WO2018040537A1 - 人pd-1基因敲除的cldn18.2 特异性嵌合抗原受体t细胞的制备方法以及应用 - Google Patents

人pd-1基因敲除的cldn18.2 特异性嵌合抗原受体t细胞的制备方法以及应用 Download PDF

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WO2018040537A1
WO2018040537A1 PCT/CN2017/076513 CN2017076513W WO2018040537A1 WO 2018040537 A1 WO2018040537 A1 WO 2018040537A1 CN 2017076513 W CN2017076513 W CN 2017076513W WO 2018040537 A1 WO2018040537 A1 WO 2018040537A1
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sequence
seq
human
gene
cells
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代红久
熊波
赵旭东
张征
周杰
李晓亮
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南京凯地生物科技有限公司
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Priority claimed from CN201610796334.3A external-priority patent/CN106399375A/zh
Priority claimed from CN201610891327.1A external-priority patent/CN106480097A/zh
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Priority to CN201780007183.1A priority Critical patent/CN109844125A/zh
Publication of WO2018040537A1 publication Critical patent/WO2018040537A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4631—Chimeric Antigen Receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C12N15/867—Retroviral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the invention relates to a preparation method and application of a human PD-1 gene knockout CLDN18.2 specific chimeric antigen receptor T cell, and belongs to the technical field of biomedicine.
  • Cancer immunotherapy mainly includes adoptive cell therapy, immunomodulators, tumor vaccines, and immunological junction blocking therapy. Among them, in the field of cell therapy, CAR-T therapy has undoubtedly become the star of research institutions and pharmaceutical companies vying for "pursuit”.
  • CAR-T Chimeric Antigen Receptor T-Cell, chimeric antigen receptor T cell
  • the principle is mainly through the patient's own T cells for genetic modification of chimeric antigen receptors, CAR-T cells can specifically recognize tumor-associated antigens (tumor cells) Markers) to direct T cells to target tumors.
  • CAR-T cells Compared with conventional immune cells, CAR-T cells have higher targeting, killing activity and persistence, and can overcome the local immunosuppressive microenvironment of tumors and break the host immune tolerance state.
  • the therapy has significant efficacy in the treatment of acute leukemia and non-Hodgkin's lymphoma and is considered to be one of the most promising treatments for cancer.
  • CAR-T immunotherapy has problems such as "off-target”. Studying a broader spectrum, efficient and safe CAR-T immunotherapy method is the future development trend.
  • the key to the application of CAR-T technology is to identify at least one tumor-associated antigen that is highly expressed on the surface of tumor cells without expression or low expression on the surface of normal cells.
  • Claudin 18 (CLAUDIN18, The CLDN18) molecule is an integrated transmembrane protein (four-transmembrane protein, Tetraspanin) with four transmembrane hydrophobic regions and two extracellular loops (loop 1 is surrounded by hydrophobic region 1 and hydrophobic region 2; ring 2 is hydrophobic) Area 3 and the hydrophobic zone 4 is surrounded by).
  • CLDN18 is present in two different splice variants: splice variant 1 (CLDN 18.1) and splice variant 2 (CLDN 18.2) with molecular weights of 27.9/27.72 kD, respectively.
  • CLDN 18.1 and CLDN 18.2 differ in the N-terminal portion comprising the first transmembrane (TM) region and loop 1, while the C-terminal protein primary sequence is identical.
  • CLDN18.1 is selectively expressed in cells of normal lung, whereas CLDN18.2 is expressed only on gastric cells. Expression of CLDN 18.2 in normal stomach is then restricted to differentiated gastric epithelial cells. Studies have shown that CLDN18.2 is expressed in a variety of tumor tissues, such as pancreatic cancer, esophageal cancer, gastric cancer, bronchial cancer, breast cancer, and ENT tumor.
  • CLDN18.2 The differential expression of CLDN18.2 between cancer cells and normal cells, its membrane localization, its absence in most toxic-associated normal tissues, etc., makes CLDN18.2 an attractive target for cancer immunotherapy, and Targeting CLDN18 in cancer therapy. Specific therapy based on antigen chimeric receptor T cells is possible.
  • cancer cells have developed a set of escape mechanisms in the long-term evolution process, for example, when the surface of cancer cells appears "PD-L1" (Programmed Death Like 1) an antigen that binds to the PD-1 receptor on the surface of T cells, and when they bind, they transmit a negative regulatory signal to T cells, inducing T cells to enter a resting state, allowing T cells Not only can the cancer cells not be recognized, but also the proliferation of self-proliferation or apoptosis, resulting in the inability to activate the body's own immune response, so cancer cells can easily escape T cells, and thus "into the room", unscrupulous expansion.
  • PD-L1 Programmed Death Like 1
  • PD-L1 proteins exist on the surface of many cancer cells, including breast cancer, lung cancer, stomach cancer, intestinal cancer, esophageal cancer, ovarian cancer, cervical cancer, kidney cancer, bladder cancer, pancreatic cancer, and nerve glue. Tumor, melanoma, etc.
  • the present invention provides a method for preparing a human PD-1 gene knockout CLDN18.2-specific chimeric antigen receptor T cell, which is based on CRISPR -Cas9 technology to knock out the human PD-1 gene, in which the target sequence of the human PD-1 gene sequence simultaneously meets the following rules, rule 1): the target sequence conforms to the sequence arrangement rule of 5'-N(20)NGG, And the sequence of "N(20)" is a target sequence, wherein N is A or T or C or G; rule 2): the target sequence is located in the exon of the human PD-1 gene; and rule 3) : the targeting of the gRNA complementary to the target sequence on the human PD-1 gene is unique; and the nucleotide sequence of the CLDN18.2-specific chimeric antigen receptor molecule employed in the preparation method is: from 5 From the end to the 3' end, the leader sequence, the scFv sequence, the human CD8 hinge region sequence
  • the human CD8 hinge region sequence is SEQ ID No. 42; the human CD8 transmembrane region sequence is shown in SEQ ID No. 43; the human 4-1BB intracellular domain sequence is as SEQ ID. Shown as No. 44; and the CD3 ⁇ domain sequence is shown in SEQ ID No. 45.
  • the target sequence adopts any one of the sequences shown in SEQ ID No. 6 to SEQ ID No. 11.
  • the DNA primer set for the target sequence as shown in SEQ ID No. 6 the forward sequence is SEQ ID As shown in No. 12, the reverse sequence is as shown in SEQ ID No. 13; or
  • DNA primer set for the target sequence as shown in SEQ ID No. 7 its forward sequence is SEQ ID As shown in No. 14, the reverse sequence is as shown in SEQ ID No. 15; or
  • DNA primer set against the target sequence set forth in SEQ ID No. 8 its forward sequence is SEQ ID As shown in No. 16, the reverse sequence is as shown in SEQ ID No. 17; or
  • DNA primer set against the target sequence set forth in SEQ ID No. 9 its forward sequence is SEQ ID As shown in No. 18, the reverse sequence is as shown in SEQ ID No. 19; or
  • DNA primer set for the target sequence set forth in SEQ ID No. 10 its forward sequence is SEQ ID As shown in No. 20, the reverse sequence is as shown in SEQ ID No. 21; or
  • DNA primer set for the target sequence as shown in SEQ ID No. 11 its forward sequence is SEQ ID As shown in No. 22, the reverse sequence is shown in SEQ ID No. 23.
  • the preparation method comprises the step of constructing a recombinant plasmid expressing a gRNA targeting a human PD-1 gene and a CLDN18.2-specific chimeric antigen receptor, the construction process comprising the steps of: (a1) based on the target The sequence synthesizes a DNA primer set corresponding thereto, the DNA primer set comprising a single-stranded DNA of a forward sequence and a single-stranded DNA of a reverse sequence; (a2) the single-stranded DNA of the forward sequence and the reverse The single-stranded DNA of the sequence is annealed to obtain double-stranded DNA; (a3) the double-stranded DNA is ligated to the restriction endonuclease BsmBI of the lentiGuide-Puro plasmid to obtain a target for targeting the PD-1 gene.
  • a recombinant plasmid for gRNA (a4) a nucleotide sequence for synthesizing the CLDN18.2-specific chimeric antigen receptor molecule, which is ligated by molecular cloning to the recombinant plasmid expressing the gRNA targeting the PD-1 gene
  • a recombinant plasmid expressing a gRNA targeting a human PD-1 gene and a CLDN18.2-specific chimeric antigen receptor is obtained; the preparation method further comprises obtaining Cas9 The step of the nuclease plasmid.
  • the preparation method further comprises transfecting the 293T cell with the recombinant plasmid expressing the gRNA targeting the human PD-1 gene and the CLDN18.2 specific chimeric antigen receptor to obtain the first virus solution;
  • the preparation method further comprises transfecting the Cas9 nuclease plasmid into 293T cells to obtain a second virus solution; mixing the first virus solution and the second virus solution to infect T cells, and obtaining the cells from the infected cells.
  • the nucleotide sequence of the CLDN18.2-specific chimeric antigen receptor gene is passed as SEQ ID
  • the EF1 alpha promoter shown in No. 47 was expressed.
  • kits comprising the above-described recombinant plasmid expressing a gRNA targeting a human PD-1 gene and a CLDN18.2-specific chimeric antigen receptor, and a Cas9 nuclease plasmid.
  • a further aspect of the present invention provides a kit for obtaining a human PD-1 gene knockout CLDN18.2-specific chimeric antigen receptor T cell, the kit comprising the above-described kit and kit instructions, The preparation method described above is described in the kit manual.
  • the present invention provides the use of the human PD-1 gene knockout CLDN18.2-specific chimeric antigen receptor T cell obtained by the above preparation method for the preparation of a product for treating a tumor.
  • the tumor comprises a tumor cell that positively expresses a CLDN18.2 protein.
  • the use refers to the use of a medicament for the treatment of pancreatic cancer and gastric cancer.
  • the invention utilizes CRISPR/Cas9 genome editing technology and chimeric antigen receptor modified T cell technology to prepare human PD-1 gene knockout CLDN18.2 specific chimeric antigen receptor T cells, and the preparation method has simple steps and obtained
  • the novel CAR-T cells can specifically recognize tumor cells, target tumor cells more effectively, have a high killing rate on tumor cells, and can be used to prepare tumor-treating products, especially for the preparation of therapeutic expression of CLDN18.2. Drugs for tumor cells.
  • Example 1 is a flow cytometric test result of a control group in the identification step of Example 6;
  • Example 2 is a flow cytometric test result of the experimental group in the identification step of Example 6;
  • Figure 3 is a flow cytometric test result of the experimental + Cas9 group in the identification step of Example 6;
  • Example 4 is a result of detecting the expression of Cas9 nuclease by western blotting in the identification step of Example 6;
  • Example 5 is a result of detecting the expression of PD-1 protein by western blotting in the identification step of Example 6;
  • Figure 6 is a test result of tumor cell killing rate using pancreatic cancer tumor cell BxPC-3 as a target cell
  • Figure 7 shows the results of tumor cell killing rate test using gastric cancer tumor cell NUGC-4 as a target cell.
  • lentiGuide-Puro lentiviral vector plasmid is a CRISPR/Cas9 plasmid vector; the gRNA expressed by the recombinant vector encoding the gRNA synthetic DNA sequence inserted into the plasmid has a guiding effect and can correspond to the genomic target gene to be edited. The position was combined and played a guiding role, and the plasmid was purchased from Gensript.
  • Example 1 Construction of an expression plasmid for gRNA targeting human PD-1 gene
  • Step 1 Determination of the target sequence of the gene to be knocked out
  • the inventors Using the human PD-1 gene sequence as the gene to be knocked out, the inventors used the relevant software to sequence the gene (the inventors set relevant correlation parameters according to experience) to obtain dozens of candidate sequences, and then the inventors screened among these candidate sequences. A suitable target sequence.
  • the target sequence conforms to the sequence alignment rule of 5'-N(20)NGG, and the sequence of the "N(20)" is a target sequence, wherein N is A or T or C or G;
  • the target sequence is located in the exon of the human PD-1 gene
  • gRNA complementary to the target sequence on the human PD-1 gene is unique. "Unique” here means that any gRNA that conforms to the rule targets only the human PD-1 gene and does not target other genes.
  • the inventors designed six target sequences located on five exons of the human PD-1 gene.
  • exon 1 the sequence is SEQ ID No. 1
  • exon 2 the sequence of which is shown in SEQ ID No. 2
  • exon 3 the sequence of which is shown by SEQ ID No. 3
  • exon 4 the sequence of which is SEQ. ID No. 4
  • exon 5 the sequence of which is shown in SEQ ID No. 5
  • target sequence 1 is located on exon 2, and the sequence of target sequence 1 is as shown in SEQ ID No. 6;
  • the target sequence 2 is located on exon 2, and the sequence of target sequence 2 is as shown in SEQ ID No. 7.
  • the target sequence 3 is located on exon 3, and the sequence of target sequence 3 is as shown in SEQ ID No. 8.
  • the target sequence 4 is located on exon 5, and the sequence of target sequence 4 is as shown in SEQ ID No. 9.
  • the target sequence 5 is located on exon 1, and the sequence of target sequence 5 is as shown in SEQ ID No. 10.
  • the target sequence 6 is located on exon 5, and the sequence of target sequence 6 is shown in SEQ ID No. 11.
  • the corresponding DNA oligo primer sets were designed as follows:
  • DNA oligo primer set for target sequence 1 DNA oligo primer set for target sequence 1:
  • Reverse sequence 1 aaacCACGAAGCTCTCCGATGTGTc (see SEQ ID No.13);
  • DNA oligo primer set for target sequence 2 DNA oligo primer set for target sequence 2:
  • Reverse sequence 2 aaacCGCTCATGTGGAAGTCACGCc (see SEQ ID) No.15);
  • DNA oligo primer set for target sequence 3 DNA oligo primer set for target sequence 3:
  • Reverse sequence 3 aaacGGCCAGCCGGCCAGTTCCAAc (see SEQ ID) No.17);
  • DNA oligo primer set for target sequence 4
  • Reverse sequence 4 aaacGAGCAGACGGAGTATGCCACc (see SEQ ID) No.19);
  • DNA oligo primer set for target sequence 5
  • Reverse sequence 5 aaacACAGGCGCCCTGGCCAGTCGc (see SEQ ID) No.21);
  • Reverse sequence 6 aaacAGCGGAATGGGCACCTCATCc (see SEQ ID No.23).
  • Step 3 Oligonucleotide single strand annealing to obtain double-stranded oligonucleotide
  • double-stranded oligonucleotides 2-6 of target sequences 2-6 were obtained, respectively.
  • Annealing reaction system DNA of any one of the target sequences in step 2.
  • Oligo primer set (wherein, forward sequence: concentration 100 uM, 5 ⁇ l, reverse sequence: concentration 100 uM, 5 ⁇ l), PCR buffer (without magnesium ion) 40 ul;
  • Annealing reaction conditions The above annealing reaction system is uniformly mixed, placed in a PCR machine, and operated at 95 ° C for 3 minutes, then turned off, and naturally cooled to room temperature.
  • the lentiGuide-Puro plasmid was extracted using the Omega Plasmid Extraction Kit (D6922-02), and then digested with restriction endonuclease BsmBI at 37 ° C for 3-5 hours.
  • the enzymatically digested plasmid was subjected to 1% agarose gel electrophoresis to detect the enzymatic cleavage effect and the gel was recovered.
  • the gel recovery was carried out using an Omega agarose gel recovery kit (D2500-01). For details, see the kit manual.
  • the annealing product (any one of the double-stranded oligonucleotides 1-6) recovered by the gel is linked to the enzyme-removed plasmid of the above-mentioned gel, and the conditions of the specific system and the ligation reaction are as follows.
  • Ligation system gel-recovered PCR annealing product (any one of double-stranded oligonucleotides 1-6) 2 ⁇ l, gel-recovered BsmBI digested lentiGuide-Puro plasmid 2 ⁇ l; T4 ligase 0.5 ⁇ l; 10x ligase buffer 1 ⁇ l, the reaction reaction system volume 10 ⁇ l;
  • 5 ⁇ l of the ligation product obtained in step 4 was added to 50 ⁇ l of competent cells (Stbl3, purchased from Invitrogen, USA), ice bath for 30 min, 42 ° C for 45 s, ice bath for 2 min, and then 500 ⁇ l of anti-LB-free liquid medium, 37 Celsius, cultured at 200 rpm for 40 min, coated with ampicillin-resistant LB solid plates, overnight in a 37 ° C incubator. After waiting for a single colony to appear, pick 5 medium-sized colonies, extract the plasmid, and send it to commercial sequencing company (Shanghai Biotech) for sequencing.
  • Stbl3 purchased from Invitrogen, USA
  • Sequencing revealed that the sequences of the fragments inserted at the BsmBI of each lentiGuide-Puro plasmid are their respective gRNA sequence counterparts, ie, the DNA of the respective target sequences in step 2)
  • the forward sequence of the primer set of oligo corresponds.
  • Step 6 Extraction and purification of expression plasmid of gRNA targeting human PD-1 gene
  • the correctly cloned clones were expanded and cultured, and the kit purchased from Shanghai Youningwei Company Qiagen Plasmid was used.
  • the Purification Kit extracts the plasmid in exactly the manner described in the kit.
  • the Cas9-NLS-Flag nuclease sequence can be selected as SEQ ID No. 24, SEQ ID Any one of No. 25, SEQ ID No. 26 or SEQ ID No. 27.
  • the Cas9-NLS-Flag nuclease sequence was ligated into a lentiviral vector by molecular cloning to obtain a Cas9 nuclease virus vector.
  • the specific procedure is as follows: the Cas9-NLS-Flag nuclease sequence is passed through a forward primer (forward sequence 5'-aagtacccgggtaggtcttgaaaggagtgggaattggctcc-3', such as SEQ ID No.
  • PCR amplification was carried out using the vector lentiCas9-Blast (Genscript) as a template, and the amplified PCR product and the lentiviral vector lentiGuide-Puro (Genscript) were linked by restriction endonucleases SmaI and MluI.
  • the ligation product was transformed with competent cell Stbl3, and the positive clone was detected by PCR and sent to a commercial sequencing company for sequencing (Shanghai Shenggong) to obtain a positive Cas9 nuclease virus vector, ie, a Cas9 nuclease plasmid.
  • Example 3 The sequence of the CLDN18.2-specific chimeric antigen receptor was cloned into the expression plasmid of the gRNA targeting the PD-1 gene obtained in Example 1.
  • Step 1) Determination of amino acid sequence and nucleotide sequence of CLDN18.2 specific chimeric antigen receptor
  • a CLDN18.2 specific chimeric antigen receptor was constructed.
  • the molecular structure of the chimeric antigen receptor (CAR) is mainly composed of an extramembranous antigen-binding region (scFv, an extracellular single-chain antibody portion), a hinge region (transmembrane protein portion), and an intracellular signaling region.
  • scFv extramembranous antigen-binding region
  • a hinge region transmembrane protein portion
  • intracellular signaling region intracellular signaling region.
  • CLDN18.1 and CLDN18.2 have 96% homology in sequence, it is the key to construct the CAR molecule to identify scFv fragments that specifically recognize CLDN18.2 but not CLDN18.1.
  • the amino acid sequence of the CLDN18.2 specific CAR molecule from the amino terminus to the carboxy terminus, by the leader sequence (eg SEQ ID No. 30), scFv sequence (select any one of the sequences shown in SEQ ID No. 31 or 32 or 33), human CD8 hinge region sequence (such as SEQ ID No. 34), human CD8 transmembrane region sequence (as shown in SEQ ID No. 35), human 4-1BB intracellular domain sequence (such as SEQ ID)
  • the sequence shown in No. 36) and the CD3 ⁇ domain sequence (shown as SEQ ID No. 37) are sequentially connected in series.
  • the nucleotide sequence of the CLDN18.2 specific CAR molecule from the 5' end to the 3' end, by the leader sequence (eg SEQ ID No. 38), scFv sequence (as shown in SEQ ID No. 39 or 40 or 41), human CD8 hinge region sequence (such as SEQ ID No. 42), human CD8 transmembrane region sequence (as shown in SEQ ID No. 43), human 4-1BB intracellular domain sequence (eg SEQ ID)
  • the sequence shown in No. 44) and the CD3 ⁇ domain sequence (shown as SEQ ID No. 45) are sequentially connected in series.
  • the complete nucleotide sequence of the CLDN18.2 specific CAR molecule is SEQ ID Shown as No. 46 (wherein the scFv sequence adopts the sequence shown in SEQ ID No. 40).
  • the specific operation is as follows: construct a single coding frame of the nucleotide sequence expression cassette of the CLDN18.2-specific CAR molecule (SEQ ID No. 46), using the EF1 alpha promoter (SEQ ID NO: SEQ ID No. 47) was expressed.
  • the scFv amino acid sequence was first optimized by human codon, and then the guide sequence, scFv sequence, CD8 hinge region, CD8 transmembrane region, 4-1BB intracellular domain, CD3 ⁇ domain CAR molecule sequence were included.
  • Synthetic (general organism), primers amplify the synthetic CAR molecule sequence, and digest the expression plasmid (lenti-PD1-sgRNA) of the PD-1 gene targeting the PD-1 gene obtained by the restriction enzymes BstEII and MluI. After the product was recovered, the T4 ligase was ligated overnight at 16 degrees in the PCR instrument, and the ligated product was transformed with the competent cell Stbl3. The positive clone was screened and sequenced (Anhui, General Bio), and the sequencing result was fitted with the CLDN18.2 specific CAR.
  • the correctly cloned clones were expanded and cultured, and the kit purchased from Shanghai Youningwei Company Qiagen Plasmid was used.
  • the Purification Kit extracts the plasmid in exactly the manner described in the kit.
  • the recombinant plasmid obtained by the expression of the human PD-1 gene and the recombinant plasmid encoding the CLDN18.2-specific chimeric antigen receptor obtained in Example 3 (KD-022 CAR) lenti-PD1-sgRNA lentiviral vector) and packaging vectors PSPAX2 and VSVG were co-transfected with Lenti-X in a ratio of 10:8:5 with polyethyleneimine transfection reagent (purchased from Sigma, product model number 408727) 293T cells (see Sigma Transfection Instructions for specific transfection procedures), replaced with complete medium 6 hours after transfection (purchased from Life) Technologies, model number 11995-065), after 48 hours and 72 hours of incubation, the supernatants of the cells rich in lentiviral particles were collected, and the virus supernatant was concentrated by ultracentrifugation to obtain the specific embedded CLDN18.2.
  • Viral fluid of a lentiviral vector combining an antigen receptor molecule and a gRNA of a human PD-1 gene (hereinafter referred to as KD-022) CAR lenti-PD1-sgRNA lentiviral vector virus solution).
  • the Cas9 nuclease plasmid obtained in Example 2 and the packaging vectors PSPAX2 and VSVG were obtained in the same manner as above in the same manner as above to obtain a virus solution carrying the Cas9 nuclease virus vector.
  • Packaging Carriers by Lenti-X Packaging Single Shots (Takara) instructions were prepared; all vectors were subjected to high-purity endotoxin-free extraction using Qiagen Plasmid Purification Kit and were ready for infection.
  • peripheral blood mononuclear cells were isolated by density gradient centrifugation; paramagnetic beads conjugated with anti-CD3 antibody and anti-CD28 antibody (purchased from Invitrogen, USA)
  • Dynabeads® Human T-Activator CD3/CD28 Human T-Activator CD3/CD28
  • peripheral blood mononuclear cells are diluted to a concentration of (10 ⁇ 30) ⁇ 106 single cells/ml, and the magnetic beads and cells are 3:1. Mix in a petri dish and incubate for 2-3 hours at room temperature using a magnetic particle collector (Magnetic Particles Concentrator (abbreviated as MPC, purchased from Invitrogen, USA) enriched CD3+ T cells.
  • MPC Magnetic Particles Concentrator
  • the enriched CD3+ T cells were resuspended in the medium (purchased from American Life) Technologies, product information for OpTmizerTM T-Cell Expansion SFM), adjusted to a cell solubility of 1 x 106 cells/ml, and finally at 37 ° C, 5% Incubate for 2 days in a CO2 incubator.
  • Example 6 Preparation of human PD-1 gene knockout CLDN18.2 specific chimeric antigen receptor T cells
  • the CD3+ T cells obtained in Example 5 were inoculated into a 24-well plate at a concentration of 1 ⁇ 105 cells/ml at 37 ° C, 5%.
  • the CO2 environment is cultured for about 24 hours (the culture time is determined according to the specific practice, and generally, the cell confluence rate is between 50-70% in the case of viral liquid infection).
  • the human PD-1 gene was knocked out 48 hours after infection and the CAR-T cells targeting CLDN18.2 were simultaneously expressed (ie, human PD-1 gene knockout CLDN18.2) CAR-T cells), the next functional experiment can be performed.
  • the cells were centrifuged, washed twice with PBS and resuspended in FACS (PBS containing 0.1% sodium azide and 0.4% BSA); Biotin-labeled Antibody goat anti-mouse-F(ab)2 antibody (115-066-072, Jackson Immunoresearch) was added to the cell suspension and incubated at 4 ° C for 30 minutes; after washing the cells twice, add phycoerythrin (PE)-labeled Streptavidin (405203, Biolenged), room temperature 30 minutes; BD FacsCanto II is used to obtain stained cells, and FlowJo is used to analyze the results.
  • FACS PBS containing 0.1% sodium azide and 0.4% BSA
  • Biotin-labeled Antibody goat anti-mouse-F(ab)2 antibody 115-066-072, Jackson Immunoresearch
  • PE phycoerythrin
  • Streptavidin 405203, Biolenged
  • BD FacsCanto II is used to
  • the control group was infected with empty vector virus T cells, and almost no expression of CAR molecules was detected; as shown in Figure 2, the experimental group was infected with CLDN18.2.
  • the T cell of CAR molecular virus solution the expression rate of CAR molecule reached 36.8%; shown in Figure 3, the experimental + Cas9 group was the human PD-1 gene knockout CLDN18.2 specific chimeric antigen receptor obtained in Example 6. In T cells, the expression rate of CAR molecules reached 41.3%.
  • the expression of Cas9 nuclease was first detected by western blotting.
  • the human PD-1 gene knockout CLDN18.2-specific chimeric antigen receptor T cells obtained in Example 6 were subjected to centrifugation, washing, and lysed in a RIPA lysate containing a protease inhibitor for 1 hour, and lysed after high-speed centrifugation.
  • Supernatant with 4 x LDS Loading Buffer Blend at 4-12% Bis-Tris Precast (NP0335BOX, Life) was carried out in technologies, and then the protein was transferred to the PVDF membrane by electroporation.
  • the PVDF membrane was blocked in TBST containing 5% skim milk powder for 1 hour at 1:2500 mouse anti-Flag antibody (Anti Flag -Tag Mouse mAb, ab002-040, Incubate overnight at 4 °C in MultiSciences).
  • the membrane was washed 3 times with TBST and coupled with HRP goat anti-mouse IgG secondary antibody at 1:10000 (115-035-003, Jackson) Immunoresearch) Incubate for 1 hour at room temperature. After incubating with ECL Plus luminescence for 5 minutes, in Tanon Exposure in 9500 machines.
  • the human PD-1 gene knockout CLDN18.2-specific chimeric antigen receptor T cells obtained in Example 6 were subjected to centrifugation, washing, and lysed in a RIPA lysate containing a protease inhibitor for 1 hour, and lysed after high-speed centrifugation.
  • the PVDF membrane was blocked in TBST containing 5% skim milk powder for one hour and incubated overnight at 1:1000 in rabbit anti-human PD-1 antibody (PRS4065, Sigma) at 4 °C.
  • the membrane was washed 3 times with TBST and coupled with HRP goat anti-rabbit IgG secondary antibody at 1:10000 (111-035-003, Jackson) Immunoresearch) Incubate for 1 hour at room temperature. After incubating with ECL Plus luminescence for 5 minutes, in Tanon Exposure in 9500 machines.
  • this experimental group is infected with CLDN18.2
  • the T cells of the CAR molecular virus solution as shown in the column of the "experimental group" in Fig.
  • the protein expression amount of PD-1 is the human PD-1 gene knockout CLDN18.2 specific chimeric antigen obtained in Example 6.
  • Receptor T cells (as shown in the column labeled "Experimental Group + Cas9" in Figure 5) showed a significant decrease.
  • the human PD-1 gene knockout CLDN18.2-specific chimeric antigen receptor T cell pair obtained in Example 6 was evaluated using the 7-AAD/CFSE Cytotoxicity Test Kit (available from Biovision, product number K315-100).
  • Pancreatic cancer tumor cells BxPC-3 ATCC product, product number: CRL-1687TM
  • gastric cancer tumor cell NUGC-4 purchased from Nanjing Kezhen Biotechnology Co., Ltd., product number CBP60493
  • CSFE-labeled tumor cells 2 ⁇ 104 cells per well were plated in culture plates, and the human PD-1 gene knockout CLDN18 obtained in Example 6 was obtained at a ratio of 10:1, 5:1, and 1:1, respectively.
  • .2 specific chimeric antigen receptor T cells were added to the above tumor cells, cultured for 20 hours, centrifuged to remove the supernatant, and the cell pellet was washed and stained with 7AAD, BD. FacsCanto II was used to obtain stained cells and FlowJo was used to analyze the results.
  • T cells which are not infected with virus, T cells infected with Cas9 virus solution, and T cells infected with empty vector virus solution (Control", “Cas9” and “empty” shown in Fig. 7, respectively The result of the vector", infected with CLDN18.2
  • the T cells of the CAR molecular virus solution (the results of the "experimental group” shown in Fig. 7) have a significant killing effect on the target cell NUGC-4, and the killing rate of the target target ratio of 10:1 is 26.5%
  • Example 6 The obtained human PD-1 gene knockout CLDN18.2-specific chimeric antigen receptor T cells ("Experiment + Cas9 group” shown in Fig.
  • the present invention utilizes the CRISPR/Cas9 genome editing technique to obtain a targeted CLDN18.2 knockdown human PD-1 gene.
  • CAR-T cells can be used to prepare related products for treating tumors, for example, to prepare drugs for treating tumors expressing CLDN18.2 protein, particularly for preparing drugs for treating solid tumors such as pancreatic cancer and gastric cancer.
  • Tacggtccca aggtcaggag ggcagggctg gggttgactc
  • Atgatcaaga gatacgacga gcaccaccag gacctgaccc Tgctgaaagc tctcgtgcgg 1020
  • Ggctacattg acggcggagc cagccaggaa gagttctaca Agttcatcaa gcccatcctg 1140
  • Atgatcgcca agagcgagca ggaaatcggc aaggctaccg Ccaagtactt cttctacagc 3120
  • Atgatcaaga gatacgacga gcaccaccag gacctgaccc Tgctgaaagc tctcgtgcgg 1020
  • Ggctacattg acggcggagc cagccaggaa gagttctaca Agttcatcaa gcccatcctg 1140
  • Atgatcgcca agagcgagca ggaaatcggc aaggctaccg Ccaagtactt cttctacagc 3120
  • Atgatcaaga gatacgacga gcaccaccag gacctgaccc Tgctgaaagc tctcgtgcgg 960
  • Ggctacattg acggcggagc cagccaggaa gagttctaca Agttcatcaa gcccatcctg 1080
  • Atgatcgcca agagcgagca ggaaatcggc aaggctaccg Ccaagtactt cttctacagc 3060
  • Atgatcaaga gatacgacga gcaccaccag gacctgaccc Tgctgaaagc tctcgtgcgg 960
  • Ggctacattg acggcggagc cagccaggaa gagttctaca Agttcatcaa gcccatcctg 1080
  • Atgatcgcca agagcgagca ggaaatcggc aaggctaccg Ccaagtactt cttctacagc 3060
  • Trp Ile Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
  • Tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg Cagtgcacac gagggggctg 120
  • Gcagtgcaca cgagggggct ggacttcgcc tgtgatatct Acatctgggc gcccttggcc 960

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Abstract

提供了一种人PD-1基因敲除的CLDN18.2特异性嵌合抗原受体T细胞的制备方法，以及获得人PD-1基因敲除的CLDN18.2特异性嵌合抗原受体T细胞的质粒和试剂盒，以及该制备方法获得的人PD-1基因敲除的CLDN18.2特异性嵌合抗原受体T细胞在制备治疗肿瘤的产品方面的应用。

Description

人PD-1基因敲除的CLDN18.2 特异性嵌合抗原 受体T细胞的制备方法以及应用 技术领域
本发明涉及人PD-1基因敲除的CLDN18.2特异性嵌合抗原受体T细胞的制备方法以及应用，属于生物医药技术领域。
背景技术
随着生物技术的飞速发展，免疫细胞治疗已成为癌症治疗领域的第四大疗法。癌症免疫疗法主要包括过继性细胞治疗、免疫调节剂、肿瘤疫苗以及免疫结合点阻断治疗等。其中，在细胞治疗领域，CAR-T疗法无疑已成为研究机构和制药公司争相“追捧”的明星。
CAR-T（Chimeric Antigen Receptor T-Cell，嵌合抗原受体T细胞）免疫疗法，其原理主要是通过取病人自身T细胞进行嵌合抗原受体的基因修饰，CAR-T细胞能够特异性地识别肿瘤相关抗原（肿瘤细胞标志物），从而引导T细胞靶向肿瘤。相较于常规免疫细胞，CAR-T细胞的靶向性、杀伤活性和持久性都更高，并且可以克服肿瘤局部免疫抑制微环境并打破宿主免疫耐受状态。该疗法在急性白血病和非霍奇金淋巴瘤的治疗上有着显著的疗效，被认为是最有前景的肿瘤治疗方式之一。
然而，90%的癌症是实体瘤，更多种类的实体瘤以及更多的肿瘤表面特异性靶点抗原有待进一步确认。CAR-T免疫疗法应用于实体瘤治疗的最大难点在于CAR-T细胞对于肿瘤细胞上表达的特异性要求非常高，否则容易造成T细胞持续激活而杀伤正常细胞，或释放大量细胞因子引起严重副作用。既然CAR-T免疫疗法对于肿瘤细胞表达的特异性要求非常高，但肿瘤特异的靶抗原可选择性并不多，并且，肿瘤表达的大多数抗原不具备肿瘤特异，以肿瘤相关抗原作为靶点的CAR-T免疫疗法存在“脱靶”等问题。研究更广谱、高效、安全的CAR-T免疫治疗方法是未来的发展趋势。
因此，发挥CAR-T技术应用的关键在于确定至少一种肿瘤相关的抗原，这种抗原在肿瘤细胞表面高表达，而在正常细胞表面无表达或低表达。
密蛋白18（CLAUDIN18, CLDN18）分子是整合跨膜蛋白（四次跨膜蛋白，Tetraspanin），具有四个跨膜疏水区和两个胞外环（环1由疏水区1和疏水区2环绕而成；环2由疏水区 3和疏水区4环绕而成）。CLDN18以两种不同剪接变体存在：剪接变体1（CLDN18.1）和剪接变体2（CLDN18.2），分子量分别为27.9/27.72kD。CLDN18.1和CLDN18.2在包含第一个跨膜（TM）区和环1的N端部分中存在差异，而C端的蛋白质一级序列相同。
CLDN18.1在正常肺的细胞中选择性表达，而CLDN18.2仅在胃细胞上表达。然后CLDN18.2在正常胃中的表达局限于已分化的胃上皮细胞中。已有研究表明CLDN18.2在多种肿瘤组织中表达，例如胰腺癌、食管癌、胃癌、支气管癌、乳腺癌以及 ENT肿瘤。
CLDN18.2在癌细胞和正常细胞之间的差异表达、其膜定位、其在大多数毒性相关正常组织中不存在等特性，使得CLDN18.2成为癌症免疫治疗的有吸引力的靶标，并且在癌症治疗中使用靶向CLDN18. 2的基于抗原嵌合受体T细胞的特异性疗法成为可能。
另外，T细胞表面有一个重要的“接头”分子，他们把它称为“程序性死亡受体-1”或PD-1（Programmed Death 1），正常状态下，它都能识别出癌细胞表面的抗原，并与之结合。然而癌细胞在长期的进化过程中也发展出了一套自身的逃逸机制，例如，当癌细胞表面出现“PD-L1”（Programmed Death Like 1）的抗原，该抗原也能与T细胞表面的PD-1受体结合，并且它俩一旦结合便会向T细胞传递一种负向调控信号，诱导T细胞进入静息状态，使T细胞不但无法识别癌细胞，还会出现自身增殖减少或凋亡，导致无法启动机体的自身免疫反应，因此癌细胞可以毫不费力躲过T细胞，从而“登堂入室”、肆无忌惮地扩张。
值得一提的是，许多癌细胞表面都存在这种PD-L1蛋白，包括乳腺癌、肺癌、胃癌、肠癌、食管癌、卵巢癌、宫颈癌、肾癌、膀胱癌、胰腺癌、神经胶质瘤、黑色素瘤等。
 技术问题
科学家们开始思索如何能阻止T细胞的PD-1受体与癌细胞的PD-L1配体的结合，使得T细胞能逃逸癌细胞的抑制信号从而高效识别和攻击癌细胞。
技术解决方案
鉴于相关技术的上述问题和/或其他问题，本发明一方面提供了一种人PD-1基因敲除的CLDN18.2特异性嵌合抗原受体T细胞的制备方法，所述制备方法基于CRISPR-Cas9技术来敲除人PD-1基因，其中以人PD-1基因序列中同时符合以下规则的作为靶序列，规则1）：靶序列符合5’-N（20）NGG的序列排列规则，且所述“N（20）”所述序列为靶序列，其中N为A或T或C或G；规则2）：靶序列位于所述人PD-1基因的外显子；且规则3）：与靶序列互补的gRNA在人PD-1基因上的靶向是唯一的；以及，所述制备方法所采用的CLDN18.2特异性嵌合抗原受体分子的核苷酸序列为：从5’端到3’端，由引导序列、scFv序列、人CD8铰链区序列、人CD8跨膜区序列、人4-1BB胞内结构域序列和CD3ζ结构域序列依次串联而成；其中，所述引导序列如SEQ ID No.38所示；所述scFv序列如SEQ ID No.39或40或41任意一个所示；所述人CD8铰链区序列如SEQ ID No.42所示；所述人CD8跨膜区序列如SEQ ID No.43所示；所述人4-1BB胞内结构域序列如SEQ ID No.44所示；且所述CD3ζ结构域序列如SEQ ID No.45所示。
优选的，所述靶序列采用如SEQ ID No.6至SEQ ID No.11所示的任意一个序列。
优选的，针对所述如SEQ ID No.6所示的靶序列的DNA引物组：其正向序列如SEQ ID No.12所示，其反向序列如SEQ ID No.13所示；或
针对所述如SEQ ID No.7所示的靶序列的DNA引物组：其正向序列如SEQ ID No.14所示，其反向序列如SEQ ID No.15所示；或
针对所述如SEQ ID No.8所示的靶序列的DNA引物组：其正向序列如SEQ ID No.16所示，其反向序列如SEQ ID No.17所示；或
针对所述如SEQ ID No.9所示的靶序列的DNA引物组：其正向序列如SEQ ID No.18所示，其反向序列如SEQ ID No.19所示；或
针对所述如SEQ ID No.10所示的靶序列的DNA引物组：其正向序列如SEQ ID No.20所示，其反向序列如SEQ ID No.21所示；或
针对所述如SEQ ID No.11所示的靶序列的DNA引物组：其正向序列如SEQ ID No.22所示，其反向序列如SEQ ID No.23所示。
优选的，所述制备方法包括构建表达靶向人PD-1基因的gRNA和CLDN18.2特异性嵌合抗原受体的重组质粒的过程，该构建过程包括以下步骤：（a1）基于所述靶序列合成与之对应的DNA引物组，所述DNA引物组包括正向序列的单链DNA和反向序列的单链DNA；（a2）将所述正向序列的单链DNA与所述反向序列的单链DNA退火，获得双链DNA；（a3）将所述双链DNA连接到lentiGuide-Puro质粒的限制性内切酶BsmBI的酶切位点处，得到表达靶向PD-1基因的gRNA的重组质粒；（a4）合成所述CLDN18.2特异性嵌合抗原受体分子的核苷酸序列，通过分子克隆的方式连接到所述表达靶向PD-1基因的gRNA的重组质粒的限制性内切酶BstEII和MluI的酶切位点处，获得了表达靶向人PD-1基因的gRNA和CLDN18.2特异性嵌合抗原受体的重组质粒；所述制备方法还包括获得Cas9核酸酶质粒的步骤。
优选的，所述的制备方法还包括将所述表达靶向人PD-1基因的gRNA和CLDN18.2特异性嵌合抗原受体的重组质粒转染293T细胞，获得第一病毒液；所述的制备方法还包括将所述Cas9核酸酶质粒转染293T细胞，获得第二病毒液；将所述第一病毒液和所述第二病毒液混合后感染T细胞，从感染后的细胞中获得人PD-1基因敲除的CLDN18.2特异性嵌合抗原受体T细胞。
优选的，所述步骤（a4）中，所述CLDN18.2特异性嵌合抗原受体基因的核苷酸序列通过如SEQ ID No.47所示的EF1 alpha启动子来进行表达。
本发明另一方面提供了一种成套质粒，由上述的表达靶向人PD-1基因的gRNA和CLDN18.2特异性嵌合抗原受体的重组质粒和Cas9核酸酶质粒组成。
本发明再一方面提供了一种获得人PD-1基因敲除的CLDN18.2特异性嵌合抗原受体T细胞的试剂盒，所述试剂盒包括上述的成套质粒以及试剂盒说明书，所述试剂盒说明书记载有上述的制备方法。
本发明还一方面提供了上述的制备方法获得的人PD-1基因敲除的CLDN18.2特异性嵌合抗原受体T细胞，在制备用于治疗肿瘤的产品中的应用。
优选的，所述肿瘤包括阳性表达CLDN18.2蛋白的肿瘤细胞。
优选的，所述应用是指制备治疗胰腺癌和胃癌的药物方面的应用。
 有益效果
本发明利用CRISPR/Cas9基因组编辑技术和嵌合抗原受体修饰T细胞技术来制备人PD-1基因敲除的CLDN18.2特异性嵌合抗原受体T细胞，该制备方法步骤简单，获得的新型CAR-T细胞能特异识别肿瘤细胞，能更有效地靶向攻击肿瘤细胞，对肿瘤细胞的杀伤率高，并且可以用于制备治疗肿瘤的产品，尤其是用于制备治疗表达CLDN18.2的肿瘤细胞的药物。
附图说明
图1为实施例6的鉴定步骤中对照组的流式细胞检测结果；
图2为实施例6的鉴定步骤中实验组的流式细胞检测结果；
图3为实施例6的鉴定步骤中实验+Cas9组的流式细胞检测结果；
图4为实施例6的鉴定步骤中western blotting检测Cas9核酸酶的表达的结果；
图5为实施例6的鉴定步骤中western blotting检测PD-1蛋白的表达的结果；
图6为以胰腺癌肿瘤细胞BxPC-3为靶细胞的肿瘤细胞杀伤率测试结果；
图7为以胃癌肿瘤细胞NUGC-4为靶细胞的肿瘤细胞杀伤率测试结果。
 本发明的最佳实施方式
以下通过实施例对本发明作进一步的说明，但本发明并不限于这些具体实施方式。
下面实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
lentiGuide-Puro慢病毒载体质粒：是一种CRISPR/Cas9质粒载体；将编码gRNA人工合成DNA序列插入到该质粒后的重组载体表达的gRNA具有导向作用，可以与所要编辑的基因组靶向基因的相应位置进行结合，起到导向的作用，该质粒购买自Gensript公司。
实施例1：靶向人PD-1基因的gRNA的表达质粒的构建
步骤1、待敲除的基因靶序列的确定
以人PD-1基因序列为待敲除基因，发明人利用相关软件对该基因进行序列分析（发明人根据经验设置相关分析参数）获得数十个候选序列，然后发明人在这些候选序列中筛选合适的靶序列。
发明人经过大量的研究和测试，确定了靶序列的设计规则：
1）靶序列符合5’-N（20）NGG的序列排列规则，且所述“N（20）”所述序列为靶序列，其中N为A或T或C或G；
2）靶序列位于所述人PD-1基因的外显子；
3）与靶序列互补的gRNA在人PD-1基因上的靶向是唯一的。这里的“唯一”是指任意一条符合规则的gRNA仅靶向人PD-1基因，不会靶向其他基因。
最终，发明人设计出来了6个靶序列，位于人PD-1基因的五个外显子上。
从5’端到3’端，依次将五个外显子序列命名为外显子1（其序列如SEQ ID No.1所示）、外显子2（其序列如SEQ ID No.2所示）、外显子3（其序列如SEQ ID No.3所示）、外显子4（其序列如SEQ ID No.4所示）和外显子5（其序列如SEQ ID No.5所示）。
其中，靶序列1位于外显子2上，靶序列1的序列如SEQ ID No.6所示；
靶序列2位于外显子2上，靶序列2的序列如SEQ ID No.7所示；
靶序列3位于外显子3上，靶序列3的序列如SEQ ID No.8所示；
靶序列4位于外显子5上，靶序列4的序列如SEQ ID No.9所示；
靶序列5位于外显子1上，靶序列5的序列如SEQ ID No.10所示；
靶序列6位于外显子5上，靶序列6的序列如SEQ ID No.11所示。
步骤2、DNA oligo引物的设计
根据上面确定的6个靶序列，分别设计与它们对应的DNA oligo引物组，具体如下：
针对靶序列1的DNA oligo引物组：
正向序列1：caccgACACATCGGAGAGCTTCGTG （参见SEQ ID No.12）；
反向序列1：aaacCACGAAGCTCTCCGATGTGTc （参见SEQ ID No.13）；
针对靶序列2的DNA oligo引物组：
正向序列2：caccgGCGTGACTTCCACATGAGCG （参见SEQ ID No.14）；
反向序列2：aaacCGCTCATGTGGAAGTCACGCc （参见SEQ ID No.15）；
针对靶序列3的DNA oligo引物组：
正向序列3：caccgTTGGAACTGGCCGGCTGGCC （参见SEQ ID No.16）；
反向序列3：aaacGGCCAGCCGGCCAGTTCCAAc （参见SEQ ID No.17）；
针对靶序列4的DNA oligo引物组：
正向序列4：caccgGTGGCATACTCCGTCTGCTC （参见SEQ ID No.18）；
反向序列4：aaacGAGCAGACGGAGTATGCCACc （参见SEQ ID No.19）；
针对靶序列5的DNA oligo引物组：
正向序列5：caccgCGACTGGCCAGGGCGCCTGT （参见SEQ ID No.20）；
反向序列5：aaacACAGGCGCCCTGGCCAGTCGc （参见SEQ ID No.21）；
针对靶序列6的DNA oligo引物组：
正向序列6：caccgGATGAGGTGCCCATTCCGCT （参见SEQ ID No.22）；
反向序列6：aaacAGCGGAATGGGCACCTCATCc （参见SEQ ID No.23）。
步骤3、寡核苷酸单链退火获得双链寡核苷酸
将合成的针对靶序列1的两条DNA oligo单链（正向序列1和反向序列1）退火，获得双链寡核苷酸1。
同样的，分别获得靶序列2-6的双链寡核苷酸2-6。
退火反应体系：步骤2中的任意一个靶序列的DNA oligo引物组（其中，正向序列：浓度100uM，5μl，反向序列：浓度100uM，5μl），PCR缓冲液（不含镁离子）40ul；
退火反应条件：将上述退火反应体系混合均匀后放入PCR仪中，95摄氏度作用3分钟，然后关机，自然冷却到室温即可。
步骤4、酶切及连接反应
首先，用Omega质粒提取试剂盒（D6922-02）提取lentiGuide-Puro质粒，然后用限制性核酸内切酶BsmBI进行酶切，酶切条件为37摄氏度、3-5小时。 将酶切后的质粒进行1%琼脂糖凝胶电泳检测酶切效果并进行胶回收，胶回收采用Omega琼脂糖凝胶回收试剂盒（D2500-01），具体操作参见试剂盒说明书。
将胶回收的退火产物（双链寡核苷酸1-6中的任意一个）与上述胶回收的酶切质粒进行连接反应，具体体系和连接反应的条件如下。
连接体系：胶回收的PCR退火产物（双链寡核苷酸1-6中的任意一个）2μl，胶回收的BsmBI酶切lentiGuide-Puro质粒2μl；T4连接酶0.5μl；10x连接酶buffer 1μl，连接反应体系体积10μl；
连接条件：PCR仪中16摄氏度连接过夜。
步骤5、转化
将5μl步骤4获得的连接产物加入到50μl的感受态细胞（Stbl3，购买自美国invitrogen公司）中，冰浴30min，42℃45s，冰浴2min，然后加入500μl无抗LB液体培养基后，37摄氏度，200rpm震荡培养40min，涂布氨苄抗性的LB固体平板，在37摄氏度培养箱中过夜。等待单菌落出现后，挑取5个大小适中的菌落，提取质粒，再送往商业测序公司（上海生工）测序。
经测序表明在每一个lentiGuide-Puro质粒的酶切位点BsmBI处插入的片段的序列均为它们各自的gRNA序列对应，即与步骤2）中各自靶序列的DNA oligo的引物组的正向序列对应。
因此，证明获得了靶向PD-1基因的gRNA的表达质粒（发明人将其命名为lenti-PD1-sgRNA）。
步骤6）靶向人PD-1基因的gRNA的表达质粒的提取与纯化
将测序正确的克隆进行扩大培养，再用从上海优宁维公司购自的试剂盒Qiagen Plasmid Purification Kit提取质粒，操作方法严格按照该试剂盒的说明书进行。
 本发明的实施方式
实施例2： Cas9核酸酶质粒的构建
Cas9-NLS-Flag核酸酶序列可以选择如SEQ ID No.24、SEQ ID No.25、SEQ ID No.26或SEQ ID No.27中的任意其中一条序列。
将所述Cas9-NLS-Flag核酸酶序列通过分子克隆连接入慢病毒载体，获得Cas9核酸酶病毒载体。具体操作步骤如下：将所述Cas9-NLS-Flag核酸酶序列通过正向引物（正向序列5’-aagtacccgggtaggtcttgaaaggagtgggaattggctcc-3’，如SEQ ID No.28所示）和反向引物（反向序列：5’-acttaacgcgtttacttatcgtcatcgtctttgtaatctttcttcttct-3’，如如SEQ ID No.29所示），以载体lentiCas9-Blast（Genscript）为模板进行PCR扩增，扩增后的PCR产物和克隆慢病毒载体lentiGuide-Puro（Genscript）经过限制性内切酶SmaI和MluI相连，连接产物经感受态细胞Stbl3转化，PCR检测阳性克隆送商业测序公司测序（上海生工），获得序列完全正确的阳性Cas9核酸酶病毒载体，即Cas9核酸酶质粒。
实施例3：将CLDN18.2特异性嵌合抗原受体的序列克隆到实施例1获得的靶向PD-1基因的gRNA的表达质粒中
步骤1）CLDN18.2特异性嵌合抗原受体的氨基酸序列和核苷酸序列的确定
构建CLDN18.2特异性嵌合抗原受体。关于嵌合抗原受体（CAR）的分子结构，主要由膜外抗原结合区（scFv，胞外单链抗体部分）、铰链区（跨膜蛋白部分）和胞内信号传导区三部分构成。其中，由于CLDN18.1和CLDN18.2在序列上有96%的同源性，鉴定出专一性识别CLDN18.2而不识别CLDN18.1的scFv片段是构建该CAR分子的关键。我们对CLDN18.2进行了序列分析和抗体片段挖掘，成功筛选出与CLDN18.2具有较强亲和力而与CLDN18.1没有亲和力的scFv片段。最终获得了本发明的CLDN18.2特异性嵌合抗原受体的氨基酸序列和核苷酸序列。具体如下。
CLDN18.2特异性CAR分子的氨基酸序列：自氨基端到羧基端，由引导序列（如SEQ ID No.30所示）、scFv序列（选择如SEQ ID No.31或32或33所示其中任意一个序列）、人CD8铰链区序列（如SEQ ID No.34所示）、人CD8跨膜区序列（如SEQ ID No.35所示）、人4-1BB胞内结构域序列（如SEQ ID No.36所示）和CD3ζ结构域序列（如SEQ ID No.37所示）依次串联而成。
CLDN18.2特异性CAR分子的核苷酸序列：从5’端到3’端，由引导序列（如SEQ ID No.38所示）、scFv序列（如SEQ ID No.39或40或41所示）、人CD8铰链区序列（如SEQ ID No.42所示）、人CD8跨膜区序列（如SEQ ID No.43所示）、人4-1BB胞内结构域序列（如SEQ ID No.44所示）和CD3ζ结构域序列（如SEQ ID No.45所示）依次串联而成。
CLDN18.2特异性CAR分子的完整核苷酸序列如SEQ ID No.46所示（其中，scFv序列采用如SEQ ID No.40所示序列）。
步骤2）分子克隆
首先，全基因合成CLDN18.2特异性CAR分子的核苷酸序列（SEQ ID No.46所示），通过分子克隆的方式连接到上述实施例1所获得的靶向PD-1基因的gRNA的表达质粒中。
具体操作如下：构建单一编码框的CLDN18.2特异性CAR分子的核苷酸序列表达框（SEQ ID No.46所示），利用EF1 alpha启动子（序列表SEQ ID No.47）进行表达。为提高scFv表达效率，首先将scFv氨基酸序列进行人源密码子优化，然后将含有引导序列、scFv序列、CD8铰链区、CD8跨膜区、4-1BB胞内结构域、CD3ζ结构域CAR分子序列人工合成（通用生物），引物扩增人工合成CAR分子序列，经过限制性内切酶BstEII和MluI消化实施例1获得的靶向PD-1基因的gRNA的表达质粒（lenti-PD1-sgRNA），产物经回收后T4连接酶于PCR仪16度连接过夜，连接产物经感受态细胞Stbl3转化，筛选阳性克隆并测序（安徽、通用生物），将测序结果与拟合成的CLDN18.2特异性CAR分子序列比对证实序列完全正确，证明获得了表达靶向人PD-1基因的gRNA和CLDN18.2特异性嵌合抗原受体的重组质粒（发明人将其命名为KD-022 CAR lenti-PD1-sgRNA 慢病毒载体）。
将测序正确的克隆进行扩大培养，再用从上海优宁维公司购自的试剂盒Qiagen Plasmid Purification Kit提取质粒，操作方法严格按照该试剂盒的说明书进行。
实施例4：病毒液的制备
将实施例3获得的表达靶向人PD-1基因的gRNA和CLDN18.2特异性嵌合抗原受体的重组质粒（KD-022 CAR lenti-PD1-sgRNA慢病毒载体）和包装载体PSPAX2和VSVG，按照10：8：5的比例，用聚乙烯亚胺转染试剂（购买自Sigma公司，产品型号为408727）共转染Lenti-X 293T 细胞（具体的转染操作过程参见Sigma转染说明书），转染后6小时更换为完全培养基（购买自Life Technologies公司，产品型号为11995-065），培养48小时和72小时后，分别收集富含慢病毒颗粒的细胞上清液，病毒上清液通过超离心浓缩病毒，获得携带CLDN18.2特异性嵌合抗原受体分子和人PD-1基因的gRNA的慢病毒载体的病毒液（下面简称KD-022 CAR lenti-PD1-sgRNA慢病毒载体的病毒液）。
将实施例2获得的Cas9核酸酶质粒以及包装载体PSPAX2和VSVG，按照10：8：5的比例，和上面同样的方式获得携带Cas9核酸酶病毒载体的病毒液。
包装载体按照Lenti-X Packaging Single Shots（Takara）说明书进行制备；所有载体采用Qiagen Plasmid Purification Kit进行高纯度无内毒素抽提，已备感染。
实施例5：T细胞的分离培养
取健康供者的新鲜外周血，通过密度梯度离心分离新鲜的外周血单核细胞；再利用偶联了抗-CD3抗体和抗-CD28抗体的顺磁磁珠（购买自美国Invitrogen公司，产品信息为Dynabeads® Human T-Activator CD3/CD28）来富集CD3+T细胞，具体来说，将外周血单核细胞稀释至浓度为（10~30）×106个单个细胞/ml，再将磁珠与细胞以3:1的比例在培养皿中混合，室温下共孵育2-3小时，利用磁微粒收集器（Magnetic particles concentrator，简称MPC，购买自美国Invitrogen公司）富集CD3+T细胞。最后将富集的CD3+T细胞重悬于培养基（购买自美国Life Technologies公司，产品信息为OpTmizer™ T-Cell Expansion SFM）中，调至细胞溶度为1×106个/ml，最后在37℃、5% CO2培养箱中培养2天。
实施例6：人PD-1基因敲除的CLDN18.2特异性嵌合抗原受体T细胞的制备
取实施例5获得的CD3+T细胞，接种到24孔板，接种浓度为1×105个细胞/ml，在37℃、5% CO2环境培养约24小时（培养时间依据具体实践而定，一般来说，保证在病毒液感染时细胞汇合率介于50-70%之间）。
之后，取实施例4收集的KD-022 CAR lenti-PD1-sgRNA慢病毒载体的病毒液以及携带Cas9核酸酶病毒载体的病毒液，按照MOI=1-10的值，一同加入细胞培养瓶后，封好口，放入平角离心机后，低速（500g-1000g/min）离心1小时，然后放入培养箱中37℃培养。感染后48小时后获得敲除了人PD-1基因并且同时表达靶向CLDN18.2的CAR-T细胞（即人PD-1基因敲除的CLDN18.2 CAR-T细胞），可以进行下一步的功能实验。
鉴定
1、利用流式细胞术分析检测CAR蛋白的表达
离心细胞，用PBS清洗两次后重悬于FACS液中（含0.1%叠氮化钠和0.4%BSA的PBS）；将Biotin-labeled polyclonal goat anti-mouse-F(ab)2抗体（115-066-072, Jackson Immunoresearch）加入细胞悬液中，4℃孵育30分钟；清洗细胞两次后，加入phycoerythrin (PE)-labeled streptavidin（405203, Biolenged），室温30分钟；BD FacsCanto II用于获取染色细胞，FlowJo用于分析结果。如图1所示，对照组为感染空载体病毒液的T细胞，几乎检测不到CAR分子的表达；图2所示，实验组为感染CLDN18.2 CAR分子病毒液的T细胞，CAR分子的表达率达到36.8%；图3所示，实验+Cas9组为实施例6获得的人PD-1基因敲除的CLDN18.2特异性嵌合抗原受体T细胞中，CAR分子的表达率达到41.3%。
2、Cas9的表达以及人PD-1基因敲除的验证
首先利用western blotting检测了Cas9核酸酶的表达。
实施例6获得的人PD-1基因敲除的CLDN18.2特异性嵌合抗原受体T细胞经过离心、清洗以后在含有蛋白酶抑制剂的RIPA裂解液中裂解1小时，高速离心后得到的裂解上清液与4 x LDS上样缓冲液混合后在4-12% Bis-Tris预制胶（NP0335BOX, Life technologies）中进行分离，然后通过电转将蛋白转移到PVDF膜上。PVDF膜在含5%脱脂奶粉的TBST中封闭1小时，在1：2500的小鼠抗Flag抗体 (Anti Flag -Tag Mouse mAb, ab002-040, MultiSciences)中4℃孵育过夜。用TBST清洗膜3次，用1：10000偶联HRP的羊抗小鼠IgG二抗（115-035-003,Jackson Immunoresearch）室温孵育1小时。用ECL Plus发光液孵育5分钟后，在Tanon 9500机器中曝光。如图4所示，相对于没有感染Cas9病毒液的对照T细胞（如图4中标注 “对照”的一列结果）而言，实施例6获得的人PD-1基因敲除的CLDN18.2特异性嵌合抗原受体T细胞（如图4中标注“Cas9病毒液”的一列结果）中有很强的Cas9蛋白表达。
接下来利用western blotting检测PD-1蛋白的表达情况。实施例6获得的人PD-1基因敲除的CLDN18.2特异性嵌合抗原受体T细胞经过离心、清洗以后在含有蛋白酶抑制剂的RIPA裂解液中裂解1小时，高速离心后得到的裂解上清液与4 x LDS上样缓冲液混合后在10% Bis-Tris预制胶（NP0301BOX，Life technologies）中进行分离，然后通过电转将蛋白转移到PVDF膜上。PVDF膜在含5%脱脂奶粉的TBST中封闭一小时，在1：1000的兔抗人PD-1抗体（PRS4065，Sigma）中4℃孵育过夜。用TBST清洗膜3次，用1：10000偶联HRP的羊抗兔IgG二抗(111-035-003，Jackson Immunoresearch）室温孵育1小时。用ECL Plus发光液孵育5分钟后，在Tanon 9500机器中曝光。如图5所示，相比实验组（该实验组为感染CLDN18.2 CAR分子病毒液的T细胞，如图5中标注“实验组”的一列），PD-1的蛋白表达量在实施例6获得的人PD-1基因敲除的CLDN18.2特异性嵌合抗原受体T细胞（如图5中标注“实验组+Cas9”的一列结果）中有明显的下降。
应用例以及效果数据
利用7-AAD/CFSE细胞毒性测试试剂盒（购自Biovision公司，产品编号K315-100）评估实施例6获得的人PD-1基因敲除的CLDN18.2特异性嵌合抗原受体T细胞对胰腺癌肿瘤细胞BxPC-3（ATCC产品，产品编号：CRL-1687™）和胃癌肿瘤细胞NUGC-4（购自南京科佰生物科技有限公司，产品编号CBP60493）的杀伤情况。CSFE标记肿瘤细胞后，以每孔2×104的细胞铺于培养板中，分别以10：1、5：1和1：1的比例将实施例6获得的人PD-1基因敲除的CLDN18.2特异性嵌合抗原受体T细胞分别加入上述肿瘤细胞中，培养20小时后离心去上清，细胞沉淀清洗之后用7AAD染色，BD FacsCanto II用于获取染色细胞，FlowJo用于分析结果。
如图6所示，相对于不感染病毒的T细胞、感染Cas9病毒液的T细胞以及感染空载体病毒液的T细胞的结果（分别为图6中所示的“对照”、“Cas9”和“空载体”的结果），感染CLDN18.2 CAR分子病毒液的T细胞（图6中所示“实验组”的结果）对靶细胞BxPC-3有明显的杀伤作用，效靶比10:1时的杀伤率达到40%，以及实施例6获得的人PD-1基因敲除的CLDN18.2特异性嵌合抗原受体T细胞（图6中所示“实验+Cas9组”）对靶细胞BxPC-3有明显的杀伤作用，效靶比10:1时的杀伤率达到50.3%。通过对比实施例6的人PD-1基因敲除的CLDN18.2特异性嵌合抗原受体T细胞与感染CLDN18.2 CAR分子病毒液的T细胞的结果，可以看出，CLDN18.2 CAR-T细胞在敲除人PD-1基因后对胰腺癌肿瘤细胞BxPC-3的杀伤率显著提高。
如图7所示，相对于不感染病毒的T细胞、感染Cas9病毒液的T细胞和感染空载体病毒液的T细胞（分别为图7中所示的“对照”、“Cas9”和“空载体”的结果），感染CLDN18.2 CAR分子病毒液的T细胞（图7中所示“实验组”的结果）对靶细胞NUGC-4有明显的杀伤作用，效靶比10:1时的杀伤率达到26.5%，以及实施例6获得的人PD-1基因敲除的CLDN18.2特异性嵌合抗原受体T细胞（图7中所示“实验+Cas9组”）对靶细胞NUGC-4有明显的杀伤作用，效靶比10:1时的杀伤率达到37.2%。通过对比实施例6的人PD-1基因敲除的CLDN18.2特异性嵌合抗原受体T细胞与感染CLDN18.2 CAR分子病毒液的T细胞的结果，可以看出，CLDN18.2 CAR-T细胞在敲除人PD-1基因后对胃癌肿瘤细胞NUGC-4的杀伤率显著提高。
本发明利用CRISPR/Cas9基因组编辑技术获得的敲除人PD-1基因的靶向CLDN18.2 CAR-T细胞可用于制备治疗肿瘤的相关产品，例如制备治疗表达CLDN18.2蛋白的肿瘤的药物，特别是用于制备治疗胰腺癌和胃癌等实体瘤的药物。
应当理解，虽然本说明书按照实施方式加以描述，但并非每个实施方式仅包含一个独立的技术方案，说明书的这种叙述方式仅仅是为清楚起见，本领域技术人员应当将说明书作为一个整体，各实施方式中的技术方案也可以经适当组合，形成本领域技术人员可以理解的其他实施方式。
上文所列出的一系列的详细说明仅仅是针对本发明的可行性实施方式的具体说明，它们并非用以限制本发明的保护范围，凡未脱离本发明技艺精神所作的等效实施方式或变更均应包含在本发明的保护范围之内。
 工业实用性
序列表自由内容
<110> 南京凯地生物科技有限公司
<120> 人PD-1基因敲除的CLDN18.2特异性嵌合抗原受体T细胞的制备方法以及应用
<130> 2017-PD-1
<160> 47
<170> PatentIn version 3.5
<210> 1
<211> 146
<212> DNA
<213> 人
<400> 1
acagtttccc ttccgctcac ctccgcctga gcagtggaga aggcggcact ctggtggggc 60
tgctccaggc atgcagatcc cacaggcgcc ctggccagtc gtctgggcgg tgctacaact 120
gggctggcgg ccaggatggt tcttag 146
<210> 2
<211> 360
<212> DNA
<213> 人
<400> 2
actccccaga caggccctgg aaccccccca ccttctcccc agccctgctc gtggtgaccg 60
aaggggacaa cgccaccttc acctgcagct tctccaacac atcggagagc ttcgtgctaa 120
actggtaccg catgagcccc agcaaccaga cggacaagct ggccgccttc cccgaggacc 180
gcagccagcc cggccaggac tgccgcttcc gtgtcacaca actgcccaac gggcgtgact 240
tccacatgag cgtggtcagg gcccggcgca atgacagcgg cacctacctc tgtggggcca 300
tctccctggc ccccaaggcg cagatcaaag agagcctgcg ggcagagctc agggtgacag 360
<210> 3
<211> 156
<212> DNA
<213> 人
<400> 3
agagaagggc agaagtgccc acagcccacc ccagcccctc acccaggcca gccggccagt 60
tccaaaccct ggtggttggt gtcgtgggcg gcctgctggg cagcctggtg ctgctagtct 120
gggtcctggc cgtcatctgc tcccgggccg cacgag 156
<210> 4
<211> 35
<212> DNA
<213> 人
<400> 4
ggacaatagg agccaggcgc accggccagc ccctg 35
<210> 5
<211> 1417
<212> DNA
<213> 人
<400> 5
aaggaggacc cctcagccgt gcctgtgttc tctgtggact atggggagct ggatttccag 60
tggcgagaga agaccccgga gccccccgtg ccctgtgtcc ctgagcagac ggagtatgcc 120
accattgtct ttcctagcgg aatgggcacc tcatcccccg cccgcagggg ctcagctgac 180
ggccctcgga gtgcccagcc actgaggcct gaggatggac actgctcttg gcccctctga 240
ccggcttcct tggccaccag tgttctgcag accctccacc atgagcccgg gtcagcgcat 300
ttcctcagga gaagcaggca gggtgcaggc cattgcaggc cgtccagggg ctgagctgcc 360
tgggggcgac cggggctcca gcctgcacct gcaccaggca cagccccacc acaggactca 420
tgtctcaatg cccacagtga gcccaggcag caggtgtcac cgtcccctac agggagggcc 480
agatgcagtc actgcttcag gtcctgccag cacagagctg cctgcgtcca gctccctgaa 540
tctctgctgc tgctgctgct gctgctgctg ctgcctgcgg cccggggctg aaggcgccgt 600
ggccctgcct gacgccccgg agcctcctgc ctgaacttgg gggctggttg gagatggcct 660
tggagcagcc aaggtgcccc tggcagtggc atcccgaaac gccctggacg cagggcccaa 720
gactgggcac aggagtggga ggtacatggg gctggggact ccccaggagt tatctgctcc 780
ctgcaggcct agagaagttt cagggaaggt cagaagagct cctggctgtg gtgggcaggg 840
caggaaaccc ctccaccttt acacatgccc aggcagcacc tcaggccctt tgtggggcag 900
ggaagctgag gcagtaagcg ggcaggcaga gctggaggcc tttcaggccc agccagcact 960
ctggcctcct gccgccgcat tccaccccag cccctcacac cactcgggag agggacatcc 1020
tacggtccca aggtcaggag ggcagggctg gggttgactc aggcccctcc cagctgtggc 1080
cacctgggtg ttgggagggc agaagtgcag gcacctaggg ccccccatgt gcccaccctg 1140
ggagctctcc ttggaaccca ttcctgaaat tatttaaagg ggttggccgg gctcccacca 1200
gggcctgggt gggaaggtac aggcgttccc ccggggccta gtacccccgc cgtggcctat 1260
ccactcctca catccacaca ctgcaccccc actcctgggg cagggccacc agcatccagg 1320
cggccagcag gcacctgagt ggctgggaca agggatcccc cttccctgtg gttctattat 1380
attataatta taattaaata tgagagcatg ctaagga 1417
<210> 6
<211> 20
<212> DNA
<213> 人
<400> 6
acacatcgga gagcttcgtg 20
<210> 7
<211> 20
<212> DNA
<213> 人
<400> 7
gcgtgacttc cacatgagcg 20
<210> 8
<211> 20
<212> DNA
<213> 人
<400> 8
ttggaactgg ccggctggcc 20
<210> 9
<211> 20
<212> DNA
<213> 人
<400> 9
gtggcatact ccgtctgctc 20
<210> 10
<211> 20
<212> DNA
<213> 人
<400> 10
cgactggcca gggcgcctgt 20
<210> 11
<211> 20
<212> DNA
<213> 人
<400> 11
gatgaggtgc ccattccgct 20
<210> 12
<211> 25
<212> DNA
<213> 人
<400> 12
caccgacaca tcggagagct tcgtg 25
<210> 13
<211> 25
<212> DNA
<213> 人
<400> 13
aaaccacgaa gctctccgat gtgtc 25
<210> 14
<211> 25
<212> DNA
<213> 人
<400> 14
caccggcgtg acttccacat gagcg 25
<210> 15
<211> 25
<212> DNA
<213> 人
<400> 15
aaaccgctca tgtggaagtc acgcc
<210> 16
<211> 25
<212> DNA
<213> 人
<400> 16
caccgttgga actggccggc tggcc 25
<210> 17
<211> 25
<212> DNA
<213> 人
<400> 17
aaacggccag ccggccagtt ccaac 25
<210> 18
<211> 25
<212> DNA
<213> 人
<400> 18
caccggtggc atactccgtc tgctc 25
<210> 19
<211> 25
<212> DNA
<213> 人
<400> 19
aaacgagcag acggagtatg ccacc 25
<210> 20
<211> 25
<212> DNA
<213> 人
<400> 20
caccgcgact ggccagggcg cctgt 25
<210> 21
<211> 25
<212> DNA
<213> 人
<400> 21
aaacacaggc gccctggcca gtcgc 25
<210> 22
<211> 25
<212> DNA
<213> 人
<400> 22
caccggatga ggtgcccatt ccgct 25
<210> 23
<211> 25
<212> DNA
<213> 人
<400> 23
aaacagcgga atgggcacct catcc 25
<210> 24
<211> 4176
<212> DNA
<213> 人
<400> 24
atggacaaga agtacagcat cggcctggac atcggcacca actctgtggg ctgggccgtg 60
atcaccgacg agtacaaggt gcccagcaag aaattcaagg tgctgggcaa caccgaccgg 120
cacagcatca agaagaacct gatcggagcc ctgctgttcg acagcggcga aacagccgag 180
gccacccggc tgaagagaac cgccagaaga agatacacca gacggaagaa ccggatctgc 240
tatctgcaag agatcttcag caacgagatg gccaaggtgg acgacagctt cttccacaga 300
ctggaagagt ccttcctggt ggaagaggat aagaagcacg agcggcaccc catcttcggc 360
aacatcgtgg acgaggtggc ctaccacgag aagtacccca ccatctacca cctgagaaag 420
aaactggtgg acagcaccga caaggccgac ctgcggctga tctatctggc cctggcccac 480
atgatcaagt tccggggcca cttcctgatc gagggcgacc tgaaccccga caacagcgac 540
gtggacaagc tgttcatcca gctggtgcag acctacaacc agctgttcga ggaaaacccc 600
atcaacgcca gcggcgtgga cgccaaggcc atcctgtctg ccagactgag caagagcaga 660
cggctggaaa atctgatcgc ccagctgccc ggcgagaaga agaatggcct gttcggaaac 720
ctgattgccc tgagcctggg cctgaccccc aacttcaaga gcaacttcga cctggccgag 780
gatgccaaac tgcagctgag caaggacacc tacgacgacg acctggacaa cctgctggcc 840
cagatcggcg accagtacgc cgacctgttt ctggccgcca agaacctgtc cgacgccatc 900
ctgctgagcg acatcctgag agtgaacacc gagatcacca aggcccccct gagcgcctct 960
atgatcaaga gatacgacga gcaccaccag gacctgaccc tgctgaaagc tctcgtgcgg 1020
cagcagctgc ctgagaagta caaagagatt ttcttcgacc agagcaagaa cggctacgcc 1080
ggctacattg acggcggagc cagccaggaa gagttctaca agttcatcaa gcccatcctg 1140
gaaaagatgg acggcaccga ggaactgctc gtgaagctga acagagagga cctgctgcgg 1200
aagcagcgga ccttcgacaa cggcagcatc ccccaccaga tccacctggg agagctgcac 1260
gccattctgc ggcggcagga agatttttac ccattcctga aggacaaccg ggaaaagatc 1320
gagaagatcc tgaccttccg catcccctac tacgtgggcc ctctggccag gggaaacagc 1380
agattcgcct ggatgaccag aaagagcgag gaaaccatca ccccctggaa cttcgaggaa 1440
gtggtggaca agggcgcttc cgcccagagc ttcatcgagc ggatgaccaa cttcgataag 1500
aacctgccca acgagaaggt gctgcccaag cacagcctgc tgtacgagta cttcaccgtg 1560
tataacgagc tgaccaaagt gaaatacgtg accgagggaa tgagaaagcc cgccttcctg 1620
agcggcgagc agaaaaaggc catcgtggac ctgctgttca agaccaaccg gaaagtgacc 1680
gtgaagcagc tgaaagagga ctacttcaag aaaatcgagt gcttcgactc cgtggaaatc 1740
tccggcgtgg aagatcggtt caacgcctcc ctgggcacat accacgatct gctgaaaatt 1800
atcaaggaca aggacttcct ggacaatgag gaaaacgagg acattctgga agatatcgtg 1860
ctgaccctga cactgtttga ggacagagag atgatcgagg aacggctgaa aacctatgcc 1920
cacctgttcg acgacaaagt gatgaagcag ctgaagcggc ggagatacac cggctggggc 1980
aggctgagcc ggaagctgat caacggcatc cgggacaagc agtccggcaa gacaatcctg 2040
gatttcctga agtccgacgg cttcgccaac agaaacttca tgcagctgat ccacgacgac 2100
agcctgacct ttaaagagga catccagaaa gcccaggtgt ccggccaggg cgatagcctg 2160
cacgagcaca ttgccaatct ggccggcagc cccgccatta agaagggcat cctgcagaca 2220
gtgaaggtgg tggacgagct cgtgaaagtg atgggccggc acaagcccga gaacatcgtg 2280
atcgaaatgg ccagagagaa ccagaccacc cagaagggac agaagaacag ccgcgagaga 2340
atgaagcgga tcgaagaggg catcaaagag ctgggcagcc agatcctgaa agaacacccc 2400
gtggaaaaca cccagctgca gaacgagaag ctgtacctgt actacctgca gaatgggcgg 2460
gatatgtacg tggaccagga actggacatc aaccggctgt ccgactacga tgtggaccat 2520
atcgtgcctc agagctttct gaaggacgac tccatcgaca acaaggtgct gaccagaagc 2580
gacaagaacc ggggcaagag cgacaacgtg ccctccgaag aggtcgtgaa gaagatgaag 2640
aactactggc ggcagctgct gaacgccaag ctgattaccc agagaaagtt cgacaatctg 2700
accaaggccg agagaggcgg cctgagcgaa ctggataagg ccggcttcat caagagacag 2760
ctggtggaaa cccggcagat cacaaagcac gtggcacaga tcctggactc ccggatgaac 2820
actaagtacg acgagaatga caagctgatc cgggaagtga aagtgatcac cctgaagtcc 2880
aagctggtgt ccgatttccg gaaggatttc cagttttaca aagtgcgcga gatcaacaac 2940
taccaccacg cccacgacgc ctacctgaac gccgtcgtgg gaaccgccct gatcaaaaag 3000
taccctaagc tggaaagcga gttcgtgtac ggcgactaca aggtgtacga cgtgcggaag 3060
atgatcgcca agagcgagca ggaaatcggc aaggctaccg ccaagtactt cttctacagc 3120
aacatcatga actttttcaa gaccgagatt accctggcca acggcgagat ccggaagcgg 3180
cctctgatcg agacaaacgg cgaaaccggg gagatcgtgt gggataaggg ccgggatttt 3240
gccaccgtgc ggaaagtgct gagcatgccc caagtgaata tcgtgaaaaa gaccgaggtg 3300
cagacaggcg gcttcagcaa agagtctatc ctgcccaaga ggaacagcga taagctgatc 3360
gccagaaaga aggactggga ccctaagaag tacggcggct tcgacagccc caccgtggcc 3420
tattctgtgc tggtggtggc caaagtggaa aagggcaagt ccaagaaact gaagagtgtg 3480
aaagagctac tggggatcac catcatggaa agaagcagct tcgagaagaa tcccatcgac 3540
tttctggaag ccaagggcta caaagaagtg aaaaaggacc tgatcatcaa gctgcctaag 3600
tactccctgt tcgagctgga aaacggccgg aagagaatgc tggcctctgc cggcgaactg 3660
cagaagggaa acgaactggc cctgccctcc aaatatgtga acttcctgta cctggccagc 3720
cactatgaga agctgaaggg ctcccccgag gataatgagc agaaacagct gtttgtggaa 3780
cagcacaagc actacctgga cgagatcatc gagcagatca gcgagttctc caagagagtg 3840
atcctggccg acgctaatct ggacaaagtg ctgtccgcct acaacaagca ccgggataag 3900
cccatcagag agcaggccga gaatatcatc cacctgttta ccctgaccaa tctgggagcc 3960
cctgccgcct tcaagtactt tgacaccacc atcgaccgga agaggtacac cagcaccaaa 4020
gaggtgctgg acgccaccct gatccaccag agcatcaccg gcctgtacga gacacggatc 4080
gacctgtctc agctgggagg cgacaagcga cctgccgcca caaagaaggc tggacaggct 4140
aagaagaaga aagattacaa agacgatgac gataag 4176
<210> 25
<211> 4167
<212> DNA
<213> 人
<400> 25
atggacaaga agtacagcat cggcctggac atcggcacca actctgtggg ctgggccgtg 60
atcaccgacg agtacaaggt gcccagcaag aaattcaagg tgctgggcaa caccgaccgg 120
cacagcatca agaagaacct gatcggagcc ctgctgttcg acagcggcga aacagccgag 180
gccacccggc tgaagagaac cgccagaaga agatacacca gacggaagaa ccggatctgc 240
tatctgcaag agatcttcag caacgagatg gccaaggtgg acgacagctt cttccacaga 300
ctggaagagt ccttcctggt ggaagaggat aagaagcacg agcggcaccc catcttcggc 360
aacatcgtgg acgaggtggc ctaccacgag aagtacccca ccatctacca cctgagaaag 420
aaactggtgg acagcaccga caaggccgac ctgcggctga tctatctggc cctggcccac 480
atgatcaagt tccggggcca cttcctgatc gagggcgacc tgaaccccga caacagcgac 540
gtggacaagc tgttcatcca gctggtgcag acctacaacc agctgttcga ggaaaacccc 600
atcaacgcca gcggcgtgga cgccaaggcc atcctgtctg ccagactgag caagagcaga 660
cggctggaaa atctgatcgc ccagctgccc ggcgagaaga agaatggcct gttcggaaac 720
ctgattgccc tgagcctggg cctgaccccc aacttcaaga gcaacttcga cctggccgag 780
gatgccaaac tgcagctgag caaggacacc tacgacgacg acctggacaa cctgctggcc 840
cagatcggcg accagtacgc cgacctgttt ctggccgcca agaacctgtc cgacgccatc 900
ctgctgagcg acatcctgag agtgaacacc gagatcacca aggcccccct gagcgcctct 960
atgatcaaga gatacgacga gcaccaccag gacctgaccc tgctgaaagc tctcgtgcgg 1020
cagcagctgc ctgagaagta caaagagatt ttcttcgacc agagcaagaa cggctacgcc 1080
ggctacattg acggcggagc cagccaggaa gagttctaca agttcatcaa gcccatcctg 1140
gaaaagatgg acggcaccga ggaactgctc gtgaagctga acagagagga cctgctgcgg 1200
aagcagcgga ccttcgacaa cggcagcatc ccccaccaga tccacctggg agagctgcac 1260
gccattctgc ggcggcagga agatttttac ccattcctga aggacaaccg ggaaaagatc 1320
gagaagatcc tgaccttccg catcccctac tacgtgggcc ctctggccag gggaaacagc 1380
agattcgcct ggatgaccag aaagagcgag gaaaccatca ccccctggaa cttcgaggaa 1440
gtggtggaca agggcgcttc cgcccagagc ttcatcgagc ggatgaccaa cttcgataag 1500
aacctgccca acgagaaggt gctgcccaag cacagcctgc tgtacgagta cttcaccgtg 1560
tataacgagc tgaccaaagt gaaatacgtg accgagggaa tgagaaagcc cgccttcctg 1620
agcggcgagc agaaaaaggc catcgtggac ctgctgttca agaccaaccg gaaagtgacc 1680
gtgaagcagc tgaaagagga ctacttcaag aaaatcgagt gcttcgactc cgtggaaatc 1740
tccggcgtgg aagatcggtt caacgcctcc ctgggcacat accacgatct gctgaaaatt 1800
atcaaggaca aggacttcct ggacaatgag gaaaacgagg acattctgga agatatcgtg 1860
ctgaccctga cactgtttga ggacagagag atgatcgagg aacggctgaa aacctatgcc 1920
cacctgttcg acgacaaagt gatgaagcag ctgaagcggc ggagatacac cggctggggc 1980
aggctgagcc ggaagctgat caacggcatc cgggacaagc agtccggcaa gacaatcctg 2040
gatttcctga agtccgacgg cttcgccaac agaaacttca tgcagctgat ccacgacgac 2100
agcctgacct ttaaagagga catccagaaa gcccaggtgt ccggccaggg cgatagcctg 2160
cacgagcaca ttgccaatct ggccggcagc cccgccatta agaagggcat cctgcagaca 2220
gtgaaggtgg tggacgagct cgtgaaagtg atgggccggc acaagcccga gaacatcgtg 2280
atcgaaatgg ccagagagaa ccagaccacc cagaagggac agaagaacag ccgcgagaga 2340
atgaagcgga tcgaagaggg catcaaagag ctgggcagcc agatcctgaa agaacacccc 2400
gtggaaaaca cccagctgca gaacgagaag ctgtacctgt actacctgca gaatgggcgg 2460
gatatgtacg tggaccagga actggacatc aaccggctgt ccgactacga tgtggaccat 2520
atcgtgcctc agagctttct gaaggacgac tccatcgaca acaaggtgct gaccagaagc 2580
gacaagaacc ggggcaagag cgacaacgtg ccctccgaag aggtcgtgaa gaagatgaag 2640
aactactggc ggcagctgct gaacgccaag ctgattaccc agagaaagtt cgacaatctg 2700
accaaggccg agagaggcgg cctgagcgaa ctggataagg ccggcttcat caagagacag 2760
ctggtggaaa cccggcagat cacaaagcac gtggcacaga tcctggactc ccggatgaac 2820
actaagtacg acgagaatga caagctgatc cgggaagtga aagtgatcac cctgaagtcc 2880
aagctggtgt ccgatttccg gaaggatttc cagttttaca aagtgcgcga gatcaacaac 2940
taccaccacg cccacgacgc ctacctgaac gccgtcgtgg gaaccgccct gatcaaaaag 3000
taccctaagc tggaaagcga gttcgtgtac ggcgactaca aggtgtacga cgtgcggaag 3060
atgatcgcca agagcgagca ggaaatcggc aaggctaccg ccaagtactt cttctacagc 3120
aacatcatga actttttcaa gaccgagatt accctggcca acggcgagat ccggaagcgg 3180
cctctgatcg agacaaacgg cgaaaccggg gagatcgtgt gggataaggg ccgggatttt 3240
gccaccgtgc ggaaagtgct gagcatgccc caagtgaata tcgtgaaaaa gaccgaggtg 3300
cagacaggcg gcttcagcaa agagtctatc ctgcccaaga ggaacagcga taagctgatc 3360
gccagaaaga aggactggga ccctaagaag tacggcggct tcgacagccc caccgtggcc 3420
tattctgtgc tggtggtggc caaagtggaa aagggcaagt ccaagaaact gaagagtgtg 3480
aaagagctac tggggatcac catcatggaa agaagcagct tcgagaagaa tcccatcgac 3540
tttctggaag ccaagggcta caaagaagtg aaaaaggacc tgatcatcaa gctgcctaag 3600
tactccctgt tcgagctgga aaacggccgg aagagaatgc tggcctctgc cggcgaactg 3660
cagaagggaa acgaactggc cctgccctcc aaatatgtga acttcctgta cctggccagc 3720
cactatgaga agctgaaggg ctcccccgag gataatgagc agaaacagct gtttgtggaa 3780
cagcacaagc actacctgga cgagatcatc gagcagatca gcgagttctc caagagagtg 3840
atcctggccg acgctaatct ggacaaagtg ctgtccgcct acaacaagca ccgggataag 3900
cccatcagag agcaggccga gaatatcatc cacctgttta ccctgaccaa tctgggagcc 3960
cctgccgcct tcaagtactt tgacaccacc atcgaccgga agaggtacac cagcaccaaa 4020
gaggtgctgg acgccaccct gatccaccag agcatcaccg gcctgtacga gacacggatc 4080
gacctgtctc agctgggagg cgacaagcga cctgccgcca caaagaaggc tggacaggct 4140
aaggattaca aagacgatga cgataag 4167
<210> 26
<211> 4116
<212> DNA
<213> 人
<400> 26
atgaccgacg agtacaaggt gcccagcaag aaattcaagg tgctgggcaa caccgaccgg 60
cacagcatca agaagaacct gatcggagcc ctgctgttcg acagcggcga aacagccgag 120
gccacccggc tgaagagaac cgccagaaga agatacacca gacggaagaa ccggatctgc 180
tatctgcaag agatcttcag caacgagatg gccaaggtgg acgacagctt cttccacaga 240
ctggaagagt ccttcctggt ggaagaggat aagaagcacg agcggcaccc catcttcggc 300
aacatcgtgg acgaggtggc ctaccacgag aagtacccca ccatctacca cctgagaaag 360
aaactggtgg acagcaccga caaggccgac ctgcggctga tctatctggc cctggcccac 420
atgatcaagt tccggggcca cttcctgatc gagggcgacc tgaaccccga caacagcgac 480
gtggacaagc tgttcatcca gctggtgcag acctacaacc agctgttcga ggaaaacccc 540
atcaacgcca gcggcgtgga cgccaaggcc atcctgtctg ccagactgag caagagcaga 600
cggctggaaa atctgatcgc ccagctgccc ggcgagaaga agaatggcct gttcggaaac 660
ctgattgccc tgagcctggg cctgaccccc aacttcaaga gcaacttcga cctggccgag 720
gatgccaaac tgcagctgag caaggacacc tacgacgacg acctggacaa cctgctggcc 780
cagatcggcg accagtacgc cgacctgttt ctggccgcca agaacctgtc cgacgccatc 840
ctgctgagcg acatcctgag agtgaacacc gagatcacca aggcccccct gagcgcctct 900
atgatcaaga gatacgacga gcaccaccag gacctgaccc tgctgaaagc tctcgtgcgg 960
cagcagctgc ctgagaagta caaagagatt ttcttcgacc agagcaagaa cggctacgcc 1020
ggctacattg acggcggagc cagccaggaa gagttctaca agttcatcaa gcccatcctg 1080
gaaaagatgg acggcaccga ggaactgctc gtgaagctga acagagagga cctgctgcgg 1140
aagcagcgga ccttcgacaa cggcagcatc ccccaccaga tccacctggg agagctgcac 1200
gccattctgc ggcggcagga agatttttac ccattcctga aggacaaccg ggaaaagatc 1260
gagaagatcc tgaccttccg catcccctac tacgtgggcc ctctggccag gggaaacagc 1320
agattcgcct ggatgaccag aaagagcgag gaaaccatca ccccctggaa cttcgaggaa 1380
gtggtggaca agggcgcttc cgcccagagc ttcatcgagc ggatgaccaa cttcgataag 1440
aacctgccca acgagaaggt gctgcccaag cacagcctgc tgtacgagta cttcaccgtg 1500
tataacgagc tgaccaaagt gaaatacgtg accgagggaa tgagaaagcc cgccttcctg 1560
agcggcgagc agaaaaaggc catcgtggac ctgctgttca agaccaaccg gaaagtgacc 1620
gtgaagcagc tgaaagagga ctacttcaag aaaatcgagt gcttcgactc cgtggaaatc 1680
tccggcgtgg aagatcggtt caacgcctcc ctgggcacat accacgatct gctgaaaatt 1740
atcaaggaca aggacttcct ggacaatgag gaaaacgagg acattctgga agatatcgtg 1800
ctgaccctga cactgtttga ggacagagag atgatcgagg aacggctgaa aacctatgcc 1860
cacctgttcg acgacaaagt gatgaagcag ctgaagcggc ggagatacac cggctggggc 1920
aggctgagcc ggaagctgat caacggcatc cgggacaagc agtccggcaa gacaatcctg 1980
gatttcctga agtccgacgg cttcgccaac agaaacttca tgcagctgat ccacgacgac 2040
agcctgacct ttaaagagga catccagaaa gcccaggtgt ccggccaggg cgatagcctg 2100
cacgagcaca ttgccaatct ggccggcagc cccgccatta agaagggcat cctgcagaca 2160
gtgaaggtgg tggacgagct cgtgaaagtg atgggccggc acaagcccga gaacatcgtg 2220
atcgaaatgg ccagagagaa ccagaccacc cagaagggac agaagaacag ccgcgagaga 2280
atgaagcgga tcgaagaggg catcaaagag ctgggcagcc agatcctgaa agaacacccc 2340
gtggaaaaca cccagctgca gaacgagaag ctgtacctgt actacctgca gaatgggcgg 2400
gatatgtacg tggaccagga actggacatc aaccggctgt ccgactacga tgtggaccat 2460
atcgtgcctc agagctttct gaaggacgac tccatcgaca acaaggtgct gaccagaagc 2520
gacaagaacc ggggcaagag cgacaacgtg ccctccgaag aggtcgtgaa gaagatgaag 2580
aactactggc ggcagctgct gaacgccaag ctgattaccc agagaaagtt cgacaatctg 2640
accaaggccg agagaggcgg cctgagcgaa ctggataagg ccggcttcat caagagacag 2700
ctggtggaaa cccggcagat cacaaagcac gtggcacaga tcctggactc ccggatgaac 2760
actaagtacg acgagaatga caagctgatc cgggaagtga aagtgatcac cctgaagtcc 2820
aagctggtgt ccgatttccg gaaggatttc cagttttaca aagtgcgcga gatcaacaac 2880
taccaccacg cccacgacgc ctacctgaac gccgtcgtgg gaaccgccct gatcaaaaag 2940
taccctaagc tggaaagcga gttcgtgtac ggcgactaca aggtgtacga cgtgcggaag 3000
atgatcgcca agagcgagca ggaaatcggc aaggctaccg ccaagtactt cttctacagc 3060
aacatcatga actttttcaa gaccgagatt accctggcca acggcgagat ccggaagcgg 3120
cctctgatcg agacaaacgg cgaaaccggg gagatcgtgt gggataaggg ccgggatttt 3180
gccaccgtgc ggaaagtgct gagcatgccc caagtgaata tcgtgaaaaa gaccgaggtg 3240
cagacaggcg gcttcagcaa agagtctatc ctgcccaaga ggaacagcga taagctgatc 3300
gccagaaaga aggactggga ccctaagaag tacggcggct tcgacagccc caccgtggcc 3360
tattctgtgc tggtggtggc caaagtggaa aagggcaagt ccaagaaact gaagagtgtg 3420
aaagagctac tggggatcac catcatggaa agaagcagct tcgagaagaa tcccatcgac 3480
tttctggaag ccaagggcta caaagaagtg aaaaaggacc tgatcatcaa gctgcctaag 3540
tactccctgt tcgagctgga aaacggccgg aagagaatgc tggcctctgc cggcgaactg 3600
cagaagggaa acgaactggc cctgccctcc aaatatgtga acttcctgta cctggccagc 3660
cactatgaga agctgaaggg ctcccccgag gataatgagc agaaacagct gtttgtggaa 3720
cagcacaagc actacctgga cgagatcatc gagcagatca gcgagttctc caagagagtg 3780
atcctggccg acgctaatct ggacaaagtg ctgtccgcct acaacaagca ccgggataag 3840
cccatcagag agcaggccga gaatatcatc cacctgttta ccctgaccaa tctgggagcc 3900
cctgccgcct tcaagtactt tgacaccacc atcgaccgga agaggtacac cagcaccaaa 3960
gaggtgctgg acgccaccct gatccaccag agcatcaccg gcctgtacga gacacggatc 4020
gacctgtctc agctgggagg cgacaagcga cctgccgcca caaagaaggc tggacaggct 4080
aagaagaaga aagattacaa agacgatgac gataag 4116
<210> 27
<211> 4104
<212> DNA
<213> 人
<400> 27
atgaccgacg agtacaaggt gcccagcaag aaattcaagg tgctgggcaa caccgaccgg 60
cacagcatca agaagaacct gatcggagcc ctgctgttcg acagcggcga aacagccgag 120
gccacccggc tgaagagaac cgccagaaga agatacacca gacggaagaa ccggatctgc 180
tatctgcaag agatcttcag caacgagatg gccaaggtgg acgacagctt cttccacaga 240
ctggaagagt ccttcctggt ggaagaggat aagaagcacg agcggcaccc catcttcggc 300
aacatcgtgg acgaggtggc ctaccacgag aagtacccca ccatctacca cctgagaaag 360
aaactggtgg acagcaccga caaggccgac ctgcggctga tctatctggc cctggcccac 420
atgatcaagt tccggggcca cttcctgatc gagggcgacc tgaaccccga caacagcgac 480
gtggacaagc tgttcatcca gctggtgcag acctacaacc agctgttcga ggaaaacccc 540
atcaacgcca gcggcgtgga cgccaaggcc atcctgtctg ccagactgag caagagcaga 600
cggctggaaa atctgatcgc ccagctgccc ggcgagaaga agaatggcct gttcggaaac 660
ctgattgccc tgagcctggg cctgaccccc aacttcaaga gcaacttcga cctggccgag 720
gatgccaaac tgcagctgag caaggacacc tacgacgacg acctggacaa cctgctggcc 780
cagatcggcg accagtacgc cgacctgttt ctggccgcca agaacctgtc cgacgccatc 840
ctgctgagcg acatcctgag agtgaacacc gagatcacca aggcccccct gagcgcctct 900
atgatcaaga gatacgacga gcaccaccag gacctgaccc tgctgaaagc tctcgtgcgg 960
cagcagctgc ctgagaagta caaagagatt ttcttcgacc agagcaagaa cggctacgcc 1020
ggctacattg acggcggagc cagccaggaa gagttctaca agttcatcaa gcccatcctg 1080
gaaaagatgg acggcaccga ggaactgctc gtgaagctga acagagagga cctgctgcgg 1140
aagcagcgga ccttcgacaa cggcagcatc ccccaccaga tccacctggg agagctgcac 1200
gccattctgc ggcggcagga agatttttac ccattcctga aggacaaccg ggaaaagatc 1260
gagaagatcc tgaccttccg catcccctac tacgtgggcc ctctggccag gggaaacagc 1320
agattcgcct ggatgaccag aaagagcgag gaaaccatca ccccctggaa cttcgaggaa 1380
gtggtggaca agggcgcttc cgcccagagc ttcatcgagc ggatgaccaa cttcgataag 1440
aacctgccca acgagaaggt gctgcccaag cacagcctgc tgtacgagta cttcaccgtg 1500
tataacgagc tgaccaaagt gaaatacgtg accgagggaa tgagaaagcc cgccttcctg 1560
agcggcgagc agaaaaaggc catcgtggac ctgctgttca agaccaaccg gaaagtgacc 1620
gtgaagcagc tgaaagagga ctacttcaag aaaatcgagt gcttcgactc cgtggaaatc 1680
tccggcgtgg aagatcggtt caacgcctcc ctgggcacat accacgatct gctgaaaatt 1740
atcaaggaca aggacttcct ggacaatgag gaaaacgagg acattctgga agatatcgtg 1800
ctgaccctga cactgtttga ggacagagag atgatcgagg aacggctgaa aacctatgcc 1860
cacctgttcg acgacaaagt gatgaagcag ctgaagcggc ggagatacac cggctggggc 1920
aggctgagcc ggaagctgat caacggcatc cgggacaagc agtccggcaa gacaatcctg 1980
gatttcctga agtccgacgg cttcgccaac agaaacttca tgcagctgat ccacgacgac 2040
agcctgacct ttaaagagga catccagaaa gcccaggtgt ccggccaggg cgatagcctg 2100
cacgagcaca ttgccaatct ggccggcagc cccgccatta agaagggcat cctgcagaca 2160
gtgaaggtgg tggacgagct cgtgaaagtg atgggccggc acaagcccga gaacatcgtg 2220
atcgaaatgg ccagagagaa ccagaccacc cagaagggac agaagaacag ccgcgagaga 2280
atgaagcgga tcgaagaggg catcaaagag ctgggcagcc agatcctgaa agaacacccc 2340
gtggaaaaca cccagctgca gaacgagaag ctgtacctgt actacctgca gaatgggcgg 2400
gatatgtacg tggaccagga actggacatc aaccggctgt ccgactacga tgtggaccat 2460
atcgtgcctc agagctttct gaaggacgac tccatcgaca acaaggtgct gaccagaagc 2520
gacaagaacc ggggcaagag cgacaacgtg ccctccgaag aggtcgtgaa gaagatgaag 2580
aactactggc ggcagctgct gaacgccaag ctgattaccc agagaaagtt cgacaatctg 2640
accaaggccg agagaggcgg cctgagcgaa ctggataagg ccggcttcat caagagacag 2700
ctggtggaaa cccggcagat cacaaagcac gtggcacaga tcctggactc ccggatgaac 2760
actaagtacg acgagaatga caagctgatc cgggaagtga aagtgatcac cctgaagtcc 2820
aagctggtgt ccgatttccg gaaggatttc cagttttaca aagtgcgcga gatcaacaac 2880
taccaccacg cccacgacgc ctacctgaac gccgtcgtgg gaaccgccct gatcaaaaag 2940
taccctaagc tggaaagcga gttcgtgtac ggcgactaca aggtgtacga cgtgcggaag 3000
atgatcgcca agagcgagca ggaaatcggc aaggctaccg ccaagtactt cttctacagc 3060
aacatcatga actttttcaa gaccgagatt accctggcca acggcgagat ccggaagcgg 3120
cctctgatcg agacaaacgg cgaaaccggg gagatcgtgt gggataaggg ccgggatttt 3180
gccaccgtgc ggaaagtgct gagcatgccc caagtgaata tcgtgaaaaa gaccgaggtg 3240
cagacaggcg gcttcagcaa agagtctatc ctgcccaaga ggaacagcga taagctgatc 3300
gccagaaaga aggactggga ccctaagaag tacggcggct tcgacagccc caccgtggcc 3360
tattctgtgc tggtggtggc caaagtggaa aagggcaagt ccaagaaact gaagagtgtg 3420
aaagagctac tggggatcac catcatggaa agaagcagct tcgagaagaa tcccatcgac 3480
tttctggaag ccaagggcta caaagaagtg aaaaaggacc tgatcatcaa gctgcctaag 3540
tactccctgt tcgagctgga aaacggccgg aagagaatgc tggcctctgc cggcgaactg 3600
cagaagggaa acgaactggc cctgccctcc aaatatgtga acttcctgta cctggccagc 3660
cactatgaga agctgaaggg ctcccccgag gataatgagc agaaacagct gtttgtggaa 3720
cagcacaagc actacctgga cgagatcatc gagcagatca gcgagttctc caagagagtg 3780
atcctggccg acgctaatct ggacaaagtg ctgtccgcct acaacaagca ccgggataag 3840
cccatcagag agcaggccga gaatatcatc cacctgttta ccctgaccaa tctgggagcc 3900
cctgccgcct tcaagtactt tgacaccacc atcgaccgga agaggtacac cagcaccaaa 3960
gaggtgctgg acgccaccct gatccaccag agcatcaccg gcctgtacga gacacggatc 4020
gacctgtctc agctgggagg cgacaagcga cctgccgcca caaagaaggc tggacaggct 4080
gattacaaag acgatgacga taag 4104
<210> 28
<211> 41
<212> DNA
<213> 人
<400> 28
aagtacccgg gtaggtcttg aaaggagtgg gaattggctc c 41
<210> 29
<211> 49
<212> DNA
<213> 人
<400> 29
acttaacgcg tttacttatc gtcatcgtct ttgtaatctt tcttcttct 49
<210> 30
<211> 21
<212> PRT
<213> 人
<400> 30
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro
20
<210> 31
<211> 254
<212> PRT
<213> 人
<400> 31
Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Thr Val Thr Ala Gly
1 5 10 15
Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser
20 25 30
Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
50 55 60
Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn
85 90 95
Asp Tyr Ser Tyr Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile
100 105 110
Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
115 120 125
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
130 135 140
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr
145 150 155 160
Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
165 170 175
Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe
180 185 190
Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr
195 200 205
Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
210 215 220
Ala Arg Asp Leu Arg Arg Thr Val Val Thr Pro Arg Ala Tyr Tyr Gly
225 230 235 240
Met Asp Val Tyr Gly Gln Gly Thr Thr Val Thr Val Ser Ser
245 250
<210> 32
<211> 246
<212> PRT
<213> 人
<400> 32
Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Thr Val Thr Ala Gly
1 5 10 15
Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser
20 25 30
Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
50 55 60
Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn
85 90 95
Asp Tyr Ser Tyr Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile
100 105 110
Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
115 120 125
Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg Pro Gly Ala
130 135 140
Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
145 150 155 160
Trp Ile Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
165 170 175
Gly Asn Ile Tyr Pro Ser Asp Ser Tyr Thr Asn Tyr Asn Gln Lys Phe
180 185 190
Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr
195 200 205
Met Gln Leu Ser Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
210 215 220
Thr Arg Ser Trp Arg Gly Asn Ser Phe Asp Tyr Trp Gly Gln Gly Thr
225 230 235 240
Thr Leu Thr Val Ser Ser
245
<210> 33
<211> 249
<212> PRT
<213> 人
<400> 33
Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Thr Val Thr Ala Gly
1 5 10 15
Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser
20 25 30
Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
50 55 60
Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn
85 90 95
Asp Tyr Ser Tyr Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile
100 105 110
Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
115 120 125
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
130 135 140
Ser Val Glu Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
145 150 155 160
Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
165 170 175
Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Gly Tyr Ala Gln Lys Phe
180 185 190
Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val His
195 200 205
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
210 215 220
Ala Arg Gly Gly Tyr Ser Ser Ser Ser Asp Ala Phe Asp Ile Tyr Gly
225 230 235 240
Gln Gly Thr Met Val Thr Val Ser Ser
245
<210> 34
<211> 45
<212> PRT
<213> 人
<400> 34
Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala
1 5 10 15
Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly
20 25 30
Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp
35 40 45
<210> 35
<211> 24
<212> PRT
<213> 人
<400> 35
Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu
1 5 10 15
Ser Leu Val Ile Thr Leu Tyr Cys
20
<210> 36
<211> 42
<212> PRT
<213> 人
<400> 36
Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met
1 5 10 15
Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe
20 25 30
Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu
35 40
<210> 37
<211> 112
<212> PRT
<213> 人
<400> 37
Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly
1 5 10 15
Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr
20 25 30
Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys
35 40 45
Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys
50 55 60
Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg
65 70 75 80
Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala
85 90 95
Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
100 105 110
<210> 38
<211> 63
<212> DNA
<213> 人
<400> 38
atggccctgc ccgtcaccgc tctgctgctg ccccttgctc tgcttcttca tgcagcaagg 60
ccg 63
<210> 39
<211> 762
<212> DNA
<213> 人
<400> 39
gatattgtaa tgacccaatc accgagtagt cttacggtta ctgccggcga aaaggtaacg 60
atgagttgta aaagtagtca gagccttctt aacagcggaa accagaaaaa ctatctcacg 120
tggtaccagc agaagcccgg tcagccgcct aagttgttga tctactgggc gagtaccaga 180
gagtctggtg tgccagatcg gtttaccggg agtggttctg gcactgactt tacattgacg 240
atcagttccg tccaggcaga agaccttgcg gtttactatt gccaaaatga ctattcttat 300
ccattcacct tcggatctgg cactaagttg gaaattaagg gtggaggagg cagcggcggt 360
ggagggtctg gtggaggtgg ttctcaggtg caactcgttc agtcaggggc agaagtgaaa 420
aagccaggag cttctgtcaa ggtaagctgt aaagcaagtg gatatacgtt tacaggatac 480
tacatgcact gggtacggca agcgccaggc cagggtctgg aatggatggg gtggattaat 540
ccaaattcag gaggaaccaa ctacgctcaa aaattccagg gtcgggtcac catgactaga 600
gatacttcca tcagtacggc atacatggaa ctctcccgcc ttcggtcaga tgacacagcg 660
gtgtactatt gtgcccgcga tctgcggagg accgtagtaa ctcccagagc ttactatggg 720
atggacgttt atggacaagg tactacagtt actgtatcca gt 762
<210> 40
<211> 738
<212> DNA
<213> 人
<400> 40
gatattgtaa tgacccaatc accgagtagt cttacggtta ctgccggcga aaaggtaacg 60
atgagttgta aaagtagtca gagccttctt aacagcggaa accagaaaaa ctatctcacg 120
tggtaccagc agaagcccgg tcagccgcct aagttgttga tctactgggc gagtaccaga 180
gagtctggtg tgccagatcg gtttaccggg agtggttctg gcactgactt tacattgacg 240
atcagttccg tccaggcaga agaccttgcg gtttactatt gccaaaatga ctattcttat 300
ccattcacct tcggatctgg cactaagttg gaaattaagg gtggaggagg cagcggcggt 360
ggagggtctg gtggaggtgg ttctcaagtg cagcttcagc aaccgggggc agagttggtt 420
aggccaggcg catccgtcaa acttagctgc aaagcgagtg ggtatacatt cacatcctat 480
tggattaact gggtgaaaca gagacctgga caaggattgg aatggattgg taacatctac 540
ccaagcgatt catacacaaa ttataatcaa aagttcaaag ataaggcgac actcactgtc 600
gataaatctt catctacagc ttacatgcag ttgagttctc cgaccagcga agattccgca 660
gtatattact gtacccggtc atggagaggc aactcctttg attattgggg acagggcacg 720
acgttgacgg tcagcagc 738
<210> 41
<211> 747
<212> DNA
<213> 人
<400> 41
gatattgtaa tgacccaatc accgagtagt cttacggtta ctgccggcga aaaggtaacg 60
atgagttgta aaagtagtca gagccttctt aacagcggaa accagaaaaa ctatctcacg 120
tggtaccagc agaagcccgg tcagccgcct aagttgttga tctactgggc gagtaccaga 180
gagtctggtg tgccagatcg gtttaccggg agtggttctg gcactgactt tacattgacg 240
atcagttccg tccaggcaga agaccttgcg gtttactatt gccaaaatga ctattcttat 300
ccattcacct tcggatctgg cactaagttg gaaattaagg gtggaggagg cagcggcggt 360
ggagggtctg gtggaggtgg ttctcaggta cagctggtac agagcggagc agaggtcaaa 420
aagcccggag cgagcgttga ggtctcctgc aaggcgagtg ggtatacctt tacctcttat 480
tatatgcact gggtgcgcca ggctcccggt caagggttgg agtggatggg gatcataaac 540
ccctctggcg gctcaacagg ctacgctcaa aagttccaag ggcgagttac gatgactagg 600
gacacttcca catccacggt ccacatggag ttgtcatccc tgcgctctga ggacaccgca 660
gtctactact gcgctcgggg tggttattct tccagctctg acgcgttcga catatatggt 720
cagggtacga tggtcactgt gtctagt 747
<210> 42
<211> 135
<212> DNA
<213> 人
<400> 42
accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60
tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120
gacttcgcct gtgat 135
<210> 43
<211> 72
<212> DNA
<213> 人
<400> 43
atctacatct gggcgccctt ggccgggact tgtggggtcc ttctcctgtc actggttatc 60
accctttact gc 72
<210> 44
<211> 126
<212> DNA
<213> 人
<400> 44
aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 60
actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 120
gaactg 126
<210> 45
<211> 336
<212> DNA
<213> 人
<400> 45
agagtgaagt tcagcaggag cgcagacgcc cccgcgtaca agcagggcca gaaccagctc 60
tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 120
cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 180
gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 240
cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 300
tacgacgccc ttcacatgca ggccctgccc cctcgc 336
<210> 46
<211> 1470
<212> DNA
<213> 人
<400> 46
atggccctgc ccgtcaccgc tctgctgctg ccccttgctc tgcttcttca tgcagcaagg 60
ccggatattg taatgaccca atcaccgagt agtcttacgg ttactgccgg cgaaaaggta 120
acgatgagtt gtaaaagtag tcagagcctt cttaacagcg gaaaccagaa aaactatctc 180
acgtggtacc agcagaagcc cggtcagccg cctaagttgt tgatctactg ggcgagtacc 240
agagagtctg gtgtgccaga tcggtttacc gggagtggtt ctggcactga ctttacattg 300
acgatcagtt ccgtccaggc agaagacctt gcggtttact attgccaaaa tgactattct 360
tatccattca ccttcggatc tggcactaag ttggaaatta agggtggagg aggcagcggc 420
ggtggagggt ctggtggagg tggttctcaa gtgcagcttc agcaaccggg ggcagagttg 480
gttaggccag gcgcatccgt caaacttagc tgcaaagcga gtgggtatac attcacatcc 540
tattggatta actgggtgaa acagagacct ggacaaggat tggaatggat tggtaacatc 600
tacccaagcg attcatacac aaattataat caaaagttca aagataaggc gacactcact 660
gtcgataaat cttcatctac agcttacatg cagttgagtt ctccgaccag cgaagattcc 720
gcagtatatt actgtacccg gtcatggaga ggcaactcct ttgattattg gggacagggc 780
acgacgttga cggtcagcag caccacgacg ccagcgccgc gaccaccaac accggcgccc 840
accatcgcgt cgcagcccct gtccctgcgc ccagaggcgt gccggccagc ggcggggggc 900
gcagtgcaca cgagggggct ggacttcgcc tgtgatatct acatctgggc gcccttggcc 960
gggacttgtg gggtccttct cctgtcactg gttatcaccc tttactgcaa acggggcaga 1020
aagaaactcc tgtatatatt caaacaacca tttatgagac cagtacaaac tactcaagag 1080
gaagatggct gtagctgccg atttccagaa gaagaagaag gaggatgtga actgagagtg 1140
aagttcagca ggagcgcaga cgcccccgcg tacaagcagg gccagaacca gctctataac 1200
gagctcaatc taggacgaag agaggagtac gatgttttgg acaagagacg tggccgggac 1260
cctgagatgg ggggaaagcc gagaaggaag aaccctcagg aaggcctgta caatgaactg 1320
cagaaagata agatggcgga ggcctacagt gagattggga tgaaaggcga gcgccggagg 1380
ggcaaggggc acgatggcct ttaccagggt ctcagtacag ccaccaagga cacctacgac 1440
gcccttcaca tgcaggccct gccccctcgc 1470
<210> 47
<211> 1259
<212> DNA
<213> 人
<400> 47
tgcaaagatg gataaagttt taaacagaga ggaatctttg cagctaatgg accttctagg 60
tcttgaaagg agtgggaatt ggctccggtg cccgtcagtg ggcagagcgc acatcgccca 120
cagtccccga gaagttgggg ggaggggtcg gcaattgatc cggtgcctag agaaggtggc 180
gcggggtaaa ctgggaaagt gatgtcgtgt actggctccg cctttttccc gagggtgggg 240
gagaaccgta tataagtgca gtagtcgccg tgaacgttct ttttcgcaac gggtttgccg 300
ccagaacaca ggtaagtgcc gtgtgtggtt cccgcgggcc tggcctcttt acgggttatg 360
gcccttgcgt gccttgaatt acttccacct ggctgcagta cgtgattctt gatcccgagc 420
ttcgggttgg aagtgggtgg gagagttcga ggccttgcgc ttaaggagcc ccttcgcctc 480
gtgcttgagt tgaggcctgg cctgggcgct ggggccgccg cgtgcgaatc tggtggcacc 540
ttcgcgcctg tctcgctgct ttcgataagt ctctagccat ttaaaatttt tgatgacctg 600
ctgcgacgct ttttttctgg caagatagtc ttgtaaatgc gggccaagat ctgcacactg 660
gtatttcggt ttttggggcc gcgggcggcg acggggcccg tgcgtcccag cgcacatgtt 720
cggcgaggcg gggcctgcga gcgcggccac cgagaatcgg acgggggtag tctcaagctg 780
gccggcctgc tctggtgcct ggcctcgcgc cgccgtgtat cgccccgccc tgggcggcaa 840
ggctggcccg gtcggcacca gttgcgtgag cggaaagatg gccgcttccc ggccctgctg 900
cagggagctc aaaatggagg acgcggcgct cgggagagcg ggcgggtgag tcacccacac 960
aaaggaaaag ggcctttccg tcctcagccg tcgcttcatg tgactccacg gagtaccggg 1020
cgccgtccag gcacctcgat tagttctcga gcttttggag tacgtcgtct ttaggttggg 1080
gggaggggtt ttatgcgatg gagtttcccc acactgagtg ggtggagact gaagttaggc 1140
cagcttggca cttgatgtaa ttctccttgg aatttgccct ttttgagttt ggatcttggt 1200
tcattctcaa gcctcagaca gtggttcaaa gtttttttct tccatttcag gtgtcgtga 1259

Claims (10)

  1. 一种人PD-1基因敲除的CLDN18.2特异性嵌合抗原受体T细胞的制备方法，其特征在于：
    所述制备方法基于CRISPR-Cas9技术来敲除人PD-1基因，其中以人PD-1基因序列中同时符合以下规则的作为靶序列，
    规则1）：靶序列符合5’-N（20）NGG的序列排列规则，且所述“N（20）”所述序列为靶序列，其中N为A或T或C或G；
    规则2）：靶序列位于所述人PD-1基因的外显子；且
    规则3）：与靶序列互补的gRNA在人PD-1基因上的靶向是唯一的；以及，
    所述制备方法所采用的CLDN18.2特异性嵌合抗原受体分子的核苷酸序列为：从5’端到3’端，由引导序列、scFv序列、人CD8铰链区序列、人CD8跨膜区序列、人4-1BB胞内结构域序列和CD3ζ结构域序列依次串联而成；其中，
    所述引导序列如SEQ ID No.38所示；
    所述scFv序列如SEQ ID No.39或40或41任意一个所示；
    所述人CD8铰链区序列如SEQ ID No.42所示；
    所述人CD8跨膜区序列如SEQ ID No.43所示；
    所述人4-1BB胞内结构域序列如SEQ ID No.44所示；且
    所述CD3ζ结构域序列如SEQ ID No.45所示。
  2. 如权利要求1所述的制备方法，其特征在于：
    所述靶序列采用如SEQ ID No.6至SEQ ID No.11所示的任意一个序列。
  3. 如权利要求2所述的制备方法，其特征在于：
    针对所述如SEQ ID No.6所示的靶序列的DNA引物组：其正向序列如SEQ ID No.12所示，其反向序列如SEQ ID No.13所示；或
    针对所述如SEQ ID No.7所示的靶序列的DNA引物组：其正向序列如SEQ ID No.14所示，其反向序列如SEQ ID No.15所示；或
    针对所述如SEQ ID No.8所示的靶序列的DNA引物组：其正向序列如SEQ ID No.16所示，其反向序列如SEQ ID No.17所示；或
    针对所述如SEQ ID No.9所示的靶序列的DNA引物组：其正向序列如SEQ ID No.18所示，其反向序列如SEQ ID No.19所示；或
    针对所述如SEQ ID No.10所示的靶序列的DNA引物组：其正向序列如SEQ ID No.20所示，其反向序列如SEQ ID No.21所示；或
    针对所述如SEQ ID No.11所示的靶序列的DNA引物组：其正向序列如SEQ ID No.22所示，其反向序列如SEQ ID No.23所示。
  4. 如权利要求2所述的制备方法，其特征在于：
    所述制备方法包括构建表达靶向人PD-1基因的gRNA和CLDN18.2特异性嵌合抗原受体的重组质粒的过程，该构建过程包括以下步骤：（a1）基于所述靶序列合成与之对应的DNA引物组，所述DNA引物组包括正向序列的单链DNA和反向序列的单链DNA；（a2）将所述正向序列的单链DNA与所述反向序列的单链DNA退火，获得双链DNA；（a3）将所述双链DNA连接到lentiGuide-Puro质粒的限制性内切酶BsmBI的酶切位点处，得到表达靶向PD-1基因的gRNA的重组质粒；（a4）合成所述CLDN18.2特异性嵌合抗原受体分子的核苷酸序列，通过分子克隆的方式连接到所述表达靶向PD-1基因的gRNA的重组质粒的限制性内切酶BstEII和MluI的酶切位点处，获得了表达靶向人PD-1基因的gRNA和CLDN18.2特异性嵌合抗原受体的重组质粒；
    所述制备方法还包括获得Cas9核酸酶质粒的步骤。
  5. 如权利要求4所述的制备方法，其特征在于：
    所述的制备方法还包括将所述表达靶向人PD-1基因的gRNA和CLDN18.2特异性嵌合抗原受体的重组质粒转染293T细胞，获得第一病毒液；
    所述的制备方法还包括将所述Cas9核酸酶质粒转染293T细胞，获得第二病毒液；
    将所述第一病毒液和所述第二病毒液混合后感染T细胞，从感染后的细胞中获得人PD-1基因敲除的CLDN18.2特异性嵌合抗原受体T细胞。
  6. 一种成套质粒，由权利要求3中所述的表达靶向人PD-1基因的gRNA和CLDN18.2特异性嵌合抗原受体的重组质粒和Cas9核酸酶质粒组成。
  7. 一种获得人PD-1基因敲除的CLDN18.2特异性嵌合抗原受体T细胞的试剂盒，其特征在于：所述试剂盒包括如权利要求6所述的成套质粒以及试剂盒说明书，所述试剂盒说明书记载有如权利要求5所述的制备方法。
  8. 如权利要求1至5中任意一项所述的制备方法获得的人PD-1基因敲除的CLDN18.2特异性嵌合抗原受体T细胞，在制备治疗肿瘤的产品方面的应用。
  9. 如权利要求8所述应用，其特征在于：所述肿瘤包括阳性表达CLDN18.2蛋白的肿瘤细胞。
  10. 如权利要求9所述应用，其特征在于：所述应用是指制备治疗胰腺癌和胃癌的药物方面的应用
PCT/CN2017/076513 2016-08-31 2017-03-13 人pd-1基因敲除的cldn18.2 特异性嵌合抗原受体t细胞的制备方法以及应用 WO2018040537A1 (zh)

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CN111705085A (zh) * 2020-08-20 2020-09-25 北京百奥赛图基因生物技术有限公司 构建动物模型的方法及应用
CN113234685A (zh) * 2021-05-25 2021-08-10 临沂大学 一种双靶点cd19/cd20联用嵌合抗原受体t细胞及其制备方法和应用
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