KHL polypeptide and application thereof in preparation of TABP-EIC cells
1. A KHL polypeptide or a conjugate thereof that specifically binds to PSMA, wherein said KHL polypeptide is selected from any one of:
1) 1, polypeptide shown as SEQ ID NO;
2) 1, the derivative polypeptide which is formed by substituting, deleting or adding one or more amino acid residues of the polypeptide shown in SEQ ID NO. 1 and has basically the same function;
3) a polypeptide having more than 90% homology with the polypeptide shown in SEQ ID NO. 1, specifically, the polypeptide has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% homology with the polypeptide shown in SEQ ID NO. 1;
preferably, the KHL polypeptide is a polypeptide shown as SEQ ID NO. 1;
preferably, the conjugate of a KHL polypeptide comprises a KHL polypeptide and a detectable label;
preferably, the detectable label comprises one or more of a fluorescent dye, a fluorescent molecule, a chemiluminescent label, a dye molecule, a phosphorescent molecule, biotin, a radioisotope, a molecule that absorbs in the UV spectrum, a molecule that absorbs in the near infrared radiation, or a molecule that absorbs in the far infrared radiation;
preferably, the detectable label is a fluorescent molecule;
preferably, the fluorescent molecule is FITC.
2. A tumor antigen binding peptide comprising a specific binding region that specifically binds PSMA;
preferably, said specific binding region comprises the KHL polypeptide of claim 1;
preferably, said specific binding region is 3 repeats of the KHL polypeptide of claim 1;
preferably, said KHL polypeptide is linked between 3 repeats with GGGS;
preferably, the tumor antigen binding peptide further comprises a hinge region, a transmembrane domain and/or a signaling domain;
preferably, the hinge region is a CD8 a hinge region;
preferably, the amino acid sequence of the hinge region is shown as SEQ ID NO. 11;
preferably, the transmembrane domain is selected from a transmembrane region of 2B4 gene;
preferably, the amino acid sequence of the transmembrane domain is shown as SEQ ID NO. 2;
preferably, the signalling domain comprises a co-stimulatory domain and/or a primary signalling domain;
preferably, the co-stimulation signal is an intracellular signaling structure of 2B4 gene;
preferably, the amino acid sequence of the costimulatory signal is shown as SEQ ID NO 3;
preferably, the primary signaling domain is selected from the intracellular signaling structure of the NKG2D gene;
preferably, the amino acid sequence of the primary signaling domain is as set forth in SEQ ID NO 4;
preferably, the tumor antigen binding peptide is composed in the order KHL polypeptide-hinge region-transmembrane domain-costimulatory domain-primary signaling domain;
preferably, the amino acid sequence of the tumor antigen binding peptide is shown in SEQ ID NO. 5 at positions 22-316;
preferably, the tumor antigen binding peptide may also be linked to a signal peptide;
preferably, the amino acid sequence of the tumor antigen binding peptide connected with the signal peptide is shown as SEQ ID NO. 5.
3. A DNA molecule encoding the KHL polypeptide of claim 1, the tumor antigen binding peptide of claim 2, or the tumor antigen binding peptide of claim 2 linked to a signal peptide;
preferably, the sequence of the DNA molecule encoding the KHL polypeptide of claim 1 is shown in SEQ ID NO 6;
preferably, the DNA molecule sequence encoding the tumor antigen binding peptide of claim 2 is SEQ ID NO 10 at positions 64-948;
preferably, the DNA molecule encoding the tumor antigen binding peptide linked to a signal peptide of claim 2 has the sequence shown in SEQ ID NO. 10.
4. A vector comprising the DNA molecule of claim 3;
preferably, the vector comprises a plasmid, a lentiviral vector, an adenoviral vector, or a retroviral vector;
preferably, the vector further comprises one or more regulatory elements.
5. A host cell comprising one or more of the polypeptide of claim 1, the tumor antigen binding peptide of claim 2, the DNA molecule of claim 3, and the vector of claim 4;
preferably, the host cell comprises Escherichia coli, Streptomyces, Agrobacterium, yeast cells, plant cells, animal cells or viruses;
preferably, the virus is a lentivirus;
preferably, the animal cell is a human immune cell;
preferably, the immune cell is an NK cell.
6. A pharmaceutical composition comprising one or more of the polypeptide of claim 1, the tumor antigen binding peptide of claim 2, the DNA molecule of claim 3 and the vector of claim 4 and the host cell of claim 5;
preferably, the pharmaceutical composition further comprises an optional pharmaceutically acceptable immunomodulator.
7. A method of making the host cell of claim 5, comprising the step of introducing into the host cell one or more of a DNA molecule encoding the polypeptide of claim 1, a DNA molecule encoding the tumor antigen binding peptide of claim 2, a DNA molecule of claim 3, and a vector of claim 4;
preferably, the method of introduction into the host cell may be a particle gun method, an electrotransfer method, a viral transduction method or a heat shock method.
8. A method for detecting PSMA, comprising the step of contacting a conjugate of a KHL polypeptide of claim 1 with a sample to be detected;
preferably, the detection is for non-diagnostic purposes;
preferably, the sample to be detected is a sample suspected of containing PSMA.
9. A kit for detecting PSMA, comprising reagents for use in the method of claim 8;
preferably, the kit further comprises the instruments or devices required for the detection of PSMA.
10. An application, comprising any one of:
1) use of the polypeptide of claim 1, the tumor antigen binding peptide of claim 2, the DNA molecule of claim 3, the vector of claim 4, or the host cell of claim 5 in the preparation of the pharmaceutical composition of claim 6;
2) use of the polypeptide of claim 1, the tumor antigen binding peptide of claim 2, the DNA molecule of claim 3, or the vector of claim 4 in the preparation of the host cell of claim 5;
3) use of the DNA molecule of claim 3 for the preparation of the vector of claim 4;
4) use of the polypeptide of claim 1, the tumor antigen binding peptide of claim 2, the DNA molecule of claim 3, the vector of claim 4, the host cell of claim 5, or the pharmaceutical composition of claim 6 in the manufacture of a medicament for the treatment of cancer;
preferably, the cancer is prostate cancer;
5) use of a conjugate of the polypeptide of claim 1 for the preparation of a kit for the diagnosis of cancer;
preferably, the cancer is prostate cancer;
preferably, the diagnosis is detected PSMA;
preferably, the kit is the kit of claim 9.
Background
Tumor Chimeric Antigen Receptor (CAR) therapy refers to a novel precise targeted therapy, which has a good effect on clinical tumor therapy by optimization and improvement in recent years, and is a novel tumor immunotherapy method with a very promising possibility of curing cancer. Generally, the CAR activates immune cells through a genetic engineering technology, and is provided with a positioning navigation device CAR, common T cells and NK cells are correspondingly recombined into CAR-T cells and CAR-NK cells, and the CAR-T cells and CAR-NK cells can specifically recognize in vivo tumor cells by utilizing chimeric CAR thereof, and release a large amount of multiple effector factors through immunization, so that the tumor cells can be efficiently killed, and the purpose of treating malignant tumors is achieved. In addition, transplantation of the immune cells with the localized knockins into the CAR back into the body of the original patient can avoid the challenge of the autoimmune system.
Natural Killer (NK) cells are an important component of the non-specific immune system, cells that are critical mediators of the innate immune system response. NK cells are a broad spectrum immune cells with specific functions of rapidly discovering and destroying abnormal cells (such as cancer or virus-infected cells), and exhibit potent activity of lysing abnormal cells without the need for pre-sensitization or HLA-typing. However, the number of NK cells is small, the NK cells only account for 10% -15% of lymphocytes in peripheral blood, the content of NK in umbilical cord blood is lower, only about 5%, and the content of NK cells in normal human peripheral blood and umbilical cord blood is far from meeting the requirement of clinical treatment. The number and purity of NK cells are important influence factors of clinical curative effect, the number of NK cells is too small to achieve the curative effect, and the low purity can influence the killing effect on tumors.
Chimeric antigen receptor (abbreviated CAR) modified immune cells use genetic engineering approaches to modify immune cells to express exogenous anti-tumor genes. The CAR gene mainly includes an extracellular recognition domain and an intracellular signaling domain: the former is used for identifying tumor surface specific molecules, and the latter is used for starting immune cell response after identifying tumor surface molecules, and plays a role in cytotoxicity.
The NK cell modified by the CAR structure can efficiently identify the tumor cell, and kill the tumor cell by various means such as releasing a killing medium, inducing apoptosis of a target cell and the like. However, the CAR-NK cell prepared by the prior art has the problems of complex operation of the preparation method, insufficient activity of the prepared cell and small quantity.
The KHL polypeptide with better tumor specific binding efficiency is screened by a phage display technology, and the specificity of the polypeptide is verified. The KHL polypeptide can be used for detecting a cancer marker PSMA after being combined with a detectable marker, and TABP-EIC (Tumor Antigen Binding Peptide-engineered Immune Cell) cells prepared from the KHL polypeptide can accurately target PSMA-expressing cells.
Disclosure of Invention
The invention provides a KHL polypeptide specifically combined with PSMA (prostate specific membrane antigen), a conjugate thereof, a tumor antigen binding peptide, a DNA molecule, a vector and a host cell.
Polypeptides
In one aspect, the invention provides a KHL polypeptide that specifically binds to PSMA, said KHL polypeptide being selected from any one of:
1) 1, polypeptide shown as SEQ ID NO;
2) 1, and has PSMA specific binding function, wherein the PSMA specific binding function is formed by substituting, deleting or adding one or more (2, 3,4, 5, 6) amino acid residues of the polypeptide shown in SEQ ID NO;
3) the polypeptide has more than 90 percent of homology with the polypeptide shown in SEQ ID NO. 1, in particular, the polypeptide has 90 percent, 91 percent, 92 percent, 93 percent, 94 percent, 95 percent, 96 percent, 97 percent, 98 percent and 99 percent of homology with the polypeptide shown in SEQ ID NO. 1.
Preferably, the KHL polypeptide is a polypeptide shown as SEQ ID NO. 1.
Preferably, the KHL polypeptide may comprise one or more synthetic amino acids known in the art and including, but not limited to, aminocyclohexanecarboxylic acid, norleucine, α -amino n-decanoic acid, homoserine, S-acetamidomethyl-cysteine, trans-3-and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β -phenylserine β -hydroxyphenylalanine, phenylglycine, α -naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-2-carboxylic acid, 1,2,3, 4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, phenylglycine, α -naphthylalanine, cyclohexylalanine, cyclohexylglycine, and mixtures thereof, Aminomalonic acid monoamides, N ' -benzyl-N ' -methyl-lysine, N ' -dibenzyl-lysine, 6-hydroxylysine, ornithine, alpha-aminocyclopentanecarboxylic acid, alpha-aminocyclohexanecarboxylic acid, alpha-aminocycloheptane carboxylic acid, alpha- (2-amino-2-norbornane) -carboxylic acid, alpha, gamma-diaminobutyric acid, alpha, beta-diaminopropionic acid, homophenylalanine and alpha-tert-butylglycine.
Conjugates
In another aspect, the invention provides a conjugate of a KHL polypeptide that specifically binds to PSMA.
Preferably, the conjugate of a KHL polypeptide comprises a KHL polypeptide and a detectable label.
Preferably, the detectable label comprises one or more of a fluorescent dye, a fluorescent molecule, a chemiluminescent label, a dye molecule, a phosphorescent molecule, biotin, a radioisotope, a molecule that absorbs in the UV spectrum, a molecule that absorbs in the near infrared radiation or a molecule that absorbs in the far infrared radiation.
Preferably, the fluorescent dye includes, but is not limited to, rhodamine, p-methylaminophenol, fluorescein, thiofluorescein, aminofluorescein, carboxyfluorescein, chlorofluorescein, methylfluorescein, sulfofluorescein, amino-p-methylaminophenol, carboxy-p-methylaminophenol, chloro-p-methylaminophenol, methyl-p-methylaminophenol, sulfop-methylaminophenol, aminorhodamine, carboxyrhodamine, chlororhodamine, methylrhodamine, sulforhodamine, and sulforhodamine, cyanine, indocyanine, oxonol, thiacarbocyanine, merocyanine, cyanine dyes (e.g., cyanine 2, cyanine 3, cyanine 3.5, cyanine 5, cyanine 5.5, cyanine 7), oxadiazole derivatives, pyridyloxazole, nitrobenzoxadiazole, benzonitrobenzene, pyrene derivatives, waterfall blue, oxazine derivatives, nile red, nile blue, cresol purple, oxazine 170, acridine derivatives, proflavine, acridine orange, carboxyfluorescein, methylchlororhodamine, and sulforhodamine Acridine yellow, arylmethine derivatives, auramine, thioxanthene dyes, sulfonated thioxanthene dyes, AlexaFluor (e.g., AlexaFluor 594, AlexaFluor 633, AlexaFluor 647, AlexaFluor 700), crystal violet, malachite green, tetrapyrrole derivatives, porphyrins, phthalocyanines, bilirubin, cy5.5, indocyanine green (ICG), DyLight750, or IRdye 800.
Preferably, the fluorescent molecule includes, but is not limited to, FAM, FITC, VIC, JOE, TET, CY3, CY5, ROX, Texas Red, or LC Red 460.
Preferably, the chemiluminescent label includes, but is not limited to, peroxidase, alkaline phosphatase, luciferase, aequorin, functionalized iron-porphyrin derivatives, luminol, isoluminol, acridinium esters, sulfonamides, and the like.
Preferably, the luciferase includes, but is not limited to, a pleiones magna (Gaussia) luciferase, a Renilla (Renilla) luciferase, a dinoflagellate luciferase, a firefly luciferase, a fungal luciferase, a bacterial luciferase, and a glowworm (vargula) luciferase.
Preferably, the detectable label is a fluorescent molecule;
preferably, the fluorescent molecule is FITC.
Tumor antigen binding peptides
In another aspect, the present invention provides a tumor antigen binding peptide comprising a specific binding domain that specifically binds to PSMA.
Preferably, the specific binding domain comprises the aforementioned KHL polypeptide.
Preferably, the specific binding domain is a multiple repeat of the aforementioned KHL polypeptide.
Preferably, the plurality of times is 1 to 10 times; specifically, 1 time, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times.
Preferably, the specific binding domain is 3 repeats of the aforementioned KHL polypeptide.
Preferably, said plurality of repeats of KHL polypeptide are linked by a linker.
Preferably, the linker is GS, GGS, GGGS.
Preferably, the linker is GGGS.
Preferably, the tumor antigen binding peptide further comprises a hinge region, a transmembrane domain and/or a signaling domain;
preferably, the hinge region comprises a combination of one or more of a CD8 a hinge region, a CD28 hinge region, a CD4 hinge region, a CD5 hinge region, a CD134 hinge region, a CD137 hinge region, an ICOS hinge region.
Preferably, the hinge region is a CD8 a hinge region.
Preferably, the amino acid sequence of the hinge region is shown as SEQ ID NO. 11.
Preferably, the nucleic acid sequence of the hinge region is shown in SEQ ID NO 12.
Preferably, the transmembrane domain comprises a transmembrane domain of a protein comprising: the transmembrane region of the 2B4 gene, the α, β or ζ chain of a T cell receptor, CD28, CD3 ∈, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD123, CD134, CD137 and CD 154.
Preferably, the transmembrane domain is selected from the transmembrane region of the 2B4 gene.
Preferably, the amino acid sequence of the transmembrane domain is shown as SEQ ID NO 2.
Preferably, the nucleic acid sequence of the transmembrane domain is as shown in SEQ ID NO 7.
Preferably, the signalling domain comprises a co-stimulatory domain and/or a primary signalling domain.
Preferably, the co-stimulatory domain comprises functional signaling domains of 2B4, CD3 ζ, OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1(CD11a/CD18), ICOS (CD278) and 4-1BB (CD 137).
Preferably, the co-stimulatory signal is an intracellular signaling structure of 2B4 gene.
Preferably, the amino acid sequence of the co-stimulatory signal is shown in SEQ ID NO 3.
Preferably, the nucleic acid sequence of the co-stimulatory signal is shown as SEQ ID NO. 8;
preferably, the primary signaling domain is selected from the intracellular signaling structure of the NKG2D gene;
preferably, the amino acid sequence of the primary signaling domain is as set forth in SEQ ID NO 4.
Preferably, the nucleic acid sequence of the primary signaling domain is set forth in SEQ ID NO 9.
Preferably, the tumor antigen binding peptide is composed in the order KHL polypeptide-transmembrane domain-costimulatory domain-primary signaling domain.
Preferably, the amino acid sequence of the tumor antigen binding peptide is shown in SEQ ID NO. 5 at positions 22-316.
Preferably, the tumor antigen binding peptide may also be linked to a signal peptide.
Preferably, the amino acid sequence of the tumor antigen binding peptide connected with the signal peptide is shown as SEQ ID NO. 5.
DNA molecules
In another aspect, the present invention provides a DNA molecule encoding the aforementioned KHL polypeptide.
Preferably, the sequence of the DNA molecule for coding the KHL polypeptide is shown as SEQ ID NO. 6.
In another aspect, the present invention provides a DNA molecule encoding the aforementioned tumor antigen binding peptide.
Preferably, the DNA molecule sequence encoding the tumor antigen binding peptide of claim 2 is SEQ ID NO 10 at positions 64-948.
Preferably, the tumor antigen binding peptide may also be linked to a signal peptide.
Preferably, the DNA molecule sequence of the tumor antigen binding peptide connected with the signal peptide is shown as SEQ ID NO. 10.
Carrier
In another aspect, the present invention provides a vector comprising the aforementioned DNA molecule encoding a polypeptide and/or the aforementioned DNA molecule encoding a tumor antigen binding peptide.
Preferably, the vector comprises a plasmid (expression plasmid, cloning vector, minicircle, microcarrier, double minichromosome), lentiviral vector, adenoviral vector or retroviral vector.
Preferably, the lentiviral vector comprises a primate recombinant lentiviral vector, i.e., a recombinant Human Immunodeficiency Virus (HIV) or a recombinant Simian Immunodeficiency Virus (SIV).
Preferably, the lentiviral vector comprises a non-primate recombinant lentiviral vector, i.e., a recombinant Equine Infectious Anemia Virus (EIAV), a recombinant Feline Immunodeficiency Virus (FIV), or a recombinant Caprine Arthritis Encephalitis Virus (CAEV).
Preferably, the lentiviral vector comprises the following: pLKO 0.1-puro, pLKO 0.1-CMV-tGFP, pLKO.1-puro-CMV-tGFP, pLKO 0.1-CMV-Neo, pLKO 0.1-Neo, pLKO.1-Neo-CMV-tGFP, pLKO.1-puro-CMV-TagCFP, pLKO.1-puro-CMV-TagYFP, pLKO.l-puro-CMV-TagFP, pLKO.1-puro-CMV-TagFP635, pLKO. pLKO-puro-IPTG-1xLacO, pLKO-puro-IPTG-3xLacO, pLPl, pLP2, pLP/VSV-G, pENTR/U6, pLenti6/BLOCK-iT-DEST, pLenti 6-GW/U6-laminsham, pcDNAl, 2/V5-GW/lacZ, pLenti6.2/N-Lumio/V5-DEST, pGCSIL-GFP and Lenti 6.2/N-Lumio/V5-GW/lacZ.
Preferably, the vector further comprises one or more regulatory elements.
Preferably, the regulatory elements comprise a promoter, enhancer, ribosome binding site for translation initiation, terminator, polyadenylation sequence, selectable marker gene.
Preferably, the promoter is an inducible promoter, a constitutive promoter, a tissue specific promoter, a suicide type promoter, or any combination thereof.
Host cell
In another aspect, the present invention provides a host cell comprising one or more of the aforementioned polypeptide, the aforementioned tumor antigen binding peptide, the aforementioned DNA molecule encoding a KHL polypeptide, the aforementioned DNA molecule encoding a tumor antigen binding peptide, and the aforementioned vector.
In one embodiment, the host cell comprises one or more of E.coli, Streptomyces, Agrobacterium, yeast cells, plant cells, animal cells, or viruses.
In one embodiment, the virus is a lentivirus.
In one embodiment, the animal cell is a human cell.
Preferably, the host cell is an immune cell.
Preferably, the immune cells comprise one or more of T cells, B cells, K cells and NK cells.
Preferably, the immune cell is an NK cell.
Preferably, the immune cells are autologous or allogeneic.
Preferably, the immune cell is a commercial cell line; preferably, the cell line comprises NK-92, NKG, YT, NK-YS, HANK-1, YTS or NKL.
The host Cell prepared in the embodiments of the present invention is called TABP-EIC (Tumor Antigen Binding Peptide-engineered Immune Cell).
Pharmaceutical composition
In another aspect, the present invention provides a pharmaceutical composition comprising one or more of the aforementioned polypeptide, the aforementioned tumor antigen binding peptide, the aforementioned DNA molecule encoding a KHL polypeptide, the aforementioned DNA molecule encoding a tumor antigen binding peptide, the aforementioned vector, and the aforementioned host cell.
Preferably, the pharmaceutical composition may be a tablet (including a sugar-coated tablet, a film-coated tablet, a sublingual tablet, an orally disintegrating tablet, an oral tablet and the like), a pill, a powder, a granule, a capsule (including a soft capsule, a microcapsule), a lozenge, a syrup, a liquid, an emulsion, a suspension, a controlled release preparation (e.g., an instantaneous release preparation, a sustained release microcapsule), an aerosol, a film (e.g., an orally disintegrating film, an oral mucosa-adhesive film), an injection (e.g., subcutaneous injection, intravenous injection, intramuscular injection, intraperitoneal injection), an intravenous drip, a transdermal absorption preparation, an ointment, a lotion, an adhesive preparation, a suppository (e.g., a rectal suppository, a vaginal suppository), a pellet, a nasal preparation, a pulmonary preparation (an inhalant), an eye drop and the like, an oral or parenteral preparation (e.g., intravenous, intramuscular, subcutaneous, intraorgan, intranasal, intradermal, instillation, intracerebral, intrarectal, etc. administration to the vicinity of tumors and directly to lesions).
Preferably, the pharmaceutical composition further comprises an optional pharmaceutically acceptable immunomodulator.
Preferably, the immunomodulator may include, but is not limited to, cytokines, chemokines, stem cell growth factors, lymphotoxins, hematopoietic factors, Colony Stimulating Factors (CSF), erythropoietin, thrombopoietin, tumor necrosis factor-a (TNF), TNF-i3, granulocyte-colony stimulating factor (G-CSF), granulocyte macrophage-colony stimulating factor (GM-CSF), interferon- α, interferon- β, interferon- γ, interferon- λ, stem cell growth factor designated "S1 factor", human growth hormone, N-methionyl human growth hormone, bovine growth hormone, parathyroid hormone, thyroxine, insulin, proinsulin, relaxin, Follicle Stimulating Hormone (FSH), Thyroid Stimulating Hormone (TSH), Luteinizing Hormone (LH), hepatic growth factor, prostaglandin, fibroblast growth factor, prolactin, placental lactogen, OB protein, Mullerian-inhibiting substance (mullerian-inhibiting substance), mouse gonadotropin-related peptide, inhibin, activin, vascular endothelial growth factor, integrin, NGF-beta, platelet growth factor, TGF-a, TGF-f3, insulin-like growth factor-1, insulin-like growth factor-II, macrophage 43, IL-l, IL-la, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, alpha-beta-glucosidase, gamma-, IL-18, IL-21, IL-25, LIF, FLT-3, angiostatin, thrombospondin, endostatin or lymphotoxin.
Method
In another aspect, the present invention provides a method for producing the aforementioned host cell, which comprises the step of introducing the aforementioned DNA molecule encoding a KHL polypeptide, the aforementioned DNA molecule encoding a tumor antigen-binding peptide, or the aforementioned vector into a cell.
Preferably, the method for introducing the cells may be a particle gun method, an electrotransfer method, a viral transduction method or a heat shock method.
Preferably, the viral transduction method comprises a step of preparing a recombinant lentivirus and a step of infecting cells with the recombinant lentivirus.
Preferably, the recombinant lentivirus is obtained by transfecting a recombinant lentivirus vector into a lentivirus packaging cell and then performing cell culture;
in another aspect, the present invention provides a method for detecting PSMA, comprising the step of contacting a sample to be detected with a conjugate of the aforementioned KHL polypeptide.
Preferably, the method further comprises the step of processing the sample.
Preferably, the detection is for non-diagnostic purposes.
Preferably, the sample to be detected is a sample suspected of containing PSMA.
Reagent kit
In another aspect, the present invention provides a kit for detecting PSMA, the kit comprising reagents used in the aforementioned method for detecting PSMA;
preferably, the kit further comprises the instruments or devices required for the detection of PSMA.
Applications of
In another aspect, the present invention provides the use of the aforementioned polypeptide, tumor antigen binding peptide, the aforementioned DNA molecule encoding a KHL polypeptide, the aforementioned DNA molecule encoding a tumor antigen binding peptide, or the aforementioned vector, or the aforementioned host cell, in the preparation of the aforementioned pharmaceutical composition;
in another aspect, the present invention provides the use of the aforementioned polypeptide, tumor antigen-binding peptide, the aforementioned DNA molecule encoding a KHL polypeptide, the aforementioned DNA molecule encoding a tumor antigen-binding peptide, or the aforementioned vector, in the preparation of the aforementioned host cell;
in another aspect, the invention provides the use of the aforementioned DNA molecule encoding a KHL polypeptide, the aforementioned DNA molecule encoding a tumor antigen binding peptide, in the preparation of the aforementioned vector;
in another aspect, the invention provides the use of a polypeptide according to claim 1, a tumor antigen binding peptide according to claim 2, a DNA molecule according to claim 3, a vector according to claim 4, a host cell according to claim 5 or a pharmaceutical composition according to claim 6 for the manufacture of a medicament for the treatment of cancer;
preferably, the cancer is prostate cancer;
in another aspect, the present invention provides the use of the aforementioned polypeptide, tumor antigen binding peptide, the aforementioned DNA molecule encoding a KHL polypeptide, the aforementioned DNA molecule encoding a tumor antigen binding peptide, the aforementioned vector, or the aforementioned host cell, or the aforementioned pharmaceutical composition, in the preparation of a kit for diagnosing cancer.
Preferably, the cancer is prostate cancer.
Preferably, the diagnosis is detected PSMA.
Preferably, the kit is the aforementioned kit.
General definition:
unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.
The terms "polynucleotide", "nucleic acid" and "oligonucleotide" are used interchangeably to refer to a polymeric form of nucleotides of any length, i.e., deoxyribonucleotides or ribonucleotides or analogs thereof. The polynucleotide may be further modified after polymerization, for example by conjugation with a labeling component. The term also refers to double-stranded and single-stranded molecules.
As used herein, the term "vector" refers to a non-chromosomal nucleic acid comprising an intact replicon, such that when placed in a permissive cell, the vector can be replicated, e.g., by a transformation process. Vectors can replicate in one cell type (e.g., bacteria), but have a limited ability to replicate in another cell (e.g., mammalian cells). The vector may be viral or non-viral. Exemplary non-viral vectors for delivering nucleic acids include naked DNA; and DNA complexed with cationic lipids alone or in combination with cationic polymers; anionic and cationic liposomes; DNA-protein complexes and particles comprising DNA condensed with cationic polymers (e.g., heterogeneous polylysines, fixed length oligopeptides, and polyethylene imines), in some cases in liposomes;
as used herein, the terms "peptide," "polypeptide," and "protein" are used interchangeably and refer to a compound having amino acid residues covalently linked by: a peptide bond.
The term "expression" or "expression" refers to the production of a gene product in a cell.
Drawings
FIG. 1 shows the immunofluorescence results of Lncap cell line with higher expression of KHL and PSMA.
FIG. 2 shows the immunofluorescence results of PC3 cell line with low expression of KHL and PSMA.
FIG. 3 is a graph showing fluorescence detection of mice on day 14; a is blank control, B is control group, TABP-EIC-GTI cell, C is experimental group, TABP-EIC-KHL.
FIG. 4 is a graph showing fluorescence detection of mice on day 28; a is blank control, B is control group, TABP-EIC-GTI cell, C is experimental group, TABP-EIC-KHL.
FIG. 5 is a graph showing fluorescence detection of mice on day 42; a is blank control, B is control group, TABP-EIC-GTI cell, C is experimental group, TABP-EIC-KHL.
Detailed Description
The present invention will be further described with reference to the following examples, which are intended to be illustrative only and not to be limiting of the invention in any way, and any person skilled in the art can modify the present invention by applying the teachings disclosed above and applying them to equivalent embodiments with equivalent modifications. Any simple modification or equivalent changes made to the following embodiments according to the technical essence of the present invention, without departing from the technical spirit of the present invention, fall within the scope of the present invention.
Example 1 peptide library screening
1. Purpose of experiment
The invention adopts a Ph.D. -12 phage display peptide library kit to screen out the polypeptide KHL specifically combined with PSMA.
2. Ph.D. -12 phage display peptide library kit composition
Random dodecapeptide phage display library: 100 μ L, 1.5X 1013pfu/mL, stored in TBS solution with 50% Glycerol, complexity 2.7X 109Transforming the cells; -28gIII sequencing primers: 5 '-HOGTATGGGATTTTTGCTAAACAAC-3', 100pmol, 1 pmol/. mu.L; -96gIII sequencing primers: 5 '-HOCCCTCATAGTTAGCGTAACG-3', 100 pmol/. mu.L, 1 pmol/. mu.L; coli ER2738 host strain F' lacIq Δ (lacZ) M15 proA + B + zzf Tn10 (TetR)/fhaA 2 supE thi Δ (lac-proAB) Δ (hsdMS-mcrB)5 (rk-mk-McrBC-): the strain is provided in the form of a thallus culture containing 50% of glycerol, and non-competent cells are stored at-70 ℃; streptavidin, 1.5mg of freeze-dried powder; biotin: 10mM 100. mu.L.
3. Experimental methods
Day one
Depending on the number and type of target molecules on which library panning is to be performed simultaneously, panning experiments were performed in single sterile polystyrene petri dishes, 12 or 24 well plates, 96 well microtiter plates, per plateThe amount given in the following method is the amount of 60X 15mm culture dish, the amount of microwell plate in brackets, the other medium-sized wells adjusted accordingly, but in each case the same number of phage was added: 1.5X 1011A virus seed;
(1) a100. mu.g/mL solution of the target molecule (NaHCO dissolved in 0.1M, pH 8.6) was prepared3) If it is desired to stabilize the target molecule, other buffers of similar ionic strength (containing metal ions, etc.) may also be used;
(2) adding 1.5mL (150 μ L per well of microporous plate) of the above solution into each plate (well), and repeatedly rotating until the surface is completely wet;
(3) shaking slightly at 4 deg.C in a humidifying container (such as sealable plastic box arranged with wet paper towel), incubating overnight, and storing the plate in the container at 4 deg.C;
the next day
(4) Selecting ER2738 monoclonal (plate paved when measuring bacteriophage titer) in 10mL LB liquid culture medium, if amplifying eluted bacteriophage on the same day, also inoculating ER2738 in 20mL LB liquid culture medium, using 250mL triangular flask, shaking culture at 37 deg.C;
(5) pouring out the coating liquid in each plate, inverting the plate, patting and throwing the plate on a clean paper towel forcibly to remove residual solution, filling sealing liquid in each plate (or hole), and acting at 4 ℃ for at least 1 h;
(6) spin wash plate 6 times, spin each time to wash the bottom and edge of the plate or well, pour off buffer, shake-off upside down on clean paper towel to remove residual solution (or use an automatic plate washer);
(7) 4X 10 dilutions were made in 1mL (100. mu.L for microwell plates) of TBST buffer10The phage (i.e., 10. mu.L of the original library) were then added to the coated plate and gently shaken at room temperature for 10-60 min;
(8) pouring to remove the unbound phage, inverting the plate, and patting on a clean paper towel to remove the residual solution;
(9) wash the plate 10 times with TBST buffer as described in 6, change clean paper towel each time to avoid cross contamination;
(10) according to the intermolecular interactions studied, the bound phage were eluted with 1mL (100. mu.L for microwell plates) of the appropriate elution buffer, the known ligand for the target molecule was dissolved in TBS solution at a concentration of 0.1-1mM or the bound phage were competitively eluted from the immobilized target with free target solution (-100. mu.g/mL in TBS), gently shaken at room temperature for 10-60min, and the eluate was aspirated into another clean microfuge tube; non-specific buffers such as 0.2M Glycine-HCl (pH 2.2), 1mg/mL BSA can also be used to separate the bound molecules: gently shake for >10min, the eluate is aspirated into another clean microcentrifuge tube, and the eluate is neutralized with 150. mu.L (15. mu.L for microwells) 1M Tris-HCl (pH 9.1);
(11) the titers of the small amounts (. about.1. mu.L) of the eluates were determined as described above in the conventional M13 procedure, and plaques from the first or second round of eluate titer determination were sequenced as needed as follows: if necessary, the remaining eluate may be stored at 4 ℃ overnight and expanded the next day, in which case ER2738 may be cultured overnight in LB-Tet medium, the next day the culture 1:100 is diluted in 20mL LB (contained in a 250mL Erlenmeyer flask), the unexpanded eluate is added, and the culture is vigorously shaken at 37 ℃ for 4.5h, and step 13 is continued;
(12) amplification of the remaining eluate: adding the eluate into 20mL of ER2738 culture (the thallus is in the early stage of logarithm), and culturing at 37 ℃ for 4.5h by shaking vigorously;
(13) the culture was transferred into a centrifuge tube and then centrifuged at 10,000rpm at 4 ℃ for 10 min. Transferring the supernatant into another centrifugal tube, and centrifuging;
(14) transferring the upper 80% of the supernatant to a fresh tube, adding 1/6 volume of PEG/NaCl, and allowing the phage to precipitate at 4 ℃ for at least 60min overnight;
the third day
(15) Centrifuging PEG at 4 deg.C and 10,000rpm for 15min, discarding supernatant, centrifuging for a short time, and removing residual supernatant;
(16) the precipitate was resuspended in 1mL TBS, the suspension was transferred to a microcentrifuge tube and centrifuged at 4 ℃ for 5min to pellet the residual cells;
(17) transferring the supernatant into another fresh microfuge tube, reprecipitating with 1/6 volume of PEG/NaCl, incubating on ice for 15-60min, centrifuging at 4 deg.C for 10min, discarding supernatant, centrifuging for a short time, and removing residual supernatant with micropipette;
(18) the pellet was resuspended in 200. mu.L TBS, 0.02% NaN3Centrifuging for 1min, precipitating any residual insoluble substances, and transferring the supernatant into a fresh tube, wherein the supernatant is the eluate after amplification;
(19) titrating the amplified eluate with LB/IPTG/Xgal plates according to the conventional M13 method, and storing at 4 ℃;
(20) coating a plate or hole for the second round of elutriation;
the fourth and fifth days
(21) The titer was determined by counting the number of blue spots on the plate and this value was used to calculate a titer corresponding to 1-2X 1011The amount of pfu added; if the titer is too low, the next rounds of panning may be performed down to 109Testing the phage addition amount of pfu;
(22) and (3) carrying out a second round of panning: the eluate obtained by the first panning and amplification is 1-2X 1011Repeating steps 4-18 for the amount of phage in pfu, increasing the concentration of Tween to 0.5% (v/v) in the washing step;
(23) the titer of the eluate obtained from the second round of panning after amplification was determined on LB/IPTG/Xgal plates;
(24) coating a plate or a hole for a third round of elutriation;
day six
(25) Performing a third panning: 2X 10 of the eluate amplified by the second panning11The phage amount of pfu repeats steps 4-11, with the washing step again using 0.5% (v/v) Tween;
(26) the titers of the eluates from the third round of panning were determined on LB/IPTG/Xgal plates without amplification, and the eluates from the third round were not necessarily amplified unless a fourth round of panning was performed, and plaques obtained from the titer determination were used for sequencing: as long as the plate culture time is not longer than 18h, the culture time is too long, the loss is easy to occur, and the rest eluates are stored at 4 ℃;
(27) one ER2738 monoclonal was selected and cultured overnight in LB-Tet medium.
4. Results of the experiment
The experimental result shows that the amino acid sequence of the screened polypeptide KHL which is specifically combined with PSMA is KHLHYHSSVRYG (SEQ ID NO: 1).
Example 2 verification of KHL polypeptide specificity
KHL polypeptide is respectively subjected to immunofluorescence detection with Lncap cell line with high PSMA expression level and PC3 cell line with low PSMA expression level, and the used fluorescence marker is FITC.
The fluorescence detection of KHL polypeptide and Lncap cell line is shown in FIG. 1, and the fluorescence detection of KHL polypeptide and PC3 cell line is shown in FIG. 2.
In fig. 1, the center of the dot is the nucleus and the light color around the dot is the fluorescence exhibited by KHL in combination with the cell surface antigen PSMA. In FIG. 2 there is only staining of the nuclei. KHL-labeled cell surface fluorescence appears only in FIG. 1, demonstrating the high specificity of KHL binding to the cell surface antigen PSMA.
Example 3 demonstration of tumor-inhibiting effect of TABP-EIC-KHL
1. Preparation of NK cells
The NK cells used in the experiment are all obtained by amplifying Peripheral Blood Mononuclear Cells (PBMC).
2. Construction of tumor antigen-binding peptide expression vector
The structural sequences of the tumor antigen binding peptide are obtained by gene synthesis (general organisms), and the expression vector is pLenti-EF1a-Backbone (NN) (addendum # 27961). The restriction sites of the tumor antigen binding peptide structure insertion are BsiWI and EcoRI.
3. Lentiviral packaging
TABP-EIC backbone vector and auxiliary vector pMD2.G (addgen #12259), pMDLg/pRRE (addgen #12251) and pRSV-Rev (addgen #12253) were mixed at a ratio of 10:5:3:2, and 293T cells were transfected with 20ug of plasmid per 10ml transfection system. Then, supernatant was collected for 48 hours and 72 hours, and purified and concentrated to obtain lentivirus.
4. Lentiviral transduction
The concentrated lentivirus and NK cells are mixed according to 200ul of purified lentivirus per 100 ten thousand cells, then the mixture is placed in an incubator at 37 ℃ under the condition of 5% CO2, and the solution is completely changed after 24 hours.
5. Amplification of TABP-EIC-GTL cells
And (3) normally culturing and amplifying TABP-EIC cells obtained after lentivirus infection.
6. Detection of TABP-EIC-GTL cell tumor antigen binding peptide expression efficiency
And (4) taking part of cells to extract genome and carrying out RT-PCR detection on the seventh day after the TABP-EIC cell lentivirus is infected.
7. Results of the experiment
According to the results of RT-PCR, the infection efficiency (%) is 63.21-6.36 × Δ CT (detection group CT-control group CT) according to the formula, and generally more than 20% of the infection efficiency is required for use.
The KHL sequence in the structure of the tumor antigen binding peptide may be repeated in a plurality of instances, here in 3 repeats. The amino acid sequence of the tumor antigen binding peptide is shown as SEQ ID NO:5, the nucleic acid sequence of the tumor antigen binding peptide is shown as SEQ ID NO: shown at 10.
Mice were subjected to abdominal cavity tumor formation using GFP-labeled prostate cancer cell line (Lncap-GFP), abdominal cavity perfusion was performed on day 14 of tumor formation, and a blank control (saline), a control group (TABP-EIC-GTI cells) and TABP-EIC-KHL cells were sequentially perfused in the abdominal cavity at 500 ten thousand cells/cell, once a week, for 3 weeks.
Fluorescence imaging assays were performed on mice on days 14, 28, and 42 to observe tumor size.
Fluorescence imaging at day 14 is shown in FIG. 3, where A is blank, B is control (TABP-EIC-GTI cells), and C is TABP-EIC-KHL.
Fluorescence imaging at day 28 is shown in FIG. 4, where A is blank, B is control (TABP-EIC-GTI cells), and C is TABP-EIC-KHL.
Fluorescence imaging at day 42 is shown in FIG. 5, where A is blank, B is control (TABP-EIC-GTI cells), and C is TABP-EIC-KHL.
The experiment proves that TABP-EIC-GTL and TABP-EIC-KHL cells have obvious inhibition effect on tumors, and the treatment effect of TABP-EIC-KHL is better.
Sequence listing
<110> Shino Rev medicine science and technology (New zone of Zhuhai horizontal organ) Co., Ltd
<120> KHL polypeptide and application thereof in preparing TABP-EIC cells
<141> 2021-07-14
<160> 12
<170> SIPOSequenceListing 1.0
<210> 1
<211> 12
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 1
Lys His Leu His Tyr His Ser Ser Val Arg Tyr Gly
1 5 10
<210> 2
<211> 35
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 2
Gln Asp Cys Gln Asn Ala His Gln Glu Phe Arg Phe Trp Pro Phe Leu
1 5 10 15
Val Ile Ile Val Ile Leu Ser Ala Leu Phe Leu Gly Thr Leu Ala Cys
20 25 30
Phe Cys Val
35
<210> 3
<211> 120
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 3
Trp Arg Arg Lys Arg Lys Glu Lys Gln Ser Glu Thr Ser Pro Lys Glu
1 5 10 15
Phe Leu Thr Ile Tyr Glu Asp Val Lys Asp Leu Lys Thr Arg Arg Asn
20 25 30
His Glu Gln Glu Gln Thr Phe Pro Gly Gly Gly Ser Thr Ile Tyr Ser
35 40 45
Met Ile Gln Ser Gln Ser Ser Ala Pro Thr Ser Gln Glu Pro Ala Tyr
50 55 60
Thr Leu Tyr Ser Leu Ile Gln Pro Ser Arg Lys Ser Gly Ser Arg Lys
65 70 75 80
Arg Asn His Ser Pro Ser Phe Asn Ser Thr Ile Tyr Glu Val Ile Gly
85 90 95
Lys Ser Gln Pro Lys Ala Gln Asn Pro Ala Arg Leu Ser Arg Lys Glu
100 105 110
Leu Glu Asn Phe Asp Val Tyr Ser
115 120
<210> 4
<211> 51
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 4
Met Gly Trp Ile Arg Gly Arg Arg Ser Arg His Ser Trp Glu Met Ser
1 5 10 15
Glu Phe His Asn Tyr Asn Leu Asp Leu Lys Lys Ser Asp Phe Ser Thr
20 25 30
Arg Trp Gln Lys Gln Arg Cys Pro Val Val Lys Ser Lys Cys Arg Glu
35 40 45
Asn Ala Ser
50
<210> 5
<211> 316
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 5
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 Lys His Leu His Tyr His Ser Ser Val Arg Tyr
20 25 30
Gly Gly Gly Gly Ser Lys His Leu His Tyr His Ser Ser Val Arg Tyr
35 40 45
Gly Gly Gly Gly Ser Lys His Leu His Tyr His Ser Ser Val Arg Tyr
50 55 60
Gly Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile
65 70 75 80
Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala
85 90 95
Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Gln Asp
100 105 110
Cys Gln Asn Ala His Gln Glu Phe Arg Phe Trp Pro Phe Leu Val Ile
115 120 125
Ile Val Ile Leu Ser Ala Leu Phe Leu Gly Thr Leu Ala Cys Phe Cys
130 135 140
Val Trp Arg Arg Lys Arg Lys Glu Lys Gln Ser Glu Thr Ser Pro Lys
145 150 155 160
Glu Phe Leu Thr Ile Tyr Glu Asp Val Lys Asp Leu Lys Thr Arg Arg
165 170 175
Asn His Glu Gln Glu Gln Thr Phe Pro Gly Gly Gly Ser Thr Ile Tyr
180 185 190
Ser Met Ile Gln Ser Gln Ser Ser Ala Pro Thr Ser Gln Glu Pro Ala
195 200 205
Tyr Thr Leu Tyr Ser Leu Ile Gln Pro Ser Arg Lys Ser Gly Ser Arg
210 215 220
Lys Arg Asn His Ser Pro Ser Phe Asn Ser Thr Ile Tyr Glu Val Ile
225 230 235 240
Gly Lys Ser Gln Pro Lys Ala Gln Asn Pro Ala Arg Leu Ser Arg Lys
245 250 255
Glu Leu Glu Asn Phe Asp Val Tyr Ser Met Gly Trp Ile Arg Gly Arg
260 265 270
Arg Ser Arg His Ser Trp Glu Met Ser Glu Phe His Asn Tyr Asn Leu
275 280 285
Asp Leu Lys Lys Ser Asp Phe Ser Thr Arg Trp Gln Lys Gln Arg Cys
290 295 300
Pro Val Val Lys Ser Lys Cys Arg Glu Asn Ala Ser
305 310 315
<210> 6
<211> 36
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
aagcacctgc actaccacag cagcgtgaga tacggc 36
<210> 7
<211> 105
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
caggactgtc agaatgccca tcaggaattc agattttggc cgtttttggt gatcatcgtg 60
attctaagcg cactgttcct tggcaccctt gcctgcttct gtgtg 105
<210> 8
<211> 360
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
tggaggagaa agaggaagga gaagcagtca gagaccagtc ccaaggaatt tttgacaatt 60
tacgaagatg tcaaggatct gaaaaccagg agaaatcacg agcaggagca gacttttcct 120
ggagggggga gcaccatcta ctctatgatc cagtcccagt cttctgctcc cacgtcacaa 180
gaaccagcat atacattata ttcattaatt cagccttcca ggaagtctgg ttccaggaag 240
aggaaccaca gcccttcctt caatagcact atctatgaag tgattggaaa gagtcaacct 300
aaagcccaga accctgctcg attgagccgc aaagagctgg agaactttga tgtttattcc 360
<210> 9
<211> 153
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
atggggtgga ttcgtggtcg gaggtctcga cacagctggg agatgagtga atttcataat 60
tataacttgg atctgaagaa gagtgatttt tcaacacgat ggcaaaagca aagatgtcca 120
gtagtcaaaa gcaaatgtag agaaaatgca tct 153
<210> 10
<211> 951
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60
ccgaagcacc tgcactacca cagcagcgtg agatacggcg gcggcggcag caagcacctg 120
cactaccaca gcagcgtgag atacggcggc ggcggcagca agcacctgca ctaccacagc 180
agcgtgagat acggcaccac taccccagca ccgaggccac ccaccccggc tcctaccatc 240
gcctcccagc ctctgtccct gcgtccggag gcatgtagac ccgcagctgg tggggccgtg 300
catacccggg gtcttgactt cgcctgcgat caggactgtc agaatgccca tcaggaattc 360
agattttggc cgtttttggt gatcatcgtg attctaagcg cactgttcct tggcaccctt 420
gcctgcttct gtgtgtggag gagaaagagg aaggagaagc agtcagagac cagtcccaag 480
gaatttttga caatttacga agatgtcaag gatctgaaaa ccaggagaaa tcacgagcag 540
gagcagactt ttcctggagg ggggagcacc atctactcta tgatccagtc ccagtcttct 600
gctcccacgt cacaagaacc agcatataca ttatattcat taattcagcc ttccaggaag 660
tctggttcca ggaagaggaa ccacagccct tccttcaata gcactatcta tgaagtgatt 720
ggaaagagtc aacctaaagc ccagaaccct gctcgattga gccgcaaaga gctggagaac 780
tttgatgttt attccatggg gtggattcgt ggtcggaggt ctcgacacag ctgggagatg 840
agtgaatttc ataattataa cttggatctg aagaagagtg atttttcaac acgatggcaa 900
aagcaaagat gtccagtagt caaaagcaaa tgtagagaaa atgcatctta a 951
<210> 11
<211> 45
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 11
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> 12
<211> 135
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
accactaccc cagcaccgag gccacccacc ccggctccta ccatcgcctc ccagcctctg 60
tccctgcgtc cggaggcatg tagacccgca gctggtgggg ccgtgcatac ccggggtctt 120
gacttcgcct gcgat 135
