Application of MDH2 inhibitor in the preparation of preparations for reducing fumonisin toxicity

Abstract

The present invention discloses the application of an MDH2 inhibitor in the preparation of a preparation for reducing the toxicity of fumonisin, and belongs to the field of biotechnology. The present invention performs a fumonisin treatment experiment on cells with MDH2 gene mutants and cells with wild-type MDH2 genes. It is found that the cell viability of cells with MDH2 gene mutants is significantly improved under the condition of 100 μg / mL fumonisin concentration compared with cells expressing wild-type MDH2 genes, which can be increased by 15.2%, and the cell apoptosis rate is significantly reduced, which can be reduced by 7.76%, indicating that the MDH2 gene mutation can improve the tolerance to fumonisin. In addition, the inhibition of the MDH2 activity of wild-type IPEC‑J2 cells with the LW6 inhibitor can also improve the tolerance of cells to fumonisin. The present invention provides a potential solution for preventing adverse reactions caused by fumonisin, and provides a theoretical basis for the prevention and control of FB1 hazards to food and animal husbandry.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to application of an MDH2 inhibitor in preparing a preparation for reducing the toxicity of fumonisin. Background Art

[0002] Fumonisin B1 (FB1) is a type of mycotoxin produced by fungi such as Fusarium moniliforme, Fusarium verticillioides and Fusarium solani. It is a water-soluble metabolite. FB1 is composed of different polyhydroxy alcohols and malonic acid, with a similar diester structure. FB1 is widely distributed in soil and in the organisms of plants and animals, especially in crops such as corn, sorghum and wheat, causing crops to wilt or rot and contaminating various grains and their products.

[0003] FB1 can seriously endanger human and animal health through the food chain, and has harmful effects such as neurotoxicity, organ toxicity, immunotoxicity and carcinogenicity. Due to the global distribution of FB1 and its harm to humans and animals, international research institutions have classified it as a Class 2B hazard. Studies have shown that FB1 can damage the function of intestinal antigen-presenting cells, reduce the stimulatory function of T lymphocytes, and significantly increase the expression of IL-1β, IL-6 and TNF-α genes, indicating that FB1 can affect the body's immune function. Acute FB1 exposure can cause excessive brain excitement, increase the mitochondrial membrane potential of the cerebral cortex, affect the neurodevelopment of animals, and have a wide range of neurotoxicity. In addition, FB1 also shows toxic effects on organs and tissues such as the liver, kidneys and intestines. High doses or long-term intake of FB1 may even induce cancer. The liver is the main target organ of FB1, and liver toxicity has also become one of the most significant features of FB1 hazards.

[0004] In view of the significant impact of FB1 on the health of livestock and poultry, economic benefits and food safety, how to solve the adverse reactions caused by FB1 on the health of livestock and poultry and human body is an urgent problem to be solved. Summary of the invention

[0005] The purpose of the present invention is to provide an application of an MDH2 inhibitor in the preparation of a preparation for reducing the toxicity of fumonisin, so as to solve the problems existing in the above-mentioned prior art. The present invention provides a potential solution for preventing adverse reactions caused by fumonisin, and provides a theoretical basis for the prevention and control of FB1 hazards to food and animal husbandry.

[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 preparing a preparation for reducing the toxicity of fumonisin.

[0008] The present invention also provides the use of the MDH2 inhibitor in the preparation of a drug for improving the toxicity tolerance of fumonisin.

[0009] The present invention also provides the use of the MDH2 inhibitor in breeding plant varieties or animal varieties tolerant to fumonisin.

[0010] Furthermore, the MDH2 inhibitor includes a CRISPR / Cas9 system for knocking out or knocking down the MDH2 gene; the CRISPR / Cas9 system includes sgRNA, and the DNA sequence for synthesizing the sgRNA includes the sequence shown in SEQ ID NO.4 and the sequence shown in SEQ ID NO.5.

[0011] Furthermore, the CDS sequence of the MDH2 gene is shown in SEQ ID NO.2.

[0012] Furthermore, the amino acid sequence encoded by the MDH2 gene is shown in SEQ ID NO.3.

[0013] Furthermore, the MDH2 inhibitor includes an LW6 inhibitor.

[0014] The present invention also provides a method for improving the tolerance of IPEC-J2 cells to fumonisin toxicity, comprising knocking out the MDH2 gene of the IPEC-J2 cells or culturing the IPEC-J2 cells using a culture medium containing LW6; the CDS sequence of the MDH2 gene is shown in SEQ ID NO.2.

[0015] The present invention discloses the following technical effects:

[0016] The present invention conducts a fumonisin treatment experiment on cells with suppressed MDH2 gene expression and wild-type MDH2 gene, and the results show that the cell viability of the cells with suppressed MDH2 gene expression is significantly improved by 15.2% under the condition of 100 μg / mL fumonisin concentration, and the cell apoptosis rate is significantly reduced by 7.76%, indicating that inhibiting MDH2 gene expression can improve tolerance to fumonisin.

[0017] The present invention provides a potential solution for preventing adverse reactions caused by fumonisin and provides a theoretical basis for preventing and controlling the harm of FB1 to food and animal husbandry. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] 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.

[0019] Figure 1 This is the pSpCas9-2A-Puro(PX459)V2.0 vector map;

[0020] Figure 2 This is the sequencing map of the PX459 (MDH2-sgRNA) plasmid;

[0021] Figure 3 This is the peak diagram of CRISPR target site sequencing in MDH2 mutant IPEC-J2 cell line (MDH2-KO);

[0022] Figure 4 This is the peak diagram of CRISPR target site sequencing of MDH2 wild-type IPEC-J2 cell line;

[0023] Figure 5 It is a sequence alignment diagram of IPEC-J2 cell MDH2 wild-type and mutant monoclonal cell lines (MDH2-KO);

[0024] Figure 6 Effects of FB1 on cell viability of IPEC-J2 cells with wild-type and mutant MDH2 (MDH2-KO) and MDH2 inhibition group (IPEC-J2+LW6);

[0025] Figure 7 The effect of FB1 on apoptosis of IPEC-J2 cells with wild-type and mutant MDH2 (MDH2-KO) and MDH2 inhibition group (IPEC-J2+LW6). DETAILED DESCRIPTION

[0026] 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.

[0027] 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.

[0028] 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.

[0029] 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.

[0030] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0031] In the embodiment of the present invention, the DNA sequence of the MDH2 gene of the IPEC-J2 cell is shown as SEQ ID NO.1, the CDS sequence of the MDH2 gene of the IPEC-J2 cell is shown as SEQ ID NO.2, and the amino acid sequence encoded by it is shown as SEQ ID NO.3.

[0032] SEQ ID NO.1:

[0033]

[0034] SEQ ID NO.2:

[0035]

[0036] SEQ ID NO.3:

[0037] MLSALARPAGAALRRSFSTSAQNNAKVAVLGASGGIGQPLSLLLKNSPLVSRLTLYDIAHTPGVAADLSHIETRATVKGYLGPEQLPDCLKGCDVVVIPAGVPRKPGMTRDDLFNTNATIVATLTAACAQHCPDAMICIISNPVNSTIPITAEVFKKHGVYNPNKIFGV TTLDIVRANAFVAELKGLDPARVSVPVIGGHAGKTIIPLISQCTPKVDFPQDQLSTHTGRIQEAGTEVVKAKAGAGSATLSMAYAGARFVFSLVDAMNGKEGVVECSFVKSQETDCPYFSTPLLLGKKGIEKNLGIGKISPFEEKMIAEAIPELKASIKKGEEFVKNMK.

[0038] Example

[0039] 1. Construction of knockout vector

[0040] 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 of the porcine MDH2 gene (9126..9203). 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 forward and reverse DNA chains were annealed to form a reverse complementary double-stranded DNA fragment, and the reverse complementary double-stranded DNA fragment was cloned into the following 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.

[0041] The specific steps are as follows:

[0042] Table 1 Forward and reverse oligo DNA and sequences

[0043] name Sequence (5'-3') MDH2-F (SEQ ID NO.4) CACCGCCCGTCATTGGCGGCCACGC MDH2-R (SEQ ID NO.5) CGGGCAGTAACCGCCGGTGCGCAAA

[0044] The positive strand Oligo DNA (MDH2-F) and the negative strand Oligo DNA (MDH2-R) in Table 1 were annealed to form dsDNA. The reaction system is shown in Table 2:

[0045] Table 2 Annealing reaction system

[0046] Reverse Oligo (100μM) 1μL (final concentration 10μM) Forward Oligo (100μM) 1μL (final concentration 10μM) 10×T4 Ligation Buffer 1μL <![CDATA[H2O]]> 6.5μL T4PNK (10U / μL) 0.5μL (final concentration 0.5U / μL) Total 10μL

[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:

[0049] Table 3 Connection system

[0050] PX459 plasmid template 25ng Annealed dsDNA 1μL 10×T4 Ligation Buffer 1μL <![CDATA[H2O]]> XμL BbsI (5U / μL) 0.5μL(2.5U) T4 ligase (400U / μL) 0.5μL(200U) Total 10μL

[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 E. coli cells, place on ice for 30 minutes; heat shock in a 42°C water bath for 90 seconds; cool on ice for 5 minutes, add 500 μL of LB liquid culture medium without ampicillin (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 PCR primers

[0056]

[0057]

[0058] The sequencing results of positive clones containing PX459 (MDH2-sgRNA) plasmid are as follows Figure 2 shown.

[0059] 3. Construction of MDH2 gene-deficient cell lines

[0060] Transient transfection was performed using an electroporator to transfer the knockout vector constructed above into the nucleus of IPEC-J2 cells. Screening was performed continuously for 7 days with complete medium containing 4 μg / mL puromycin, 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 to obtain the MDH2 gene mutant IPEC-J2 cell line (MDH2-KO). The genomic DNA of the wild-type and mutant IPEC-J2 cells of the MDH2 gene were extracted respectively, and primers (F: TGAAAGGCTACCTCGGACCT, SEQ ID NO.8; R: CATCCCGTGTCATGCCTGG, SEQ ID NO.9) were designed for the sgRNA region, and high-fidelity PCR was performed, and the PCR products were sent to Sangon for sequencing. The peak diagram of CRISPR target site sequencing of the MDH2 gene mutant (MDH2-KO) IPEC-J2 cell line is shown in the figure. Figure 3 As shown, the corresponding sequencing peak diagram of the MDH2 wild-type cell line is as follows Figure 4 , the sequences of wild-type and mutant monoclonal cell lines (MDH2-KO) of IPEC-J2 cells were compared, and the results were as follows Figure 5 ,Depend on Figure 5 It can be seen that in the IPEC-J2 cell MDH2 mutant monoclonal cell line, there is a deletion of 5 bases "GGCCA" in the gene sequence, proving that the MDH2 gene knockout was successful.

[0061] 4. Cell viability assessment

[0062] Normal IPEC-J2 cells and MDH2-mutant (MDH2-KO) cell lines were cultured at 2×10 4 Inoculate 96-well culture plates at a density of 100 μg / mL and culture overnight. The next day, the cells in the normal group (IPEC-J2) and MDH2-KO group were replaced with cell growth medium containing 100 μg / mL FB1 in each well. For some normal IPEC-J2 cells, add cell growth medium containing 30 μM concentration of LW6 inhibitor for pretreatment for 2 hours in advance, and then add FB1 to serve as the MDH2 inhibition group (IPEC-J2+LW6). Thoroughly mix the liquid in the above wells in the plate and culture in a cell culture incubator at 37°C and 5% CO2 for 72 hours.

[0063] 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 placed in a 37°C cell culture incubator for 1 h. The absorbance at 450 nm was measured using a microplate reader, and the cell viability was calculated.

[0064] Cell viability % = (luminescence value of treatment group - background luminescence value) / (luminescence value of control group - background luminescence value) × 100

[0065] The results are as follows Figure 6 As shown in the figure, before FB1 treatment, there was no significant difference in cell viability among the groups; after FB1 treatment, the cell viability of each group decreased, but compared with the normal group, under the condition of 100 μg / mL FB1 concentration, the cell viability of the MDH2-KO group was extremely significantly increased by 15.20% (***P<0.001), and the cell viability of the IPEC-J2+LW6 group was extremely significantly increased by 7.19% (**P<0.01).

[0066] 5. Identification of cell apoptosis rate

[0067] Normal IPEC-J2 cells and MDH2-mutant (MDH2-KO) cell lines were cultured at 2×10 4 Inoculate 96-well culture plates at a density of 100 μg / mL and culture overnight. The next day, the cells in the normal group (IPEC-J2) and MDH2-KO group were replaced with cell growth medium containing 100 μg / mL FB1 in each well. For some normal IPEC-J2 cells, add cell growth medium containing 30 μM concentration of LW6 inhibitor for pretreatment for 2 hours in advance, and then add FB1 to serve as the MDH2 inhibition group (IPEC-J2+LW6). Thoroughly mix the liquid in the above wells in the plate and culture in a cell culture incubator at 37°C and 5% CO2 for 72 hours.

[0068] The cells were digested and separated with 0.25% trypsin without EDTA and washed twice with phosphate buffered saline (PBS). The cell pellet was resuspended in a 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 to count the cell apoptosis.

[0069] The results are as follows Figure 7As shown in the figure, before FB1 treatment, there was no significant difference in cell apoptosis among the groups; after FB1 treatment, the cell apoptosis rate of each group increased, but compared with the normal group, under the condition of 100 μg / mL FB1 concentration, the cell apoptosis rate of the MDH2-KO group was extremely significantly reduced by 12.97% (***P<0.001), and the cell apoptosis rate of the IPEC-J2+LW6 group was extremely significantly reduced by 7.76% (*P<0.05).

[0070] The above results show that inhibiting the expression of MDH2 gene can improve the tolerance of cells to FB1. The present invention provides a potential solution for preventing adverse reactions caused by FB1 and provides a theoretical basis for the prevention and control of the harm of FB1 to food and animal husbandry.

[0071] 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. MDH2 The use of an inhibitor in the preparation of a preparation for reducing the toxicity of fumonisin to porcine intestinal epithelial cells is characterized in that: Said MDH2 Inhibitors are LW6 inhibitors or used for knockout or knockdown MDH2 The CRISPR / Cas9 system of the gene; the CRISPR / Cas9 system includes sgRNA, and the DNA sequence for synthesizing the sgRNA consists of the sequence shown in SEQ ID NO.4 and the sequence shown in SEQ ID NO.

5.

2. MDH2 The use of an inhibitor in the preparation of a drug for improving the tolerance of porcine intestinal epithelial cells to fumonisin toxicity is characterized in that: Said MDH2 Inhibitors are LW6 inhibitors or used for knockout or knockdown MDH2 The CRISPR / Cas9 system of the gene; the CRISPR / Cas9 system includes sgRNA, and the DNA sequence for synthesizing the sgRNA consists of the sequence shown in SEQ ID NO.4 and the sequence shown in SEQ ID NO.

5.

3. MDH2 Use of an inhibitor in breeding plant varieties or animal varieties tolerant to fumonisin, characterized in that The animal is a pig, and the tolerance of the intestinal epithelial cells of the pig to the toxicity of fumonisin is improved; Said MDH2 Inhibitors are LW6 inhibitors or used for knockout or knockdown MDH2 The CRISPR / Cas9 system of the gene; the CRISPR / Cas9 system includes sgRNA, and the DNA sequence for synthesizing the sgRNA consists of the sequence shown in SEQ ID NO.4 and the sequence shown in SEQ ID NO.

5.

4. The use according to any one of claims 1 to 3, characterized in that: Said MDH2 The CDS sequence of the gene is shown as SEQ ID NO.

2.

5. The use according to claim 4, characterized in that: Said MDH2 The amino acid sequence encoded by the gene is shown in SEQ ID NO.

3.

6. A method for improving the tolerance of IPEC-J2 cells to fumonisin toxicity, characterized in that: The IPEC-J2 cells MDH2 The IPEC-J2 cells are cultured by knocking out the gene or using a culture medium containing LW6; MDH2 The CDS sequence of the gene is shown in SEQ ID NO.2; Knockout of the IPEC-J2 cells MDH2 Knockout or knockdown of genes MDH2 The CRISPR / Cas9 system of the gene is implemented, the CRISPR / Cas9 system includes sgRNA, and the DNA sequence for synthesizing the sgRNA consists of the sequence shown in SEQ ID NO.4 and the sequence shown in SEQ ID NO.5.

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