Metabolite screening method for promoting expression of porcine intestinal defensin pBD1 and application of metabolite screening method

Abstract

The invention discloses a metabolite screening method for promoting expression of porcine intestinal defensin pBD1 and application of the metabolite screening method. The screening method comprises the following steps: 1) constructing a porcine defensin pBD1 gene fluorescence report intestinal epithelial cell line; 2) evaluating the promotion effects of different metabolites based on the pBD1 gene fluorescence report intestinal epithelial cell line; and 3) further evaluating the promotion effects of the metabolites screened in the step 2) based on a porcine intestinal epithelial cell IPEC-J2 model. And 4) further evaluating the promotion effect of the metabolite screened in the step 3) based on the pig intestinal organ. The invention can be used for preparing or screening drugs influencing the expression of the porcine intestinal defensin pBD1. According to the application disclosed by the invention, the effect of the D-malic acid for improving the immune function of the pig intestinal tract by promoting the expression of the defensin pBD1 is found for the first time, verification is carried out on a pig intestinal tract organ-like model, and meanwhile, the optimal treatment concentration of the D-malic acid for promoting the expression of the defensin pBD1 in the pig intestinal tract is optimized.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a method for screening metabolites promoting the expression of porcine intestinal defensin pBD1 and an application thereof. Background Art

[0002] my country is a major pig-raising country in the world, with the largest breeding scale and pork consumption in the world. Pig breeding has become a pillar industry of my country's animal husbandry. However, for a long time, feed antibiotics have been used in pig breeding, which has caused a series of safety problems such as bacterial resistance, antibiotic residues in pork, and antibiotic emission pollution. Therefore, in-depth research on improving the innate immune function of pig intestines through nutritional means, thereby improving pig growth, can effectively reduce the use of feed antibiotics, which is of great significance to ensuring green pig breeding.

[0003] Antimicrobial peptides are a class of important innate immune factors widely present in animals, and together with the intestinal barrier, they form the first line of defense to prevent the invasion of pathogenic microorganisms. In addition to antibacterial effects, antimicrobial peptides may play more immunomodulatory functions in the body, such as initiating and regulating acquired immunity, promoting lymphocyte proliferation, inhibiting inflammatory responses, and exerting chemotaxis. Among them, defensin antimicrobial peptides are widely distributed in various tissues, mainly expressed in epithelial and mucosal tissues, with the advantages of small molecules, stable properties, high activity, and a wide antibacterial spectrum. pBD1 is the earliest discovered porcine defensin antimicrobial peptide, which has a good inhibitory effect on Gram-negative bacteria. At the same time, it has been proven to be a key regulatory factor in regulating cytokine expression, affecting barrier function, and maintaining the balance of intestinal flora. Promoting the expression of porcine intestinal defensin pBD1, thereby improving the immunity and anti-stress ability of pigs, has become a hot topic of concern under the current ban on antibiotics, and is of great significance to ensuring the healthy breeding of live pigs.

[0004] At present, the research on nutrients promoting the expression of porcine intestinal defensin pBD1 is in its infancy. Different studies have disclosed that short-chain fatty acids such as n-butyric acid, γ-aminobutyric acid and their metabolites, branched-chain amino acids such as leucine and indole and their metabolites have the potential to promote the expression of pBD1 in porcine intestinal epithelial cells. However, the existing research has many limitations, such as the lack of intuitive comparison of the effects of different nutrients on regulating pBD1 expression, the significant differences in the concentrations of cells treated with different nutrients, and the inability of existing methods to compare the regulatory functions of multiple nutrients at the same time, which is not conducive to the efficient screening of nutrients and functional development and practical application.

[0005] Malic acid, chemical name 2-hydroxysuccinic acid, is an important dicarboxylic acid that plays a key role in the tricarboxylic acid cycle of organisms. Malic acid can be divided into three isomers: L-malic acid, D-malic acid and DL-malic acid. L-malic acid is commonly found in fruits, D-malic acid is a metabolite of specific microorganisms, and DL-malic acid is prepared by artificially synthesizing L-malic acid and D-malic acid. Studies have reported the biological functions of L-malic acid, such as anti-oxidation, improving immunity, and maintaining electrolyte balance. There is evidence that supplementing L-malic acid has a beneficial effect in pig farming, such as intervening in the conversion of muscle fiber types in pig skeletal muscle, reducing drip loss in pork, and alleviating intestinal inflammatory reactions in weaned piglets. However, existing studies have shown that D-malic acid is mainly synthesized by intestinal microorganisms and cannot be absorbed by the animal body. There are currently no reports on the regulation of immune function by D-malic acid. Summary of the invention

[0006] In order to overcome the deficiencies of the prior art, the present invention aims to provide a method for screening metabolites that promote the expression of porcine intestinal defensin pBD1 and its application.

[0007] The objective of the present invention is achieved through the following technical solutions: A method for screening metabolites that promote the expression of porcine intestinal defensin pBD1 comprises the following steps: 1) Construction of porcine defensin pBD1 gene fluorescent reporter intestinal epithelial cell line: The genomic DNA was isolated from the small intestine sample of piglets, and the pBD1 promoter fragment was amplified by PCR technology. The pBD1 promoter fragment was cloned into the fluorescent reporter vector to obtain the pBD1 fluorescent reporter plasmid and linearized by restriction digestion. The plasmid was transfected into the pig intestinal epithelial cell IPEC-J2, and the successfully transfected positive cells were screened, which were the pig defensin pBD1 gene fluorescent reporter intestinal epithelial cell line; 2) Evaluation of the promoting effects of different metabolites based on pBD1 gene fluorescent reporter intestinal epithelial cell line: Different metabolites were used to treat the porcine defensin pBD1 gene fluorescence reporter intestinal epithelial cell line, detect luciferase expression, measure fluorescence values, and preliminarily determine the metabolites that promote the expression ability of porcine intestinal defensin pBD1; 3) Further evaluate the promoting effect of the metabolites screened in step 2) based on the porcine intestinal epithelial cell IPEC-J2 model: 4) Further evaluate the promoting effect of the metabolites screened in step 3) based on porcine intestinal organoids.

[0008] In the method, the pBD1 promoter fragment in step 1) has a sequence as shown in SEQ ID NO.1.

[0009] In the method described, the fluorescent reporter vector described in step 1) is pGL4.21.

[0010] In the method, in the transfection described in step 1), the pBD1 gene fluorescent reporter linearized vector after enzyme linearization is mixed with the transfection reagent and the cell culture medium at a ratio of 1 μg: 2 μL: 1 mL, and the intestinal epithelial cells are treated for a total of 24 hours.

[0011] In the method, the different metabolites described in step 2) include D-malic acid, arachidonic acid, indole, indoleacetic acid, glutaric acid, hydroxybutyric acid, acetylornithine, (acetylthio)acetic acid, hydroxy (methylthio)butyric acid, linoleic acid, eicosadienoic acid, eicosapentaenoic acid, docosahexaenoic acid, aminobutyric acid, ornithine, cucurbitacin B, galactitol, tyramine, vanillic acid, sebacic acid, acetyleucine, tyrosine methyl ester, acetylmethionine, acetylvaline, cyclohexanedione, trihydroxyflavanone, methoxyvinylphenol, oleyl monoethanolamine, acetylphenylalanine, hydroxybutanone, methyladipic acid, dimethyl isoflavone, methylglutaric acid, leucine alanine, acetylalanine, glutamine, hydroxyflavonoids, indoleacetamide, aminoisobutyric acid, acetyllysine and hexadecenoic acid.

[0012] The metabolites screened in step 2) of the method include D-malic acid, indole, indoleacetic acid, glutaric acid, hydroxybutyric acid, acetylornithine, (acetylthio)acetic acid, hydroxy (methylthio)butyric acid, eicosadienoic acid, docosahexaenoic acid, aminobutyric acid, ornithine, galactitol, tyramine, vanillic acid, sebacic acid, acetyleucine, tyrosine methyl ester, acetylvaline, cyclohexanedione, trihydroxyflavanone, methoxyvinylphenol, hydroxybutanone, methyladipic acid, methylglutaric acid, leucine alanine, hydroxyflavonoids, indoleacetamide and acetyl lysine, totaling 29 kinds.

[0013] The metabolites screened in step 3) of the method include D-malic acid, indole, indoleacetic acid, glutaric acid, acetylornithine, (acetylthio)acetic acid, hydroxy (methylthio)butyric acid, docosahexaenoic acid, galactitol, tyramine, vanillic acid, trihydroxyflavanone, methylglutaric acid, hydroxyflavonoids and acetyl lysine, a total of 15 kinds.

[0014] The metabolites screened in step 4) of the method include D-malic acid, galactitol, tyramine, vanillic acid and methylglutaric acid, a total of 5 kinds.

[0015] Among the metabolites screened by the method, D-malic acid has the best effect. The D-malic acid treatment concentration is 25 μmol / L and the treatment time is 24 h. The D-malic acid treatment of porcine intestinal epithelial cells can promote the expression of pBD1 gene by 1.59 times.

[0016] The metabolites screened by the method are used to prepare or screen drugs that affect the expression of porcine intestinal defensin pBD1. The drug screening model includes one or more of the porcine defensin pBD1 gene fluorescent reporter intestinal epithelial cell line, porcine intestinal epithelial cell IPEC-J2, and porcine intestinal organoids.

[0017] The beneficial effects of the present invention are: The present invention provides a method for screening the effects of a total of 41 different metabolites on the expression of porcine intestinal defensin pBD1. It is found for the first time that D-malic acid has the best effect on improving the fluorescence value of porcine intestinal defensin pBD1 fluorescent reporter cell line, porcine intestinal epithelial cells and porcine intestinal organoid pBD1 expression, while L-malic acid has no similar effect. The D-malic acid of the present invention has been successfully applied to porcine intestinal organoids to promote the immune function of the porcine intestine, laying a foundation for subsequent exploration of the role of D-malic acid in breeding production. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is the result of screening different metabolites using porcine intestinal defensin pBD1 fluorescent reporter cell line.

[0019] Figure 2 The effect of incubation with D-malic acid and L-malic acid on the fluorescence value of porcine intestinal defensin pBD1 fluorescent reporter cell line.

[0020] Figure 3 This is the result of the expression of the defensin pBD1 gene in porcine intestinal epithelial cells after incubation with different metabolites.

[0021] Figure 4 This is the result of gene expression of defensin pBD1 in porcine intestinal organoids after incubation with different metabolites.

[0022] Figure 5 The morphological changes of pig intestinal organoids after different treatments.

[0023] Figure 6 This is the proliferation of pig intestinal organoids after different treatments. DETAILED DESCRIPTION

[0024] The present invention is further described below in conjunction with the accompanying drawings and embodiments.

[0025] Example 1 Construction of pBD1 gene fluorescent reporter intestinal epithelial cell line Experimental method: The porcine defensin pBD1 gene sequence described in the present invention is designed using the NCBI online database; wherein, the pBD1 gene promoter sequence is shown in SEQ ID NO.1. 1-2 cm piglet small intestine samples were taken to isolate genomic DNA, and the pBD1 promoter fragment was amplified using PCR technology, with a length of 1843 bp, and the PCR product was cloned into the pGL4.21 fluorescent reporter vector. The competent strain Escherichia coli DH5α was further transformed by heat shock method, the plasmid was extracted and the linearized vector was obtained by enzyme digestion using KpnI-Xhol enzyme. 500 μL of the linearized vector was mixed with transfection reagent (Lipofectamine 3000, ThermoFisher) and cell culture medium (DMEM basic, Gibco) at a ratio of 1 μg: 2 μL: 1 mL, and intestinal epithelial cells IPEC-J2 were treated for a total of 24 hours, and then replaced with complete culture medium (DMEM F12, Gibco) containing 3 μg / mL puromycin for screening.

[0026] Result analysis: The positive cells that survived 7 days after screening were the pBD1 gene fluorescent reporter intestinal epithelial cell lines.

[0027] Example 2 Evaluation of the promoting effect of different metabolites based on pBD1 gene fluorescence reporter intestinal epithelial cell line Experimental method: Based on the pBD1 gene fluorescence reporter intestinal epithelial cell line obtained by the method in Example 1, the cells were incubated with 41 different metabolites, and the porcine defensin pBD1 gene fluorescence reporter intestinal epithelial cell line treated with PBS was used as the control group. The luciferase expression was detected using a luciferase detection kit, and the fluorescence value was measured using a fluorescence spectrophotometer.

[0028] Result analysis: Figure 1 As shown, compared with the control group, 29 metabolites including D-malic acid, arachidonic acid, indole, indoleacetic acid, glutaric acid, acetylornithine, (acetylthio)acetic acid, hydroxy (methylthio)butyric acid, eicosadienoic acid and docosahexaenoic acid can significantly increase the cell fluorescence value (relative fluorescence>1.5).

[0029] Example 3 Evaluation of the promoting effect of D-malic acid and L-malic acid based on pBD1 gene fluorescence reporter intestinal epithelial cell line Experimental method: Based on the pBD1 gene fluorescence reporter intestinal epithelial cell line obtained by the method in Example 1, the cells were incubated with 25 μmol / L D-malic acid and 25 μmol / L L-malic acid, respectively. The porcine defensin pBD1 gene fluorescence reporter intestinal epithelial cell line treated with PBS was used as the control group. The luciferase expression was detected using a luciferase detection kit, and the fluorescence value was measured using a fluorescence spectrophotometer.

[0030] Result analysis: Figure 2 As shown, compared with the control group, incubation with 25 μmol / L D-malic acid can significantly increase the cell fluorescence value, while incubation with 25 μmol / L L-malic acid has no effect on the cell fluorescence value.

[0031] Example 3 Evaluation of the effect of different metabolites on the expression of pBD1 gene in porcine intestinal epithelial cells Experimental method: Based on the 29 metabolites that can significantly increase the cell fluorescence value obtained by preliminary screening in Example 2, porcine intestinal epithelial cells IPEC-J2 were further incubated and the cells were treated for a total of 24 hours.

[0032] Sample collection and determination of gene expression levels: TRIzol (purchased from Invitogen) was used to collect cells and extract RNA, and RevertAid RT Reverse Transcription Kit (purchased from Thermo) was used to reverse transcribe and synthesize cDNA. The expression of pBD1 gene was determined by RT-PCR technology.

[0033] Result analysis: Using 2 -△△Ct The expression of pBD1 gene in cells was calculated by the method. Compared with the control group, 29 metabolites can increase the expression of pBD1 gene, which proves the accuracy of the fluorescent reporter intestinal epithelial cell line screening. The results of 15 metabolites that can significantly increase the gene expression of pBD1 (P < 0.05) are as follows Figure 3 As shown, 25 μmol / L D-malic acid significantly increased the expression of cellular pBD1 gene (4.48 times), which was higher than that of other metabolites.

[0034] Example 4 Evaluation of the effect of different metabolites on the expression of pBD1 gene in porcine intestinal organoids Experimental method: A pig intestinal organoid model was incubated with 15 metabolites that were further screened in Example 3 and that could significantly increase the expression of pBD1 gene in pig intestinal epithelial cells.

[0035] Sample collection and gene expression level determination: as described in Example 3; Results analysis: The results of the five metabolites that can significantly promote the expression of pBD1 gene in pig intestinal organoids are as follows Figure 4 As shown, compared with the control group, 25 μmol / L D-malic acid had the best effect in promoting the expression of pBD1 gene (1.59 times).

[0036] Example 5 Evaluation of the effect of D-malic acid on alleviating TNF-α-induced inflammation in porcine intestinal organoids Construction of pig intestinal organoid model: same as described in Example 4.

[0037] Sample treatment and observation: The control group received no treatment, the D-malic acid group was added with 25 μmol / LD-malic acid from the second day, the TNF-α group was added with 0.12 μg / mL TNF-α from the second day, and the TNF-α+D-malic acid group was added with 0.12 μg / mL TNF-α and 25 μmol / LD-malic acid from the second day. The morphology of organoids in each group was observed under an optical microscope at a fixed time every day; the organoids from the above groups on the fourth day were collected for EdU staining, and after fixation with paraformaldehyde, the morphology and staining of the organoids were observed under a laser confocal microscope.

[0038] Result analysis: Figure 5 As shown in the figure, compared with the control group, organoids treated with D-malic acid were larger and had more buds, but TNF-α treatment had obvious content outflow and fewer buds from the third day, which caused inflammatory damage to the organoids. There was no obvious content outflow in the organoids of the TNF-α+D-malic acid group, and the organoid growth and budding were normal, indicating that D-malic acid treatment alleviated TNF-α-induced organoid inflammatory damage. Figure 6 As shown in the figure, compared with the control group, the number of green fluorescent cells in the organoids increased after D-malic acid treatment, but TNF-α treatment caused organoid structural damage, content outflow, and a decrease in the number of green fluorescent cells. The organoid structure and growth of the TNF-α+D-malic acid group were normal, and the number of fluorescent cells did not decrease significantly, indicating that D-malic acid may alleviate TNF-α-induced organoid inflammatory damage by promoting the proliferation of intestinal stem cells.

[0039] Finally, it should be noted that the above examples are only some specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments, and there may be many variations and equivalent substitutions. All variations and equivalent substitutions that can be directly derived or associated with the contents disclosed by a person skilled in the art from the present invention are within the scope of protection of the present invention.

Claims

1. A method for screening metabolites that promote the expression of porcine intestinal defensin pBD1, characterized in that: The following steps are involved: 1) Construction of porcine defensin pBD1 gene fluorescent reporter intestinal epithelial cell line: The genomic DNA was isolated from the small intestine sample of piglets, and the pBD1 promoter fragment was amplified by PCR technology. The pBD1 promoter fragment was cloned into the fluorescent reporter vector to obtain the pBD1 fluorescent reporter plasmid and linearized by restriction digestion. The plasmid was transfected into the pig intestinal epithelial cell IPEC-J2, and the successfully transfected positive cells were screened, which were the pig defensin pBD1 gene fluorescent reporter intestinal epithelial cell line; 2) Evaluation of the promoting effects of different metabolites based on pBD1 gene fluorescent reporter intestinal epithelial cell line: Different metabolites were used to treat the porcine defensin pBD1 gene fluorescence reporter intestinal epithelial cell line, detect luciferase expression, measure fluorescence values, and preliminarily determine the metabolites that promote the expression ability of porcine intestinal defensin pBD1; 3) Further evaluate the promoting effect of the metabolites screened in step 2) based on the porcine intestinal epithelial cell IPEC-J2 model: 4) Further evaluate the promoting effect of the metabolites screened in step 3) based on porcine intestinal organoids.

2. The method according to claim 1, characterized in that The pBD1 promoter fragment described in step 1) has a sequence as shown in SEQ ID NO.

1.

3. The method according to claim 1, characterized in that The fluorescent reporter vector described in step 1) is pGL4.

21.

4. The method according to claim 1, characterized in that In the transfection described in step 1), the pBD1 gene fluorescent reporter linearized vector after enzyme digestion and linearization was mixed with the transfection reagent and cell culture medium at a ratio of 1 μg: 2 μL: 1 mL, and the intestinal epithelial cells were treated for a total of 24 hours.

5. The method according to claim 1, characterized in that The different metabolites described in step 2) include D-malic acid, arachidonic acid, indole, indoleacetic acid, glutaric acid, hydroxybutyric acid, acetylornithine, (acetylthio)acetic acid, hydroxy(methylthio)butyric acid, linoleic acid, eicosadienoic acid, eicosapentaenoic acid, docosahexaenoic acid, aminobutyric acid, ornithine, cucurbitacin B, galactitol, tyramine, vanillic acid, sebacic acid, acetyleucine, tyrosine methyl ester, acetylmethionine, acetylvaline, cyclohexanedione, trihydroxyflavanone, methoxyvinylphenol, oleyl monoethanolamine, acetylphenylalanine, hydroxybutanone, methyladipic acid, dimethyl isoflavone, methylglutaric acid, leucine alanine, acetylalanine, glutamine, hydroxyflavonoids, indoleacetamide, aminoisobutyric acid, acetyllysine and hexadecenoic acid.

6. The method according to claim 5, characterized in that Step 2) The metabolites screened included D-malic acid, indole, indoleacetic acid, glutaric acid, hydroxybutyric acid, acetylornithine, (acetylthio)acetic acid, hydroxy(methylthio)butyric acid, eicosadienoic acid, docosahexaenoic acid, aminobutyric acid, ornithine, galactitol, tyramine, vanillic acid, sebacic acid, acetyleucine, tyrosine methyl ester, acetylvine, cyclohexanedione, trihydroxyflavanone, methoxyvinylphenol, hydroxybutanone, methyladipic acid, methylglutaric acid, leucine alanine, hydroxyflavonoids, indoleacetamide and acetyl lysine, a total of 29 types.

7. The method according to claim 6, characterized in that Step 3) The metabolites screened included D-malic acid, indole, indoleacetic acid, glutaric acid, acetylornithine, (acetylthio)acetic acid, hydroxy (methylthio)butyric acid, docosahexaenoic acid, galactitol, tyramine, vanillic acid, trihydroxyflavanone, methylglutaric acid, hydroxyflavonoids and acetyl lysine, a total of 15 types.

8. The method according to claim 7, characterized in that Step 4) The metabolites obtained by screening include D-malic acid, galactitol, tyramine, vanillic acid and methylglutaric acid, a total of 5 types.

9. The use of the metabolites screened by the method of claim 8, characterized in that: The application effect of D-malic acid is the best. The D-malic acid treatment concentration is 25 μmol / L and the treatment time is 24 h. The D-malic acid treatment of porcine intestinal epithelial cells can promote the expression of pBD1 gene by 1.59 times.

10. Use of the metabolites screened by the method according to claim 1, characterized in that: Used for preparing or screening drugs that affect the expression of porcine intestinal defensin pBD1, the drug screening model includes one or more of the porcine defensin pBD1 gene fluorescent reporter intestinal epithelial cell line, porcine intestinal epithelial cell IPEC-J2, and porcine intestinal organoids.

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