Application of ssc-miR-146a-5p in the preparation of products promoting intestinal development in piglets

Abstract

The present invention belongs to the field of animal husbandry technology, and specifically relates to the application of ssc‑miR‑146a‑5p in the preparation of products that promote intestinal development in piglets. The nucleotide sequence of the ssc‑miR‑146a‑5p is shown in SEQ ID NO.1. The present invention is based on the ssc‑miR‑146a‑5p that is key to promoting intestinal cell proliferation screened from the breast milk of sows with high and low piglet growth performance. The ssc‑miR‑146a‑5p lentiviral overexpression vector is constructed and injected into suckling piglets in the form of three-point intraperitoneal injection. It is found that overexpression of ssc‑miR‑146a‑5p can increase the expression of intestinal cell proliferation genes and intestinal mucin genes in suckling piglets, promote intestinal development in suckling piglets, improve intestinal morphology and mucosal barrier, and improve the growth performance of suckling piglets during lactation. The ssc-miR-146a-5p provided by the present invention can significantly improve the intestinal development and intestinal health of suckling piglets, and provide a feasible solution for solving the production problem of piglet morbidity and mortality caused by delayed intestinal development in suckling piglets.

Description

Technical Field

[0001] The invention belongs to the technical field of animal husbandry, and particularly relates to the application of ssc-miR-146a-5p in preparing a product for promoting intestinal development of piglets. Background Art

[0002] Intestinal problems are one of the main causes of morbidity and mortality in suckling piglets. As an important organ for piglets to digest food and absorb nutrients, the health of the intestine directly affects their growth performance during lactation. In addition to digestion and absorption functions, the intestine is also involved in the body's immunity. The intestinal development of piglets during lactation and weaning is not yet mature, and they are more susceptible to the external environment and pathogens, leading to the occurrence of intestinal diseases such as diarrhea. In addition, imperfect intestinal barrier function may allow toxins to invade, triggering inflammatory reactions and overreactions of the immune system. Furthermore, changes in the barn environment and feed ingredients during the weaning period also bring many challenges to the intestinal health of piglets. The food source has also changed from single breast milk to commercial solid feed, and the intestine will be stressed due to the short-term drastic changes in the environment and food. According to statistics, the mortality rate of piglets during lactation and nursery periods is as high as 20%-30%. Therefore, promoting intestinal development is particularly important for improving the health of lactating piglets.

[0003] The English name of microRNA is microRNA, miRNA. It is a type of non-coding RNA molecule that exists in animals and plants. It is about 22 nucleotides in length. They silence the translation of mRNA or cause its degradation by binding or complementary pairing with the 3'UTR part of the target gene mRNA, thereby regulating the expression level of the target gene and reducing the formation of specific proteins. In recent years, experiments have shown that microRNA plays an important role in disease warning. However, the use of microRNA in promoting intestinal development in piglets is unclear. Therefore, it is necessary to carry out application research on microRNA in promoting intestinal development in piglets. Summary of the invention

[0004] In order to carry out new application research of microRNA in promoting the intestinal development of piglets, the present invention provides the application of ssc-miR-146a-5p in the preparation of products promoting the intestinal development of piglets.

[0005] To achieve the above purpose, the present invention adopts the following technical solution.

[0006] The present invention provides an application of ssc-miR-146a-5p in the preparation of a product for promoting intestinal development in piglets. The present invention develops a new application of ssc-miR-146a-5p derived from pig milk in promoting intestinal development in piglets, which is a research on the application of related products of microRNA in promoting intestinal development in piglets, solving the problem of unclear use of microRNA in promoting intestinal development in piglets.

[0007] Wherein, the ssc-miR-146a-5p is a miRNA derived from pig milk. The nucleotide sequence of the ssc-miR-146a-5p is shown in SEQ ID NO.1.

[0008] The present invention has found through research that the characteristics of ssc-miR-146a-5p in promoting the intestinal development of suckling piglets are to improve the growth performance of piglets during lactation, increase the intestinal weight and length of suckling piglets, increase the length of intestinal villi and the depth of crypts, and promote the expression of intestinal cell proliferation-related genes and mucin-related genes.

[0009] Preferably, the piglets are suckling piglets.

[0010] The inventors of the present invention compared the differences in miRNA carried in the milk of sows with high and low piglet growth performance during lactation. Differential miRNAs were obtained by high-throughput sequencing methods, and the effects and mechanisms of key miRNAs on the growth performance, intestinal development and intestinal health of lactating piglets were screened and verified. The inventors of the present invention found that pig milk ssc-miR-146a-5p is a key effector factor in promoting intestinal development and growth performance of piglets during lactation. In the lactating piglet experiment, the present invention constructed an ssc-miR-146a-5p lentiviral overexpression vector and injected it intraperitoneally into the lactating piglets at a specific dose, and found that it can significantly improve the intestinal development, intestinal health and growth performance of the lactating piglets. Therefore, the present application provides a related application of ssc-miR-146a-5p to promote intestinal development in piglets.

[0011] Preferably, the product is used to improve the intestinal morphology and intestinal mucosal barrier of suckling piglets, thereby enhancing the early intestinal resistance to pathogens and improving intestinal health.

[0012] This includes upregulating genes related to cell proliferation in the intestine CCND-1 , PCNA , c-Myc and EGFR expression.

[0013] The ssc-miR-146a-5p is used to improve the intestinal mucosal barrier of suckling piglets by upregulating intestinal mucosal factors in the intestine. ZO-1 , Occludin expression.

[0014] Preferably, improving the intestinal morphology of suckling piglets refers to increasing the length of villi and the depth of crypts in the jejunum of suckling piglets, thereby effectively enhancing the intestinal immune function and digestion and absorption.

[0015] Preferably, the product is used to increase the weight and length of the small intestine and the entire intestinal tract of suckling piglets, and promote the digestion and absorption of nutrients.

[0016] Preferably, the product is a medicine or a feed additive.

[0017] The drug is prepared with the ssc-miR-146a-5p or the agonist of the ssc-miR-146a-5p as the active ingredient and supplemented with pharmaceutically acceptable excipients.

[0018] The feed additive is prepared by using the ssc-miR-146a-5p or the agonist of the ssc-miR-146a-5p as an active ingredient and supplemented with pharmaceutically acceptable excipients.

[0019] The medicine or feed additive provided by the present invention can be used to promote early intestinal development and intestinal health of piglets.

[0020] Preferably, the agonist of ssc-miR-146a-5p comprises an overexpression vector containing ssc-miR-146a-5p. The agonist of ssc-miR-146a-5p mainly functions to promote the expression of ssc-miR-146a-5p in vivo.

[0021] Preferably, the overexpression vector containing ssc-miR-146a-5p is the ssc-miR-146a-5p lentiviral overexpression vector, which is obtained through the steps of plasmid construction and transformation, lentiviral packaging and concentration, and lentiviral titration.

[0022] The ssc-miR-146a-5p lentiviral overexpression vector is injected by three-point intraperitoneal injection.

[0023] The time of three-point intraperitoneal injection of the ssc-miR-146a-5p lentiviral overexpression vector is the first day after the piglets are born.

[0024] The injection volume of the ssc-miR-146a-5p lentiviral overexpression vector at each point of the three-point intraperitoneal injection was 1×10 9 TU transduction unit.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] 1. The present invention provides an application of ssc-miR-146a-5p in the preparation of a product that promotes the intestinal development of piglets, and provides a new method for promoting the intestinal development of piglets. The present invention has developed a new application of ssc-miR-146a-5p derived from pig milk in promoting the intestinal development of piglets. It is a research on the application of related products of microRNA in promoting the intestinal development of piglets, and solves the problem of unclear use of microRNA in promoting the intestinal development of piglets.

[0027] 2. The ssc-miR-146a-5p provided by the present invention can significantly improve the intestinal development and mucosal barrier of suckling piglets, promote the growth performance of suckling piglets, and provide a feasible solution for reducing the production problems of piglet morbidity and mortality caused by slow intestinal development in suckling piglets. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The quantitative results of ssc-miR-146a-5p in jejunal tissue of the ssc-miR-146a-5p lentivirus overexpression group, lentivirus control group and PBS blank group in the embodiment of the present invention are compared.

[0029] Figure 2 This is a comparison of the average body weight of piglets in the ssc-miR-146a-5p lentivirus overexpression group, lentivirus control group and PBS blank group during the experimental period in the examples of the present invention.

[0030] Figure 3 The length and weight of the small intestine and the whole intestine of the piglets in the ssc-miR-146a-5p lentivirus overexpression group, the lentivirus control group and the PBS blank group in the embodiment of the present invention are compared: wherein, Figure 3 Figure A shows the length of the small intestine and the entire intestine; Figure 3 Panel B shows the weight of the small intestine and the entire intestine.

[0031] Figure 4 The representative pictures of the intestinal morphology of the H&E stained jejunal tissue of the piglets in the ssc-miR-146a-5p lentivirus overexpression group, lentivirus control group and PBS blank group in the embodiment of the present invention are as follows: Figure 4 Figure A in the figure is the PBS blank group; Figure 4 Figure B in the figure is the lentivirus control group; Figure 4 Figure C shows the ssc-miR-146a-5p lentivirus overexpression group.

[0032] Figure 5 This is a comparison of the villus intestine and crypt depth of the jejunum morphology observed in the ssc-miR-146a-5p lentivirus overexpression group, the lentivirus control group and the PBS blank group in the examples of the present invention.

[0033] Figure 6 This is a comparison of the expression of genes related to the proliferation of piglet jejunal tissue cells in the ssc-miR-146a-5p lentivirus overexpression group, the lentivirus control group and the PBS blank group in the examples of the present invention.

[0034] Figure 7 This is a comparison of the expression of intestinal mucosal barrier-related genes in the jejunum tissue of piglets in the ssc-miR-146a-5p lentivirus overexpression group, the lentivirus control group and the PBS blank group in the examples of the present invention. DETAILED DESCRIPTION

[0035] The present invention is described in detail below in conjunction with the accompanying drawings and specific examples, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0036] Example 1: Construction of ssc-miR-146a-5p lentiviral overexpression vector

[0037] The transcript number of ssc-miR-146a-5p in the present invention is MIMAT0022963. The nucleotide sequence of ssc-miR-146a-5p is shown in SEQ ID NO.1:

[0038] 5'-UGAGAACUGAAUUCCAUGGGUU-3'.

[0039] The name of the overexpression vector containing ssc-miR-146a-5p is: pLv3ltr-ecMv-mcherry (2A) Puro-U6, P202303AZ, which was purchased from Beijing Qingke Biotechnology Co., Ltd.

[0040] Lentivirus packaging plasmids were pSPAX2 and pMD2G, which were purchased from Beijing Qingke Biotechnology Co., Ltd.

[0041] The ssc-miR-146a-5p lentiviral overexpression vector of the present invention is obtained by the steps of plasmid construction and transformation, lentiviral packaging and concentration, and lentiviral titration. The specific steps are as follows:

[0042] 1) Construction of plasmid

[0043] Refer to the sequence information of ssc-miR-146a-5p shown in SEQ ID NO.1, and add EcoR Ⅰ enzyme and XOtⅠ restriction site, specific primers of ssc-miR-146a-5p sequence were designed by Primer Premier 5.0 software, PCR amplification was performed with porcine intestinal epithelial cell cDNA as template, and PCR amplification was performed using Shanghai Biotech Taq DNA polymerase.

[0044] Wherein, the specific primers include primer F and primer R, and the primer sequences are as follows:

[0045] The nucleotide sequence of primer F is shown in SEQ ID NO.2:

[0046] 5'-ggaattccACGAACCAGGGTGAGAACT-3';

[0047] The nucleotide sequence of primer R is shown in SEQ ID NO.3:

[0048] 5'-ccgctcgagAACCCATGGAATTCAGTTC-3'.

[0049] Primer F and primer R were synthesized by Beijing Qingke Biotechnology Co., Ltd.

[0050] The steps of PCR amplification are as follows: the total volume of the PCR reaction system is 50 μL, which includes 0.25 μL of Biyuntian Taq DNA polymerase, 2 μL of upstream primer, 2 μL of downstream primer, 2 μL of cDNA template, 5 μL of 10× PCR buffer, 4 μL of dNTP deoxynucleotide and 34.75 μL of double distilled water.

[0051] The reaction conditions of PCR amplification were as follows: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 1 min, 30 cycles, extension at 72°C for 10 min, and termination at 4°C.

[0052] The PCR amplification product was sent to Beijing Qingke Biotechnology Co., Ltd. for gene sequencing to verify the accuracy of the sequence. Then, 15 μL of the PCR amplification product was mixed with 15 μL of pLv3ltr-ecMv-mcherry (2A) Puro-U6, 1 μL of EcoR Ⅰ enzyme and 1 μL XOt Ⅰ enzyme mixture, react at 37°C for 3h, and obtain a recombinant plasmid mixture with initial ssc-miR-146a-5p overexpression.

[0053] 2) Recovery and purification of plasmid

[0054] Use 0.5×TBE buffer and agarose according to the weight ratio to prepare a 1% agarose gel plate, put it into the electrophoresis tank and add 0.5×TBE buffer until the gel plate is completely immersed. Load the recombinant plasmid mixture with initial ssc-miR-146a-5p overexpression obtained in the previous step into the gel comb hole, set the voltage to 75V and the current to 40mA or more for electrophoresis.

[0055] When bromophenol blue migrates to more than 2 cm, observe under long-wave ultraviolet light, and use a cleaned blade to cut a gel block of uniform width and about 2 cm in length in front of the target fragment. Place the cut gel block in the recovery gel tank, add low-melting point agarose gel to seal the incision, and put it back into the electrophoresis tank after solidification to continue electrophoresis. After the target band completely enters the low-melting point gel, cut the gel strip containing the target DNA with a cleaned blade under ultraviolet light, put it into a sterilized 1.5mL Eppendorf centrifuge tube, add 300µL of 1×TB conversion buffer, and dissolve it in a 65℃ water bath for 10 minutes. Then add an equal amount of Tris-Cl saturated phenol with pH=8.0, mix thoroughly, and centrifuge at 12000rpm for 5 minutes, remove the aqueous phase to a new 1.5mL Eppendorf centrifuge tube, add 3 times the volume of pre-cooled anhydrous ethanol, mix well, and centrifuge for 5 minutes. Place the Eppendorf centrifuge tube in liquid nitrogen for 3 minutes, take it out and place it at -20℃ for 3 minutes, and centrifuge it again at 12000rpm for 10 minutes. The supernatant was removed to obtain the purified recombinant plasmid at the bottom, which was washed with anhydrous ethanol and then dried at 55°C for 10 min until no ethanol remained. Finally, 20 µL of ultrapure water was added to dissolve it to obtain the purified ssc-miR-146a-5p overexpression recombinant plasmid.

[0056] 3) Plasmid transformation

[0057] In a new 1.5mL Eppendorf tube, add auxiliary plasmid pSPAX2, auxiliary plasmid pMD2G and purified ssc-miR-146a-5p overexpression recombinant plasmid according to the weight ratio of the three plasmids 3:1:4, add 5U / μL T4 DNA ligase and connect overnight at 16℃ to obtain a three-plasmid mixture. Subsequently, 100μL of the obtained three-plasmid mixture was added to DH5α competent cells, ice-bathed for 30min, heat-shocked at 42℃ for 45s, and then ice-bathed for 2min to obtain a competent cell mixture. Among them, overnight refers to a time ≥12h.

[0058] Prepare LB solid medium and LB liquid medium. The formula of LB solid medium is: 100g LB powder plus 1.5g agar powder and 100mL ultrapure water, sterilize at 120℃ for 30min, add ampicillin to 100μg / mL when cooled to 60℃, pour into a plate until solidified. LB liquid medium is 100g LB powder dissolved in 100mL ultrapure water, also sterilize and cool for use.

[0059] Add 100 μL of competent cell mixture obtained in the previous step to 900 μL of LB liquid medium and shake in a 37°C constant temperature shaker for 1 hour. Then, take 100 μL of culture solution and spread it on LB solid medium containing 100 μg / mL ampicillin and culture it in a 37°C incubator overnight. Pick a single colony and inoculate it into a new LB liquid medium and culture it for 16 hours. Then use Tiangen Bio's endotoxin-free plasmid extraction kit DP117 to extract the plasmid, and the resulting ssc-miR-146a-5p overexpression recombinant plasmid.

[0060] Among them, overnight culture refers to a culture time ≥ 12h.

[0061] 4) ssc-miR-146a-5p overexpression lentivirus coating and concentration

[0062] 1.0×10 5 HEK293T cells with a density of 100 / mL were inoculated into a 100 mm culture dish and cultured at 37°C and 5% CO2 for 12 hours. When the cell density reached 80%, lentivirus coating was performed. The specific operation is as follows:

[0063] Add 10μg of auxiliary plasmid pSPAX2, 10μg of auxiliary plasmid pMD2G and 10μg of ssc-miR-146a-5p overexpression recombinant plasmid to 500μL serum-free medium; take another 500μL serum-free DMEM medium, add 20μL Lipofectamine2000, let it stand for 5min, mix it with the three plasmid mixture medium, let it stand for 20min, then slowly add it to HEK293T cells, shake it well and place it in a 37℃, 5% CO2 incubator for culture. After 12h, replace it with DMEM medium containing 10%wt fetal bovine serum and continue to culture. Collect the supernatant at 24h, 48h and 72h, and add fresh DMEM medium containing 10%wt fetal bovine serum. The supernatants at each time point were mixed, centrifuged at 4°C, 2500 rpm for 10 min to remove cell debris, transferred to an ultracentrifuge tube and centrifuged at 4°C, 5000 rpm for 2 h, and the supernatant was collected, i.e., the unconcentrated ssc-miR-146a-5p overexpression lentiviral solution.

[0064] 5×PEG8000 solution was prepared using 8.766g NaCl, 50g PEG8000 and 200mL sterilized water. After mixing, the mixture was sterilized by high pressure for 30min to obtain 5×PEG8000 stock solution. The unconcentrated ssc-miR-146a-5p overexpression lentiviral solution was filtered with a 0.45μm filter head, and 7.5mL of 5×PEG8000 stock solution was added to every 30mL. The mixture was mixed 5 times with an interval of 20min. After standing at 4°C overnight, the mixture was centrifuged at 4000g for 20min, and the supernatant was carefully removed. The remaining was the concentrated virus precipitate. 1mL of PBS solution with a concentration of 0.01mol / L and a pH of 7.4 was added to dissolve the precipitate to obtain the ssc-miR-146a-5p overexpression lentiviral stock solution.

[0065] HEK293T cells, human embryonic kidney cell line, were purchased from Beijing Huakang Innovation Technology Co., Ltd.

[0066] 5) Titer determination of ssc-miR-146a-5p overexpression lentivirus

[0067] Take 50 μL of ssc-miR-146a-5p overexpression lentivirus stock solution and mix it with 100 μL of DMEM medium containing 10%wt fetal bovine serum as the starting solution. Use DMEM medium to dilute the ssc-miR-146a-5p overexpression lentivirus in the starting solution, and dilute it at a 10-fold concentration. The steps are as follows: take 100 μL of the starting solution and add it to 900 μL of DMEM medium, which is a 10-fold dilution of the starting solution. The 10-fold dilution of the next gradient is analogous, and the steps are consistent. A total of 6 dilution gradients are diluted to obtain 6 dilution gradients of ssc-miR-146a-5p overexpression lentivirus dilutions.

[0068] 1.0×10 5HEK293T cells with a concentration of 100 μL / mL were inoculated in a 96-well plate, and 100 μL of cell suspension was added to each well. The cells were cultured in a 37°C, 5% CO2 incubator for 24 h, and the assay was performed after the cell density reached 80%. The 96-well plate was taken out, and the old culture medium was aspirated with a pipette. The group with the ssc-miR-146a-5p overexpression lentivirus dilution was the assay group. One gradient was one assay group, with a total of 6 assay groups. Each assay group had 6 replicates, each replicate had 1 well, and each well was added with 100 μL of lentivirus dilution of the corresponding concentration. In addition, a blank group was set up to add DMEM culture medium. The blank group also had 6 replicates, each replicate had 1 well, and each well was added with 100 μL of DMEM culture medium. After the assay group and the blank group were cultured for 72 h, 100 μL of DMEM culture medium containing 10% wt fetal bovine serum and 1.5 μg / mL puromycin was added to each well, and the culture was continued for 5 days. The results were observed using a fluorescence microscope, and the wells with a live cell percentage of 25% were selected for virus titer calculation.

[0069] The formula for calculating the lentiviral titer is as follows:

[0070] Virus titer = number of cells × percentage of positive clones × MOI multiplicity of infection × virus dilution times × 10³;

[0071] Among them, MOI multiplicity of infection = total number of virus particles / total number of cells;

[0072] The unit of virus titer is TU / mL.

[0073] Example 2: Study on the effect of ssc-miR-146a-5p on promoting intestinal development and intestinal health in suckling piglets

[0074] 1) Experimental design

[0075] Three sows with similar body shape, parity and reproductive performance were selected. On the first day of delivery, six piglets were selected from each litter based on the principle of half male and half female and similar weight. A total of 18 piglets were selected from three litters for the experiment. Each litter of piglets was evenly divided into three groups: PBS blank group, lentivirus control group and ssc-miR-146a-5p lentivirus overexpression group, with 6 replicates in each group and 1 piglet in each replicate. Among them, the sows were derived from dual-purpose sows bred from Landrace boars and Large White sows.

[0076] After each group of piglets consumed colostrum, they were given intraperitoneal injections at three sites in a triangle with the abdominal cavity as the center point.

[0077] The piglets in the PBS blank group were injected with 1 mL of PBS, which was purchased from Beijing Solebao Biotechnology with a concentration of 0.01 mol / L and a pH of 7.4.

[0078] The injection concentration of each site in the lentivirus control group and the ssc-miR-146a-5p lentivirus overexpression group was 10 9 TU transduction unit / mL of recombinant lentivirus, totaling 1mL.

[0079] The recombinant lentivirus refers to a lentivirus solution containing a ssc-miR-146a-5p lentivirus overexpression vector; the lentivirus solution is prepared / obtained by diluting the lentivirus stock solution to 10% using a PBS solution with a concentration of 0.01 mol / L and a pH of 7.4. 9 TU transduction units / mL.

[0080] All experimental piglets were fed in their original litter after injection, and breast milk was the only energy source for the piglets during lactation.

[0081] 1) Sample collection

[0082] The test period was 14 days, and the piglets in each group were weighed every 2 days. On the 14th day of the test period, all piglets were anesthetized by intramuscular injection of sodium pentobarbital at a dose of 30 mg / kg. After anesthesia, a 3 cm incision was made in the piglet's neck with a scalpel, and the scalpel was inserted into the anterior cavity until the heart, and then the blood was let out and dissected. The abdomen was cut open with a scalpel from the lower edge of the ribs to the anus, along the midline of the abdomen, and the piglet intestines were removed, the small intestine and the whole intestine were separated, and the length and weight of the small intestine and jejunum were measured respectively.

[0083] The whole intestine includes the small intestine and the large intestine.

[0084] Among them, the small intestine includes the duodenum, jejunum and ileum.

[0085] At the same time, use sterile scissors to cut 1 cm in the middle of the jejunum. 2 The intestinal tissue of 1 cm in length was washed with saline to remove the sticky contents on the tissue, and then quickly placed in liquid nitrogen and transferred to a -80°C refrigerator for RNA extraction and gene expression quantification. The jejunal tissue of 1 cm in length was taken, washed with saline to remove the contents, and then fixed in 10% neutral paraformaldehyde for paraffin section preparation and histological observation.

[0086] 2) Index determination

[0087] The steps for RNA extraction and gene expression quantification are as follows:

[0088] Total RNA extraction: The total RNA in the tissue was extracted using the Trizol method. About 100 mg of tissue sample was cut into pieces and added to a centrifuge tube containing 1 mL of Trizol solution and grinding beads. The sample was placed in a refrigerated grinding homogenizer for crushing and homogenization to fully mix the lysate and cells to ensure complete cell lysis. The crushing and homogenization conditions were: 3000 × g, 4 ° C, 3 min.

[0089] After adding 0.5 mL of chloroform, shake vigorously for 15 seconds and let stand at room temperature for 3 minutes. Subsequently, centrifuge at 4°C and 12,000×g for 15 minutes. Carefully transfer the upper aqueous phase to a new centrifuge tube, add an equal volume of isopropanol, mix well and let stand at room temperature for 10 minutes, and centrifuge again at 4°C and 12,000×g for 15 minutes to precipitate RNA; pour out the supernatant, wash the RNA precipitate with 75%wt ethanol solution, gently blow and mix well, and centrifuge at 7,500×g for 5 minutes; discard the supernatant, open the lid, let stand in air for 10 minutes to dry the RNA precipitate, then dissolve the RNA with 50μL of RNase-free water, aliquot, and store at -80°C.

[0090] Reverse transcription reaction cDNA: First, after measuring RNA quality and concentration using NanoDrop 2000 nucleic acid spectrophotometer, the RNA sample was diluted to 400 ng / μL by using RNase-free water.

[0091] Among them, NanoDrop 2000 nucleic acid spectrophotometer: Thermo, USA.

[0092] The reverse transcription of mRNA was performed according to the instructions of the PrimeScript RT reagent Kit, and a two-step reverse transcription reaction was performed on a common PCR instrument.

[0093] Among them, PrimeScript RT reagent Kit: Takara, Japan.

[0094] Ordinary PCR instrument: Thermo, USA.

[0095] The two-step mRNA reverse transcription steps are as follows: prepare the reverse transcription system in a 0.2mL RNase-free centrifuge tube. The reaction system for the first step: 5×genomic DNA removal enzyme buffer: 2.0μL; genomic DNA removal enzyme: 1.0μL; RNase-free water: 4.0μL; RNA: 3.0μL; reaction conditions: 42℃, 2min. The reaction system for the second step: 10.0μL of step 1 reaction solution; reverse transcriptase mixture I: 1.0μL; reverse transcription primer mixture: 1.0μL; 5×deoxynucleotide + magnesium ion mixture solution: 4.0μL; RNase-free water: 4.0μL; reaction conditions are 37℃, 15min, 85℃, 5s. After the reaction, add 180μL of RNase-free water and store at -20℃ for mRNA quantification.

[0096] The reverse transcription of miRNA was performed according to the instructions of the Mir-X™ miRNA First-Strand Synthesis Kit, and a one-step reverse transcription reaction was performed on a common PCR instrument.

[0097] Among them, the kit Mir-X™ miRNA First-Strand Synthesis Kit: Takara, Japan.

[0098] Ordinary PCR instrument: Thermo, USA.

[0099] The steps of miRNA one-step reverse transcription are as follows: prepare the reverse transcription system in a 0.2mL RNase-free centrifuge tube. The miRNA reverse transcription system is: 2×mRQ solution: 5.0μL; RNA solution: 3.75μL; mRQ enzyme: 1.25μL; reaction conditions: incubate at 37℃ for 60min; inactivate enzyme activity at 85℃ for 5min; after the reaction, add 90μL of RNase-free water and store at -20℃ for miRNA quantification.

[0100] Real-time fluorescence quantitative PCR reactions were performed on the Roche LightCycler 96 Real-time Quantitative PCR System according to the instructions of the TaKaRa TB Green® Premix Ex Taq™ II Kit.

[0101] Among them, Roche LightCycler® 96 Real-time Quantitative PCR System: Roche, Germany.

[0102] TaKaRa TB Green® Premix Ex Taq™ II Kit: Takara, Japan.

[0103] The total amount of the reaction system was 10 μL, including upstream primer: 0.4 μL; downstream primer: 0.4 μL; Premix Ex Taq™ II premix with DNA fluorescent dye and deoxynucleotide: 5 μL; cDNA template: 1 μL; RNase-free water: 3.2 μL. The PCR reaction conditions were as follows: pre-denaturation: 95°C, 10 min; denaturation: 95°C, 10 s; annealing temperature Tm: 60°C, 10 s; extension: 72°C, 10 s. Three replicates were set for each sample. The primer table is shown in Table 1. Quantification of mRNA and miRNA used 2 −△△t The relative expression of genes was calculated.

[0104] Table 1 Sequence list of primers for mRNA and miRNA quantification

[0105]

[0106] The steps of paraffin section preparation and histological observation are as follows:

[0107] Tissue dehydration: The fixed tissues were immersed in 75%, 85%, 90%, and 95% alcohol (V / V) for 4 h, 2 h, 2 h, and 1 h, respectively; then, they were dehydrated with anhydrous ethanol for 1 h twice; and then, they were transparentized with xylene for 10 min twice.

[0108] Wax dipping and embedding: Place the transparent sample in melted paraffin and replace fresh paraffin at 65℃ several times to ensure sufficient wax dipping. Place the wax-dipping sample in a paraffin embedding box and pour in liquid paraffin, waiting for it to cool and solidify. After cooling, take the wax block out of the embedding box and trim the wax block.

[0109] Sectioning and dewaxing: After the trimmed wax block is cooled at -20℃, a rotary slicer is used to cut the paraffin block into 5μm thick slices. The slices are floated on 40℃ warm water to flatten the tissue, and then transferred to a slide. The slide is placed in an oven at 60℃ to remove excess paraffin and allow the slices to adhere to the slide.

[0110] H&E staining: Place the dehydrated sections in a 1% W / V hematoxylin stain solution, and the staining time is usually 10 minutes. Hematoxylin mainly stains the cell nucleus, making it dark purple. Rinse the sections with distilled water to remove excess hematoxylin stain. Place the hematoxylin-stained sections in a 0.5% W / V eosin stain solution, and the staining time is usually 5 minutes. Eosin mainly stains the cytoplasm, making it pink. Rinse the sections with distilled water to remove excess eosin stain. Soak the stained sections in 75%, 85%, 90%, 95% and 100% concentration gradient alcohol solutions in turn to dehydrate the tissue. Soak the sections in xylene to make them transparent. Add neutral gum to the sections for sealing, and then cover with a coverslip for tissue morphology observation.

[0111] Histomorphological observation: Observe and take pictures under a fluorescence microscope at 100 times magnification.

[0112] 4) Results and analysis

[0113] Figure 1 This is a comparison of the quantitative results of ssc-miR-146a-5p in jejunal tissue of the ssc-miR-146a-5p lentivirus overexpression group, the lentivirus control group and the PBS blank group in Example 2.

[0114] Figure 1 The results showed that the expression level of ssc-miR-146a-5p in the jejunum tissue of piglets in the ssc-miR-146a-5p lentivirus overexpression group was significantly higher than that in the lentivirus control group and the PBS blank group, P<0.001, indicating that the construction of the ssc-miR-146a-5p lentivirus overexpression vector in Example 1 was successful, and ssc-miR-146a-5p could be successfully overexpressed in the jejunum of suckling piglets.

[0115] Figure 2 The average weight of piglets in the ssc-miR-146a-5p lentivirus overexpression group, lentivirus control group and PBS blank group during the trial period in Example 2 was compared. 14 days after intraperitoneal injection of ssc-miR-146a-5p lentivirus, it was found that the average weight of piglets in the ssc-miR-146a-5p overexpression group was significantly higher than that in the lentivirus control group, P<0.05, and there was an increasing trend compared with the weight of piglets in the PBS blank group, P=0.07.

[0116] Figure 3 This is a comparison of the length and weight of the small intestine and the whole intestine of piglets in the ssc-miR-146a-5p lentivirus overexpression group, the lentivirus control group and the PBS blank group in Example 2.

[0117] Figure 3 The results showed that the length of the small intestine and the whole intestine of the piglets in the ssc-miR-146a-5p lentivirus overexpression group was significantly increased, P<0.05; compared with the lentivirus control group, the length of the small intestine of the piglets in the ssc-miR-146a-5p lentivirus overexpression group had an increasing trend, P=0.07. At the same time, the weight of the small intestine of the piglets in the ssc-miR-146a-5p lentivirus overexpression group was significantly higher than that of the piglets in the lentivirus control group, P<0.05.

[0118] Figure 4 These are representative images of H&E stained intestinal morphology microscopic observation of piglet jejunum tissue in the ssc-miR-146a-5p lentivirus overexpression group, lentivirus control group and PBS blank group in Example 2.

[0119] Figure 5 This is a comparison of the villus intestinal morphology and crypt depth of the piglets in the jejunum of the ssc-miR-146a-5p lentivirus overexpression group, the lentivirus control group, and the PBS blank group in Example 2. It can be seen that the villus length and crypt depth of the jejunum of the piglets in the ssc-miR-146a-5p lentivirus overexpression group were significantly higher than those in the lentivirus control group and the PBS blank group, P<0.05.

[0120] Figure 6 Figure 2 shows the expression of cell proliferation-related genes in the jejunum of piglets in the ssc-miR-146a-5p lentivirus overexpression group, the lentivirus control group, and the PBS blank group in Example 2. It can be seen that the Wnt pathway downstream genes c-Myc and cell cycle proteins in the jejunum of piglets in the ssc-miR-146a-5p lentivirus overexpression group CCND-1 , proliferating cell nuclear antigen PCNA Growth hormone receptor EGFR The gene expression was significantly higher than that in the lentivirus control group and PBS blank group, P<0.05.

[0121] Figure 7 This is a comparison of the expression of intestinal mucosal barrier-related genes in the jejunum tissue of piglets in the ssc-miR-146a-5p lentivirus overexpression group, the lentivirus control group and the PBS blank group in Example 2.

[0122] Figure 7 The results showed that the intestinal mucin in the jejunum tissue of piglets in the ssc-miR-146a-5p lentivirus overexpression group Occludin and ZO-1 The gene expression level was significantly higher than that in the lentivirus control group and PBS blank group, P<0.05.

[0123] The above results indicate that ssc-miR-146a-5p can improve the growth performance of suckling piglets, promote intestinal development of suckling piglets, and improve intestinal barrier and health functions.

[0124] The application of ssc-miR-146a-5p provided by the present invention in the preparation of a product for promoting intestinal development in piglets is a related product use of ssc-miR-146a-5p derived from pig milk to promote intestinal development in piglets. The present invention develops a new application of ssc-miR-146a-5p derived from pig milk in promoting intestinal development in piglets, which is a related product application research on microRNA in promoting intestinal development in piglets, solving the problem of unclear use of microRNA in promoting intestinal development in piglets.

[0125] It should be noted that when the present invention involves a numerical range, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes a preferred embodiment.

[0126] Although preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once the basic inventive concepts are known, and all such changes and modifications fall within the scope of the present invention.

Claims

1. Application of ssc-miR-146a-5p in the preparation of a product for promoting intestinal development in piglets, characterized in that: The nucleotide sequence of the ssc-miR-146a-5p is shown in SEQ ID NO.1; The piglets are suckling piglets; The product is used to improve the intestinal morphology of suckling piglets; Improving the intestinal morphology of suckling piglets refers to increasing the length of villi and the depth of crypts in the jejunum of suckling piglets; The product is used to increase the weight and length of the small intestine and the entire intestine of suckling piglets.

2. The use of ssc-miR-146a-5p according to claim 1 in preparing a product for promoting intestinal development in piglets, characterized in that: The product is a medicine or a feed additive.

3. The use of ssc-miR-146a-5p according to claim 2 in preparing a product for promoting intestinal development in piglets, characterized in that: The product contains the ssc-miR-146a-5p or an agonist of the ssc-miR-146a-5p as an active ingredient.

4. The use of ssc-miR-146a-5p according to claim 3 in preparing a product for promoting intestinal development in piglets, characterized in that: The ssc-miR-146a-5p agonist includes an overexpression vector containing ssc-miR-146a-5p.

5. The use of ssc-miR-146a-5p according to claim 4 in preparing a product for promoting intestinal development in piglets, characterized in that: The overexpression vector containing ssc-miR-146a-5p is the ssc-miR-146a-5p lentiviral overexpression vector.

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