CN117486986A - G-type clostridium perfringens antigen protein EntB and preparation and application thereof - Google Patents

G-type clostridium perfringens antigen protein EntB and preparation and application thereof Download PDF

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CN117486986A
CN117486986A CN202311465532.8A CN202311465532A CN117486986A CN 117486986 A CN117486986 A CN 117486986A CN 202311465532 A CN202311465532 A CN 202311465532A CN 117486986 A CN117486986 A CN 117486986A
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entb
protein
clostridium perfringens
immunoprotection
perfringens type
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石火英
李泽伟
陈逸飞
谢海萍
李�权
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Yangzhou University
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Abstract

The invention relates to the technical field of genetic engineering, in particular to a G clostridium perfringens antigen protein EntB, and preparation and application thereof; the amino acid of the G clostridium perfringens EntB protein is shown as SEQ ID No.1, and the preparation method comprises the following steps: designing a primer to amplify an EntB gene according to the clostridium perfringens total gene sequence, and constructing a recombinant expression vector pET28a-EntB and prokaryotic expression recombinant protein; the prepared EntB protein of the G type clostridium perfringens can obviously relieve clinical symptoms caused by the G type clostridium perfringens, and the EntB protein which is screened by the invention and has an immunoprotection effect on necrotic enteritis of chickens has potential for developing subunit vaccine of necrotic enteritis of chickens.

Description

G-type clostridium perfringens antigen protein EntB and preparation and application thereof
Technical Field
The invention relates to the technical field of genetic engineering, in particular to preparation of an EntB protein of clostridium perfringens G and application of the EntB protein as subunit vaccine.
Background
Clostridium perfringens (Clostridium perfringens, CP) is a gram-positive, anaerobic and spore-forming bacterium that is widely distributed in various environments, among which G-type clostridium perfringens mainly causes necrotic enteritis (Necrotic enteritis, NE) that has a significant economic impact on the poultry industry worldwide. Typically clostridium perfringens is part of the gut microbiota population, however, several causative factors, such as coccidial infection, high feeding density, immunosuppression and gut dysbiosis, lead to rapid proliferation of clostridium perfringens and development of NE.
Previously, antibacterial Growth Promoters (AGPs) have been used to improve productivity and control the incidence of infectious diseases in poultry production; however, due to the advent of AGP usage bans, the prevalence of NEs has increased significantly. Therefore, research into alternative products for AGP, such as prebiotics, synbiotics, vaccines, etc., is highly necessary. At present, the research on necrotic enteritis vaccines is mainly focused on alpha and NetB toxins, but at present, no corresponding vaccine is approved to be marketed in China.
EntB is a protein found to be immunogenic in the highly virulent Clostridium perfringens type G exotoxin and is a protein consisting of 4 bacterial SH 3-like domains (SH 3 b), 1 cell wall-associated NlpC/P60 cysteine peptidase domains. The protein is obtained from early antigen screening immunoprecipitation, can generate specific immune reaction with clostridium perfringens type G exotoxin antibody, proves the potential of the protein as clostridium perfringens subunit vaccine antigen, and has no report on clostridium perfringens EntB protein at present.
Disclosure of Invention
In order to solve the problems, the clostridium perfringens type G EntB protein with immunoprotection is provided.
The aim of the invention is realized by the following technical scheme:
in a first aspect, the invention provides a clostridium perfringens type G EntB protein with immunoprotection, wherein the amino acid of the clostridium perfringens type G EntB protein is shown in SEQ ID No. 1.
In a second aspect, the present invention provides a method for preparing clostridium perfringens type G EntB protein having immunoprotection, said method comprising:
step 1, designing a primer according to a gene sequence of EntB in a clostridium perfringens str.13 (GenBank ID: BA 000016.3) complete gene sequence, and amplifying an EntB gene by PCR, wherein the gene sequence of the EntB is shown as SEQ ID No. 2;
step 2, inserting the gene amplified in the step 1 into an expression vector pET28a by using a homologous recombination method to form a recombinant expression plasmid pET28a-EntB;
step 3, transforming the identified correct recombinant expression plasmid into an escherichia coli engineering bacterium BL21, picking a clone strain, identifying the correct clone strain as a positive clone strain through PCR and sequencing, and naming the positive clone strain as BL21 (pET 28 a-EntB);
and 4, expressing, purifying and identifying the expression strain BL21 (pET 28 a-EntB) of the protein in the step 3 to obtain the purified clostridium perfringens type G EntB protein.
Further, the primer sequence in step 1 was EntB-F:5'-atgggtcgcggatccgaattcACAGAACATAAAAATTCTAATCAATTAGAAGA-3', entB-R:5'-gtggtggtggtggtgctcgagAAGAACTCTTCTTGCTCTTCCTATTTT-3', and the amplification procedure was 98℃for 2nin,98℃for 10s,55℃for 10s, and 72℃for 30s,35 cycles, and 5min.
Further, the homologous recombination method described in step 2 was 100ng of the gene amplification product in step 1, 50ng of pET28a linearized plasmid, 2. Mu.L of homologous recombination enzyme, 4. Mu.L of homologous recombination buffer, and the reaction was carried out at 37℃for 30min with the addition of ultrapure water to make up to 20. Mu.L.
Further, the specific steps for expressing the protein expression strain BL21 (pET 28 a-EntB) in the step 4 are as follows:
(1) The resulting recombinant E.coli BL21 (pET 28 a-EntB) was cultured to OD in 100mL of LB liquid medium 600 Isopropyl- β -D-thiogalactoside (IPTG) was added to a final concentration of 0.5mM at 0.6 and induced to express for 8 hours on a 25 ℃ shaker;
(2) BL21 (pET 28 a-EntB) thalli is collected by centrifuging the bacterial liquid for 10min at 8000rpm/min, and 16mL of balance buffer solution is used for resuspension of the thalli;
(3) After the thalli are crushed by an ultrasonic crusher, centrifuging at 9000rpm/min for 10min at 4 ℃ to collect supernatant, and re-suspending the precipitate by 16mLPBS for later use;
(4) Taking 20 mu L of the supernatant and the sediment heavy suspension obtained in the step (3), respectively adding 5 mu L of loading buffer solution into each 20 mu L of the supernatant and the sediment heavy suspension, uniformly mixing, and boiling in boiling water at 100 ℃ for 10min; the expression was identified by electrophoresis on a 12% SDS-PAGE gel.
Further, the formulation of the equilibration buffer is: 60mM NaH 2 PO 4 ,、300mM NaCl,、10mM imidazole,pH 8.0。
Further, the PBS formulation was: KCl 0.2g, naCl 8g, na 2 HPO 4 1.44g、KH 2 PO 4 0.24g,1000mL distilled water, pH 7.6.
Further, the loading buffer formulation is: 1M Tris-HCl, pH 6.8 1mL,200mM DDT 0.31g,4%SDS 0.4g,0.2% bromophenol blue 0.02g,20% glycerol 2mL,7mL ultrapure water.
In a third aspect, the invention provides the use of the clostridium perfringens type G EntB protein with immunoprotection as a subunit vaccine.
The application method is as follows: the EntB protein was emulsified in equal volumes using the adjuvant Montanide ISA201VG, at 100 μg per chicken EntB protein and immunized by the leg intramuscular route.
The invention has the following beneficial effects: the invention relates to a clostridium perfringens type G EntB protein with immunoprotection. Obtaining an EntB gene fragment through PCR amplification, and preparing and obtaining recombinant EntB protein by using a prokaryotic expression method; animal experiments were used to evaluate their immunoprotection. As a result, the clostridium perfringens type G entB protein has good immunoreactivity, and can obviously relieve clinical symptoms caused by clostridium perfringens type G. The clostridium perfringens type G EntB protein with immunoprotection function has potential for developing clostridium perfringens type G subunit vaccine.
Drawings
FIG. 1, bacterial liquid PCR identification of BL21 (pET 28 a-EntB).
FIG. 2, entB protein expression SDS-PAGE.
FIG. 3, entB protein purified SDS-PAGE identification, wherein lane 1: a pre-purification sample; lane 2: purified flow through sample lane 3: purifying the washing liquid sample; lanes 4-7: purifying the eluent sample; lane 8: pET28a control.
FIG. 4, western blot identification of EntB protein.
Fig. 5, protective effect of enchyma c to SPF chicken by EntB after challenge with clostridium perfringens necrotic enteritis lesions (< p < 0.01).
Figure 6, intestinal pathology after clostridium perfringens g challenge.
Detailed Description
Example 1: cloning and prokaryotic expression of the EntB Gene of Clostridium perfringens G
Primers are designed according to the gene sequence of EntB in the clostridium perfringens str.13 (GenBank ID: BA 000016.3) total gene sequence, and the EntB gene is amplified by PCR, wherein the gene sequence of the EntB is shown in SEQ ID NO.2, and the primer sequence is as follows: entB-F (SEQ ID No. 3):
5'-atgggtcgcggatccgaattcACAGAACATAAAAATTCTAATCAATTAGAAGA-3',
EntB-R(SEQ ID No.4):
5'-gtggtggtggtggtgctcgagAAGAACTCTTCTTGCTCTTCCTATTTT-3' the amplification procedure was 98℃pre-denaturation 2nin,98℃denaturation 10s,55℃annealing 10s,72℃extension 30s,35 cycles, 5min extension;
inserting the amplified gene into an expression vector pET28a by using a homologous recombination method to form a recombinant expression plasmid pET28a-EntB; the method comprises the following steps: 100ng of gene amplification product, 50ng of pET28a linearization plasmid, 2 mu L of homologous recombination enzyme and 4 mu L of homologous recombination buffer, adding ultrapure water to complement to 20 mu L, and reacting for 30min at 37 ℃; the recombinant plasmid was transformed into E.coli engineering bacteria DH 5. Alpha. And PCR was performed using pET28a universal T7 primer (F (SEQ ID No. 5): 5'-TAATACGACTCACTATAGGG-3', R (SEQ ID No. 6): 5'-GCTAGTTATTGCTCAGCGG-3').
Then the recombinant expression plasmid with correct identification is transformed into escherichia coli engineering bacterium BL21, a clone is selected, the correct clone is identified as a positive clone through PCR and sequencing identification, and the positive clone is named BL21 (pET 28 a-EntB). As shown in FIG. 1, the PCR results were shown that the pET28a universal T7 primer (F: 5'-TAATACGACTCACTATAGGG-3', R: 5'-GCTAGTTATTGCTCAGCGG-3') amplified a fragment of the expected size, about 1900bp, confirming the successful acquisition of BL21 (pET 28 a-EntB) strain.
The steps for identifying whether the EntB protein is expressed are as follows:
(1) The resulting recombinant E.coli BL21 (pET 28 a-EntB) and BL21 (pET 28 a) were cultured to OD in 100mL of LB liquid medium 600 Isopropyl- β -D-thiogalactoside (IPTG) was added at 0.6 to a final concentration of 0.5mM and expression was induced on a 25℃shaker for 8 hours.
(2) BL21 (pET 28 a-EntB) cells were collected by centrifugation at 8000rpm/min for 10min, and 16mL of the equilibration buffer (60 mM NaH) was used 2 PO 4 300mM NaCl,10mM imidazole,pH 8.0) the cells were resuspended.
(3) After the cells were disrupted by an ultrasonic disrupter, the supernatant was collected by centrifugation at 9000rpm/min for 10min at 4℃and the pellet was resuspended in 16mL PBS for use.
(4) 20. Mu.L of each of the supernatant and the precipitated resuspension obtained in the step (3) was mixed with 5. Mu.L of a loading buffer (1M Tris-HCl (pH 6.8) 1mL,200mM DDT 0.31g,4%SDS 0.4g,0.2% bromophenol blue 0.02g,20% glycerol 2mL,7mL of ultrapure water) and boiled in boiling water at 100℃for 10 minutes. The expression was identified by electrophoresis on a 12% SDS-PAGE gel. As a result, as shown in FIG. 2, at about 59kDa, the band of interest appears in both supernatant and pellet, whereas BL21 (pET 28 a) lane does not.
Example 2: purification of clostridium perfringens EntB protein
The supernatant sample obtained after crushing in example 1 was further purified using a High-Affinity Ni-NTA Resin as follows:
(1) 5mL of Ni-NTA metal chelating His protein purification media packing (available from gold Style Co.) was added to the affinity column;
(2) 12mL ddH was added to the affinity column 2 O washing;
(3) Filtering the supernatant obtained after crushing in example 1 by a 0.45 μm pore size filter, adding the filtered supernatant into an affinity chromatography column, and collecting a flow-through liquid;
(4) Add equilibration buffer (60 mM NaH) 2 PO 4 300mM NaCl,10mM imidazole,pH8.0) balance column
(5) 50mL of wash buffer (60 mM NaH) was added 2 PO 4 300mM NaCl,50mM imidazole,pH 8.0) washing off the impurity protein, and collecting the impurity washing liquid;
(6) 12mL of elution buffer (60 mM NaH) was added 2 PO 4 300mM NaCl,500mM imidazole,pH 8.0) eluting the target protein, collecting the eluent by a branch pipe, and collecting 6 pipes in total;
(7) Respectively adding 20 mu L of the sample before purification, the flow-through liquid, the impurity washing liquid and the eluent into 5 mu L of the loading buffer solution and boiling for 10min;
(8) Preparing 12% SDS-PAGE polyacrylamide gel, adding the four samples processed in the step (7) into a hole for electrophoresis, taking down the gel, staining with coomassie brilliant blue overnight, decoloring, and identifying whether EntB is already present in the eluent. The results showed that the higher purity of the EntB protein was present in the eluate, with a size of 59kDa (fig. 3);
(9) The eluate containing the target protein was dialyzed and concentrated through a 10kDa ultrafiltration tube, and the buffer was replaced with PBS (formula: KCl 0.2g, naCl 8g, na) 2 HPO 4 1.44g，KH 2 PO 4 0.24g,1000mL of distilled water, pH 7.6) to obtain an EntB protein suspension dissolved in PBS.
Example 3: identification of clostridium perfringens EntB protein G
The purified protein obtained in example 2 was further identified by Western blot. The purified recombinant protein was dissolved in an equilibration buffer and subjected to electrophoresis. Proteins were electrotransferred to nitrocellulose membranes using a microdialyzer (Bio-Rad). Membranes were incubated overnight in blocking buffer (PBST with 5% nonfat milk powder) at 4 ℃ and after washing with PBST, the correct expression of clostridium perfringens G EntB protein was further verified using Enhanced Chemiluminescence (ECL) substrate detection signals with His-Tag as primary antibody, HRP-labeled goat anti-mouse IgG as secondary antibody, primary antibody dilution of 1:5000, secondary antibody dilution of 1:5000 (fig. 4).
Example 4: determination of immunoprotection efficacy of clostridium perfringens EntB protein subunit vaccine G
The 18 SPF chickens at 1 day of age were randomly divided into three groups, namely, a clostridium perfringens protein G-immunized group, an challenge control group and a blank control group, each group having 6 animals. The clostridium perfringens EntB protein immunization group was injected intramuscularly with 100 μg clostridium perfringens EntB protein per chicken leg (protein isovolumetric emulsification with adjuvant Montanide ISA201 VG), and was boosted 2 weeks later and 3 weeks later, respectively, using the method described above; meanwhile, in the challenge control group, each SPF chicken was injected with an equal volume of PBS (Montanide ISA201VG equal volume emulsion). The EntB protein immune group and the challenge control group are subjected to G-type clostridium perfringens infection one week after the last immunization, and the stomach is infused and the challenge is carried out twice a day in the afternoon for 3 consecutive days, wherein the challenge dosage is 1 multiplied by 10 9 5 days before the virus attack, each chicken irrigates stomach 10000 to poison Eimeria oocysts; the blank group was not subjected to any treatment.
The chickens were euthanized 4 hours after the last challenge, the intestinal lesions of the chickens were observed after dissection and scored according to the lesions, ranging from duodenum to merkel diverticulum, with the scoring criteria: 0 minutes-no lesions are seen in the intestine; 1 minutes-the intestinal mucosa has a small amount of mucus attached, loses tension, and the intestinal wall becomes thin or fragile; 2 minutes-focal necrosis or ulceration; 3 minutes-intestinal mucosa is shed, and intestinal tract internal bleeding. As shown in FIG. 5, the intestinal surface of the challenge control group was attached with a large amount of inflammatory exudates, the exudates were gently peeled off to see the intestinal mucosa falling off, and the EntB protein immune challenge group was attached with the blank intestinal mucosa intact or with only a small amount of exudates. By the comprehensive score, the enteropathy score of the EntB protein immune challenge group is significantly lower than that of the challenge control group. The results of the histopathological section are shown in fig. 6, the glands of the toxicity attack control group are greatly proliferated, intestinal villi disintegrate and lose normal intestinal physiological structure, the EntB protein immune toxicity attack group is relatively normal, and only the surface of the intestinal villi is adhered with exudates. The result shows that the EntB protein immunization can relieve the clinical symptoms of necrotic enteritis caused by clostridium perfringens infection, and has good immune protection effect.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Meanwhile, the above embodiments are only for illustrating the technical idea of the present invention, and the protection scope of the present invention is not limited thereto, and any modification made on the basis of the technical scheme according to the technical idea of the present invention falls within the protection scope of the present invention.
SEQ ID NO.1
MTEHKNSNQLEENNQTRSVKKGQVINVSTNLRIRKSPSTSSDVIGYLTNGEIFN
IDGKEGSWYKINANGKVGYIHGDYVKEVSGNSNSSSNNSGSNSNLDTSLAGK
KGTVVNVSTSLRVRQSPSTSSSVVGSLRGGQTFEIKGKSGSWYYINANGLTGY
IHGDYVQVGENSSNNGGQSSGNNGQSSENNSGMDTSLAGKTGKVVNVSTSL
RIRQSPSTSSSVVGSLSAGQTFKINGKNGAWYNIDAQGTKGHVHGDYVQVLS
GNESSNSGSNNNQSGSQNNNLDESYNGKAGKVVNVTTNLRLRSQPSTSSSVL
AYLLPNERFTLQGKTSSGWFKVNYNGKIGYLHEDYVKIVSSNEGSNGGVNEN
LGSSQNGSTSGGQVNQSKYEQVLSIMKSQIGSPYIYGGAGETLTSSLLSSLRRT
FPDHAARGFYDIPSNYLNGNYRAFDCSGLMQWSFRQAGISLGRTTWDQINNG
YEVSPSNAKPGDLLFFSNLGHVGMYIGNGQWIEAPNKGKFVSITSVPWSKIGR
ARRVL
SEQ ID NO.2
ATGACAGAACATAAAAATTCTAATCAATTAGAAGAGAATAATCAAACTAGAT
CGGTAAAAAAAGGACAAGTTATAAATGTAAGTACAAATTTAAGAATTAGAA
AGAGTCCAAGTACAAGCAGTGATGTTATTGGATATTTAACTAATGGTGAAAT
TTTTAATATAGATGGAAAAGAAGGTTCATGGTATAAAATAAATGCTAATGGT
AAAGTTGGCTATATTCATGGAGATTATGTTAAAGAAGTAAGTGGAAATTCTA
ATAGTAGCAGCAATAATAGTGGAAGTAATTCAAATTTAGATACTTCATTAGC
TGGGAAAAAGGGTACTGTTGTAAACGTAAGTACTTCTTTAAGAGTTAGACA
ATCGCCAAGCACAAGTAGCTCAGTTGTAGGTAGCCTAAGAGGTGGTCAGA
CATTTGAGATAAAGGGTAAAAGTGGAAGTTGGTATTATATAAATGCAAATGG
ACTTACAGGATATATTCATGGAGATTATGTACAAGTAGGTGAAAATAGTTCA
AATAATGGTGGGCAATCATCAGGAAATAATGGACAATCATCAGAAAATAAC
TCAGGAATGGATACTTCATTAGCAGGAAAAACTGGTAAGGTTGTAAATGTA
AGTACTTCTTTAAGAATTAGACAATCACCAAGTACAAGTAGTTCAGTTGTTG
GATCATTAAGTGCAGGCCAAACTTTTAAGATAAATGGTAAAAATGGAGCTT
GGTATAATATTGATGCACAAGGAACAAAAGGACATGTTCATGGAGATTATGT
ACAAGTATTAAGTGGAAACGAAAGTTCTAATAGTGGAAGTAATAATAACCA
AAGTGGAAGCCAAAATAACAATTTAGATGAATCTTATAATGGAAAAGCTGG
AAAGGTTGTAAATGTAACAACTAATTTAAGACTTAGAAGTCAACCTAGTAC
TAGTAGTTCTGTTTTAGCATATTTATTACCTAATGAAAGATTTACTTTACAAG
GAAAGACTTCTTCAGGATGGTTTAAAGTAAATTATAATGGAAAAATTGGTTA
TTTACATGAAGATTATGTTAAAATAGTATCTTCAAATGAAGGTTCAAATGGA
GGCGTAAATGAAAATTTAGGTTCAAGCCAAAATGGAAGCACAAGTGGTGG
ACAAGTTAACCAAAGTAAATATGAACAAGTTTTAAGTATTATGAAATCTCAA
ATTGGTTCTCCATATATTTATGGAGGAGCTGGAGAAACACTAACTTCAAGCT
TATTAAGTAGCTTAAGAAGAACTTTCCCAGATCATGCTGCAAGAGGATTTTA
TGATATACCATCAAACTATTTAAATGGAAATTACAGAGCCTTTGATTGCTCTG
GATTAATGCAATGGAGTTTTAGACAAGCTGGAATTAGTTTAGGAAGAACAA
CTTGGGATCAAATAAACAATGGATATGAAGTTTCTCCAAGTAATGCTAAGCC
AGGAGACTTATTATTCTTTAGTAATTTAGGCCATGTAGGTATGTATATAGGAA
ATGGTCAATGGATAGAGGCTCCTAACAAAGGTAAATTTGTTTCTATTACATC
TGTTCCATGGAGTAAAATAGGAAGAGCAAGAAGAGTTCTTTAA

Claims (10)

1. The clostridium perfringens type G EntB protein with the immunoprotection function is characterized in that the amino acid of the clostridium perfringens type G EntB protein is shown as SEQ ID No. 1.
2. The process for the preparation of an immunoprotected clostridium perfringens type G EntB protein according to claim 1 characterized by the following:
step 1, designing a primer according to a gene sequence of EntB in a clostridium perfringens complete gene sequence and amplifying an EntB gene by PCR, wherein the gene sequence of the EntB is shown as SEQ ID No. 2;
step 2, inserting the gene amplified in the step 1 into an expression vector pET28a by using a homologous recombination method to form a recombinant expression plasmid pET28a-EntB;
step 3, transforming the identified correct recombinant expression plasmid into an escherichia coli engineering bacterium BL21, picking a clone strain, identifying the correct clone strain as a positive clone strain through PCR and sequencing, and naming the positive clone strain as BL21 (pET 28 a-EntB);
and 4, expressing, purifying and identifying the expression strain BL21 (pET 28 a-EntB) of the protein in the step 3 to obtain the purified clostridium perfringens type G EntB protein.
3. The method for preparing the protein EntB of clostridium perfringens type G with immunoprotection according to claim 2 wherein the primer sequence in step 1 is EntB-F5'-atgggtcgcggatccgaattcACAGAACATAAAAATTCTAATCAATTAGAAGA-3', entB-R5'-gtggtggtggtggtgctcgagAAGAACTCTTCTTGCTCTTCCTATTTT-3', the amplification procedure is 98 ℃ pre-denaturation 2nin,98 ℃ denaturation 10s,55 ℃ annealing 10s,72 ℃ extension 30s,35 cycles, 5min extension.
4. The method for preparing the protein EntB of clostridium perfringens type G with immunoprotection according to claim 2 wherein said homologous recombination method in step 2 is 100ng of the gene amplification product in step 1, 50ng of pET28a linearized plasmid, 2. Mu.L of homologous recombination enzyme, 4. Mu.L of homologous recombination buffer, adding ultrapure water to make up to 20. Mu.L, and reacting at 37℃for 30min.
5. The method for preparing the protein EntB of clostridium perfringens type G with immunoprotection according to claim 2, wherein said expression of the protein expression strain BL21 (pET 28 a-EntB) in step 4 is specifically carried out as follows:
(1) The resulting recombinant E.coli BL21 (pET 28 a-EntB) was cultured to OD in 100mL of LB liquid medium 600 isopropyl-beta-D-thiogalactoside was added at 0.6 to a final concentration of 0.5mM and induced to express for 8 hours on a 25℃shaker;
(2) BL21 (pET 28 a-EntB) thalli is collected by centrifuging the bacterial liquid for 10min at 8000rpm/min, and 16mL of balance buffer solution is used for resuspension of the thalli;
(3) After the thalli are crushed by an ultrasonic crusher, centrifuging at 9000rpm/min for 10min at 4 ℃ to collect supernatant, and re-suspending the precipitate by 16mLPBS for later use;
(4) Taking 20 mu L of the supernatant and the sediment heavy suspension obtained in the step (3), respectively adding 5 mu L of loading buffer solution into each 20 mu L of the supernatant and the sediment heavy suspension, uniformly mixing, and boiling in boiling water at 100 ℃ for 10min; the expression was identified by electrophoresis on a 12% SDS-PAGE gel.
6. The method for preparing the protein EntB of clostridium perfringens type G with immunoprotection according to claim 5, wherein the formulation of the balancing buffer solution is: 60mM NaH 2 PO 4 ,、300mM NaCl,、10mM imidazole,pH 8.0。
7. The method for preparing the protein EntB of clostridium perfringens type G with immunoprotection according to claim 5 wherein said PBS formulation comprises: KCl 0.2g, naCl 8g, na 2 HPO 4 1.44g，KH 2 PO 4 0.24g,1000mL distilled water, pH 7.6.
8. The method for preparing the protein EntB of clostridium perfringens type G with immunoprotection according to claim 5, wherein the loading buffer solution is prepared by the following formula: 1M Tris-HCl, pH 6.8 1mL,200mM DDT 0.31g,4% SDS 0.4g,0.2% bromophenol blue 0.02g,20% glycerol 2mL,7mL ultrapure water.
9. Use of the clostridium perfringens type G EntB protein with immunoprotection according to claim 1 as subunit vaccine.
10. The use according to claim 9, characterized in that the method of use is as follows: the EntB protein was emulsified in equal volumes using the adjuvant Montanide ISA206VG, at 100 μg per chicken EntB protein, and immunized by leg intramuscular injection.
CN202311465532.8A 2023-11-07 2023-11-07 G-type clostridium perfringens antigen protein EntB and preparation and application thereof Pending CN117486986A (en)

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