Poultry disease-resistant related protein S100A9 as well as coding gene and application thereof

Abstract

The invention belongs to the technical field of genetic engineering of animal molecular breeding, and aims to disclose a poultry disease-resistant related protein S100A9 as well as a coding gene and application thereof. Through accurate screening and analysis of chicken embryo lung tissue sequencing data, it is determined that S100A9 plays a key regulation role in MG infection, and CCRI and TLR signal pathways in which S100A9 participates are immune pathways in which chicken embryos and chicks jointly resist MG infection. The discovery provides important theoretical basis and practical support for poultry breeding for disease resistance and prevention and treatment of mycoplasma gallisepticum.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering of animal molecular breeding, and specifically relates to a poultry disease resistance-related protein S100A9, a coding gene thereof and an application thereof. Background Art

[0002] Disease resistance breeding is the use of genetic basis to cultivate poultry breeds with strong disease resistance. Disease resistance in poultry is a polygenic trait regulated by a complex gene network. The proteins encoded by these genes can directly or indirectly participate in the disease resistance process of poultry and regulate the resistance of poultry to pathogens. Therefore, screening and identifying the expression characteristics of disease resistance genes is of great significance for breeding poultry with stronger disease resistance.

[0003] S100A9, with a size of 14KDa, was originally named migration inhibitor-related protein and later named myeloid-related protein MRP14 because it was first discovered in myeloid-derived macrophages. S100A9 has a strong affinity for calcium ions and has significant calcium ion binding ability. S100A9 can also exert antibacterial activity by chelating zinc ions. Inflammation regulation is the main function of S100A9. S100A9 is a member of DAMPs and can mediate NFκB activation by binding to receptors RAGE or TLRs, and participate in regulating the activation of inflammatory responses. S100A9 is expressed in large quantities in leukocytes and secreted by leukocytes, especially neutrophils, and plays a role in infection-induced innate immune responses. A large number of literatures have confirmed that S100A9 plays an important role in the progression of a variety of inflammatory lung diseases. Summary of the invention

[0004] The technical solution adopted by the present invention is a poultry disease resistance-related protein S100A9, the amino acid sequence of the S100A9 protein is SEQ ID NO: 2, the gene encoded by the S100A9 is S100A9, and the DNA sequence is SEQ ID NO: 1.

[0005] The present invention also provides a recombinant overexpression vector, which is recombined by inserting the DNA sequence of the S100A9 gene into pcDNA3.1 through HindIII and NotI restriction sites.

[0006] The present invention also provides a recombinant engineered bacterium, which is prepared by transforming the above-mentioned recombinant overexpression vector into a host bacterium, and the selected host bacterium is Escherichia coli TM5α.

[0007] The present invention provides a poultry disease resistance-related protein S100A9 and its encoding gene and application, which specifically comprises the following steps:

[0008] 1) Cultivation of poultry alveolar type II epithelial cells, macrophages, chicken embryos and chicks;

[0009] 2) using the E. coli transformation method, using the recombinant engineered bacteria constructed above to extract the recombinant overexpression vector and transfect the alveolar type II epithelial cells and macrophages obtained in step 1);

[0010] 3) RNA extraction

[0011] ① Take out the lung, bursa of Fabricius, spleen and thymus tissues of chicken organs from the MG infection group and the control group at -80℃, take 100 mg from them, add 1 mL of TRIzol, and homogenize them in a homogenizer 4 times, each time for 20 seconds to ensure that the tissues are completely broken. After the homogenization, place the samples at -80℃ for low-temperature lysis for more than 0.5 h;

[0012] ② Centrifuge at 4°C, 12,000 rpm for 10 min;

[0013] ③ Add 200 μL of chloroform, shake vigorously for 15 seconds, let stand at 4°C for 5 minutes, and centrifuge at 4°C and 12,000 r / min for 15 minutes;

[0014] ④ Pipette the supernatant into a new 1.5 mL RNase-free centrifuge tube, add an equal volume of pre-cooled isopropanol, mix by inversion several times, let stand at -20°C for 15 min, centrifuge at 4°C and 12,000 r / min for 15 min, and discard the supernatant;

[0015] ⑤ Add 1 mL of 75% anhydrous ethanol, gently flick the precipitate to rinse the precipitate, centrifuge at 4°C and 12,000 r / min for 5 min, and discard the supernatant; repeat this step twice;

[0016] ⑥ According to the amount of precipitation, add an appropriate amount of DEPC water to dissolve the RNA precipitation. After gently mixing and dissolving, NanoDrop-2000 was used to determine the concentration and purity of the extracted total RNA. Take 2 μL of total RNA and perform electrophoresis on 1.2% agarose gel. The remaining RNA was stored in a -80℃ refrigerator for later use. The three bands of 28s rRNA, 18s rRNA, and 5.8s rRNA were observed to determine the quality of RNA extraction.

[0017] ⑦ Determination of RNA purity and concentration: RNA has the largest absorption peak at 230nm wavelength. The concentration of total RNA is measured by 230nm wavelength spectrophotometry. The OD260 / OD280 ratio of pure RNA samples is about 2.0, so the purity of RNA can be estimated based on the OD260 / OD280 ratio.

[0018] 4) PCR primer design

[0019] The chicken S100A9 CDS region was searched in the NCBI database, and primers capable of PCR amplification of the full length of the CDS region were designed based on the search. According to the pcDNA3.1 plasmid sequence, appropriate restriction sites were selected and added to the 5' end of the primers. Primers were designed and synthesized using the primer design software primer5.0, and then fluorescent quantitative PCR was performed after synthesis; the primer sequences were

[0020] S100A9CDS-F: 5'-CCCAAGCTTATGAGCAAGGCTGCCAGA-3'

[0021] S100A9CDS-R: 5'-AAATAT GCGGCCGC TCAGTGGTTGTGCTGATGT-3'

[0022] 5) qPCR detection and western blot analysis were performed on poultry alveolar type II epithelial cells, macrophages, chicken embryos and chick organs such as lung, bursa of Fabricius, spleen and thymus tissue.

[0023] The beneficial effects of the present invention are: by screening and determining a differential gene S100A9 after MG infection through gene localization, the gene can be determined to regulate the disease resistance of poultry at the transcriptional level through knockdown experiments and overexpression experiments, indicating that the S100A9 gene is a poultry disease resistance-related gene, and as the expression level of the S100A9 gene increases, the MG infection becomes more serious, and the discovery of this gene provides a theoretical basis and scientific basis for poultry disease resistance breeding. In the process of verifying the function of the OsITAND gene, the present invention constructs a recombinant overexpression vector and a recombinant engineering bacterium: Escherichia coli DH5α strain. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 is the expression level of the S100A9 gene of the present invention in AECII cells and HD11 cells infected with different doses of MG

[0025] Figure 2 The recombinant overexpression vector containing the CDS sequence of the S100A9 gene constructed in the present invention Figure 3 The expression efficiency of the recombinant overexpression vector containing the CDS sequence of the S100A9 gene in AECII cells and HD11 cells is Figure 4 The inhibition efficiency of the SiRNA of the S100A9 gene in AECII cells and HD11 cells

[0026] Figure 5 The effect of S100A9 gene on the replication of MG in AECII cells and HD11 cells Figure 6This is a comparative analysis of the expression of the S100A9 gene in different chicken breeds in the present invention. Figure 7 This is an analysis diagram of the correlation between the MMP7 gene expression in lung tissue and the thickness of chicken tracheal mucosa. DETAILED DESCRIPTION

[0029] The technical solution of the present invention is further explained below in conjunction with the accompanying drawings and specific embodiments. Those skilled in the art may make modifications and substitutions to the technical solution of the present invention based on a full understanding of the technical solution of the present invention, which shall all fall within the protection scope of the present invention.

[0030] Example 1 Construction of recombinant overexpression vector

[0031] According to the chicken S100A9 CDS region sequence retrieved from the NCBI database, PCR primers were designed and the amplified product was purified. The purified S100A9 gene was inserted into the linearized vector pcDNA3.1 treated with HindIII and NotI restriction endonucleases. The specific steps are as follows:

[0032] 1) Amplification of the full length of the S100A9 CDS region:

[0033] Using chicken lung tissue cDNA as template, FastPfu DNA Polymerase amplifies the target fragment, and the PCR reaction system is as follows:

[0034] S100A9-CDS-F: 1.0 μL

[0035] S100A9-CDS-R: 1.0 μL

[0036] 5×Pfu Buffer:10.0μL

[0037] DNTPs: 5.0 μL

[0038] FastPfu high-fidelity DNA polymerase: 1.0 μL

[0039] MGSO4: 1.0 μL

[0040] Template cDNA: 2.0 μL

[0041] ddH2O: 29.0 μL

[0042] Total volume: 50.0 μL

[0043] The concentration of FastPfu high-fidelity DNA polymerase was 5U / μL, and the amount of template cDNA was about 100ng; after mixing, the PCR reaction procedure was as follows: 94℃; 2min: (94℃: 30sec; 55℃: 30sec, 72℃: 30sec) × 35; 72℃: 5min: 15℃: 10sec. After the PCR reaction was completed, the amplification results were detected by 1.0% agarose gel electrophoresis. (3) Gel recovery of the full-length amplified product of the S100A9 CDS region

[0044] According to the EasyPure Quick Gel Extraction Kit instructions, the S100A9 CDS region amplification product was recovered. The specific steps are as follows:

[0045] ① Under ultraviolet light, use a clean scalpel to quickly cut out the gel block containing the target DNA from the agarose gel, put it into a clean 2mL centrifuge tube and weigh it. If the gel weight is 100mg, it can be regarded as 100μL (100mg≈100μL);

[0046] ② Add 3 times the volume of GSB solution and melt the gel in a 55℃ dry bath for 10 minutes. When the agar block is completely melted, observe the color of the solution. If the color is purple, add an appropriate amount of 3M sodium acetate to adjust the color to the same as GSB;

[0047] ③ Place at room temperature until the melted gel mixture cools to room temperature, add it to the adsorption column and let it stand for 1 minute, centrifuge at 10,000r / min for 1 minute, and discard the filtrate;

[0048] ④ Add 650 μL Wash buffer solution, centrifuge at 10,000 r / min for 1 min., and discard the filtrate;

[0049] ⑤ Centrifuge at 10,000r / min for 1-2min to remove the residual solution;

[0050] ⑥ Place the adsorption column in a 1.5 mL clean centrifuge tube, add 30 μL of 55°C EB or deionized water (PH>7.0) in the center of the column, and let it stand at room temperature for 1 min;

[0051] ⑦ Centrifuge at 10,000r / min for 1 min to elute the DNA, and temporarily store the eluted DNA at -20℃.

[0052] (4) Enzyme digestion of S100A9-CDS and pcDNA3.1 complete plasmid

[0053] Using HindIII and NotI enzymes, double-digest S100A9-CDS, pcDNA3.1 and the complete plasmid.

[0054] The enzyme digestion reaction system is as follows:

[0055] 10×H buffer: 5.0 μL

[0056] NotI: 5.0 μL

[0057] HindIII: 2.5 μL

[0058] pcDNA3.1 plasmid: 15.0 μL

[0059] ddH2O: 25.0 μL

[0060] Total volume: 50.0 μL

[0061] The enzyme digestion was carried out overnight (8-12 h) in a 37°C biochemical incubator, and the enzyme digestion results were detected by 1.0% agarose gel electrophoresis.

[0062] (5) Connection of recovered products

[0063] The recovered S100A9 CDS fragment was connected to the overexpression vector pcDNA3.1 fragment. The connection reaction system was as follows:

[0064] pcDNA3.1: 4.0 μL

[0065] S100A9CDS: 4.0 μL

[0066] T4 ligase: 1.0 μL

[0067] 10×T4 buffer: 1.0 μL

[0068] Total volume: 10.0 μL

[0069] The PCR reaction procedure was 37°C for 15 min. After the reaction was completed, the product was stored at 4°C for future use.

[0070] (6) Transformation of Ligation Product

[0071] ①Take out 50 μL of Trelief TM5α Chemically Competent Cell from -80℃ refrigerator, thaw on ice, and

[0072] Add 10 μL of the ligation product and mark the tube accordingly;

[0073] ② Gently mix the mixture, place it on ice for 5 minutes, heat shock it at 42℃ for 45 seconds, and quickly place it on ice for 2 minutes;

[0074] ③Evenly apply 55 μL of the liquid in step ② on a LA plate (containing 1% ampicillin);

[0075] ④ Place the coated plate upright in a 37°C constant temperature incubator for 15 minutes, then invert and incubate overnight.

[0076] ⑤Pick a single colony into a 1.5mL sterile centrifuge tube, add 1mL of LA liquid culture medium containing 1‰ ampicillin, and culture at 37℃, 220r / min with shaking for 4h.

[0077] Example 2

[0078] A poultry disease resistance-related protein S100A9, its encoding gene and application, specifically comprising the following steps:

[0079] 1) Cultivation of poultry alveolar type II epithelial cells, macrophages, chicken embryos and chicks;

[0080] 2) using the E. coli transformation method, using the recombinant engineered bacteria constructed above to extract the recombinant overexpression vector and transfect the alveolar type II epithelial cells and macrophages obtained in step 1);

[0081] 3) RNA extraction

[0082] ① Select chicken embryo lung tissues from the MG infection group and the control group of Jingfen No. 6, Hailanbai, Qingjiao Ma chicken, Tianlu Ma chicken and Guangxi native chicken, take 100 mg from them, add 1 mL of TRIzol, and homogenize in a homogenizer 4 times, each time for 20 seconds to ensure that the tissue is completely broken. After the homogenization, place the sample at -80℃ for low-temperature lysis for more than 0.5h;

[0083] ② Centrifuge at 4°C, 12,000 rpm for 10 min;

[0084] ③ Add 200 μL of chloroform, shake vigorously for 15 seconds, let stand at 4°C for 5 minutes, and centrifuge at 4°C and 12,000 r / min for 15 minutes;

[0085] ④ Pipette the supernatant into a new 1.5 mL RNase-free centrifuge tube, add an equal volume of pre-cooled isopropanol, mix by inversion several times, let stand at -20°C for 15 min, centrifuge at 4°C and 12,000 r / min for 15 min, and discard the supernatant;

[0086] ⑤ Add 1 mL of 75% anhydrous ethanol, gently flick the precipitate to rinse the precipitate, centrifuge at 4°C and 12,000 r / min for 5 min, and discard the supernatant; repeat this step twice;

[0087] ⑥ According to the amount of precipitation, add an appropriate amount of DEPC water to dissolve the RNA precipitation. After gently mixing and dissolving, NanoDrop-2000 was used to determine the concentration and purity of the extracted total RNA. Take 2 μL of total RNA and perform electrophoresis on 1.2% agarose gel. The remaining RNA was stored in a -80℃ refrigerator for later use. The three bands of 28s rRNA, 18s rRNA, and 5.8s rRNA were observed to determine the quality of RNA extraction.

[0088] ⑦ Determination of RNA purity and concentration: RNA has the largest absorption peak at 230nm wavelength. The concentration of total RNA is measured by 230nm wavelength spectrophotometry. The OD260 / OD280 ratio of pure RNA samples is about 2.0, so the purity of RNA can be estimated based on the OD260 / OD280 ratio.

[0089] 4) PCR primer design

[0090] The chicken S100A9 CDS region was searched in the NCBI database, and primers capable of PCR amplification of the full length of the CDS region were designed based on the search. According to the pcDNA3.1 plasmid sequence, appropriate restriction sites were selected and added to the 5' end of the primers. Primers were designed and synthesized using the primer design software primer5.0, and then fluorescent quantitative PCR was performed after synthesis; the primer sequences were

[0091] S100A9 CDS-F: 5'-CCCAAGCTTATGAGCAAGGCTGCCAGA-3'

[0092] S100A9 CDS-R: 5'-AAATAT GCGGCCGC TCAGTGGTTGTGCTGATGT-3'

[0093] 5) Detection of S100A9 gene expression

[0094] The expression of S100A9 gene was detected in lung tissues of Jingfen No. 6, Hailanbai, Qingjiao Ma chicken, Tianlu Ma chicken and Guangxi native chicken, as well as AECII and HD11 cells. The reaction conditions of fluorescence quantitative PCR were: 95℃ pre-denaturation for 5min; 95℃ denaturation for 30s, 62℃ annealing for 30s, 72℃ extension for 30s, and 38 cycles.

[0095] 6) Determination of tracheal mucosal thickness in MG-infected chicks

[0096] The thickness of tracheal mucosa of Jingfen No. 6, Hailanbai, Qingjiao Ma chicken, Tianlu Ma chicken and Guangxi native chicken after MG infection was measured.

[0097] like Figure 6-Figure 7The expression level of S100A9 gene in lung tissues of Jingfen No. 6, Hailanbai, Qingjiao Ma chicken, Tianlu Ma chicken and Guangxi native chicken was detected, and its correlation with mucosal thickness was analyzed.

[0098] The present invention designs primers for the S100A9 gene of chicken, performs PCR amplification, detects the expression changes of the mRNA of the S100A9 gene in AECII and HDII cells infected with different MG concentrations, and the correlation between the expression and the body weight and tracheal mucosal thickness of MG infected with different breeds. The expression shows that the chickens with high S100A9 expression are more susceptible to infection with Mycoplasma gallisepticum, which provides an immune theoretical basis for the formulation of MG infection prevention and control strategies and the cultivation of disease-resistant breeding strains using molecular marker-assisted selection methods.

[0099]

[0100]

Claims

1. A poultry disease resistance-related protein S100A9, characterized in that: The amino acid sequence of the S100A9 protein is SEQ ID NO: 2, the gene encoded by the S100A9 protein is S100A9, and the DNA sequence is SEQ ID NO:

1.

2. A recombinant overexpression vector, characterized in that: The recombinant overexpression vector is formed by inserting the DNA sequence in claim 1 into pcDNA3.1 through HindIII and NotI restriction sites.

3. A recombinant engineered bacterium, characterized in that: The recombinant engineered bacteria are prepared by transforming the recombinant overexpression vector in claim 2 into a host bacteria.

4. A recombinant engineered bacterium according to claim 3, characterized in that: The host bacteria is Escherichia coli TM5α strain.

5. A poultry disease resistance-related protein S100A9, its encoding gene and application, characterized in that: The specific steps include: 1) Cultivation of poultry alveolar type II epithelial cells, macrophages, chicken embryos and chicks; 2) using the recombinant engineered bacteria described in claims 3-4 to extract the recombinant overexpression vector and transfect the alveolar type II epithelial cells and macrophages obtained in step 1); 3) RNA extraction ① Take out the lung, bursa of Fabricius, spleen and thymus tissues of chicken organs from the MG infection group and the control group at -80℃, take 100 mg from them, add 1 mL of TRIzol, and homogenize them in a homogenizer 4 times, each time for 20 seconds to ensure that the tissues are completely broken. After the homogenization, place the samples at -80℃ for low-temperature lysis for more than 0.5 h; ② Centrifuge at 4°C, 12,000 rpm for 10 min; ③ Add 200 μL of chloroform, shake vigorously for 15 seconds, let stand at 4°C for 5 minutes, and centrifuge at 4°C and 12,000 r / min for 15 minutes; ④ Pipette the supernatant into a new 1.5 mL RNase-free centrifuge tube, add an equal volume of pre-cooled isopropanol, mix by inversion several times, let stand at -20°C for 15 min, centrifuge at 4°C and 12,000 r / min for 15 min, and discard the supernatant; ⑤ Add 1 mL of 75% anhydrous ethanol, gently flick the precipitate to rinse the precipitate, centrifuge at 4°C and 12,000 r / min for 5 min, and discard the supernatant; repeat this step twice; ⑥ According to the amount of precipitation, add an appropriate amount of DEPC water to dissolve the RNA precipitation. After gently mixing and dissolving, NanoDrop-2000 was used to determine the concentration and purity of the extracted total RNA. Take 2 μL of total RNA and perform electrophoresis on 1.2% agarose gel. The remaining RNA was stored in a -80℃ refrigerator for later use. The three bands of 28s rRNA, 18s rRNA, and 5.8s rRNA were observed to determine the quality of RNA extraction. ⑦ Determination of RNA purity and concentration: RNA has the largest absorption peak at 230nm wavelength. The concentration of total RNA is measured by 230nm wavelength spectrophotometry. The OD260 / OD280 ratio of pure RNA samples is about 2.0, so the purity of RNA can be estimated based on the OD260 / OD280 ratio. 4) PCR primer design The chicken S100A9 CDS region was searched in the NCBI database, and primers capable of PCR amplification of the full length of the CDS region were designed based on the search. According to the pcDNA3.1 plasmid sequence, appropriate restriction sites were selected and added to the 5' end of the primers. Primers were designed and synthesized using the primer design software primer5.0, and then fluorescent quantitative PCR was performed after synthesis. The primer sequence is S100A9 CDS-F: 5'-CCCAAGCTrATGAGCAAGGGCTGCCAGA-3' S100A9 CDS-R: 5'-AAATAT GCGGCCGC TCAGTGGTTGTGCTGATGT-3' 5) Detect the expression of S100A9 gene in cells The expression of S100A9 gene was detected in AECII and HD11 cells infected with different concentrations of MG.The reaction conditions of fluorescence quantitative PCR were: 95℃ pre-denaturation for 5min; 95℃ denaturation for 30s, 62℃ annealing for 30s, 72℃ extension for 30s, 38 cycles. 6) Detection of the expression level of S100A9 gene in chicken embryos The expression of S100A9 gene was detected in lung tissue, immune organ spleen, thymus and bursa of MG infected chicken embryos.The reaction conditions of fluorescence quantitative PCR were: 95℃ pre-denaturation for 5min; 95℃ denaturation for 30s, 62℃ annealing for 30s, 72℃ extension for 30s, 38 cycles. 7) Detection of the expression level of S100A9 gene in chicks The expression of S100A9 gene was detected in lung tissue, immune organ spleen, thymus and bursa of Fabricius of MG infected chickens.The reaction conditions of fluorescence quantitative PCR were: 95℃ pre-denaturation for 5min; 95℃ denaturation for 30s, 62℃ annealing for 30s, 72℃ extension for 30s, 38 cycles.

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