A mutant protein related to notch signaling pathway and use thereof

Abstract

The present application relates to the field of biological medicine, in particular to a mutant protein related to Notch signal pathway and use thereof. The mutant protein provided by the present application is a mutant Notch1 protein, a mutant DLL4 protein or a mutant JAG1 protein. The mutant protein related to Notch signal pathway provided by the present application: the mutant Notch1 protein has strong activation signal after binding with its ligands DLL4 and JAG1, and is used for regulating the disorder of Notch signal pathway, and can provide a basis for the occurrence mechanism research and development of treatment strategies of developmental disorders, cancers and other diseases.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to a mutant protein associated with the Notch signaling pathway and its uses. Background Technology

[0002] The Notch signaling pathway is a unique pathway that transduces signals via cell-to-cell contact through the interaction of Notch receptors on the cell membrane with ligands such as JAG1 or DLL4. It was discovered by Mohr in Drosophila in 1919. Subsequently, it was found that the Notch signaling pathway is highly conserved from nematodes to mammals. In over a century of research, the Notch signaling pathway has been recognized as one of the most important signal transduction pathways, significantly involved in cell proliferation, death, fate determination, and individual development.

[0003] Dysregulation of the Notch signaling pathway is associated with various developmental disorders and cancers, such as developmental syndromes, Alzheimer's disease, cardiovascular disease, leukemia, and squamous cell carcinoma. The second step of Notch cleavage is crucial in signal transduction and is the most prone to dysregulation. This second step relies on the interaction between the ligand and Notch, which alters the stress-sensitive NRR (negative regulatory region) domain of Notch, exposing the S2 site, which is deeply embedded within the NRR and cleavable by ADAM, thus enabling a second cleavage.

[0004] Therefore, the interaction between the ligand and Notch is considered a crucial step in regulating the second enzymatic cleavage. Existing research indicates that Notch can sense stress generated during intercellular interactions. This stress can promote prolonged, low-affinity interactions between the ligand and Notch, forming a catch bond, which effectively promotes ADAM cleavage and drives downstream Notch signal transduction. The molecular basis for the catch bond formation between Notch and the ligand may lie in the formation of new hydrogen bonds and salt bridges during the dynamic interaction of Notch and the ligand under stress regulation. Summary of the Invention

[0005] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide a mutant protein related to the Notch signaling pathway and its uses, in order to solve the problems in the prior art.

[0006] To achieve the above and other related objectives, the present invention provides a mutant protein related to the Notch signaling pathway, wherein the mutant protein is a mutant Notch1 protein, a mutant DLL4 protein, or a mutant JAG1 protein.

[0007] The mutant Notch1 protein has a mutation at amino acid position 470 and / or position 421 relative to the wild-type Notch1 protein, and the wild-type Notch1 protein contains a polypeptide with the amino acid sequence shown in SEQ ID No. 1;

[0008] The mutant DLL4 protein has a mutation at amino acid position 215 and / or position 114 relative to the wild-type DLL4 protein, and the wild-type DLL4 protein contains a polypeptide with an amino acid sequence as shown in SEQ ID No. 2.

[0009] The mutant JAG1 protein has a mutation at position 93 of the amino acid sequence compared to the wild-type JAG1 protein, and the wild-type JAG1 protein contains a polypeptide with the amino acid sequence shown in SEQ ID No. 3.

[0010] The present invention also provides an isolated polynucleotide that encodes the aforementioned mutant protein.

[0011] The present invention also provides a nucleic acid construct comprising the aforementioned polynucleotide and backbone plasmid.

[0012] The present invention also provides a viral vector containing the aforementioned polynucleotide or the aforementioned nucleic acid construct.

[0013] The present invention also provides the use of the aforementioned mutant protein, the aforementioned polynucleotide, the aforementioned nucleic acid construct or the aforementioned viral vector in the preparation of disease treatment products, stem cell treatment products or organoid culture products.

[0014] As described above, the Notch signaling pathway-related mutant protein and its uses according to the present invention have the following beneficial effects:

[0015] The mutant Notch1 protein provided by this invention has a strong activation signal after binding with its ligands DLL4 and JAG1, which can regulate the dysregulation of the Notch signaling pathway and can provide a basis for studying the pathogenesis of diseases such as developmental disorders and cancer, as well as developing treatment strategies. Attached Figure Description

[0016] Figure 1 The results shown are from the co-culture of Notch1 mutant and DLL4 wild type in this invention.

[0017] Figure 2 The results shown are from the co-culture of Notch1 mutant and JAG1 wild-type in this invention.

[0018] Figure 3 The results shown are from the co-culture of Notch1 wild-type and DLL4 mutant in this invention.

[0019] Figure 4 The results shown are from the co-culture of Notch1 wild-type and JAG1 mutant in this invention. Detailed Implementation

[0020] This invention provides a mutant protein related to the Notch signaling pathway, wherein the mutant protein is a mutant Notch1 protein, a mutant DLL4 protein, or a mutant JAG1 protein.

[0021] The mutant Notch1 protein has a mutation at amino acid position 470 and / or position 421 relative to the wild-type Notch1 protein, and the wild-type Notch1 protein contains a polypeptide with the amino acid sequence shown in SEQ ID No. 1;

[0022] The mutant DLL4 protein has a mutation at amino acid position 215 and / or position 114 relative to the wild-type DLL4 protein, and the wild-type DLL4 protein contains a polypeptide with an amino acid sequence as shown in SEQ ID No. 2.

[0023] The mutant JAG1 protein has a mutation at position 93 of the amino acid sequence compared to the wild-type JAG1 protein, and the wild-type JAG1 protein contains a polypeptide with the amino acid sequence shown in SEQ ID No. 3.

[0024] In some specific embodiments, the mutant Notch1 protein, relative to the wild-type Notch1 protein, has mutations at amino acid positions 311, 322, 323, 324, 325, 353, 355, 356, 357, 368, 369, 378, 388, 389, 390, 391, 392, 397, 415, 418, 419, 420, 422, 424, 435, 436, 448, 450, 451, 452, 466, 468, 469, 477, 480, and / or 496.

[0025] In some specific embodiments, the mutant DLL4 protein has mutations relative to the wild-type DLL4 protein at amino acid positions 71, 76, 109, 110, 179, 185, 186, 187, 189, 191, 195, 216, 218, 65, 66, 78, and / or 108; preferably, the mutant DLL4 protein has mutations relative to the wild-type DLL4 protein at amino acid positions 187, 76, 78, 195, 108, and / or 71.

[0026] In some specific embodiments, the mutant JAG1 protein has mutations relative to the wild-type JAG1 protein at amino acid positions 81, 87, 94, 125, 126, 127, 191, 197, 199, 201, 203, 205, 206, 207, 210, 255, 269, 280, 283, 287, 289, 290, 291, 292, 297, 298, 301, 311, 312, 314, 315, 316 and / or 317.

[0027] In some specific embodiments, the mutation is the change from the amino acid corresponding to the wild-type Notch1 protein, wild-type DLL4 protein, or wild-type JAG1 protein to a polar or charged amino acid.

[0028] Further, the polar or charged amino acid is selected from any one of the following: arginine, asparagine, aspartic acid, glutamine, glutamic acid, histidine, lysine, or serine. Preferably, the polar or charged amino acid is histidine.

[0029] Furthermore, the mutation includes any one or more of the following:

[0030] 1) Mutant Notch1 proteins: T311H, V322H, C323H, V324H, N325H, R353H, A355H, S356H, F357H, L368H, L369H, S378H, D388H, T389H, N390H, P391H, V392H, I397H, E415H, L418H, G419H, A420H, N421H, P422H, E424H, S435H, F436H, R448H, E450H, I451H, D452H, T466H, L468H, D469H, Q470H, I477H, P480H, or S496H;

[0031] 2) Mutant DLL4 proteins: E71H, F76H, F109H, T110H, Y179H, S185H, R186H, L187H, K189H, K191H, F195H, K215H, Y216H, D218H, Y65H, Q66H, N78H, N108H, or T114H;

[0032] 3) Mutant JAG1 proteins: E81H, R87H, S93H, F94H, S125H, F126H, A127H, Y191H, N197H, F199H, R201H, R203H, D205H, F206H, F207H, Y210H, Y255H, P269H, W280H, L283H, N287H, G289H, G290H, Q291H, L292H, L297H, N298H, G301H, T311H, C312H, N314H, T315H, G316H, or P317H.

[0033] In some specific embodiments, the mutant Notch1 protein, mutant DLL4 protein, or mutant JAG1 protein may also be a polypeptide fragment having 90% or more sequence identity with SEQ ID No. 1, 2, or 3, provided that it has the aforementioned mutation.

[0034] Further, the polypeptide fragment specifically refers to a polypeptide fragment obtained by substituting, deleting, or adding one or more (specifically 1-50, 1-30, 1-20, 1-10, 1-5, or 1-3) amino acids to the amino acid sequence shown in SEQ ID No. 1, 2, or 3, under the premise of having the aforementioned mutation, or by adding one or more (specifically 1-50, 1-30, 1-20, 1-10, 1-5, or 1-3) amino acids to the N-terminus and / or C-terminus, and having the function of the polypeptide fragment with the amino acid sequence shown in SEQ ID No. 1, wherein the amino acid sequence of the mutant protein is identical to any of SEQ ID No. 1, 2, or 3 with a sequence identity of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more.

[0035] In some specific embodiments, the mutant protein includes a histidine tag. A histidine tag, or multiple histidine tags, is a sequence of 2 to 20 histidine residues attached to a DNA polymerase. Histidine tags may contain 2 to 20 histidine residues, 5 to 15 histidine residues, 5 to 18 histidine residues, 5 to 16 histidine residues, 5 to 15 histidine residues, 5 to 14 histidine residues, 5 to 13 histidine residues, 5 to 12 histidine residues, 5 to 11 histidine residues, 5 to 10 histidine residues, 6 to 12 histidine residues, 6 to 11 histidine residues, or 7 to 10 histidine residues. Histidine tags can be used to purify proteins using chromatographic methods with nickel-based chromatographic media.

[0036] In some specific embodiments, the mutant protein comprises a polypeptide fragment encoding a fluorescent protein. Specifically, the fluorescent protein may be selected from green fluorescent protein (GFP), red fluorescent protein (RFP), yellow fluorescent protein (YFP), or blue fluorescent protein (BFP).

[0037] The present invention also provides an isolated polynucleotide encoding the aforementioned mutant protein.

[0038] The present invention also provides a nucleic acid construct comprising the aforementioned polynucleotide and backbone plasmid.

[0039] In this application, the backbone plasmid, also referred to as a "plasmid backbone," is a circular or linear DNA molecule that can autonomously replicate and express the inserted target gene within the cell. The backbone plasmid may contain regulatory sequences, such as promoters, replicons, and transcription and translation initiation and termination codons. The backbone plasmid is typically linked with the target gene to form a complete expression vector capable of expressing a specific product within the cell.

[0040] The present invention also provides a viral vector containing the aforementioned nucleic acid construct or the aforementioned polynucleotide.

[0041] In some specific embodiments, the viral vector may be selected from adeno-associated virus, adenovirus, or lentivirus.

[0042] The present invention also provides the use of the aforementioned mutant protein, the aforementioned polynucleotide, the aforementioned nucleic acid construct or the aforementioned viral vector in the preparation of disease treatment products, stem cell treatment products or organoid culture products.

[0043] In some specific embodiments, the disease may be selected from developmental disorders or tumors.

[0044] Furthermore, the tumor may be selected from one or more of the following: adrenocortical carcinoma, bladder urothelial carcinoma, breast cancer, cervical squamous cell carcinoma, cervical endogenous adenocarcinoma, bile duct carcinoma, colonic adenocarcinoma, lymphoid tumor, esophageal cancer, glioblastoma multiforme, head and neck squamous cell carcinoma, renal chromophobe carcinoma, renal clear cell carcinoma, renal papillary cell carcinoma, acute myeloid leukemia, low-grade glioma of the brain, hepatocellular carcinoma, mesothelial cell carcinoma, ovarian cancer, pancreatic cancer, pheochromocytoma, paraganglioma, prostate cancer, rectal cancer, malignant sarcoma, melanoma, gastric cancer, testicular germ cell tumor, thyroid cancer, thymic carcinoma, endometrial cancer, chronic myeloid leukemia, lung cancer, anal cancer, and retinoblastoma.

[0045] The present invention also provides a method for treating a disease, wherein the method comprises administering the aforementioned mutant protein, the aforementioned polynucleotide, the aforementioned nucleic acid construct or the aforementioned viral vector to a patient.

[0046] In some specific embodiments, the dosage is 1-1000 mg / kg / day. Specifically, the dosage is 1-3 mg / kg / day, 3-5 mg / kg / day, 5-10 mg / kg / day, 10-20 mg / kg / day, 20-30 mg / kg / day, 30-40 mg / kg / day, 40-60 mg / kg / day, 60-80 mg / kg / day, 80-100 mg / kg / day, 100-200 mg / kg / day, 200-500 mg / kg / day, or 500 mg-1000 mg / kg / day.

[0047] In some specific embodiments, the object of the method can be a mammal; preferably, the object of the method is a human.

[0048] In this invention, the terms "homology," "identity," or "similarity" refer to the sequence similarity between two peptides or two nucleic acid molecules. Homology can be determined by comparing corresponding positions in different polypeptide or nucleic acid molecules. When the same position in the sequence of the compared molecules is occupied by the same base or amino acid in different sequences, then the molecules are homologous at that position. The degree of homology between sequences is determined as a function of the number of common matching or homologous positions. An "unrelated" or "non-homologous" sequence should have less than 20% homology with one of the sequences disclosed in this invention.

[0049] A percentage of sequence homology (e.g., 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99%) between a polynucleotide or polynucleotide region (or polypeptide or polypeptide region) and another polynucleotide or polynucleotide region (or polypeptide or polypeptide region) means that, at the time of alignment, the two sequences being aligned contain that percentage of identical bases (or amino acids). This alignment and percentage homology or sequence identity can be determined using software programs and methods known in the art.

[0050] In this invention, the term "backbone plasmid" can also be referred to as "plasmid backbone," which is a circular or linear DNA molecule that can autonomously replicate and express the inserted target gene within a cell. The backbone plasmid may contain regulatory sequences, such as promoters, replicons, and transcription and translation initiation and termination codons. The backbone plasmid is typically linked with the target gene to form a complete expression vector capable of expressing a specific output within the cell.

[0051] In this invention, the terms "polynucleotide" and "oligonucleotide" are used interchangeably, and they refer to polymeric forms of nucleotides of any length, whether deoxyribonucleotides, ribonucleotides, or analogs thereof. Polynucleotides can have any three-dimensional structure and can perform any function, known or unknown. Examples of polynucleotides include, but are not limited to, the following: genes or gene fragments (including probes, primers, EST or SAGE tags), exons, introns, messenger RNA, transfer RNA, ribosomal RNA, ribozymes, cDNA, dsRNA, siRNA, miRNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Polynucleotides also include modified nucleotides, such as methylated nucleotides and nucleotide analogs. If modifications are present on the polynucleotide, these modifications can be conferred before or after assembly. Nucleotide sequences can be broken down by non-nucleotide components. Polynucleotides can be further modified after polymerization, for example, by coupling with labeled components. The term refers to both double-stranded and single-stranded polynucleotide molecules. Unless otherwise stated or required, any embodiment of the polynucleotide disclosed in this invention includes its double-stranded form and any one of two complementary single-stranded forms known or predicted to constitute the double-stranded form.

[0052] In this invention, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably and refer to a polymeric form of amino acids of any length, which may include encoded and non-coding amino acids, chemically or biochemically modified or derived amino acids, and polypeptides having a modified peptide backbone.

[0053] In this invention, the term "DNA sequence encoding a specific RNA" refers to a DNA nucleic acid sequence transcribed into RNA. DNA polynucleotides can encode RNA (mRNA) that is translated into protein, or they can encode RNA that is not translated into protein (e.g., tRNA, rRNA, or gRNA; also called "non-coding" RNA or "ncRNA"). A "protein-coding sequence," or a sequence encoding a specific protein or polypeptide, is a nucleic acid sequence that, under the control of appropriate regulatory sequences, is transcribed into mRNA (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vivo or in vitro.

[0054] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0055] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention; in the specification and claims of the present invention, unless otherwise expressly stated in the text, the singular forms "a", "an" and "this" include the plural forms.

[0056] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0057] The specific sequences used in this application are as follows:

[0058] SEQ ID No.1 (Full length 1-2531)

[0059] MPRLLAPLLCLTLLPALAARGLRCSQPSGTCLNGGRCEVANGTEACVCSGAFVGQRCQDPS

[0060] PCLSTPCKNAGTCYVVDHGGIVDYACSCPLGFSGPLCLTPLANACLANPCRNGGTCDLLTLT

[0061] EYKCRCPPGWSGKSCQQADPCASNPCANGGQCLPFESSYICGCPPGFHGPTCRQDVNECSQ

[0062] NPGLCRHGGTCHNEIGSYRCACRATHTGPHCELPYVPCSPSPCQNGGTCRPTGDTTHECAC

[0063] LPGFAGQNCEENVDDCPGNNCKNGGACVDGVNTYNCRCPPEWTGQYCTEDVDECQLMP

[0064] NACQNGGTCHNSHGGYNCVCVNGWTGEDCSENIDDCASAACFQGATCHDRVASFYCECP

[0065] HGRTGLLCHLNDACISNPCNEGSNCDTNPVNGKAICTCPSGYTGPACSQDVDECALGANPC

[0066] EHAGKCLNTLGSFECQCLQGYTGPRCEIDVNECISNPCQNDATCLDQIGEFQCICMPGYEGV

[0067] YCEINTDECASSPCLHNGRCVDKINEFLCQCPKGFSGHLCQYDVDECASTPCKNGAKCLDG

[0068] PNTYTCVCTEGYTGTHCEVDIDECDPDPCHYGLCKDGVATFTCLCQPGYTGHHCETNINEC

[0069] HSQPCRHGGTCQDRDNYYLCLCLKGTTGPNCEINLDDCASNPCDSGTCLDKIDGYECACEP

[0070] GYTGSMCNVNIDECAGSPCHNGGTCEDGIAGFTCRCPEGYHDPTCLSEVNECNSNPCIHGA

[0071] CRDGLNGYKCDCAPGWSGTNCDINNNECESNPCVNGGTCKDMTSGYVCTCREGFSGPNC

[0072] QTNINECASNPCLNQGTCIDDVAGYKCNCPLPYTGATCEVVLAPCATSPCKNSGVCKESED

[0073] YESFSCVCPTGWQGQTCEIDINECVKSPCRHGASCQNTNGSYRCLCQAGYTGRNCESDIDD

[0074] CRPNPCHNGGSCTDGVNAAFCDCLPGFQGAFCEEDINECASNPCQNGANCTDCVDSYTCT

[0075] CPTGFNGIHCENNTPDCTESSCFNGGTCVDGINSFTCLCPPGFTGSYCQYDVNECDSRPCLH

[0076] GGTCQDSYGTYKCTCPQGYTGLNCQNLVRWCDSAPCKNGGKCWQTNTQYHCECRSGWT

[0077] GFNCDVLSVSCEVAAQKRGIDVTLLCQHGGLCVDEEDKHYCHCQAGYTGSYCEDEVDECS

[0078] PNPCQNGATCTDYLGGFSCKCVAGYHGSNCSEEINECLSQPCQNGGTCIDLTNTYKCSCPR

[0079] GTQGVHCEINVDDCHPPLDPASRSPKCFNNGTCVDQVGGYTCTCPPGFVGERCEGDVNEC

[0080] LSNPCDPRGTQNCVQRVNDFHCECRAGHTGRRCESVINGCRGKPCRNGGVCAVASNTARG

[0081] FICRCPAGFEGATCENDARTCGSLRCLNGGTCISGPRSPTCLCLGSFTGPECQFPASSPCVGSN

[0082] PCYNQGTCEPTSESPFYRCLCPAKFNGLLCHILDYSFTGGAGRDIPPPQIEEACELPECQEDA

[0083] GNKVCNLQCNNHACGWDGGDCSLNFNDPWKNCTQSLQCWKYFSDGHCDSQCNSAGCLF

[0084] DGFDCQLTEGQCNPLYDQYCKDHFSDGHCDQGCNSAECEWDGLDCAEHVPERLAAGTLV

[0085] LVVLLPPDQLRNNSFHFLRELSHVLHTNVVFKRDAQGQQMIFPYYGREEELRKHPIKRSAV

[0086] GWATTSLLPGTNGGRQRRELDPMDIHGSIVYLEIDNRQCVQSSSQCFQSATDVAAFLGALAS

[0087] LGSLNIPYKIEAVKSETVEPPLPSQLHLMYVAAAAFVLLFFVGCGVLLSRKRRRQHGQLWFP

[0088] EGFKVSEASKKKRREPLGEDSVGLKPLKNASDGALMDDNQNEWGDEDLETKKFRFEEPV

[0089] VLPDLDDQTDHRQWTQQHLDAADLRVSAMAPTPPQGEVDADCMDVNVRGPDGFTPLMI

[0090] ASCSGGGLETGNSEEEEDAPAVISDFIYQGASLHNQTDRTGETALHLAARYSRSDAAKRLLE

[0091] ASADANIQDNMGRTPLHAAVSADAQGVFQILLRNRATDLDARMHDGTTPLILAARLAVEG

[0092] MLEDLINSHADVNAVDDLGKSALHWAAAVNNVDAAVVLLKNGANKDMQNNKEETPLFL

[0093] AAREGSYETAKVLLDHFANRDITDHMDRLPRDIAQERMHHDIVRLLDEYNLVRSPQLHGTA

[0094] LGGTPTLSPTLCSPNGYLGNLKSATQGKKARKPSTKGLACSSKEAKDLKARRKKSQDGKG

[0095] CLLDSSSMLSPVDSLESPHGYLSDVASPPLLPSPFQQSPSMPLSHLPGMPDTHLGISHLNVAA

[0096] KPEMAALAGGSRLAFEPPPPRLSHLPVASSASTVLSTNGTGAMNFTVGAPASLNGQCEWLP

[0097] RLQNGMVPSQYNPLRPGVTPGTLSTQAAGLQHGMMGPIHSSLSTNTLSPIIYQGLPNTRLAT

[0098] QPHLVQTQQVQPQNLQIQPQNLQPPSQPHLSVSSAANGHLGRSFLSGEPSQADVQPLGPSSL

[0099] PVHTILPQESQALPTSLPSSMVPPMTTTQFLTPPSQHSYSSSPVDNTPSHQLQVPEHPFLTPSP

[0100] ESPDQWSSSSPHSNISDWSEGISSPPTSMPSQITHIPEAFK

[0101] SEQ ID No.2(Full length 1 - 685)

[0102] MAAASRSASGWALLLLVALWQQRAAGSGIFQLRLQEFANERGMLANGRPCEPGCRTFFRIC

[0103] LKHYQATFSEGPCTFGNVSTPVLGTNSFVIRDKNSGSGRNPLQLPFNFTWPGTFSLNIQAWH

[0104] TPGDDLRPETSPGNSLISQIIIQGSLAVGKNWKSDEQNNTLTRLRYSYRVVCSDNYYGDSCS

[0105] RLCKKRDDHFGHYECQPDGSLSCLPGWTGKYCDQPICLSGCHEQNGYCSKPDECNCRPG

[0106] WQGPLCNECIPHNGCRHGTCTIPWQCACDEGWGGLFCDQDLNYCTHHSPCKNGSTCSNSG

[0107] PRGYTCTCLPGYTGEHCELELSKCASNPCRNGGSCKDHENSYHCLCPPGYYGQHCEHSTLT

[0108] CADSPCFNGGSCRERNQGASYACECPPNFTGSNCEKKVDRCTSNPCANGGQCLNRGPSRTC

[0109] RCRPGFTGTHCELHISDCARSPCAHGGTCHDLENGPVCTCPAGFSGRRCEVRITNDACASGP

[0110] CFNGATCYTGLSPNNFVCNCPYGFVGSRCEFPVGLPPSFPWVAVSLGVGLVVLLVLLVMVAV

[0111] AVRQLRLRRPDDDSREAMNNLSDFQKDNLIPAAQLKNTNQKKELEVDCGLDKSNCGKLQ

[0112] NHTLDYNLAPGFLGRGSTPGKYPHSDKSLGEKVPLRLHSEKPACRISAICSPRDSMYQSVCL

[0113] ISEERNECVIATEV

[0114] SEQ ID No.3 (Full length 1 - 1219)

[0115] MRSPRTRGRSGRPLSLLLALLCALRAKVCGASGQFELEILSMQNVNGELQNGNCCGGARN

[0116] PGDRKCTRDECDTYFKVCLKEYQSRVTAGGPCSFGSGSTPVIGGNTFNLKASRGNDRNRIV

[0117] LPFSFAWPRSYTLLVEAWDSSNDTIQPDSIIEKASHSGMINPSRQWQTLKQNTGIAHFEYQIR

[0118] VTCDDHYYGFGCNKFCRPRDDFFGHYACDQNGNKTCMEGWMPGPECNKAICRQGCSPKH

[0119] GSCKLPGDCRCQYGWQGLYCDKCIPHPGCVHGTCNEPWQCLCETNWGGQLCDKDLNYC

[0120] GTHQPCLNRGTCSNTGPDKYQCSCPEGYSGPNCEIAEHACLSDPCHNRGSCKETSSGFECE

[0121] CSPGWTGPTCSTNIDDCSPNNCSHGGTCQDLVNGFKCVCPPQWTGKTCQLDANECEAKPC

[0122] VNARSCKNLIASYYCDCLPGWMGQNCDININDCLGQCQNDASCRDLVNGYRCICPPGYAG

[0123] DHCERDIDECASNPCLNGGHCQNEINRFQCLCPTGFSGNLCQLDIDYCEPNPCQNGAQCYN

[0124] RASDYFCKCPEDYEGKNCSHLKDHCRTTPCEVIDSCTVAMASNDTPEGVRYISSNVCGPHG

[0125] KCKSESGGKFTCDCNKGFTGTYCHENINDCEGNPCTNGGTCIDGVNSYKCICSDGWEGAH

[0126] CENNINDSCSQNPCHYGGTCRDLVNDFYCDCKNGWKGKTCHSRDSQCDEATCNNGGTCYD

[0127] EVDTFKCMCPGGWEGTTCNIARNSSCLPNPCHNGGTCVVNGDSFTCVCKEGWEGPICTQN

[0128] TNDCSPHPCYNSGTCVDGDNWYRCECAPGFAGPDCRININECQSSPCAFGATCVDEINGYQ

[0129] CICPPGHSGAKCHEVSGRSCITMGRVILDGAKWDDDCNTCQCLNGRVACSKVWCGPRPCQ

[0130] LHKGHGECPNGQSCIPVLDDQCFVRPCTGAGECRSSSLQPVKTKCTSDSYYQDNCANITFT

[0131] FNKEMMSPGLTTEHICSELRNLNILKNVSAEYSIYIACEPSLSANNEIHVAISAEDIRDDGNPV

[0132] KEITDKIIDLVSKRDGNSSLIAAVAEVRVQRRPLKNRTDFLVPLLSSVLTVAWVCCLVTAFYW

[0133] CVRKRRRKPSSHTHSAPEDNTTNNVREQLNQIKNPIEKHGANTVPIKDYENKNSKMSKIRT

[0134] HNSEVEEDDMDKHQQKVRFAKQPVYTLVDREEKAPSGTPTKHPNWTNKQDNRDLESAQS

[0135] LNRMEYIV

[0136] SEQ ID No.4

[0137] MAAASRSASGWALLLLVALWQQRAAGSGIFQLRLQEFANERGMLANGRPCEPGCRTF

[0138] FRICLKHYQATFSEGPCTFGNVSTPVLGTNSFVIRDKNSGSGRNPLQLPFNFTWPGTFSLNIQ

[0139] AWHTPGDDLRPETSPGNSLISQIIIQGSLAVGKNWKSDEQNNTLTRLRYSYRVVCSDNYYGD

[0140] SCSRLCKKRDDHFGHYECQPDGSLSCLPGWTGKYCDQPICLSGCHEQNGYCSKPDECNCR

[0141] PGWQGPLCNECIPHNGCRHGTCTIPWQCACDEGWGGLFCDQDLNYCTHHSPCKNGSTCSN

[0142] SGPRGYTCTCLPGYTGEHCE

[0143] Example 1: Construction of wild-type / mutant cell lines of Notch1 / DLL4 / JAG1

[0144] Using existing gene sequences Notch1 (NM_001105721.1), JAG1 (NM_019147.3), and DLL4 (NM_001107760.1), primers for single-site histidine mutations were designed (mutant primers are shown in Table 1), amplified, and cloned into the pHR plasmid. Subsequently, the virus was packaged using the pHR, psPAX2, and pMD2G three-plasmid system. Next, K562 cells equipped with the UAS-eBFP-PGK-mCherry reporter system were infected with Notch1 wild-type / mutant virus, and ordinary K562 cells were infected with DLL4 / JAG1 wild-type / mutant virus. After appropriate culture time, flow cytometry was performed to enrich positive cells.

[0145] Table 1. Design of Mutant Primers

[0146]

[0147]

[0148]

[0149]

[0150]

[0151] Example 2: Co-culture and activation of Notch1 and DLL4 / JAG1

[0152] The co-culture was divided into four groups: ① mutNotch1s + wtDLL4, ② mutNotch1s + wtJAG1, ③ mutDLL4s + wtNotch1, and ④ mutJAG1s + wtNotch1. The cell culture medium was changed the day before co-culturing to ensure the cells were in optimal condition for the next day's co-culture. 2 × 10⁶ cells were used. 4 One Notch receptor cell, 1×1025 One DLL4 / JAG1 ligand cell (i.e., a receptor cell to ligand cell ratio of 1:5) was added to a 96-well plate, and culture medium was added to a final volume of 200 μL. After static co-culturing for 24 hours, flow cytometry was used to detect the activation and expression of BFP (blue fluorescent protein) in Notch receptor cells.

[0153] The results of the above experiment are as follows Figures 1-4 As shown, where Figure 1 Results: In the co-culture group of Notch1 mutant and DLL4 wild type, Notch1-N421H / Q470H had a relatively strong post-modification activation signal, indicating the success of histidine mutation and Notch1 mutation modification. Figure 2 Results: In the co-culture group of Notch1 mutant and JAG1 wild type, Notch1-Q470H had a relatively strong post-modification activation signal, indicating the success of histidine mutation and Notch1 mutation modification. Figures 3-4 The results showed that the DLL4 mutants had effects at the L187, F76, N78, F195, N108, and E71 sites, while the K215 and T114 sites had significant effects.

[0154] Example 3: Optical tweezers measurement of Notch1 receptor cells and DLL4

[0155] The N-EGF3 region of DLL4 was cloned into the pd649 plasmid and transfected with Expi293F to generate DLL4 (N-EGF3) protein. The purified DLL4 (N-EGF3) protein (amino acid sequence shown in SEQ ID No. 4) was obtained after purification using Ni-NTA and molecular sieve chromatography (Superdex 200 increase, Cytiva). Subsequently, the force-time relationship of Notch1 receptor cells and DLL4 (N-EGF3) protein was analyzed using M-Crap chromatography, and an inverse bond-locking curve was fitted.

[0156] The above embodiments are for illustrating the implementation schemes disclosed in this invention and should not be construed as limiting the invention. Furthermore, various modifications and variations of the methods listed herein will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been specifically described in conjunction with various specific preferred embodiments, it should be understood that the invention should not be limited to these specific embodiments. In fact, various modifications as described above that are obvious to those skilled in the art to obtain the invention should be included within the scope of this invention.

Claims

1. A mutant protein associated with the Notch signaling pathway, characterized in that, The mutant protein is a mutant Notch1 protein, a mutant DLL4 protein, or a mutant JAG1 protein; The mutant Notch1 protein has a mutation at amino acid position 470 and / or 421 relative to the wild-type Notch1 protein, and the wild-type Notch1 protein contains a polypeptide with the amino acid sequence shown in SEQ ID No. 1; and / or, the mutant DLL4 protein has a mutation at amino acid position 215 and / or 114 relative to the wild-type DLL4 protein, and the wild-type DLL4 protein contains a polypeptide with the amino acid sequence shown in SEQ ID No. 2; and / or, the mutant JAG1 protein has a mutation at amino acid position 93 relative to the wild-type JAG1 protein, and the wild-type JAG1 protein contains a polypeptide with the amino acid sequence shown in SEQ ID No.

3.

2. The mutant protein according to claim 1, characterized in that, The mutant Notch1 protein, relative to the wild-type Notch1 protein, has mutations at amino acid positions 311, 322, 323, 324, 325, 353, 355, 356, 357, 368, 369, 378, 388, 389, 390, 391, 392, 397, 415, 418, 419, 420, 422, 424, 435, 436, 448, 450, 451, 452, 466, 468, 469, 477, 480, and / or 496. And / or, the mutant DLL4 protein has mutations relative to the wild-type DLL4 protein at amino acid positions 71, 76, 109, 110, 179, 185, 186, 187, 189, 191, 195, 216, 218, 65, 66, 78, and / or 108; preferably, the mutant DLL4 protein has mutations relative to the wild-type DLL4 protein at amino acid positions 187, 76, 78, 195, 108, and / or 71. And / or, the mutant JAG1 protein has mutations relative to the wild-type JAG1 protein at amino acid positions 81, 87, 94, 125, 126, 127, 191, 197, 199, 201, 203, 205, 206, 207, 210, 255, 269, 280, 283, 287, 289, 290, 291, 292, 297, 298, 301, 311, 312, 314, 315, 316 and / or 317.

3. The mutant protein according to claim 1 or 2, characterized in that, The mutation is the change of the amino acid corresponding to the wild-type Notch1 protein, wild-type DLL4 protein, or wild-type JAG1 protein to a polar or charged amino acid.

4. The mutant protein according to claim 3, characterized in that, The polar or charged amino acid is selected from any of the following: arginine, asparagine, aspartic acid, glutamine, glutamic acid, histidine, lysine, or serine.

5. The mutant protein according to claim 4, characterized in that, The mutation includes any one or more of the following: 1) Mutant Notch1 proteins: T311H, V322H, C323H, V324H, N325H, R353H, A355H, S356H, F357H, L368H, L369H, S378H, D388H, T389H, N390H, P391H, V392H, I397H, E415H, L418H, G419H, A420H, N421H, P422H, E424H, S435H, F436H, R448H, E450H, I451H, D452H, T466H, L468H, D469H, Q470H, I477H, P480H, or S496H; 2) Mutant DLL4 proteins: E71H, F76H, F109H, T110H, Y179H, S185H, R186H, L187H, K189H, K191H, F195H, K215H, Y216H, D218H, Y65H, Q66H, N78H, N108H, or T114H; 3) Mutant JAG1 proteins: E81H, R87H, S93H, F94H, S125H, F126H, A127H, Y191H, N197H, F199H, R201H, R203H, D205H, F206H, F207H, Y210H, Y255H, P269H, W280H, L283H, N287H, G289H, G290H, Q291H, L292H, L297H, N298H, G301H, T311H, C312H, N314H, T315H, G316H, or P317H.

6. An isolated polynucleotide, characterized in that, The polynucleotide encodes the mutant protein as described in any one of claims 1-5.

7. A nucleic acid construct, characterized in that, The nucleic acid construct comprises the polynucleotide and backbone plasmid as described in claim 6.

8. A viral vector, characterized in that, The viral vector contains the polynucleotide of claim 6 or the nucleic acid construct of claim 7.

9. Use of the mutant protein of any one of claims 1-5, the polynucleotide of claim 6, the nucleic acid construct of claim 7, or the viral vector of claim 8 in the preparation of disease treatment products, stem cell treatment products, or organoid culture products.

10. The use according to claim 9, characterized in that, The disease is selected from developmental disorders or tumors; preferably, the tumor is selected from one or more of the following: adrenocortical carcinoma, bladder urothelial carcinoma, breast cancer, cervical squamous cell carcinoma, cervical endogenous adenocarcinoma, bile duct carcinoma, colonic adenocarcinoma, lymphoid tumor, esophageal cancer, glioblastoma multiforme, head and neck squamous cell carcinoma, renal chromophobe carcinoma, renal clear cell carcinoma, renal papillary cell carcinoma, acute myeloid leukemia, low-grade glioma of the brain, hepatocellular carcinoma, mesothelial cell carcinoma, ovarian cancer, pancreatic cancer, pheochromocytoma, paraganglioma, prostate cancer, rectal cancer, malignant sarcoma, melanoma, gastric cancer, testicular germ cell tumor, thyroid cancer, thymic carcinoma, endometrial cancer, chronic myeloid leukemia, lung cancer, anal cancer, and retinoblastoma.

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