Effect of SERHL2 gene in AFB1 pathopoiesis and application of SERHL2 gene

Abstract

The invention discloses an effect of an SERHL2 gene in AFB1 pathopoiesis and application of the SERHL2 gene, and belongs to the technical field of biology. Pig intestine epithelial cells (IPEC-J2) are knocked out through SERHL2 gene editing, and after AFB1 treatment and SERHL2 knockout, the activity of the pig intestine epithelial cells is increased, cell death is reduced, and toxicity resistance is increased. Based on this, it is found for the first time that SERHL2 gene knockout can resist intestinal cytotoxicity caused by AFB1 toxin, and the SERHL2 gene knockout can become a therapeutic target for treating AFB1 mycotoxin and an important target for serving animal and human health.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering, specifically relating to the application of SERHL2 gene knockout to promote tolerance of porcine intestinal epithelial cells to aflatoxin B1 and its potential drug target for related diseases. Background Technology

[0002] Aflatoxin enteric toxicity: Aflatoxin is a fungal toxin widely distributed in food and feed, possessing extremely high toxicity and carcinogenicity. Its toxicological mechanisms in the intestine mainly include affecting nutrient absorption and immune function, leading to damage and dysfunction of intestinal epithelial cells. Aflatoxin exerts direct toxic effects on the intestine by affecting lipid metabolism, inducing apoptosis, and disrupting cell membrane integrity. Furthermore, it can also affect intestinal health by inhibiting the activity of intestinal immune cells, reducing the intestine's resistance to pathogens.

[0003] The SERHL2 gene (Serine Hydrolase Like 2) encodes a protein belonging to the α / β-hydrolase superfamily, which may possess serine hydrolase activity. It is presumed to be located in the cytoplasm, near transnuclear vesicles, and peroxisomes. In humans, this gene is located on chromosome 22q (42,553,617–42,574,382, GRCh38). Current research suggests that SERHL2 may be involved in muscle hypertrophy and skeletal muscle metabolism regulation. Particularly under exercise-induced conditions, demethylation of its promoter region enhances the binding activity of Nr4a transcription factors, thereby upregulating SERHL2 expression, promoting lipid metabolism and lipid droplet formation, and maintaining muscle cell energy homeostasis. Gene functional annotation suggests that SERHL2 possesses potential hydrolase activity, but its specific biological functions in different tissues and its role in metabolic-related diseases require further investigation.

[0004] CRISPR / Cas9 gene editing technology: Clustered Regularly Interspaced Short Palinmic Repeats gene editing system is widely used to knock out genes in cells, mice, pigs and other animals because it is characterized by efficient and precise gene knockout. Summary of the Invention

[0005] The technical problem to be solved by this invention is to provide an editable target gene SERHL2 that can affect the tolerance of porcine intestinal epithelial cells to aflatoxin B1, and its potential application in the treatment and application of related diseases as a potential drug target.

[0006] The technical solution of the present invention is: a method for improving the tolerance of cells to AFB1 toxin by gene editing to knock out the SERHL2 gene in cells, thereby improving the tolerance of cells to AFB1 toxin.

[0007] Furthermore, the gene editing knockout is a targeted knockout of the SERHL2 gene using the CRISPR / Cas9 editing system.

[0008] Furthermore, the sgRNA sequence of the CRISPR / Cas9 editing system is shown in SEQ ID No. 1.

[0009] Furthermore, the cells are porcine small intestinal epithelial cells.

[0010] An anti-AFB1 toxin cell model was obtained by the method described above.

[0011] An sgRNA, the sequence of which is shown in SEQ ID No. 1.

[0012] The above-mentioned application of sgRNA in improving cellular AFB1 toxin tolerance.

[0013] The above-mentioned application of sgRNA in the preparation of drugs against AFB1 toxin.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] This invention is the first to discover and demonstrate that knockout of the SERHL2 gene reduces AFB1 toxin-induced cell death and promotes cell survival. It reveals a direct link between the SERHL2 gene and an animal's ability to adapt to AFB1 toxicity. This invention provides a novel key functional gene target for mycotoxin resistance adaptation research and has potential applications in animal breeding and the treatment of AFB1 poisoning-related diseases.

[0016] This invention establishes an AFB1 toxicity tolerance cell model by knocking out the SERHL2 gene, which can be widely used in scientific research and drug development related to toxicity tolerance. Attached Figure Description

[0017] Figure 1 Sanger sequencing base sequences of normal and SERHL2 knockout porcine intestinal epithelial cells. In the figure, A represents the sequencing peaks of normal cells; B represents the sequencing peaks of cells after SERHL2 knockout.

[0018] Figure 2 Changes in cell viability of normal cells and SERHL2 knockout cells after treatment with different aflatoxin concentrations for 48 h;

[0019] Figure 3Optical micrographs of SERHL2 gene knockout cells and normal cells after treatment with 40 μM aflatoxin for 48 hours. Detailed Implementation

[0020] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were all purchased from commercial sources.

[0021] Example 1: SERHL2 gene knockout

[0022] 1. An sgRNA targeting the porcine SERHL2 gene (CDS sequence shown in SEQ ID No. 4) was designed using the website chopchop (https: / / chopchop.cbu.uib.no / ), and the corresponding DNA sequence of this sgRNA was ligated into the LentiCRISPR-V2 vector. Subsequently, the successfully ligated LentiCRISPR-V2 vector was packaged into lentiviruses using 293T cells. The sgRNA nucleotide sequence is: F:5'-GGTGGTAATACGGGAAACCT-3' (SEQ ID No. 1).

[0023] 2. Subsequently, the IPEC-J2 (porcine small intestinal epithelial cell) cell line was infected with lentivirus and screened for resistance to puromycin. After screening, cell samples were collected for subsequent knockout identification. Cell DNA was extracted using a cell DNA extraction kit, and DNA was extracted from normal IPEC-J2 cells as a control. PCR amplification was performed using knockout identification primers, followed by sequencing and alignment analysis. The nucleotide sequences of the knockout identification primers are shown in SEQ ID No. 2 and SEQ ID No. 3.

[0024] F:5'- ACGGCTGGCTGGACAATG -3' (SEQ ID No.2)

[0025] R:5'-TCCTGGCAGAACTGAGGC-3' (SEQ ID No.3)

[0026] Sequencing results as follows Figure 1 As shown, based on the sequencing results, it was found that ( Figure 1 In the SERHL2 knockout group (SERHL2-KO), compared with the normal group, 7 bases were missing at the sgRNA target sequence, indicating that the SERHL2 gene knockout was successful.

[0027] Example 2: The preventive effect of SERHL2 gene knockout on aflatoxin poisoning in porcine intestinal cells.

[0028] 1. Porcine intestinal epithelial cells from the SERHL2 knockout group (SERHL2-KO) and the control group (IEPC-J2-V2-control) were seeded into 96-well plates with 5×10³ cells per well and 6 replicates. The cells were cultured at 37°C in a 5% CO2 incubator until the cell confluence reached 80%.

[0029] 2. Add basal culture medium containing aflatoxin B1 at final concentrations of 0, 20, and 40 μM to the SERHL2 knockout group (SERHL2-KO) and the control group (IEPC-J2-V2-control), respectively, and incubate at 37°C in a 5% CO2 incubator for 48 hours.

[0030] 3. After 48 hours, the treatment was terminated. Basal culture medium containing 10% CCK-8 reagent was added to each well. After incubation at 37°C in the dark for one hour, the absorbance was measured at a wavelength of 450 nm.

[0031] 4. Calculate the cell survival rate according to the following formula: Cell survival rate (%) = [(As-Ab) / (Ac-Ab)] × 100%, where As represents the absorbance of the experimental well, Ac represents the absorbance of the control well, and Ab represents the absorbance of the blank well.

[0032] Cell viability results as follows Figure 2 As shown, SERHL2 gene knockout did not affect the cell viability of porcine intestinal epithelial cells, therefore, SERHL2 gene knockout does not affect normal cell viability. However, the cell viability of the SERHL2 knockout group (SERHL2-KO) was significantly higher than that of the control group (IEPC-J2-V2-control) under different concentrations of aflatoxin treatment, indicating that SERHL2 gene knockout can significantly improve the tolerance of porcine intestinal epithelial cells to aflatoxin.

[0033] Example 3: Effect of SERHL2 gene knockout on aflatoxin tolerance in porcine intestinal cells

[0034] 1. SERHL2 knockout porcine intestinal epithelial cells and normal porcine intestinal epithelial cells were seeded into 6-well plates, 1 × 10⁶ cells per well. 5 Cells were cultured in triplicate wells at 37°C in a 5% CO2 incubator until the cell confluence reached 80%.

[0035] 2. Add basal medium containing 40 μM aflatoxin B1 to SERHL2 knockout porcine intestinal epithelial cells and porcine intestinal epithelial cells transfected with lentiCRISPR-V2 empty vector. Incubate at 37℃ in a 5% CO2 incubator for 48 hours and observe changes in cell morphology. The cell morphology of SERHL2 knockout porcine intestinal epithelial cells treated with 40 μM aflatoxin for 48 hours is as follows. Figure 3 As shown, no changes in cell morphology or cell mass were observed in porcine intestinal epithelial cells after treatment with 40 μM aflatoxin for 48 hours compared to normal cells, indicating that SERHL2 knockout porcine intestinal epithelial cells can effectively resist the toxicological effects of aflatoxin and develop tolerance to aflatoxin.

Claims

1. A method for improving cellular tolerance to AFB1 toxin, characterized in that, By knocking out the SERHL2 gene in cells through gene editing, the cells' tolerance to AFB1 toxin can be improved.

2. The method according to claim 1, characterized in that, The gene editing knockout refers to the targeted knockout of the SERHL2 gene using the CRISPR / Cas9 editing system.

3. The method according to claim 2, characterized in that, The sgRNA sequence of the CRISPR / Cas9 editing system is shown in SEQ ID No.

1.

4. The method according to claim 1, characterized in that, The cells in question are porcine small intestinal epithelial cells.

5. An anti-AFB1 toxin cell model, obtained by the method described in any one of claims 1-4.

6. An sgRNA having the sequence shown in SEQ ID No.

1.

7. The application of the sgRNA according to claim 6 in enhancing cellular AFB1 toxin tolerance.

8. The use of the sgRNA according to claim 6 in the preparation of a drug against AFB1 toxin.

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