JP4521519B2 - Partial histone tail type immunoporter - Google Patents

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]
BACKGROUND OF THE INVENTION
The present invention relates to a fusion protein for transporting nucleic acid into cells, a method for producing the same, and a method for transporting nucleic acid using the same.
[0002]
[Prior art]
Known methods for transporting nucleic acids into cells include calcium phosphate method, electroporation, virus infection such as retrovirus and adenovirus, lipofection, cell fusion, and the like. Recently, a method called a recombinant immunogene method, which utilizes a recombinant immunoporter as a carrier for nucleic acid transport, has been developed (see, for example, Patent Document 1).
[0003]
A recombinant immunoporter is a single-chain antibody having a charged peptide tail, and in particular, a fusion protein of a single-chain antibody against a cell surface antigen and a peptide having a nucleic acid binding ability is effective. In gene transfer, a recombinant immunoporter to which a nucleic acid to be transported into a cell is bound is administered to a cell having the surface antigen. The antibody moiety binds to its surface antigen and the cell takes up the recombinant immunoporter along with the bound nucleic acid into the cell by endocytosis. Thus, the target nucleic acid can be transported into the cell 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 with high nucleic acid transport efficiency in the 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, wherein the single-chain antibody against the surface antigen of the cell is bound 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. The histone is particularly preferably H1, but since the histone is originally a DNA-binding protein, the partial histone polypeptide for binding to the nucleic acid is not limited to histone H1, and may be derived from any histone.
[0007]
In the fusion protein of the present invention, the partial histone polypeptide may be 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]
The fusion protein of the present invention may be characterized in that the single chain antibody is a human single chain antibody.
[0010]
In any 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 It may be a fusion protein that specifically binds to a cell surface antigen and transports the nucleic acid into the cell.
[0011]
Furthermore, 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, and a step of preparing a fusion cDNA, Expressing and isolating a fusion protein comprising said single chain antibody and said partial histone polypeptide.
[0012]
Furthermore, the nucleic acid transport method according to the present invention is a nucleic acid transport method for transporting a nucleic acid into a cell, wherein the nucleic acid is mixed with any one of the above fusion proteins to produce a nucleic acid-protein mixture, Administering a nucleic acid-protein mixture to the cells.
[0013]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, embodiments of the present invention will be described in detail with reference to examples.
J. Sambrook, EF Fritsch & T. Maniatis (Ed.), Molecular cloning, a laboratory manual (2nd edition), Cold Spring Harbor Press, Cold Spring Harbor, New York (1989); FM Ausubel, R. Brent, Standard protocol collections such as RE Kingston, DD Moore, JG Seidman, JA Smith, K. Struhl (Ed.), Current Protocols in Molecular Biology, John Wiley & Sons Ltd. The method described therein can be easily carried out by those skilled in the art. Therefore, in the case of using the methods described in these standard protocols, or modified or modified methods thereof, no detailed description is given in the embodiments and examples of this specification. In addition, when using commercially available reagent kits and measuring apparatuses, those skilled in the art normally use the attached protocol, and unless otherwise specified, use the attached protocol.
[0014]
The objects, features, advantages, and ideas of the present invention will be apparent to those skilled in the art from the description of the present specification, and those skilled in the art can easily reproduce the present invention from the description of the present 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 explanation, and the present invention is not limited to them. It is not limited. It will be apparent to those skilled in the art that various modifications and variations can be made based on the description of the present specification within the spirit and technical scope of the invention disclosed herein.
[0015]
The fusion protein according to the present invention constitutes a fusion protein for transporting a nucleic acid into a cell as one form of a recombinant immunoporter, a single-chain antibody against a cell surface antigen, and a peptide for binding to the nucleic acid Includes a partial histone polypeptide as a tail. When nucleic acid is mixed with this fusion protein, the nucleic acid binds to a portion of the partial histone polypeptide by charge. When this nucleic acid-recombinant immunoporter conjugate is administered to a cell, the single-chain antibody portion of the recombinant immunoporter specifically recognizes and binds to the surface antigen of the cell. The cell capping the antigen-recombinant immunoporter complex, and the complex is taken into the cell by endocytosis. At the same 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 introduction, particularly gene introduction for gene therapy can be performed. In the case of RNA, for example, RNAi can be introduced. For example, by suppressing a gene related to cancer cell growth with RNAi, it can be used for suppressing cancer cell growth. As in these examples, the cells may be in vivo, but are also applicable to cells in tissue culture systems and cell culture systems.
[0017]
Target cell surface antigens include, for example, receptors for diffusion factors such as growth factors, growth factors, cytokines, and protein factors such as membrane proteins, receptors for chemical molecules such as neurotransmitters and nicotine, cells Any protein that can be recognized by an antibody, such as a protein or sugar chain expressed on the cell membrane of a target cell, such as a membrane protein capable of intercellular interaction that functions for adhesion or information transmission, may be used. Furthermore, it is also possible to use an antibody in which the cell surface (antigen) is labeled with another antibody, a peptide, or a low molecular substance such as biotin, and recognizes it. In the following examples, EGFR (EGFreceptor; epidermal growth factor receptor) was used as a target protein on the cell membrane to produce a single chain antibody against EGFR.
[0018]
Single-chain antibodies provide variable region (VH) and variable chain region (VL) on a single molecule by extracting variable regions from the heavy and light chains of an 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, single-chain antibodies are obtained by isolating mRNA from a mouse hybridoma expressing a monoclonal antibody that recognizes the target protein, and using the mRNA from the cDNA library prepared using the mRNA. It can be obtained by isolating cDNAs encoding the chain and L chain, constructing recombinant DNA encoding a single-chain antibody by genetic manipulation, and expressing it in cultured cells. In addition, without preparing a cDNA library, the necessary part of the gene encoding the H chain and L chain is amplified directly from mRNA using RT-PCT, and the amplified fragment is used for genetic manipulation. It is also possible to construct a recombinant DNA encoding a single chain antibody.
[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. This recombinant DNA library is displayed on the phage surface by, for example, a phage display method, and using a target cell membrane surface antigen, a phage expressing an antibody against the surface antigen is identified, Recombinant DNA encoding the single chain antibody possessed by the phage is recovered.
[0021]
The single-chain antibody prepared in this way may be a mouse-type antibody derived from a mouse, but since it has a problem of rejection due to administration of a heterologous protein, such as when administered to a human as a pharmaceutical composition, it is derived from a human. Human antibodies may be preferred. At that time, when a recombinant DNA library encoding various single-chain antibodies is constructed in advance and the target antibody is screened, the tissue used as the source of mRNA may be first derived from human.
[0022]
However, when producing a human single-chain antibody using a monoclonal antibody derived from a mouse hybridoma, it is necessary to convert the mouse antibody into a human 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 the three-dimensional structure of the antibody molecule. The framework structure region (FWR) having the function to be held has a structure arranged in a mosaic shape as FWR1-CDR1-FWR2-CDR2-FWR3-CDR3-FWR4. Since the amino acid sequence of FWR is conserved depending on the species, the single chain antibody can be changed from the mouse type sequence to the human type sequence by changing the amino acid sequence of this region from the mouse type to the human type.
[0023]
In the present invention, a partial sequence of histone H1 was used as a charged peptide tail that is a nucleic acid binding site. Since nucleic acids are negatively charged, using positively charged peptide tails such as histone H1 subsequences, these molecules bind to each other and the recombinant immunoporter is taken up into the cell. Nucleic acids are also transported into the cell.
Hereinafter, the present invention will be described in detail with specific examples.
[0024]
【Example】
== Production of single chain antibody gene ==
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 monoclonal antibody B4G7 against EGFR, reverse transcription was performed using oligoT as a primer, and cDNA was synthesized. A DNA fragment encoding the VH domain of B4G7 was amplified by PCR using a mixed primer from HB1 to HB19 and a mixed primer from HF1 to HF4, and a mixed primer of LB1 to LB17 and lamdaB, and LF1 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 5 ′ end and the 3 ′ end of each of the 3 ′ end of the VH fragment and the 5 ′ end of the VL fragment. A recombinant DNA called VH-linker-VL was prepared and cloned into pCANTAB5E.
[0025]
Since it is originally a recombinant DNA derived from the monoclonal antibody B4G7, there should be only one kind, but because it contains primer degeneracy and PCR errors, the recombinant DNA is a heterogeneous molecular population. Therefore, the mouse type single chain antibody gene was cloned using the phage display method as follows. First, a library in which recombinant DNA was expressed on the phage surface was prepared. On the other hand, A431 cells overproducing EGFR were fixed with PBS containing 10% formalin (3.6% formaldehyde) for 10 minutes, washed, and then used with a plastic dish on which A431 cells blocked with PBS containing 3% BSA were adsorbed. The phage library was panned 5 times to concentrate phages displaying antibodies against EGFR. Next, the concentrated antibody-displayed phage was screened by ELISA using immobilized A431 cells as an antigen, and mouse type single chain antibody-expressing phage scBH was cloned.
[0026]
Table 1: Primers used to produce single chain antibodies
VH back
HB1
GGCGGCGGCGGCTCCGGTGGTGGTGGATCCGAKGTRMAGCTTCAGGAGTC [SEQ ID NO: 4]
HB2
GGCGGCGGCGGCTCCGGTGGTGGTGGATCCGAGGTBCAGCTBCAGCAGTC [SEQ ID NO: 5]
HB3
GGCGGCGGCGGCTCCGGTGGTGGTGGATCCCAGGTGCAGCTGAAGSASTC [SEQ ID NO: 6]
HB4
GGCGGCGGCGGCTCCGGTGGTGGTGGATCCGAGGTCCARCTGCAACARTC [SEQ ID NO: 7]
HB5
GGCGGCGGCGGCTCCGGTGGTGGTGGATCCCAGGTYCAGCTBCAGCARTC [SEQ ID NO: 8]
HB6
GGCGGCGGCGGCTCCGGTGGTGGTGGATCCCAGGTYCARCTGCAGCAGTC [SEQ ID NO: 9]
HB7
GGCGGCGGCGGCTCCGGTGGTGGTGGATCCCAGGTCCACGTGAAGCAGTC [SEQ ID NO: 10]
HB8
GGCGGCGGCGGCTCCGGTGGTGGTGGATCCGAGGTGAASSTGGTGGAATC [SEQ ID NO: 11]
HB9
GGCGGCGGCGGCTCCGGTGGTGGTGGATCCGAVGTGAWGYTGGTGGAGTC [SEQ ID NO: 12]
HB10
GGCGGCGGCGGCTCCGGTGGTGGTGGATCCGAGGTGCAGSKGGTGGAGTC [SEQ ID NO: 13]
HB11
GGCGGCGGCGGCTCCGGTGGTGGTGGATCCGAKGTGCAMCTGGTGGAGTC [SEQ ID NO: 14]
HB12
GGCGGCGGCGGCTCCGGTGGTGGTGGATCCGAGGTGAAGCTGATGGARTC [SEQ ID NO: 15]
HB13
GGCGGCGGCGGCTCCGGTGGTGGTGGATCCGAGGTGCARCTTGTTGAGTC [SEQ ID NO: 16]
HB14
GGCGGCGGCGGCTCCGGTGGTGGTGGATCCGARGTRAAGCTTCTCGAGTC [SEQ ID NO: 17]
HB15
GGCGGCGGCGGCTCCGGTGGTGGTGGATCCGAAGTGAARSTTGAGGAGTC [SEQ ID NO: 18]
HB16
GGCGGCGGCGGCTCCGGTGGTGGTGGATCCCAGGTTACTCTRAAAGWGTSTG [SEQ ID NO: 19]
HB17
GGCGGCGGCGGCTCCGGTGGTGGTGGATCCCAGGTCCAACTVCAGCARCC [SEQ ID NO: 20]
HB18
GGCGGCGGCGGCTCCGGTGGTGGTGGATCCGATGTGAACTTGGAAGTGTC [SEQ ID NO: 21]
HB19
GGCGGCGGCGGCTCCGGTGGTGGTGGATCCGAGGTGAAGGTCATCGAGTC [SEQ ID NO: 22]
VH FOR
SCFOR GGAATTCGGCCCCCGAG [SEQ ID NO: 23]
HF1
GGAATTCGGCCCCCGAGGCCGAGGAAACGGTGACCGTGGT [SEQ ID NO: 24]
HF2
GGAATTCGGCCCCCGAGGCCGAGGAGACTGTGAGAGTGGT [SEQ ID NO: 25]
HF3
GGAATTCGGCCCCCGAGGCCGCAGAGACAGTGACCAGAGT [SEQ ID NO: 26]
HF4
GGAATTCGGCCCCCGAGGCCGAGGAGACGGTGACTGAGGT [SEQ ID NO: 27]
VL BACK
SCBACK TTACTCGCGGCCCAGCCGGCCATGGCGGACTACAAAG [SEQ ID NO: 28]
LB1
GCCATGGCGGACTACAAAGAYATCCAGCTGACTCAGCC [SEQ ID NO: 29]
LB2
GCCATGGCGGACTACAAAGAYATTGTTCTCWCCCAGTC [SEQ ID NO: 30]
LB3
GCCATGGCGGACTACAAAGAYATTGTGMTMACTCAGTC [SEQ ID NO: 31]
LB4
GCCATGGCGGACTACAAAGAYATTGTGYTRACACAGTC [SEQ ID NO: 32]
LB5
GCCATGGCGGACTACAAAGAYATTGTRATGACMCAGTC [SEQ ID NO: 33]
LB6
GCCATGGCGGACTACAAAGAYATTMAGATRAMCCAGTC [SEQ ID NO: 34]
LB7
GCCATGGCGGACTACAAAGAYATTCAGATGAYDCAGTC [SEQ ID NO: 35]
LB8
GCCATGGCGGACTACAAAGAYATYCAGATGACACAGAC [SEQ ID NO: 36]
LB9
GCCATGGCGGACTACAAAGAYATTGTTCTCAWCCAGTC [SEQ ID NO: 37]
LB10
GCCATGGCGGACTACAAAGAYATTGWGCTSACCCAATC [SEQ ID NO: 38]
LB11
GCCATGGCGGACTACAAAGAYATTSTRATGACCCARTC [SEQ ID NO: 39]
LB12
GCCATGGCGGACTACAAAGAYRTTKTGATGACCCARAC [SEQ ID NO: 40]
LB13
GCCATGGCGGACTACAAAGAYATTGTGATGACBCAGKC [SEQ ID NO: 41]
LB14
GCCATGGCGGACTACAAAGAYATTGTGATAACYCAGGA [SEQ ID NO: 42]
LB15
GCCATGGCGGACTACAAAGAYATTGTGATGACCCAGWT [SEQ ID NO: 43]
LB16
GCCATGGCGGACTACAAAGAYATTGTGATGACACAACC [SEQ ID NO: 44]
LB17
GCCATGGCGGACTACAAAGAYATTTTGCTGACTCAGTC [SEQ ID NO: 45]
LAMDAB
GCCATGGCGGACTACAAAGATGCTGTTGTACTCAGGAATC [SEQ ID NO: 46]
VL FOR
LF1
GGAGCCGCCGCCGCCAGAACCACCACCACCAGAACCACCACCACCACGTTTGATTTCCAGCTTGG [SEQ ID NO: 47]
LF2
GGAGCCGCCGCCGCCAGAACCACCACCACCAGAACCACCACCACCACGTTTTATTTCCAGCTTGG [SEQ ID NO: 48]
LF4
GGAGCCGCCGCCGCCAGAACCACCACCACCAGAACCACCACCACCACGTTTTATTTCCAACTTTG [SEQ ID NO: 49]
LF5
GGAGCCGCCGCCGCCAGAACCACCACCACCAGAACCACCACCACCACGTTTCAGCTCCAGCTTGG [SEQ ID NO: 50]
LAMDAF
GGAGCCGCCGCCGCCAGAACCACCACCACCAGAACCACCACCACCACCTAGGACAGTCAGTTTGG [SEQ ID NO: 51]
[0027]
== Conversion of mouse antibody scBH to human antibody CaC ==
The base sequence of the gene encoding the mouse type single chain antibody scBH was determined, and the amino acid sequence was deduced. A human antibody FWR similar to the FWR amino acid sequence of mouse scBH is searched in a database, and residues that are conserved at the VL / VH interface are conserved as much as possible. The human antibody sequence is selected, and a human type single chain antibody in which the amino acid sequence of the 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 number 4-107), primer BX-VH2r (corresponding to base number 87-192), primer BX-VH3 (corresponding to base number 182-276) and primer BX-VH4r (base) 50 μl of a PCR reaction solution (Expand High Fidelity PCR system, currently Roche Diagnostics) containing 1 μM each, and treating at 94 ° C. for 2 minutes, [94 ° C. for 30 seconds, 50 ° C. 20 Second, 68 ° C. for 10 seconds] was performed 30 times. By these reactions, a DNA fragment corresponding to base numbers 28-216 and a DNA fragment corresponding to base numbers 206-369 are obtained. 2 μl each of the reaction product, primer fNcoHr (corresponding to nucleotide sequence 1-23, having NcoI restriction enzyme site and initiation codon on the 5 ′ side) and primer VHBsp (corresponding to nucleotide sequence 326-351, at the 5 ′ end) (BspHI restriction enzyme site) is added to each new PCR reaction solution in 50 μl, treated at 94 ° C. for 2 minutes, and then subjected to a reaction cycle of [94 ° C. for 30 seconds, 50 ° C. for 20 seconds, 68 ° C. for 20 seconds] 30 times. It was. By this reaction, a VH fragment corresponding to the base number 1-351 and having an initiation codon is obtained. The PCR products were separated and purified by 3% agarose gel electrophoresis.
[0029]
A VL fragment was prepared in the same manner. That is, primer BX-VL1 (corresponding to base numbers 406-499), 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 the reaction product (corresponding to 630-717), the reaction product (DNA fragment corresponding to base numbers 406-573 and 554-717) and primer fBamVL (corresponding to base numbers 400-425, at the 5 'end). VL fragment (corresponding to base numbers 400-753) is amplified using BamHI restriction enzyme site) and rHLkEco (corresponding to base numbers 699-753 and having EcoRI restriction enzyme site and stop codon on the 5 'side). And purified in the same manner.
[0030]
The VH fragment and VL fragment were digested with NcoI and BspHI, BamHI and EcoRI, respectively, and then separated and purified by 3% agarose gel electrophoresis.
Next, the linkers Lk-Up and rLk-Dn were mixed in 50 μl of 10 mM TrisHCL (pH 7.4) -50 mM NaCl-1 mM EDTA so as to be 5 μM each, heated to 94 ° C., and then naturally cooled to room temperature. Annealed to create a double stranded linker. End-digested VH, VL DNA fragments and double-stranded linkers are mixed in equimolar amounts and ligated with pET22b (Novagen) digested with NcoI-EcoRI to clone full-length CaC into pET22b, and the nucleotide sequence It was confirmed.
[0031]
Table 2: Primer sequences used for conversion from mouse antibody scBH to humanized antibody CaC (all 5 ′ → 3 ′). Upper case letters represent the same bases as the original sequence, and lower case letters represent bases different from the original sequence due to linkers, restriction enzyme site introduction, displacement introduction, and the like.
[0032]
BX-VH1:
GTTCAACTTGTACAGTCTGGTGCTGAAGTTAAGAAACCAGGAGCTTCAGTTAAGGTATCTTGTAAGGCTTCCGGTTTCAACATCAAAGACACTCTGATGCATTG [SEQ ID NO: 52]
BX-VH2r:
GAATTTGCGTACGTACTTGGTATTACCGTCTTCTGGATCAATCCATCCCATCCACTCAAGACCTTGTCCTGGAGCCTGACGAACCCAATGCATCAGAGTGTCTTTG [SEQ ID NO: 53]
BX-VH3:
CCAAGTACGTACGCAAATTCCAGGGTCGTGTTACTATGACCCGTGACACTTCAACCTCTACTGTTTATATGGAGCTTTCTAGTCTGCGATCAGAAGATACCGCT [SEQ ID NO: 54]
BX-VH4r [g / a]:
AGTAACAGTAGTACCTTGTCCCCAATAAGCCCAGTTACCGTAAGATCCACCACGARCACAATAGTATACAGCGGTATCTTCTGATCGCA [SEQ ID NO: 55]
fNcoH: catgccATGGGAcatcaccatcaccatcacCAGGTTCAACTTGTACAGTCTGG [SEQ ID NO: 56]
rVHBsp: acctccggaAGATACAGTAACAGTAGTACCTTGTC [SEQ ID NO: 57]
Lk-Up: ccggaggtggcggatctggcggtggaggttcaggaggtggcg [SEQ ID NO: 58]
rLk-Dn: gatccgccacctcctgaacctccaccgccagatccgccacct [SEQ ID NO: 59]
fBamVL: ggcggatccGACATTCAGATGACCCAATCACCATC [SEQ ID NO: 60]
rHLkEco: ggaattcTTAAGAACCGCCACCgtgatggtgatggtgatgTTTGATGTCAACAGTGGTGCCT [SEQ ID NO: 61]
BX-VL1:
CAGATGACCCAATCACCATCTAGTCTGTCAGCTTCCGTTGGAGATCGTGTTACTATTACCTGTCGCACTTCTGAGAACATCTACTCCTACTTTG [SEQ ID NO: 62]
BX-VL2r:
TACACCTTCAGCCAGGGTCTTAGCATTGTAAATAAGGAGCTTTGGAGCTTTTCCTGACTTCTGTTGATACCAAGCAAAGTAGGAGTAGATGTTC [SEQ ID NO: 63]
BX-VL3:
AGACCCTGGCTGAAGGTGTACCGTCTCGTTTCAGTGGATCTGGTTCTGGAACTGAGTTCACCCTCACTATTTCATCCCTTCAACCAGAAGACTTCG [SEQ ID NO: 64]
BX-VL4r:
GATGTCAACAGTGGTGCCTCCACCGAAAGTGTGCAGAGATCCGTAATGGTGCTGACAGAAATAGGTAGCGAAGTCTTCTGGTTGAAGG [SEQ ID NO: 65]
[0033]
== Preparation 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 synthetic oligo DNA and PCR method. The modified α-factor signal sequence was linked to the C-terminus of the immunoporter in an attempt to improve secretion efficiency.
[0034]
First, the human single chain antibody gene C-terminal linker sequence was modified. PCR reaction solution containing 1 μM each of primers α-Factor-f and VL-Lk-R using pPIC9Kdel (see Japanese Patent Application Laid-Open No. 2001-333780) in which a human type single chain antibody gene of D4Sx5 type tail is incorporated as a template 50 μl of Expand High Fidelity PCR System (currently Roche Diagnostics) was prepared, treated at 94 ° C. for 2 minutes, and then subjected to a reaction cycle of [94 ° C. for 30 seconds, 50 ° C. for 20 seconds, 68 ° C. for 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 DNA fragment 1.3L-1
50 μl of a PCR reaction solution (Expand High Fidelity PCR system, currently Roche Diagnostics) containing 5 μM each of primers 1.3L-1f and 1.3L-1r was prepared, treated at 94 ° C. for 2 minutes, then [94 ° C. for 30 seconds, 50 The reaction cycle of 20 ° C. for 20 seconds and 68 ° C. for 10 seconds] was repeated 30 times to obtain a recombinant DNA fragment 1.3L-1 having the following sequence.
1.3L-1 sequence:
CTACTTCCCATGGTTCTGGTAAATCTTCCGAAGGTACCCCGAAGAAAAGCATCAAAAAGACTCCTAAGAAGGTAAAGAAGCCAGCAACCGCTGCTGGGACCAAGAAAGTGGCCAAGAGTGCGAAAAAGGTGAAAACACCTCAGCCAAAAAAAGCTGCCA [SEQ ID NO: 66]
[0036]
Similarly to 1.3L-1, a recombinant DNA fragment 1.3L-2 having the following sequence was obtained using primers 1.3L-2f and 1.3L-2r.
1.3L-2 sequence:
GCGAAAAAGGTGAAAACACCTCAGCCAAAAAAAGCTGCCAAGAGTCCAGCTAAGGCCAAAGCCCCTAAGCCCAAGGCGGCCAAGCCTAAGTCGGGGAAGCCGAAGGTTACAAAGGCAAAGAAGGCAGCTCCGAAGAAAAAGAGTTGAGTTAACGAATTCGC [SEQ ID NO: 67]
[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, 1 μM each of the primers common F and H1.3L-totalR was added, treated at 94 ° C. for 2 minutes, [94 The reaction cycle of 30 ° C. for 30 seconds, 50 ° C. for 20 seconds, and 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 reaction products using pro1-f and pro1-r, pro2-f and pro2-r, and using Pro / EcoRI-f and Pro / AvrII-r. The α-factor signal sequence was amplified, digested with EcoRI, and separated and purified by 3% agarose gel electrophoresis. Since the method and conditions at each stage are the same as in the case of PCR product 1.3L, detailed description is omitted.
[0039]
(4) Preparation of partial histone H1 tail type immunoporter gene
Ligation was performed by mixing equimolar amounts of the single-chain antibody gene fragment that had been treated with the restriction enzyme, 1.3 L of the PCR product, and the modified α-factor signal sequence. 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 NhoI cleavage site in the neomycin resistance region and the NcoI cleavage site in the His4 region were replaced with cttgag and ccatag from pPIC9K: see JP-A-2001-333780), and cloned. The base 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.a1st- 50 μl of a PCR reaction solution (Expand High Fidelity PCR system, currently Roche Diagnostics) containing 5 μM each of R is prepared, treated at 94 ° C. for 2 minutes, and then [94 ° C. for 30 seconds, 50 ° C. for 20 seconds, 68 ° C. for 10 seconds] This cycle was repeated 30 times to obtain the following DNA fragment 28a.a1st. Prepare 2 μl of the reaction solution containing this DNA fragment 28a.a1st and 50 μl of a PCR reaction solution (Expand High Fidelity PCR system) containing 1 μM each of the following primers, 28a.a1st-F, 28a.a2nd-R, and 94 ° C. for 2 minutes After the treatment, a reaction cycle of [94 ° C. for 30 seconds, 50 ° C. for 20 seconds, 68 ° C. for 10 seconds] was performed 30 times to obtain DNA fragment 28a.a2nd.
This DNA fragment was digested with NcoI and separated and purified by 3% agarose gel electrophoresis.
[0041]
The restriction enzyme-treated single-chain antibody gene fragment (prepared in the same manner as in the production of the partial histone H1 tail region) and the 28a.a2nd sequence were mixed in an equimolar amount to carry out a ligation reaction. 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 incorporated and cloned into the XhoI-EcoRI site of pPIC9Kdd-CaCH1.3Lpro by a conventional method, and the nucleotide sequence was confirmed. This plasmid was designated as pPIC9Kdd-CaCH28aa.
[0042]
Similarly, DNA fragments 23a.a2nd were obtained using the following primers, 23a.a1st-F, 23a.a1st-R, and 23a.a2nd-R. Thereafter, it was similarly amplified with a-Factor-f and FLAG / common R, and cloned into pPIC9Kdd-CaCH1.3Lpro to prepare plasmid pPIC9Kdd-CaCH23aa.
[0043]
Table 3. PCR primers used for the production of partial histone H1 tail type immunoporter genes (all 5 ′ → 3 ′). Upper case letters represent the same bases as the original sequence, and lower case letters represent bases different from the original sequence due to linkers, restriction enzyme site introduction, displacement introduction, and the like.
[0044]
α-Factor-f: TACTATTGCCAGCATTGCTGC [SEQ ID NO: 68]
VL-Lk-R: aaccatgggaagtagaaccTTTGATGTCAACAGTGGTGC [SEQ ID NO: 69]
1.3L-1f:
CTACTTCCCATGGTTCTGGTAAATCTTCCGAAGGTACCCCGAAGAAAAGCATCAAAAAGACTCCTAAGAAGGTAAAGAAGCCAGCAACCGCTGCTG [SEQ ID NO: 70]
1.3L-1r:
TAAGAAGGTAAAGAAGCCAGCAACCGCTGCTGGGACCAAGAAAGTGGCCAAGAGTGCGAAAAAGGTGAAAACACCTCAGCCAAAAAAAGCTGCCA [SEQ ID NO: 71]
1.3L-2f:
GCGAAAAAGGTGAAAACACCTCAGCCAAAAAAAGCTGCCAAGAGTCCAGCTAAGGCCAAAGCCCCTAAGCCCAAGGCGGCCAAGCCTAAGTCGGGG [SEQ ID NO: 72]
1.3L-2r:
CCTAAGCCCAAGGCGGCCAAGCCTAAGTCGGGGAAGCCGAAGGTTACAAAGGCAAAGAAGGCAGCTCCGAAGAAAAAGAGTTGAGTTAACGAATTCGC [SEQ ID NO: 73]
common F: ctacttcccatggTTCTGGTAAATCTTCCG [SEQ ID NO: 74]
H1.3L-totalR: gcgaattcgttaactcaACTCTTTTTCTTCGGAGCT [SEQ ID NO: 75]
pro1-f:
CTAGtctaGAGGCTGAAGCTgcAccTgtTaaTacAacTacCgaGgaCgaaacTgcCcaaatCccAgcAgaagcAgtTatAggttaTAG [SEQ ID NO: 76]
pro1-r:
cAttGttAgtTGAAttACTGaaAggTaaGacTgcGacGtcAaaGtcGccttcCaaGtcACTAtaaccTatAacTgcttcTgcTggGatttg [SEQ ID NO: 77]
pro2-f:
gtCttAccTttCAGTaaTTCAacTaaCaaTggAttGttAttCatCaaCactacAatCgcTagTatCgctgcAaaGgaGgaaggTgtTtcA [SEQ ID NO: 78]
pro2-r:
ttccgcggccgctatggccgacGTCGACagcttcagcTtctctCttctcTagTgaAacAccttcCtcCttTgcagcGatActA [SEQ ID NO: 79]
Pro / EcoRI-f:
CCGGAATTCGAGGCTGAAGCTGCACCTGTTAATACAAC [SEQ ID NO: 80]
Pro / AvrII-r:
CACCTAGGTCAAGCTTCAGCTTCTCTCTTCTCTAGTGA [SEQ ID NO: 81]
Modified α-factor signal sequence:
ccgGAATTCGAGGCTGAAGCTgcAccTgtTaaTacAacTacCgaGgaCgaaacTgcCcaaatCccAgcAgaagcAgtTatAggttaTAGTgaCttGgaaggCgaCttTgaCgtCgcAgtCttAccTttCAGTaaTTCAacTaaCaaTggAttGttAttCatCaaCactacAatCgcTagTatCgctgcAaaGgaGgaaggTgtTtcActAgagaaGagagaAgctgaagctTGACCTAGGtg [SEQ ID NO: 82]
28a.a1st-F: CTACTTCccatggTTCTGGTAAATCTTCCGAAGGTACCCCGAAGAAAAGCATCAAAAAGACTCCt [SEQ ID NO: 83]
28a.a1st-R:
GGCCACTTTCTTGGTCCcagcagcggttgctggcttctttaccttcttaGGAGTCTTTTTGATGC [SEQ ID NO: 84]
28a.a2nd-R:
ccgGAATTCcgTCATCAagagtcatctttataatcACTCTTGGCCACTTTCTTGGTCCc [SEQ ID NO: 85]
28a.a2nd sequence: CTACTTCccatggTTCTGGTAAATCTTCCGAAGGTACCCCGAAGAAAAGCATCAAAAAGACTCCtaagaaggtaaagaagccagcaaccgctgctgGGACCAAGAAAGTGGCCAAGAGTgattataaagatgactctTGATGAcgGAATTCcgg [SEQ ID NO: 86]
23a.a1st-F:
CTACTTCccatggTTCTGGTAAATCTTCCGAAGGTACCCCGAAGAAAAGCATCAAAAAGACTCC [SEQ ID NO: 87]
23a.a1st-R:
AGACTTGGTCCcagcagcggttgctggcttctttaccttcttaGGAGTCTTTTTGATGC [SEQ ID NO: 88]
23a.a2nd-R:
ccgGAATTCcgTCATCAagagtcatctttataatcAGACTTGGTCCc [SEQ ID NO: 89]
FLAG / common R: ccgGAATTCcgTCATCAagagtc [SEQ ID NO: 90]
23a.a2nd sequence:
CTACTTCccatggTTCTGGTAAATCTTCCGAAGGTACCCCGAAGAAAAGCATCAAAAAGACTCCtaagaaggtaaagaagccagcaaccgctgctgGGACCAAGTCTgattataaagatgactctTGATGAcgGAATTCcgg [SEQ ID NO: 91]
[0045]
== Expression and Preparation of Partial Histone H1 Tail Type Immunoporter ==
Using a Pichia expression kit (InVitrogen), a partial histone H1 tail immunoporter was prepared as follows.
The plasmid encoding the partial histone H1 tail type immunoporter prepared as described above was digested with the restriction enzyme BglII, introduced into the Pichia pastris GS115 strain by electroporation, and selected with a minimal medium to obtain a transformed strain. . This transformed strain was cultured in a liquid medium containing 0.5% methanol and 0.5M NaCl to induce expression of a partial histone H1 tail type immunoporter. The culture supernatant was diluted 3 times with 20 mM Tris (pH 7.5) -250 mM NaCl, and the partial histone H1 tail type immunoporter was adsorbed on the ion exchange resin DTREAMLINE-S, washed with the same buffer, and then washed with 20 mM Tris (pH 7.5). Elution with -1M NaCl. Thereafter, the partial histone H1 tail type immunoporter was adsorbed again on the TALON resin (Clontech) and eluted with 100 mM EDTA (pH 8.0).
[0046]
In order to prevent adsorption to the filtration membrane, 1/9 amount 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 gel filtration column of superdex75 (Pharmacia) equilibrated with 1M Arginine HCl-5% Glycerol. For each fraction fractionated by gel filtration, each peak fraction and several fractions before and after that were subjected to SDS-PAGE with 12% acrylamide gel using the absorbance at 280 nm as an index, and the fraction containing the target immunoporter was obtained by silver staining. Identified. Three large peak fractions containing the desired immunoporter were collected. Each fraction is 50 mM NaPO _Four Dialyzed against -500 mM NaCl pH 7.4 and concentrated by ultrafiltration.
[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 diluted with distilled water. When 3 μl of 1 mM PEI and 1 μg of DNA were mixed, calculation was carried out as (PEI free amino group) / (number of nucleic acid phosphate groups) = N / P = 1. A431 cells used for gene transfer were seeded to be approximately 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 immunoporter were mixed, diluted with distilled water to a total volume of 50 μl, and left at room temperature for 30 minutes. Then, 1 mM PEI was added so that it might become each N / P ratio, it mixed rapidly, and also left still for 30 minutes at room temperature. The total amount of the complex solution is added to each well containing 1 ml of DMEM medium containing 10% FCS, and 5% CO ₂ The gene was introduced by culturing at 37 ° C. in the presence.
[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.
In FIG. 5, the experimental result using a partial histone H1 tail type | mold immunoporter as an immunoporter is shown. As a control, the experiment was carried out under conditions without an immunoporter. As N / P increases, nonspecific adsorption to cells and the like increases, and target cell specificity for gene transfer decreases. Therefore, N / P is preferably small. However, the smaller the N / P, the lower the gene transfer efficiency itself into the cell. Conventionally, for example, as a peptide tail (AspAspAspAspSer) ₅ In the case of D4Sx5 tail type immunoporter (hereinafter referred to as CaCD20) having N / P = 10 to 20 or more, a sufficient amount (10 in this example) ⁵ In this example, N / P = 8 and 10 transfections were not observed. ⁵ Levels of gene transfer were 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. Conventional CaCD20 was used for control. 10 at N / P = 3 and 5 ⁵ Excellent gene transfer efficiency above the level. This indicates that the nucleic acid transport efficiency according to the present invention is quite high.
[0053]
【The invention's effect】
An object of the present invention is to provide a system with high nucleic acid transport efficiency in the recombinant immunogene method.
[0054]
[Sequence Listing]

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

Claims (5)

A fusion protein for transporting nucleic acids into cells,
A single chain antibody to the cell surface antigen;
Comprising a partial histone polypeptide for binding to a nucleic acid,
The fusion protein, wherein the partial histone polypeptide is a polypeptide having any one of SEQ ID NOs: 1 to 3. A fusion protein for transporting nucleic acids into cells,
A single chain antibody to the cell surface antigen;
Comprising a partial histone polypeptide for binding to a nucleic acid,
The partial histone polypeptide is a partial sequence of SEQ ID NO: 1,
Fusion protein.   The fusion protein according to claim 1 or 2, wherein the single chain antibody is a human type single chain antibody. A method for producing a fusion protein for transporting a nucleic acid into a cell,
Using a mRNA isolated from a hybridoma capable of producing a monoclonal antibody against a cell surface antigen to produce a cDNA encoding a single chain antibody against the surface antigen;
Adding a cDNA encoding a partial histone polypeptide to the single chain antibody cDNA to produce a fusion cDNA;
Expressing the fusion cDNA and isolating the fusion protein comprising the single chain antibody and the partial histone polypeptide;
Including
The method for producing a fusion protein, wherein the partial histone polypeptide is a polypeptide having any one of SEQ ID NOs: 1 to 3 or a partial sequence of SEQ ID NO: 1 . A nucleic acid transport method for transporting nucleic acid into a cell, comprising:
Mixing the nucleic acid with the fusion protein according to any one of claims 1 to 3 to produce a nucleic acid-protein mixture;
The nucleic acid - the step of administering a protein mixture to the cells and (except for administration to cells in a human individual)
A nucleic acid transport method comprising:

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