と同様、最後の数字はゲル濾過におけるピーク分画を表す。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a fusion protein for transporting a nucleic acid into a cell, a method for producing the same, and a method for transporting a nucleic acid using the same.
[0002]
[Prior art]
As a method for transporting a nucleic acid into a cell, a calcium phosphate method, electroporation, infection with a virus such as a retrovirus or an adenovirus, lipofection, cell fusion, and the like are known. Recently, a method called a recombinant immunogene method using a recombinant immunoporter as a carrier for nucleic acid transport has been developed (for example, see Patent Document 1).
[0003]
The recombinant immunoporter is a single-chain antibody having a charged peptide tail. In particular, a fusion protein of a single-chain antibody against a cell surface antigen and a peptide having nucleic acid binding ability is effective. In gene transfer, a recombinant immunoporter to which a nucleic acid to be transferred into a cell is bound is administered to a cell having the surface antigen. The antibody portion binds to its surface antigen, and the cell takes up the recombinant immunoporter together with the bound nucleic acid into the cell by endocytosis. In this way, the target nucleic acid can be transported into cells using the recombinant immunoporter.
[0004]
[Patent Document 1]
JP 2001-333780 A
[0005]
[Problems to be solved by the invention]
An object of the present invention is to provide a system having high nucleic acid transport efficiency in a recombinant immunogene method.
[0006]
[Means for Solving the Problems]
The fusion protein of the main invention of the present invention for achieving the above object is a fusion protein for transporting a nucleic acid into a cell, and a single-chain antibody against the surface antigen of the cell, for binding to the nucleic acid. Includes partial histone polypeptides. Here, the partial histone polypeptide refers to a polypeptide that is a part of a histone protein. It is particularly preferable that the histone is H1, but since the histone is essentially a DNA binding protein, the partial histone polypeptide for binding to the nucleic acid is not limited to the histone H1, but may be derived from any histone.
[0007]
Further, the fusion protein of the present invention may be characterized in that the partial histone polypeptide is any one of SEQ ID NOS: 1 to 3.
[0008]
The fusion protein of the present invention may be characterized in that the partial histone polypeptide is a partial sequence of SEQ ID NO: 1.
[0009]
Further, the fusion protein of the present invention may be characterized in that the single-chain antibody is a human-type single-chain antibody.
[0010]
In any one of the above fusion proteins, one or several amino acid residues are deleted, substituted, or added, wherein the partial histone polypeptide binds to a nucleic acid, and the single-chain antibody is It may be a fusion protein that specifically binds to a cell surface antigen and transports the nucleic acid into a cell.
[0011]
Further, the method for producing a fusion protein of the present invention is a method for producing a fusion protein for transporting a nucleic acid into a cell, using mRNA isolated from a hybridoma capable of producing a monoclonal antibody against a cell surface antigen. A step of preparing a cDNA encoding a single-chain antibody against the surface antigen; a step of adding a cDNA encoding a partial histone polypeptide to the single-chain antibody cDNA to prepare a fusion cDNA; Expressing and isolating a fusion protein containing the single-chain antibody and the partial histone polypeptide.
[0012]
Further, the nucleic acid delivery method according to the present invention is a nucleic acid delivery method for delivering a nucleic acid into a cell, wherein the nucleic acid-protein mixture is prepared by mixing the nucleic acid with any one of the above fusion proteins, Administering a nucleic acid-protein mixture to said cells.
[0013]
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, embodiments of the present invention will be described in detail with reference to examples.
In addition, J. Sambrook, E .; F. Fritsch & T. Maniatis (Ed.), Molecular cloning, a laboratory manual (2nd edition), Cold Spring Harbor Press, Cold Spring Harbor, New York (1989); M. Ausubel, R.A. Brent, R .; E. FIG. Kingston, D.S. D. Moore, J .; G. FIG. See Seidman, J .; A. Smith, K.M. Struhl (Ed.), Current Protocols in Molecular Biology, John Wiley & Sons Ltd. Such standard protocol collections are naturally referred to by those skilled in the art, and the methods described therein can be easily implemented by those skilled in the art. Therefore, when using the methods described in these standard protocols, or when modifying or modifying the methods, the embodiments and examples in this specification are not particularly described in detail. In addition, when a commercially available reagent kit or measuring device is used, those skilled in the art usually use the protocol attached thereto, and unless otherwise described, use the attached protocol.
[0014]
Further, the objects, features, advantages, and ideas of the present invention will be apparent to those skilled in the art from the description of this specification, and those skilled in the art will be able to easily reproduce the present invention from the description in this specification. it can. The embodiments and specific examples of the invention described below show preferred embodiments of the present invention, and are shown for illustration or description. There is no limitation. It will be apparent to those skilled in the art that various modifications and variations can be made based on the description in the present specification within the spirit and scope of the present invention disclosed herein.
[0015]
The fusion protein according to the present invention, as a form of a recombinant immunoporter, constitutes a fusion protein for transporting a nucleic acid into a cell, a single-chain antibody against a cell surface antigen, and a peptide for binding to the nucleic acid. Includes partial histone polypeptide as a tail. When a nucleic acid is mixed with the fusion protein, the nucleic acid binds to a part of the partial histone polypeptide by charge. When the nucleic acid-recombinant immunoporter conjugate is administered to a cell, the single-chain antibody portion of the recombinant immunoporter specifically recognizes a cell surface antigen and binds to the surface antigen. The cell causes capping of the antigen-recombinant immunoporter complex, and takes up the complex into the cell by endocytosis. At this time, the nucleic acid bound to the recombinant immunoporter is simultaneously taken into the cell.
[0016]
Here, the nucleic acid may be DNA or RNA. In the case of DNA, according to the present invention, for example, gene transfer, particularly gene transfer for gene therapy, can be performed. In the case of RNA, for example, introduction of RNAi can be performed. For example, by suppressing a gene involved in cancer cell growth with RNAi, it can be used for suppressing cancer cell growth. As in these examples, the cells may be in a living body, but are also applicable to cells in a tissue culture system or a cell culture system.
[0017]
Cell surface antigens to be targeted include, for example, receptors for diffusion factors such as growth factors, growth factors and cytokines, receptors for protein factors such as membrane proteins, receptors for neurotransmitters and chemical molecules such as nicotine, and cells. Any protein, such as a protein or sugar chain expressed on the cell membrane of the target cell, such as a membrane protein involved in cell-cell interaction that functions for adhesion or information transmission, may be used as long as it can be recognized by the antibody. Furthermore, an antibody that recognizes the cell surface (antigen) by labeling it with another antibody, a peptide, or a low-molecular substance such as biotin can also be used. In the following examples, single-chain antibodies against EGFR were produced using EGFR (EGF receptor; epidermal growth factor receptor) as a target protein on the cell membrane.
[0018]
A single-chain antibody is obtained by providing a variable region of H chain (VH) and a variable region of L chain (VL) on one molecule by extracting variable regions from the H chain and L chain of the antibody molecule and linking them with a linker. , An antibody having the same antibody recognition ability as an antibody molecule consisting of an H chain and an L chain.
[0019]
Basically, a single-chain antibody is obtained by isolating mRNA from a mouse hybridoma expressing a monoclonal antibody recognizing a protein of interest, and obtaining the monoclonal antibody H from a cDNA library prepared using the mRNA. It can be obtained by isolating the cDNAs encoding the chain and the L chain, constructing a recombinant DNA encoding the single-chain antibody by genetic manipulation, and expressing it in cultured cells. Also, without preparing a cDNA library, a necessary portion of the gene encoding the H chain and the L chain is directly amplified from the mRNA using RT-PCT, and the fragment thus amplified is subjected to genetic manipulation. Alternatively, a recombinant DNA encoding a single-chain antibody can be constructed.
[0020]
In addition, a recombinant DNA library encoding various single-chain antibodies can be constructed using peripheral blood, spleen, or the like, instead of a specific hybridoma, in the same manner as described above. The recombinant DNA library is displayed on the phage surface by, for example, a phage display method, and a phage expressing an antibody against the surface antigen is identified using a target cell membrane surface antigen, The recombinant DNA encoding the single-chain antibody of the phage is recovered.
[0021]
The single-chain antibody thus prepared may be a mouse-type antibody derived from a mouse, but is derived from a human because it has a problem of rejection due to administration of a heterologous protein, such as when administered to a human as a pharmaceutical composition. Human antibodies may be preferred. At this time, when constructing a recombinant DNA library encoding various single-chain antibodies in advance and screening the target antibody, the tissue used as the source of mRNA may be human-derived at first.
[0022]
However, when a human-type single-chain antibody is prepared using a mouse hybridoma-derived monoclonal antibody, it is necessary to convert the mouse-type antibody to a human-type antibody. As described above, a single-chain antibody is a molecule in which VH and VL are linked by a linker. Each region has a hypervariable region (CDR) that determines the specificity for an antigen and a three-dimensional structure of the antibody molecule. A framework structure region (FWR) having a function of holding has a mosaic structure such as FWR1-CDR1-FWR2-CDR2-FWR3-CDR3-FWR4. Since the amino acid sequence of the FWR is conserved among species, the single chain antibody can be changed from a mouse sequence to a human sequence by changing the amino acid sequence in this region from a mouse type to a human type.
[0023]
In the present invention, a partial sequence of histone H1 was used as a chargeable peptide tail which is a nucleic acid binding site. Because nucleic acids are negatively charged, using a positively charged charged peptide tail, such as the histone H1 subsequence, allows these molecules to bind to each other and to be taken up by the recombinant immunoporter into the cell. , Nucleic acids are also transported into cells.
Hereinafter, the present invention will be described in detail with reference to specific examples.
[0024]
【Example】
== Single-chain antibody gene preparation ==
The single-chain antibody gene was prepared as follows using a recombinant single-chain antibody preparation kit (Pharmacia). MRNA was isolated from mouse hybridoma cells producing the monoclonal antibody B4G7 against EGFR, and reverse transcribed using oligoT as a primer to synthesize cDNA. A DNA fragment encoding the VH domain of B4G7 was amplified by PCR using the mixed primers HB1 to HB19 and the mixed primers HF1 to HF4, and the mixed primer of LB1 to LB17 and lamdaB was added to the cDNA. Using a mixed primer of LF2, LF4, LF5 and lamdaF, a DNA fragment encoding the VL domain was amplified by PCR. After gel purification of the obtained DNA fragment, PCR was performed by adding a linker capable of hybridizing with homology at the 3 ′ end of the VH fragment and the 5 ′ end of the VL fragment and the 5 ′ end and 3 ′ end respectively. , VH-linker-VL was prepared and cloned into pCANTAB5E.
[0025]
Originally, since it is a recombinant DNA derived from a monoclonal antibody called B4G7, there should be only one kind, but the recombinant DNA is a heterogeneous molecular population because it contains primer degeneracy and PCR errors. Thus, a mouse single-chain antibody gene was cloned using the phage display method as follows. First, a library in which the recombinant DNA was expressed on the phage surface was prepared. On the other hand, A431 cells that overproduce EGFR were fixed with 10% formalin (3.6% formaldehyde) -containing PBS for 10 minutes, washed, and then washed with a plastic dish on which A431 cells blocked with 3% BSA-containing PBS were adsorbed. The phage library was panned five times to concentrate phages displaying antibodies to EGFR. Next, the concentrated antibody-displaying phage was screened by ELISA using the immobilized A431 cells as an antigen, and a mouse-type single-chain antibody-expressing phage scBH was cloned.
[0026]
Table 1: Primers used for preparing single-chain antibodies
VH back
HB1
GGCGGGCGGCGGCTCCGGTGGTGGGTGGATCCGAKGTRMAGCTTCAGGGAGTC [SEQ ID NO: 4]
HB2
GGCGGGCGGGCGCTCCGGTGGTGGTGGATCCGAGGTBCAGCTBCAGCAGTC [SEQ ID NO: 5]
HB3
GGCGGGCGGCGGCTCCGGTGGTGGTGGATCCCAGGTGCAGCTGAAGASTC [SEQ ID NO: 6]
HB4
GGCGGGCGGCGGCTCCGGTGGTGGTGGATCCGAGGTCCARCCTGCAAARTC [SEQ ID NO: 7]
HB5
GGCGGGCGGCGGCTCCGGTGGTGGTGGATCCCAGGTYCAGCTBCAGARTC [SEQ ID NO: 8]
HB6
GGCGGGCGGCGGCTCCGGTGGTGGTGGATCCCAGGGTYARCCTGCAGCAGTC [SEQ ID NO: 9]
HB7
GGCGGGCGGCGGCTCCGGTGGTGGTGGATCCCAGGTCCACGTGAAGCAGTC [SEQ ID NO: 10]
HB8
GGCGGGCGGCGGCTCCGGTGGGTGTGGATCCGAGGTGAASSTGGGTGGAATC [SEQ ID NO: 11]
HB9
GGCGGGCGGGGCTCCGGTGGTGGGTGATCCGAVGGTGAWGYTGGTGGAGGTC [SEQ ID NO: 12]
HB10
GGCGGGCGGGGCTCCGGTGGTGGGTGATCCGAGGTGCAGSKGGTGGGAGTC [SEQ ID NO: 13]
HB11
GGCGGGCGGGGCTCCGGTGGTGGGTGATCCGAKGTGCAMCTGGTGGAGGTC [SEQ ID NO: 14]
HB12
GGCGGGCGGCGGCTCCGGTGGTGGTGGATCCGAGGTGAAGCTGATGGARTC [SEQ ID NO: 15]
HB13
GGCGGGCGGCGGCTCCGGTGGTGGTGGATCCCGAGGTGARCCTTGTTGAGTC [SEQ ID NO: 16]
HB14
GGCGGGCGGCGGCTCCGGTGGTGGTGGATCCGARGTRAAGCTTCTCGAGGTC [SEQ ID NO: 17]
HB15
GGCGGGCGGCGGCTCCGGTGGTGGTGGATCCCGAAGTGAARSTTGAGGGAGTC [SEQ ID NO: 18]
HB16
GGCGGGCGGGGCTCCGGTGGTGGGTGATCCCAGGTTACTCTRAAAGWGTSTG [SEQ ID NO: 19]
HB17
GGCGGCGGCGGCTCCGGTGGTGGGTGATCCCAGGTCCAACTVCAGARCCC [SEQ ID NO: 20]
HB18
GGCGGGCGGCGGCTCCGGTGGTGGTGGATCCGATGTGAACTTGGAAGTGTC [SEQ ID NO: 21]
HB19
GGCGGGCGGGGCTCCGGTGGTGGGTGGATCCGAGGTGAAGGTCATCGAGTC [SEQ ID NO: 22]
VH FOR
SCFOR GGAATTCGGCCCCCGAG [SEQ ID NO: 23]
HF1
GGAATTCGGCCCCCGAGGCCGAGGAAACGGTGACCGTGGGT [SEQ ID NO: 24]
HF2
GGAATTCGGCCCCCGAGGCCGAGGAGACTGTGAGAGTGGGT [SEQ ID NO: 25]
HF3
GGAATTCGGCCCCGAGGCCGCAGAGACAGTGACCAGAGT [SEQ ID NO: 26]
HF4
GGAATTCGGCCCCCGAGGCCGAGGAGACGGTGACTGAGGGT [SEQ ID NO: 27]
VL BACK
SCBACK TTACTCGCGGCCAGCCCGGCCATGGCGGGACTACAAAG [SEQ ID NO: 28]
LB1
GCCATGGCGGACTACAAAAGAYATCCAGCTGACTCAGCC [SEQ ID NO: 29]
LB2
GCCATGGCGGACTACAAAAGAYATTGTTCTCWCCCAGTC [SEQ ID NO: 30]
LB3
GCCATGGCGGACTACAAAAGAYATTGTGMTMACTCAGTC [SEQ ID NO: 31]
LB4
GCCATGGCGGACTACAAAAGAYATTGTGYTRACACAGTC [SEQ ID NO: 32]
LB5
GCCATGGCGGACTACAAAAGAYATTGTRATGACMCAGTC [SEQ ID NO: 33]
LB6
GCCATGGCGGACTACAAAAGAYATTMAGATRAMCCAGTC [SEQ ID NO: 34]
LB7
GCCATGGCGGACTACAAAAGAYATTCAGATGAYDCAGTC [SEQ ID NO: 35]
LB8
GCCATGGCGGGACTACAAAAGAYATYCAGATGACACAGAC [SEQ ID NO: 36]
LB9
GCCATGGCGGGACTACAAAAGAYATTGTTCTCAWCCAGTC [SEQ ID NO: 37]
LB10
GCCATGGCGGACTACAAAAGAYATTGWGCTSACCCAATC [SEQ ID NO: 38]
LB11
GCCATGGCGGACTACAAAAGAYATTSTRATGACCCARTC [SEQ ID NO: 39]
LB12
GCCATGGCGGACTACAAAAGAYRTTKTGATGAACCCARAC [SEQ ID NO: 40]
LB13
GCCATGGCGGACTACAAAAGAYATTGTGATGACBCAGKGC [SEQ ID NO: 41]
LB14
GCCATGGCGGACTACAAAAGAYATTGTGATAACYCAGGA [SEQ ID NO: 42]
LB15
GCCATGGCGGACTACAAAAGAYATTGTGATGACCCAGWT [SEQ ID NO: 43]
LB16
GCCATGGCGGACTACAAAAGAYATTGTGATGACACAACC [SEQ ID NO: 44]
LB17
GCCATGGCGGACTACAAAAGAYATTTTGCTGACTCAGTC [SEQ ID NO: 45]
LAMDAB
GCCATGGCGGACTACAAAGATGCTGTTGTACTCAGGAATC [SEQ ID NO: 46]
VL FOR
LF1
GGAGCCGCCGCCGCCAGAACCACCACCACCAGAACCACCACCACCACGTTTGATTTTCCAGCTTGG [SEQ ID NO: 47]
LF2
GGAGCCGCCGCCGCCAGAACCACCACCACCAGAACCACCACCACCACGTTTTATTTCCCAGCTTGG [SEQ ID NO: 48]
LF4
GGAGCCGCCGCCGCCAGAACCACCACCACCAGAACCACCACACCACGTTTATTATTTCCAACTTTG [SEQ ID NO: 49]
LF5
GGAGCCGCCGCCGCCAGAACCACCACCCACCAGAACCACCACCACCACGTTTCAGCTCCAGCTTGG [SEQ ID NO: 50]
LAMDAF
GGAGCCGCCGCCGCCAGAACCACCACCACACCAGAACCACCACCACCACCTAGGACAGTCAGTTTGG [SEQ ID NO: 51]
[0027]
== Conversion of mouse antibody scBH to human antibody CaC ==
The nucleotide sequence of the gene encoding the mouse single-chain antibody scBH was determined, and the amino acid sequence was estimated. A human antibody FWR similar to the amino acid sequence of the mouse scBH FWR was searched in a database, and residues believed to affect the structure of the CDR and residues conserved at the VL / VH interface were conserved as much as possible. A human antibody sequence is selected, and a human-type single-chain antibody in which the amino acid sequence of FWR of scBH is converted to this human-type sequence is prepared (see FIG. 1). The VL region and VH region of this humanized single-chain antibody are connected by a linker to obtain a humanized single-chain antibody CaC. Hereinafter, a method for producing CaC will be described in detail.
[0028]
First, primer BX-VH1 (corresponding to base numbers 4-107), primer BX-VH2r (corresponding to base numbers 87-192), primer BX-VH3 (corresponding to base numbers 182-276) and primer BX-VH4r (base No. 254-345) (1 μM each) was prepared (Expand High Fidelity PCR System, currently Roche Diagnostics, Inc.) 50 μl, treated at 94 ° C. for 2 minutes, [94 ° C. for 30 seconds, 50 ° C. 20 Second, 68 ° C., 10 seconds]. By these reactions, a DNA fragment corresponding to base numbers 28 to 216 and a DNA fragment corresponding to base numbers 206 to 369 are obtained. Each 2 μl of the reaction product, primer fNcoHr (corresponding to nucleotide sequence 1-23 and having an NcoI restriction enzyme site and an initiation codon on the 5 ′ side) and primer VHBsp (corresponding to nucleotide sequence 326-351 and 5 ′ (With BspHI restriction enzyme site) in 1 μM each in 50 μl of a new PCR reaction solution, and after treatment at 94 ° C. for 2 minutes, a reaction cycle of [94 ° C. 30 seconds, 50 ° C. 20 seconds, 68 ° C. 20 seconds] was performed 30 times. Was. By this reaction, a VH fragment corresponding to base number 1-351 and having an initiation codon is obtained. This PCR product was separated and purified by 3% agarose gel electrophoresis.
[0029]
A VL fragment was similarly prepared. That is, primer BX-VL1 (corresponding to base numbers 406-499) and primer BX-VL2r (corresponding to base numbers 480-573), primer BX-VL3 (corresponding to base numbers 554-649) and primer BX-VL4r (base number PCR was performed using 630-717), and the reaction product (DNA fragment corresponding to base numbers 406-573 and 554-717) and primer fBamVL (corresponding to base numbers 400-425, 5 ′ A VL fragment (corresponding to base numbers 400-753) was amplified using BamHI restriction enzyme site) and rHLkEco (corresponding to base numbers 699-753 and having an EcoRI restriction enzyme site and a stop codon on the 5 'side). And purified in the same manner.
[0030]
The VH fragment and the VL fragment were each digested with NcoI and BspHI, BamHI and EcoRI at their ends, and then separated and purified by 3% agarose gel electrophoresis.
Next, the linkers Lk-Up and rLk-Dn were mixed with 50 μl of 10 mM TrisHCL (pH 7.4) -50 mM NaCl-1 mM EDTA at 5 μM each, heated to 94 ° C., and allowed to cool to room temperature. Anneal to create a double-stranded linker. The end-digested VH and VL DNA fragments and the double-stranded linker were mixed in equimolar amounts and ligated together with NcoI-EcoRI-digested pET22b (Novagen), thereby cloning full-length CaC into pET22b to obtain a nucleotide sequence. It was confirmed.
[0031]
Table 2: Primer sequences used for conversion from mouse antibody scBH to humanized antibody CaC (all 5 ′ → 3 ′). Uppercase letters indicate bases identical to the original sequence, and lowercase letters indicate bases different from the original sequence due to linkers, introduction of restriction enzyme sites, introduction of displacements, and the like.
[0032]
BX-VH1:
GTTTCAACTTGTACAGTCTGGTGCTGAAGTTAAGAAACCAGGAGCTCAGGTTAAGGTATCTTGTAAGGCTCCGGTTTCACACATCAAAGACACTCTGATGCATTG [SEQ ID NO: 52]
BX-VH2r:
GAATTTGCGTACGTACTTGGTATTACCGTCTTCTGGATCAATCCATCCCATCCACTCAAGACCTTGTCCTGGAGCCTGACGAACCCAATGCATCAGAGTGTCTTTTG [SEQ ID NO: 53]
BX-VH3:
CCAAGTACGTACGCAAATTCCAGGGTCGTGTTACTATGACCCTGGACACTTCAACCTCACTGTTTATATGGAGCTTTCTAGGTCTGCGATCAGAAGATACCGCT [SEQ ID NO: 54]
BX-VH4r [g / a]:
AGTAACAGTAGTACCTTGTCCCAAATAAGCCCAGTTACGTAAGATCCACCACGARCACAATAGTATACAGCGGTATCTCTCTGATCGCA [SEQ ID NO: 55]
fNcoH: catgccATGGGAcatcaccatccatcatcCAGGTTCAACTTGTACAGTCTGGG [SEQ ID NO: 56]
rVHBsp: acctccggaAGATACAGTAACAGTAGTACCTTGTC [SEQ ID NO: 57]
Lk-Up: ccgggaggtggcgggatctggcgggtgggggttcagggggtggcg [SEQ ID NO: 58]
rLk-Dn: gatccgccacctcctgaacctccaccgccagatccgccacct [SEQ ID NO: 59]
fBamVL: ggcggatccGACATCAGATGACCCAATCCATCAT [SEQ ID NO: 60]
rHLkEco: ggaattcTTAAGAACCGCCCACCgtgatgtgatgggtgatgTTTGATGTCAACAGTGGTGCCT [SEQ ID NO: 61]
BX-VL1:
CAGATGACCCAATCACCATCTAGTCTGTCAGCTTCCGGTTGGAGATCGTGTTACTATTACCTGTGCACTCTCTGAGAACATCTACTCCTACTTTG [SEQ ID NO: 62]
BX-VL2r:
TACACCTTCAGCCAGGGTCTTAGCATTGTAAATAAGGAGCTTTGGAGCTTTTCCTGACTTCTGTTGATACCAAGCAAAGTAGGAGGTAGATGTTC [SEQ ID NO: 63]
BX-VL3:
AGACCCTGGCTGAAGGTGTACCGTCTCGTTTCAGGTGATCTGGTTCTGGAACTGAGTTCACCCTCACTATTTCCATCTCTCAACCAGAGACTTCG [SEQ ID NO: 64]
BX-VL4r:
GATGTCAACAGTGGGGCCTCCACCGAAAGTGTGCAGAGATCCGTAATGGTGCTGACAGAAATAGGGTAGGAAGTCTTCTGGTTGAAGG [SEQ ID NO: 65]
[0033]
== Production of partial histone H1 tail-type immunoporter gene ==
(1) Preparation of single-chain antibody gene fragment having modified α-factor signal sequence
Using the humanized single-chain antibody gene CaC, a partial histone H1 tail-type immunoporter gene was prepared as follows by a synthetic oligo DNA and PCR method. To improve the secretion efficiency, a modified α-factor signal sequence was linked to the C-terminal of the immunoporter.
[0034]
First, the human single-chain antibody gene C-terminal linker sequence was modified. A PCR reaction solution containing primers α-Factor-f and VL-Lk-R and 1 μM each using pPIC9Kdel (see Japanese Patent Application Laid-Open No. 2001-333780) into which a human-type single-chain antibody gene of D4Sx5 type tail was incorporated (see JP-A-2001-333780). (Expand High Fidelity PCR System, currently Roche Diagnostics) (50 μl) was prepared, treated at 94 ° C. for 2 minutes, and then subjected to 30 reaction cycles of [94 ° C. 30 seconds, 50 ° C. 20 seconds, 68 ° C. 10 seconds] 30 times.
The PCR product was digested with NcoI, and the single-chain antibody gene fragment was separated and purified by 1% agarose gel electrophoresis.
[0035]
(2) Preparation of 1.3L-1 DNA fragment
50 μl of a PCR reaction solution (Expand High Fidelity PCR System, currently Roche Diagnostics) containing 5 μM of each of the primers 1.3L-1f and 1.3L-1r was prepared, treated at 94 ° C. for 2 minutes, and then treated at 94 ° C. for 30 seconds. , 50 ° C. for 20 seconds, 68 ° C. for 10 seconds] 30 times to obtain a recombinant DNA fragment 1.3L-1 having the following sequence.
1.3L-1 sequence:
CTACTTCCCCATGGTTCTGGTAAATCTTCCGAAGGTACCCCGAAGAAAAGCATCAAAAAGACTCCTAAAGAGTAAAGAAGCCAGCAACCGCTGCTGGGACCAAGAAAGTGGCCAAGATGGCGAAAAAGGTCGAAGAACACCACCAGAGACACACCACCAGAGACACACCACCAGAGACAGACCAGAGACACACCACCAGAGACAGACCACAGACCAGACCACAGACCACAGACCACAGACCACACCAGACC sequence
[0036]
Similarly to 1.3L-1, a recombinant DNA fragment 1.3L-2 having the following sequence was obtained using the primers 1.3L-2f and 1.3L-2r.
1.3L-2 sequence:
GCGAAAAAGGGTGAAAACCCTCAGCCAAAAAAAGCTGCCAAAGAGTCCCAGCTAAGGCCAAAGCCCCTAAGCCCAAGGCGGCCAAGCCTAAGTCGGGGGAAGCCGAAGGTTACAAGGCAAAGAAGGCAGCTAGGAATGA sequence AGAGAAGATAGGA
[0037]
2 μl of the PCR reaction solution containing the DNA fragments 1.3L-1 and 1.3L-2 was mixed with 50 μl of the new PCR reaction solution, and the primers common F and H1.3L-totalR were added at 1 μM each, and treated at 94 ° C. for 2 minutes. A reaction cycle of [94 ° C. for 30 seconds, 50 ° C. for 20 seconds, 68 ° C. for 20 seconds] was performed 30 times.
Then, 1.3 L of the PCR product was digested with NcoI and EcoRI, and separated and purified by 3% agarose gel electrophoresis.
[0038]
(3) Preparation of modified α-factor signal sequence
The modified α-factor signal sequence is obtained by mixing PCR products using pro1-f and pro1-r, pro2-f and pro2-r, and using Pro / EcoRI-f and Pro / AvrII-r It was prepared by amplifying the α-factor signal sequence, digesting with EcoRI, separating and purifying by 3% agarose gel electrophoresis. Since the method and conditions of each step are the same as those of the PCR product of 1.3 L, detailed description will be omitted.
[0039]
(4) Preparation of partial histone H1 tail immunoporter gene
The restriction enzyme-treated single-chain antibody gene fragment, 1.3 L of PCR product and the modified α-factor signal sequence were mixed in equimolar amounts and ligated. The ligation reaction solution was similarly amplified with primers: α-Factor-f and Pro / AvrII-r, digested with XhoI and AvrII, and separated and purified by 3% agarose gel electrophoresis. This was incorporated into the XhoI-AvrII site of PPIC9Kdd (a plasmid in which the XhoI cleavage site of the neomycin resistance region and the NcoI cleavage site of the His4 region were replaced with cttgag and ccatag from pPIC9K: see JP-A-2001-333780), and cloned. The nucleotide sequence was confirmed. This plasmid was designated as pPIC9Kdd-CaCH1.3Lpro.
[0040]
(5) Preparation of 23 amino acid residue histone H1 tail immunoporter gene (FIG. 3) and 28 amino acid residue histone H1 tail immunoporter gene (FIG. 4) The following primers, 28a. a1st-F and 28a. 50 μl of a PCR reaction solution (Expand High Fidelity PCR system, currently Roche Diagnostics) containing 5 μM each of a1st-R was prepared, treated at 94 ° C. for 2 minutes, and then treated at 94 ° C. for 30 seconds, 50 ° C. for 20 seconds, 68 ° C. for 10 seconds. Second cycle] 30 times, and the following DNA fragment 28a. a1st was obtained. This DNA fragment 28a. a1st-containing reaction solution (2 μl) and the following primers, 28a. a1st-F, 28a. a2nd-R A 50 µl PCR reaction solution (Expand High Fidelity PCR system) containing 1 µM each was prepared, treated at 94 ° C for 2 minutes, and subjected to 30 reaction cycles of [94 ° C for 30 seconds, 50 ° C for 20 seconds, 68 ° C for 10 seconds]. Times, the DNA fragment 28a. a2nd was obtained.
This DNA fragment was digested with NcoI and separated and purified by 3% agarose gel electrophoresis.
[0041]
A single-chain antibody gene fragment that has been treated with a restriction enzyme (prepared in the same manner as when the partial histone H1 tail region was prepared); and 28a. The a2nd sequence was mixed in equimolar amounts and a ligation reaction was performed. The reaction solution was similarly amplified with primers α-Factor-f and FLAG / common R, digested with XhoI and EcoRI, and separated and purified by 1% agarose gel electrophoresis. This was inserted into the XhoI-EcoRI site of pPIC9Kdd-CaCH1.3Lpro by a conventional method and cloned to confirm the nucleotide sequence. This plasmid was designated as pPIC9Kdd-CaCH28aa.
[0042]
Similarly, the following primers, 23a. a1st-F and 23a. a1st-R, 23a. a2nd-R, the DNA fragment 23a. a2nd was obtained. Thereafter, it was similarly amplified with a-Factor-f and FLAG / common R, and cloned into pPIC9Kdd-CaCH1.3Lpro to prepare a plasmid pPIC9Kdd-CaCH23aa.
[0043]
Table 3 PCR primers used for the preparation of the partial histone H1 tail immunoporter gene (all 5 ′ → 3 ′). Uppercase letters indicate bases identical to the original sequence, and lowercase letters indicate bases different from the original sequence due to linkers, introduction of restriction enzyme sites, introduction of displacements and the like.
[0044]
α-Factor-f: TACTATTGCCAGCATTGCTGC [SEQ ID NO: 68]
VL-Lk-R: accatggggagagttagaccccTTTGATGTCAACAGTGGTGC [SEQ ID NO: 69]
1.3L-1f:
CTACTTCCCATGGTTCTGGTAAATCTTCCGAAGGTACCCGAAGAAAAGCATCAAAAAGACTCCTAAGAAGGTAAAGGAAGCCAGCAACCGCTGCTG [SEQ ID NO: 70]
1.3L-1r:
TAAGAAGGGTAAAGAAGCCAGCAACCGCTGCTGGGACCAAGAAAGTGGCCAAGAGTGCGAAAAAGGTTGAAAACACCTCAGCCAAAAAAAGCTGCCA [SEQ ID NO: 71]
1.3L-2f:
GCGAAAAGGTGAAAACCCTCAGCCAAAAAAAGCTGCCAAGAGTCCCAGCTAAGGCCAAAGCCCCTAAGCCCAAGGCGGCCAAGCCTAAGTCGGGGG [SEQ ID NO: 72]
1.3L-2r:
CCTAAGCCCAAGGCGGCCAAGCCTAAGTCCGGGGAAGCCGAAGGTTACAAAGGCAAAGAAGCAGCTCCGAAGAAAAAGAGTTGAGTTAACGAATTCGC [SEQ ID NO: 73]
common F: ctactcccatggTTCTGGTAAATCTTTCCG [SEQ ID NO: 74]
H1.3L-totalR: gcgaattcgttaactcaACTCTTTTTCTTCGGAGCT [SEQ ID NO: 75]
pro1-f:
CTAGtctaGAGGCTGAAGCTgcAccTgtTaaTacAacTacCgaGgaCgaacTgcCcaatCccAgAgaagcAgtTatAggtttaTAG [SEQ ID NO: 76]
pro1-r:
cAttGttAgtTGAAttACTGaaAggTaaGacTgcGacGtcAaaaGtcGccttcCaaGtcACTAtaaccTatAacTgcttcTgcTggGattttg [SEQ ID NO: 77]
pro2-f:
gtCttAccTttCAGTaaTTCAacTaaCaaTggAttGttAttCatCaaCactacAatCgcTagTatCgctgcAaaGgaGgaagTgtTtcA [SEQ ID NO: 78]
pro2-r:
ttccgcgggccgctatgggccgacGTCGACagcttcagcTtctctCttctcTagTgaAacAccttCtcCttTgcagcGatActA [SEQ ID NO: 79]
Pro / EcoRI-f:
CCGGAATTCGAGGCTGAGCTGCACCTGTTAATACAAC [SEQ ID NO: 80]
Pro / AvrII-r:
CACTAGGTCAAGCTTCAGCTTCTCTCTCTCTCTAGGTGA [SEQ ID NO: 81]
Modified α-factor signal sequence:
ccgGAATTCGAGGCTGAAGCTgcAccTgtTaaTacAacTacCgaGgaCgaaacTgcCcaaatCccAgcAgaagcAgtTatAggttaTAGTgaCttGgaaggCgaCttTgaCgtCgcAgtCttAccTttCAGTaaTTCAacTaaCaaTggAttGttAttCatCaaCactacAatCgcTagTatCgctgcAaaGgaGgaaggTgtTtcActAgagaaGagagaAgctgaagctTGACCTAGGtg [SEQ ID NO: 82]
28a. a1st-F: CTACTTCccatggTTCTGGTAAATCTTCCGAAGGTACCCCCGAAAGAAGCATCAAAAAAGACTCCt [SEQ ID NO: 83]
28a. a1st-R:
GGCCACTTTCTTGGTCCcagcagcggttgctggcttcttttacctctcttaGGAGCTCTTTTTGATGC [SEQ ID NO: 84]
28a. a2nd-R:
ccgGAATTCcTCATCAagagtcatctttataatcACTCTTGGGCCACTTTCTTGGTCCc [SEQ ID NO: 85]
28a. a2nd array: CTACTTCccatggTTCTGGTAAATCTTCCGAAGGTACCCCGAAGAAAAGCATCAAAAAGACTCCtaagaaggtaaagaagccagcaaccgctgctgGGACCAAGAAAGTGGCCAAGAGTgattataaagatgactctTGATGAcgGAATTCcgg [SEQ ID NO: 86]
23a. a1st-F:
CTACTTCccatggTTCTGGTAAATCTTCCGAAGGTACCCGAAGAAAAGCATCAAAAAGAACTCC [SEQ ID NO: 87]
23a. a1st-R:
AGACTTGGTCCcagcagggttgctgggttcttttacctttcttaGGAGTCTTTTTTGATGC [SEQ ID NO: 88]
23a. a2nd-R:
ccgGAATTCcgTCATCAagagtcattttataatcAGACTTGGTCCc [SEQ ID NO: 89]
FLAG / common R: cggGAATTCcgTCATCAagagtc [SEQ ID NO: 90]
23a. a2nd sequence:
CTACTTCccatggTTCTGGTAAATCTTCCGAAGGTACCCCGAAGAAAAGCATCAAAAAGACTCCtaagaaggtaaagaagcccccaccgctgctgGGACCAGTCTgattattagaatgctctGATCtGATCTGATGATCTGATGATCTCGATCTGATGATCTGATGATCTCGATCGATCTGATGATCTGTCGATCTCGATCTGTCGATCGTGATCTCGATCGATCTGTCGATCTGTCGATCTGTCGATCCTGATCGATCTGTCGATCCTGATCGATCTGTCGATCCTGATCGATCTGCTGATCCTCTCCccatggTTCTGGTAAATCTTCGAAGGTACCCCGAAGAAAAGCATCAAAAAGACTCCtaagaaggtaaagaagcccccaccgctgctgGGCCAAGTCTgattattaagattgATCGTCGTGATCTCGA
[0045]
== Expression and preparation of partial histone H1 tail immunoporter ==
Using a Pichia expression kit (InVitrogen), a partial histone H1 tail-type immunoporter was prepared as follows.
The plasmid encoding the partial histone H1 tail-type immunoporter, prepared as described above, was cut with the restriction enzyme BglII, introduced into Pichia pastris GS115 by electroporation, and selected with a minimal medium to obtain a transformant. . By culturing this transformant in a liquid medium containing 0.5% methanol and 0.5 M NaCl, the expression of the partial histone H1 tail immunoporter was induced. The medium supernatant was diluted 3-fold with 20 mM Tris (pH 7.5) -250 mM NaCl, and the partial histone H1 tail-type immunoporter was adsorbed to the ion exchange resin DTREAMLINE-S. After washing with the same buffer, the 20 mM Tris (pH 7) .5) Elution with 1M NaCl. Thereafter, the partial histone H1 tail immunoporter was adsorbed again to TALON resin (Clontech), and was eluted with 100 mM EDTA (pH 8.0).
[0046]
In order to prevent adsorption to the filtration membrane, 1/9 volume of 5M NaCl was added to the eluted sample to adjust the final concentration to 0.5M NaCl, and ultrafiltration was performed.
[0047]
To prevent adsorption to the resin, the concentrated sample was purified on a superdex 75 (Pharmacia) gel filtration column equilibrated with 1 M Arginine HCl-5% Glycerol. For each fraction fractionated by gel filtration, each peak fraction and several fractions before and after the peak fraction were subjected to SDS-PAGE on a 12% acrylamide gel using the absorbance of 280 nm as an index, and the fraction containing the desired immunoporter was subjected to silver staining. Identified. Three large peak fractions containing the desired immunoporter were collected. Each fraction was treated with 50 mM NaPO ₄ Dialysis was performed against -500 mM NaCl pH 7.4, and ultrafiltration was performed.
[0048]
== Gene transfer by partial histone H1 tail type immunoporter ==
45 mg of polyethyleneimine (PEI: Aldrich) having an average molecular weight of 25 kD was dissolved in 10 ml of distilled water, neutralized with hydrochloric acid, and sterilized with a filter to obtain a 100 mM solution. For gene transfer experiments, PEI was used after dilution with distilled water. In addition, when 3 μl of 1 mM PEI and 1 μg of DNA were mixed, the calculation was performed assuming that (free amino group of PEI) / (number of phosphate groups of nucleic acid) = N / P = 1. A431 cells to be used for gene transfer were seeded so as to be about 60% confluent using a 24-well plate the day before the experiment, and used for the experiment the next day.
[0049]
0.25 μg of the luciferase expression vector pSRD-GL3 and 1 μg (about 5 μl) of the immunoporter were mixed, diluted with distilled water to a total volume of 50 μl, and left at room temperature for 30 minutes. Thereafter, 1 mM PEI was added to each N / P ratio, mixed quickly, and left at room temperature for 30 minutes. The entire amount of the complex solution was added to each well containing 1 ml of DMEM medium containing 10% FCS, and 5% CO 2 was added. ₂ The cells were cultured at 37 ° C. in the presence, and gene transfer was performed.
[0050]
After 24 hours, luciferase activity was measured using a luciferase assay system (Promega) and a luminometer MicroLumat Plus LB96V (Berthold). For each condition, experiments were performed in 3-4 wells, and the mean and standard deviation were calculated.
[0051]
The results are shown in FIGS.
FIG. 5 shows the results of an experiment using a partial histone H1 tail-type immunoporter as the immunoporter. As a control, the experiment was performed under the condition without an immunoporter. The higher the N / P, the higher the non-specific adsorption to cells and the like, and the lower the target cell specificity of gene transfer. Therefore, the smaller the N / P, the better. However, the smaller the N / P, the lower the efficiency of gene transfer into cells. Conventionally, for example, as a peptide tail (AspAspAspAspSer) ₅ In a D4Sx5 tail type immunoporter (hereinafter referred to as CaCD20) having N / P, N / P = 10-20 or more is sufficient (in this example, 10/20). ⁵ In this example, N / P = 8 and 10 ⁵ A level of gene transfer was observed.
[0052]
FIG. 6 shows the results of experiments using a 23 amino acid residue histone H1 tail immunoporter and a 28 amino acid residue histone H1 tail immunoporter. As a control, conventional CaCD20 was used. N / P = 10 even at 3 and 5 ⁵ Excellent gene transfer efficiency above the level was demonstrated. This indicates that the nucleic acid transport efficiency according to the present invention is considerably high.
[0053]
【The invention's effect】
An object of the present invention is to provide a system having high nucleic acid transport efficiency in a recombinant immunogene method.
[0054]
[Sequence list]

[Brief description of the drawings]
FIG. 1 shows the nucleotide sequence of a recombinant DNA encoding human single-chain antibody CaC (SEQ ID NO: 92) and the amino acid sequence of CaC (SEQ ID NO: 93) in one example of the present invention.
FIG. 2 is a diagram showing the nucleotide sequence of a recombinant DNA encoding a partial histone H1 tail-type immunoporter (SEQ ID NO: 94) and the amino acid sequence of the immunoporter (SEQ ID NO: 95) in one example of the present invention.
FIG. 3 shows the nucleotide sequence of a recombinant DNA encoding a 23-amino acid residue histone H1 tail-type immunoporter gene (SEQ ID NO: 96) and the amino acid sequence of an immunoporter (SEQ ID NO: 97) in one example of the present invention. It is.
FIG. 4 shows the nucleotide sequence of a recombinant DNA encoding a 28-amino acid residue histone H1 tail-type immunoporter gene (SEQ ID NO: 98) and the amino acid sequence of an immunoporter (SEQ ID NO: 99) in one example of the present invention. It is.
FIG. 5 is a graph showing the results of a gene transfer experiment using a partial histone H1 tail-type immunoporter in one example of the present invention. The y-axis shows the measured luciferase activity per unit amount. The number after H3L- represents the peak fraction in gel filtration, and no gene transfer was observed in the first fraction, but this fraction was a fraction that was somewhat degraded during processing. Seem.
FIG. 6 is a graph showing the results of a gene transfer experiment using a histone H1 tail-type immunoporter of 23 amino acid residues and 28 amino acid residues in one example of the present invention. The y-axis shows the measured luciferase activity per unit amount as in FIG. In this figure, as in FIG. 5, the last numeral represents the peak fraction in gel filtration.

Claims (9)

A fusion protein for transporting a nucleic acid into a cell,
A single-chain antibody against the cell surface antigen,
A fusion protein comprising a partial histone polypeptide for binding to a nucleic acid. The fusion protein according to claim 1, wherein the partial histone polypeptide is the polypeptide of SEQ ID NO: 1. The fusion protein according to claim 1, wherein the partial histone polypeptide is a partial sequence of SEQ ID NO: 1. The fusion protein according to claim 3, wherein the partial histone polypeptide is the polypeptide of SEQ ID NO: 2. The fusion protein according to claim 3, wherein the partial histone polypeptide is the polypeptide of SEQ ID NO: 3. The fusion protein according to claim 1, wherein the single-chain antibody is a human-type single-chain antibody. The fusion protein according to any one of claims 2, 4, and 5, wherein one or several amino acid residues are deleted, substituted, or added,
The partial histone polypeptide binds to a nucleic acid,
The single-chain antibody specifically binds to a surface antigen of the cell,
A fusion protein for transporting the nucleic acid into a cell. A method for producing a fusion protein for transporting a nucleic acid into a cell,
Using mRNA isolated from a hybridoma capable of producing a monoclonal antibody against a cell surface antigen, preparing a cDNA encoding a single-chain antibody against the surface antigen,
A step of adding a cDNA encoding a partial histone polypeptide to the single-chain antibody cDNA to prepare a fusion cDNA,
Expressing the fusion cDNA and isolating a fusion protein comprising the single-chain antibody and the partial histone polypeptide;
A method for producing a fusion protein comprising: A nucleic acid transport method for transporting a nucleic acid into a cell,
A step of preparing a nucleic acid-protein mixture by mixing the nucleic acid with the fusion protein according to any one of claims 1 to 7,
Administering the nucleic acid-protein mixture to the cells;
A nucleic acid transport method comprising:

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