Application of MDH2 knockout system or inhibitor in alleviating T-2 toxin toxicity

Abstract

The present invention discloses the application of an MDH2 knockout system or inhibitor in reducing the toxicity of T-2 toxin, and belongs to the technical field of biological preparations. The present invention constructs an IPEC-J2 cell line with MDH2 knockout by a CRISPR / Cas9 gene editing system, and finds that the cell viability of the cell line is significantly improved under the condition of a T-2 toxin concentration of 20ng / mLT-2, and the cell mortality rate is significantly reduced, and the cell has obvious resistance to T-2 toxin. Treatment of cells with the MDH2 inhibitor LW6 can also enhance the resistance of cells to T-2 toxin. It is explained that the present invention can reduce the toxicity of T-2 toxin in cells by knocking out the MDH2 gene in IPEC-J2 cells and inhibiting the activity of MDH2, improve the tolerance of cells to T-2 toxin, and provide a theoretical basis for the prevention and control of the harm of T-2 toxin to humans and animals.

Description

Technical Field

[0001] The present invention relates to the technical field of biological preparations, and in particular to the application of an MDH2 knockout system or an inhibitor in reducing the toxicity of T-2 toxin. Background Art

[0002] T-2 toxin (T-2) is a secondary metabolite of Fusarium (such as Fusarium sporeans, Fusarium pyrifolium, Fusarium graminearum and Fusarium solani), and is the most toxic type A trichothecene mycotoxin. T-2 toxin contamination covers a wide range of plants, including nuts, grains, fruits and herbs around the world. Due to its stable chemical and physical properties, T-2 toxin is difficult to completely remove from food and feed. Once ingested from contaminated feed and enters the body, it will threaten the health of animals and cause poisoning. Therefore, T-2 toxin poses a great threat to human and animal health and is one of the most dangerous mycotoxins.

[0003] The main toxicity of T-2 toxin is cytotoxicity, from which other toxic effects are derived, including genotoxicity, metabolic regulation, immunotoxicity, liver toxicity and gastrointestinal toxicity, etc. It is characterized by inhibiting the synthesis of proteins and nucleic acids, changing the cell cycle, inducing oxidative stress, apoptosis and necrosis, and can be easily absorbed by the animal body through the skin, intestines and lung mucosa. Animal experimental studies have shown that the skin, kidneys, liver, brain, hematopoietic, lymphatic, gastrointestinal, bone marrow and reproductive systems can all undergo pathological changes due to T-2 toxin infection, causing multi-organ damage. When animals ingest T-2 toxin, it will cause a variety of acute toxicological effects in livestock, including immunotoxicity, vomiting, anorexia, neuroendocrine disorders and growth retardation. Anorexia is a typical clinical symptom of acute poisoning, which can lead to decreased appetite, gastrointestinal damage and decreased immunity in livestock and poultry, and has a significant impact on the growth and development of animals and economic benefits. Therefore, the prevention and control of T-2 toxin is of great significance in the livestock and poultry breeding industry. Summary of the invention

[0004] The purpose of the present invention is to provide an MDH2 knockout system or an inhibitor for use in reducing the toxicity of T-2 toxin, so as to solve the problems existing in the above-mentioned prior art. By knocking out the MDH2 gene in IPEC-J2 cells, the tolerance of cells to T-2 toxin can be significantly improved, and the damage of T-2 toxin to cells can be reduced, thereby providing a theoretical basis for the prevention and control of the harm of T-2 toxin to the pig production industry.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides an application of an MDH2 knockout system or an inhibitor in any of the following:

[0007] (1) Application in the preparation of drugs for reducing the toxicity of T-2 toxin to pigs;

[0008] (2) Application in breeding pig breeds tolerant to T-2 toxin;

[0009] (3) Application in the preparation of drugs for improving the resistance of IPEC-J2 cells to T-2 toxin;

[0010] (4) Use in the preparation of drugs for reducing the toxicity of T-2 toxin in IPEC-J2 cells;

[0011] (5) Application in the preparation of a drug for increasing the viability of IPEC-J2 cells in T-2 toxin;

[0012] (6) Use in the preparation of drugs for reducing apoptosis of IPEC-J2 cells in T-2 toxin;

[0013] The MDH2 gene sequence is shown in SEQ ID NO:1.

[0014] Preferably, the MDH2 knockout system is a CRISPR / Cas9 gene editing system for targeted knockout of the MDH2 gene.

[0015] Preferably, the CRISPR / Cas9 gene editing system includes an sgRNA for targeted knockout of the MDH2 gene, and the DNA sequence corresponding to the sgRNA is the sense strand shown in SEQ ID NO: 3 and the antisense strand shown in SEQ ID NO: 4.

[0016] Preferably, the MDH2 inhibitor is LW6, which can inhibit the activity of MDH2 protein.

[0017] The present invention also provides a method for improving the tolerance of IPEC-J2 cells to T-2 toxin, comprising the following steps: using the CRISPR / Cas9 gene editing system to target and knock out the MDH2 gene in the IPEC-J2 cells, so that the tolerance of the IPEC-J2 cells to T-2 toxin is improved; wherein the MDH2 gene sequence is shown in SEQ ID NO: 1.

[0018] Preferably, the sgRNA for targeted knockout of the MDH2 gene in IPEC-J2 cells comprises a sense strand as shown in SEQ ID NO: 3 and an antisense strand as shown in SEQ ID NO: 4.

[0019] The present invention also provides a method for improving the tolerance of IPEC-J2 cells to T-2 toxin, comprising the step of culturing the IPEC-J2 cells with a culture medium containing LW6 to improve the tolerance of the IPEC-J2 cells to T-2 toxin.

[0020] The present invention discloses the following technical effects:

[0021] The present invention utilizes CRISPR / Cas9 gene editing system to establish IPEC-J2 cell line with knockout of target gene MDH2. Experimental results show that, compared with wild-type IPEC-J2 cell line, MDH2 mutant IPEC-J2 cell line (MDH2-KO) has significantly increased cell viability by 69.04% (***P<0.001) under 20ng / mL T-2 toxin concentration, and significantly reduced cell mortality by 45.06% (***P<0.001), and has obvious tolerance to T-2 toxin. At the same time, MDH2-KO treated with 30μM concentration of LW6 inhibitor, compared with wild-type IPEC-J2 cell line, has significantly increased cell viability by 27.05% (***P<0.001) under 20ng / mL T-2 toxin condition, and significantly reduced cell mortality by 12.40% (***P<0.001), and significantly improved tolerance to T-2 toxin. The present invention can reduce the toxicity of T-2 toxin and improve the tolerance of cells to T-2 toxin by knocking out the MDH2 gene in the IPEC-J2 cell line. In addition, the MDH2 activity of wild-type IPEC-J2 cells is inhibited by the LW6 inhibitor, which can also improve the tolerance of cells to T-2 toxin. The present invention provides a potential solution for preventing adverse reactions caused by T-2 toxin, and provides a theoretical basis for the prevention and control of the harm of T-2 toxin to the pig production industry; the present invention also provides a theoretical basis for breeding pig breeds with tolerance to T-2 toxin based on the MDH2 gene, or developing drugs with the MDH2 gene and its encoded RNA or protein as targets to improve the tolerance to T-2 toxin. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0023] Figure 1 This is the pSpCas9-2A-Puro(PX459)V2.0 vector map;

[0024] Figure 2 This is the sequencing map of the PX459 (MDH2-sgRNA) plasmid;

[0025] Figure 3 This is the peak diagram of CRISPR target site sequencing of MDH2 wild-type IPEC-J2 cell line;

[0026] Figure 4 This is the peak diagram of CRISPR target site sequencing in MDH2 mutant IPEC-J2 cell line (MDH2-KO);

[0027] Figure 5 It is a sequence alignment diagram of IPEC-J2 cell MDH2 wild-type and mutant monoclonal cell lines (MDH2-KO);

[0028] Figure 6 The effect of T-2 toxin on the cell viability of IPEC-J2 cells with wild-type and mutant MDH2 (MDH2-KO) and MDH2 inhibition group (IPEC-J2+LW6);

[0029] Figure 7 The effect of T-2 toxin on apoptosis of IPEC-J2 cells with wild-type and mutant MDH2 (MDH2-KO) and MDH2 inhibition group (IPEC-J2+LW6). DETAILED DESCRIPTION

[0030] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0031] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0032] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0033] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0034] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0035] LW6 is an inhibitor of MDH2, with CAS number 934593-90-5 and the structural formula is as follows:

[0036]

[0037] Example 1

[0038] 1. Construction of knockout vector

[0039] The DNA corresponding to the sgRNA shown in Table 1 was designed and synthesized for the exon (open reading frame ORF) of the MDH2 gene (NCBI Gene ID: 397039, CDS sequence as shown in SEQ ID NO: 1), wherein the sgRNA action site is located in the exon region of the porcine MDH2 gene (9126..9203), CACCG was added to the 5' end of the DNA sequence corresponding to the sgRNA to form a forward OligoDNA (MDH2-F), C was added to the 5' end of the reverse OligoDNA (MDH2-R), and CAAA was added to the 3' end. The reverse complementary double-stranded DNA fragment formed after annealing the forward and reverse DNA chains was cloned into Figure 1 The pSpCas9(BB)-2A-Puro(PX459)V2.0 vector shown in Figure 1 was used to construct a plasmid that simultaneously expresses sgRNA and Cas9 protein. The constructed vector was sent to Qingke and sequenced using the U6 promoter. The sequencing results are shown in Figure 1. Figure 2 As shown, the MDH2 gene vector was successfully constructed.

[0040] The specific steps are as follows:

[0041] Table 1 sgRNA sequences

[0042] name Sequence number Sequence (5'-3') MDH2-F SEQ ID NO: 3 CACCGCCCGTCATTGGCGGCCACGC MDH2-R SEQ ID NO: 4 CGGGCAGTAACCGCCGGTGCGCAAA

[0043] The positive-strand Oligo DNA (MDH2-F) and the negative-strand Oligo DNA (MDH2-R) were annealed to form dsDNA.

[0044] The reaction system is shown in Table 2:

[0045] Table 2 Reaction system

[0046]

[0047] The above reaction system was mixed, centrifuged and placed in a PCR reactor according to the following reaction program: 37°C for 30 min; 95°C for 5 min, gradient down to 25°C, 5°C / min.

[0048] The dsDNA formed after annealing was connected to the pSpCas9-2A-Puro(PX459)V2.0 vector. The connection system is shown in Table 3 below.

[0049] Table 3 Connection system

[0050]

[0051] The above reaction system was placed in a PCR reactor and reacted according to the following reaction program: 37°C for 5 min; 23°C for 5 min, 25 cycles.

[0052] 2. Transformation and screening of PX459 (MDH2-sgRNA) positive clones

[0053] Take 2 μL of the above ligation product and add it to the competent cells, place it on ice for 30 minutes; heat shock in a 42°C water bath for 90 seconds; cool it on ice for 5 minutes, add 500 μL of LB liquid culture medium without ampicillin (Amp) to the tube, mix well and culture at 37°C for 1 hour to restore the bacteria to normal growth state and express the Amp resistance gene encoded by the plasmid; take the above 100 μL of bacterial solution and evenly spread it on the LB solid culture plate containing Amp, place it on the front until the bacterial solution is completely absorbed by the culture medium, and invert the culture dish and culture it at 37°C for 16 hours.

[0054] Use a sterilized pipette to pick up several single colonies, inoculate them into 1 mL of LB liquid medium containing Amp, and culture them at 37°C and 200 rpm for 2-3 hours; design primers, identify positive clones by PCR, and send them to Qingke for sequencing using the U6 promoter. PCR primers are shown in Table 4 below.

[0055] Table 4 Primers

[0056] name Sequence number Sequence (5'-3') ZT-MDH2-F SEQ ID NO: 5 GATACAAGGCTGTTAGAGAGATAATT ZT-MDH2-R SEQ ID NO: 6 AAACGGGCAGTAACCGCCGGTGCGC

[0057] 3. Construction of MDH2 gene-deficient cell lines

[0058] Transient transfection was performed using an electroporator to transfer the knockout vector constructed above into the cell nucleus. Complete medium containing 4 μg / mL puromycin was used for continuous screening for 7 days, and fresh complete medium containing puromycin was replaced every two days. Subsequently, monoclonal dilution was performed, and the obtained monoclonal cells were transferred from the 96-well plate to a 24-well plate for further expansion and culture. Genomic DNA of different monoclonal cells was extracted, primers were designed for the sgRNA region, and high-fidelity PCR was performed. The PCR products were sent to Bioengineering for sequencing.

[0059] The peak diagram of CRISPR target site sequencing in MDH2 wild-type IPEC-J2 cell line is shown in Figure 3As shown; the peak diagram of CRISPR target site sequencing of MDH2 mutant IPEC-J2 cell line (MDH2-KO) is shown Figure 4 As shown; Sequence comparison of IPEC-J2 cell MDH2 wild-type and mutant monoclonal cell lines (MDH2-KO) is shown in Figure 5 The sequencing results show that the target sequence of the MDH2 gene mutant IPEC-J2 cell line of the present invention is missing. Compared with the wild-type IPEC-J2 cell line, it lacks 5 consecutive bases of "GGCCA", so a frameshift mutation occurs, thereby achieving MDH2 gene knockout.

[0060] 4. Cell viability assessment

[0061] Normal IPEC-J2 cells and MDH2-KO cell lines were cultured at 2×10 4 The cells were inoculated at a density of 100 μM in a 96-well culture plate and cultured overnight. On the second day, the cells in the IPEC-J2 group and the MDH2-KO group were replaced with a cell growth medium containing T-2 toxin in each well. For some normal IPEC-J2 cells, a cell growth medium containing 30 μM LW6 inhibitor was added in advance for pretreatment for 2 hours, and then T-2 toxin was added to serve as the MDH2 inhibition group (IPEC-J2+LW6). The liquid in the above wells of the plate was fully mixed and cultured in a cell culture incubator at 37°C and 5% CO2 for 72 hours.

[0062] Cell viability was determined using CCK8 reagent. Five wells without cells were selected from a 96-well plate and 100 μL of DMEM / F12 complete medium was added to each well to obtain background luminescence values. 10 μL of CCK8 solution was added to each well with liquid in the 96-well plate, the culture plate was incubated in a 37°C cell culture incubator for 1 hour, and the absorbance at 450 nm was measured using a microplate reader.

[0063] Cell viability % = (luminescence value of the treatment group - background luminescence value) / (luminescence value of the control group - background luminescence value).

[0064] The results are as follows Figure 6 As shown, compared with the control group, the cell viability of the MDH2 gene mutant (MDH2-KO) was extremely significantly increased by 69.04% (***P<0.001) under the condition of 20 ng / mL T-2 toxin concentration; compared with the control group, the cell viability of the MDH2 inhibition group treated with 30 μM concentration of LW6 inhibitor was extremely significantly increased by 27.05% (***P<0.001) under the condition of 20 ng / mL T-2 toxin.

[0065] 5. Identification of cell apoptosis rate

[0066] Normal IPEC-J2 cells and MDH2-mutant (MDH2-KO) IPEC-J2 cells were cultured at 6.25×10 5 The cells were inoculated at a density of 100 μM in a 6-well culture plate and cultured overnight. On the second day, the cells in the IPEC-J2 group and the MDH2-KO group were replaced with a cell growth medium containing T-2 toxin in each well. For some normal IPEC-J2 cells, a cell growth medium containing 30 μM LW6 inhibitor was added in advance for pretreatment for 2 hours, and then T-2 toxin was added to serve as the MDH2 inhibition group (IPEC-J2+LW6). The liquid in the above wells of the plate was fully mixed and cultured in a cell culture incubator at 37°C and 5% CO2 for 72 hours.

[0067] The cells were detached by digestion with 0.25% trypsin without EDTA and washed twice with phosphate buffered saline (PBS). The cell pellet was resuspended in binding buffer containing Annexin V-FITC and PI staining solution. The cells were incubated at room temperature in the dark for 20 minutes and then analyzed on a flow cytometer using the fluorescence (FITC) green channel and (PI) red fluorescence channel.

[0068] The results are as follows Figure 7 As shown, the cell death rate of the MDH2 gene mutant (MDH2-KO) IPEC-J2 cell line was extremely significantly reduced by 45.06% (***P<0.001) under the condition of 20 ng / mL T-2 toxin concentration compared with the control group; the cell death rate of the MDH2 inhibition group treated with 30 μM concentration of LW6 inhibitor was extremely significantly reduced by 12.40% (***P<0.001) under the condition of 20 ng / mL T-2 toxin compared with the control group.

[0069] CDS sequence of IPEC-J2 cell MDH2 gene (SEQ ID NO: 1):

[0070]

[0071] Amino acid sequence of IPEC-J2 cell MDH2 gene (SEQ ID NO: 2):

[0072] MLSALARPAGAALRRSFSTSAQNNAKVAVLGASGGIGQPLSLLLKNSPLVSRLTLYDIAHTPGVAADLSHIETRATVKGYLGPEQLPDCLKGCDVVVIPAGVPRKPGMTRDDLFNTNATIVATLTAACAQHCPDAMICIISNPVNSTIPITAEVFKKHGVYNPNKIFGVT TLDIVRANAFVAELKGLDPARVSVPVIGGHAGKTIIPLISQCTPKVDFPQDQLSTHTGRIQEAGTEVVKAKAGAGSATLSMAYAGARFVFSLVDAMNGKEGVVECSFVKSQETDCPYFSTPLLLGKKGIEKNLGIGKISPFEEKMIAEAIPELKASIKKGEEFVKNMK*.

[0073] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. Use of MDH2 knockout system or inhibitor in any of the following: (1) Application in the preparation of drugs for improving the tolerance of IPEC-J2 cells to T-2 toxin; (2) Use in the preparation of drugs for reducing the toxicity of T-2 toxin in IPEC-J2 cells; (3) Application in the preparation of a drug for increasing the viability of IPEC-J2 cells in T-2 toxin; (4) Application in the preparation of drugs for reducing apoptosis of IPEC-J2 cells in T-2 toxin; The MDH2 gene sequence is shown in SEQ ID NO: 1; The knockout system of the MDH2 gene is a CRISPR / Cas9 gene editing system that targets and knocks out the MDH2 gene; The CRISPR / Cas9 gene editing system includes an sgRNA that targets and knocks out the MDH2 gene, and the DNA sequence corresponding to the sgRNA is a sense strand as shown in SEQ ID NO: 3 and an antisense strand as shown in SEQ ID NO: 4; The MDH2 inhibitor is LW6.

2. A method for improving the tolerance of IPEC-J2 cells to T-2 toxin, characterized in that: The following steps are involved: The MDH2 gene in the IPEC-J2 cells is targetedly knocked out using the CRISPR / Cas9 gene editing system, so that the tolerance of the IPEC-J2 cells to T-2 toxin is improved; wherein the MDH2 gene sequence is shown in SEQ ID NO: 1; The sgRNA used for targeted knockout of the MDH2 gene in IPEC-J2 cells is the sense strand shown in SEQ ID NO: 3 and the antisense strand shown in SEQ ID NO:

4.

3. A method for improving the tolerance of IPEC-J2 cells to T-2 toxin, characterized in that: The method comprises the step of culturing the IPEC-J2 cells with a culture medium containing LW6 to improve the tolerance of the IPEC-J2 cells to T-2 toxin.

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