Lin28B gene upstream open reading frame coded protein and application thereof

Abstract

The invention relates to the technical field of biological medicines, and provides a protein coded by an upstream open reading frame of a Lin28B gene and application thereof, and the polypeptide has an amino acid sequence shown as SEQ ID NO: 2 or 4 or an amino acid sequence modified by a conservative form of the amino acid sequence shown as SEQ ID NO: 2 or 4. According to the embodiment of the invention, the polypeptide is obtained by expression of an upstream open reading frame of the Lin28B gene, can effectively regulate mutual transformation of an originating state and an original state of the pluripotent stem cells, and has important significance in treatment of embryo implantation defect related diseases.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a protein encoded by an upstream open reading frame of a Lin28B gene and an application thereof. Background Art

[0002] At present, the existing functional reports of non-canonical open reading frames (ORFs) are mainly concentrated on the ORFs of long noncoding RNA (lncRNA). The function of the upstream open reading frame (uORF) of mRNA is mainly as a cis-regulatory element to inhibit the translation of the downstream main open reading frame (mORF), while there are few reports on the functions of microproteins encoded by the upstream open reading frame (uORF) of mRNA.

[0003] There are two main pluripotency states of pluripotent stem cells, the naive state and the primed state, which correspond to different stages of embryonic development in vivo. Compared with cells in the primed state, cells in the naive state have embryonic chimerism ability and are an important source of seed cells for xenogeneic organ reconstruction in the future. Regulating the transition between the two pluripotency states has important research value for basic research and regenerative medicine. The existing effective factors that promote the transition between different pluripotency states are mostly transcription factors, and their efficiency is relatively low. There are no reports on the research on the microproteins encoded by uORFs regulating the transition between the naive state and the naive state. Summary of the invention

[0004] The present invention aims to solve one of the technical problems in the related art at least to a certain extent.

[0005] The function of the upstream open reading frame (uORF) of mRNA is mainly as a cis-regulatory element to inhibit the translation of the downstream main open reading frame (mORF). There are few reports on the function of the microprotein encoded by the upstream open reading frame (uORF) of mRNA. However, in the present application, the inventors found through a large number of experimental studies that Lin28B uORF can encode a new microprotein (named 85aa / 87aa-uORF, where 87aa in humans and 85aa in mice) to regulate the fate of pluripotent stem cells, and can regulate the transformation of primed pluripotent stem cells to primitive pluripotent stem cells, and the regulation efficiency is close to 100%, and homogenized primitive or primed pluripotent stem cells can be obtained.

[0006] To this end, in the first aspect of the present invention, the present invention proposes a polypeptide. According to an embodiment of the present invention, the polypeptide has an amino acid sequence shown in SEQ ID NO: 2 or 4 or an amino acid sequence modified in a conservative form thereof. The polypeptide according to an embodiment of the present invention is obtained by encoding Lin28BuORF, and the polypeptide can effectively regulate the primitive state and the initial state of pluripotent stem cells, plays an important role in embryo implantation, and can effectively treat diseases related to embryo implantation defects.

[0007] In the second aspect of the present invention, the present invention provides a nucleic acid. According to an embodiment of the present invention, the nucleic acid encodes the polypeptide described in the first aspect. The polypeptide encoded by the nucleic acid according to the embodiment of the present invention can effectively regulate the primitive state and the initial state of pluripotent stem cells, and can effectively treat diseases related to embryo implantation defects.

[0008] According to an embodiment of the present invention, the above nucleic acid may further include at least one of the following additional technical features:

[0009] According to an embodiment of the present invention, the nucleic acid has a nucleotide sequence shown in SEQ ID NO: 1 or 3.

[0010] In the third aspect of the present invention, the present invention provides an expression vector. According to an embodiment of the present invention, the expression vector comprises the nucleic acid described in the second aspect, or expresses the polypeptide described in the first aspect. According to an embodiment of the present invention, the expression vector carries the aforementioned nucleic acid molecule. Thus, the expression of the aforementioned polypeptide is effectively achieved, thereby achieving a large amount of in vitro acquisition of the aforementioned polypeptide.

[0011] In a fourth aspect of the present invention, the present invention provides a recombinant cell. According to an embodiment of the present invention, the recombinant cell carries the aforementioned nucleic acid or expression vector, or expresses the aforementioned polypeptide. The recombinant cell can effectively express the aforementioned polypeptide in the cell under suitable conditions.

[0012] According to an embodiment of the present invention, the above-mentioned recombinant cell may further include at least one of the following additional technical features:

[0013] According to an embodiment of the present invention, the cells include mammalian pluripotent stem cells.

[0014] According to an embodiment of the present invention, the cells include mammalian embryonic stem cells or induced pluripotent stem cells.

[0015] In the fifth aspect of the present invention, the present invention proposes a biological model. According to an embodiment of the present invention, the biological model does not express or low-expresses the polypeptide described in the first aspect. As mentioned above, the polypeptide according to the embodiment of the present invention is obtained by encoding the Lin28B uORF, and the polypeptide can effectively regulate the primitive state and the initial state of pluripotent stem cells, and plays an important role in embryo implantation. When the polypeptide is not expressed or low-expressed in the biological model, for pluripotent stem cells, the pluripotent stem cells will transform into the direction of primitive pluripotent stem cells, and embryo implantation defects are prone to occur. At this time, the biological model can be a biological model related to embryo implantation defects, which can be used in subsequent disease treatment research or other scientific research.

[0016] According to an embodiment of the present invention, the biological model is a cell model, an organoid model or an animal model. Those skilled in the art can select and prepare a cell model, an organoid model or an animal model according to needs.

[0017] A cell model refers to a model of a cell that patterns its structure and shape, and is proposed to facilitate the clarification of cell-to-cell interactions and the relationship between function, structure and morphology, artificial particles such as ribosomes that have some cell functions, or cells that have their membrane structure and soluble part of the cytoplasm removed while maintaining the structure (contractile structure) and function involved in cell movement. Organoid models are miniature three-dimensional structures constructed through cell engineering technology that simulate the key functions and microenvironments of animal or human organs. These miniature organs can be cultured from patients' cells and can replicate the physiological and pathological characteristics of organs, providing researchers with new tools for studying complex disease mechanisms, drug screening and the development of treatment methods. Animal models refer to animals with human disease-mimicking manifestations established in various scientific studies.

[0018] In the sixth aspect of the present invention, the present invention proposes the use of an agent for changing the polypeptide described in the first aspect in pluripotent stem cells in the preparation of high-purity primed state pluripotent stem cells or naive state pluripotent stem cells. As mentioned above, the polypeptide can effectively regulate the naive state and primed state of pluripotent stem cells. When the expression of the polypeptide is downregulated or inhibited, it can effectively promote the conversion of pluripotent stem cells into naive state pluripotent stem cells, especially the conversion of primed state pluripotent stem cells into naive state pluripotent stem cells. Overexpression of the polypeptide can effectively convert pluripotent stem cells into primed state pluripotent stem cells, and the conversion efficiency can reach 100%. Thus, high-purity naive state pluripotent stem cells or primed state pluripotent stem cells with a unified state can be obtained.

[0019] According to an embodiment of the present invention, the above-mentioned use may also include at least one of the following additional technical features:

[0020] According to an embodiment of the present invention, the change comprises overexpressing the polypeptide in the pluripotent stem cells, or downregulating or inhibiting the expression of the polypeptide in the pluripotent stem cells.

[0021] According to an embodiment of the present invention, the reagent is a reagent based on at least one of shRNA, antisense nucleic acid, ribozyme, CRISPR-Cas9, CRISPR-Cpf1 and zinc finger nuclease.

[0022] According to an embodiment of the present invention, the reagent further comprises an overexpression vector or an agonist of the polypeptide.

[0023] According to an embodiment of the present invention, the agonist comprises at least one of a small molecule, a drug, an enzyme or a hormone.

[0024] According to an embodiment of the present invention, the primed pluripotent stem cells are prepared by overexpressing the polypeptide described in the first aspect in the pluripotent stem cells, thereby obtaining highly pure primed pluripotent stem cells.

[0025] According to an embodiment of the present invention, the naive pluripotent stem cells are prepared by down-regulating or inhibiting the expression of the polypeptide described in the first aspect in the pluripotent stem cells, thereby obtaining high-purity naive pluripotent stem cells.

[0026] According to an embodiment of the present invention, the pluripotent stem cells include mammalian embryonic stem cells or induced pluripotent stem cells.

[0027] In the seventh aspect of the present invention, the present invention proposes the use of an agent for overexpressing the polypeptide described in the first aspect in pluripotent stem cells in the preparation of high-purity primed pluripotent stem cells. The polypeptide according to the embodiment of the present invention can effectively transform pluripotent stem cells into primed pluripotent stem cells, and, according to the examples of the present application, the transformation efficiency can reach 100%, therefore, overexpressing the polypeptide described in the first aspect can effectively prepare high-purity primed pluripotent stem cells.

[0028] According to an embodiment of the present invention, the reagent includes an overexpression vector or an agonist of the polypeptide. Those skilled in the art will appreciate that any reagent that can overexpress the polypeptide and does not affect the survival of the pluripotent stem cells can be used.

[0029] According to an embodiment of the present invention, the agonist comprises at least one of a small molecule, a drug, an enzyme or a hormone.

[0030] According to an embodiment of the present invention, the pluripotent stem cells include mammalian embryonic stem cells or induced pluripotent stem cells. It can be understood by those skilled in the art that any pluripotent cells can be used herein.

[0031] In the eighth aspect of the present invention, the present invention proposes the use of an agent for down-regulating or inhibiting the expression of the polypeptide described in the first aspect in pluripotent stem cells in the preparation of naive pluripotent stem cells. As mentioned above, the polypeptide can effectively regulate the naive state and the primed state of pluripotent stem cells. When the expression of the polypeptide is down-regulated or inhibited, it can effectively promote the conversion of pluripotent stem cells into naive pluripotent stem cells, especially the primed pluripotent stem cells into naive pluripotent stem cells, and the conversion efficiency can reach 100%. Thus, high-purity naive pluripotent stem cells with a uniform state can be obtained.

[0032] According to an embodiment of the present invention, the reagent is a reagent based on at least one of shRNA, antisense nucleic acid, ribozyme, CRISPR-Cas9, CRISPR-Cpf1 and zinc finger nuclease.

[0033] According to an embodiment of the present invention, the pluripotent stem cells include mammalian embryonic stem cells or induced pluripotent stem cells. It can be understood by those skilled in the art that any mammalian cell with pluripotency can be used herein.

[0034] In the ninth aspect of the present invention, the present invention proposes a method for preparing high-purity primed state pluripotent stem cells or primitive state pluripotent stem cells. According to an embodiment of the present invention, the method comprises: contacting a reagent that changes the polypeptide described in the first aspect with a pluripotent stem cell. As previously mentioned, the polypeptide can effectively regulate the primitive state and the primed state of pluripotent stem cells. When the expression of the polypeptide is downregulated or inhibited, it can effectively promote the conversion of pluripotent stem cells into primitive state pluripotent stem cells, especially the conversion of primed state pluripotent stem cells into primitive state pluripotent stem cells. Overexpression of the polypeptide can effectively convert pluripotent stem cells into primed state pluripotent stem cells, and the conversion efficiency can reach 100%. Thus, high-purity primitive state pluripotent stem cells or primed state pluripotent stem cells with a unified state can be obtained.

[0035] According to an embodiment of the present invention, the above method may further include at least one of the following additional technical features:

[0036] According to an embodiment of the present invention, the change comprises overexpressing the polypeptide in the pluripotent stem cells, or downregulating or inhibiting the expression of the polypeptide in the pluripotent stem cells.

[0037] According to an embodiment of the present invention, the reagent is a reagent based on at least one of shRNA, antisense nucleic acid, ribozyme, CRISPR-Cas9, CRISPR-Cpf1 and zinc finger nuclease.

[0038] According to an embodiment of the present invention, the reagent further comprises an overexpression vector or agonist of the polypeptide. Those skilled in the art will appreciate that any reagent capable of overexpressing the polypeptide without affecting the survival of the pluripotent stem cells can be used.

[0039] According to an embodiment of the present invention, the agonist comprises at least one of a small molecule, a drug, an enzyme or a hormone.

[0040] According to an embodiment of the present invention, the method comprises: obtaining the primed pluripotent stem cells by overexpressing the polypeptide described in the first aspect in the pluripotent stem cells or contacting the pluripotent stem cells with the polypeptide described in the first aspect, thereby obtaining highly pure primed pluripotent stem cells.

[0041] According to an embodiment of the present invention, the method comprises: obtaining the naive pluripotent stem cells by down-regulating or inhibiting the expression of the polypeptide described in the first aspect in the pluripotent stem cells, thereby obtaining high-purity naive pluripotent stem cells.

[0042] According to an embodiment of the present invention, the pluripotent stem cells include mammalian embryonic stem cells or induced pluripotent stem cells.

[0043] In the tenth aspect of the present invention, the present invention proposes a method for preparing high-purity primed pluripotent stem cells. According to an embodiment of the present invention, the method comprises: contacting the polypeptide described in the first aspect with pluripotent stem cells, or contacting the reagent that overexpresses the polypeptide described in the first aspect with the pluripotent stem cells. As mentioned above, the polypeptide can effectively regulate the primitive state and the primed state of pluripotent stem cells, and when the expression of the polypeptide is upregulated or when the pluripotent stem cells in the primitive state are contacted with the above-mentioned polypeptide, it can effectively promote the conversion of the pluripotent stem cells in the primitive state into the pluripotent stem cells in the primed state, thereby obtaining high-purity primitive pluripotent stem cells with a uniform state.

[0044] According to an embodiment of the present invention, the reagent includes an overexpression vector or an agonist of the polypeptide. Those skilled in the art will appreciate that any reagent that can overexpress the polypeptide and does not affect the survival of the pluripotent stem cells can be used.

[0045] According to an embodiment of the present invention, the agonist comprises at least one of a small molecule, a drug, an enzyme or a hormone.

[0046] According to an embodiment of the present invention, the pluripotent stem cells include mammalian embryonic stem cells or induced pluripotent stem cells.

[0047] In the eleventh aspect of the present invention, the present invention proposes a method for preparing high-purity primitive state pluripotent stem cells. According to an embodiment of the present invention, the method comprises: contacting the pluripotent stem cells with an agent that down-regulates or inhibits the expression of the polypeptide described in the first aspect. As mentioned above, the polypeptide can effectively regulate the primitive state and the primed state of pluripotent stem cells. When the expression of the polypeptide is down-regulated or completely inhibited, it can effectively promote the conversion of primed state pluripotent stem cells into primitive state pluripotent stem cells, thereby obtaining high-purity primitive state pluripotent stem cells with a uniform state.

[0048] According to an embodiment of the present invention, the reagent is a reagent based on at least one of shRNA, antisense nucleic acid, ribozyme, CRISPR-Cas9, CRISPR-Cpf1 and zinc finger nuclease.

[0049] According to an embodiment of the present invention, the pluripotent stem cells include mammalian embryonic stem cells or induced pluripotent stem cells.

[0050] In the twelfth aspect of the present invention, the present invention proposes the use of the biological model described in the fifth aspect in screening drugs for treating diseases related to embryo implantation defects. According to an embodiment of the present invention, the disease includes at least one of threatened abortion, habitual abortion, infertility, endometriosis, endometrial weakness, endometritis, uterine malformation, ovarian polycystic disease and corpus luteum insufficiency. As mentioned above, the polypeptide according to the embodiment of the present invention can effectively regulate the primitive state and the initial state of pluripotent stem cells, and plays an important role in embryo implantation. When the polypeptide is not expressed or low-expressed in the biological model, for pluripotent stem cells, the pluripotent stem cells will be transformed in the direction of primitive pluripotent stem cells, and embryo implantation defects are likely to occur. At this time, the biological model is a biological model related to embryo implantation defects, which can be used in subsequent disease treatment research or other scientific research, such as screening drugs.

[0051] In the thirteenth aspect of the present invention, the present invention proposes the use of the polypeptide described in the first aspect, the nucleic acid described in the second aspect, the expression vector described in the third aspect, or the recombinant cell described in the fourth aspect in the preparation of a drug. According to an embodiment of the present invention, the drug is used to prevent, alleviate and / or treat diseases related to embryo implantation defects. As mentioned above, the polypeptide according to the embodiment of the present invention can effectively regulate the original state and the primed state of pluripotent stem cells, and when the polypeptide is in contact with the pluripotent stem cells in the original state, it can effectively promote the transformation of the pluripotent stem cells in the original state into the pluripotent stem cells in the primed state.

[0052] Naive pluripotent stem cells and primed pluripotent stem cells are two different stem cell states, corresponding to different stages of embryonic development. Naive pluripotent stem cells have stronger plasticity and can simulate the stage of embryonic development before implantation in vivo, which is 3.5 to 4.5 days after the formation of mouse fertilized eggs, while primed pluripotent stem cells correspond to the development stage of post-implantation embryos, which is 5.5 to 7.5 days after the formation of mouse fertilized eggs, naive pluripotent stem cells correspond to 6 to 8 days after the formation of human fertilized eggs, and primed pluripotent stem cells correspond to 11 to 12 days after the formation of human fertilized eggs. Therefore, drugs containing the aforementioned polypeptides, nucleic acids, expression vectors or recombinant cells play an important role in embryo implantation.

[0053] According to an embodiment of the present invention, the disease includes at least one of threatened abortion, habitual abortion, infertility, endometriosis, endometrial weakness, endometritis, uterine malformation, polycystic ovary disease and luteal insufficiency.

[0054] In the fourteenth aspect of the present invention, the present invention proposes a drug for preventing, alleviating and / or treating diseases related to embryo implantation defects. According to an embodiment of the present invention, the drug comprises the polypeptide described in the first aspect, the nucleic acid described in the second aspect, the expression vector described in the third aspect, or the recombinant cell described in the fourth aspect. As mentioned above, the polypeptide can effectively regulate the transition between the initial state and the original state of pluripotent stem cells, thereby facilitating embryo implantation. Therefore, products related to the polypeptide, such as the nucleic acid, expression vector, and recombinant cells, have the same therapeutic effect as the polypeptide. Furthermore, drugs containing the above products can also be used for diseases related to embryo implantation defects.

[0055] According to an embodiment of the present invention, the above-mentioned medicine may also include at least one of the following additional technical features:

[0056] According to an embodiment of the present invention, the medicament comprises a pharmaceutically acceptable excipient.

[0057] According to an embodiment of the present invention, the disease includes at least one of threatened abortion, habitual abortion, infertility, endometriosis, endometrial weakness, endometritis, uterine malformation, polycystic ovary disease and luteal insufficiency.

[0058] In the fifteenth aspect of the present invention, the present invention proposes the use of a preparation in the preparation of a drug for preventing, alleviating and / or treating diseases related to embryo implantation defects. According to an embodiment of the present invention, the preparation causes the polypeptide described in the first aspect to be overexpressed in a cell. As previously mentioned, the polypeptide can effectively regulate the transition between the primordial state and the original state of pluripotent stem cells, thereby facilitating embryo implantation, and therefore, the preparation related to the overexpression of the polypeptide can promote the transformation of the original state pluripotent stem cells to the primordial state, which is beneficial to embryo implantation, and therefore, the preparation can be used for diseases related to embryo implantation defects.

[0059] According to an embodiment of the present invention, the cells include mammalian pluripotent stem cells.

[0060] According to an embodiment of the present invention, the pluripotent stem cells include embryonic stem cells or induced pluripotent stem cells.

[0061] According to an embodiment of the present invention, the preparation includes an overexpression vector or an agonist of the polypeptide;

[0062] According to an embodiment of the present invention, the agonist comprises at least one of a small molecule, a drug, an enzyme or a hormone.

[0063] In the sixteenth aspect of the present invention, the present invention proposes the use of a reagent for detecting the polypeptide described in the first aspect or the nucleic acid encoding the polypeptide described in the first aspect in the preparation of a kit. According to an embodiment of the present invention, the kit is used to detect diseases related to embryo implantation defects. As mentioned above, the polypeptide can effectively regulate the transition between the initial state and the original state of pluripotent stem cells. When the polypeptide is not expressed or lowly expressed in the pluripotent stem cells, the pluripotent stem cells will transform in the direction of the original state pluripotent stem cells, for example, from the initial state pluripotent stem cells to the original state pluripotent stem cells. When the polypeptide is overexpressed in the pluripotent stem cells, the pluripotent stem cells will transform to the initial state, for example, from the original state pluripotent stem cells to the initial state pluripotent stem cells. Therefore, the reagent for detecting the polypeptide described in the first aspect or a product related thereto, such as a nucleic acid, can effectively judge the state of the pluripotent stem cells, and the kit can be used for scientific research, such as studying the expression level changes of the polypeptide or nucleic acid in cells and tissues, or performing qualitative or quantitative detection thereof.

[0064] According to an embodiment of the present invention, the diseases related to embryo implantation defects include at least one of threatened abortion, habitual abortion, infertility, endometriosis, endometrial weakness, endometritis, uterine malformation, polycystic ovary and corpus luteum insufficiency.

[0065] According to an embodiment of the present invention, the reagents include antibodies, probes, primers and mass spectrometry detection reagents against the polypeptide or the nucleic acid encoding the polypeptide.

[0066] In the seventeenth aspect of the present invention, the present invention provides a kit, characterized in that the kit comprises a reagent for detecting the polypeptide described in the first aspect, or a nucleic acid encoding the polypeptide described in the first aspect. The kit of the present invention can specifically bind to the polypeptide, can effectively detect the polypeptide, or detect the nucleic acid encoding the polypeptide, thereby determining the state of cells in the sample to be detected or whether they are diseased.

[0067] According to an embodiment of the present invention, the kit comprises an antibody for detecting the polypeptide of the first aspect.

[0068] According to an embodiment of the present invention, the kit further comprises a probe, a primer and / or a mass spectrometry detection reagent for amplifying a nucleic acid encoding the polypeptide described in the first aspect. The kit can detect a nucleic acid encoding the polypeptide, and those skilled in the art will appreciate that when detecting a nucleic acid sequence, a primer corresponding to the nucleic acid is required to amplify the nucleic acid sequence.

[0069] The beneficial effects of this application are at least:

[0070] (1) The polypeptide described in the present application (microprotein 85aa / 87aa-uORF encoded by Lin28B uORF, 87aa in humans and 85aa in mice) can regulate the original state and the primed state of pluripotent stem cells with nearly 100% high efficiency. When the polypeptide is not expressed or lowly expressed in the pluripotent stem cells, the pluripotent stem cells will be transformed into the original state pluripotent stem cells, for example, from the primed state pluripotent stem cells to the original state pluripotent stem cells. When the polypeptide is overexpressed in the pluripotent stem cells, the pluripotent stem cells will be transformed into the primed state, for example, from the original state pluripotent stem cells to the primed state pluripotent stem cells, thereby obtaining homogenized original state or primed state pluripotent stem cells;

[0071] (2) Naive pluripotent stem cells and primed pluripotent stem cells are two different stem cell states, corresponding to different stages of embryonic development. Naive pluripotent stem cells have stronger plasticity and can simulate the stage of embryonic development before implantation in vivo, while primed pluripotent stem cells correspond to the development stage of embryos after implantation. Since the polypeptide can regulate the naive and primed states of pluripotent stem cells, it plays an important role in embryo implantation and can be used for scientific research, treatment and diagnosis in related fields.

[0072] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0073] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0074] Figure 1 The endogenous expression and identification of the 85aa-uORF-encoded microprotein of the embodiment of the present invention are shown, wherein:

[0075] Figure A: Schematic diagram of the location of 85aa-uORF on Lin28B mRNA.

[0076] Figure B: The amino acid sequence of the 85aa-uORF is highly conserved in many mammals, including humans and mice.

[0077] Figure C: 85aa-uORF starts translation with ATG.

[0078] Figure D: 85aa-uORF is highly expressed in primed pluripotent stem cells;

[0079] Figure 2 The 85aa-uORF knockout cell line (KO) was established by the CRISPR-Cas9 system in an embodiment of the present invention, wherein:

[0080] Figure A: Schematic diagram of the location of sgRNA targeting 85aa-uORF sequence,

[0081] Figure B: Sequencing results showed that the 85aa-uORF knockout cell line (KO) had one base added or four bases deleted, resulting in a frameshift mutation.

[0082] Figure C: Western blot showed that 85aa-uORF was successfully knocked out in OG2-EpiSCs.

[0083] Panel D: 85aa-uORF knockout does not affect the translational expression of Lin28B;

[0084] Figure 3 It shows that the knockout of 85aa-uORF in the present invention significantly promotes Primed-to- Transformation (PNT), PNT efficiency is close to 100%;

[0085] Figure 4 The results show that 87aa-uORF knockout in the present invention promotes the Primed-to- The three AP staining images on the left show the transformation of the control group (sgCTRL) and the 87aa-uORF knockout (sguORF1 / 2) into The clone diagram of the state, the statistical diagram on the right counts the number of clones in the control group and the experimental group;

[0086] Figure 5 It is shown that overexpression of 85aa-uORF promotes -to-Primed transition (NPT), where:

[0087] Figure A: Overexpression of 85aa-uORF promotes Schematic diagram of the transition of ESC cells to Primed state EpiSC cells.

[0088] Figure B: ESC and ESC overexpressing 85aa-uORF at transformation day 5 and primed pluripotency marker gene expression;

[0089] Figure 6 The construction of the 85aa-uORF gene knockout (MP85- / -) model mouse in Example 7 of the present invention is shown, wherein:

[0090] Figure A: Schematic diagram of designing sgRNA targeting sequence.

[0091] Figure B: Genome sequencing of MP85- / - mice showed that 4 bases were deleted and a frameshift mutation occurred.

[0092] Figure C: Western blot shows that MP85- / - genotype mice were successfully constructed;

[0093] Figure 7 It is shown that the 85aa-uORF in the embodiment of the present invention promotes the normal development of mouse embryos, promotes blastocyst implantation, and improves the fertility rate of offspring. DETAILED DESCRIPTION

[0094] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be understood as limiting the present invention.

[0095] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. Further, in the description of the present invention, unless otherwise specified, the meaning of "plurality" is two or more.

[0096] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0097] In order to make the present invention more easily understood, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which the present invention belongs.

[0098] In this document, the terms “include” or “comprising” are open expressions, that is, including the contents specified in the present invention but not excluding other contents.

[0099] As used herein, the terms "optionally", "optional" or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0100] It should be noted that the "nucleic acid" described in the present application can be any polymer containing deoxyribonucleotides or ribonucleotides, including but not limited to modified or unmodified DNA and RNA, and its length is not subject to any special restrictions. For constructs used to construct recombinant cells, it is preferred that the nucleic acid is DNA, because DNA is more stable and easy to operate compared to RNA. The "nucleic acid" described in the present application actually includes any one or two of the complementary double strands. For convenience, in this specification and claims, although only one strand is given in most cases, the other strand complementary to it is actually disclosed. For example, mentioning SEQ ID NO: 1 and 3 actually includes their complementary sequences. Those skilled in the art can also understand that one strand can be used to detect another strand, and vice versa.

[0101] In this article, the term "expression vector" generally refers to a nucleic acid molecule that can be inserted into a suitable host and replicates itself, and transfers the inserted nucleic acid molecule into and / or between host cells. The expression vector may include a vector that is mainly used to insert DNA or RNA into a cell, a vector that is mainly used to replicate DNA or RNA, and a vector that is mainly used for the expression of transcription and / or translation of DNA or RNA. The expression vector also includes vectors with multiple of the above functions. The expression vector can be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, the expression vector can produce a desired expression product by culturing a suitable host cell containing the expression vector.

[0102] In this article, the term "recombinant cell" generally refers to the use of genetic engineering technology or cell fusion technology to modify or reorganize the genetic material of the host cell to obtain a cell with a unique trait of stable inheritance. Among them, the term "host cell" refers to a prokaryotic cell or eukaryotic cell that can be introduced into a recombinant expression vector. The terms "transformed" or "transfected" used herein refer to the introduction of nucleic acids (such as vectors) into cells by various techniques known in the art. Suitable host cells can be transformed or transfected with the DNA sequence of the present invention, and can be used for the expression and / or secretion of target proteins. Examples of suitable host cells that can be used in the present invention include immortalized hybridoma cells, NS / 0 myeloma cells, 293 cells, Chinese hamster ovary (CHO) cells, HeLa cells, Cap cells (cells derived from human amniotic fluid) and CoS cells.

[0103] Single-base gene editing refers to a gene editing technology that can cause a single base change in the genome. The basic principle is to fuse cytosine deaminase (APOBEC) or adenosine deaminase with existing Cas9n (D10A). It relies on the CRISPR principle to modify a single base at positions 4 to 7 away from the PAM end of the target.

[0104] Zinc finger nuclease (ZFN) is composed of a DNA recognition domain and a non-specific nuclease. The DNA recognition domain is composed of a series of Cys2-his2 zinc finger proteins in series. Each zinc finger protein recognizes and binds to a specific triplet base. For example, the most classic zinc finger nuclease is a fusion of a non-specific nuclease FokI and a domain containing zinc fingers. Its purpose is naturally to cut specific sequences. The cut DNA can be deleted by the excision repair mechanism so that the single-stranded part of the cut is deleted and then reconnected. This method can be used to delete specific fragments on the chromosome, thereby constructing mutants or completing treatment.

[0105] Transcription activator-like (TAL) effector nucleases (TALENs) are enzymes that can target and modify specific DNA sequences. They use TAL effectors, a natural protein secreted by plant bacteria, to recognize specific DNA base pairs. TAL effectors can be designed to recognize and bind to all target DNA sequences. TALENs are generated by attaching a nuclease to the TAL effector. TAL effector nucleases can bind to DNA and cut the DNA chain at specific sites, thereby introducing new genetic material.

[0106] The CRISPR / Cas9 system is widely present in prokaryotic genes and is an acquired immune defense mechanism that bacteria and archaea have evolved to cope with constant attacks from viruses and plasmids. In these organisms, foreign genetic material from bacteriophages is acquired and integrated into CRISPR sites; these sequence-specific fragments are transcribed into short CRISPR RNAs (CRISPR-derived RNAs), which bind to tracrRNA (trans-activating RNA) through base pairing to form double-stranded RNAs. The tracrRNA / crRNA complex then guides the Cas9 protein to cut the double-stranded DNA for gene editing.

[0107] Human induced pluripotent stem cells (iPSCs) can be induced to become almost any type of cell. Combining iPSCs with CRISPR / Cas9 gene editing technology can not only repair pathogenic sites in patient-derived iPSCs, but also introduce disease-causing mutations into healthy WT iPSCs.

[0108] Adeno-associated virus (AAV) has low immunogenicity and is considered the most promising gene therapy vector due to its high safety and stable long-term expression. Using AAV to deliver the CRISPR / Cas9 system can achieve highly efficient gene editing.

[0109] In this article, the term "drug" generally refers to a unit dosage form and can be prepared by any of the methods well known in the pharmaceutical art. All methods include the step of combining the active ingredient with a carrier that constitutes one or more accessory ingredients. Generally, the composition is prepared by uniformly and thoroughly combining the active compound with a liquid carrier, a finely divided solid carrier, or both.

[0110] In this article, the term "pharmaceutically acceptable excipient" may include any solvent, solid excipient, diluent or other liquid excipient, etc., suitable for a specific target dosage form. Except for any conventional excipients incompatible with the compound of the present invention, such as any adverse biological effect produced or any other component of the pharmaceutically acceptable composition that interacts in a harmful manner, their use is also within the scope of the present invention.

[0111] For ease of understanding, the following is an overview of the contents of this application:

[0112] The upstream open reading frame (uORF) is a sequence located in the 5' untranslated region (5'UTR) of mRNA, which contains a start codon (usually AUG) and a stop codon, which can be recognized by the ribosome and start the translation process. In the present application, the inventors found that there is an upstream open reading frame (uORF) in the 5'UTR region of the Lin28B gene, which can encode the microprotein 85aa / 87aa-uORF, which is 87aa in humans and 85aa in mice. The amino acid sequence of the microprotein is highly conserved in multiple mammals such as humans and mice, and is highly expressed in primed pluripotent stem cells. The Lin28B gene uORF can not only act as a cis-regulatory element, but also encode new proteins to play a role.

[0113] Then, the inventors used CRISPR / Cas9 to introduce a frameshift mutation in the region where the uORF does not overlap with the main open reading frame (mORF), established a primed-to-primed mouse EpiSC cell line with 85aa-uORF knockout, and demonstrated by Western blot that 85aa-uORF knockout did not affect the translation of its Lin28B mORF. The inventors found that 85aa-uORF knockout promoted the primed-to-primed transition. The present invention has created a new method for promoting the transition of pluripotent stem cells from the primed state to the primitive state through the microprotein encoded by uORF, and greatly improved the efficiency to nearly 100%. It can efficiently obtain uniform and high-purity primitive pluripotent stem cells, which has important application value in the field of regenerative medicine.

[0114] The present invention also established a naive cell line overexpressing 85aa-uORF, and found that overexpressing 85aa-uORF significantly promoted the naive-to-primed transition (NPT). The present invention revealed that 85aa-uORF plays an important role in the naive / primed transition of pluripotency. The present invention explored a new method for uORFs encoding microproteins to promote the transition from naive to primed states.

[0115] Furthermore, the inventors constructed genetically engineered mice with 85aa-uORF knockout and found that the loss of 85aa-uORF led to embryo implantation defects in mice and a reduction in the number of offspring. The present invention discovered an important new protein 85aa-uORF related to embryo implantation, which not only provides a new research model for embryo implantation defects, but also has application value in the prevention, alleviation or treatment of diseases related to embryo implantation defects.

[0116] Thus, in some specific embodiments, the present invention provides the use of the aforementioned polypeptide in preparing high-purity naive pluripotent stem cells and primed pluripotent stem cells. As mentioned above, the polypeptide can regulate the naive state and primed state of pluripotent stem cells, and the conversion efficiency is 100%, overexpressing the polypeptide in pluripotent stem cells to obtain the primed pluripotent stem cells, downregulating or inhibiting the expression of the polypeptide in pluripotent stem cells to obtain the naive pluripotent stem cells, and the bidirectional conversion efficiency is 100%.

[0117] In some specific embodiments, the present invention also provides a biological model, which highly expresses the aforementioned polypeptide. The biological model is a cell model, an organoid model or an animal model. As mentioned above, the polypeptide can effectively regulate the initial state and the original state of pluripotent stem cells. The biological model according to the embodiment of the present invention can be a well-developed pluripotent stem cell or its tissue, or a fertilized egg with a development time of less than 14 days. The biological model can be used to conduct scientific research in the fields of embryonic development, such as studying the mechanism of embryonic development or screening drugs that are beneficial to or prevent further development of the embryo.

[0118] The above biological model may also include at least one of the following additional technical features:

[0119] According to a specific embodiment of the present invention, the cells include mammalian pluripotent stem cells.

[0120] According to a specific embodiment of the present invention, the cells include mammalian embryonic stem cells or induced pluripotent stem cells.

[0121] The scheme of the present disclosure will be explained below in conjunction with the examples. Those skilled in the art will appreciate that the following examples are only used to illustrate the present disclosure and should not be considered to limit the scope of the present disclosure. Where specific techniques or conditions are not indicated in the examples, the techniques or conditions described in the literature in this area or the product instructions are used. Where the manufacturers of reagents or instruments are not indicated, they are all conventional products that can be obtained commercially.

[0122] Example 185 Endogenous Expression and Identification of Microprotein Encoded by aa-uORF

[0123] In this example, an open reading frame (ORF) was found in the 5'UTR region of the mouse Lin28B gene (NM_001031772.2) through ORF finder (https: / / www.ncbi.nlm.nih.gov / orffinder / ) to encode a microprotein of 85 amino acids, which was named: (Mus-)85aa-uORF, wherein the nucleotide sequence of 85aa-uORF is shown in SEQ ID NO: 1, and the amino acid sequence of the microprotein encoded by 85aa-uORF is shown in SEQ ID NO: 2. The 5'UTR region of human Lin28B also has an open reading frame that can encode an 87aa microprotein, which is named (Homo-)87aa-uORF herein, wherein the nucleotide sequence of 87aa-uORF is shown in SEQ ID NO: 3, and the amino acid sequence of the microprotein encoded by 87aa-uORF is shown in SEQ ID NO: 4. In this example, the upstream open reading frame of mouse Lin28B (SEQ ID NO: 1) was constructed and its start codon ATG was mutated to TAA, and a mutant with a Flag tag was added. The Western blot method was used to prove that 85aa-uORF started translation with ATG, while the mutated TAA could not translate 85aa-uORF. In addition, a polyclonal antibody that specifically recognized the microprotein and could be detected by the Western blot method was designed based on the amino acid sequence of 85aa-uORF, and the expression of 85aa-uORF was detected in naive and primed pluripotent stem cells and somatic cells, confirming that 85aa-uORF was highly expressed in naive pluripotent stem cells. The specific experimental operation is as follows:

[0124] By chemically synthesizing peptides, a peptide segment ESFHLWSLK (SEQ ID NO: 5) or the entire amino acid sequence in 85aa-uORF was synthesized as an immunogen, and the immunogen was injected into New Zealand rabbits by subcutaneous injection. Four weeks after the initial immunization, booster immunization was started and performed once every week, for a total of 4 immunizations. When the antibody titer reached the expected level, antiserum was prepared by bleeding, and specific polyclonal antibodies were separated from the antiserum using antigen-specific affinity purification. The experimental operations of immunization and separation of polyclonal antibodies can be achieved by conventional experimental techniques in the art. The purified antibodies were detected by western to detect the endogenous expression and changes of the microprotein encoded by 85aa-uORF in different cells, and western detection was performed by conventional techniques in the art.

[0125] The nucleotide sequence of Mus-85aa-uORF (258 nt) is as follows:

[0126] Atgcgaactaaatttgtgatcgcacaaaatcaagatgttagactgatgctgaagatcactccggtccaaagggaaagttttcatctctgg agtttgaagctgagggccggtggggcaacatggccgaaggcggggcaagcaaaggtgaagagccagaaaaactgcccgggctggca gaggacgaaccccaggttctgcatggcactggccactgtaaatggttcaacgtgcgcatgggattcggattcatctccatga (SEQ ID NO: 1).

[0127] The amino acid sequence of the Mus-85aa-uORF microprotein (Lin28B-85aa) is as follows:

[0128] MRTKFVIAQNQDVRLMLKITPVQRESFHLWSLKLRAGGATWPKAGQAKVKSQKNCPGWQRTNPRFCMALATVNGSTCAWDSDSSP (SEQ ID NO: 2).

[0129] The nucleotide sequence of Homo-87aa-uORF (264 nt) is as follows:

[0130] ATGCGAGCTAAATTTGTGATCGCACAAAAATCAAGATGTTAGATTGATGCAGAAGATCACTCC

[0131] GTTCCAAAGGGAAAGTTTTCATCTCACGAGTTTGGAGCTGAGGGCCCGTGGGGCAACATGGCC

[0132] GAAGGCGGGGCTAGCAAAGGTGGTGGAGAAGAGCCCGGGAAGCTGCCGGAGCCGGCAGAGG

[0133] AGGAATCCCAGGTTTTGCGCGGAACTGGCCACTGTAAGTGGTTCAATGTGCGCATGGGATTTG

[0134] GATTCATCTCCATGA (SEQ ID NO:3).

[0135] The amino acid sequence of the Homo-87aa-uORF microprotein (Lin28B-87aa) is as follows:

[0136] MRAKFVIAQNQDVRLMQKITPFQRESFHLTSLELRARGATWPKAGLAKVVEKSPGSCRSRQRRNPRFCAELATVSGSMCAWDLDSSP (SEQ ID NO: 4).

[0137] 1) Construction of start codon ATG mutant

[0138] The nucleotide sequence of the 5'UTR of Lin28B was analyzed using genetic software, and 85aa-uORF and two mutant plasmids were constructed: a) the start codon ATG of 85aa-uORF was mutated to TAA (85aa-uORF-mut1); b) the UTR region before ATG of 85aa-uORF was retained (85aa-uORF-mut2); then the 3Flag tag protein sequence was added to the C-terminus of the above mutants, and cloned into the pLVX-EF1α-IRES-Puro vector (purchased from Takara, 631988) using the ECORI and BamHI restriction sites. The constructed pLVX-TAA-85aa-uORF-3Flag (85aa-uORF-mut1), UTR-85aa-uORF-3Flag (85aa-uORF-mut2), and 85aa-uORF-3Flag (wild type) plasmids were transfected into HEK293T cells respectively. The specific steps of cell transfection are as follows: 1 ml of serum-free diluent Opti-MEM (purchased from Gibco), 8 μg of plasmid DNA, and 64 μL of polyethyleneimine transfection (PEI) reagent were mixed evenly to form a nucleic acid-PEI complex, and allowed to stand at room temperature for 15 minutes. Subsequently, the configured nucleic acid-PEI complex was added to HEK293T cells that were inoculated into a 10 cm cell culture dish one day in advance and grown to a density of 70%-80%, and fresh culture medium was replaced after 8 to 10 hours of culture, and the protein expression of Lin28B microprotein was detected by Western blot after further culture for 36 hours.

[0139] The nucleotide sequence of 85aa-uORF-mut1 is as follows:

[0140] Aagaaggaaagcacattagacctaacgaactaaatttgtgatcgcacaaaatcaagatgttagactgatgctgaagatcactccggtc

[0141] caaagggaaagttttcatctctggagtttgaagctgagggccggtggggcaacatggccgaaggcggggcaagcaaaggtgaagagcca

[0142] gaaaaactgcccgggctggcagaggacgaaccccaggttctgcatggcactggccactgtaaatggttcaacgtgcgcatgggattcggat

[0143] tcatctccatga (SEQ ID NO:9).

[0144] The nucleotide sequence of 85aa-uORF-mut2 is as follows:

[0145] aagaaggaaagcacattagaccatgcgaactaaatttgtgatcgcacaaaatcaagatgttagactgatgctgaagatcactccggtc

[0146] caaagggaaagttttcatctctggagtttgaagctgagggccggtggggcaacatggccgaaggcggggcaagcaaaggtgaagagcca

[0147] gaaaaactgcccgggctggcagaggacgaaccccaggttctgcatggcactggccactgtaaatggttcaacgtgcgcatgggattcggat

[0148] tcatctccatga (SEQ ID NO: 10).

[0149] 2) Establishment of stable cell line overexpressing 85aa-uORF

[0150] The 85aa-uORF-3Flag plasmid constructed as described above and the lentiviral packaging plasmid psPAX2 (purchased from Addgene, 12260) and pMD2g (purchased from Addgene, 12259) were transfected into HEK293T cells at a ratio of 4:3:1 according to the above method. After 48 hours of transfection, the lentiviral supernatant was collected and concentrated by centrifugation, diluted with EpiSC culture medium, and then infected with EpiSC cells. The cells were selected with puromycin (about 3 days) and then amplified normally.

[0151] 3) Cell culture

[0152] The transfected HEK-293T cells obtained in part 2) were cultured in DMEM (purchased from Gibco) medium supplemented with 10% fetal bovine serum (FBS, NTC), GlutaMax (1x, Gibco) and NEAA (1x, Gibco), and cultured in a cell culture incubator at 5% CO2 and 37°C.

[0153] and Primed cells are derived from mouse embryos at 4.5 and 6.5 days of embryonic development, respectively. The cells were cultured in DMEM (purchased from Hyclone) + 15% fetal bovine serum (purchased from Gibco) + L-GlutaMax (1×, purchased from Gibco) + NEAA (1×, purchased from Gibco) + Sodium pyruvate (1×, purchased from Gibco) + Penicillin-Streptomycin antibiotics (1×, purchased from Hyclone) + 0.1 mM β-mercaptoethanol (purchased from Gibco) + Lif (homemade) + 3 μM CHIR99021 (purchased from Selleck) + 1 μM PD0325901 (purchased from Selleck). Primed cells were cultured in 50% Neurobasal (purchased from Gibco) + 50% F12 (Gibco) + N2 (0.5×, purchased from Gibco) + B27 (0.5×, purchased from Gibco) + glutamine L-GlutaMax (1×, purchased from Gibco) + non-essential amino acid NEAA (1×, purchased from Gibco) + 0.1 mM β-mercaptoethanol (purchased from Gibco) + 5% BSA (purchased from Sigma) + 20 ng / mL bFGF (purchased from PeproTech) + 20 ng / mL activin A (purchased from PeproTech), and 1 μM XAV939 (XAV; purchased from Selleck) or 0.5 μM IWR-endo (purchased from Selleck) was added to prevent cell differentiation.

[0154] 4) Western blot

[0155] Total cell protein was extracted by RIPA method, quantified by BCA protein quantification method, and 15% lower gel and 5% upper gel were prepared by Ya enzyme one-step rapid gel preparation kit. Protein can be loaded after half an hour. Protein electrophoresis was performed at 80V for 30 minutes and 130V for 1 hour; 250mA transfer membrane was performed for 50 minutes; 5% skim milk was blocked for 1.5 hours; 85aa-uORF rabbit anti-antibody (universal for human and mouse) (dilution ratio of 1:500), Flag antibody (dilution ratio of 1:1000, purchased from Abcam,) β-actin antibody (dilution ratio of 1:3000, purchased from Abcam) were used to incubate at 4°C overnight. The next day, rabbit secondary antibody (dilution ratio of 1:5000) was used for incubation at room temperature for 1 hour, TBST was washed for 5 minutes / time, a total of 5 times, and then luminescence development was performed.

[0156] Figure 1 A shows the location of the Lin28B upstream open reading frame 85aa-uORF on mouse Lin28B mRNA. Figure 1B shows that the amino acid sequence of 85aa-uORF is highly conserved in many mammals such as humans and mice. Figure 1 C shows that the start codon of 85aa-uORF is ATG, which cannot be translated if it mutates to TAA. Figure 1 D shows that the 85aa-uORF microprotein is highly expressed in mouse primed pluripotent stem cells, whereas It is almost not expressed in pluripotent stem cells and embryonic fibroblast (MEF) cells, suggesting that this microprotein may have the function of maintaining the pluripotency of Primed cells and affecting cell fate transformation.

[0157] Example 285aa-uORF microprotein regulates the transition between the primed state and the naive state of pluripotent stem cells

[0158] 1) Establishment of CRISPR / Cas9 knockout cell lines

[0159] According to the sequence of 85aa-uORF, several gRNAs targeting different regions of 85aa-uORF were designed, and frameshift mutations were introduced into a section of Lin28B uORF that did not overlap with mORF using CRISPR / Cas9 technology, which did not affect the translation and expression of Lin28B mORF, and an EpiSC cell line with 85aa-uORF knockout was obtained. Similarly, several gRNAs were designed according to the sequence of 87aa-uORF, and lentiviral vector plasmids were constructed to infect human pluripotent stem cells to construct an EpiSC cell line with 87aa-uORF knockout.

[0160] The specific gRNA sequence information is as follows:

[0161] 85aa-sgRNA1: 5'-TGAAGATCACTCCGGTCCAA-3' (SEQ ID NO: 5);

[0162] 85aa-sgRNA2: 5'-GAAGATCACTCCGGTCCAAA-3' (SEQ ID NO: 6);

[0163] 87aa-sgRNA1: 5'-AGAAGATCACTCCGTTCCAA-3' (SEQ ID NO: 7);

[0164] 87aa-sgRNA2: 5'-GAAGATCACTCCGTTCCAAA-3' (SEQ ID NO: 8);

[0165] After the above sgRNAs (SEQ ID NO: 5 and SEQ ID NO: 6) were synthesized, they were cloned into pSpCas9(BB)-2A-Puro(PX459) V2.0 vectors (purchased from Addgene, 62988) through the BbsI restriction site, and the above vectors were transformed into OG2-EpiSC cells (OG2: Oct4-ΔPE-GFP as Pluripotency reporter line), EpiSC transfected with 85aa-sgRNA1 and 2 were selected with puromycin for three days, and then positive clones were selected, and genome sequencing was extracted to obtain two strains of EpiSC cells with 85aa-uORF knockout, of which KO1 was a frameshift mutation with a G base inserted, and KO2 was a frameshift mutation with four bases missing GGTC. Similarly, the above sgRNA (SEQ ID NO: 7 and SEQ ID NO: 8) was synthesized, cloned into lentiCRISPR v2 (purchased from Addgene, 52961) through the BbsI restriction site, packaged with lentivirus to infect human pluripotent stem cells H9, and the cell line with 87aa-uORF knockout was obtained after three days of puromycin selection of infected H9 cells.

[0166] 2) Flow cytometry detection of the primed-to-naive transition of 85aa-uORF knockout mouse pluripotent stem cells transition, PNT) efficiency

[0167] OG2-mEpiSC (wild type, WT) and 85aa-uORF knockout OG2-mEpiSC (KO) were seeded in 12-well plates, with 30,000 cells per well. Each cell type was replicated in 3 wells and the experiment was performed 3 times. WT and KO cells were induced to express in PNT medium containing BMP4+iCD1. Change of direction, successful transformation The cells emit green fluorescence and can be detected by fluorescence microscopy and flow cytometry. The WT and KO cells on day 6 and day 10 of the culture transformation were detected, analyzed and the efficiency was statistically analyzed using a C6 flow cytometer (BD Biosciences).

[0168] 3) AP staining and qPCR detection of PNT efficiency of human pluripotent stem cells with 87aa-uORF (sguORF1 / 2) knockout

[0169] Wild-type and 87aa-uORF knockout human pluripotent stem cell lines (sguORF1 and sguORF2) were seeded in 12-well plates, with 30,000 cells per well, and 3 replicates were made for each well. After 9 days of culture in the culture system (PMID: 29669738), the cells were stained with alkaline phosphatase (AP) detection reagent and the number of clones of the three types of cells was counted.

[0170] 4) Overexpression of 85aa-uORF promotes the transformation of mouse embryonic stem cells from the primitive state to the primed state ( -primed transition, NPT)

[0171] According to the above method, the constructed pLVX-85aa-uORF-3Flag mass and lentiviral packaging plasmid were transfected into HEK293T cells to package lentivirus. The lentiviral supernatant was collected and concentrated by centrifugation, diluted with culture medium and infected with mouse embryonic stem cells (mESC). The cells were selected with puromycin (about 3 days) and then amplified normally to obtain 85aa-uORF overexpression cells. Cell lines were compared with control The cells were seeded on a 6-well plate, with 50,000 cells per well and 3 replicates. The culture medium of primed cells was used to induce them to transition to the primed state. After 5 days, photos were taken under an optical microscope and samples were collected to extract RNA for analysis.

[0172] Sequencing and Western blot results showed that the 85aa-uORF knockout OG2-mEpiSC (KO) cell line was successfully constructed ( Figure 2 A-2C) and does not affect the translation and expression of the major open reading frame of Lin28B ( Figure 2 D). Flow cytometry experiments demonstrated that the PNT efficiency of 85aa-uORF KO was significantly higher than that of WT on the sixth day, and the efficiency of KO in promoting PNT was close to 100% on the tenth day ( Figure 3 ). Knocking out 87aa-uORF in human pluripotent stem cells also significantly promoted PNT efficiency, reflecting the high conservation of the amino acid sequence of Lin28B uORF in different species such as humans and mice and its function ( Figure 4 In addition, overexpression of 85aa-uORF significantly promoted Differentiation of cells in the primed state reduces Nanog, Klf4, Esrrb, Tfcp2l1, Dpp5a, Sox2, etc. Pluripotency marker gene expression, improve the expression of Primed pluripotency marker genes such as Fgf5, Otx2, Dnmt3b ( Figure 5 ).

[0173] Example 385aa-uORF microprotein regulates embryo implantation

[0174] 1) Western blot detection of the expression of 85aa-uORF microprotein in different organs of mice

[0175] Adult mice were killed by cervical dislocation, and brain, heart, liver, lung, kidney, intestine, spleen, testis and ovary tissues were taken, ground and ultrasonicated, and the supernatant was taken after centrifugation to prepare protein samples. Electrophoresis, transfer, blocking, primary antibody (rabbit anti-85aa-uORF, 1:500) incubation at 4°C overnight, TBST washing, secondary antibody (mouse anti-, 1:5000) incubation for 1 hour, washing, developing, and photographing for analysis.

[0176] The experimental results are as follows Figure 6 As shown, the microprotein encoded by 85aa-uORF is normally expressed in the brain, heart, liver, kidney, intestine and testis of wild-type (MP85+ / +) mice, and the 85aa-uORF knockout mouse model (MP85- / -) was successfully constructed.

[0177] 2) Construction of 85aa-uORF knockout mice (MP85- / -)

[0178] sgRNA was designed for the non-overlapping region of Lin28B uORF and mORF. After in vitro cleavage to verify the cleavage efficiency, the sgRNA with the highest efficiency (ACTGATGCTGAAGATCACTCCGG (SEQ ID NO: 11)) and Cas9 were transcribed in vitro, and the mixed Cas9 and sgRNA were microinjected into mouse fertilized eggs. The frameshift mutation introduced by base deletion or insertion was repaired by non homologous recombination (NHEJ) after double-strand breaks. After genotyping, F0 generation mice with frameshift mutations were obtained, and F1 generation mice were further bred and used in experiments after genotyping.

[0179] 3) Chicago Sky Blue 6B to detect the number of implantation sites in 85aa-uORF knockout mouse embryos

[0180] Superovulation experiments were performed on 7-8 week old wild-type (MP85+ / +) female mice and 85aa-uORF knockout female mice (MP85- / -). 7.5 IU pregnant mare serum gonadotropin (PMSG) was injected intraperitoneally, and 7.5 IU human chorionic gonadotropin (hCG) was injected 48 hours later. They were mated with adult male mice of the same genotype, and the female mice with vaginal plugs the next day were recorded as 0.5 dpc (0.5 days after mating). 1% Chicago Sky Blue 6B was injected into the tail vein at 5.5 dpc, and the mice were killed 5 minutes later, and photos were taken and the uterine blastocyst implantation sites were counted.

[0181] The experimental results are as follows Figure 7As shown, the mouse blastocyst implantation points were dyed blue using Chicago Sky Blue 6B, and photographed and counted. It was found that the number of implantation points of MP85- / - genotype mice at 5.5dpc was significantly less than that of MP85+ / + genotype mice, indicating that the number of mouse blastocyst implantation would be affected after the knockout of 85aa-uORF. In order to exclude the influence of MP85- / - female mice themselves, mating female mice with the genotype MP85- / - with wild-type male mice with MP85+ / + did not significantly affect the number of female mouse blastocyst implantation points, indicating that the loss of 85aa-uORF would lead to embryonic development defects and affect blastocyst implantation.

[0182] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", "some implementation schemes" or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, unless they are contradictory.

[0183] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A polypeptide, characterized in that The polypeptide has the amino acid sequence shown in SEQ ID NO: 2 or 4 or a conservatively modified amino acid sequence thereof.

2. An expression vector, characterized in that: The expression vector expresses the polypeptide according to claim 1.

3. A recombinant cell, characterized in that Carrying the expression vector of claim 2, or expressing the polypeptide of claim 1; Optionally, the cells comprise mammalian pluripotent stem cells; Optionally, the cells comprise mammalian embryonic stem cells or induced pluripotent stem cells.

4. A biological model, characterized in that The biological model does not express or lowly expresses the polypeptide according to claim 1; Optionally, the biological model is a cell model, an organoid model or an animal model.

5. Use of an agent for changing the polypeptide of claim 1 in pluripotent stem cells in the preparation of high-purity primed pluripotent stem cells or naive pluripotent stem cells; Optionally, the change comprises overexpressing the polypeptide in the pluripotent stem cells, or downregulating or inhibiting the expression of the polypeptide in the pluripotent stem cells; Optionally, the reagent is a reagent based on at least one of shRNA, antisense nucleic acid, ribozyme, CRISPR-Cas9, CRISPR-Cpf1 and zinc finger nuclease; Optionally, the reagent further comprises an overexpression vector or an agonist of the polypeptide; Optionally, the agonist comprises at least one of a small molecule, a drug, an enzyme or a hormone.

6. The use according to claim 5, characterized in that The primed pluripotent stem cells are prepared by overexpressing the polypeptide according to claim 1 in the pluripotent stem cells; Optionally, the naive pluripotent stem cells are prepared by down-regulating or inhibiting the expression of the polypeptide according to claim 1 in the pluripotent stem cells; Optionally, the pluripotent stem cells include mammalian embryonic stem cells or induced pluripotent stem cells.

7. A method for preparing high-purity primed pluripotent stem cells or naive pluripotent stem cells, characterized in that: include: contacting a reagent that changes the polypeptide of claim 1 with pluripotent stem cells; Optionally, the change comprises overexpressing the polypeptide in the pluripotent stem cells, or downregulating or inhibiting the expression of the polypeptide in the pluripotent stem cells; Optionally, the agent is an agent based on at least one of shRNA, antisense nucleic acid, ribozyme, CRISPR-Cas9, CRISPR-Cpf1 and zinc finger nuclease; Optionally, the reagent further comprises an overexpression vector or an agonist of the polypeptide; Optionally, the agonist comprises at least one of a small molecule, a drug, an enzyme or a hormone.

8. The method according to claim 7, characterized in that include: Obtaining the primed pluripotent stem cells by overexpressing the polypeptide of claim 1 in the pluripotent stem cells or contacting the pluripotent stem cells with the polypeptide of claim 1; Optionally, the method comprises: obtaining the naive pluripotent stem cells by down-regulating or inhibiting the expression of the polypeptide of claim 1 in the pluripotent stem cells; Optionally, the pluripotent stem cells include mammalian embryonic stem cells or induced pluripotent stem cells.

9. Use of the biological model according to claim 4 in screening drugs for treating diseases related to embryo implantation defects, characterized in that: The disease comprises at least one of threatened abortion, habitual abortion, infertility, endometriosis, endometrial weakness, endometritis, uterine malformation, polycystic ovary disease and luteal insufficiency.

10. Use of the polypeptide according to claim 1, the nucleic acid encoding the polypeptide according to claim 1, the expression vector according to claim 2 or the recombinant cell according to claim 3 in preparing a drug, characterized in that: The drug is used to prevent, alleviate and / or treat diseases related to embryo implantation defects; Optionally, the disease comprises at least one of threatened abortion, habitual abortion, infertility, endometriosis, endometrial weakness, endometritis, uterine malformation, polycystic ovary disease and luteal insufficiency.

11. A drug for preventing, alleviating and / or treating diseases related to embryo implantation defects, characterized in that: The drug comprises the polypeptide of claim 1, a nucleic acid encoding the polypeptide of claim 1, an expression vector of claim 2 or a recombinant cell of claim 3; Optionally, the disease comprises at least one of threatened abortion, habitual abortion, infertility, endometriosis, endometrial weakness, endometritis, uterine malformation, polycystic ovary disease and luteal insufficiency.

12. Use of the preparation in the preparation of a drug for preventing, alleviating and / or treating diseases related to embryo implantation defects, characterized in that: The preparation causes the polypeptide of claim 1 to be overexpressed in a cell; Optionally, the cells comprise mammalian pluripotent stem cells; Optionally, the pluripotent stem cells include embryonic stem cells or induced pluripotent stem cells; Optionally, the formulation includes an overexpression vector or an agonist of the polypeptide; Optionally, the agonist comprises at least one of a small molecule, a drug, an enzyme or a hormone.

13. Use of a reagent for detecting the polypeptide of claim 1 or a nucleic acid encoding the polypeptide of claim 1 in preparing a kit for detecting diseases related to embryo implantation defects.

14. A kit, characterized in that: The kit comprises a reagent for detecting the polypeptide of claim 1 or detecting a nucleic acid encoding the polypeptide of claim 1.

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