A subunit vaccine against Clostridium perfringens in birds

Abstract

The present invention provides a subunit vaccine against avian Clostridium perfringens, which is obtained by truncating CPA and NetB proteins, and adding auxiliary protease Fba, and fusing three gene sequences in series through Linker to obtain a recombinant protein. Through the immunogenicity test of N‑C‑F recombinant protein, it was found that N‑C‑F recombinant protein can induce SPF chickens to produce antibodies. The results of the immune protection test showed that the subunit vaccine prepared by N‑C‑F protein and adjuvant was found to achieve 100% immune protection effect for SPF chickens at 100μg and 200μg / head, and can be used as a subunit vaccine against multiple toxin types.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering vaccines, and in particular relates to a subunit vaccine against avian Clostridium perfringens. Background Art

[0002] Clostridium perfringens (CP) is a Gram-positive bacterium of the Bacillaceae family and the genus Clostridium. It is one of the important foodborne pathogens. After infection, the bacterium can decompose muscle and connective tissue and produce a large amount of gas. When it grows in a serum-containing culture medium in humans and animals, it forms a capsule, so it is named Clostridium perfringens. According to the difference in its secretion of toxins, it can be divided into 7 toxin types (A, B, C, D, E, F, G, Table 1). The bacteria are prevalent worldwide and cause serious harm to the breeding industry. Among them, type A and type F toxin types of Clostridium perfringens can infect humans, easily causing food poisoning, vomiting, diarrhea, etc. through meat products, or causing gas gangrene at the wound site leading to systemic poisoning, which can easily cause shock and death in patients, threatening human health and leading to serious public health events.

[0003] Table 1: Clostridium perfringens toxin typing table

[0004]

[0005] Clostridium perfringens disease of poultry is a necrotic enteritis and gangrenous dermatitis caused by Clostridium perfringens. Its clinical manifestations include depression, decreased appetite, bloody diarrhea, bleeding in the small intestinal mucosa, and obvious flatulence in the necrotic intestine, which affects the production performance and feed efficiency of poultry. The economic losses caused to the global poultry industry each year are as high as 6 billion US dollars, causing serious economic losses to the global poultry industry. Its main pathogen is certain toxin types of Clostridium perfringens (mainly type A and type G, and some type C infections).

[0006] Clinically, antibiotics are usually used to treat and prevent poultry Clostridium perfringens disease. However, with the increasing calls to reduce and limit the use of antibiotics, the prevention and control of Clostridium perfringens disease has brought huge challenges. This requires the adoption of a new prevention and control strategy that focuses on prevention and treatment. Summary of the invention

[0007] The invention provides a subunit vaccine against avian Clostridium perfringens, which can effectively prevent and treat the harm caused by Clostridium perfringens disease.

[0008] The present invention first provides an antigen fusion protein, wherein the antigen fusion protein is constructed by connecting truncated CPA protein and NetB protein of avian Clostridium perfringens via a linker sequence;

[0009] Furthermore, the antigen fusion protein also contains a partial fragment of fructokinase Fba;

[0010] As a specific description of the embodiment, the truncated CPA protein of avian Clostridium perfringens has an amino acid sequence of SEQ ID NO: 2, and a nucleotide fragment encoding the gene is SEQ ID NO: 1;

[0011] The truncated NetB protein has an amino acid sequence of SEQ ID NO:4, and a nucleotide fragment encoding the gene is SEQ ID NO:3;

[0012] The linker sequence has an amino acid sequence of a protein as SEQ ID NO:6 and a nucleotide fragment encoding a gene as SEQ ID NO:5;

[0013] The fructose kinase Fba has an amino acid sequence of a protein of SEQ ID NO:8 and a nucleotide fragment of a coding gene of SEQ ID NO:7.

[0014] Furthermore, the antigen fusion protein has a specific amino acid sequence of SEQ ID NO: 10, and the corresponding nucleotide fragment of the encoding gene is SEQ ID NO: 9.

[0015] In yet another aspect, the present invention provides a recombinant expression vector, into which a nucleotide fragment encoding an antigen fusion protein is inserted.

[0016] The present invention also provides a recombinant host bacterium, which carries the recombinant expression vector.

[0017] The antigen fusion protein of the present invention is used for preparing subunit vaccine.

[0018] In order to ensure that the expressed recombinant protein can effectively stimulate the body to produce antibodies, the present invention links Fba fructokinase with two toxin proteins. Compared with the effect of simple immunization through toxin protein fusion, the immune effect of NCF is significantly enhanced. In addition, by selecting a suitable prokaryotic expression vector and inducing expression conditions, the recombinant protein can be expressed in large quantities in the supernatant, and at the same time, the buffer solution is screened during the purification process, and finally a recombinant protein with a higher concentration is obtained. In the attack and protection test, the protective efficacy of the vaccine was evaluated by intestinal lesion scoring and it was found that the intestinal score of the NCF immunization group was significantly lower than that of the control group. This result shows that the vaccine has an effective protective effect on Clostridium perfringens. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1: pET-30-NCF recombinant protein structure prediction diagram;

[0020] Figure 2 :pET-30-NCF recombinant expression plasmid map;

[0021] Figure 3 : PCR amplification results of NCF gene in positive plasmid, where M: DL2000 DNA marker, 1: NCF gene fragment, 2: negative control.

[0022] Figure 4 : Expression and identification of NCF recombinant protein, the left figure is SDS PAGE electrophoresis, the right figure is Western blot, lane 1: protein molecular standard; lane 2: 25℃-Rossetta-0.2mM IPTG; lane 3: 25℃-Rossetta-0.5mM IPTG; lane 4: 25℃-Rossetta-1mM IPTG; lane 5: 25℃-BL21-0.2mMIPTG; lane 6: 25℃-BL21-0.5mM IPTG; lane 7: 25℃-BL21-1mM IPTG; lane 8: 16℃-Rossetta-0.2mM IPTG; lane 9: 16℃-Rossetta-0.5mM IPTG; lane 10: 16℃-Rossetta-1mM IPTG; lane 11: 16℃-BL21-0.2mM IPTG; Lane 12: 16°C-BL21-0.5 mM IPTG; Lane 13: 16°C-BL21-1 mM IPTG.

[0023] Figure 5 : The results of NCF recombinant protein purification, the left figure is the SDS PAGE electrophoresis figure, the right figure is the Western blot figure, wherein lane 1: protein molecule standard amount; lane 2: NCF stock solution; lane 3: flow-through solution; lane 4: 20mM imidazole 1; lane 5: 20mM imidazole 3; lane 6: 50mM imidazole 1; lane 7: 50mM imidazole 3; lane 8: 80mM imidazole 1; lane 9: 80mM imidazole 3; lane 10: 90mM imidazole 1; lane 11: 90mM imidazole 3; lane 12: 250mM imidazole 1; lane 13: 50mM imidazole 2; lane 14: 250mM imidazole 3.

[0024] Figure 6 : PAGE gel results of NCF protein concentrate;

[0025] Figure 7 :Results of serum antibody identification of NCF recombinant protein;

[0026] Figure 8 :Clostridium perfringens 10 -6 and 10 -7 Dilution plate result diagram;

[0027] Fig. 9 :Analysis of weight gain rate of control group and immune group;

[0028] Fig.10 :The results of intestinal autopsy after bacterial attack;

[0029] Fig.11 :Intestinal score analysis of control group and immune group;

[0030] Fig.12 :Identification diagram of positive colonies of Clostridium perfringens. DETAILED DESCRIPTION

[0031] The present invention constructs a subunit vaccine that can simultaneously contain two or more toxins or extracellular enzymes by establishing an animal model for evaluating avian Clostridium perfringens vaccine, and evaluates the efficacy, safety, and immune protection of the constructed avian Clostridium perfringens subunit vaccine. The constructed subunit vaccine can prevent infection by multiple types of Clostridium perfringens, laying a foundation for the prevention and control of avian Clostridium perfringens.

[0032] The materials used in the embodiments of the present invention include NETB-CPA-Fba (hereinafter referred to as NCF) pet-30a (+) plasmid, BL21 (DE3) Escherichia coli competent cells, Rosetta (DE3) Escherichia coli competent cells, DH5α Escherichia coli competent cells and poultry Clostridium perfringens.

[0033] The main reagents and preparation methods used in the specific examples are as follows:

[0034] (1) LB liquid medium: Weigh 12.5 g of LB broth medium, add 500 mL of ultrapure water to dissolve the high pressure, cool and place in a 4°C refrigerator for later use. This is referred to as LB liquid medium below.

[0035] (2) Kana-resistant liquid LB medium: Take 50 mL of LB liquid medium without resistance, add 50 μL of kana-resistant solution with a final concentration of 100 μg / mL, vortex mix, and place in a 4°C refrigerator for later use. This medium is hereinafter referred to as LB / Kana liquid medium.

[0036] (3) Antibiotic-free LB solid medium: Weigh 8 g of LB broth agar, add 500 mL of ultrapure water and dissolve by high pressure. After cooling slightly, place in sterile culture dishes, seal and label, and place in a 4°C refrigerator for later use. This is referred to as LB solid medium below.

[0037] (4) Kanana resistance LB solid medium: Cool the autoclaved LB solid medium slightly to below 50°C, add kanamycin at a final concentration of 100 μg / mL, dispense into sterile culture dishes, seal and label, and place in a 4°C refrigerator for later use. Hereinafter, they are referred to as LB / kana solid medium.

[0038] (5) 50% glycerol: Take 100 mL of glycerol and add ultrapure water to make up to 200 mL. After high-pressure sterilization, cool to room temperature and store at 4°C for later use.

[0039] (6) 500 mM imidazole pH 7.4: Weigh 17.06 g of imidazole and add 500 mL of PBS to dissolve and set aside.

[0040] 20mM imidazole pH 7.4: Take 2mL of 500mM imidazole, add PBS to make up to 50mL, filter through a 0.22μm pore size filter and set aside.

[0041] 50 mM imidazole pH 7.4: Take 5 mL of 500 mM imidazole, add PBS to make up to 50 mL, filter through a 0.22 μm pore size filter and set aside.

[0042] 80 mM imidazole pH 7.4: Take 8 mL of 500 mM imidazole, add PBS to make up to 50 mL, filter through a 0.22 μm pore size filter and set aside.

[0043] 90 mM imidazole pH 7.4: Take 9 mL of 500 mM imidazole, add PBS to make up to 50 mL, filter through a 0.22 μm pore size filter and set aside.

[0044] 250mM imidazole pH 7.4: Take 25mL of 500mM imidazole, add PBS to make up to 50mL, filter through a 0.22μm pore size filter and set aside.

[0045] (7) MES buffer pH 5.0: Prepare 500 mL of 20 mM N-morpholine 2-ethanesulfonic acid (MES) and 0.1 M sodium chloride by adding ultrapure water.

[0046] (8) 20% ethanol: Take 10 mL of anhydrous ethanol and add ultrapure water to make up to 5 mL.

[0047] TSC solid culture medium: weigh 47 g TSC culture medium, add 1 L ultrapure water to dissolve the high pressure, and add 5% cycloserine (20 mL cycloserine for 250 mL culture medium) before dispensing into sterile culture dishes.

[0048] FTG liquid culture medium: weigh 29.3 g FTG culture medium, add 1 L ultrapure water to dissolve the high pressure, cool and place in a 4 ° C refrigerator for use.

[0049] Coomassie Brilliant Blue staining solution: weigh 1g of Coomassie Brilliant Blue, add 100mL of glacial acetic acid and 250mL of isopropanol, and dilute to 1L with deionized water.

[0050] Decolorizing solution: Measure 50 mL of anhydrous ethanol and 100 mL of glacial acetic acid, and make up to 1 L with deionized water.

[0051] The present invention is described in detail below in conjunction with embodiments and drawings.

[0052] Example 1: Construction of amino acid sequence of subunit vaccine and optimization of coding gene sequence

[0053] The toxin types of avian Clostridium perfringens are mainly type A and type G. The main secreted toxin of type A is α(CPA), and the main secreted toxins of type G are α(CPA) and NetB. Since CPA and NetB proteins are cytotoxic, the non-toxic and highly antigenic gene sequences were obtained through DNAstar biological software and previous experimental research and analysis. The CPA truncated sequence is from nucleotide position 756 to nucleotide position 1062 (seq-1), the NetB truncated sequence is from nucleotide position 1162 to nucleotide position 1693 (seq-2), and the rigid link sequence is 66bp long (seq-3).

[0054] However, the recombinant expression experiment found that the subunit vaccine prepared by fusing only two toxin proteins could not effectively stimulate the body to produce higher antibodies. Therefore, the present invention fuses the nucleotide sequence of the auxiliary protein fructose kinase (fba class II fructose-1,6-bisphosphate aldolase, Fba) 867bp (seq-4) in length into the toxin protein composed of the truncated sequence of CPA and the truncated sequence of NetB, ensuring that the toxin protein enters the body to exert its biological function and stimulate the body to produce effective protective antibodies. The connection sequence of the prepared subunit vaccine is Net B (756-1062)-CPA (1693-1162)-Fba (hereinafter referred to as NCF), and the predicted structure of its F recombinant protein is shown in the figure below. Figure 1 shown.

[0055] The nucleotide sequence of the gene encoding the CPA truncated polypeptide Seq1 is as follows (SEQ ID NO: 1):

[0056] AGTGAACTAGTAGCTTACATAGGGACTAGTGGTGGGGAAGATGCTGGAACAGATGAAAACAACAGTAAAGGAATCAAAACAAAGGATGGCCCCCCTCAAGAATGGGAAATGGACAACCCAGGAAATGATTTTATGACTGGAAGTTGCGATACTTATACTTTCAAATTAAAAGATGAAAATCTAAAAATTGATGATATACAAAATATGTGGATTAGAAAAAGAAAAGTAACAGCATTCCCAGATGCTTATAAGCCAGAAAACATAAAGGTAATAGCAAATGGAAAAGTTGTAGTGGACAAGGATATAAATGAGTGGATTTCAGGAAATTCAACTTATAATATAAAA;

[0057] The amino acid sequence of the encoded protein is as follows (SEQ ID NO: 2): SELVAYIGTSGGEDAGTDENNSKGIKTKDGPPQEWEMDNPGNDFMTGSCDT YTFKLKDENLKIDDIQNMWIRKRKVTAFPDAYKPENIKVIANGKVVVDKDINEWISGNSTYNIK。

[0058] The nucleotide sequence of the gene encoding the truncated polypeptide Seq2 of NetB is as follows (SEQ ID NO: 3): ;

[0059] The amino acid sequence of the encoded protein is as follows (SEQ ID NO: 4): EVSASIGYSIGGNISVEGKTAGAGINASSNVKPTTNYEQPDFRTIQRKDDANLASWDIKFVETKDGYNIDSYHAIYGNQLFMKSRLYNNGDKNFTDDRDLSTLISGGFSPNMALALTAPKNAKESVDIVEYQRFDNDYKKNWETTQWRGTNKLSSTSEYNEFMFKINWQDHKIEYYL

[0060] The nucleotide sequence of the rigid link sequence Seq3 is as follows (SEQ ID NO: 5):

[0061] GCGGAAGCGGCGGCGAAGGAAGCGGCGGCGAAGGAAGCGGCGGCG AAGGAAGCGGCGGCGAAGGCG;

[0062] The amino acid sequence of the encoded polypeptide is as follows (SEQ ID NO: 6):

[0063] AEAAAKEAAAKEAAAKEAAAKA.

[0064] The nucleotide sequence of the coding gene for protein fructokinase Fba (Seq4) is as follows (SEQ ID NO:7): ATGGCATTAGTTAACGCAAAAGAAATGTTAAATAAAGCAAGAGAAGGAAAATACGCTGTTGGTCAATTCAACATAAACAACTTAGAATGGACAAAAGCTATATTATTAACTGCTCAAGAAAATAACTCACCAGTTATATTAGGAGTATCAGAAGGTGCTGCTAAATACATGTGTGGATTCAAAACAATAGTTGGAATGGTTAACGGAATGTTAGAAGAATTAAAAATAACTGTTCCTGTAGCATTACACTTAGATCACGGTAGCTACCAAGGAGCTATAGATGCTATGGATGCTGGATTCTCATCAGTAATGTTCGATGGATCACACTACTCAATCGAAGAAAACATAGTTAAAACTAAAGAAATAATCAACTTAGCTGCTGCTAAAAACGTATCAGTTGAAGCTGAAGTTGGATCAATCGGTGGAGAAGAAGACGGTGTTGTTGGAGCTGGTGAAATCGCTGATCCTGCTGAATGTAAACAAATCGCTGAATTAGGAGTTACTATGTTAGCTGCTGGTATCGGAAACATTCACGGAAAATACCCTGCAAACTGGGCTGGATTAAACTTCGAAGCTTTAGCTAACATTAAAGCTGCTACTGGAGATATGCCTTTAGCAAGTGAATATAACGAATTTATGTTTAAAATAAATTGGCAAGATCATAAAATATTAGGAGTATCAAAAATAAATGTTAATACTGAGTGTCAATTATCATTTGCTGAAGCTACTCGTAAATATATAGAAGCTGGAAAAGACTTAGAAGGAAAAGGATTTGACCCAAGAAAATTATTAAATCCTGGATTCGAAGCTATAAAAGCTACAGTTAAAGAAAAAATGGAATTATTTGGTTCAGTAAACAGAGCTTAA;

[0065] The amino acid sequence of the translated polypeptide is as follows (SEQ ID NO: 8): MALVNAKEMLNKAREGKYAVGQFNINNLEWTKAILLTAQENNSPVILGVSEGAAKYMCGFKTIVGMVNGMLEELKITVPVALHLDHGSYQGAIDAMDAGFSSVMFDGSHYSIEENIVKTKEIINLAAAKNVSVEAEVGSIGGEEDGVVGAGEIADPAECKQIAELGVTMLAAGIGNIHGKYPANWAGLNFEALANIKAATGDMPLASEYNEFMFKINWQDHKILGVSKINVNTECQLSFAEATRKYIEAGKDLEGKGFDPRKLLNPGFEAIKATVKEKMELFGSVNRA.

[0066]

[0067] The amino acid sequence of the translated polypeptide is as follows (SEQ ID NO: 10):.

[0068] The corresponding nucleotide fusion sequence of the coding gene was sent to a synthesis company for synthesis, and the synthesized NCF nucleotide fragment was inserted into the pET-30a(+) vector ( Figure 2 ).

[0069] Example 2: Recombinant expression of subunit vaccines

[0070] 1. Extraction of recombinant plasmid

[0071] Take 50 μL of plasmid-containing glycerol bacteria and inoculate them into 5 mL LB / Kana liquid medium, and culture them overnight at 37°C in a constant temperature shaker at 200 r / min. Extract the plasmid according to the instructions of the TIANGEN plasmid extraction kit, collect the synthesized plasmid, determine the concentration, and store it in a -20°C refrigerator for later use.

[0072] The extracted plasmid was amplified by PCR, and the amplified product was analyzed by 1% agarose gel electrophoresis. The results showed that the amplified fragment of about 1900 bp was consistent with the expected result ( Figure 3 ).

[0073] 2. Expression and purification of recombinant plasmid in host bacteria

[0074] 1) Transformation of recombinant plasmid

[0075] The extracted recombinant plasmids were transformed into BL21 (DE3) Escherichia coli competent cells and Rosetta (DE3) Escherichia coli competent cells, respectively. The bacterial solution was frozen for later use. After transformation, the plates were stored at 4°C.

[0076] 2.1. Exploration of expression conditions of recombinant plasmids in host bacteria

[0077] (1) Take 50 μL of glycerol bacteria containing plasmids and place them in 5 mL of LB / Kana liquid medium. Incubate in a constant temperature shaker at 200 r / min and 37°C until cloudy.

[0078] (2) Isopropyl-β-D-thiogalactoside (IPTG) was added to set the final concentration to 0.2 / 0.5 / 1 mM, the induction temperature to 16 / 25°C, and the induction was carried out at 150 r / min for 16 h.

[0079] (3) Add 2 mL of the induced bacterial solution to a 2 mL EP tube, centrifuge at 12,000 rpm for 1 min, discard the supernatant, repeat twice, add 500 μL of high-pressure PBS solution to the bacterial pellet, and re-vortex the bacterial cells.

[0080] (4) The mixed sample was ultrasonically disrupted on ice for 5 min.

[0081] Ultrasonic crushing conditions: No. 2 horn, ultrasound on for 3s, off for 5s, power 25%

[0082] (5) After ultrasonic disruption, centrifuge the protein lysate at 12,000 rpm for 10 min and collect the supernatant.

[0083] (6) Collect 50 μL of the supernatant from step (5), add 17 μL of 4× Protein Loading Buffer, centrifuge, boil in boiling water for 15 min, centrifuge, cool and set aside

[0084] (7) Take 10 μL of the prepared sample and 5 μL of protein marker and identify them by SDS-PAGE gel electrophoresis. After the electrophoresis is completed, stain with Coomassie Brilliant Blue for 1 hour and then decolorize overnight, and then take pictures.

[0085] Electrophoresis conditions: 80V for stacking gel and 120V for separation gel.

[0086] (8) After SDS gel electrophoresis, the excess gel was removed and Western-blot detection was performed using a His-tagged primary antibody and a goat anti-mouse secondary antibody.

[0087] The supernatant of pET30a-NCF after in vitro induction expression was analyzed by 12% SDS-PAGE gel electrophoresis. The results showed that there was an expected size of about 70KDa target band ( Figure 4 ).

[0088] 3. The recombinant plasmid is expressed in large quantities and purified in the host bacteria.

[0089] (1) Take 50 μL of glycerol bacteria containing plasmids and place them in 5 mL of LB / Kana liquid culture medium. Place them in a constant temperature shaker at 200 r / min and culture at 37°C until the bacterial liquid becomes cloudy.

[0090] (2) The activated bacterial solution was transferred to 200 mL LB / Kana liquid culture medium and cultured in a constant temperature shaker at 200 r / min at 37°C until the OD600 reached 0.4-0.6, and an appropriate volume of isopropyl-β-D-thiogalactopyranoside (IPTG) was added.

[0091] (3) After inducing expression for 16 hours at 25°C on a pre-cooled constant temperature shaker, collect the bacterial solution into 50 mL centrifuge tubes, centrifuge at 12000 r / min for 1 min to collect the bacteria, and resuspend the bacteria in 25 mL of high-pressure PBS solution.

[0092] (4) Use an ultrasonic disruptor on ice to disrupt the cells until they are clear, then centrifuge at 12,000 rpm for 10 min at 4°C to collect the supernatant.

[0093] Ultrasonic disruption conditions: No. 6 horn, ultrasound on for 3 s, off for 5 s, power 25%, 15 min each time, repeated multiple times.

[0094] After exploring the IPTG concentration, induction time and expression host bacteria, the conditions for higher expression of the recombinant plasmid were finally determined as shown in Table 2.

[0095] Table 2: Optimal expression conditions of recombinant plasmids in E. coli

[0096]

[0097] The supernatant after the fragmentation was further purified using a His affinity column. The specific method is as follows:

[0098] (1) The supernatant after the above disruption was filtered through a 0.45 μm pore size filter and stored at 4°C for later use.

[0099] (2) Centrifuge at 700 × g for 2 min to remove the storage solution in the purification column, and add 9 mL of filtered equilibration buffer (5 mM imidazole, PBS) for equilibration twice.

[0100] (3) Each time, add two column volumes of filtered supernatant and mix with nickel filler, mix at 4°C for 40 min, centrifuge at 700×g for 2 min, repeat several times, collect all flow-through, and take 50 μL for sample preparation. (4) Add two column volumes of filtered wash buffer with different concentration gradients, centrifuge at 700×g for 2 min, repeat several times, and collect 50 μL of wash buffer for sample preparation.

[0101] (5) Add one column volume of filtered elution buffer and centrifuge at 700 × g for 2 min to elute the target protein. Repeat at least three times, collect all of the elution buffer and take 50 μL of each for sample preparation.

[0102] (6) After purification, add two column volumes of MES Buffer to the purification column, centrifuge at 700 × g for 2 min, and repeat 5 times; add two column volumes of filtered ultrapure water, centrifuge at 700 × g for 2 min, and repeat 5 times; add an appropriate amount of 20% ethanol and store the purification column at 4°C.

[0103] (7) Select representative pre-purification samples, flow-through, wash solution, and eluate for SDS PAGE polyacrylamide gel electrophoresis and Western-blot detection.

[0104] After exploring the purification conditions of the recombinant protein, the results showed that a relatively pure target protein could be obtained ( Figure 5 The conditions with better recombinant protein purification effect are shown in Table 3.

[0105] Table 3: Optimal purification conditions for recombinant plasmids in E. coli

[0106]

[0107]

[0108] 4. Concentration and concentration determination of recombinant protein

[0109] (1) Select a 10KD ultrafiltration tube, add an appropriate amount of purified protein solution into the ultrafiltration inner tube, place it in a 4°C precooled centrifuge, and centrifuge at 4000r / min for 15-20min.

[0110] (2) After centrifugation, discard the liquid in the outer tube. Use a pipette to gently blow the inner tube protein sample evenly, then add an appropriate amount of purified protein solution again and repeat step (1); repeat multiple times until all the purified protein solution is completely concentrated.

[0111] (3) Add high-pressure PBS solution to the ultrafiltration inner tube containing the concentrated protein sample, place it in a precooled centrifuge at 4°C, centrifuge at 4000r / min for 15-20min, and repeat three times. After blowing evenly, collect all the liquid in the ultrafiltration inner tube into a 2mL EP tube and temporarily store it at 4°C.

[0112] (4) Take an appropriate amount of protein sample and dilute it according to 1:10 / 1:50 / 1:100 and mix it thoroughly. Set up three groups of replicates and use the BCA protein quantification kit to measure the protein concentration and take the average value.

[0113] The recombinant protein purified three times was concentrated and the protein concentration was determined by BCA method. The concentration of the recombinant protein was 405 μg / mL. The concentration identification results were as follows: Figure 6 shown.

[0114] Example 2: Identification of immunogenicity of subunit vaccine prepared by recombinant protein

[0115] 1. Vaccine preparation

[0116] The recombinant protein was emulsified with Freund's incomplete adjuvant in a 1:1 ratio for the first and second immunizations, and the recombinant protein was emulsified with Freund's complete adjuvant in a 1:1 ratio for the third immunization.

[0117] 2. Immunization and grouping

[0118] Twelve SPF chickens were divided into three groups and immunized with recombinant protein NCF and PBS respectively. Each chicken was immunized twice by intramuscular injection of 100 μg, 200 μg and PBS into the leg muscle. After immunization, serum was collected by blood sampling from the subwing vein every week. One SPF chicken was selected from each group to collect serum by blood sampling from the heart one week after three immunizations.

[0119] 3. Serum antibody testing

[0120] After immunization, blood was collected from the wing vein every week to separate serum.

[0121] 1) Place the collected blood sample in a 37°C incubator for 1 hour, and then place it in a 4°C refrigerator for 1 hour until the serum precipitates;

[0122] 2) Collect the serum into an Eppendorf tube, centrifuge at 5000 r / min for 1 min, collect the clean serum, and store it at -20°C for later use.

[0123] Western-blot detection was performed using the serum to be tested diluted 1:20 in 2% BSA as the primary antibody and rabbit anti-chicken secondary antibody.

[0124] 3. Identification of the immune protection efficacy of recombinant protein

[0125] Eighteen SPF chickens were divided into three groups and injected with recombinant protein NCF and PBS solution (control group) respectively. NCF protein was injected intramuscularly at 100 μg and 200 μg protein respectively, and there were 6 chickens in the control group.

[0126] Table 4: Immunization group and control group

[0127]

[0128] 4. Serum antibody testing

[0129] One week after the second immunization, blood was collected from the wing vein to separate serum, and blood was collected from the heart before autopsy to collect serum. Some serum was randomly selected for Western-blot detection using serum primary antibody diluted 1:20 in 2% BSA and rabbit anti-chicken secondary antibody.

[0130] The serum of immunized SPF chickens was collected and Western blot analysis revealed that 100 μg and 200 μg of recombinant protein NCF could effectively induce the body to produce corresponding antibodies, and the bands of the 200 μg immunization group were thicker and more obvious, while no specific bands were detected in the negative serum ( Figure 7 ). The results showed that the recombinant protein NCF had good immunogenicity.

[0131] 5. Bacteria attack test

[0132] 5.1 Cultivation of Clostridium perfringens

[0133] (1) Take 50 μL of GZ1 Clostridium perfringens glycerol culture and evenly spread it on TSC solid culture medium and culture it overnight.

[0134] (2) Pick a single colony and place it in 2 mL of FTG liquid culture medium. After culture, transfer it to a 5 mL centrifuge tube and activate it for 2 h.

[0135] (3) The activated bacterial solution was transferred into a large centrifuge tube and cultured anaerobically for 10 h.

[0136] (4) Concentrate the cultured bacterial solution.

[0137] (5) Take 100 μL of concentrated bacterial solution and add 900 μL of high pressure PBS solution to dilute it to 10 -7 .

[0138] (6) Take 10 -6 / 10 -7 100 μL of the diluted bacterial solution was evenly spread on TSC solid culture medium, cultured anaerobically at 42°C, and then counted.

[0139] The Clostridium perfringens solution was concentrated five, six, seven, and eight times respectively, and 10 -6 , 10-7 100 μL of the diluted bacterial solution was plated and counted. The results showed that the bacterial count could reach 1×10 9 CFU / mL, meet the bacterial content requirements of attacking bacteria ( Figure 8 ).

[0140] Table 5: Results of the exploration of the amount of Clostridium perfringens in poultry

[0141]

[0142] The experimental chickens were infected by gavage at 28 days of age, and the clinical symptoms of SPF chickens after infection were observed. The chickens were killed after 7 days.

[0143] (1) Weigh the body weight of each SPF chicken before and after infection, and calculate the weight gain rate and analyze it.

[0144] Weight gain rate = (weight after infection - weight before infection) / weight before infection.

[0145] The chickens were weighed before infection and autopsy, and the weight gain rate was calculated. The differences between the immunized NC-F 100 μg and 200 μg groups and the control group were significant. The results showed that the NCF subunit vaccine can effectively resist the effect of Clostridium perfringens on the weight loss of chickens ( Fig. 9 ).

[0146] (2) After autopsy, intestinal tissue was observed and scored.

[0147] After the SPF chickens were killed and autopsied, the control group showed symptoms such as intestinal bloating, intestinal thinning, and bleeding; the immunized groups, 100μg and 200μg NCF immunized groups had no obvious lesions. Fig.10 shown.

[0148] According to the results of previous studies, when the intestinal lesion score of chickens is ≥2 points, they are considered NE positive; when the intestinal lesion score of the immunized group is ≤1 point, the vaccine is considered to be protective; or when there is a significant difference in the intestinal scores between the control group and the immunized group, the vaccine is also considered to be protective.

[0149] The intestinal scores of the immunized group and the control group were analyzed. The intestinal scores of the immunized group with NCF 100μg and 200μg were significantly different from those of the control group ( Fig.11 ). The results showed that NCF subunit vaccine has an effective protective effect against avian Clostridium perfringens, and its protection rate can reach 100%.

[0150] 5.2 Isolation and identification of Clostridium perfringens from intestinal tissue and anal swabs

[0151] (1) Take an appropriate amount of intestinal tissue with obvious lesions after autopsy and place it in a 2 mL grinding tube, and add an appropriate amount of sterile PBS solution.

[0152] (2) Grind the tissue using a high-throughput tissue grinder, and centrifuge at 500 × g for 5-10 min after the tissue is evenly ground.

[0153] (3) Take 100 μL of the supernatant and spread it evenly on TSC solid culture medium, culture it anaerobically at 42°C, and count the cells.

[0154] (4) Take a small amount of the supernatant and inoculate it into 2 mL of FTG liquid culture medium and culture it anaerobically at 42°C.

[0155] (5) Randomly select some intestinal tissues and corresponding culture fluids, extract the genome using a kit, and then perform PCR identification.

[0156] Reaction system (25 μL): 2×Taq Master Mix 12.5 μL, DNA template 2 μL, upstream and downstream primers 1 μL each (primers in the system are mixed in equal proportions), ddH2O 8.5 μL.

[0157] Reaction procedure: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s; annealing at 55°C for 30 s; extension at 72°C for 30 s; 30 cycles; extension at 72°C for 10 min; storage at 4°C.

[0158] Take 5 μL of PCR product and run electrophoresis on 1% agarose gel at 200V for 20 min. Observe on gel imager with DL 2000 DNA Marker as reference. Positive colonies with the same size as the target band can be identified as Clostridium perfringens ( Fig.12 ).

[0159] In summary, the avian Clostridium perfringens subunit prepared by the present invention can induce the body to produce corresponding antibodies, generate immune response, and improve the body's immunity, and can be used as a candidate subunit vaccine for multiple toxin types of avian Clostridium perfringens.

Claims

1. An antigen fusion protein, characterized in that: The antigen fusion protein is constructed by connecting truncated CPA protein and NetB protein of avian Clostridium perfringens through a linker sequence, and the antigen fusion protein also contains a partial fragment of fructokinase Fba; the amino acid sequence of the antigen fusion protein is SEQ ID NO:

10.

2. A recombinant expression vector, characterized in that: A nucleotide fragment encoding the antigen fusion protein according to claim 1 is inserted into the recombinant expression vector.

3. A recombinant host bacterium, characterized in that: The recombinant host bacteria carries the recombinant expression vector according to claim 2.

4. Use of the antigen fusion protein according to claim 1 as an antigen in the preparation of a product for preventing or treating avian Clostridium perfringens.

5. A subunit vaccine, characterized in that: The subunit vaccine uses the antigen fusion protein described in claim 1 as an antigen.

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