Application of LIMD1 protein or coding gene thereof in improvement of disease resistance of poultry

Abstract

The invention discloses an application of a protein 1 (LIMD1) based on an LIM structural domain, which is used for inhibiting the replication of mycoplasma gallisepticum (MG) in a host cell so as to resist diseases caused by MG replication and improve the resistance of a chronic respiratory disease (CRD). The invention further discloses a preparation method of the protein 1 (LIMD1) based on the LIM structural domain, and the application of the protein 1 (LIMD1) based on the LIM structural domain is used for inhibiting the replication of the MG in the host cell. The invention finds that the LIMD1 gene is associated with CRD to a certain degree, and the expression quantity of the LIMD1 gene is obviously reduced along with the prolonging of MG infection time. The LIMD1 gene is overexpressed in chicken macrophages (HD11), shows a relatively strong inhibition effect on MG inoculation, effectively reduces cell apoptosis and inflammatory response caused by MG infection, and remarkably improves the MG resistance of the chicken macrophages. Therefore, the LIMD1 gene can effectively improve the MG resistance of HD11 cells, can be used as a potential disease-resistant gene, provides a gene resource for cultivation of disease-resistant poultry varieties, and has important significance in the field of animal breeding.

Description

Technical Field

[0001] The present invention relates to the technical field of animal breeding, and particularly relates to the application of LIMD1 protein or its encoding gene in improving the disease resistance of poultry. Background Art

[0002] Chronic respiratory disease (CRD) caused by Mycoplasma gallisepticum (MG) is a highly contagious disease that is widely prevalent globally and has become one of the major infectious diseases currently endangering the development of the chicken industry, causing huge economic losses to the poultry industry. After MG invades the chicken body, it binds to the receptors on the surface of respiratory mucosal epithelial cells through its surface adhesion proteins (such as GapA), multiplies locally in large numbers, causes respiratory inflammatory reactions, recruits lymphocytes, macrophages, and heterophilic cells to the lamina propria of the mucosa, and produces a large number of cytokines and chemokines to regulate the immune response. An overly strong immune response causes inflammatory damage. If there is no timely and effective treatment, MG enters the blood circulation through the host respiratory mucosal barrier, causing systemic pathological damage. MG is also an inducing factor for infections by other bacteria (such as Escherichia coli) and viruses (such as avian influenza virus), causing more serious harm to the poultry industry. Currently, the prevention and control measures for MG mainly rely on antibiotics and vaccines. Drugs can only relieve clinical symptoms and are not easy to eliminate mycoplasma, resulting in endogenous persistent infections. In particular, once an intensive farm is infected, it is difficult to eliminate the disease. In addition, the abuse of antibiotics has led to the widespread emergence of MG drug-resistant strains, and the drug resistance of some strains to commonly used antibiotics is as high as over 70%, and it also causes food safety problems. Due to the variability of MG, vaccines cannot prevent the continuous colonization of pathogens in the poultry flock. Therefore, from the perspective of the host, breeding disease-resistant varieties will become the most effective measure to solve the harm of CRD. However, currently, key host disease-resistant genes are severely lacking, and exploring host susceptible / resistant genes has become one of the key research focuses urgently needed.

[0003] LIM domain-containing protein 1 (LIMD1) is a multifunctional scaffold protein that is considered a key tumor suppressor in cancer and slows down tissue remodeling by inhibiting epithelial-mesenchymal transition in fibrotic diseases. In this study, RNA-seq was used to find that the expression level of LIMD1 decreased significantly at different time points (3h, 6h, 12h, and 24h) in MG-infected chicken macrophages (HD11), and this result was further confirmed by immunofluorescence (IF) and Western Blot. However, the role of LIMD1 protein in MG infection is still unclear. Summary of the Invention

[0004] The object of the present invention is to provide the application of LIMD1 protein or its encoding gene in improving the disease resistance of poultry.

[0005] In order to achieve the above object of the present invention, the present invention provides the following technical solutions:

[0006] The application of LIMD1 protein or its encoding gene in improving the disease resistance of poultry, characterized in that the amino acid sequence of the LIMD1 protein is as shown in SEQ ID NO.1, and the CDS sequence of the encoding gene of the LIMD1 protein is as shown in SEQ ID NO.2.

[0007] The present invention finds that the expression level of the LIMD1 gene is closely related to the infection time of MG. After MG infection, the expression level of the LIMD1 gene gradually decreases with the prolongation of the infection time. Further, by overexpressing the LIMD1 gene in HD11 cells, it is obtained that the LIMD1 gene is a key gene for anti-MG infection.

[0008] The research of the present invention shows that the LIMD1 gene is a key gene for anti-MG infection. Overexpressing the gene LIMD1 in HD11 cells inhibits the adhesion and proliferation of MG in cells and alleviates the inflammatory response and apoptosis caused by MG infection.

[0009] Further, the process of overexpressing the LIMD1 gene in HD11 cells is to insert the LIMD1 gene into the expression vector pcDNA3.1 to construct the overexpression vector pcDNA3.1-LIMD1 of the LIMD1 gene; and transfect the constructed overexpression vector pcDNA3.1-LIMD1 into HD11 cells to obtain HD11 cells overexpressing the LIMD1 gene.

[0010] The present invention has the following beneficial effects:

[0011] The present invention discovers a LIMD1 protein closely related to Mycoplasma gallisepticum. The expression level of the LIMD1 protein gradually decreases with the time of MG infection of HD11 cells and the adhesion of MG. By overexpressing the encoding gene of the LIMD1 protein in HD11 cells, the ability of cells to resist MG can be effectively improved, the copy number of MG in cells and the expression level of the MG adhesion protein GapA can be reduced, and the apoptosis and inflammatory response induced by MG can be significantly alleviated, thereby preventing and treating MG infection. Therefore, LIMD1 can be used as a potential target for anti-MG infection and has important clinical application value. Description of the Drawings

[0012] Figure 1 To detect the mRNA expression levels of GapA and LIMD1 at different time points after inoculating MG in HD11 cells by using qPCR technology.

[0013] Figure 2 To detect the expression level of LIMD1 protein at different time points after inoculating MG in HD11 cells using immunofluorescence.

[0014] Figure 3 After transfecting HD11 cells with the overexpression vector pcDNA3.1-LIMD1, qPCR was used to detect the copy numbers of LIMD1 gene, MG, and the expression level of GapA in LIMD1 gene overexpressing cells and wild-type cells.

[0015] Figure 4 To detect the expression levels of inflammatory factors TNF-α, IL-6, IL-10, and TGF-β in the LIMD1 gene overexpression group and the control group using qPCR technology.

[0016] Figure 5 To detect the apoptosis situation in the LIMD1 gene overexpression group and the control group using a TUNEL kit. Specific implementation manners

[0017] 1. The technical solutions of the present invention will be further described in detail below in conjunction with examples and related drawings. Based on the examples in the present invention, all other examples obtained by those of ordinary skill in the art without creative efforts fall within the scope of protection of the present invention.

[0018] 2. The experimental methods in the following examples are all conventional methods unless otherwise specified; the test materials used in the following examples are all purchased from conventional biochemical reagent companies or are conventional biological materials well-known to those skilled in the art unless otherwise specified.

[0019] 3. Experimental materials

[0020] The pcDNA3.1 vector was purchased from Invitrogen Corporation, and the MG strain and HD11 cells were provided by the State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, China.

[0021] 4. Example 1: Detection of the expression amount of LIMD1 protein at different time points after inoculating MG in HD11 cells by immunofluorescence

[0022] (1) Resuscitation of HD11 cells

[0023] Take out the cryopreserved cells from the liquid nitrogen tank and quickly thaw the cells in a 37°C water bath. During the thawing process, continuously shake the cryotube to enable the cells to pass through the ice crystal stage. Transfer the thawed cell mixture into a 15 mL centrifuge tube, add 10 mL of cell culture medium DMEM, centrifuge at 1000 r / min for 5 minutes, and discard the supernatant. Add 1 mL of complete cell culture medium, resuspend the cells, inoculate them into a culture flask for culture, and supplement the culture medium to about 3 mL. Gently shake the culture flask to evenly spread the cells on the bottom of the flask, place it in a 37°C, 5% CO2 incubator for culture, and observe the cell growth status at any time.

[0024] (2) Subculture of HD11 cells

[0025] Observe the cells. When the cells in the culture flask are basically confluent and the cell density reaches more than 90%, the cells need to be subcultured. The basic subculture operations are as follows: Aspirate the cell culture medium in the culture flask, add PBS to wash the cells, and repeat the operation twice. After discarding the PBS, add an appropriate amount of 0.25% trypsin to cover the bottom of the flask, place the cells in the incubator for 1 minute, observe the cell digestion under a microscope. When the cells begin to shrink, become round and small, add cell culture medium with twice the volume of trypsin to terminate the digestion. Pipette the cells in the culture flask to form a single-cell suspension and collect the cells. Transfer the cell suspension into a 15 mL centrifuge tube, centrifuge at 1000 r / min for 5 minutes, and discard the supernatant. Add complete cell culture medium, pipette to resuspend the cells, transfer the cells into a new culture flask, supplement an appropriate amount of cell culture medium, place it in a 37°C, 5% CO2 constant temperature incubator for culture, and observe the cell growth status at any time.

[0026] (3) Viral challenge of HD11 cells

[0027] One day before infection, inoculate an appropriate number of cells on cell slides. When the cell density reaches 60%-70% and the cell state is good, conduct a cell viral challenge test and set up a normal cell control group. Aspirate the original cell culture medium, wash 3 times with PBS buffer to remove as much of the original cell culture medium as possible. According to the MG virulence test results, select an appropriate viral challenge dose and supplement the cell culture medium (without penicillin and streptomycin) to 2 mL. Place the cell slides in a 37°C, 5% CO2 constant temperature incubator for 24 hours. Aspirate the original cell culture medium, wash 3 times with PBS buffer to remove as much of the original cell culture medium as possible.

[0028] (4) Immunofluorescence staining

[0029] ① Wash the cells twice with PBS and fix them with 4% paraformaldehyde at room temperature for 15 minutes; wash 3 times with PBS, 5 minutes each time;

[0030] ② Add 0.5% Triton 100 and permeabilize at room temperature for 15 minutes; wash 3 times with PBS, 5 minutes each time;

[0031] ③ Block with 1% BSA at 37°C for 1 hour; incubate with the primary antibody overnight at 4°C;

[0032] ④ Continue to incubate with the primary antibody at 37°C for 1 hour; wash 3 times with PBS, 5 minutes each time;

[0033] ⑤ Incubate with the secondary antibody at 37°C for 1 hour; wash 3 times with PBS, 5 minutes each time;

[0034] ⑥ Incubate with phalloidin at room temperature for 30 minutes; wash 3 times with PBS, 5 minutes each time;

[0035] ⑦ Counterstain with DAPI at room temperature for 5 minutes; wash 3 times with PBS, 5 minutes each time;

[0036] ⑧ Mount with glycerol and observe by photographing under a fluorescence microscope.

[0037] The experimental results are as Figure 2 shown, and the expression level of LIMD1 protein gradually decreases with the prolongation of MG infection time.

[0038] 5. Example 2: Construction of an overexpression vector for the LIMD1 gene. The following steps are included:

[0039] Design pcDNA3.1-LIMD1-F (5’-CCGCTCGAGCGGCAGGATGGATAAGTACGA and pcDNA3.1-LIMD1-R (5’-CCGCTCGAGCGGACGTTGGCTAGTAGTGAT-3’) for amplifying the full-length chicken LIMD1 gene, and amplify the full-length CDS region containing the chicken LIMD1 gene from HD11 cells by PCR amplification. Recover the LIMD1 CDS region amplification product by gel extraction, and ligate the PCR product and the pcDNA3.1 vector treated with XhoI restriction endonuclease by homologous recombination to construct the pcDNA3.1-LIMD1 vector. Then transform the recombinant plasmid into Escherichia coli DH5α competent cells, pick a single colony and inoculate it into LB liquid medium containing ampicillin, culture it with shaking at 37°C, and after correct Sanger sequencing, extract the plasmid using an endotoxin-free plasmid extraction kit.

[0040] 6. Example 3: LIMD1 gene inhibits the proliferation of Mycoplasma gallisepticum

[0041] (1) Preparation of HD11 cells overexpressing the LIMD1 gene

[0042] When the cell density reaches 50%-60% and the cell state is good, transfection is carried out. Taking a 6-well plate as an example, the transfection steps are as follows:

[0043] ① One hour before transfection, aspirate and discard the cell culture medium, wash the cells twice with PBS without double antibiotics, aspirate and discard the PBS, and add 2 mL of Opti-MEM® I Reduced Serum Medium;

[0044] ② Pipette 1.5 μL of LipofectamineTM 2000 and mix it into 50 μL of Opti-MEM® I Reduced Serum Medium;

[0045] ③ Pipette 2500 ng of pcDNA3.1-LIMD1 and mix it into 50 μL of Opti-MEM® I Reduced Serum Medium, and let it stand at room temperature for 5 minutes;

[0046] ④ Mix the mixtures in ② and ③, and let it stand at room temperature for 20 minutes to form a complex;

[0047] ⑤ Pipette 100 μL of the mixed solution into the prepared cells respectively, gently shake the cell culture plate to mix them evenly, and replace the complete medium (containing 1% double antibiotics) 4-6 hours after transfection;

[0048] ⑥ Incubate in a cell culture incubator at 37 °C and 5% CO2 for 24-72 hours;

[0049] (2) According to the sequence of MG 16S rRNA published by NCBI, primers are designed for its highly conserved region, and the primer sequences are as follows:

[0050] 16S rRNA-F AGCTAATCTGTAAAGTTGGTC

[0051] 16S rRNA-R CGCTTCCTTGCGGTTAGCAAC

[0052] Extract DNA by phenol-chloroform extraction method, recover the target product by gel extraction, and ligate the PCR product and pMD-18T vector by TA cloning ligation method. Then transform the ligated plasmid into competent Escherichia coli DH5α cells, pick a single colony and inoculate it into LB liquid medium containing ampicillin, culture it with shaking at 37 °C, and after correct Sanger sequencing, extract the plasmid using an endotoxin-free plasmid extraction kit. Determine the concentration of the recombinant plasmid by a NanoDrop 2000 ultra-micro spectrophotometer, convert the plasmid concentration into molecular copy number according to the molecular weight and Avogadro's constant, and dilute the recombinant plasmid to the original concentration and store it at -80 °C for later use. Then perform 10-fold serial dilution to prepare standards. Using these standards as templates and setting negative controls, perform fluorescence quantitative PCR under the optimized best conditions. Obtain the Ct values of different template concentrations respectively, record the Ct values, and draw a standard curve.

[0053] Molecular copy number = DNA mass concentration / DNA molecular weight

[0054] DNA molecular weight = number of DNA bases × 324.5

[0055] DNA mass concentration = absorbance at 260 nm

[0056] The experimental results are as Figure 3 shown, the up-regulation of LIMD1 gene content leads to a decrease in the copy number of Mycoplasma gallisepticum in HD11 cells.

[0057] 7. Example 4: LIMD1 gene alleviates cell apoptosis caused by Mycoplasma gallisepticum infection

[0058] After treating HD-11 cells with overexpression of LIMD1, analyze the apoptosis of HD-11 cells using a TUNEL kit from Elabscience (Wuhan, China). The specific steps are as follows:

[0059] Cell treatment:

[0060] ① Immerse the air-dried cell slides in 4% paraformaldehyde fixative, fix at room temperature for 15 - 20 minutes or at 4 °C for 1 - 2 hours.

[0061] ② Immerse the fixed samples in PBS and rinse 3 times, 5 minutes each time.

[0062] ③ Immerse the samples in 0.2% Triton × 100 permeabilization solution and incubate at 37 °C for 10 minutes.

[0063] ④ Immerse the permeabilized samples in PBS and rinse 3 times, 5 minutes each time.

[0064] TUNEL staining:

[0065] ① Prepare the TdT enzyme reaction solution: Calculate the sample volume and prepare it centrally. The dosage for each sample is as follows: Add 5 μL of the fluorescent labeling solution and 5 μL of the TdT enzyme to 35 μL of the equilibration solution (1×), mix well thoroughly, and use it immediately after preparation.

[0066] ② Add 100 μL of TdT Equilibration Buffer to each sample and react at 37 °C for 30 minutes.

[0067] ③ Use absorbent paper to remove the TdT Equilibration Buffer (note not to dry the slices). Add 50 μL of the labeling working solution to each sample and place it in a humid box to react at 37 °C in the dark for 60 minutes. Note: If the signal intensity is weak, the incubation time of the DNA labeling reaction can be extended. Some systems may require a 4-hour reaction at 37 °C.

[0068] ④ Immerse the samples in PBS and rinse 3 times, 5 minutes each time.

[0069] ⑤ After drying with absorbent paper, add the DAPI working solution and incubate at room temperature in the dark for 5 minutes to counterstain the cell nuclei. Immerse the samples in PBS and rinse 4 times, 5 minutes each time.

[0070] ⑥ Use absorbent paper to blot dry the excess liquid and mount the slides with a mounting medium containing an anti-fluorescence quencher (prepared by yourself).

[0071] (3) Microscopic examination: Observe under a fluorescence microscope. The maximum excitation wavelength of AF488 is 495 nm, and the maximum emission wavelength is 519 nm (green fluorescence).

[0072] The experimental results are as Figure 5 shown. MG infection induces a large number of green positive apoptotic signals in HD11 cells, and overexpression of LIMD1 can significantly reduce the apoptosis of HD11 cells induced by MG infection.

[0073] SEQ ID NO.1

[0074] Amino acid sequence of the LIMD1 protein:

[0075] MDKYDDLGLEASKFIEDLNMYEASKDGLFRVDRAAGNNPEFEETRRVFATKMAKIHLQQQQQQQQQEMLMATASLNGGAALGAPRPGLPAPRGAKPPLGSPAGPLPQVAAEGPGSGRPGTCRAFPMMPGEPQCPGSQAGRRGWEAEPRGDPQENGSDDRRGSQGQELKPAGQRSSSFSQVRGPVGPSDGAPAATLQHRSSFSASAAQPSPGFLALTEPCGPRAGVDGPTTDGGQLWYQGGSRPFPPSSAPGGPSVDYQQAGIHPSPSSHFVAHGHNHRPNGGPELGASHLAAARGPTSDAPKQPFKEGTSSLHPDGGHQEGCGVAKVKLPCQTLQQPEQGPSAAELKLEALTQRLEQEMDARPKADYFGTCVKCSKGVYGANQACQAMGNLYHDGCFTCGACSRKLRGKAFYFVNGKVFCEEDFLYSGFQQSADRCFICGHLIMDMILQALGKSYHPGCFRCVVCNECLDGVPFTVDSENKIYCVRDYHKVLAPKCAACGLPILPSEGSDETIRVVSMDKDYHVECYHCEDCGMELNDEDGHRCYPLDDHLLCHSCHLKHIENGTTPPAAVYQHHY

[0076] SEQ ID NO.2

[0077] CDS sequence of the LIMD1 coding gene:

[0078]

Claims

1. Use of LIMD1 protein or its coding gene in improving disease resistance of poultry, characterized in that, The amino acid sequence of the LIMD1 protein is shown in SEQ ID NO.1, and the CDS sequence of the coding gene of the LIMD1 protein is shown in SEQ ID NO.

2.

2. The overexpression sequence is designed according to the CDS region of the LIMD1 gene as described in claim 1, characterized in that, The primer sequences are pcDNA3.1-LIMD1-F (5’-CCGCTCGAGCGGCAGGATGGATAAGTACGA) and pcDNA3.1-LIMD1-R (5’-CCGCTCGAGCGGACGTTGGCTAGTAGTGAT-3’).

3. The application according to claim 2, wherein, The substance capable of overexpressing LIMD1 is the LIMD1 overexpression vector pcDNA3.1-LIMD1, which is recombined by inserting the CDS region amplified by the overexpression primers in Claim 2 into pcDNA3.1 through the XhoI restriction site.

4. The application according to claim 3, characterized in that The process of overexpressing the LIMD1 gene in chicken macrophages is as follows: inserting the LIMD1 gene into the expression vector pcDNA3.1 to construct the LIMD1 gene overexpression vector pcDNA3.1-LIMD1; transfecting the constructed overexpression vector pcDNA3.1-LIMD1 into chicken macrophages to obtain chicken macrophages overexpressing the LIMD1 gene.

5. The application according to claim 1, characterized in that Inoculate Mycoplasma gallisepticum into chicken macrophages, and detect the dynamic changes of the LIMD1 gene and the Mycoplasma gallisepticum adhesion protein GapA in the cells by fluorescence quantitative PCR technology and detect the dynamic changes of the LIMD1 protein in the cells by immunofluorescence technology.

6. The application according to claim 1, characterized in that, Overexpressing the gene LIMD1 in chicken macrophages inhibits the adhesion and proliferation of Mycoplasma gallisepticum in the cells. Further, it alleviates the inflammatory response and macrophage apoptosis caused by Mycoplasma gallisepticum infection.

7. Use of the LIMD1 protein or its coding gene according to claim 1 in enhancing the disease resistance of poultry, characterized in that Improve the resistance of chickens to Mycoplasma gallisepticum and breed new disease-resistant chicken varieties against chronic respiratory disease.

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