CN113699153A

CN113699153A - miRNA related to sheep F17 escherichia coli and application thereof

Info

Publication number: CN113699153A
Application number: CN202111197413.XA
Authority: CN
Inventors: 孙伟; 葛玲; 邹双霞; 袁泽湖
Original assignee: Yangzhou University
Current assignee: Yangzhou University
Priority date: 2021-10-14
Filing date: 2021-10-14
Publication date: 2021-11-26
Also published as: NL2033164B1; NL2033164A

Abstract

The present invention proposes a molecular marker miR-299-5p related to sheep F17 Escherichia coli, the targeting sheep beta defensin 2 gene ( SBD2 ) of the miR-299-5p, and miRNA in the study of sheep small intestinal epithelial cells The application in anti-F17 Escherichia coli infection, the NCBI sequence number of the SBD2 gene is NM_001198545.1, and miR‑299‑5p can improve the ability of small intestinal epithelial cells to resist F17 Escherichia coli infection of small intestinal epithelial cells. The invention can not only regulate the infection of small intestinal epithelial cells against F17 Escherichia coli by negatively regulating the target gene, but also can further explore the role of SBD2 in Hu sheep small intestinal epithelial cells against F17 Escherichia coli infection and its regulatory mechanism. , to provide a theoretical basis for the application of defensins in production. The miR-299-5p disclosed in the present invention can also be applied to provide a new drug target for developing antibacterial drugs for diseases caused by Escherichia coli F17, and can be used to evaluate and improve the economic benefits of sheep farming.

Description

miRNA related to sheep F17 escherichia coli and application thereof

Technical Field

The invention relates to miRNA related to sheep F17 escherichia coli and application thereof, belonging to the technical field of genetic engineering.

Background

It is known that E.coli of diarrheal type is one of the common pathogens which are capable of causing diarrhea in a wide range of humans and animals. The F17 Escherichia coli is a gram-negative bacterium, can cause large-scale diarrhea of newborn lambs, and has high lethality rate. With the rapid modernization and large-scale development of the sheep raising industry, colibacillosis becomes one of the important factors restricting the rapid development of the sheep industry. In order to improve the survival rate of lambs and promote the rapid and healthy development of the Chinese sheep industry, the pathogenesis of colibacillosis is overcome from the genetic aspect, and therefore the problem of improving the resistance of lambs to colibacillosis is a problem which is urgently needed to be solved at present.

MicroRNA (miRNA) is an endogenous single-stranded non-coding small-molecule RNA consisting of about 19-22 nucleotides and is involved in almost all the life activity process of cells. It has been shown that mirnas affect several major biological pathways by modulating most protein-encoding genes. However, miRNA markers that regulate ovine F17 colibacillosis are currently lacking.

Disclosure of Invention

The invention aims to solve the technical problem that miRNA related to sheep F17 escherichia coli and application thereof are provided to overcome the defects of the prior art, and the infection of small intestine epithelial cells against F17 escherichia coli can be regulated and controlled through negative regulation and control of target genes; the research on the function of SBD2 in resisting F17 escherichia coli infection of sheep small intestine epithelial cells and the regulation mechanism thereof can be further deeply researched, a certain theoretical basis is provided for the application of defensins in production, the F17 escherichia coli resistance of lambs is improved, and the economic benefit of sheep farming is improved.

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention provides a miRNA related to sheep F17 Escherichia coli, wherein the target gene of the miRNA is SBD2, the NCBI accession number of the miRNA is NM-001198545.1, and the nucleotide sequence of the miRNA is shown in SEQ ID NO. 1.

The invention also provides a miRNA related to sheep F17 escherichia coli, wherein the miRNA is miR-299-5p, and the nucleotide sequence of the miRNA is shown in SEQ ID NO. 2.

The invention detects the gene expression condition after the Hu sheep lamb small intestine epithelial cells infect F17 escherichia coli for about 6 hours, screens out F17 escherichia coli key gene SBD2, and then predicts miRNA with a targeting relation with gene SBD2 through bioinformatics, namely: miR-299-5 p.

The invention also provides application of the miRNA related to the sheep F17 escherichia coli in resisting the sheep F17 escherichia coli infection.

The sheep are Hu sheep, and the small intestinal epithelial cells of Hu sheep lambs of 3-5 days old are preferably selected. The time for infecting the lamb small intestine epithelial cells by the F17 escherichia coli is 0h and 6 h.

The miR-299-5p related to the sheep F17 escherichia coli is applied to research on F17 escherichia coli infection resistance of the Hu sheep small intestine epithelial cells, and the miR-299-5p regulates and controls the F17 escherichia coli infection resistance of the Hu sheep small intestine epithelial cells through negative regulation and control of a target gene SBD 2.

According to the invention, qRT-PCR verification shows that the relative expression quantity of miR-299-5p after Hu sheep small intestine epithelial cells are infected with F17 escherichia coli for about 6 hours is strongly and negatively correlated with the relative expression quantity of gene SBD2, and the existence of a targeting relationship between miR-299-5p and gene SBD2 is preliminarily judged.

The application comprises that miR-299-5p reduces the number of F17 escherichia coli adhered to the small intestine epithelial cells of Hu sheep.

The application comprises the capability of miR-299-5p for inhibiting the small intestine epithelial cells from infecting Escherichia coli F17. Thus, miR-299-5p can improve the capability of the small intestine epithelial cells in resisting the infection of the small intestine epithelial cells by F17 Escherichia coli.

The miRNA including the miR-299-5p can be used for preparing a novel molecular drug target for preventing, relieving or treating sheep F17 colibacillosis.

Compared with the prior art, the invention has the advantages that the molecular marker miR-299-5p related to sheep F17 escherichia coli is provided, the infection of small intestine epithelial cells F17 escherichia coli can be controlled through the negative regulation and control of target genes SBD2, the function and the regulation and control mechanism of SBD2 in the Hu sheep small intestine epithelial cell F17 escherichia coli infection resistance can be further deeply researched, a certain theoretical basis is provided for the application of defensin in production, the F17 escherichia coli disease resistance of lambs is further improved, and the economic benefit of sheep farming is improved. The miR-299-5p disclosed by the invention is also applied to providing a new drug target for researching and developing antibacterial drugs for diseases caused by F17 escherichia coli, and can be used for evaluating and improving the economic benefit of sheep raising industry.

Drawings

FIG. 1 is a map of a psiCHECK-2 plasmid of the present invention.

FIG. 2 is a schematic diagram of miR-299-5p expression levels before and after Hu sheep small intestine epithelial cells are infected by F17 Escherichia coli in the invention. Wherein, compared to the control group, denotes 0.01< P <0.05, denotes P < 0.01.

FIG. 3 is a schematic diagram of the verification result of miR-299-5p mimics transfection efficiency in the invention.

FIG. 4 is a schematic diagram of the verification result of miR-299-5p inhibitor transfection efficiency in the invention.

FIG. 5 is a diagram showing the identification result of a wild-type vector in the present invention. Wherein A represents psiCHECK-2, B represents a wild-type vector, C represents psiCHECK-2 double enzyme digestion, D to E represent wild-type vector double enzyme digestion, F represents SBD 2-3' -UTR double enzyme digestion, and 1, 2 and 3 represent wild-type vector colony PCR.

FIG. 6 is a diagram of miR-299-5p target site prediction in the invention.

FIG. 7 shows the sequencing peaks of the wild-type and mutant vectors of the present invention.

FIG. 8 is a diagram showing the results of the dual luciferase activity assay of the present invention.

FIG. 9 is a schematic diagram showing the effect of miR-299-5p on the relative expression amount of SBD2 in the invention.

FIG. 10 is a schematic diagram of the effect of miR-299-5p on SBD2 protein expression in the invention.

FIG. 11 is a schematic diagram of the ability of miR-299-5p to resist F17 Escherichia coli to infect small intestine epithelial cells in the invention.

Detailed Description

The technical scheme of the invention is further explained in detail by combining the embodiment as follows: the present embodiment is implemented on the premise of the technical solution of the present invention, and a detailed implementation and a specific operation process are given, but the protection authority of the present invention is not limited to the implementation described below. Experimental procedures, for which specific conditions are not noted in the following examples, were selected according to methods and conditions conventional in the art, or according to commercial instructions. The reagents and materials involved in the following examples are commercially available and are not listed here.

Example 1miRNA prediction and screening

Infecting F17 escherichia coli on the small intestinal epithelial cells of the Hu sheep lambs of 3-5 days old, detecting the gene expression condition of the Hu sheep lambs after about 6 hours by adopting a qRT-PCR technology, and screening out a key gene SBD 2. And predicting miRNAs of the SBD2 in a targeted manner by using bioinformation software such as TargetScan and RNAhybrid, wherein the miRNAs are oar-miR-299-5p, oar-miR-370-3p, oar-miR-412-5p and oar-miR-541-5p respectively, and simultaneously performing primary screening on the predicted miRNAs by using a qRT-PCR technology. The specific screening method of the target miRNA of SBD2 is as follows: with a concentration of 1X 10⁷F17 escherichia coli of CFU/mL respectively infects Hu sheep small intestine epithelial cells, total RNA of the cells is extracted after 6 hours, and after reverse transcription to cDNA, qRT-PCR is carried out to detect the relative expression quantity of oar-miR-299-5p, oar-miR-370-3p, oar-miR-412-5p and oar-miR-541-5 p. The data obtained were analyzed by SPSS 25.0 and plotted by Graphpad Prsim 6 (see fig. 2). The result shows that oar-miR-541-5P has no obvious change before and after the F17 escherichia coli infects small intestine epithelial cells and has no statistical significance (P)>0.05), the relative expression level of oar-miR-370-3P is remarkably increased after F17 escherichia coli infects small intestine epithelial cells (P17 escherichia coli)<0.01), the relative expression amounts of oar-miR-299-5P and oar-miR-370-3P are extremely reduced after the F17 escherichia coli infects small intestine epithelial cells (P)<0.01)。

Example 2 amplification of the 3' -UTR region of SBD2

2.1 primer design

According to the 3 '-UTR sequence of sheep beta-defensin 2(SBD2) in a GeneBank library, primers are designed by using Primer 5.0 software, and Xho I and Not I enzyme cutting sites are respectively inserted into the upstream and downstream 5' ends of the set primers. The primers were obtained from Oncorks Biotechnology (Beijing) Ltd, and the design of the primers is shown in Table 1.

TABLE 1 primers for SBD 2-3' -UTR

2.2PCR amplification of SBD2 fragment

Using DNA of Hu sheep small intestine epithelial cell as template

Max DNA Polymerase high fidelity enzyme (Takara) was used for PCR amplification, and the specific reaction solution was prepared as shown in Table 2.

TABLE 2PCR reaction solution

The preparation of the PCR reaction solution is operated on ice, and after the reaction solution is prepared, the reaction solution is instantaneously centrifuged and placed in a PCR instrument for the following reactions:

the obtained PCR product was purified and recovered with a SanPrep column PCR product purification kit (Bio-engineering), and then sent to the department of biotechnology (Beijing) Ltd for sequencing.

Example 3 Dual luciferase reporter vector construction

3.1psiCHECK-2 plasmid extraction

Sucking psiCHECK-2 Plasmid (Takara), diluting, coating on LB solid culture medium containing Amp +, placing in an incubator at 37 ℃ for culturing for 12-16h, picking single colony to LB liquid culture medium sterilizing centrifuge tube containing Amp +, shaking for 12-16h by a shaking table, extracting Plasmid according to an endotoxin-free Plasmid minim Plasmid Kit II (Tiangen), and sending to Protechinque (Beijing) Limited company for sequencing, wherein the map is shown in figure 1.

3.2 cleavage of SBD 2-3' -UTR with psiCHECK-2 plasmid

The target SBD 2-3' -UTR and psiCHECK-2 plasmids recovered above were used in Quickcut^TMXho I and Quickcut^TMNotI enzyme was used for double digestion, and the reaction solution was prepared as shown in Table 3.

TABLE 3 double digestion reaction

The reaction system is placed in a PCR instrument and incubated at 37 ℃ for 30min, and then purified and recovered again by using a SanPrep column type PCR product purification kit.

3.3 ligation of the fragment of interest and the plasmid

The purified product obtained in the above step was purified by DNA Ligation Kit Ver.2.1 Kit (Takara) according to the plasmid psiCHECK-2: the target fragments were mixed at a molar ratio of 1:8, centrifuged instantaneously and ligated overnight in a PCR instrument at 16 ℃ to obtain the ligation products, the specific mixing ratios are shown in table 4.

TABLE 4 plasmid ligation reaction solution

3.4 transformation of ligation products and Positive clone identification

(1) The competent cells DH5 alpha (Takara) were thawed on ice in advance, and the water bath was opened to 42 ℃ to melt the SOC medium in water bath.

(2) Pipette 10. mu.L of ligation product into 100. mu.L of competent cells, and place on ice for 30 min.

(3) And (3) thermally shocking in a water bath kettle at 42 ℃ for 1min, immediately taking out and carrying out ice bath for 2min, adding 1mL of preheated SOC culture medium, and shaking in a shaking table at 37 ℃ for 1h until the SOC culture medium is completely and uniformly mixed.

(4) Sucking 50 mu L of bacterial liquid, coating the bacterial liquid on an LB solid culture medium containing Amp +, and placing the bacterial liquid in an incubator for culturing for 12-16h at 37 ℃.

(5) And (4) picking a plurality of single colonies into a sterilized centrifuge tube of LB liquid culture medium containing Amp +, and shaking the shaker for 12-16 h.

(6) Extracting plasmids by using an endotoxin-free Plasmid small and medium extraction Kit EndoFree Plasmid Kit II (Tiangen), carrying out enzyme digestion identification, and sending the positive plasmids to the Qin Biotechnology (Beijing) Co.

Construction and identification of psiCHECK-2 wild-type and mutant vectors: PCR amplification of the 3' -UTR sequence of the target gene SBD2 and use of Quickcut^TMNot I and Quickcut^TMXho I restriction enzyme cuts a target product with good specificity, is connected to a psiCHECK-2 dual-luciferase reporter vector, and plasmid sequencing detects whether a 3' -UTR fragment of a target gene SBD2 is successfully inserted into the vector. The result is shown in fig. 5, after electrophoresis, the PCR amplification verification product of the bacterial liquid shows that the position of the band is consistent with the position of the target fragment, and the obtained sequencing sequence is consistent with the comparison result of the original sequence, which indicates that the psiCHECK-2 report vector is successfully constructed.

Designing a target sequence mutation primer on the basis of a wild-type vector by utilizing a PCR mutation method:

SBD2-WUT–F:

GTCCTTAGACAGAGCGTCTAAAATTTCACCCGGAATAAATTTT

G

SBD2-WUT-R:

CAAATTATTCCGGGTGAATTTTTAAGACGCTCTGTCTAAGGACmiR-299-5 p target site prediction and mutation sites are shown in FIG. 6:

mutation of TAAACCA into TCACCCG is carried out by Fast Site-Directed Mutagenesis Kit (Tiangen) mutation, and the specific steps are as follows:

and amplifying the plasmid containing the mutation site by using a wild type vector as a template, and then amplifying by using FastAlternation DNA Polymerase to finally obtain the mutant plasmid with a gap. The methylated template plasmid is digested with Dpn I, leaving the mutated plasmid just amplified, and after transformation into a recipient bacterium, the nicks in the mutated plasmid are repaired, allowing replication. The plasmid was sent to Nanjing Ongjingkidaceae Biotechnology Co., Ltd for sequencing and identification, and the sequencing peak diagram is shown in FIG. 7.

Example 4 validation of targeting relationship of miR-299-5p and SBD2

4.1293T cell recovery and passage

Cell resuscitation

(1) The 293T cells were taken out from the liquid nitrogen holding bucket, put into PE gloves, and quickly shaken in a 37 ℃ water bath until the frozen solution was thawed.

(2) And transferring the cells in the frozen tube to a sterilized centrifuge tube, adding an equal amount of culture solution, and repeatedly blowing and beating.

(3) Centrifuge at 1200rpm for 5 min. The supernatant was discarded, and the cells were gently washed 3 times with PBS buffer, centrifuged, and the supernatant was discarded.

(4) The precipitated cells were resuspended in the culture medium, and transferred to a culture flask (a small amount of culture medium was added to the flask in advance) to make up to 5mL of the culture medium.

(5) Observing with an inverted microscope, adding CO₂Culturing in an incubator, and changing the culture solution after 24 h.

Cell passage

(1) Old culture medium was carefully aspirated, gently washed 3 times with PBS buffer, and waste liquid was discarded.

(2) Adding pancreatin 0.5mL (covering the surface of the culture dish) to digest the cells, and placing in CO₂Digesting in an incubator for 5min, adding an equal amount of culture solution along the wall of a culture flask, repeatedly blowing adherent cells, stopping digestion, and transferring to a sterilized centrifuge tube.

(3) Centrifuging at 1200rpm for 5min, discarding supernatant, washing with PBS for 3 times, centrifuging, and discarding waste liquid.

(4) Adding culture solution, slightly blowing and beating to prepare cell suspension, transferring the cell suspension into a culture bottle, and supplementing the cell suspension to 5 mL.

(5) Observing under an inverted microscope, adding CO₂An incubator.

4.2 Dual luciferase reporter Gene detection

And (3) inoculating 293T cells into a cell culture plate, and performing cell transfection when the cell growth fusion degree reaches 60-80%. According to the jetPRIME transfection reagent (Polyplus) and miRNA specification (gimerap gene), the specific transfection experiments were grouped as follows:

a first group: psiCHECK-2-SBD2-MUT + miR-299-5p mimics

Second group: psiCHECK-2-SBD2-MUT + miR-299-5p mimics-NC

Third group: psiCHECK-2-SBD2-WT + miR-299-5p mimics

And a fourth group: psiCHECK-2-SBD2-WT + miR-299-5p mimics-NC

(1) The medium in the transfection plate was discarded and washed 3 times with PBS buffer without the double antibody.

(2) Before each use, 5 × Cell Lysis Buffer: ddH is added₂The mixture was kept on ice until the mixture was 1: 4.

(3) The Luciferase Substrate was removed from-70 ℃ and mixed with Stop & Reaction Buffer (Renilla Substrate: 50: 1) and kept in the dark at room temperature.

(4) Discarding the cleaning solution, adding 100 mu L of 1 × Cell Lysis Buffer into each hole, standing at room temperature for 5-10 min, blowing Cell Lysis products, collecting into a 1.5mL sterile centrifuge tube, centrifuging at 12000rpm for 2min, and taking the supernatant for subsequent detection.

(5) And (3) balancing 100 mu l of Luciferase Substrate to room temperature, adding the Substrate to the ELISA plate, carefully absorbing 20 mu l of cell lysate to the ELISA plate, rapidly mixing the Substrate uniformly, and immediately detecting the activity of the Firely Luciferase reporter gene in a fluorescence detector.

(6) 100 μ l of freshly prepared Renilla Substrate working solution is added into the reaction solution, and the activity of the Renilla Substrate reporter gene is detected after the mixture is rapidly and uniformly mixed.

Targeting relationships were verified using a dual luciferase reporter system: the successfully constructed wild type psiCHECK-2-SBD2-WT and mutant psiCHECK-2-SBD2-MUT dual-luciferase reporter vectors are transfected to 293T cells respectively with miR-299-5P mimics and Negative Control, and the dual-luciferase fluorescence activity is detected by a microplate reader, so that the results are shown in figure 8, the luciferase activity of the group transfected with miR-299-5P and psiCHECK-2-SBD2-WT plasmids is remarkably different from that of the luciferase of a Control group (P is less than 0.01), the luciferase activity of the group transfected with the psiCHECK-2-MUT plasmids is remarkably different from that of the luciferase of the Control group, and the difference is not remarkable by combining the results, so that the target gene of miR-299-5P is SBD 2.

Example 5 functional validation of miRNA

5.1 culture and transfection of Hu sheep small intestine epithelial cells

Uniformly inoculating the small intestine epithelial cells with good growth state on a cell culture plate, and respectively transfecting miR-299-5p mimics, miR-299-5p mimics-NC, miR-299-5p inhibitors and miR-299-5p inhibitors-NC to the small intestine epithelial cells according to a jetPRIME transfection reagent specification and an miRNA use specification when the cell growth fusion degree is 30% -50%, wherein the specific operation is as follows:

(1) discarding the original culture medium in the cell plate, washing with PBS for 2 times, each time for about 30s, and adding 1mL culture medium into each hole;

(2) diluting miRNA to

Vortex in buffer for 5 s;

(3) adding 4 mu L of jetPRIME transfection reagent, whirling for 1s, and centrifuging for a short time; incubating at room temperature for 15 min;

(4) uniformly dripping the transfection mixed solution into a cell plate;

(5) gently shake the cell plate back and forth and side to side and incubate at 37 ℃.

5.2 Total RNA extraction from cells

The method is characterized by extracting with a TRNzol total RNA extraction reagent (Tiangen), and comprises the following specific steps:

(1) the old culture solution in the cell culture plate is discarded, the cell culture plate is lightly washed 3 times by PBS buffer solution without double antibody, TRNzol lysate is added, and the cell culture plate is placed at room temperature for lysis for 15 min. Collect the lysed cell fluid into an enzyme-free centrifuge tube.

(2) Add 200. mu.L chloroform to each 1mL TRNzol lysate, vortex for 15s, and then let stand at room temperature for 5 min.

(3) The refrigerated centrifuge was pre-cooled to 4 ℃ by idling in advance, and the cell sap in the enzyme-free centrifuge tube was centrifuged at 12000rpm at 4 ℃ for 15 min.

(4) The sample was now divided into three layers and the upper aqueous phase was transferred to a new 1.5mL enzyme free centrifuge tube.

(5) Adding isopropanol into the centrifuge tube, mixing, and standing at room temperature for 10 min.

(6) Then, the mixture was centrifuged at 12000rpm at 4 ℃ for 10min, and the supernatant was discarded.

(7) Adding 75% ethanol (prepared with 0.1% DEPC water) to the adherent, washing upside down, centrifuging at 12000rpm at 4 deg.C for 5min, discarding supernatant, centrifuging instantaneously, and sucking out the rest liquid with a tip.

(8) Standing at room temperature for 2-3min, air drying, adding RNase free water (30-60 μ L), repeatedly beating, mixing, and dissolving RNA completely.

(9) The concentration is measured by a microplate reader, and the product is stored at the temperature of minus 80 ℃.

5.3 reverse transcription of miRNA and qRT-PCR

The upstream primer and stem-loop primer of miR-299-5p are synthesized in the official network design of Novozan Biotechnology GmbH (Table 5), Qincao Biotechnology (Beijing) GmbH, and the downstream Universal primer is miRNA Universal

The qPCR Master Mix kit (nuozazan) was self-contained. And detecting the expression level of miRNA by using the U6 gene as an internal reference. The primer sequences are as follows:

TABLE 5 primer information Table

The reaction system using miRNA 1st Strand cDNA Synthesis Kit (by stem-loop) (Novozam) Kit instructions is as follows:

(1) preparing a mixed solution of a genomic DNA removal system (ice operation):

gently shaking and mixing, and incubating for 2min at 42 ℃;

(2) preparing a reverse transcription reaction system (ice operation):

(3) and (3) PCR reaction conditions: 5min at 25 ℃, 15min at 50 ℃ and 5min at 85 ℃;

(4) the PCR product was stored at-20 ℃ for subsequent qRT-PCR detection.

Reference miRNA Universal

qPCR Master Mix kit instructions, reaction system as follows (prepared on ice):

setting a reaction program: at 95 ℃ for 5min, at 95 ℃ for 10s, at 60 ℃ for 15s, for 40 cycles; melt Curve.

5.4 recovery and culture of Escherichia coli strain F17

When the strains are recovered, the glycerol strains are taken out of the ultra-low temperature refrigerator, the glycerol strains are placed in an ice bath at low temperature or 0 ℃ when in use, a small amount of frozen strains are picked by using an inoculating loop or a toothpick and put on a plate, and the plate is cultured for 8-12 hours at 37 ℃. After the strain is used, the strain is put back into an ultra-low temperature refrigerator as soon as possible, and the melted part on the surface is picked up during inoculation and can not be completely dissolved, and cell walls can be broken due to repeated freezing and melting. Picking single bacterial colony with a sterilizing gun head, feeding into a sterilizing centrifuge tube added with a culture medium, sealing the tube opening, and culturing at 37 ℃ by a shaking table at 200 r/min.

5.5 adhesion test

When the epithelial cells of the small intestine grow to 30% -50% confluence, respectively transfecting miR-299-5p mimics, miR-299-5p mimics-NC, miR-299-5p mimics and miR-299-5p inhibitors-NC to the epithelial cells of the small intestine according to the jetPRIME transfection reagent instruction and the miRNA use instruction, after 24 hours, centrifuging the F17 escherichia coli liquid cultured in the step 5.4 at 4 ℃ for 10 minutes, removing the culture medium, and adding an F12 culture medium without serum and antibiotics to dilute the F17 escherichia coli to 1 × 10⁷CFU/mL, 1mL of inoculum was added to each well. The plates were incubated at 37 ℃ in 5% CO₂Culturing in incubator for 3 hr, collecting supernatant, and storing at-80 deg.C. The column was washed with PBS buffer for 3 times and the waste solution was discarded. To each well was added 300. mu.L of 0.5% Triton X-100, cracking the lysate for 30min, collecting the lysate, adding 200 mu L of the lysate, washing, collecting washing liquid for 2 times, collecting 700 mu L of the lysate in a 1.5mL sterile centrifuge tube, uniformly mixing the lysate and the washing liquid by oscillation, diluting by multiple times, coating the diluent on an LB culture dish, inverting the culture dish in a constant-temperature incubator at 37 ℃ for overnight culture, and counting the number of colonies attached to cells.

5.6 enzyme-linked immunosorbent assay (ELISA)

Referring to the specification of a sheep beta defensin 2 (beta-Defensins 2) enzyme-linked immunosorbent assay (ELISA) detection kit, the specific experimental steps are as follows:

(1) the stored sample was taken out from the-80 ℃ ultra-low temperature refrigerator in step 5.5, centrifuged for about 20min (2000-3000rpm), and the supernatant was collected.

(2) Sample adding of the standard: the standard wells were each filled with 50. mu.L of standard at different concentrations.

(3) Sample adding: and a blank hole and a sample hole to be detected are respectively arranged. Adding 50 mu L of sample diluent into a blank hole on the enzyme-labeled coated plate, adding 40 mu L of sample diluent into a sample hole to be detected, then adding 10 mu L of samples to be detected with different concentration gradients and time gradients, and slightly shaking and uniformly mixing.

(4) Adding an enzyme: add enzyme labeling reagent 100. mu.L to each well except for blank wells.

(5) And (3) incubation: the plates were sealed with a sealing plate and incubated at 37 ℃ for 60 min.

(6) Preparing liquid: diluting the 20 times of concentrated washing solution with 20 times of distilled water for later use.

(7) Washing: carefully uncovering the sealing plate membrane, discarding liquid, spin-drying, filling washing liquid into each hole, standing for 30s, discarding, repeating the steps for 5 times, and patting dry.

(8) Color development: adding 50 μ L color-developing agent A into each well, adding 50 μ L color-developing agent B, shaking gently, mixing, and developing at 37 deg.C in dark for 15 min.

(9) And (4) terminating: the reaction was stopped by adding 50. mu.L of stop solution to each well.

(10) And (3) determination: the absorbance (OD value) of each well was measured sequentially at a wavelength of 450nm with the blank well being zeroed. The determination should be performed within 15min after the addition of the stop solution.

Effect of miR-299-5p on SBD2 expression: transfecting the miR-299-5p mimics and inhibitors to small intestine epithelial cells, extracting total RNA of the cells, carrying out reverse transcription to obtain cDNA, and carrying out qRT-PCR (quantitative reverse transcription-polymerase chain reaction) to detect the relative expression of SBD 2. The data obtained were analyzed by SPSS 25.0 and plotted by Graphpad Prsim 6 (see fig. 9 and 10). As shown in FIG. 9, compared with the control group, the mimics of miR-299-5P has the relative expression amount of SBD2 extremely reduced (P <0.01), and compared with the control group, the inhibitor of miR-299-5P has the relative expression amount of SBD2 extremely increased (P < 0.01). As shown in FIG. 10, compared with the control group, the mimics of miR-299-5P has the advantages that the protein expression of SBD2 is extremely reduced (P <0.01), and compared with the control group, the inhibitor of miR-299-5P has the advantages that the relative protein expression of SBD2 is extremely increased (P < 0.01).

The ability of miR-299-5p to resist F17 Escherichia coli to infect epithelial cells of small intestine: the mimic and the inhibitor of the miR-299-5p are transfected to the small intestine epithelial cells to carry out adhesion experiments, and the ability of the miR-299-5p to the small intestine epithelial cells to resist F17 escherichia coli infection is detected by calculating the colony number adhered to the small intestine epithelial cells. The results are shown in fig. 11, the number of colonies adhered to the small intestine epithelial cells of the transfected mimics group is significantly higher than that of the transfected control group (P <0.01), the number of colonies adhered to the small intestine epithelial cells of the transfected inhibitor group is significantly lower than that of the transfected control group (P <0.01), and the ability of the miR-299-5P to regulate the small intestine epithelial cell to resist the F17 escherichia coli infection is demonstrated.

The above description is only an embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can understand that the modifications or substitutions within the technical scope of the present invention are included in the scope of the present invention, and therefore, the scope of the present invention should be subject to the protection scope of the claims.

Sequence listing

<110> Yangzhou university

<120> miRNA related to sheep F17 escherichia coli and application thereof

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1888

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

tataaagcgg cattcgcagc ctcttctcca gcatcagctg cagagctcgt gacgccaaca 60

tgaggctcca tcacctgctc ctcgtgctct tcttcgtggt cctgtctgct gggtcaggtg 120

agctcgtggg agcccctggg ggagtcgcgg gctctctctc ctgtttctac ctccttctgt 180

cctgctaccc tcatctacac gtggtcagac taaacccacc gcatttgatg cttctgagaa 240

gccaccgctg agtccttagt agcgagaggg gtctgagaac tgtcctgcaa aatttctggc 300

tgtttctaag ccactctagc gagccaaagc ttctgagccc gtctcctcac tggtttcatg 360

aggaggaaac cgaagatgcc tctgataagt gactctcctt tttttctttt tcgtaaaagc 420

aagatatttg attttgtccc atgacagaag cgcagaatgt tgctttataa atctttctat 480

ttgaagggtg gtggctctag gtgaccggag aagtccagag gagagtcagg ccgagcctgg 540

gcttcagagg ctgcccttga gacgctccgg gctgagtctc ctcgcaggac acgtcgctga 600

tgatgcgacc ctcgctccac agctgggagg cagcgcatcc aaagcagtgt gcaggatcgg 660

ctgtctaatg ttcatcttac ccttgatatt tccagaaatt ggatgaaaat atgtaggaag 720

gaaggaggga gggaggaagg gagagagagg atgcggagac actgagacac ctgattaagt 780

gtcagaaatc aacttgaaaa gcccttgtct ggtcagtgtt gtttatggct gtaacccaac 840

tcttagcaca gtggcaggga gagaggacag gggagtaagg aagacggcct ctcagtgccc 900

ggggcctggg ttcaatccct cggggaggaa ctagagaggg accagaggtg ccttgtggcc 960

aaaagagaga aaggggtgtg aggaagacgg gaaggcagcg gctggtcagg aggaaaccac 1020

acagggagga ggagaggggc tgactcgcac cagcgctggg agcgatccga gccagtggag 1080

acaatggtac aaaacttttt ctctgaacca tcgtcaccgt cagctgtaag ttcctgtgag 1140

agtaagctct ctttaaacag ttcttacact ctcagcggca tcttttcctc ctatctcagc 1200

tccactgctt gactgactcc atgaggtgga aaactaaagg gtgaagatag acccccgcgg 1260

gccgcttctg actcctggcg ggagggtgga tgcagcagag cttcccggtc tttgccctcg 1320

tggtggagct gaccccgcac acaaccgggg tcctcacgcc ccagtccctc ggcctctggt 1380

tctggaaaca tgagggtcca ccagagcctg ggcagggtca gcgtctgtct ggaagctgag 1440

ccacggggtc ctggactcac atcttgttgt catgaagccg tgtccacatc tcagcacaga 1500

gccccagggc cttgctcaga ggggacacag ctggccttct cgagatgccg ctttcctgct 1560

gacacgttta tccctctttg ttctcttttc aggatttact catggagtaa cagatagtct 1620

aagctgccgt tggaagaaag gcatctgtgt gctgaccagg tgccctggaa ccatgagaca 1680

gattggcacc tgtttcgggc ccccagtaaa atgctgcaga ctgaagtaac agaaggcgaa 1740

gacgcggccg ggaccgatgc ggagtcagaa actgcgtcct tagacagagc gtctaaaatt 1800

taaaccagaa taaattttgt tcaaagttaa agaatcttgc ccactggtca ttgaggttgt 1860

tgtgtggtgt ctgatcccag gtgaattc 1888

<210> 2

<211> 22

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

uacauacacc cugccauuug gu 22

Claims

1. An ovine F17 E.coli-associated miRNA characterized by: the target gene of the miRNA isSBD2Its NCBI accession number is NM-001198545.1.

2. An ovine F17 E.coli-associated miRNA characterized by: the miRNA is miR-299-5p, and the nucleotide sequence of the miRNA is shown in SEQ ID NO. 2.

3. Use of a miRNA according to any one of claims 1 to 2 for ovine resistance to F17 e.

4. The use of the miRNA according to claim 3 in research of Hu sheep small intestine epithelial cells against F17 Escherichia coli infection, wherein: the miRNA is used for treating a target geneSBD2The negative regulation of the expression vector can regulate the resistance of the Hu sheep small intestine epithelial cells to the infection of the F17 escherichia coli.

5. The use of the miRNA of claim 4, wherein: the applications include that mirnas reduce the number of F17 e.coli adhering to small intestinal epithelial cells of hu sheep.

6. The use of the miRNA of claim 5, wherein: the applications include the ability of mirnas to inhibit infection of small intestine epithelial cells with F17 e.

7. The use of the miRNA of claim 5, wherein: the miRNA is used as a drug target for preventing, relieving or treating sheep F17 colibacillosis.

8. The use of the miRNA of claim 3, wherein: the sheep is a Hu sheep.

9. The use of the miRNA of claim 8, wherein: the Hu sheep preferably select small intestinal epithelial cells of Hu sheep lambs of 3-5 days old.

10. The use of the miRNA of claim 9, wherein: the time for the F17 Escherichia coli to infect the small intestine epithelial cells is 0h and 6 h.