WO2024239392A1 - 靶向降解kras的嵌合泛素连接酶及其制备方法和应用 - Google Patents

靶向降解kras的嵌合泛素连接酶及其制备方法和应用 Download PDF

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WO2024239392A1
WO2024239392A1 PCT/CN2023/099965 CN2023099965W WO2024239392A1 WO 2024239392 A1 WO2024239392 A1 WO 2024239392A1 CN 2023099965 W CN2023099965 W CN 2023099965W WO 2024239392 A1 WO2024239392 A1 WO 2024239392A1
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kras
amino acid
seq
acid sequence
ubiquitin ligase
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蒋兴宇
李轩宇
李家安
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Southern University of Science and Technology
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    • C12N9/93—Ligases (6)
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  • the present invention belongs to the field of biomedicine, and specifically relates to a chimeric ubiquitin ligase for targeted degradation of KRAS, and a preparation method and application thereof.
  • RAS is a common mutated gene in tumors. In normal cells, the activation of RAS is in the downstream pathway of the cell membrane growth factor receptor family.
  • RAS protein is a membrane-bound GTP/GDP binding protein (GTPase), which is activated when bound to GTP and inactive when bound to GDP.
  • GTPase membrane-bound GTP/GDP binding protein
  • RAS isomers are mainly encoded by three genes: KRAS, NRAS and HRAS.
  • KRAS is the most common mutant type in the RAS family and is associated with 22% of human tumors. It is a high-frequency mutated gene in the three major fatal cancers in humans, lung cancer (17%), colorectal cancer (33%) and pancreatic cancer (61%).
  • Common KRAS mutations mainly occur in codons 12, 13, 61 and 146 in exon 2, of which mutations occurring at sites 12 and 13 account for 90% of the total number of mutations.
  • degrading protein accumulation in cells at the protein level is also an important strategy to reduce protein expression, among which protein degradation is an important method to reduce its accumulation in cells.
  • the mainstream protein targeted degradation technology is to connect the target protein and E3 ligase together by constructing chimeric molecules, thereby promoting the degradation of the target protein.
  • the typical representative technology is PROTAC (protein degradation targeted chimera). It is reported that there are currently many proteins that can be rapidly degraded by PROTAC technology. However, one PROTAC in this technology can only degrade one mutant KRAS, and cannot degrade multiple mutant KRAS, and is not generally applicable.
  • the present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art. To this end, the present invention provides a chimeric ubiquitin ligase for targeted degradation of KRAS, which can degrade different mutant KRAS.
  • the present invention also provides a nucleic acid molecule.
  • the present invention also provides a vector.
  • the present invention also provides a host cell.
  • the present invention also provides a method for preparing the chimeric ubiquitin ligase that targets and degrades KRAS.
  • the present invention also proposes the use of the chimeric ubiquitin ligase for targeted degradation of KRAS in the preparation of products.
  • a chimeric ubiquitin ligase for targeted degradation of KRAS comprising a KRAS binding domain and an E3 ubiquitin ligase binding protein; the KRAS binding domain comprises at least one of RAF1 (52-188), ARAF, PIP5K1A, RASSF2, APP, KRAS, and RAF1;
  • amino acid sequence shown in SEQ ID NO:1 is modified by substitution, deletion or addition of one or more amino acids and has the same or similar functions;
  • amino acid sequence shown in SEQ ID NO:5 is modified by substitution, deletion or addition of one or more amino acids and has the same or similar functions;
  • the amino acid sequence of KRAS is:
  • SEQ ID NO:6 ASTEYKLVVVGAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLCVFAINNTKS or
  • amino acid sequence shown in SEQ ID NO: 6 is modified by substitution, deletion or addition of one or more amino acids and has the same or similar functions;
  • the amino acid sequence of the RAF1 is:
  • amino acid sequence shown in SEQ ID NO:7 is modified by substitution, deletion or addition of one or more amino acids and has the same or similar functions.
  • all or part of the RAF1 protein, ARAF, PIP5K1A, RASSF2, APP, and KRAS proteins can be used as natural binding proteins that specifically bind to activated KRAS.
  • the RAF1 protein is a downstream protein of KRAS, which only binds to activated KRAS, and the mutant KRAS is basically in an activated state.
  • mutant KRAS can bind to the RAF1 domain, that is, the chimeric ubiquitin ligase including the RAF1 domain can effectively degrade KRAS, solving the problem that the inhibitor (PROTAC) in the prior art reduces the ability to bind to KRAS due to KRAS mutation.
  • the chimeric ubiquitin ligase including the KRAS binding domain can effectively degrade different mutant KRAS, so that it can be used to treat different cancers and has a wide range of applications.
  • the E3 ubiquitin ligase is selected from any one of the HECT domain family, RING domain family, and U.box domain family E3 ubiquitin ligases.
  • the E3 ubiquitin ligase is selected from the RING domain family E3 ubiquitin ligase. Specifically, the E3 ubiquitin ligase is a linker protein/Cullin protein/E2 ubiquitin conjugase recruitment domain.
  • the E3 ubiquitin ligase binding protein is part or all of the adaptor protein.
  • E3 ubiquitin ligase binding protein is part of the adaptor protein, and its amino acid sequence is:
  • amino acid sequence shown in SEQ ID NO:8 is modified by substitution, deletion or addition of one or more amino acids and has the same or similar functions.
  • the chimeric ubiquitin ligase also includes a connexin, whose amino acid sequence is shown in SEQ ID NO:9:SGSGSG or SEQ ID NO:10:SGSG.
  • amino acid sequence of the chimeric ubiquitin ligase is:
  • amino acid sequence shown in SEQ ID NO:11 is modified by substitution, deletion or addition of one or more amino acids and has the same or similar functions.
  • the ARAF and the E3 ubiquitin ligase binding protein constitute a chimeric protein.
  • the amino acid sequence of the ubiquitin ligase is:
  • the amino acid sequence of the chimeric ubiquitin ligase composed of the PIP5K1A and the E3 ubiquitin ligase binding protein is:
  • the amino acid sequence of the chimeric ubiquitin ligase composed of the RASSF2 and the E3 ubiquitin ligase binding protein is:
  • the amino acid sequence of the chimeric ubiquitin ligase composed of the APP and the E3 ubiquitin ligase binding protein is:
  • SEQ ID NO:15 MGDYKDDDDKASLPGLALLLLAAWTARALEVPTDGNAGLLAEPQIAMFCGRLNMHMNVQNGKWDSDPSGTKTCIDTKEGILQYCQEVYPELQITNVVEANQPVTIQNWCKRGR KQCKTHPHFVIPYRCLVGEFVSDALLVPDKCKFLHQERMDVCETHLHWHTVAKETCSEKSTNLHDYGMLLPCGIDKFRGVEFVCCPLAEESDNVDSADAEEDDSDVWWGGADTDYADGSEDKVVV AEEEEVAEVEEEEEEADDDEDDEDGDEVEEEAEEPYEEEATERTTSIATTTTTTTESVEEVVREVCSEQAETGPCRAMISRWYFDVTEGKCAPFFYGGCGGNRNNFDTEEYCMAVCGSAMSQSLLKTTQ EPLARDPVKLPTTAASTPDAVDKYLETPGDENEHAHFQKAKERLEAKHRERMSQVMREWEEAERQAKNLPKADKKAVIQHFQE
  • a host cell comprising the nucleic acid molecule or the vector, wherein the host cell is a prokaryotic cell or a eukaryotic cell.
  • HT29 cells 1 ⁇ 10 6 HT29 cells were seeded on a six-well plate, and cell culture medium (McCoy's 5A supplemented with 10% fetal bovine serum FBS and 1 ⁇ penicillin/streptomycin) was added, and cultured in a 5% CO 2 , 37°C incubator. After overnight, the McCoy's 5A culture medium was replaced with 1 mL of OPTI culture medium, and 2 ⁇ g of S1, DS13 expression vectors and RS14 expression vectors were transfected into HT29 cells using the commercial transfection reagent Lipofectamine MessengerMAX. After 6 hours of transfection, total cell protein was extracted for Western blotting analysis. The analysis results are shown in Figure 1.
  • C is the blank control group without transfection;
  • S1 is the control group, in which SPOP (167-374) was transfected;
  • DS13 (-) was transfected with DS13 expression vector without MG132 (proteasome inhibitor);
  • DS13 (MG132) was transfected with DS13 expression vector with MG132 added;
  • RS14 (-) was transfected with RS14 expression vector without MG132;
  • RS14 (MG132) was transfected with RS14 expression vector with MG132 added;
  • Flag-tag indicates the protein expression of S1, DS13 and RS14 (because S1, DS13 and RS14 all have Flag-tag labels);
  • HSP90 is used as the internal reference protein.
  • RS14 can significantly reduce the grayscale of the KRAS band, while the reduction effect of DS13 is not obvious. It shows that RS14 can knock down the G12V mutant KRAS, while DS13 has no obvious knockdown effect on the G12V mutant KRAS.
  • A549 cells were planted on a six-well plate, cell culture medium (Ham's F-12K with 10% FBS and 1 ⁇ penicillin/streptomycin) was added, and cultured in a 5% CO 2 , 37°C incubator. After overnight, the Ham's F-12K culture medium was replaced with 1 mL of OPTI culture medium, and 2 ⁇ g of S1, DS13 expression vectors and RS14 expression vectors were transfected into A549 cells using the commercial transfection reagent Lipofectamine MessengerMAX. After 6 hours of transfection, total cell protein was extracted for Western blotting analysis. The analysis results are shown in Figure 3, and the specific markings are the same as in Example 2.
  • RS14 can significantly reduce the grayscale of the KRAS band, while the reduction effect of DS13 is not obvious. It shows that RS14 can knock down G12S mutant KRAS, while DS13 has no obvious knockdown effect on G12S mutant KRAS.
  • RS14 can degrade various mutant KRAS but not wild-type KRAS, which may be because RAF1 protein is more likely to bind to activated KRAS (ie, mutant KRAS).

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Abstract

公开了靶向降解KRAS的嵌合泛素连接酶及其制备方法和应用。该嵌合泛素连接酶包括KRAS结合结构域和E3泛素连接酶结合蛋白；KRAS结合结构域包括RAF1(52-188)、ARAF、PIP5K1A、RASSF2、APP、KRAS、RAF1中的至少一种。还公开了编码上述嵌合泛素连接酶的核酸分子；包括上述核酸分子的载体；包括上述核酸分子或载体的宿主细胞；嵌合泛素连接酶的制备方法；及该酶在制备降解KRAS蛋白的制剂或治疗KRAS相关癌症的药物中的应用。嵌合泛素连接酶能降解不同的突变型KRAS。

Description

靶向降解KRAS的嵌合泛素连接酶及其制备方法和应用 技术领域
本发明属于生物医药领域，具体涉及靶向降解KRAS的嵌合泛素连接酶及其制备方法和应用。
背景技术
RAS是肿瘤中常见的突变基因。正常细胞中，RAS的激活处于细胞膜生长因子受体家族下游通路中。RAS蛋白是一种膜结合型的GTP/GDP结合蛋白(GTPase)，当其结合GTP时为活化状态，结合GDP时为失活状态。RAS异构体主要由三种基因编码：KRAS、NRAS和HRAS。KRAS是RAS家族中最主要的突变型，并且与22％的人类肿瘤相关，是人类三大致命性癌症，肺癌(17％)，结直肠癌(33％)和胰腺癌(61％)中高频突变基因。常见的KRAS突变主要发生在2号外显子中12、13、61和146等密码子上，其中发生在第12和13位点的突变占总突变数量的90％。
目前，对蛋白质活性的抑制主要是针对目标蛋白质的活性位点进行小分子抑制剂的开发，然而研究表明不包括KRAS蛋白在内，85％的蛋白质是“不可成药蛋白”，这类蛋白质因为结构松散易变、细胞内部其它小分子竞争结合能力强、活性位点容易发生突变等原因使得很难对其进行抑制剂的开发。即使目前有开发出部分针对KRAS的小分子抑制剂，但这些抑制剂与KRAS的结合面积太小，KRAS稍有突变，构象改变，就会影响小分子抑制剂的结合。
此外，在蛋白质水平降解蛋白质在细胞中的积累也是减弱蛋白质表达的重要策略，其中蛋白质降解是减少其在细胞中积累的重要方法。主流的蛋白质靶向降解技术是通过构建嵌合分子将目标蛋白和E3连接酶连接在一起，进而促进目标蛋白的降解，典型代表技术为PROTAC(蛋白降解靶向嵌合体)。据报道，目前已有多种蛋白质能够通过PROTAC技术被迅速降解。然而，该技术中一种PROTAC只能降解一种突变型KRAS，并不能降解多种突变型KRAS，不具有普遍适用性。
发明内容
本发明旨在至少解决上述现有技术中存在的技术问题之一。为此，本发明提出一种靶向降解KRAS的嵌合泛素连接酶，能够降解不同的突变型KRAS。
本发明还提出一种核酸分子。
本发明还提出一种载体。
本发明还提出一种宿主细胞。
本发明还提出所述靶向降解KRAS的嵌合泛素连接酶的制备方法。
本发明还提出所述靶向降解KRAS的嵌合泛素连接酶在制备产品中的应用。
根据本发明的一个方面，提出了一种靶向降解KRAS的嵌合泛素连接酶，包括KRAS结合结构域和E3泛素连接酶结合蛋白；所述KRAS结合结构域包括RAF1(52-188)、ARAF、PIP5K1A、RASSF2、APP、KRAS、RAF1中的至少一种；
所述RAF1(52-188)的氨基酸序列为：
SEQ ID NO:1：ASSKTSNTIRVFLPNKQRTVVNVRNGMSLHDCLMKALKVRGLQPECCAVFRLLHEHKGKKARLDWNTDAASLIGEELQVDFLDHVPLTTHNFARKTFLKLAFCDICQKFLLNGFRCQTCGYKFHEHCSTKVPTMCVDW；或
SEQ ID NO:1所示的氨基酸序列经一个或多个氨基酸的取代、缺失或添加修饰后，且功能相同或相似的氨基酸序列；
所述ARAF的氨基酸序列为：
SEQ ID NO:2：ASEPPRGPPANGAEPSRAVGTVKVYLPNKQRTVVTVRDGMSVYDSLDKALKVRGLNQDCCVVYRLIKGRKTVTAWDTAIAPLDGEELIVEVLEDVPLTMHNFVRKTFFSLAFCDFCLKFLFHGFRCQTCGYKFHQHCSSKVPTVCVDMSTNRQQPSRFYHSVQDLSGGSRQHEAPSNRPLNELLTPQGPSPRTQHCDPEHFPFPAPANAPLQRIRSTSTPNVHMVSTTAPMDSNLIQLTGQSFSTDAAGSRGGSDGTPRGSPSPASVSSGRKSPHSKSPAEQRERKSLADDKKKVKNLGYRDSGYYWEVPPSEVQLLKRIGTGSFGTVFRGRWHGDVAVKVLKVSQPTAEQAQAFKNEMQVLRKTRHVNILLFMGFMTRPGFAIITQ WCEGSSLYHHLHVADTRFDMVQLIDVARQTAQGMDYLHAKNIIHRDLKSNNIFLHEGLTVKIGDFGLATVKTRWSGAQPLEQPSGSVLWMAAEVIRMQDPNPYSFQSDVYAYGVVLYELMTGSLPYSHIGCRDQIIFMVGRGYLSPDLSKISSNCPKAMRRLLSDCLKFQREERPLFPQILATIELLQRSLPKIERSASEPSLHRTQADELPACLLSAARLVPG；或
SEQ ID NO:2所示的氨基酸序列经一个或多个氨基酸的取代、缺失或添加修饰后，且功能相同或相似的氨基酸序列；
所述PIP5K1A的氨基酸序列为：
SEQ ID NO:3：ASASASSGPSSSVGFSSFDPAVPSCTLSSAASGIKRPMASEVLEARQDSYISLVPYASGMPIKKIGHRSVDSSGETTYKKTTSSALKGAIQLGITHTVGSLSTKPERDVLMQDFYVVESIFFPSEGSNLTPAHHYNDFRFKTYAPVAFRYFRELFGIRPDDYLYSLCSEPLIELCSSGASGSLFYVSSDDEFIIKTVQHKEAEFLQKLLPGYYMNLNQNPRTLLPKFYGLYCVQAGGKNIRIVVMNNLLPRSVKMHIKYDLKGSTYKRRASQKEREKPLPTFKDLDFLQDIPDGLFLDADMYNALCKTLQRDCLVLQSFKIMDYSLLMSIHNIDHAQREPLSSETQYSVDTRRPAPQKALYSTAMESIQGEARRGGTMETDDHMGGIPARNSKGERLLLYIGIIDILQSYRFVKKLEHSWKALVHDGDTVSVHRPGFYAERFQRFMCNTVFKKIPLKPSPSKKFRSGSSFSRRAGSSGNSCITYQPSVSGEHKAQVTTKAEVEPGVHLGRPDVLPQTPPLEEISEGSPIPDPSFSPLVGETLQMLTTSTTLEKLEVAESEFTHG；或
SEQ ID NO:3所示的氨基酸序列经一个或多个氨基酸的取代、缺失或添加修饰后，且功能相同或相似的氨基酸序列；
所述RASSF2的氨基酸序列为：
SEQ ID NO:4：ASDYSHQTSLVPCGQDKYISKNELLLHLKTYNLYYEGQNLQLRHREEEDEFIVEGLLNISWGLRRPIRLQMQDDNERIRPPPSSSSWHSGCNLGAQGTTLKPLTVPKVQISEVDAPPEGDQMPSSTDSRGLKPLQEDTPQLMRTRSDVGVRRRGNVRTPSDQRRIRRHRFSINGHFYNHKTSVFTPAYGSVTNVRINSTMTTPQVLKLLLNKFKIENSAEEFALYVVHTSGEKQKLKATDYPLIARILQGPCEQISKVFLMEKDQVEEVTYDVAQYIKFEMPVLKSFIQKLQEEEDREVKKLMRKYTVLRLMIRQRLEEIAETPATIG；或
SEQ ID NO:4所示的氨基酸序列经一个或多个氨基酸的取代、缺失或添加修饰后，且功能相同或相似的氨基酸序列；
所述APP的氨基酸序列为：
SEQ ID NO:5：ASLPGLALLLLAAWTARALEVPTDGNAGLLAEPQIAMFCGRLNMHMNVQNGKWDSDPSGTKTCIDTKEGILQYCQEVYPELQITNVVEANQPVTIQNWCKRGRKQCKTHPHFVIPYRCLVGEFVSDALLVPDKCKFLHQERMDVCETHLHWHTVAKETCSEKSTNLHDYGMLLPCGIDKFRGVEFVCCPLAEESDNVDSADAEEDDSDVWWGGADTDYADGSEDKVVEVAEEEEVAEVEEEEADDDEDDEDGDEVEEEAEEPYEEATERTTSIATTTTTTTESVEEVVREVCSEQAETGPCRAMISRWYFDVTEGKCAPFFYGGCGGNRNNFDTEEYCMAVCGSAMSQSLLKTTQEPLARDPVKLPTTAASTPDAVDKYLETPGDENEHAHFQKAKERLEAKHRERMSQVMREWEEAERQAKNLPKADKKAVIQHFQEKVESLEQEAANERQQLVETHMARVEAMLNDRRRLALENYITALQAVPPRPRHVFNMLKKYVRAEQKDRQHTLKHFEHVRMVDPKKAAQIRSQVMTHLRVIYERMNQSLSLLYNVPAVAEEIQDEVDELLQKEQNYSDDVLANMISEPRISYGNDALMPSLTETKTTVELLPVNGEFSLDDLQPWHSFGADSVPANTENEVEPVDARPAADRGLTTRPGSGLTNIKTEEISEVKMDAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIATVIVITLVMLKKKQYTSIHHGVVEVDAAVTPEERHLSKMQQNGYENPTYKFFEQMQNG；或
SEQ ID NO:5所示的氨基酸序列经一个或多个氨基酸的取代、缺失或添加修饰后，且功能相同或相似的氨基酸序列；
所述KRAS的氨基酸序列为：
SEQ ID NO:6：ASTEYKLVVVGAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHHYREQIKRVKDSEDVPMVLVGNKCDLPSRTVDTKQAQDLARSYGIPFIETSAKTRQGVDDAFYTLVREIRKHKEKMSKDGKKKKKKSKTKCVIMG；或
SEQ ID NO:6所示的氨基酸序列经一个或多个氨基酸的取代、缺失或添加修饰后，且功能相同或相似的氨基酸序列；
所述RAF1的氨基酸序列为：
SEQ ID NO:7：ASEHIQGAWKTISNGFGFKDAVFDGSSCISPTIVQQFGYQRRASDDGKLTDPSKTSNTIRVFLPNKQRTVVNVRNGMSLHDCLMKALKVRGLQPECCAVFRLLHEHKGKKARLDWNTDAASLIGEELQVDFLDHVPLTTHNFARKTFLKLAFCDICQKFLLNGFRCQTCGYKFHEHCSTKVPTMCVDWSNIRQLLLFPNSTIGDSGVPALPSLTMRRMRESVSRMPVSSQHRYSTPHAFTFNTSSPSSEGSLSQRQRSTSTPNVHMVSTTLPVDSRMIEDAIRSHSESASPSALSSSPNNLSPTGWSQPKTPVPAQRERAPVSGTQEKNKIRPRGQRDSSYYWEIEASEVMLSTRIGSGSFGTVYKGKWHGDVAVKILKVVDPTPEQFQAFRNEVAVLRKTRHVNILLFMGYMTKDNLAIVTQWCEGSSLYKHLHVQETKFQMFQLIDIARQTAQGMDYLHAKNIIHRDMKSNNIFLHEGLTVKIGDFGLATVKSRWSGSQQVEQPTGSVLWMAPEVIRMQDNNPFSFQSDVYSYGIVLYELMTGELPYSHINNRDQIIFMVGRGYASPDLSKLYKNCPKAMKRLVADCVKKVKEERPLFPQILSSIELLQHSLPKINRSASEPSLHRAAHTEDINACTLTTSPRLPVFG；或
SEQ ID NO:7所示的氨基酸序列经一个或多个氨基酸的取代、缺失或添加修饰后，且功能相同或相似的氨基酸序列。
根据本发明的一种优选的实施方式，至少具有以下有益效果：
本发明提出的嵌合泛素连接酶中，RAF1蛋白的全部或部分、ARAF、PIP5K1A、RASSF2、APP、KRAS蛋白都可以作为特异性结合激活KRAS的天然结合蛋白。其中，RAF1蛋白为KRAS的下游蛋白，只结合激活的KRAS，而突变型KRAS基本上都是处于激活状态的。意味着大部分的突变型KRAS都能与RAF1结构域结合，即包括RAF1结构域的嵌合泛素连接酶可以有效降解KRAS，解决了现有技术中的抑制剂(PROTAC)因KRAS突变而降低与KRAS结合能力的问题。另外，由于本发明中的KRAS结合结构域与多种突变型KRAS的结合能力强，使得包括KRAS结合结构域的嵌合泛素连接酶可以有效降解不同的突变型KRAS，从而可用于治疗不同的癌症，适用范围广。
在本发明的一些实施方式中，所述E3泛素连接酶选自HECT结构域家族、RING结构域家族、U.box结构域家族E3泛素连接酶中的任一种。
在本发明的一些优选的实施方式中，所述E3泛素连接酶选自RING结构域家族E3泛素连接酶。具体地，所述E3泛素连接酶为接头蛋白/Cullin蛋白/E2泛素接合酶招募结构域。
具体地，所述E3泛素连接酶结合蛋白为部分或全部所述接头蛋白。
更具体地，所述E3泛素连接酶结合蛋白为部分所述接头蛋白，其氨基酸序列为：
a)SEQ ID NO:8：MGLDYKDDDDKASGSVNISGQNTMNMVKVPECRLADELGGLWENSRFTDCCLCVAGQEFQAHKAILAARSPVFSAMFEHEMEESKKNRVEINDVEPEVFKEMMCFIYTGKAPNLDKMADDLLAAADKYALERLKVMCEDALCSNLSVENAAEILILADLHSADQLKTQAVDFINYHASDVLETSGWKSMVVSHPHLVAEAYRSLASAQCPFLGPPRKRLKQSGS；或
b)SEQ ID NO:8所示的氨基酸序列经一个或多个氨基酸的取代、缺失或添加修饰后，且功能相同或相似的氨基酸序列。
在本发明的一些实施方式中，所述嵌合泛素连接酶还包括连接蛋白，其氨基酸序列如SEQ ID NO:9：SGSGSG或SEQ ID NO:10：SGSG所示。
在本发明的一些实施方式中，所述嵌合泛素连接酶的氨基酸序列为：
a)SEQ ID NO:11：MGDYKDDDDKASSKTSNTIRVFLPNKQRTVVNVRNGMSLHDCLMKALKVRGLQPECCAVFRLLHEHKGKKARLDWNTDAASLIGEELQVDFLDHVPLTTHNFARKTFLKLAFCDICQKFLLNGFRCQTCGYKFHEHCSTKVPTMCVDWSGSGSGSVNISGQNTMNMVKVPECRLADELGGLWENSRFTDCCLCVAGQEFQAHKAILAARSPVFSAMFEHEMEESKKNRVEINDVEPEVFKEMMCFIYTGKAPNLDKMADDLLAAADKYALERLKVMCEDALCSNLSVENAAEILILADLHSADQLKTQAVDFINYHASDVLETSGWKSMVVSHPHLVAEAYRSLASAQCPFLGPPRKRLKQSGS；或
b)SEQ ID NO:11所示的氨基酸序列经一个或多个氨基酸的取代、缺失或添加修饰后，且功能相同或相似的氨基酸序列。
在本发明的一些实施方式中，所述ARAF与所述E3泛素连接酶结合蛋白构成的嵌 合泛素连接酶的氨基酸序列为：
SEQ ID NO:12：MGDYKDDDDKASEPPRGPPANGAEPSRAVGTVKVYLPNKQRTVVTVRDGMSVYDSLDKALKVRGLNQDCCVVYRLIKGRKTVTAWDTAIAPLDGEELIVEVLEDVPLTMHNFVRKTFFSLAFCDFCLKFLFHGFRCQTCGYKFHQHCSSKVPTVCVDMSTNRQQPSRFYHSVQDLSGGSRQHEAPSNRPLNELLTPQGPSPRTQHCDPEHFPFPAPANAPLQRIRSTSTPNVHMVSTTAPMDSNLIQLTGQSFSTDAAGSRGGSDGTPRGSPSPASVSSGRKSPHSKSPAEQRERKSLADDKKKVKNLGYRDSGYYWEVPPSEVQLLKRIGTGSFGTVFRGRWHGDVAVKVLKVSQPTAEQAQAFKNEMQVLRKTRHVNILLFMGFMTRPGFAIITQWCEGSSLYHHLHVADTRFDMVQLIDVARQTAQGMDYLHAKNIIHRDLKSNNIFLHEGLTVKIGDFGLATVKTRWSGAQPLEQPSGSVLWMAAEVIRMQDPNPYSFQSDVYAYGVVLYELMTGSLPYSHIGCRDQIIFMVGRGYLSPDLSKISSNCPKAMRRLLSDCLKFQREERPLFPQILATIELLQRSLPKIERSASEPSLHRTQADELPACLLSAARLVPGSGSGSVNISGQNTMNMVKVPECRLADELGGLWENSRFTDCCLCVAGQEFQAHKAILAARSPVFSAMFEHEMEESKKNRVEINDVEPEVFKEMMCFIYTGKAPNLDKMADDLLAAADKYALERLKVMCEDALCSNLSVENAAEILILADLHSADQLKTQAVDFINYHASDVLETSGWKSMVVSHPHLVAEAYRSLASAQCPFLGPPRKRLKQSGS；或
SEQ ID NO:12所示的氨基酸经一个或多个氨基酸的取代、缺失或添加后，且功能相同或相似的氨基酸序列。
在本发明的一些实施方式中，所述PIP5K1A与所述E3泛素连接酶结合蛋白构成的嵌合泛素连接酶的氨基酸序列为：
SEQ ID NO:13：MGDYKDDDDKASASASSGPSSSVGFSSFDPAVPSCTLSSAASGIKRPMASEVLEARQDSYISLVPYASGMPIKKIGHRSVDSSGETTYKKTTSSALKGAIQLGITHTVGSLSTKPERDVLMQDFYVVESIFFPSEGSNLTPAHHYNDFRFKTYAPVAFRYFRELFGIRPDDYLYSLCSEPLIELCSSGASGSLFYVSSDDEFIIKTVQHKEAEFLQKLLPGYYMNLNQNPRTLLPKFYGLYCVQAGGKNIRIVVMNNLLPRSVKMHIKYDLKGSTYKRRASQKEREKPLPTFKDLDFLQDIPDGLFLDADMYNALCKTLQRDCLVLQSFKIMDY SLLMSIHNIDHAQREPLSSETQYSVDTRRPAPQKALYSTAMESIQGEARRGGTMETDDHMGGIPARNSKGERLLLYIGIIDILQSYRFVKKLEHSWKALVHDGDTVSVHRPGFYAERFQRFMCNTVFKKIPLKPSPSKKFRSGSSFSRRAGSSGNSCITYQPSVSGEHKAQVTTKAEVEPGVHLGRPDVLPQTPPLEEISEGSPIPDPSFSPLVGETLQMLTTSTTLEKLEVAESEFTHGSGSGSVNISGQNTMNMVKVPECRLADELGGLWENSRFTDCCLCVAGQEFQAHKAILAARSPVFSAMFEHEMEESKKNRVEINDVEPEVFKEMMCFIYTGKAPNLDKMADDLLAAADKYALERLKVMCEDALCSNLSVENAAEILILADLHSADQLKTQAVDFINYHASDVLETSGWKSMVVSHPHLVAEAYRSLASAQCPFLGPPRKRLKQSGS；或
SEQ ID NO:13所示的氨基酸经一个或多个氨基酸的取代、缺失或添加后，且功能相同或相似的氨基酸序列。
在本发明的一些实施方式中，所述RASSF2与所述E3泛素连接酶结合蛋白构成的嵌合泛素连接酶的氨基酸序列为：
SEQ ID NO:14：MGDYKDDDDKASDYSHQTSLVPCGQDKYISKNELLLHLKTYNLYYEGQNLQLRHREEEDEFIVEGLLNISWGLRRPIRLQMQDDNERIRPPPSSSSWHSGCNLGAQGTTLKPLTVPKVQISEVDAPPEGDQMPSSTDSRGLKPLQEDTPQLMRTRSDVGVRRRGNVRTPSDQRRIRRHRFSINGHFYNHKTSVFTPAYGSVTNVRINSTMTTPQVLKLLLNKFKIENSAEEFALYVVHTSGEKQKLKATDYPLIARILQGPCEQISKVFLMEKDQVEEVTYDVAQYIKFEMPVLKSFIQKLQEEEDREVKKLMRKYTVLRLMIRQRLEEIAETPATIGSGSGSVNISGQNTMNMVKVPECRLADELGGLWENSRFTDCCLCVAGQEFQAHKAILAARSPVFSAMFEHEMEESKKNRVEINDVEPEVFKEMMCFIYTGKAPNLDKMADDLLAAADKYALERLKVMCEDALCSNLSVENAAEILILADLHSADQLKTQAVDFINYHASDVLETSGWKSMVVSHPHLVAEAYRSLASAQCPFLGPPRKRLKQSGS；或
SEQ ID NO:14所示的氨基酸经一个或多个氨基酸的取代、缺失或添加后，且功能相同或相似的氨基酸序列。
在本发明的一些实施方式中，所述APP与所述E3泛素连接酶结合蛋白构成的嵌合泛素连接酶的氨基酸序列为：
SEQ ID NO:15：MGDYKDDDDKASLPGLALLLLAAWTARALEVPTDGNAGLLAEPQIAMFCGRLNMHMNVQNGKWDSDPSGTKTCIDTKEGILQYCQEVYPELQITNVVEANQPVTIQNWCKRGRKQCKTHPHFVIPYRCLVGEFVSDALLVPDKCKFLHQERMDVCETHLHWHTVAKETCSEKSTNLHDYGMLLPCGIDKFRGVEFVCCPLAEESDNVDSADAEEDDSDVWWGGADTDYADGSEDKVVEVAEEEEVAEVEEEEADDDEDDEDGDEVEEEAEEPYEEATERTTSIATTTTTTTESVEEVVREVCSEQAETGPCRAMISRWYFDVTEGKCAPFFYGGCGGNRNNFDTEEYCMAVCGSAMSQSLLKTTQEPLARDPVKLPTTAASTPDAVDKYLETPGDENEHAHFQKAKERLEAKHRERMSQVMREWEEAERQAKNLPKADKKAVIQHFQEKVESLEQEAANERQQLVETHMARVEAMLNDRRRLALENYITALQAVPPRPRHVFNMLKKYVRAEQKDRQHTLKHFEHVRMVDPKKAAQIRSQVMTHLRVIYERMNQSLSLLYNVPAVAEEIQDEVDELLQKEQNYSDDVLANMISEPRISYGNDALMPSLTETKTTVELLPVNGEFSLDDLQPWHSFGADSVPANTENEVEPVDARPAADRGLTTRPGSGLTNIKTEEISEVKMDAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIATVIVITLVMLKKKQYTSIHHGVVEVDAAVTPEERHLSKMQQNGYENPTYKFFEQMQNGSGSGSVNISGQNTMNMVKVPECRLADELGGLWENSRFTDCCLCVAGQEFQAHKAILAARSPVFSAMFEHEMEESKKNRVEINDVEPEVFKEMMCFIYTGKAPNLDKMADDLLAAADKYALERLKVMCEDALCSNLSVENAAEILILADLHSADQLKTQAVDFINYHASDVLETSGWKSMVVSHPHLVAEAYRSLASAQCPFLGPPRKRLKQSGS；或
SEQ ID NO:15所示的氨基酸经一个或多个氨基酸的取代、缺失或添加后，且功能相同或相似的氨基酸序列。
在本发明的一些实施方式中，所述KRAS与所述E3泛素连接酶结合蛋白构成的嵌合泛素连接酶的氨基酸序列为：
SEQ ID NO:16：MGDYKDDDDKASTEYKLVVVGAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHHYREQIKRVKDSEDVPMVLVGNKCDLPSRTVDTKQAQDLARSYGIPFIETSAKTRQGVDDAFYTLVREIRKHKEKMSKDGKKKKKKSKTKCVIMGSGSGSVNISGQNTM NMVKVPECRLADELGGLWENSRFTDCCLCVAGQEFQAHKAILAARSPVFSAMFEHEMEESKKNRVEINDVEPEVFKEMMCFIYTGKAPNLDKMADDLLAAADKYALERLKVMCEDALCSNLSVENAAEILILADLHSADQLKTQAVDFINYHASDVLETSGWKSMVVSHPHLVAEAYRSLASAQCPFLGPPRKRLKQSGS；或
SEQ ID NO:16所示的氨基酸经一个或多个氨基酸的取代、缺失或添加后，且功能相同或相似的氨基酸序列。
在本发明的一些实施方式中，所述RAF1与所述E3泛素连接酶结合蛋白构成的嵌合泛素连接酶的氨基酸序列为：
SEQ ID NO:17：MGDYKDDDDKASEHIQGAWKTISNGFGFKDAVFDGSSCISPTIVQQFGYQRRASDDGKLTDPSKTSNTIRVFLPNKQRTVVNVRNGMSLHDCLMKALKVRGLQPECCAVFRLLHEHKGKKARLDWNTDAASLIGEELQVDFLDHVPLTTHNFARKTFLKLAFCDICQKFLLNGFRCQTCGYKFHEHCSTKVPTMCVDWSNIRQLLLFPNSTIGDSGVPALPSLTMRRMRESVSRMPVSSQHRYSTPHAFTFNTSSPSSEGSLSQRQRSTSTPNVHMVSTTLPVDSRMIEDAIRSHSESASPSALSSSPNNLSPTGWSQPKTPVPAQRERAPVSGTQEKNKIRPRGQRDSSYYWEIEASEVMLSTRIGSGSFGTVYKGKWHGDVAVKILKVVDPTPEQFQAFRNEVAVLRKTRHVNILLFMGYMTKDNLAIVTQWCEGSSLYKHLHVQETKFQMFQLIDIARQTAQGMDYLHAKNIIHRDMKSNNIFLHEGLTVKIGDFGLATVKSRWSGSQQVEQPTGSVLWMAPEVIRMQDNNPFSFQSDVYSYGIVLYELMTGELPYSHINNRDQIIFMVGRGYASPDLSKLYKNCPKAMKRLVADCVKKVKEERPLFPQILSSIELLQHSLPKINRSASEPSLHRAAHTEDINACTLTTSPRLPVFGSGSGSVNISGQNTMNMVKVPECRLADELGGLWENSRFTDCCLCVAGQEFQAHKAILAARSPVFSAMFEHEMEESKKNRVEINDVEPEVFKEMMCFIYTGKAPNLDKMADDLLAAADKYALERLKVMCEDALCSNLSVENAAEILILADLHSADQLKTQAVDFINYHASDVLETSGWKSMVVSHPHLVAEAYRSLASAQCPFLGPPRKRLKQSGS；或
SEQ ID NO:17所示的氨基酸经一个或多个氨基酸的取代、缺失或添加后，且功能相同或相似的氨基酸序列。
根据本发明的第二个方面，提出了一种核酸分子，包括编码所述靶向降解KRAS的嵌合泛素连接酶的DNA或mRNA。
根据本发明的第三个方面，提出了一种载体，包括所述核酸分子。
在本发明的一些实施方式中，所述载体为表达载体。
具体地，包括所述核酸分子的载体可用于克隆或用于生产表达载体，所述载体可以是质粒、病毒、噬菌体或任何其他遗传工程中通常使用的载体。
具体地，所述载体除包括所述核酸分子外，还可包括用于控制表达的真核或原核元件，如用于启动和终止转录和/或翻译的调节序列、增强子、启动子、信号序列等。
在本发明的一些实施方式中，所述载体包括细菌、真菌、昆虫、病毒或哺乳动物载体。
根据本发明的第四个方面，提出了一种宿主细胞，包括所述核酸分子或所述载体，所述宿主细胞为原核细胞或真核细胞。
具体地，所述核酸分子或所述载体可被引入合适的宿主细胞。所述核酸分子可被引入所述宿主细胞的基因组；所述载体可在胞质中以染色体外的形式存在或被并入所述宿主细胞的染色体中。
在本发明的一些实施方式中，所述原核细胞为细菌细胞。
在本发明的一些实施方式中，所述真核细胞包括酵母细胞、丝状真菌细胞、哺乳动物细胞或昆虫细胞。
根据本发明的第五个方面，提出了所述靶向降解KRAS的嵌合泛素连接酶的制备方法，包括以下步骤：培养第四方面的宿主细胞，得所述嵌合泛素连接酶。
根据本发明的第六个方面，提出了所述靶向降解KRAS的嵌合泛素连接酶在制备产品中的应用，所述产品为如下任一种：
a)降解KRAS蛋白的制剂；
b)治疗KRAS相关癌症的药物。
在本发明的一些实施方式中，所述KRAS蛋白包括野生型KRAS蛋白和突变型 KRAS蛋白中的至少一种。
在本发明的一些实施方式中，所述突变型KRAS蛋白包括在G12、G13、S17、P34、A59、Q61、A146的至少一个氨基酸位置处发生突变的KRAS蛋白。
在本发明的一些实施方式中，所述KRAS相关癌症包括但不限于结直肠癌、肺癌、胰腺癌、肝癌、乳腺癌中的至少一种。
附图说明
下面结合附图和实施例对本发明做进一步的说明，其中：
图1为本发明实施例2的Western blotting检测结果图；其中，C-空白对照组，未进行转染；S1-对照组，转入SPOP(167-374)；DS13(-)-转入DS13表达载体，未加MG132(蛋白酶体抑制剂)；DS13(MG132)-转入DS13表达载体，加入MG132；RS14(-)-转入RS14表达载体，未加MG132；RS14(MG132)-转入RS14表达载体，加入MG132；Flag-tag指示S1、DS13和RS14的蛋白表达；HSP90-内参蛋白；
图2为本发明实施例3的Western blotting检测结果图；其中，C-空白对照组，未进行转染；S1-对照组，转入SPOP(167-374)；DS13(-)-转入DS13表达载体，未加MG132；DS13(MG132)-转入DS13表达载体，加入MG132；RS14(-)-转入RS14表达载体，未加MG132；RS14(MG132)-转入RS14表达载体，加入MG132；Flag-tag指示S1、DS13和RS14的蛋白表达；HSP90-内参蛋白；
图3为本发明实施例4的Western blotting检测结果图；其中，C-空白对照组，未进行转染；S1-对照组，转入SPOP(167-374)；DS13(-)-转入DS13表达载体，未加MG132；DS13(MG132)-转入DS13表达载体，加入MG132；RS14(-)-转入RS14表达载体，未加MG132；RS14(MG132)-转入RS14表达载体，加入MG132；Flag-tag指示S1、DS13和RS14的蛋白表达；HSP90-内参蛋白；
图4为本发明实施例5的Western blotting检测结果图；其中，C-空白对照组，未进行转染；S1-对照组，转入SPOP(167-374)；DS13(-)-转入DS13表达载体，未加MG132；DS13(MG132)-转入DS13表达载体，加入MG132；RS14(-)-转入RS14表达载体，未加MG132；RS14(MG132)-转入RS14表达载体，加入MG132；Flag-tag指示S1、 DS13和RS14的蛋白表达；HSP90-内参蛋白。
具体实施方式
下面详细描述本发明的实施例，描述的实施例是示例性的，仅用于解释本发明，而不能理解为对本发明的限制。
本发明的描述中，除非另有明确的限定，转染、培养等词语应做广义理解，所属技术领域技术人员可以结合技术方案的具体内容合理确定上述词语在本发明中的具体含义。
本发明的描述中，参考术语“一个实施例”、“一些实施例”等的描述意指结合该实施例描述的具体特征、材料或者特点包含于本发明的至少一个实施例中。在本说明书中，对上述术语的示意性表述不一定指的是相同的实施例。而且，描述的具体特征、材料或者特点可以在任何的一个或多个实施例中以合适的方式结合。
实施例中所使用的试验方法如无特殊说明，均为常规方法；所使用的材料、试剂等，如无特殊说明，均可从商业途径得到的试剂和材料。
实施例1
本实施例制备了一种靶向降解KRAS的嵌合泛素连接酶RS14，具体过程为：
(1)从PDB数据库中查找可以结合KRAS的蛋白RAF1，并截取与KRAS结合的蛋白片段RAF1(52-188)(氨基酸序列如SEQ ID NO:1：ASSKTSNTIRVFLPNKQRTVVNVRNGMSLHDCLMKALKVRGLQPECCAVFRLLHEHKGKKARLDWNTDAASLIGEELQVDFLDHVPLTTHNFARKTFLKLAFCDICQKFLLNGFRCQTCGYKFHEHCSTKVPTMCVDW所示)；将RAF1(52-188)与接头蛋白SPOP(167-374)(简称S1，该接头蛋白作为E3泛素连接酶结合蛋白，用于与E3泛素连接酶(接头蛋白/Cullin蛋白/E2泛素接合酶招募结构域)中的Cullin蛋白结合；其氨基酸序列如SEQ ID NO:8：MGLDYKDDDDKASGSVNISGQNTMNMVKVPECRLADELGGLWENSRFTDCCLCVAGQEFQAHKAILAARSPVFSAMFEHEMEESKKNRVEINDVEPEVFKEMMCFIYTGKAPNLDKMADDLLAAADKYALERLKVMCEDALCSNLSVENAAEILILADLHSADQLKTQAVDFINYHASDVLETSGWKSMVVSHPHLVAEAYRSLASAQCPFLGPPRKRLKQSGS所示)连接， 中间用连接蛋白(氨基酸序列如SEQ ID NO:9：SGSGSG所示)连接，形成嵌合泛素连接酶RS14(其氨基酸序列如SEQ ID NO:11：MGDYKDDDDKASSKTSNTIRVFLPNKQRTVVNVRNGMSLHDCLMKALKVRGLQPECCAVFRLLHEHKGKKARLDWNTDAASLIGEELQVDFLDHVPLTTHNFARKTFLKLAFCDICQKFLLNGFRCQTCGYKFHEHCSTKVPTMCVDWSGSGSGSVNISGQNTMNMVKVPECRLADELGGLWENSRFTDCCLCVAGQEFQAHKAILAARSPVFSAMFEHEMEESKKNRVEINDVEPEVFKEMMCFIYTGKAPNLDKMADDLLAAADKYALERLKVMCEDALCSNLSVENAAEILILADLHSADQLKTQAVDFINYHASDVLETSGWKSMVVSHPHLVAEAYRSLASAQCPFLGPPRKRLKQSGS所示)。
根据嵌合泛素连接酶RS14的氨基酸序列推导出对应的mRNA序列，如SEQ ID NO:18：5’-ATGGGCGACTACAAAGATGACGACGATAAAGCCTCCAGCAAGACCAGCAACACCATCAGAGTGTTCCTGCCTAACAAGCAGAGGACTGTGGTGAACGTGAGAAACGGCATGTCTCTGCATGACTGCCTGATGAAGGCCCTGAAGGTGAGAGGCCTGCAGCCCGAGTGCTGTGCCGTGTTCAGACTGCTGCACGAGCACAAGGGCAAGAAGGCCAGACTGGACTGGAACACCGATGCCGCCAGCCTGATCGGCGAGGAGCTGCAGGTGGATTTCCTGGACCATGTGCCTCTGACAACTCACAACTTTGCCCGGAAAACCTTTCTGAAGCTCGCATTCTGCGACATCTGCCAGAAGTTTCTGCTGAACGGGTTCAGGTGTCAGACCTGCGGCTATAAATTCCACGAGCACTGCTCTACCAAGGTGCCAACCATGTGTGTGGACTGGAGCGGCAGCGGATCTGGCAGCGTGAACATTAGTGGCCAGAACACCATGAATATGGTTAAAGTCCCAGAGTGCAGACTGGCCGACGAGCTGGGCGGCCTGTGGGAGAACTCTCGCTTCACCGACTGTTGCCTGTGCGTGGCCGGCCAGGAGTTCCAGGCCCACAAGGCCATTCTGGCCGCACGCAGCCCCGTGTTCTCCGCCATGTTTGAGCACGAGATGGAGGAGAGCAAGAAAAACCGGGTGGAAATCAACGATGTGGAACCTGAGGTGTTCAAGGAGATGATGTGCTTCATCTACACAGGCAAGGCCCCAAACCTCGACAAAATGGCCGACGACCTGCTGGCCGCCGCCGACAAATATGCTCTGGAAAGGCTGAAGGTGATGTGCGAGGATGCTCTGTGCAGTAATCTGAGCGTGGAGAACGCAGCCGA GATCCTGATTCTGGCCGACCTGCACAGCGCCGACCAGCTGAAGACCCAGGCCGTGGACTTTATCAACTATCATGCTTCAGACGTGCTGGAAACTAGTGGCTGGAAGAGCATGGTGGTTAGCCACCCACACCTGGTGGCTGAGGCCTATAGGTCCCTGGCCTCTGCTCAGTGCCCATTTCTGGGCCCTCCCCGGAAGAGACTGAAGCAGTCTGGAAGCTGA-3’所示。将该mRNA序列插入质粒中，形成RS14表达载体。使用该RS14表达载体进行以下试验。
实施例2
本实施例测试了RS14能否敲降HT29细胞(结肠癌细胞)中的野生型KRAS，具体过程为：
在测试时，还使用构建的另外一种嵌合泛素连接酶DS13来进行验证，其中，DS13是将可以结合KRAS的蛋白DARPin.K27和SPOP(167-374)连接，得到嵌合泛素连接酶DS13(其氨基酸序列如SEQ ID NO:19：MGASGSDLGKKLLEAARAGQDDEVRILMANGADVNAHDTFGFTPLHLAALYGHLEIVEVLLKNGADVNADDSYGRTPLHLAAMRGHLEIVEVLLKYGADVNAADEEGRTPLHLAAKRGHLEIVEVLLKNGADVNAQDKFGKTAFDISIDNGNEDLAEILQKLNGSGSGSGSVNISGQNTMNMVKVPECRLADELGGLWENSRFTDCCLCVAGQEFQAHKAILAARSPVFSAMFEHEMEESKKNRVEINDVEPEVFKEMMCFIYTGKAPNLDKMADDLLAAADKYALERLKVMCEDALCSNLSVENAAEILILADLHSADQLKTQAVDFINYHASDVLETSGWKSMVVSHPHLVAEAYRSLASAQCPFLGPPRKRLKQSGSDYKDDDDKS所示)。其对应的mRNA序列如SEQ ID NO:20：5’-ATGGGCGCCTCCGGAAGCGACCTCGGCAAGAAGCTGCTGGAGGCCGCCAGAGCCGGCCAGGACGATGAAGTGAGAATCCTGATGGCCAATGGAGCCGACGTGAACGCCCACGACACCTTCGGATTCACACCTCTGCACCTGGCCGCTCTGTACGGCCACCTGGAGATCGTGGAGGTGCTGCTGAAAAACGGCGCTGACGTGAATGCCGATGACAGCTACGGCAGAACACCCCTGCACCTCGCCGCCATGAGAGGGCACCTGGAGATTGTGGAGGTGCTGCTGAAGTACGGGGCCGACGTGAACGCCGCCGATGAAGAGGGAAGGACTCCACTGCACCTGGCCGCAAAGCGCGGGCACCTGGAAATCGTGGAGGTGCTGCTGA AGAATGGGGCAGACGTGAACGCTCAGGACAAGTTCGGCAAAACAGCCTTCGACATCTCCATCGACAACGGCAACGAAGACCTGGCCGAAATCCTGCAGAAGCTGAATGGCAGCGGCAGCGGATCTGGCAGCGTGAACATTAGTGGCCAGAACACCATGAATATGGTTAAAGTCCCAGAGTGCAGACTGGCCGACGAGCTGGGCGGCCTGTGGGAGAACTCTCGCTTCACCGACTGTTGCCTGTGCGTGGCCGGCCAGGAGTTCCAGGCCCACAAGGCCATTCTGGCCGCACGCAGCCCCGTGTTCTCCGCCATGTTTGAGCACGAGATGGAGGAGAGCAAGAAAAACCGGGTGGAAATCAACGATGTGGAACCTGAGGTGTTCAAGGAGATGATGTGCTTCATCTACACAGGCAAGGCCCCAAACCTCGACAAAATGGCCGACGACCTGCTGGCCGCCGCCGACAAATATGCTCTGGAAAGGCTGAAGGTGATGTGCGAGGATGCTCTGTGCAGTAATCTGAGCGTGGAGAACGCAGCCGAGATCCTGATTCTGGCCGACCTGCACAGCGCCGACCAGCTGAAGACCCAGGCCGTGGACTTTATCAACTATCATGCTTCAGACGTGCTGGAAACTAGTGGCTGGAAGAGCATGGTGGTTAGCCACCCACACCTGGTGGCTGAGGCCTATAGGTCCCTGGCCTCTGCTCAGTGCCCATTTCTGGGCCCTCCCCGGAAGAGACTGAAGCAGTCTGGAAGCGACTACAAGGACGACGATGATAAGAGTTGA-3’所示。将该mRNA序列插入质粒中，形成DS13表达载体，进行如下试验。
在六孔板上种1×106个HT29细胞，加入细胞培养液(添加10％胎牛血清FBS和1×青霉素/链霉素的McCoy’s 5A)，置于5％CO2，37℃的培养箱中培养。过夜后将McCoy’s5A培养液替换成1mL的OPTI培养液，将2μg的S1、DS13表达载体和RS14表达载体分别用商用转染试剂Lipofectamine MessengerMAX转染到HT29细胞中。转染6h后，提取细胞总蛋白用于进行Western blotting分析。分析结果如图1所示，C为空白对照组，未进行转染；S1为对照组，转入SPOP(167-374)；DS13(-)为转入DS13表达载体，未加MG132(蛋白酶体抑制剂)；DS13(MG132)为转入DS13表达载体，加入MG132；RS14(-)为转入RS14表达载体，未加MG132；RS14(MG132)为转入RS14表达载体，加入MG132；Flag-tag指示S1、DS13和RS14的蛋白表达(因为S1、DS13和RS14均有Flag-tag标签)；HSP90作为内参蛋白。可以看出，与对照组相比，DS13能显著降 低KRAS的条带灰度，而RS14的降低效果不明显。表明DS13能敲降野生型KRAS，而RS14对野生型KRAS的敲降作用不明显。
实施例3
本实施例测试了RS14能否敲降SW480细胞(人结肠癌细胞)中的G12V突变型KRAS，具体过程为：
在六孔板上种1×106个SW480细胞，加入细胞培养液(添加10％FBS和1×青霉素/链霉素的Leibovitz’s L-15)，置于5％CO2，37℃的培养箱中培养。过夜后将Leibovitz’s L-15培养液替换成1mL的OPTI培养液，将2μg的S1、DS13表达载体和RS14表达载体分别用商用转染试剂Lipofectamine MessengerMAX转染到SW480细胞中。转染6h后，提取细胞总蛋白用于进行Western blotting分析。分析结果如图2所示，具体标记同实施例2。可以看出，与对照组相比，RS14能显著降低KRAS的条带灰度，而DS13的降低效果不明显。表明RS14能敲降G12V突变型KRAS，而DS13对G12V突变型KRAS的敲降作用不明显。
实施例4
本实施例测试了RS14能否敲降A549细胞(人非小细胞肺癌细胞)中的G12S突变型KRAS，具体过程为：
在六孔板上种1×106个A549细胞，加入细胞培养液(添加10％FBS和1×青霉素/链霉素的Ham’s F-12K)，置于5％CO2，37℃的培养箱中培养。过夜后将Ham’s F-12K培养液替换成1mL的OPTI培养液，将2μg的S1、DS13表达载体和RS14表达载体分别用商用转染试剂Lipofectamine MessengerMAX转染到A549细胞中。转染6h后，提取细胞总蛋白用于进行Western blotting分析。分析结果如图3所示，具体标记同实施例2。可以看出，与对照组相比，RS14能显著降低KRAS的条带灰度，而DS13的降低效果不明显。表明RS14能敲降G12S突变型KRAS，而DS13对G12S突变型KRAS的敲降作用不明显。
实施例5
本实施例测试了RS14能否敲降PANC1细胞(人胰腺癌细胞)中的G12D突变型 KRAS，具体过程为：
在六孔板上种1×106个PANC1细胞，加入细胞培养液(添加10％FBS和1×青霉素/链霉素的DMEM)，置于5％CO2，37℃的培养箱中培养。过夜后将DMEM培养液替换成1mL的OPTI培养液，将2μg的S1、DS13表达载体和RS14表达载体分别用商用转染试剂Lipofectamine MessengerMAX转染到PANC1细胞中。转染6h后，提取细胞总蛋白用于进行Western blotting分析。分析结果如图4所示，具体标记同实施例2。可以看出，与对照组相比，RS14降低KRAS条带灰度的效果较DS13的效果更明显。表明RS14能敲降G12D突变型KRAS。
从以上实施例可以看出，RS14可以降解多种突变型KRAS，而不降解野生型KRAS，这可能是由于RAF1蛋白更易与激活的KRAS(即突变型KRAS)结合。
上面对本发明实施例作了详细说明，但是本发明不限于上述实施例，在所属技术领域普通技术人员所具备的知识范围内，还可以在不脱离本发明宗旨的前提下作出各种变化。此外，在不冲突的情况下，本发明的实施例及实施例中的特征可以相互组合。

Claims (10)

  1. 一种靶向降解KRAS的嵌合泛素连接酶，其特征在于，包括KRAS结合结构域和E3泛素连接酶结合蛋白；所述KRAS结合结构域包括RAF1(52-188)、ARAF、PIP5K1A、RASSF2、APP、KRAS、RAF1中的至少一种；
    所述RAF1(52-188)的氨基酸序列为：SEQ ID NO:1；或SEQ ID NO:1所示的氨基酸经一个或多个氨基酸的取代、缺失或添加修饰后，且功能相同或相似的氨基酸序列；
    所述ARAF的氨基酸序列为：SEQ ID NO:2；或SEQ ID NO:2所示的氨基酸序列经一个或多个氨基酸的取代、缺失或添加修饰后，且功能相同或相似的氨基酸序列；
    所述PIP5K1A的氨基酸序列为：SEQ ID NO:3；或SEQ ID NO:3所示的氨基酸序列经一个或多个氨基酸的取代、缺失或添加修饰后，且功能相同或相似的氨基酸序列；
    所述RASSF2的氨基酸序列为：SEQ ID NO:4；或SEQ ID NO:4所示的氨基酸序列经一个或多个氨基酸的取代、缺失或添加修饰后，且功能相同或相似的氨基酸序列；
    所述APP的氨基酸序列为：SEQ ID NO:5；或SEQ ID NO:5所示的氨基酸序列经一个或多个氨基酸的取代、缺失或添加修饰后，且功能相同或相似的氨基酸序列；
    所述KRAS的氨基酸序列为：SEQ ID NO:6；或SEQ ID NO:6所示的氨基酸序列经一个或多个氨基酸的取代、缺失或添加修饰后，且功能相同或相似的氨基酸序列；
    所述RAF1的氨基酸序列为：SEQ ID NO:7；或SEQ ID NO:7所示的氨基酸序列经一个或多个氨基酸的取代、缺失或添加修饰后，且功能相同或相似的氨基酸序列。
  2. 根据权利要求1所述的嵌合泛素连接酶，其特征在于，所述E3泛素连接酶结合蛋白的氨基酸序列为：
    SEQ ID NO:8；或SEQ ID NO:8所示的氨基酸序列经一个或多个氨基酸的取代、缺失或添加修饰后，且功能相同或相似的氨基酸序列。
  3. 根据权利要求2所述的嵌合泛素连接酶，其特征在于，所述嵌合泛素连接酶的氨基酸序列为：
    a)SEQ ID NO:11：MGDYKDDDDKASSKTSNTIRVFLPNKQRTVVNVRNGMSLH DCLMKALKVRGLQPECCAVFRLLHEHKGKKARLDWNTDAASLIGEELQVDFLDHVPLTTHNFARKTFLKLAFCDICQKFLLNGFRCQTCGYKFHEHCSTKVPTMCVDWSGSGSGSVNISGQNTMNMVKVPECRLADELGGLWENSRFTDCCLCVAGQEFQAHKAILAARSPVFSAMFEHEMEESKKNRVEINDVEPEVFKEMMCFIYTGKAPNLDKMADDLLAAADKYALERLKVMCEDALCSNLSVENAAEILILADLHSADQLKTQAVDFINYHASDVLETSGWKSMVVSHPHLVAEAYRSLASAQCPFLGPPRKRLKQSGS；或
    b)SEQ ID NO:11所示的氨基酸序列经一个或多个氨基酸的取代、缺失或添加修饰后，且功能相同或相似的氨基酸序列。
  4. 一种核酸分子，其特征在于，所述核酸分子包括编码权利要求1～3任一项所述嵌合泛素连接酶的DNA或mRNA。
  5. 一种载体，其特征在于，所述载体包括权利要求4所述的核酸分子。
  6. 根据权利要求5所述的载体，其特征在于，所述载体包括细菌、真菌、昆虫、病毒或哺乳动物载体。
  7. 一种宿主细胞，其特征在于，所述宿主细胞包括权利要求4所述核酸分子或权利要求5～6任一项所述载体，所述宿主细胞为原核细胞或真核细胞。
  8. 权利要求1～3任一项所述嵌合泛素连接酶的制备方法，其特征在于，包括以下步骤：培养权利要求7所述的宿主细胞，得所述嵌合泛素连接酶。
  9. 权利要求1～3任一项所述嵌合泛素连接酶在制备产品中的应用，其特征在于，所述产品为如下任一种：
    a)降解KRAS蛋白的制剂；
    b)治疗KRAS相关癌症的药物。
  10. 根据权利要求9所述的应用，其特征在于，所述KRAS蛋白包括野生型KRAS蛋白和突变型KRAS蛋白中的至少一种；
    优选地，所述突变型KRAS蛋白包括在G12、G13、S17、P34、A59、Q61、A146的至少一个氨基酸位置处发生突变的KRAS蛋白。
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