Application of MDH2 gene and inhibitor thereof in resisting ZEA toxin

Abstract

The invention discloses an application of an MDH2 gene and an inhibitor thereof in resisting ZEA toxin, and relates to the technical field of biology. An MDH2 gene deletion cell line is obtained through CRISPR gene editing, and compared with a wild type MDH2 gene expression cell, under the influence of ZEA, the cell activity of the MDH2 gene deletion cell line is remarkably improved, and the cell apoptosis rate is remarkably reduced. In addition, the LW6 inhibitor is used for inhibiting the MDH2 activity of wild type IPEC-J2 cells, and the tolerance of the cells to ZEA can also be improved. Therefore, through knockout, mutation or activity inhibition of the MDH2 gene, the ZEA resistance of people, animals and plants can be improved. The invention provides a potential solution way for solving untoward effects caused by ZEA (Zinc Ethylenediamine).

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to application of MDH2 gene and its inhibitor in resisting ZEA toxin. Background Art

[0002] Mycotoxin contamination is an environmental problem that cannot be ignored, especially in some humid, temperate regions, which provide good conditions for the growth of fungi. It is estimated that about 25% of crops worldwide are contaminated with mycotoxins. Zearalenone (ZEA), with a chemical formula of C 18 H 22 O5 is a non-steroidal estrogenic mycotoxin produced mainly by Fusarium species such as Rose Fusarium, Fusarium graminearum and Fusarium tricyclicum through secondary metabolic pathways. It is widely found in cereals such as wheat, corn, sorghum, barley, soybeans and dairy products. ZEA contamination of crops is a serious problem worldwide. As one of the most polluting mycotoxins, ZEA has caused huge economic losses. In addition to the significant economic losses of crops, mycotoxins also frequently contaminate food and animal feed. In the list of carcinogens published by the World Health Organization's International Agency for Research on Cancer in 2017, toxins (zearalenone, deoxynivalenol, zearalenol and fusarone X) derived from Fusarium graminearum, Fusarium culmorum and Fusarium cruzi were listed.

[0003] Studies have shown that ZEA has strong mutagenicity, teratogenicity, neurotoxicity, reproductive toxicity and carcinogenicity in animals and humans, and can cause hepatotoxicity, immunotoxicity, hematotoxicity and genotoxicity. ZEA can induce oxidative stress, mitochondrial damage and apoptosis, leading to cell death. It is cytotoxic by inhibiting cell viability and apoptosis, and induces stress response in different cultured cell lines. Oxidative damage is the main cause of cell damage and cell death. It can also produce reactive oxygen species (ROS) in mammals, inhibit the activity and expression of antioxidant enzymes, and lead to apoptosis.

[0004] ZEA has estrogen effects, mainly acting on the reproductive system, competing with the 17-β estrogen receptor in humans and animals, causing hyperestrogenism in livestock and experimental mice, triggering a series of reproductive toxicity, genetic toxicity, carcinogenic toxicity and immunotoxicity, and can cause miscarriage, stillbirth and malformation in pregnant animals or humans who ingest ZEA. Studies have shown that among different livestock, pigs are highly sensitive to the effects of ZEA through contaminated feed. Corn is the main energy feed in pig diets and is very susceptible to contamination by Fusarium toxins. Therefore, the harm of ZEA has a significant impact on the health and economic benefits of livestock and poultry and human food safety. It is necessary to develop drugs to improve tolerance to ZEA. Summary of the invention

[0005] The purpose of the present invention is to provide an application of the MDH2 gene and its inhibitor in resistance to ZEA toxin to solve the problems existing in the above-mentioned prior art. The present invention has found that knocking out, mutating or down-regulating the expression of the MDH2 gene can improve the ability of humans, animals and plants to resist ZEA. The present invention provides a potential solution to the adverse reactions caused by ZEA.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides the use of an MDH2 inhibitor in the preparation of a medicine for improving the tolerance of an organism to the toxicity of zearalenone.

[0008] Furthermore, the organism is a pig; the CDS sequence of the MDH2 gene is shown as SEQ ID NO.1.

[0009] Furthermore, the MDH2 inhibitor is LW6.

[0010] Furthermore, the MDH2 inhibitor is a CRISPR / Cas9 gene editing system for knocking out the MDH2 gene.

[0011] The present invention also provides the use of an MDH2 inhibitor in breeding a pig breed tolerant to zearalenone toxicity, wherein the MDH2 inhibitor is a CRISPR / Cas9 gene editing system for knocking out the MDH2 gene;

[0012] The CDS sequence of the MDH2 gene is shown in SEQ ID NO.1.

[0013] The present invention also provides the use of the MDH2 inhibitor in improving the tolerance of IPEC-J2 cells to the toxicity of zearalenone.

[0014] Furthermore, the MDH2 inhibitor is LW6.

[0015] Furthermore, the MDH2 inhibitor is a CRISPR / Cas9 gene editing system for knocking out the MDH2 gene.

[0016] The present invention also provides a method for improving the tolerance of IPEC-J2 cells to the toxicity of zearalenone, comprising the steps described in (1) or (2):

[0017] (1) knocking out the MDH2 gene of the IPEC-J2 cell; the CDS sequence of the MDH2 gene is shown in SEQ ID NO.1;

[0018] (2) The IPEC-J2 cells are cultured using a culture medium containing LW6.

[0019] Furthermore, the MDH2 gene was knocked out using the CRISPR / Cas9 gene editing system.

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

[0021] The present invention obtains a MDH2 gene-deficient cell line by CRISPR gene editing. Compared with wild-type MDH2 gene-expressing cells, under the influence of ZEA, the cell viability is significantly improved and the cell apoptosis rate is significantly reduced. In addition, the use of LW6 inhibitor to inhibit the MDH2 activity of wild-type IPEC-J2 cells can also improve the tolerance of cells to ZEA. It can be seen that knocking out, mutating or inhibiting the activity of the MDH2 gene can improve the ability of humans, animals and plants to resist ZEA. The present invention provides a potential solution to the adverse reactions caused by ZEA. 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 the IPEC-J2 cell MDH2 wild-type and mutant monoclonal cell lines (MDH2-KO);

[0028] Figure 6 Statistical graph of cell viability test results for different experimental groups;

[0029] Figure 7 Statistical graph of cell apoptosis rates in different experimental groups. 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] Terminology Note:

[0036] MDH2 gene refers to the gene for malate dehydrogenase 2;

[0037] LW6 is an inhibitor of MDH2, with CAS number 934593-90-5 and the structural formula is as follows:

[0038]

[0039] Example 1

[0040] 1. Experimental Methods

[0041] 1.1 Construction of knockout vector

[0042] The DNA sequence of sgRNA was designed and synthesized for the exon (open reading frame ORF) of the MDH2 gene (NCBI Gene ID: 397039), where the sgRNA action site was located in the exon region (9126..9203) of the porcine MDH2 gene. CACCG was added to the 5' end of the DNA sequence of the sgRNA to form a forward oligo DNA (MDH2-F), and C was added to the 5' end and CAAA was added to the 3' end of the reverse oligo DNA (MDH2-R). The reverse complementary double-stranded DNA fragment formed after annealing of the forward and reverse DNA chains was cloned into Figure 1 In the pSpCas9(BB)-2A-Puro(PX459)V2.0 vector shown, a plasmid that simultaneously expresses sgRNA and Cas9 protein was constructed, and the constructed vector was sent to Qingke for sequencing using the U6 promoter.

[0043] The specific steps are as follows:

[0044] Table 1 Forward and reverse DNA strands of sgRNA

[0045] name DNA sequence (5'-3') Sequence number MDH2-F CACCGCCCGTCATTGGCGGCCACGC SEQ ID NO.3 MDH2-R CGGGCAGTAACCGCCGGTGCGCAAA SEQ ID NO.4

[0046] (1) The forward oligo DNA (MDH2-F) and reverse oligo DNA (MDH2-R) were annealed to form dsDNA. The reaction system is shown in Table 2.

[0047] Table 2 Annealing reaction system

[0048] Reagents Addition amount Reverse Oligo (100μM) 1μL Forward Oligo (100μM) 1μL 10×T4 Ligation Buffer 1μL <![CDATA[H2O]]> 6.5μL T4PNK (10U / μL) 0.5μL Total 10μL

[0049] Mix the reaction system in Table 2, centrifuge it and place it in a PCR reaction instrument, and react according to the following reaction program: 37°C for 30 min; 95°C for 5 min, and then decrease the gradient to 25°C at 5°C / min.

[0050] (2) The dsDNA formed after annealing was connected to the pSpCas9-2A-Puro (PX459) V2.0 vector. The connection system is shown in Table 3.

[0051] Table 3 Connection system

[0052]

[0053]

[0054] Place the ligation system in Table 3 in a PCR reaction instrument and react according to the following reaction program: 37°C for 5 min; 23°C for 5 min, 25 cycles.

[0055] (3) Transformation and screening of PX459 (MDH2-sgRNA) positive clones

[0056] Take 2 μL of the ligation product of step (2) above, add it to the competent cells, and place it on ice for 30 minutes; heat shock it 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 Amp to the tube, mix it well, and culture it 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 face up 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.

[0057] Pick several single colonies with a sterilized pipette tip and inoculate them into 1 mL of LB liquid medium containing Amp, and culture them at 37°C and 200 rpm for 2 hours; design primers, identify positive clones by PCR, and send them to Qingke for sequencing using the U6 promoter. The sequencing map of PX459 (MDH2-sgRNA) plasmid is shown in Figure 2 PCR primers are shown in Table 4 below.

[0058] Table 4 PCR identification primer sequences

[0059] name DNA sequence (5'-3') Sequence number ZT-MDH2-F GATACAAGGCTGTTAGAGAGATAATT SEQ ID NO.5 ZT-MDH2-R AAACGGGCAGTAACCGCCGGTGCGC SEQ ID NO.6

[0060] 1.2 Construction of MDH2 gene-deficient cell lines

[0061] Transient transfection was performed using an electroporator, and the knockout vector PX459 (MDH2-sgRNA) constructed above was transferred into IPEC-J2 cells. The 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 the 24-well plate for further expansion and culture. The 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 Sangon for sequencing. IPEC-J2 cells with MDH2 gene mutations were named MDH2-KO.

[0062] The CRISPR target site sequencing peaks of the MDH2 wild-type IPEC-J2 cell line and the MDH2 mutant IPEC-J2 cell line (MDH2-KO) are shown in Figure 2. Figure 3 and Figure 4 , the sequence alignment of the two cells is shown in Figure 5 .

[0063] 1.3 Cell viability assessment

[0064] The experiment was divided into control group (IPEC-J2), MDH2 inhibition group (IPEC-J2+LW6) and MDH2 gene mutant group (MDH2-KO). Normal IPEC-J2 cells and MDH2 gene mutant (MDH2-KO) cell lines were cultured at 2×10 4 The cells were inoculated at a density of 100 μg / mL in a 96-well culture plate and cultured overnight. The next day, the cells in the IPEC-J2 group and the MDH2-KO group were replaced with a cell growth medium containing 40 μg / mL ZEA in each well. One part of the normal IPEC-J2 cells was used as the control group, and the other part of the normal IPEC-J2 cells was pretreated with a cell growth medium containing a 30 μM concentration of LW6 inhibitor for 2 hours in advance, and then ZEA (final concentration of 40 μg / mL) was added 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 48 hours.

[0065] Cell viability was determined using CCK8 reagent. Five cell-free wells 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, and 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.

[0066] Cell viability (%) = (luminescence value of the treatment group - background luminescence value) / (luminescence value of the control group - background luminescence value) × 100%.

[0067] 1.4 Identification of cell apoptosis rate

[0068] The experiment was divided into control group (IPEC-J2), MDH2 inhibition group (IPEC-J2+LW6) and MDH2 gene mutant group (MDH2-KO). Normal IPEC-J2 cells and MDH2 gene mutant (MDH2-KO) cell lines were cultured at 6.25×10 5 The cells were inoculated at a density of 100 μg / mL in a 6-well culture plate and cultured overnight. The next day, the cells in the IPEC-J2 group and the MDH2-KO group were replaced with a cell growth medium containing 40 μg / mL ZEA in each well. One part of the normal IPEC-J2 cells was used as the control group, and the other part of the normal IPEC-J2 cells was pretreated with a cell growth medium containing a 30 μM concentration of LW6 inhibitor for 2 hours in advance, and then ZEA (final concentration of 40 μg / mL) was added 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 48 hours.

[0069] 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.

[0070] 2. Results

[0071] The present invention utilizes the CRISPR / Cas9 gene editing system to establish an IPEC-J2 cell line with a target gene knockout. The DNA sequence of sgRNA is designed for the target gene MDH2, and the plasmid pSpCas9(BB)-2A-Puro(pX459) is used as a vector. The annealed double-stranded DNA fragment is connected to the linearized pX459 vector to obtain a knockout vector. The constructed vector is delivered to IPEC-J2 cells by transient transfection, and a monoclonal IPEC-J2 cell line with a MDH2 gene mutation is obtained by puromycin pressure screening, and its tolerance to ZEA is studied. Figure 6 and Figure 7 As shown in Figure 2, compared with the control group, the cell viability of the MDH2-KO group was significantly increased by 26.99% under the condition of 40 μg / mL ZEA concentration ( *** P<0.001, the apoptosis rate was significantly reduced by 22.53% ( *** P<0.001), and showed significant tolerance to ZEA. In addition, the cell viability of the MDH2 inhibition group treated with 30 μM LW6 inhibitor was significantly increased by 8.17% under 40 μg / mL ZEA conditions compared with the control group ( ** P<0.01, the apoptosis rate was significantly reduced by 7.90% ( * P<0.05), and the tolerance to ZEA was significantly improved.

[0072] CDS sequence of IPEC-J2 cell MDH2 gene (SEQ ID NO.1):

[0073]

[0074] Amino acid sequence of IPEC-J2 cell MDH2 protein (SEQ ID NO.2):

[0075] MLSALARPAGAALRRSFSTSAQNNAKVAVLGASGGIGQPLSLLLKNSPLVSRLTLYDIAHTPGVAADLSHIETRATVKGYLGPEQLPDCLKGCDVVVIPAGVPRKPGMTRDDLFNTNATIVATLTAACAQHCPDAMICIISNPVNSTIPITAEVFKKHGVYNPNKIFGV TTLDIVRANAFVAELKGLDPARVSVPVIGGHAGKTIIPLISQCTPKVDFPQDQLSTHTGRIQEAGTEVVKAKAGAGSATLSMAYAGARFVFSLVDAMNGKEGVVECSFVKSQETDCPYFSTPLLLGKKGIEKNLGIGKISPFEEKMIAEAIPELKASIKKGEEFVKNMK.

[0076] 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. The use of MDH2 inhibitors in the preparation of drugs for improving the tolerance of organisms to the toxicity of zearalenone.

2. The use according to claim 1, characterized in that: The organism is a pig; the CDS sequence of the MDH2 gene is shown as SEQ ID NO.

1.

3. The use according to claim 1, characterized in that: The MDH2 inhibitor is LW6.

4. The use according to claim 1, characterized in that: The MDH2 inhibitor is a CRISPR / Cas9 gene editing system for knocking out the MDH2 gene.

5. The use of an MDH2 inhibitor in breeding pig breeds tolerant to zearalenone toxicity, characterized in that: The MDH2 inhibitor is a CRISPR / Cas9 gene editing system for knocking out the MDH2 gene; The CDS sequence of the MDH2 gene is shown in SEQ ID NO.

1.

6. Application of MDH2 inhibitors in improving the tolerance of IPEC-J2 cells to zearalenone toxicity.

7. The use according to claim 6, characterized in that: The MDH2 inhibitor is LW6.

8. The use according to claim 6, characterized in that: The MDH2 inhibitor is a CRISPR / Cas9 gene editing system for knocking out the MDH2 gene.

9. A method for improving the tolerance of IPEC-J2 cells to zearalenone toxicity, characterized in that: The method comprises the steps of (1) or (2) below: (1) knocking out the MDH2 gene of the IPEC-J2 cell; the CDS sequence of the MDH2 gene is shown in SEQ ID NO.1; (2) The IPEC-J2 cells are cultured using a culture medium containing LW6.

10. The method according to claim 9, characterized in that The MDH2 gene was knocked out using the CRISPR / Cas9 gene editing system.

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