Chicken mtrf1l protein monoclonal antibody and application thereof

Abstract

The application discloses a chicken MTRF1L protein monoclonal antibody and application thereof, and belongs to the technical field of biotechnology.The MTRF1L recombinant protein is used for immunizing animals, a sensitized B cell with the ability of secreting specific antibodies and a myeloma cell with unlimited reproduction capacity are fused into a B cell hybridoma through a hybridoma technology, and a chicken MTRF1L protein monoclonal antibody is obtained through screening.The application screens a high-quality nucleotide coding sequence of the MTRF1L gene through bioinformatics analysis, and uses the nucleotide coding sequence as an antigen to immunize BABE mice, a sensitized B cell with the ability of secreting specific MTRF1L antibodies and a myeloma cell with unlimited reproduction capacity are fused into a B cell hybridoma through a hybridoma technology, and a positive hybridoma cell is obtained through screening, and the cell supernatant is used for Western Blot and indirect immunofluorescence detection, and it is found that the monoclonal antibody has good specificity and high sensitivity, which lays a foundation for detection of the MTRF1L protein and research on pathological mechanisms of virus infection diseases.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a monoclonal antibody against chicken MTRF1L protein and its application. Background Technology

[0002] Infectious bursal disease (IBD) is an acute, febrile, highly contagious, immunosuppressive disease caused by the infectious bursal disease virus (IBDV). IBD first occurred in Gambro, Delaware, USA in 1962, hence its alternative name, "Gambro disease." It is now prevalent in chicken-farming countries and regions worldwide, and was introduced to my country in the late 1970s, becoming one of the most serious infectious diseases threatening my country's poultry industry. IBD primarily affects chickens, especially chicks around 5 weeks old. Its main pathological feature is acute inflammation and destruction of the bursa of Fabricius, leading to suppression of the chicken's immune system and reduced immunity. This makes the flock susceptible to secondary infections from other pathogens (such as E. coli and avian influenza viruses). Infected flocks often experience numerous complications, secondary diseases, and vaccine failure, further exacerbating the spread of the disease and mortality, causing severe economic losses to the global poultry industry. Clinical symptoms of IBD usually appear 2 to 3 days after infection, including depression, loss of appetite, ruffled feathers, and diarrhea. In severe cases, young chickens may die suddenly. During autopsy, swelling and darkening of the bursa of Fabricius, and even necrosis, can be observed. Hemorrhage at the junction of the bursa of Fabricius, leg muscles, pectoral muscles, proventriculus, and gizzard are characteristic pathological changes.

[0003] The chicken MTRF1L protein is a key regulator located primarily in mitochondria and responsible for promoting mitochondrial division. It plays a crucial role in cellular energy metabolism, health status, stress response, and cell fate determination by maintaining mitochondrial homeostasis.

[0004] However, there is currently a lack of research on chicken MTRF1L protein antibodies and their application in disease detection. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide a monoclonal antibody against chicken MTRF1L protein and its application.

[0006] To achieve the above objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides a recombinant chicken MTRF1L protein, the amino acid sequence of which is shown in SEQ ID NO: 1.

[0007] Secondly, the present invention provides a method for expressing the above-mentioned recombinant chicken MTRF1L protein, comprising the following steps: The chicken MTRF1L gene was constructed into an expression vector, and recombinant chicken MTRF1L protein was obtained through expression.

[0008] As a preferred embodiment, the chicken MTRF1L gene sequence is shown in SEQ ID NO: 2.

[0009] As preferred options, prokaryotic expression and eukaryotic expression are included; The prokaryotic expression was performed using the prokaryotic expression vector pET-28a; the eukaryotic expression was performed using the eukaryotic expression vector pCMV.

[0010] Thirdly, the present invention provides a method for preparing a monoclonal antibody against chicken MTRF1L protein, comprising the following steps: Immunized mice were constructed using the above recombinant chicken MTRF1L protein to obtain immune serum, and then recombinant chicken MTRF1L protein monoclonal antibody was obtained through hybridoma technology.

[0011] Fourthly, the present invention provides the application of the chicken MTRF1L protein monoclonal antibody prepared by the above-described preparation method in products for detecting chicken infection with IBDV.

[0012] Preferably, the product includes chips, probes, and reagent kits.

[0013] It contains at least the following beneficial technical effects: Animals were immunized with recombinant MTRF1L protein. Using hybridoma technology, sensitized B cells capable of secreting specific antibodies were fused with myeloma cells capable of unlimited proliferation to form B-cell hybridomas. After screening, chicken MTRF1L protein monoclonal antibodies were obtained. This invention screened for high-quality nucleotide coding sequences of the MTRF1L gene using bioinformatics analysis, and used these sequences as antigens to immunize Babesia mice. Hybridoma technology was then used to fuse sensitized B cells capable of secreting specific MTRF1L antibodies with myeloma cells capable of unlimited proliferation to form B-cell hybridomas. Positive hybridoma cells were obtained after screening, and their supernatant was used for Western blotting and indirect immunofluorescence detection. The results showed that the monoclonal antibody had good specificity and high sensitivity. This lays the foundation for subsequent research on the detection of MTRF1L protein and the pathological mechanisms of viral infections. Attached Figure Description

[0014] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0015] Figure 1 Technical roadmap for the preparation of monoclonal antibodies against chicken MTRF1L protein.

[0016] Figure 2 Results of constructing the recombinant plasmid for expression: A: Polymerase chain reaction amplification product of MTRF1L gene (729bp); M is DL 2000 Maker; Lanes 1, 2, 3, 4: PCR amplification product of MTRF1L gene; B: Bacterial PCR; M is DL2000 Maker; Lanes 1, 2, 3, 4: Polymerase chain reaction amplification product (729bp); Lane 5: Negative control; Figure 3 Results of induction, expression, and purification of recombinant MTRF1L protein: A: IPTG-induced expression of recombinant MTRF1L protein; Lane 1: After induction at 37℃ for 6 hours... MTRF1L total protein; lane 2: induced at 16℃ for 6 hours MTRF1L total protein; lane 3: 37℃ induction for 6 hours MTRF1L inclusion body lysis buffer supernatant after lysis; M: DL 2000 Maker; B: Optimal imidazole concentration of recombinant protein purification elution buffer; Lane 1: After induction at 37℃ for 6 hours MTRF1L total protein; Lane 2: Supernatant after lysis; Lane 3: Supernatant after inclusion body lysis buffer; Lanes 4-11: Elution of target protein with elution buffers of different imidazole concentrations (20mM, 50mM, 100mM, 200mM, 300mM, 400mM, 500mM, 800mM); C: Detection of MTRF1L protein by His-tagged antibody using Western blotting; Lane 1: Purified MTRF1L protein (approximately 32kDa); Lane 2: Purified PET-28a empty vector protein; Standard protein marker ( 200kDa); Figure 4 Results of ultrafiltration purification of MTRF1L protein and preparation of polyclonal antibody serum titers: A: Ultrafiltration purification of MTRF1L protein, concentration 3.75 mg / ml; Lane 1: Ultrafiltration purified MTRF1L protein; Lane 2: Purified PET-28a empty carrier protein; B: Indirect enzyme-linked immunosorbent assay (ELISA) to determine the titer of the prepared antibody serum; X-axis represents the dilution gradient of anti-MTRF1L serum; Y-axis represents the absorbance of the polyclonal antibody at 450 nm wavelength; Positive serum represents anti-MTRF1L serum; Negative serum is pre-immunization serum; Figure 5 Results of indirect immunofluorescence screening for positive hybridoma cells: Four strongly positive monoclonal hybridoma cell lines, namely No. 25, No. 58, No. 59, and No. 73, were selected. Figure 6 To select four strong positive monoclonal antibodies, the results of Western blotting and quantitative real-time PCR were performed on chicken DF-1 cells after IBDV infection in the normal group and after infection with infectious bursal virus: A: Western blotting to detect the expression level of MTRF1L protein in DF-1 cells after IBDV infection; B: Quantitative real-time PCR to detect the expression level of MTRF1L protein. Detailed Implementation

[0017] Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this invention. The invention will now be described with reference to specific embodiments. It should be noted that these embodiments are merely descriptive and do not limit the invention in any way.

[0018] Example 1 1. Construction of the PET-28a-MTRF1L recombinant expression vector 1.1 Sequence optimization synthesis and primer design The reported chicken-derived MTRF1L gene (XM_040666977.2) was retrieved from GenBank. High-quality epitope protein sequences and their corresponding nucleotide sequences (729 bp) were screened using bioinformatics analysis (see Table 1). Specific primers were then designed and synthesized (see Table 2).

[0019] Table 1. MTRF1L truncated nucleotide / amino acid sequence Table 2 PCR reaction primers 1.2 PCR amplification of the target gene PCR was then performed to amplify the MTRF1L gene by binding to the template using specific primers. The total PCR reaction volume for the MTRF1L gene was 25 μL (see Table 3). The PCR reaction program was as follows: 95℃ pre-denaturation for 5 min, 95℃ denaturation for 30 s, 56℃ annealing for 30 s, 72℃ extension for 1 min, for 35 cycles, followed by a final extension at 72℃ for 10 min.

[0020] Table 3 Total PCR reaction system Depend on Figure 2 According to A, when the PCR reaction product is electrophoresed in a 1% nucleic acid gel, a clear, single, and specific band can be seen at a position with a molecular weight of approximately 729 bp.

[0021] 1.3 Double enzyme digestion The PCR amplification products of the above target gene and the PET-28a(+) expression vector were digested with EcoRI and XhoRI in a 10 μL digestion system (see Table 4), and then reacted at 37℃ for 1 hour.

[0022] Table 4. Double enzyme digestion reaction system 1.4 Connection The target fragment after double enzyme digestion was ligated into the PET-28a(+) expression vector in a ligation volume of 10 μL (see Table 5), and incubated overnight at 16°C.

[0023] Table 5 Connection Reaction System 1.5 Transformation The ligation product was gently mixed with 50 μL of competent cells and incubated on ice for 15 min. Then, it was heat-shocked at 42 °C for 90 s, incubated on ice again for 10 min, and then evenly spread on Kana-resistant solid medium and cultured at 37 °C for 12 h.

[0024] 1.6 Single-clone culture, bacterial PCR detection and sequencing Single plaques were picked from the solid culture medium and cultured in Kana-resistant liquid medium for 6 hours, followed by bacterial PCR detection. The total volume of the bacterial PCR reaction was 25 μL (see Table 6). The PCR reaction program was: 95℃ pre-denaturation for 5 min, 95℃ denaturation for 30 s, 56℃ annealing for 30 s, 72℃ extension for 1 min, 25 cycles, and a final extension at 72℃ for 10 min. Figure 2 As can be seen from the results of PCR, all three selected monoclonal bacterial cultures were positive. The positive monoclonal clones were sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing, and the sequencing results further confirmed that the positive clones were the correct PET-28a-MTRF1L clones.

[0025] Table 6 Total system for bacterial PCR reaction Subsequently, plasmid extraction was performed on the correct clone using a plasmid miniprep kit (purchased from Beijing Tiangen Biotech Co., Ltd.) to obtain the PET-28a-MTRF1L recombinant expression vector plasmid.

[0026] 2. Preparation and purification of MTRF1L protein 2.1 The successfully constructed PET-28a-MTRF1L expression vector was transformed into *E. coli* BL21 competent cells and cultured with IPTG at a final concentration of 1 mM on a shaker at 37℃ / 16℃ and 200 rpm / min for 6 hours to induce expression of the recombinant protein. The obtained recombinant protein was subjected to 12% SDS-PAGE electrophoresis to identify whether it was the desired target protein.

[0027] Depend on Figure 3 As shown in Figure A, the recombinant MTRF1L protein was highly expressed under the conditions of 37℃ and 200rpm / min, and was mainly expressed in the form of inclusion bodies in E. coli.

[0028] 2.2 The expressed MTRF1L recombinant protein was purified using a nickel affinity column. Elution conditions were optimized using imidazole concentrations of 20 mM, 50 mM, 100 mM, 200 mM, 300 mM, 400 mM, 500 mM, and 800 mM, respectively. Figure 3 As shown in section B, the optimal elution concentration is 800 mM imidazole, and the molecular weight of the MTRF1L recombinant protein obtained after purification by a nickel affinity column is approximately 32 kDa, which is consistent with the expected size.

[0029] Simultaneously, Western blot analysis using His-tagged antibodies revealed a distinct specific band at approximately 32 kDa. Figure 3 C. The MTRF1L recombinant protein was further purified by ultrafiltration to a concentration of 3.75 mg / ml, see [reference needed]. Figure 4 A. The protein was stored at -20°C and later used for mouse immunization.

[0030] 2.3 The purified MTRF1L recombinant protein was mixed with an equal volume of Freund's complete adjuvant and injected subcutaneously at multiple sites into Babes mice. The mice were boosted with immunization every 10 days (the booster immunization required the MTRF1L protein to be mixed with Freund's incomplete adjuvant). Blood was collected from the eyeballs 30 days after immunization and centrifuged to obtain immune serum.

[0031] 3. Quality Validation Methods for MTRF1L Protein Monoclonal Antibodies 3.1 Enzyme-linked immunosorbent assay (ELISA) The purified MTRF1L recombinant protein was diluted to 2.5 μg / mL as antigen and plated on 96-well plates (100 μL / well) and incubated overnight at 4°C. After washing three times with PBS-T, 200 μL of 5% skim milk powder was added to each well for blocking, and the plates were incubated at 37°C for 2 h. After washing, serially diluted (1:2000-1:256000) anti-MTRF1L serum and pre-immune serum of the same dilution gradient (100 μL / well) were added, and the plates were incubated at 37°C for 1 h. After washing, horseradish peroxidase-labeled goat anti-rabbit IgG diluted 1:3000 (120 μL / well) was added and incubated for 1 h. After washing with PBS-T, soluble single-component TMB substrate solution was added and reacted in the dark for 15 min. The reaction was terminated with concentrated sulfuric acid, and the absorbance was measured at 450 nm using a microplate reader to calculate the actual titer of the MTRF1L polyclonal antibody serum.

[0032] Depend on Figure 4 According to preliminary experiments, an antigen coating concentration of 2.5 μg / mL yielded the optimal antibody titer. Based on the formula OD-positive / OD-negative ≥ 2.1, the serum titer against MTRF1L reached 1:128000. The next step is to use hybridoma technology to fuse sensitized B cells capable of secreting MTRF1L-specific antibodies with myeloma cells possessing unlimited proliferative capacity to create B-cell hybridomas.

[0033] 4. Screening for positive monoclonal hybridoma cells 4.1 Indirect immunofluorescence detection DF-1 cells were seeded in 96-well plates and fixed with 4% paraformaldehyde solution the following day. After antigen retrieval, the plates were blocked with 5% BSA solution for 30-40 min. After blocking, the 96-well plates were incubated overnight at 4°C with anti-MTRF1L protein antibody (1:200 dilution) and pre-immunization serum (1:200 dilution), respectively. The plates were washed three times with PBS-T (5 min each time). The 96-well plates were then incubated with 1:500 dilution of CoraLite594-conjugated Goat Anti-Mouse IgG (H+L) incubation solution at room temperature in the dark for 1 hour. After washing three times with PBS-T (5 min each time), the plates were incubated with DAPI solution (C0065, Beijing Solarbio Science & Technology Co., Ltd.) in the dark for 10 min. After washing three times, the plates were immersed in PBS-T solution and examined under a Zeiss inverted fluorescence microscope for localization.

[0034] like Figure 5As shown, DF-1 cells were incubated with pre-immunization serum and monoclonal hybridoma cell supernatant as primary antibodies. Compared with the normal group, the experimental group showed a bright green fluorescent signal, which was considered positive, indicating that the monoclonal antibody can specifically bind to the MTRF1L protein in DF-1 cells.

[0035] 5. Detection method for MTRF1L expression level in IBDV-infected chicken DF-1 cells 5.1 Western Blot Detection Total protein was extracted from DF-1 cells in the normal group and after infection with infectious bursal virus (IBDV). After determining the protein concentration using the BCA method, 25 μg of protein was subjected to 12% SDS-polyacrylamide gel electrophoresis. Using the stably expressed gene GAPDH as an internal reference, the electrophoretic bands were transferred to a nitrocellulose membrane using a semi-dry transfer method. The membrane was blocked in 5% skim milk at room temperature for 30 min. Then, it was incubated overnight at 4°C on a shaker with anti-MTRF1L serum (1:1000 dilution) and pre-immunization serum (1:1000 dilution), respectively. The membrane was washed three times with PBS-T (10 min each time). The membrane was then incubated with horseradish peroxidase-labeled goat anti-mouse IgG (1:5000 dilution) at room temperature for 40 min. Finally, it was washed three times, and the protein bands were detected using a high-sensitivity ECL chemiluminescence assay kit (Shanghai Biotechnology Co., Ltd.) to detect the protein level of this gene in the tissue.

[0036] Depend on Figure 6 Studies have shown that the supernatant of anti-MTRF1L positive monoclonal hybridoma cells, used as a primary antibody, can specifically bind to MTRF1L protein in spleen tissue, and the MTRF1L protein level in the infected group is significantly reduced (P≤0.001). Compared with the uninfected group, the MTRF1L expression level in DF-1 cells infected with IBDV is significantly downregulated.

[0037] 5.2 Quantitative Real-Time PCR Detection Chicken DF-1 cell samples were collected from both the normal group and the group infected with infectious bursal virus. Total RNA was extracted and reverse transcribed to obtain total cDNA, which was then detected by quantitative real-time PCR.

[0038] like Figure 6 As shown in Figure B, the MTRF1L mRNA level in the pathological group was significantly lower than that in the normal group (P≤0.001), which is consistent with the results of Western Blot analysis.

[0039] The experimental data were processed using SPSS 25.0 software. Measurements are expressed as mean ± standard deviation (M ± SD). Analysis of variance was used for inter-group comparisons. *: p < 0.05; **: p ≤ 0.01; ***: p ≤ 0.001, indicating highly significant difference.

[0040] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A recombinant chicken MTRF1L protein, characterized in that, Its amino acid sequence is shown in SEQ ID NO:

1.

2. The method for expressing recombinant chicken MTRF1L protein according to claim 1, characterized in that, Includes the following steps: The chicken MTRF1L gene was constructed into an expression vector, and recombinant chicken MTRF1L protein was obtained through expression.

3. The expression method according to claim 2, characterized in that, The chicken MTRF1L gene sequence is shown in SEQ ID NO:

2.

4. The expression method according to claim 2, characterized in that, Including prokaryotic expression and eukaryotic expression; The prokaryotic expression was performed using the prokaryotic expression vector pET-28a; the eukaryotic expression was performed using the eukaryotic expression vector pCMV.

5. A method for preparing a monoclonal antibody against chicken MTRF1L protein, characterized in that, Includes the following steps: Immunized mice were constructed using the recombinant chicken MTRF1L protein of claim 2 to obtain immune serum, and then a monoclonal antibody against the recombinant chicken MTRF1L protein was obtained using hybridoma technology.

6. The application of the chicken MTRF1L protein monoclonal antibody prepared by the preparation method according to claim 5 in products for detecting chicken infection with IBDV.

7. The application according to claim 6, characterized in that, The products include chips, probes, and reagent kits.

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