SubA mutant and application thereof in construction of endoplasmic reticulum stress state cell model

Abstract

The invention provides a SubA mutant and application thereof in construction of an endoplasmic reticulum stress state biological model, and particularly provides application of polypeptide in preparation of the biological model, the biological model is in an endoplasmic reticulum stress state, and compared with SEQ ID NO: 3, the polypeptide has p.Ser272Ala mutation. It is found that when Ser272 residues in SubA are mutated into Ala residues, a protein only having degenerative enzyme activity can be generated, cell death cannot be caused, meanwhile, a Tet-on system is added to regulate and control efficient expression of SubA272, and therefore a tumor biological model in an endoplasmic reticulum stress state can be efficiently prepared; a research tool is provided for mechanism research of endoplasmic reticulum stress and tumor occurrence and proliferation and tumor treatment.

Description

Technical Field

[0001] The present invention relates to the field of cell biotechnology, and particularly relates to SubA mutants and their applications in constructing cell models in an endoplasmic reticulum stress state. Background Art

[0002] The endoplasmic reticulum (ER) is an organelle with a large dynamic membrane structure inside cells, where transmembrane proteins and secretory proteins are synthesized and folded. Properly folded proteins are transported out of the ER to perform their functions. Multiple factors disturbing cellular homeostasis, such as hypoxia, low glucose, infection, and chemotherapy, can lead to impaired ER function, manifested as the accumulation of misfolded and unfolded proteins in the ER lumen and the disorder of intracellular calcium (Ca 2+ ) balance, which is called ERS. During the occurrence and development of cancer, due to the rapid growth of tumors, tumor cells are exposed to low glucose, low vascularization, and hypoxic environments, and intracellular ERS-related proteins will be overexpressed. To respond to ERS, cells initiate corrective mechanisms such as UPR to promote protein folding and degrade abnormally folded proteins, restore cellular homeostasis, and promote cell survival. Currently, there are three UPR sensors in higher mammalian cells, all of which are single-pass transmembrane proteins on the endoplasmic reticulum, including IRE1 (inositol-requiring enzyme 1), PERK (PKR-like ER-resident kinase), and ATF6 (activating transcription factor 6), but only the IRE1α sensor exists in yeast.

[0003] Under non-stress conditions, IRE1 and PERK, which belong to type I transmembrane proteins located on the endoplasmic reticulum membrane, and ATF6, which belongs to type II transmembrane proteins, bind to the molecular chaperone protein on the endoplasmic reticulum - binding immunoglobulin protein (BiP), also known as glucose regulated protein 78 (GRP78), blocking the signal pathway related to UPR. Due to the occurrence of stress caused by the massive accumulation of unfolded or misfolded proteins in the ER lumen, the sensors IRE1, PERK, and ATF6 dissociate from GRP78 / BiP and are correspondingly activated, and then the UPR and downstream cascade signal pathways are initiated. A large number of studies have shown that when ERS occurs in intestinal epithelial cells, the expression level of GRP78 / BiP often increases significantly.

[0004] The endoplasmic reticulum stress response can be coupled with the intracellular inflammatory response signal transduction pathway, which is the main cause of the inflammatory response triggered by non-infectious pathogens. Therefore, endoplasmic reticulum stress and the inflammatory response are related to the pathogenesis of many diseases such as respiratory, cardiovascular, neurodegenerative diseases, cancer, and diabetes.

[0005] Numerous studies have confirmed that the occurrence and development of tumors and cancers are closely related. Many types of cancer cells rely on the high protein synthesis and folding ability of endoplasmic reticulum chaperones to ensure the growth, proliferation, and spread of cancer cells. The microenvironment characteristics of hypoxia, redox imbalance, pH fluctuations, and insufficient nutrient supply during the growth of tumor cells induce the occurrence of UPR. In addition, it has been found that the UPR signal transduction in tumor cells increases. Some key molecules in the endoplasmic reticulum stress response have been proven to be essential for tumorigenesis. For example, the GRP78 / BiP protein is highly expressed in various cancers such as prostate cancer, lung cancer, melanoma, and colon cancer cells. Studies have shown that the GRP78 / BiP protein is essential for tumorigenesis. GRP78 / BiP can enhance the growth and proliferation of tumor cells and inhibit the apoptosis of tumor cells by increasing the protein folding ability of the endoplasmic reticulum. In addition, studies have shown that GRP78 can dissolve resistance to CTL and TNFα, assisting cancer cells to evade the surveillance of the body's immune system, which plays an important role in the spread and metastasis of cancer cells. The PERK pathway of UPR also plays an important role in the proliferation and survival of tumor cells. Under extremely hypoxic conditions, mutations in the kinase domain of PERK or the formation of phosphorylation resistance of eIF2α lead to the inactivation of PERK, damaging cell survival. In addition, PERK can limit oxidative stress damage to DNA by activating Nrf2, further promoting the growth and proliferation of tumor cells. Therefore, UPR is expected to become a new effective therapeutic target for cancer. Establishing a cell model that can maintain the endoplasmic reticulum stress state for a long time can systematically study the relationship and mechanism of action between UPR and cancer. Summary of the Invention

[0006] Currently, the methods for establishing endoplasmic reticulum stress cell models can be roughly divided into three categories: chemical induction method: commonly used polyvinyl alcohol, deamino acids, and DTT to induce cells to produce endoplasmic reticulum stress; genetic engineering method: constructing specific cell lines through gene editing technology; culturing cells under specific in vitro conditions to induce cells to produce endoplasmic reticulum stress, such as hypoxia and low nutrition. Each of the above three methods has its own deficiencies. Among them, the chemical induction method has a short action time, cannot simulate the long-term endoplasmic reticulum stress state, and chemical reagents may affect other cell pathways, thus misjudging endoplasmic reticulum stress; the genetic engineering method has different efficiencies in different cells, it is difficult to obtain a stable cell model, and it may affect non-target genes and affect the experimental results; the in vitro culture method cannot fully simulate the in vivo environment, resulting in insufficient understanding of the endoplasmic reticulum stress mechanism.

[0007] Subtilase cytotoxin (SubAB) is present in Shiga toxin-producing Escherichia coli. SubA, a subunit of subtilase cytotoxin, can cause cell death independently of the SubB subunit. SubA triggers cell death by cleaving the molecular chaperone BiP / GRP78 in the endoplasmic reticulum (ER). The present invention discovers that when the Ser272 residue in SubA is mutated to an Ala (SubAA272) residue, a protein with only degenerate enzyme activity is produced, which maintains cells in a state of endoplasmic reticulum stress for a long time without causing cell death.

[0008] Thus, in the first aspect of the present invention, the present invention provides an isolated polypeptide. According to an embodiment of the present invention, compared with the amino acid sequence shown in SEQ ID NO:3, the polypeptide has a p.Ser272Ala mutation. This polypeptide is obtained by self-mutation in the present invention. The present invention discovers that the expression of this polypeptide can keep cells in a state of endoplasmic reticulum stress for a long time, and this polypeptide can be used for research and applications related to endoplasmic reticulum stress.

[0009] In the second aspect of the present invention, the present invention provides a nucleic acid molecule. According to an embodiment of the present invention, the nucleic acid molecule encodes the isolated polypeptide described in the first aspect.

[0010] In the third aspect of the present invention, the present invention provides the use of the isolated polypeptide described in the first aspect in the preparation of a biological model for diseases caused by endoplasmic reticulum stress.

[0011] When stress factors in the internal and external environments of cells (such as nutrient deficiency, metabolic disorders, pathogen invasion, ischemia and hypoxia) stimulate the endoplasmic reticulum, the number of unfolded and misfolded proteins in the endoplasmic reticulum increases, and the calcium ion level is imbalanced, thereby inducing endoplasmic reticulum stress (ERS). Moderate ERS is a stress response that protects eukaryotic cells from damage, but long-term or severe ERS can cause cell apoptosis. For example, continuous endoplasmic reticulum stress in intestinal epithelial cells can cause damage to the intestinal mucosa of animals and induce inflammatory bowel disease (IBD).

[0012] According to an embodiment of the present invention, the biological model is a cell model, an organoid model or an animal model.

[0013] According to an embodiment of the present invention, the diseases caused by endoplasmic reticulum stress include at least one of tumors and inflammatory bowel disease.

[0014] According to an embodiment of the present invention, the tumor includes colon cancer or cervical cancer.

[0015] According to an embodiment of the present invention, the biological model is an MC38, Mode-k or Hela cell model.

[0016] In a fourth aspect of the present invention, there is provided a method for preparing a biological model for diseases associated with endoplasmic reticulum stress. According to an embodiment of the present invention, it includes: causing the biological model to express the isolated polypeptide described in the first aspect. As described above, the expression of this polypeptide will keep cells in a state of long-term endoplasmic reticulum stress, and the long-term endoplasmic reticulum stress state will lead to the occurrence of related diseases.

[0017] According to an embodiment of the present invention, the biological model is a cell model, an organoid model or an animal model.

[0018] According to an embodiment of the present invention, the diseases associated with endoplasmic reticulum stress include at least one of tumors and inflammatory bowel disease.

[0019] According to an embodiment of the present invention, the tumor includes colon cancer or cervical cancer.

[0020] According to an embodiment of the present invention, the biological model is an MC38, Mode-k or Hela cell model.

[0021] According to an embodiment of the present invention, causing the biological model to express the isolated polypeptide is achieved by introducing an expression vector carrying the nucleic acid molecule described in the second aspect into the biological model.

[0022] According to an embodiment of the present invention, the type of the expression vector is not particularly limited, and the expression vector is a eukaryotic expression vector or a prokaryotic expression vector.

[0023] In a fifth aspect of the present invention, there is provided the use of a biological model in screening for drugs for treating diseases associated with endoplasmic reticulum stress. According to an embodiment of the present invention, the biological model carries the nucleic acid molecule described in the second aspect, or expresses the isolated polypeptide described in the first aspect.

[0024] According to an embodiment of the present invention, the biological model is a cell model, an organoid model or an animal model.

[0025] According to an embodiment of the present invention, the diseases associated with endoplasmic reticulum stress include at least one of tumors and inflammatory bowel disease.

[0026] According to an embodiment of the present invention, the tumor includes colon cancer or cervical cancer.

[0027] According to an embodiment of the present invention, the biological model is an MC38, Mode-k or Hela cell model.

[0028] In a sixth aspect of the present invention, there is provided the use of a reagent for detecting the isolated polypeptide described in the first aspect or the nucleic acid molecule described in the second aspect in the preparation of a kit for diagnosing diseases associated with endoplasmic reticulum stress.

[0029] According to an embodiment of the present invention, the diseases associated with endoplasmic reticulum stress include at least one of tumor and inflammatory bowel disease.

[0030] According to an embodiment of the present invention, the tumor includes colon cancer or cervical cancer.

[0031] It should be noted that the reagent includes antibodies, probes, primers and mass spectrometry detection reagents directed against the polypeptide described in the first aspect or the nucleic acid molecule described in the second aspect. The types of reagents according to the embodiments of the present invention are not particularly limited as long as they can detect the aforementioned nucleic acid or polypeptide. Among them, the antibody is a protein that can specifically bind to the polypeptide, and the mass spectrometry detection reagent is a reagent used for detecting the polypeptide by protein mass spectrometry technology. The types of the mass spectrometry detection reagents used are not particularly limited as long as they can be used to detect the polypeptide by a protein mass spectrometer.

[0032] In a seventh aspect of the present invention, there is provided the use of a reagent for specifically altering the isolated polypeptide described in the first aspect or the nucleic acid molecule described in the second aspect in the preparation of a drug for preventing, alleviating and / or treating diseases associated with endoplasmic reticulum stress.

[0033] According to an embodiment of the present invention, the specific alteration is to revert the nucleic acid or polypeptide to the wild type, and the reagent is based on at least one of gene editing methods including single-base gene editing, zinc finger nuclease, transcription activator-like effector nuclease, CRISPR / Cas9, and CRISPR / Cas9 combined with iPSC and AAV vector technology.

[0034] According to an embodiment of the present invention, the diseases associated with endoplasmic reticulum stress include at least one of tumor and inflammatory bowel disease.

[0035] According to an embodiment of the present invention, the tumor includes colon cancer or cervical cancer.

[0036] In an eighth aspect of the present invention, there is provided a drug. According to an embodiment of the present invention, the drug is used for treating diseases associated with endoplasmic reticulum stress and contains: a reagent for specifically altering the isolated polypeptide described in the first aspect or the nucleic acid molecule described in the second aspect.

[0037] According to an embodiment of the present invention, the specific change is to restore the nucleic acid or protein to the wild type, and the reagent is a reagent based on at least one of gene editing methods including single-base gene editing, zinc finger nuclease, transcription activator-like effector nuclease, CRISPR / Cas9, and the combination of CRISPR / Cas9 with iPSC and AAV vector technologies.

[0038] According to an embodiment of the present invention, the diseases associated with endoplasmic reticulum stress include at least one of tumors and inflammatory bowel disease.

[0039] According to an embodiment of the present invention, the tumor includes colon cancer or cervical cancer.

[0040] In the ninth aspect of the present invention, the present invention provides a construct. According to an embodiment of the present invention, the construct carries the nucleic acid molecule described in the second aspect. When the construct according to an embodiment of the present invention is introduced into a recipient cell, the mutant protein described above can be expressed in the recipient cell, and then the biological model described above can be obtained, which can be used for scientific research, such as drug screening.

[0041] In the tenth aspect of the present invention, the present invention provides a recombinant cell. According to an embodiment of the present invention, the recombinant cell is obtained by transforming a recipient cell with the construct described in the ninth aspect or by expressing the protein described in the first aspect. The recombinant cell according to an embodiment of the present invention can be used as a cell model for scientific research, such as drug screening or studying the pathogenic mechanism of endoplasmic reticulum stress.

[0042] The beneficial effects of the present invention compared with the prior art at least include:

[0043] (1) The SubAA272 polypeptide was discovered and isolated.

[0044] (2) It was found that the SubAA272 polypeptide can cause cells to be in a long-term endoplasmic reticulum stress state. Using this polypeptide, an endoplasmic reticulum stress cell model can be efficiently constructed, and the expression of endoplasmic reticulum stress-related signaling proteins, such as GRP78 and IRE1α, is significantly increased in this model.

[0045] The additional aspects and advantages of the present invention will be partially given in the following description, partially become apparent from the following description, or be understood through the practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] The above and / or additional aspects and advantages of the present invention will become apparent and be readily understood from the description of the embodiments in conjunction with the following drawings, wherein:

[0047] Figure 1 Shows the expression of SubA in Example 1 of the present invention A272The map of the ppb-ef1a-mcherry-t2a-subaa272-ha-kedl-cmv-egfpouro-pa plasmid and the map of the control vector;

[0048] Figure 2 The figure shows the results of detecting the expression levels of SubA and GRP78 cleavage proteins in MC38 cells transfected for 14 days by Western Blot in Example 2 of the present invention. Detailed implementation manners

[0049] The embodiments of the present invention are described in detail below. The following described embodiments are exemplary and are only used to explain the present invention, and should not be construed as a limitation to the present invention.

[0050] It should be noted that the terms "first" and "second" are only used for descriptive purposes and cannot be construed as indicating or implying relative importance or implicitly specifying the quantity of the indicated technical features. Thus, the features defined with "first" and "second" may explicitly or implicitly include one or more of such features. Further, in the description of the present invention, unless otherwise stated, the meaning of "a plurality" is two or more.

[0051] In the ranges disclosed herein, the endpoints and any values are not limited to the exact ranges or values, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, between the endpoint values of each range, between the endpoint values of each range and individual point values, and between individual point values, they can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed herein.

[0052] To make the present invention easier to understand, 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 of ordinary skill in the art to which the present invention pertains.

[0053] In this document, the term "comprising" or "including" is an open expression, that is, it includes the content specified by the present invention, but does not exclude other aspects.

[0054] In this document, the terms "optionally", "optional" or "option" generally mean that the subsequent events or conditions may or may not occur, and this description includes the cases where such events or conditions occur and the cases where such events or conditions do not occur.

[0055] As used herein, the term "cell model" refers to a model of a cell that patterns the structure and shape of the cell, an artificial particle having partial cell functions such as ribosomes, or a cell in which the membrane structure and the soluble portion of the cytoplasm are removed while maintaining the structure (contractile structure) and function involved in cell movement.

[0056] As used herein, the term "organoid model" refers to a tissue analogue with a certain spatial structure formed by in vitro three-dimensional (3D) culture of adult stem cells, pluripotent stem cells or somatic cells. These organoids can mimic some characteristics of real organs in terms of structure and function, although they are not real human organs in the true sense.

[0057] As used herein, the term "animal model" refers to an animal established in various scientific studies that exhibits manifestations simulating human diseases.

[0058] It should be noted that the "nucleic acid" described in this 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 particularly limited. For constructs used to construct recombinant cells, it is preferred that the nucleic acid is DNA because DNA is more stable and easier to operate compared to RNA. The "nucleic acid" described in this application actually includes either one or both of the complementary double strands. For convenience, in this specification and the claims, although only one strand is given in most cases, the other complementary strand is actually also disclosed. For example, referring to SEQ ID NO:1 actually includes its complementary sequence. Those skilled in the art can also understand that one strand can be used to detect the other strand, and vice versa.

[0059] It should be noted that the "construct" described in this application refers to a genetic vector that contains a specific nucleic acid sequence and can transfer the target nucleic acid sequence into a host cell to obtain a recombinant cell. According to the embodiments of the present invention, the form of the construct is not particularly limited. According to the embodiments of the present invention, it can be at least one of a plasmid, a phage, an artificial chromosome, a cosmid, and a virus, preferably a plasmid. As a genetic vector, a plasmid has the properties of simple operation and can carry large fragments, which is convenient for operation and processing. The form of the plasmid is not particularly limited either. It can be a circular plasmid or a linear plasmid, that is, it can be single-stranded or double-stranded. Those skilled in the art can make a choice according to their needs.

[0060] In this article, "single-base gene editing" refers to gene editing technology that can cause single base changes 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.

[0061] In this article, "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 with a domain containing zinc fingers. Its purpose is naturally to cut a specific sequence; the cut DNA can be repaired by the excision mechanism to delete the single-stranded part of the cut, and then reconnected together. This method can be used to delete specific fragments on the chromosome, thereby constructing mutants or completing treatment.

[0062] In this article, "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.

[0063] In this article, the "CRISPR / Cas9" system is widely present in prokaryotic genes and is an acquired immune defense mechanism evolved by bacteria and archaea in response to 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, and then the tracrRNA / crRNA complex guides the Cas9 protein to cut double-stranded DNA for gene editing.

[0064] The nucleic acid or amino acid sequences used in this application are shown in Table 1.

[0065] Table 1:

[0066]

[0067]

[0068] The present invention will be described below with reference to specific embodiments. It should be noted that these embodiments are merely descriptive and do not limit the present invention in any way.

[0069] Unless otherwise specified, the technical means used in the embodiments are conventional means well-known to those skilled in the art, and can be referred to the third edition of "Molecular Cloning Experiment Guide" or related products. The reagents and products used are also commercially available. The various processes and methods not described in detail are conventional methods well-known in the art. The source, trade name of the reagents used and those necessary to list their components are indicated at the first occurrence. For the same reagents used later, if there is no special explanation, they are the same as the content indicated at the first occurrence.

[0070] Example 1 Expression of SubA A272 Preparation of MC38 cells expressing the toxin

[0071] 1.1 Construction of the plasmid carrying the target gene

[0072] In this example, the A subunit (SubA) of the subtilase cytotoxin (SubAB) found in Shiga toxin Escherichia coli was mutated. Specifically, the 272nd amino acid residue Ser of SubA was mutated to Ala to obtain the SubA A272 subunit, and then MC38 cells expressing this subunit were constructed. The specific experimental operations are as follows:

[0073] The nucleotide sequence (SEQ ID NO:1) encoding SubA A272 toxin (SEQ ID NO:2) was ligated to the nucleotide sequence encoding the ha tag through the self-cleaving T2A peptide to mCherry fluorescent protein to facilitate visualization of the cells expressing SubA A272 The target gene is regulated by the Tet-on system. tTA is a protein formed by the fusion of TetR and the viral transcriptional activation domain VP16. rtTA is formed by mutating 4 amino acids of TA, and its phenotype is opposite to that of TA. Under physiological conditions, rtTA does not bind to TRE. Since PminCMV lacks an enhancer +, the Suba gene is not expressed. After giving DOX, the complex of DOX and rtTA binds to TRE, thereby initiating the expression of the Suba gene. The specific structure of the plasmid is as Figure 1 shown.

[0074] 1.2. Cell transfection

[0075] 1) Cell seeding:

[0076] One day before transfection, use high-glucose DMEM medium (500 ml) (purchased from Cisco Jie, product number: CA0004-500ML): fetal bovine serum (purchased from Novizan, product number: F101-01): penicillin-streptomycin mixture (100X) (purchased from Solarbio, product number P1400) = 45:5:1 medium (gifted by the Zhu Shu Laboratory of the Institute of Health Sciences, Hefei Comprehensive National Science Center) to seed MC38 cells into the cell plate, culture and make the cell density reach 50% at the time of transfection the next day. When plating, digest the cells evenly to avoid cell aggregation growth.

[0077] 2) Transfection reagent:

[0078] The transfection reagent used in this invention is Lipofectamine TM 3000 Transfection Reagent, with the product number L3000001;

[0079] Reagent preparation steps:

[0080] Dilute Lipofectamine TM 3000 reagent with Opti-MEM TM medium and mix well;

[0081] Dilute the prepared plasmid expressing the target gene or the control plasmid with Opti-MEM TM medium respectively to prepare a DNA premix, and then add P3000 TM reagent and mix well;

[0082] Add the diluted DNA (volume ratio 1:1) to each tube of diluted Lipofectamine TM 3000 reagent; incubate for 10 - 15 min; after incubation, add the obtained DNA-lipid complex to the above-cultured MC38 cells, and culture the cells at 37°C and 5% CO2 for 1 - 2 days, and observe the cell status every 24 h.

[0083] 3) Puromycin screening:

[0084] The puromycin reagent used in this invention is puromycin solution (10 mg / ml) (purchased from Biosharp, product number: BL528A), and the screening steps are as follows:

[0085] When screening, the density of MC38 cells does not exceed 25%. The MC38 cells transfected or infected for 48 hours obtained in part 2) of this experiment were placed in fresh medium containing an appropriate concentration of the above puromycin (this medium uses high-glucose DMEM medium (500 ml) (purchased from Cisco Jie): fetal bovine serum (purchased from Novizan, product number: F101-01): penicillin-streptomycin mixture (100X) (purchased from Solarbio, product number P1400) = 45:5:1) for culture to obtain the treatment group. A group in which the above untransfected MC38 cells were treated with the above fresh medium without puromycin was set as the control group. During the screening process, the medium containing puromycin was replaced every 2 - 3 days, and the cell growth status was observed daily. After 7 days of screening, 100% of the normal cells in the control group died, and the surviving cells in the treatment group were the cells expressing the puro gene. Then, according to the experimental purpose, polyclonal or monoclonal cells were screened to obtain a cell line with stable expression.

[0086] Example 2 Detection of the endoplasmic reticulum stress state of the cell line by Western Blot

[0087] In this example, the expression levels of GRP78 and IRE1α were detected by Western Blot to evaluate the endoplasmic reticulum stress situation. Vinculin, a cytoskeletal protein and focal adhesion component protein, is mainly distributed in cells. During endoplasmic reticulum stress (ERS), to maintain cell homeostasis and normal endoplasmic reticulum function, the endoplasmic reticulum makes a series of regulatory responses to reduce the excessive accumulation of unfolded or misfolded proteins. The most classical signaling pathway among them is the unfolded protein response (UPR). In animal cells, the UPR signaling pathway is mainly initiated by three endoplasmic reticulum transmembrane protein sensors: inositol-requiring enzyme 1 (IRE1), protein kinase receptor-like endoplasmic reticulum kinase (PERK), and activating transcription factor 6 (ATF6). Under non-stress conditions, IRE1 and PERK, which belong to type I transmembrane proteins located on the endoplasmic reticulum membrane, and ATF6, which belongs to type II transmembrane proteins, bind to the molecular chaperone protein on the endoplasmic reticulum - binding immunoglobulin (BiP), also known as glucose-regulated protein 78 (GRP78), blocking the signal pathway related to UPR. In this example, after the plasmid containing the mutated SubA described in Example 1 was transfected into the target cells, the expressions of ERS-related genes IRE1α and GRP78 were detected to detect whether the constructed cells were an endoplasmic reticulum stress cell model.

[0088] The specific experimental operations are as follows:

[0089] Homogenize and resuspend the protein extract of small intestinal tissue or cells in immunoprecipitation buffer (20 mM Tris pH 8.0, 150 mM NaCl, 1 mM EDTA, 1% Triton X-, and 1X protease inhibitor).

[0090] Load 20 mg of soluble protein onto an 8% SDS-PAGE gel and run it at 100 V for 90 minutes. Transfer it to a nitrocellulose membrane (Thomas Scientific) at 300 mA for 90 minutes in a cold room at 4°C. Block the membrane with TBS-based Odyssey Blocking buffer (LI-COR Biosciences), and incubate it overnight at 4°C with the primary antibody diluted in Odyssey Blocking buffer and 0.1% Tween. After washing, incubate it with the IRDye 800CW secondary antibody (diluted in Odyssey Blocking buffer and 0.1% Tween) for 1 hour at room temperature. Visualize the results using an Odyssey Fc imaging system (LI-COR Biosciences), and quantify the results using Image Studio Software (LI-COR Biosciences).

[0091] The experimental results are as Figure 2 shown. In the cells treated with Dox, the expressions of GRP78 and IRE1α are higher than those in the cells without Dox treatment. Vinculin, a cytoskeletal protein and a component of focal adhesions, is mainly distributed in cells. GRP78 and IRE1α are endoplasmic reticulum stress-related signaling proteins, indicating that the endoplasmic reticulum stress cell model is successfully constructed.

[0092] In the description of this specification, the description referring to terms such as "one embodiment", "some embodiments", "example", "specific example", "some implementation manners" or "some examples", etc. means that the specific features, structures, materials or characteristics described in connection 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 can be combined in a suitable manner in any one or more embodiments or examples. In addition, without contradiction, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples.

[0093] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention.

Claims

1. An isolated polypeptide, characterized in that, Compared with the amino acid sequence shown in SEQ ID NO: 3, the polypeptide has a p.Ser272Ala mutation.

2. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the isolated polypeptide recited in claim 1.

3. Use of the isolated polypeptide recited in claim 1 in the preparation of a biological model for diseases related to endoplasmic reticulum stress.

4. The use according to claim 3, characterized in that, The biological model is a cell model, an organoid model or an animal model; Optionally, the diseases related to endoplasmic reticulum stress include at least one of tumor and inflammatory bowel disease; Preferably, the tumor includes colon cancer or cervical cancer; Preferably, the biological model is an MC38, Mode-k or Hela cell model.

5. A method for preparing a biological model of endoplasmic reticulum stress-induced related diseases, characterized in that, Comprising: Making the biological model express the isolated polypeptide recited in claim 1.

6. The method according to claim 5, wherein The biological model is a cell model, an organoid model or an animal model; Optionally, the diseases related to endoplasmic reticulum stress include at least one of tumor and inflammatory bowel disease; Preferably, the tumor includes colon cancer or cervical cancer; Preferably, the biological model is an MC38, Mode-k or Hela cell model; Optionally, making the biological model express the isolated polypeptide is achieved by introducing an expression vector carrying the nucleic acid molecule recited in claim 2 into the biological model; Optionally, the expression vector is a eukaryotic expression vector or a prokaryotic expression vector.

7. Use of a biological model in screening drugs for treating diseases related to endoplasmic reticulum stress, characterized in that, The biological model carries the nucleic acid molecule recited in claim 2, or expresses the isolated polypeptide recited in claim 1; Optionally, the biological model is a cell model, an organoid model or an animal model; Optionally, the diseases related to endoplasmic reticulum stress include at least one of tumor and inflammatory bowel disease; Preferably, the tumor includes colon cancer or cervical cancer; Preferably, the biological model is an MC38, Mode-k or Hela cell model.

8. Use of a reagent for detecting the isolated polypeptide recited in claim 1 or the nucleic acid molecule recited in claim 2 in the preparation of a kit for diagnosing diseases related to endoplasmic reticulum stress; Optionally, the diseases related to endoplasmic reticulum stress include at least one of tumor and inflammatory bowel disease; Preferably, the tumor includes colon cancer or cervical cancer.

9. Use of a reagent for specifically altering the isolated polypeptide recited in claim 1 or the nucleic acid molecule recited in claim 2 in the preparation of a drug for preventing, alleviating and / or treating diseases related to endoplasmic reticulum stress; Optionally, the specific alteration is to revert the nucleic acid or polypeptide to the wild type, and the reagent is a reagent based on at least one of gene editing methods including single-base gene editing, zinc finger nuclease, transcription activator-like effector nuclease, CRISPR / Cas9, and CRISPR / Cas9 combined with iPSC and AAV vector technology; Optionally, the diseases related to endoplasmic reticulum stress include at least one of tumor and inflammatory bowel disease; Preferably, the tumor includes colon cancer or cervical cancer.

10. A drug, characterized in that, The drug for treating diseases related to endoplasmic reticulum stress contains: An agent that specifically modifies the isolated polypeptide according to claim 1 or the nucleic acid molecule according to claim 2; Optionally, the specific modification is to restore the nucleic acid or protein to the wild type, and the agent is an agent based on at least one of gene editing methods including single-base gene editing, zinc finger nuclease, transcription activator-like effector nuclease, CRISPR / Cas9, and CRISPR / Cas9 combined with iPSC and AAV vector technology; Optionally, the diseases associated with endoplasmic reticulum stress include at least one of tumors and inflammatory bowel disease; Preferably, the tumor includes colon cancer or cervical cancer.

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