Clostridium perfringens mbp gene and application thereof
By preparing and purifying Clostridium perfringens Mbp protein, recombinant strain BL21 (pET28a-Mbp) was constructed, solving the problem of the lack of alternative antibiotic control methods for necrotizing enteritis in poultry and achieving effective immune protection.
Patent Information
- Application Number
- CN202410078622.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-01-19
- Publication Date
- 2025-12-30
- Estimated Expiration
- 2044-01-19
AI Technical Summary
Current technologies lack effective alternatives to antibiotics for the prevention and control of necrotizing enteritis in poultry, especially against necrotizing enteritis caused by Clostridium perfringens. Furthermore, the problem of necrotizing enteritis in poultry has become increasingly serious since the European Union banned the use of growth-promoting antibiotics.
The Mbp gene of Clostridium perfringens in chickens, along with its recombinant plasmid and protein, were provided. The recombinant Mbp protein was prepared using a prokaryotic expression method for the preparation of subunit vaccines. The Mbp gene was inserted into the expression vector pET28a using a homologous recombination method to construct the recombinant bacterium BL21 (pET28a-Mbp), followed by protein purification and immunoprotection.
The prepared recombinant Mbp protein exhibited good immunoreactivity, significantly reduced clinical symptoms caused by Clostridium perfringens, showed potential as a subunit vaccine, and significantly reduced lesions in avian necrotic enteritis.
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Abstract
Description
Technical Field
[0001] This invention relates to an Mbp gene of Clostridium perfringens in chickens and its application, belonging to the field of genetic engineering. Background Technology
[0002] Clostridium perfringens (CP) is a Gram-positive, facultative anaerobic, spore-forming rod-shaped bacterium. The G-type of CP can cause necrotic enteritis (NE) in poultry. Its acute clinical form can lead to sudden death in broilers aged 2-4 weeks, while the subclinical form often causes intestinal mucosal damage, hemorrhage, and decreased production performance. Furthermore, necrotic enteritis in poultry is easily affected by predisposing factors such as coccidiosis, high-protein feed, and immunosuppressive diseases. Damage to the intestines can lead to secondary infections by other poultry pathogens, resulting in more severe disease and economic losses.
[0003] The main strategy for controlling necrotic enteritis in poultry is through the supplementation of feed with antibiotic additives for prevention and treatment. However, in response to the emergence of antibiotic-resistant strains that threaten human and animal health, the European Union (EU) banned the use of growth-promoting antibiotics in 1999, and the problem of necrotic enteritis infection in poultry has become increasingly serious. Therefore, there is an urgent need for alternative methods of control to replace antibiotics. The design and development of novel vaccines, such as subunit vaccines, is currently a hot research topic.
[0004] Mbp is a maltose ABC transporter-binding protein, reported as an immunogenic protein in *Streptococcus suis*. In Gram-positive bacteria, it typically functions as a membrane-anchored lipoprotein involved in the uptake and utilization of maltose and maltodextrin. Furthermore, it is used as an adjuvant in fusion expression with other protective proteins for subunit vaccine applications. There are currently no reports on the *Clostridium perfringens* Mbp protein. Summary of the Invention
[0005] Purpose of the invention: The technical problem to be solved by the present invention is to provide a Clostridium perfringens Mbp gene with good immunoreactivity and its application.
[0006] Technical solution: To solve the above technical problems, the present invention provides a Clostridium perfringens Mbp gene, the nucleotide sequence of which is shown in SEQ ID NO.3.
[0007] The present invention also provides a protein expressing the Clostridium perfringens Mbp gene, the amino acid sequence of which is shown in SEQ ID NO.4.
[0008] The present invention also provides a recombinant plasmid, expression cassette or recombinant cell containing the aforementioned Clostridium perfringens Mbp gene.
[0009] The present invention also provides a method for constructing the recombinant plasmid, comprising the following steps: inserting the Clostridium perfringens Mbp gene into the expression vector pET28a to obtain the recombinant plasmid pET28a-Mbp.
[0010] In this method, the Mbp gene of Clostridium perfringens was inserted into the expression vector pET28a using homologous recombination.
[0011] The homologous recombination system consists of: amplified fragment, pET28a plasmid, Exnase II, 5×CEII Buffer, and ddH2O.
[0012] The present invention also provides a recombinant bacterium containing the aforementioned Clostridium perfringens Mbp gene or the aforementioned recombinant plasmid, expression cassette or recombinant cell.
[0013] The present invention also provides a method for constructing the recombinant bacteria, comprising the following steps: transforming the recombinant plasmid into Escherichia coli BL21 to obtain recombinant bacteria BL21 (pET28a-Mbp).
[0014] The present invention also provides the application of the aforementioned Clostridium perfringens Mbp gene, the aforementioned protein, the aforementioned recombinant plasmid, expression cassette, or recombinant cell or the aforementioned recombinant bacteria in the preparation of a vaccine for the prevention of necrotizing enteritis in poultry.
[0015] The vaccine in question is a subunit vaccine.
[0016] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: 1. The present invention extracts the whole genome from Clostridium perfringens, designs primers to amplify the Mbp gene, and prepares recombinant Mbp protein for the first time using prokaryotic expression method; 2. Clostridium perfringens protein Mbp has good immunoreactivity and can significantly alleviate the clinical symptoms caused by Clostridium perfringens in chickens; 3. The Clostridium perfringens protein Mbp with immunoprotective effect provided by the present invention has the potential to be used as a subunit vaccine for Clostridium perfringens in chickens. Attached Figure Description
[0017] Figure 1 PCR identification of BL21(pET28a-Mbp) bacterial culture;
[0018] Figure 2 SDS-PAGE identification of Mbp protein expression;
[0019] Figure 3 SDS-PAGE identification of Mbp protein purification;
[0020] Figure 4Western blot identification of Mbp protein;
[0021] Figure 5 Observation of clinical symptoms induced by Clostridium perfringens challenge (*p<0.05): A is a representative image of chicken intestinal tissue; B is a statistical chart of scores for each group;
[0022] Figure 6 The pathological histological observation was performed after challenge with Clostridium perfringens (*p<0.05). Detailed Implementation
[0023] The technical solution of the present invention will be further described below with reference to the accompanying drawings.
[0024] Example 1 Cloning and Prokaryotic Expression of Clostridium perfringens Mbp Gene
[0025] 1. Preparation of the Mbp gene from Clostridium perfringens
[0026]
[0027] The amplified target gene was inserted into the expression vector pET28a(+) (Solarbio, P3110) using homologous recombination to form the recombinant expression plasmid pET28a-Mbp. The homologous recombination system consisted of: 1 μL of amplified fragment, 1 μL of pET28a plasmid, 1 μL of Exnase II, 2 μL of 5×CE II Buffer, and 5 μL of ddH2O. The homologous recombination reaction program was 37℃ for 30 min. The homologous recombination reagent was purchased from Nanjing Novizan Biotechnology Co., Ltd., and the pET28a plasmid was stored in our laboratory. Subsequently, the recombinant expression plasmid was transformed into engineered Escherichia coli BL21(DE3) (purchased from Nanjing Novizan Biotechnology Co., Ltd.). Clones were picked and identified by colony PCR and sequencing. Correct clones were identified as positive clones and named BL21(pET28a-Mbp). The PCR system consisted of: 6 μL T7 Promoter Primer, 6 μL T7 Terminator Primer, 120 μL Taq enzyme, and 108 μL ddH2O. The PCR reaction program was: 95℃ for 5 min; 95℃ for 30 s, 55℃ for 1 min, and 72℃ for 1 min, for a total of 30 cycles; extension at 72℃ for 5 min. The amplification primers were the pET28a universal T7 primers (SEQ ID NO. 5: F: 5'-TAATACGACTCACTATAGGG-3'; SEQ ID NO. 6: R: 5'-GCTAGTTATTGCTCAGCGG-3'). The PCR results are as follows: Figure 1 As shown, the amplified fragment includes 294 bp at the N-terminus and C-terminus of pET28a, and the Mbp target fragment of 1149 bp, totaling 1443 bp, which is consistent with the expected fragment size, confirming the successful acquisition of the BL21 (pET28a-Mbp) strain. The negative control contained no template, only primers, enzymes, and water; pET28a represents the amplification result after transforming the expression vector pET28a into *E. coli* BL21. The truncated Mbp protein gene sequence of *Clostridium perfringens* strain 13 is shown in SEQ ID NO. 3.
[0028] 2. Identification of Mbp protein expression
[0029] (1) The obtained recombinant Escherichia coli BL21(pET28a-Mbp) and BL21(pET28a) were cultured in 100 mL LB liquid medium until OD. 600When the concentration was 0.7, isopropyl-β-D-thiogalactoside (IPTG) (Takara, catalog number CB4601B) was added to a final concentration of 0.5 mM (5 mL of recombinant Escherichia coli BL21 (pET28a-Mbp) culture without IPTG was used as a control), and expression was induced on a shaker at 37°C for 4 hours.
[0030] (2) The bacterial culture was centrifuged at 8000 rpm for 10 min to collect BL21 (pET28a-Mbp) cells, and the cells were resuspended in 16 mL of PBS solution (PBS solution formula: KCl 0.2 g, NaCl 8 g, Na2HPO4 1.44 g, KH2PO4 0.24 g, 1000 mL distilled water, pH 7.6).
[0031] (3) After the bacterial cells were broken by ultrasonic disruption, the supernatant was collected by centrifugation at 9000 rpm / min for 10 min at 4℃. The precipitate was then resuspended in 16 mL of the above PBS solution for later use.
[0032] (4) Take 20 μL of the precipitate resuspended from the whole bacteria obtained in step (3), add 5 μL of loading buffer (1.25 mL of 1 M Tris-HCl (pH 6.8), 2.5 mL of glycerol, 0.5 g of sodium dodecyl sulfate (SDS), and 25 mg of bromophenol blue, dissolved in distilled water and brought to a final volume of 5 mL, aliquot into 500 μL portions and store at room temperature. Before use, add 25 μL of 2-ME (β-mercaptoethanol), 2 mL of 20% glycerol, and 7 mL of ultrapure water to each portion, mix well, and boil in 100 °C for 10 min. Identify expression using 12% SDS-PAGE gel electrophoresis. Results are as follows: Figure 2 As shown, after IPTG induction, the target band appeared at approximately 44 kDa in the whole BL21(pET28a-Mbp) strain, while this band was not observed in the BL21(pET28a) lane. Here, M represents a 180 kDa protein marker; pET28a control is BL21(pET28a); uninduced whole bacteria are BL21(pET28a-Mbp) whole bacteria not induced by IPTG; and induced whole bacteria are BL21(pET28a-Mbp) whole bacteria induced by IPTG.
[0033] Example 2 Purification of Mbp protein from Clostridium perfringens in chickens
[0034] The supernatant obtained after lysis in Example 1 was further purified using High-Affinity Ni-NTA Resin (purchased from Genscript Biotech Inc., L00250), as follows:
[0035] (1) The supernatant obtained after the crushing in Example 1 was filtered through a 0.45 μm pore size filter and added to an affinity chromatography column (purchased from Biosharp, BS-AC-012) and the flow-through was collected; before the experiment, 5 mL of Ni-NTA metal chelate His protein purification medium (purchased from Genscript Biotech Co., Ltd.) was added to the affinity chromatography column and washed with 12 mL of ddH2O;
[0036] (2) Add 20 mL of equilibration buffer (60 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0) to equilibrate the column;
[0037] (3) Add 50 mL of washing buffer (60 mM NaH2PO4, 300 mM NaCl, 50 mM imidazole, pH 8.0) to wash away impurities and collect the washing solution;
[0038] (4) Add 12 mL of elution buffer (60 mM NaH2PO4, 300 mM NaCl, 500 mM imidazole, pH 8.0) to elute the target protein, and collect the elution in separate tubes, for a total of 6 tubes.
[0039] (5) Take 20 μL of each of the pre-purification sample (supernatant and precipitate after induction), flow-through buffer, washing buffer and elution buffer, add 1.25 mL of 1 M Tris-HCl (pH 6.8), 2.5 mL of glycerol, 0.5 g of sodium dodecyl sulfate (SDS) and 25 mg of bromophenol blue, dissolve and bring to a final volume of 5 mL with distilled water, aliquot into 500 μL and store at room temperature. Before use, add 25 μL of 2-ME (β-mercaptoethanol), 2 mL of 20% glycerol and 7 mL of ultrapure water to each aliquot, mix well and boil for 10 min.
[0040] (6) After preparing a 12% SDS-PAGE gel, the five samples prepared in step (5) were added to the wells for electrophoresis. After electrophoresis, the gel was removed, stained with Coomassie Brilliant Blue overnight, and then destained to determine whether Mbp was present in the elution buffer. The results showed that the elution buffer contained Mbp protein with high purity and a size of 44 kDa. Figure 3 Wherein, M: 180kDa protein marker; lane 1: supernatant sample after induction; lane 2: precipitate sample after induction; lane 3: flow-through sample; lane 4: washing sample; lane 5: elution sample (Mbp); lane 6: BL21 (pET28a) control;
[0041] (7) The eluent containing the target protein was dialyzed and concentrated through a 10kDa ultrafiltration tube to obtain the purified recombinant protein. Then the buffer was replaced with PBS solution to obtain the Mbp protein suspension dissolved in PBS.
[0042] Example 3: Identification of Mbp protein in Clostridium perfringens (chicken)
[0043] The purified recombinant protein obtained in Example 2 was further identified by Western blot. 20 μL of the purified recombinant protein was added to 5 μL of loading buffer and electrophoresis was performed. The protein was electrotransferred onto a nitrocellulose membrane using a microdialyzer (Bio-Rad). The membrane was blocked overnight in blocking buffer (PBST containing 5% skim milk) at 4°C. After washing with PBST, His-Tag (Boster Biological, M30975) was used as the primary antibody, and HRP-labeled goat anti-mouse IgG (Boster Biological, BA1050) was used as the secondary antibody. The primary antibody dilution was 1:3000, and the secondary antibody dilution was 1:5000. The signal was detected using an enhanced chemiluminescence (ECL) substrate (Tanon, 180-501), further verifying the correct expression of the Clostridium perfringens protein Mbp. Figure 4 Mbp is the purified recombinant protein; pET28a is the control BL21 (pET28a).
[0044] Example 4: Determination of the immunoprotective efficacy of Clostridium perfringens Mbp protein subunit vaccine in chickens.
[0045] Fifteen one-day-old SPF chickens (SPF embryos purchased from Boehringer Ingelheim, and the SPF chickens were hatched in our laboratory) were divided into three groups: an immunization group immunized with PBS containing adjuvant followed by a challenge group (challenge control); an immunization group immunized with Clostridium perfringens Mbp protein followed by a challenge group (Mbp immunization group); and a blank control group without immunization or challenge, with five chickens in each group. In the Mbp immunization group: the recombinant Mbp protein was diluted with PBS and emulsified with an equal volume of Montanide™ ISA 201VG adjuvant (purchased from SEPPIC). Each SPF chicken was intramuscularly injected with the emulsion containing Clostridium perfringens Mbp protein (100 μg of Clostridium perfringens Mbp protein). Two and three weeks later, booster immunizations were performed using the same method, i.e., an intramuscular injection of the same amount of Clostridium perfringens Mbp protein was administered again. In the challenge control group: each SPF chicken was injected with an equal volume of PBS emulsion (PBS and Montanide™ ISA 201VG emulsified in equal volumes). On day 23 following the first intramuscular injection, both the Mbp immunization group and the challenge control group were infected with Eimerianecatrix oocysts via sporulation (Cloning and characterization of an Eimerianecatrix gene encoding a gametocyte protein and associated with oocyst wall formation, Parasit Vectors. 2014 Jan 15:7:27. doi:10.1186 / 1756-3305-7-27, https: / / pubmed.ncbi.nlm.nih.gov / 24428893 / ). The inoculation dose was 1×10⁻⁶. 4 Each individual / animal was infected with Clostridium perfringens on day 28 after the first intramuscular injection, with infection repeated for three consecutive days. The challenge dose was 1×10⁻⁶. 9 CFU / animal, all administered via gavage. The blank control group received no treatment.
[0046] Four hours after the final viral challenge, the chickens were euthanized. Dissection revealed intestinal lesions, which were scored according to the extent of the lesions, extending from the duodenum to Merkel's diverticulum. The scoring criteria were: 0 points - no intestinal lesions; 1 point - small amount of mucus adhering to the intestinal mucosa, loss of tone, thinning or fragility of the intestinal wall; 2 points - focal necrosis or ulceration; 3 points - intestinal mucosal sloughing, intestinal hemorrhage. Results are as follows: Figure 5 As shown, the intestinal surface of mice in the challenge control group was covered with a large amount of inflammatory exudate. Gently peeling away the exudate revealed sloughing of the intestinal mucosa. In contrast, the intestinal mucosa of mice in the Mbp immunization group and the blank control group remained intact or had only a small amount of exudate. Based on the comprehensive scoring, the intestinal lesion score of the Mbp immunization group was significantly lower than that of the challenge control group. Histopathological results are shown below. Figure 6 As shown, the challenged control group exhibited significant glandular hyperplasia and intestinal villi disintegration, losing its normal intestinal physiological structure. In contrast, the Mbp immunized group remained relatively normal, with only exudate adhering to the surface of the intestinal villi. These results indicate that Mbp protein immunization can alleviate the clinical symptoms of necrotizing enterocolitis caused by Clostridium perfringens type G infection, demonstrating a good immunoprotective effect.
Claims
1. Use of a Clostridium perfringens Mbp gene with a nucleotide sequence as shown in SEQ ID NO. 3 or a protein with an amino acid sequence as shown in SEQ ID NO. 4 in the preparation of a vaccine for preventing avian necrotic enteritis.
2. Use of a recombinant plasmid, expression cassette or recombinant cell containing the Mbp gene of Clostridium perfringens type A for the preparation of a vaccine for the prevention of necrotic enteritis in poultry, characterized in that, The nucleotide sequence of the Clostridium perfringens Mbp gene is as shown in SEQ ID NO.
3.
3. Use of a recombinant bacterium for the preparation of a vaccine for the prevention of necrotic enteritis in poultry, characterized in that, The recombinant bacteria contain a Clostridium perfringens Mbp gene or a recombinant plasmid or expression cassette containing a Clostridium perfringens Mbp gene, and the nucleotide sequence of the Clostridium perfringens Mbp gene is as shown in SEQ ID NO.
3.
4. The use according to any one of claims 1 to 3, characterized in that, The vaccine includes a subunit vaccine.