Chicken MFN1 antigenic determinant and its application

Abstract

The present invention provides a chicken MFN1 antigenic determinant and its application. The present invention analyzes the chicken MFN1 protein and screens the antigenic determinant of the chicken MFN1 protein; obtains the MFN1-E coding gene by amplification, inserts it into the pET-28a(+) vector, and constructs the pET-28a-Mfn1-E prokaryotic expression plasmid; transforms the constructed pET-28a-Mfn1-E prokaryotic expression plasmid into competent cells, and uses IPTG to induce the expression of the MFN1-E protein; prepares the purified MFN1-E protein as an antigen and immunizes mice to obtain anti-chicken MFN1 polyclonal antibodies. The prepared polyclonal antibodies can be used for quantitative and localization analysis of chicken MFN1 protein. The anti-chicken MFN1 polyclonal antibodies prepared by the present invention have good reactivity with the chicken MFN1 protein, provide important tools and technical means for further research on avian mitochondrial dynamics, and have high practical application value.

Description

Technical Field

[0001] The invention belongs to the technical field of animal molecular biology, and particularly relates to a chicken MFN1 antigenic determinant and application thereof. Background Art

[0002] Mitochondria are double-membrane organelles that physiologically exist in a dynamic equilibrium between fusion and fission. Mitochondrial fusion is primarily coordinated by the proteins MFN1 (mitofusin 1), MFN2, and OPA1 (optic atrophy). Outer membrane fusion is primarily mediated by MFN1 and MFN2, while inner membrane fusion is primarily mediated by OPA1. Literature reports indicate that knocking out Mfn1 causes embryonic lethality in mice, indicating that Mfn1 is crucial for embryonic development. In mouse embryonic fibroblasts, double knockout of both Mfn1 and Mfn2 completely blocks mitochondrial fusion, while overexpression of both Mfn1 and Mfn2 promotes mitochondrial fusion. The grain toxin deoxynivalenol upregulates the expression of mitochondrial fission proteins while downregulating the expression of mitochondrial fusion proteins (MFN1 and MFN2), leading to excessive mitochondrial fission and impaired intestinal barrier function in pigs. As previously reviewed, mitochondrial fusion proteins such as MFN1 are important targets for studying mitochondrial dynamics. However, there are currently no commercially available anti-chicken MFN1 antibodies available, and human or mouse MFN1 antibodies have low cross-reactivity with the chicken MFN1 protein. Therefore, the development of anti-chicken MFN1 antibodies would provide an important tool and technical means for studying avian mitochondrial dynamics and possesses high practical application value. Summary of the Invention

[0003] The present invention aims to overcome the current lack of antibodies that can specifically bind to chicken MFN1 and provides a chicken MFN1 antigenic determinant, named MFN1-E, comprising or consisting of amino acids 238 to 469 of the chicken MFN1 protein as shown in SEQ NO.1.

[0004] The chicken MFN1 antigenic determinant of the present invention is obtained by the following method:

[0005] 1. Screening of chicken MFN1 antigenic determinants:

[0006] (a) The physicochemical properties of chicken MFN1 protein were analyzed. DNAstar was used to predict antigenic determinants, and an amino acid sequence with a high antigenic index and no signal peptide or transmembrane region was identified, which was named MFN1-E.

[0007] (b) The chicken MFN1 antigenic determinant is the amino acid sequence at positions 238 to 469 of the chicken MFN1 protein.

[0008] 2. Construction of pET-28a-Mfn1-E prokaryotic expression plasmid

[0009] (a) Design Mfn1-E-specific primers containing HindIII and XhoI restriction sites: the upstream primer SEQ NO. 2 is: 5'-CTCCGTCGACAAGCTAATAGATGGGATGCCTCTG-3', and the downstream primer SEQ NO. 3 is: 5'-GGTGGTGGTGCTCGACGACTCATTGACTTCACTG-3';

[0010] (b) Using PCR technology, a Mfn1-E DNA fragment with homology arms was amplified using chicken intestinal cDNA as a template; this DNA fragment was purified and homologously recombined with the pET-28a(+) vector digested with HindIII and XhoI;

[0011] (c) The recombinant product was transformed into Escherichia coli DH5α using the heat shock method. After sequencing verification, the plasmid was extracted to obtain the pET-28a-Mfn1-E prokaryotic expression plasmid.

[0012] 3. Induced expression and purification of MFN1-E protein:

[0013] The recombinant plasmid pET-28a-Mfn1-E was transformed into Escherichia coli BL21 competent cells, cultured to the logarithmic growth phase, added with IPTG at a final concentration of 1 mM, and cultured at 37°C with shaking for 3-4 hours. Afterwards, the bacterial pellet was collected by centrifugation and resuspended in 40 mL of protein denaturing lysis buffer, ultrasonically disrupted, and centrifuged. The pellet and supernatant were subjected to SDS-PAGE electrophoresis, respectively. The supernatant was selected for Ni column purification based on the test results.

[0014] 4. Preparation of antigens and polyclonal antibodies:

[0015] MFN1-E protein solution was mixed with Freund's complete adjuvant in equal proportions and emulsified on ice. The mixture was then injected subcutaneously at multiple points on the back of mice. Thereafter, MFN1-E protein solution was mixed with Freund's incomplete adjuvant in equal proportions, and booster immunizations were performed every 10 days for a total of three times. Finally, whole blood was collected to obtain antiserum, which was then purified to obtain polyclonal antibodies.

[0016] Another object of the present invention is to provide the application of the chicken MFN1 antigenic determinant in the quantitative and localization analysis of chicken MFN1 protein, wherein the chicken MFN1 antigenic determinant comprises or consists of the sequence of amino acids 238 to 469 of the chicken MFN1 protein as shown in SEQ NO.1.

[0017] A recombinant plasmid capable of expressing a chicken MFN1 protein antigenic determinant is disclosed. Specific primers are designed as shown in SEQ NO. 2: 5'-CTCCGTCGACAAGCTAATAGATGGGATGCCTCTG-3' and SEQ NO. 3: 5'-GGTGGTGGTGCTCGACGACTCATTGACTTCACTG-3'. A DNA fragment encoding the chicken MFN1 antigenic determinant is amplified using chicken intestinal tissue cDNA as a template. The prokaryotic expression plasmid is used to induce expression of the MFN1 antigenic determinant and emulsify it into an antigen. Non-avian animals are immunized to obtain antiserum, and polyclonal or monoclonal antibodies against chicken MFN1 are further prepared. The prepared polyclonal antibodies can be used for quantitative and localization analysis of the chicken MFN1 protein.

[0018] The present invention studies the chicken MFN1 protein, screens its antigenic determinants, constructs recombinant plasmids, and inducibly expresses the chicken MFN1 antigenic determinant polypeptide. This antigen is then used to immunize non-avian animals. Polyclonal antibodies prepared using this antigenic determinant can specifically bind to the chicken MFN1 protein. The resulting anti-chicken MFN1 polyclonal antibodies can be used for quantitative and localized analysis of the chicken MFN1 protein, providing new tools and approaches for studying chicken mitochondrial dynamics. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the chicken MFN1 antigenic determinant screened by the present invention, wherein the boxed area (238-469aa) at the bottom of the figure is the selected antigenic determinant.

[0020] Figure 2 This is a map of the pET-28a-Mfn1-E recombinant plasmid constructed in the present invention.

[0021] Figure 3 The chicken MFN1-E protein induced and expressed by the present invention and its purification results are shown, wherein M is a protein molecular weight standard; 1 is a column flow-through; 2-3 is a gradient concentration imidazole washing solution; 4-7 is a gradient concentration imidazole elution solution.

[0022] Figure 4 The results of Western blot analysis of MFN1 protein levels in chicken small intestine, where M represents protein molecular weight standard; 1-2 represents total protein in chicken intestinal tissue; and 3-4 represents total protein in chicken crypts.

[0023] Figure 5 The results of immunofluorescence staining were used to analyze the localization of MFN1 protein in chicken granulosa cells. DETAILED DESCRIPTION

[0024] The present invention is further described with reference to the accompanying drawings and embodiments.

[0025] Example 1: Screening of MFN1 antigenic determinants

[0026] The readable sequence NM_001012931.3 of the chicken Mfn1 gene was selected from NCBI to obtain the amino acid sequence of its protein. The physicochemical properties of the protein were analyzed using Prot Param (https: / / web.expasy.org / protparam / ). The results showed that the theoretical isoelectric point of the chicken MFN1 protein was 6.20; the number of amino acids was 740; the theoretical molecular weight was 84.00 kDa; the instability coefficient was 43.17, indicating that it was an unstable protein; the hydrophilicity was -0.299, indicating that it was a hydrophilic protein. The antigenic determinant cluster was predicted using DNAstar, and a region with a high antigenic index of 238 to 469 aa was screened out (see Figure 1 , sequence see SEQ NO.1), and named MFN1-E; NovoPro (https: / / novopro.cn / tools / signalp) was used to analyze the protein signal peptide, and the results showed that the selected segment had no signal peptide; Detaibio (http: / / www.detaibio.com / tools / transmembrane.html) was used to predict the protein transmembrane region, and the results showed that the selected segment had no transmembrane region.

[0027]

[0028] Example 2: Construction of pET-28a-Mfn1-E recombinant plasmid

[0029] The Mfn1-E specific primers containing HindⅢ and Xho I restriction sites were designed: F: 5'-CTCCGTCGACAAGCTAATAGATGGGATGCCTCTG-3'; R: 5'-GGTGGTGGTGCTCGACGACTCATTGACTTCACTG-3'. Using PCR technology, the Mfn1-E DNA fragment with homology arms was amplified using chicken intestinal segment cDNA as a template. The PCR system was 25 μL 2×Phanta Max Master Mix, 2 μL upstream primer, 2 μL downstream primer, 5 μL cDNA, and 16 μL ddH2O. The PCR pre-denaturation temperature was 95°C for 3 min, followed by 35 cycles of denaturation at 95°C for 15 s, annealing at 60°C for 15 s, and extension at 72°C for 15 s, followed by extension at 72°C for 5 min, and finally storage at 4°C. The PCR amplification product was purified and then homologously recombined with the pET-28a(+) vector double-digested with HindⅢ and Xho I. The reagents used included 0.5 μL vector, 1 μL PCR product, 2 μL 5×CE II Buffer, 1 μL Exnase II, and 5.5 μL ddH2O. The reaction procedure was 37°C for 30 min. The recombinant product was transformed into competent cells DH5α by heat shock method, 100 μL of bacterial solution was taken and spread on LB solid medium containing kanamycin, cultured at 37°C overnight, and single clones were picked and transferred to 1 ml LB liquid medium containing kanamycin, cultured at 37°C for 8-12 hours, and the single clones verified by sequencing were expanded and the pET-28a-Mfn1-E prokaryotic expression plasmid ( Figure 2 ).

[0030] Example 3: Expression and purification of MFN1-E protein

[0031] On LB solid medium containing kanamycin, the pET-28a-Mfn1-E prokaryotic expression plasmid was transformed into Escherichia coli BL21 competent cells, and a single clone was selected and cultured in 20 mL LB liquid medium containing kanamycin at 37°C and 220 rpm for 12-14 h. After that, 4 mL of the bacterial solution was added to 400 mL of LB liquid medium containing kanamycin. The cells were cultured in LB medium flasks at 37°C, 120 rpm until the bacteria reached the logarithmic growth phase and the OD600 value was 0.4-0.6. IPTG was then added at a final concentration of 1 mM and cultured at 37°C with shaking for 3-4 h. The induced bacterial solution was dispensed into 50 ml centrifuge tubes and centrifuged at 4°C, 4000 rpm for 5 min. The bacterial pellets were collected and 40 mL of protein denaturation lysis buffer was added. After ultrasonic disruption (efficiency 50%, ultrasonication for 2 s, pause for 6 s, for a total of 40 min) and centrifugation (4°C, 5000 rpm for 15 min), the pellets and supernatant were subjected to SDS-PAGE electrophoresis, respectively.

[0032] The recombinant protein was purified using Ni-NTA. The steps were as follows: 4 mL of protein supernatant was added to the equilibrated purification column, mixed, placed on ice, shaken on a horizontal shaker for 60 min, incubated at 4°C overnight, and the sample was loaded and flowed through 3 to 5 times; 5 column volumes of denaturing lysis buffer were added, and the column was equilibrated twice; 5 column volumes of washing buffer were added, washed twice, 1 column volume of elution buffer was added, and eluted 8 times. 8 tubes of eluate were collected for SDS-PAGE electrophoresis detection. The eluate in tubes 1 to 3 contained more target protein. Figure 3 The figure shows the induced expression of MFN1-E protein and its purification results.

[0033] Example 4: Western blot detection of MFN1 polyclonal antibody specificity

[0034] Total protein from the duodenum of laying hens was extracted and subjected to 10% SDS-PAGE electrophoresis, membrane transfer, and blocking with 5% skim milk at room temperature for 2 hours. The anti-chicken MFN1 polyclonal antibody provided by the present invention was then added dropwise and incubated at 4°C overnight. The membrane was then washed three times with TBST and incubated with HRP-goat anti-mouse IgG at room temperature for 1 hour. The membrane was then washed three times with TBST and developed using a high-sensitivity ECL luminescence kit. The results are shown in Figure 2. Figure 4 As shown, there is a clear band at 85 kDa, which is the chicken MFN1 protein.

[0035] Example 5: Immunofluorescence analysis of the localization of MFN1 in chicken granulosa cells

[0036] Cultured chicken granulosa cells were washed with 0.1 mol / L PBS buffer, fixed with 4% paraformaldehyde, soaked in PBS with 0.3% Triton X-100 (i.e., PBST), and blocked with goat serum at room temperature. The anti-chicken MFN1 polyclonal antibody provided by the present invention was then added dropwise and incubated at 4°C overnight. The cells were then washed with PBST, incubated in the dark with 488-goat anti-mouse IgG for 1 hour, washed with PBST, stained with DAPI, and washed with PBST. Finally, the cells were observed using a fluorescence microscope. Figure 5 As shown, MFN1 protein is located in the cytoplasm of chicken granulosa cells.

Claims

1. A recombinant plasmid capable of expressing the antigenic determinant of chicken MFN1 protein, characterized in that: Specific primers were designed as shown in SEQ ID NO. 2: 5'-CTCCGTCGACAAGCTAATAGATGGGATGCCTCTG-3' and SEQ ID NO. 3: 5'-GGTGGTGGTGCTCGACGACTCATTGACTTCACTG-3'. Using chicken intestinal tissue cDNA as a template, a DNA fragment encoding the chicken MFN1 protein antigenic determinant was amplified. The protein antigenic determinant consists of the sequence of amino acids 238 to 469 of the chicken MFN1 protein as shown in SEQ ID NO.

1.

2. Use of the recombinant plasmid of the chicken MFN1 protein antigenic determinant according to claim 1 in the preparation of a reagent for quantitative and localization analysis of chicken MFN1 protein.

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