CN115850404A - Recombinant erysipelothrix rhusiopathiae surface protection antigen A with tandem dominant epitopes and application thereof - Google Patents
Recombinant erysipelothrix rhusiopathiae surface protection antigen A with tandem dominant epitopes and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of biology, and particularly relates to a tandem dominant epitope recombinant erysipelothrix rhusiopathiae surface protective antigen A (SpaA) and application thereof. The amino acid sequence of the recombinant erysipelothrix rhusiopathiae surface protection antigen A with the tandem dominant epitope is shown as SEQ ID No.1, and the nucleotide sequence for coding the protein is shown as SEQ ID No.2. The invention further constructs an expression vector containing the nucleotide sequence, and the sequence is shown as SEQ ID No. 3. Transforming colibacillus to obtain recombinant expression strain. The recombinant erysipelothrix rhusiopathiae surface protection antigen A with the tandem dominant epitope is uniformly mixed with an adjuvant, and can induce an organism to generate an immune response after a mouse is immunized, so that a 100% immune protection effect for resisting 1000MLD virulent attack can be provided for an attacking mouse; after the pig is immunized, 67% of immune protection effect for resisting 2MLD virulent attack can be provided for the attacking pig, and 100% of immune protection effect for resisting 1MLD virulent attack can be provided for the attacking pig.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a recombinant erysipelothrix rhusiopathiae surface protection antigen A with tandem dominant epitopes and application thereof.
Background
Swine erysipelas is caused by erysipelas suis (erysipelas rhusiopathiae), and is also called "diamond dermatosis" as a symptom of marked diamond-shaped skin damage, commonly called "fire marks". Acute type is marked by septicemia with diamond lesions of the skin, and chronic type is marked by non-suppurative arthritis and proliferative endocarditis. The swine erysipelas is easy to be caused in the high-temperature and humid periods of summer and autumn, and the swine with the age of 2-6 months is most susceptible. Pig erysipelas is high in morbidity and mortality rate and often presents an aggregated outbreak situation, so that pig death and pig ketone body derogation caused by chronic symptoms cause huge losses to farmers and breeding enterprises, and negative effects are generated on meat product supply and national economy.
The erysipelothrix rhusiopathiae is a gram-positive bacterium, is slender or slightly bent, has blunt and round two ends, is not encapsulated, does not form spores and cannot move. The bacterium is sensitive to penicillin, streptomycin, tylosin, tetracycline and cephalosporins, but the antibiotic therapy has poor effect on chronic infection. Erysipelothrix rhusiopathiae is heat sensitive but can survive in marinated, smoked, frozen and dried meat and can be transmitted through the respiratory tract, digestive tract and blood. In addition, contaminated feed, drinking water, land, greenhouses and the like are indirect transmission media of the bacteria. The erysipelothrix rhusiopathiae has wide hosts, and is currently reported to be separated from mice, turkeys, chickens, ducks, cattle, sheep, deer, moose and musk deer. Erysipelothrix rhusiopathiae can also infect people through skin wounds to cause local cellulitis, namely rhodophyta-like disease (Eutasipeloid), and part of infected bacteria in patients spread through blood diffusion to finally cause joint and organ damage.
The main means for preventing and controlling swine erysipelas is vaccine immunity, and the current commercial swine erysipelas vaccines in China comprise swine erysipelas inactivated vaccines and swine erysipelas live vaccines (G4T 10 strains and GC42 strains respectively). The immune effect of the swine erysipelas live vaccine is influenced by antibiotics, and the swine erysipelas live vaccine has certain influence on the establishment and implementation of an immune program and medication, so that the raising end is more inclined to select the swine erysipelas inactivated vaccine. However, the swine erysipelas inactivated vaccine also has some defects in the using process, such as certain batch difference of immune protection effect, mainly because the content of key protective antigen components has certain difference when the swine erysipelas bacillus inactivated antigen is produced by using the traditional bacterial inactivated vaccine production process. Therefore, the development of subunit vaccines based on key protective antigenic components would be an important development direction in the field of swine erysipelas control.
Surface protective antigen A (SpaA) is a key protective antigen of erysipelothrix rhusiopathiae. Different erysipelothrix rhusiopathiae strains have different SpaA lengths, generally have consistent N-terminal lengths, and have the main difference that the repetition times of C-terminal repetitive sequences are different. Most erysipelothrix rhusiopathiae strains SpaA full-length 626aa,1-27aa are signal peptides, and the molecular weight of the extracellularly secreted SpaA is about 69kDa. Mice and pigs immunized with the full-length SpaA protein and the N terminal can generate high-level erysipelothrix rhusiopathiae protective antibodies, and the C terminal cannot generate high-level erysipelothrix rhusiopathiae protective antibodies. Borarathybay and the like construct a swine erysipelas SpaA deletion strain to carry out infection experiments, and the results show that the pathogenicity and complement tolerance of the SpaA deletion strain to mice are obviously lower than those of a wild strain, thereby proving that SpaA plays a key role in the infection process of swine erysipelas. These researches prove that the erysipelothrix rhusiopathiae surface antigen SpaA has excellent immunogenicity, and an important theoretical basis is established for the erysipelothrix rhusiopathiae surface antigen SpaA as a vaccine candidate antigen. In order to simplify the immunization procedure, reduce the stress response of the frequent vaccination to the pigs and reduce the labor cost of vaccination, the combined vaccine has more market advantages than the single vaccine. The dominant epitope is connected in series, the signal peptide, the N-terminal redundant sequence and the C-terminal are removed, the length of the swine erysipelas bacillus key protective antigen is compressed to the utmost extent, and a larger space can be reserved for fusion expression of other pathogenic protective antigens.
Disclosure of Invention
In order to overcome the defects and shortcomings of the existing commercial vaccine, the invention mainly aims to provide the recombinant erysipelothrix rhusiopathiae surface protection antigen A with tandem dominant epitopes.
Another objective of the invention is to provide a nucleotide sequence of the recombinant erysipelothrix rhusiopathiae surface protection antigen A for encoding the tandem dominant epitope, wherein the sequence is artificially modified.
The third purpose of the invention is to provide a recombinant expression plasmid containing the nucleotide sequence of the recombinant erysipelothrix rhusiopathiae surface protection antigen A which codes the tandem dominant epitope.
The fourth purpose of the invention is to provide a recombinant escherichia coli which can express the recombinant erysipelothrix rhusiopathiae surface protection antigen A with tandem dominant epitopes.
The fifth purpose of the invention is to provide a swine erysipelas subunit vaccine.
The sixth purpose of the invention is to provide the application of the recombinant erysipelothrix rhusiopathiae surface protection antigen A with the tandem dominant epitope.
The purpose of the invention is realized by the following technical scheme:
a recombinant erysipelothrix rhusiopathiae surface protection antigen A with tandem dominant epitopes has an amino acid sequence shown in SEQ ID No. 1.
The nucleotide sequence of the recombinant erysipelothrix rhusiopathiae surface protection antigen A for coding the tandem dominant epitope is shown as SEQ ID No.2.
A recombinant expression plasmid comprises the nucleotide sequence of the recombinant erysipelothrix rhusiopathiae surface protection antigen A of the coding tandem dominant epitope, and the nucleotide sequence of the recombinant expression plasmid is shown as SEQ ID No. 3.
The starting vector of the recombinant expression plasmid is pET-28b (+).
The recombinant expression plasmid is pET-28b (+) -spaAE, namely the nucleotide sequence SEQ ID No.2 of the recombinant erysipelothrix rhusiopathiae surface protection antigen A of the coding tandem dominant epitope is inserted into a multiple cloning site.
A recombinant Escherichia coli is obtained by transforming Escherichia coli BL21 (DE 3) with the recombinant expression plasmid. The recombinant Escherichia coli is named as pET-28b (+) -spaAE BL21 (DE 3), the preservation number is CGMCC No.25901, and the classification and the name are as follows: escherichia coli (Escherichia coli) which has been deposited in the general microbiological center of the China Committee for culture Collection of microorganisms 10.12.10.2022 at the institute of microbiology, no.3 of Xilu-Beichen, north-ward-Yang district, beijing, institute of sciences, china.
A preparation method of a recombinant erysipelothrix rhusiopathiae surface protection antigen A with tandem dominant epitopes comprises the following steps:
(1) Carrying out optimization of preferred codons of escherichia coli on a nucleotide sequence SEQ ID No.2 of a recombinant erysipelothrix rhusiopathiae surface protection antigen A coding the tandem dominant epitope, wherein the optimized nucleotide sequence is shown as SEQ ID No.6, artificially synthesizing, and inserting a pET-28b (+) vector to obtain a recombinant expression plasmid pET-28b (+) -spaAE;
(2) Transforming the recombinant expression plasmid into escherichia coli BL21 (DE 3 strain) to obtain a recombinant expression strain;
(3) The recombinant erysipelothrix rhusiopathiae surface protection antigen A with the tandem dominant epitope is obtained by carrying out fermentation culture, induction expression, thallus crushing, inclusion body denaturation, ion chromatography and dialysis renaturation on the recombinant expression strain;
the recombinant expression plasmid is a nucleotide sequence of a recombinant erysipelothrix rhusiopathiae surface protection antigen A which codes a tandem dominant epitope and is inserted into a pET-28b (+) vector cloning site.
A swine erysipelas subunit vaccine is prepared by performing fermentation culture, induction expression, thallus crushing, inclusion body denaturation, ion chromatography and dialysis renaturation on recombinant escherichia coli, collecting target protein (recombinant erysipelas swine surface protection antigen A with tandem dominant epitopes), and mixing the target protein with an immunologic adjuvant.
The immune Adjuvant is a Manganese Jelly (MnJ) Adjuvant colloidal Manganese Adjuvant.
The temperature for inducing expression is preferably 25 ℃.
The induction expression time is preferably 6h.
The IPTG induced expression concentration is preferably 0.5mmol/L.
The recombinant erysipelothrix rhusiopathiae surface protection antigen A with the tandem dominant epitope is applied to the preparation of erysipelothrix rhusiopathiae vaccines.
Compared with the prior art, the invention has the following advantages and effects:
(1) The invention artificially designs and synthesizes a nucleotide sequence for coding a recombinant erysipelothrix rhusiopathiae surface protection antigen A containing a plurality of dominant antigen epitopes according to the preference of escherichia coli codons, and the sequence is shown as SEQ ID No.2.
(2) The subunit vaccine prepared by the recombinant erysipelothrix rhusiopathiae surface protection antigen A of the tandem dominant epitope expressed by the recombinant escherichia coli can induce an organism to generate immune response after a mouse is immunized, and can provide 100 percent immune protection effect for resisting 1000MLD virulent attack for an attacking mouse; after the pig is immunized, 67% of immune protection effect for resisting 2MLD virulent attack can be provided for the attacking pig, and 100% of immune protection effect for resisting 1MLD virulent attack can be provided for the attacking pig. The subunit vaccine provided by the invention can be used as an effective and safe swine erysipelas subunit vaccine candidate for preventing swine erysipelas bacillus infection.
Drawings
FIG. 1 is a graph showing the results of rER-SpaAE protein purification.
1: marker;2: not inducing; 3: crushing thallus and supernatant; 4: the thalli is crushed and precipitated and Trition-100 is washed to obtain supernatant; 5: the thalli crushing precipitation Trition-100 is cleaned for the second supernatant; 6: the thalli is crushed and deposited, PBS is used for washing the supernatant; 7: washing to complete inclusion body (precipitation); 8: LB effluent liquid; 9: WB effluent liquid; 10: EB effluent liquid; 11: supernatant of the dialysis solution; 12: and precipitating the dialysis completion solution.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.
Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
Unless otherwise indicated, reagents and materials used in the following examples are commercially available.
In the examples, the erysipelothrix rhusiopathiae virulent attacking strain was preserved under the supervision of Chinese veterinary drugs.
EXAMPLE 1 construction of recombinant expression vectors
(1) Gene sequence design and synthesis of recombinant protein
The specific method comprises the following steps: the sequence is shown as SEQ ID No.4 on the basis of the amino acid sequence (AB 019124.1) of erysipelothrix rhusiopathiae SpaA recorded in GenBank.
Analyzing the SpaA amino acid sequence with bioinformatics software to determine the position of signal peptide (MKKKKHLFPKVSLMSCLLTAMPLQTA) and C-terminal repeat sequence (EKSGGMATGGWKKVADKWYYLLDTGAIVKKVANKWYYLLEKSGAMMA TGWKKVSWYLENGYLENGAMMA GYLENGWYLENGYLENGWYLENGYLENGKWAKKGYLENGWYLENGWYNKWYYL), epitope analysis was performed on the sequences between these 2 regions, determining dominant epitopes (IGEQ, PVLPGTGVHAQYNKMT, NQKVKP, EPKGYQS, EEIN, ELKNEGMS, IPELDEAY, VKYGKVKK, DRIRS, PEAHE, LVSDSEYNEYNKLN, RRNRQ, VYPNLER, SLKTIKDDIKQRGKKLQ, QRGDVRKPDV, KYVVVDEEKLQDYLESDISSDSYSVEKSVEK IRNKEI, AQSISEIK, FQNEESDSKVESPVKKVEVVEKPEEKDQKKLVDQQSQKSKP SNSKSKWIKKDN K). Intercepting dominant epitope sequence, combining with open reading frame coding protein amino acid sequence of pET-28b (+) vector expression exogenous protein, designing recombinant erysipelothrix rhusiopathiae surface protection antigen A amino acid sequence with tandem dominant epitope, and its concrete sequence is shown in SEQ ID No. 1. IGEQPVLPGTGVHAQEYNKMT, NQKVKP, EPKGYQS, EEINELKNEGMS, IPELDEAYY, VKYGKVKKGRADRIRS, PEAHELVSSEYNEKDLN, and combinations thereof RRNRQVYPNLER SLKTIKDDIKQRGKKLQRQRSGLDVRKPDVKYQSVDEEKNKLQDYLESDIDIFSY SVDGEKIRNKEIAQSISEIK,
FQNEESDSKVESPVKVEKPVDEEKPKDQKKLVDQSKPESNSKEGWIKKDN K is the dominant epitope of erysipelothrix rhusiopathiae surface protection antigen A, and corresponds to the dominant epitope of natural protein. The nucleotide sequence is matched and encoded with the amino acid sequence from the first dominant epitope to the last dominant epitope, the specific sequence is shown as SEQ ID No.5, the nucleotide sequence is subjected to preferred codon optimization of escherichia coli, the sequence is shown as SEQ ID No.6, and the nucleotide sequence is synthesized by adopting a Seamless Cloning Kit (product number C5891) by Zhongmeitai and Biotechnology (Beijing) Limited company according to a product instruction.
(2) Construction of recombinant expression plasmids
(1) Inserting the nucleotide sequence of the artificially synthesized recombinant erysipelothrix rhusiopathiae surface protection antigen A for coding the tandem dominant epitope into a multiple cloning site of a pET-28b (+) expression vector, and selecting positive clone for carrying out full-length sequencing identification on the plasmid.
(2) The nucleotide sequence of the recombinant expression plasmid is shown as SEQ ID No.3, wherein the nucleotide sequence of the recombinant erysipelothrix rhusiopathiae surface protection antigen A which codes the tandem dominant epitope is between 5071 and 5976 bases, the nucleotide sequence consists of 906 bases, and the specific sequence is shown as SEQ ID No.2. In the nucleotide sequence SEQ ID No.2, the initial end sequence (atgggcagca gccatca tcatcatcac agcagcggcgtcc tgggtgccgcggcagcgatccat atggctagca tgactggggg acagaaaatggtcgggac) and the end sequence (gcggcggcac tcgagagagcacca ccacccacccacactga) are respectively the 5 'and 3' end sequences of an open reading frame for expressing pET-28b (+) exogenous target proteins, and the sequences between the two are nucleotide sequences for coding the tandem dominant epitope of the surface protection antigen A of the erysipelothrix rhusiopathiae. The terminal sequence gcggccgcac tcgagcacca ccacccac cactga has a 6 × His coding sequence, i.e., cacca ccacca caccac. The nucleotide sequence of the open reading frame of the recombinant expression plasmid for expressing the exogenous target protein is consistent with that of SEQ ID No.2, and is consistent with the expectation. The recombinant expression plasmid was designated as pET-28b (+) -spaAE.
Example 2 obtaining of recombinant Escherichia coli and expression of recombinant protein
(1) Obtaining of recombinant Escherichia coli
(1) Transforming the successfully identified recombinant expression plasmid pET-28b (+) -spaAE into BL21 (DE 3) competent cells, gently blowing and uniformly mixing the cells by using a pipette gun, and standing the cells on ice for 25min; (2) taking out the competent cells from the ice bath, quickly putting the competent cells into a water bath at 42 ℃ for heat shock for 45s, taking out the competent cells and incubating the competent cells in the ice bath for 2min; (3) adding 900 μ L SOC culture medium into EP tube, and shake culturing at 37 deg.C in a bacterial culture shaker at 200r/min for 4h; (4) gradient dilution of transformants with SOC Medium (10) -1 、10 -2 、10 -3 ) The diluted transformed bacteria were each pipetted at 100. Mu.L and spread on an LB solid medium (30. Mu.g/mL kanamycin, after solidification, 16. Mu.L of 50mg/mL isopropyl-. Beta. -D-thiogalactoside (IPTG) and 40. Mu.L of 20mg/mL X-gal were added to the surface, and the mixture was spread uniformly using a sterile bent L-shaped spreading bar; (5) and (3) culturing the flat plate in a bacterial incubator at 37 ℃ for 48 hours, observing the color of colonies, wherein white colonies are recombinant escherichia coli and are named as escherichia coli pET-28b (+) -spaAE/BL21 (DE 3) strains, carrying out amplification culture, freeze-drying and storing at the temperature below 0 ℃. The preservation number of pET-28b (+) -spaAE BL21 (DE 3) is CGMCC No.25901, the strain is preserved in the common microorganism center of China Committee for culture Collection of microorganisms at 10 months and 12 days in 2022, and the preservation address is No.3 of the national institute of sciences, north Chen West Lu No.1 of the Chaoyang district in Beijing.
(2) Recombinant protein expression
Escherichia coli pET-28b (+) -spaAE/BL21 (DE 3) strain was inoculated into 8mL of LB liquid medium containing 30. Mu.g/mL kanamycin, incubated at 37 ℃ with shaking at 200r/min to OD 600 When the concentration is 0.6-0.8, adding IPTG with final concentration of 0.5mmol/L to induce expression for 6h at 25 ℃, respectively, centrifugally collecting thalli after the completion of bacterial liquid culture, adding 10mL of lysis buffer solution [50mmol/L Tris-HCl,100mmol/L NaCl,5mmol/L EDTA (pH 8.5) according to the wet weight of each gram of thalli]Resuspending the thalli according to the proportion, crushing the thalli for 20min in an ice bath homogenizer under the conditions that: the operation lasts for 30s, the intermission lasts for 30s, and the homogenizing rotating speed is 18000r/min. And centrifuging the crushed bacterial liquid at 4 ℃ and 8000r/min for 10min, and respectively collecting supernatant and precipitate. Get 40mu.L of the lysate was mixed with 10. Mu.L of 5 XSDS-PAGE loading buffer in a 1.5ml EP tube, boiled in boiled water for 10min, centrifuged at 12000r/min for 5min, and subjected to 10% SDS-PAGE electrophoresis. The protein expression temperature is determined to be 25 ℃, and the target protein is expressed in the inclusion body.
And centrifuging the crushed bacteria liquid, and collecting supernatant and precipitate respectively. And (3) uniformly mixing the cracking sample with SDS-PAGE loading buffer solution, boiling, denaturing, centrifuging, and carrying out SDS-PAGE electrophoresis detection to determine that the protein expression temperature is 25 ℃, wherein the target protein is expressed in the inclusion body.
(3) Recombinant protein expression condition optimization
Further optimizing the concentration and time of induced expression, escherichia coli pET-28b (+) -spaAE/BL21 (DE 3) strain was inoculated into LB liquid medium containing kanamycin and cultured at 37 ℃ with shaking at 200r/min to OD 600 When the concentration is 0.6-0.8, IPTG with final concentration of 0.1, 0.5 and 1.0mmol/L is added to induce expression at 25 deg.C and 37 deg.C for 6h and 18h respectively, and then cell treatment and SDS-PAGE detection are performed by the same method. The optimal conditions for final determination of expression were 25 ℃ and 0.5mmol/L IPTG induced expression for 6 hours.
(4) Purification of recombinant proteins
Inoculating seed liquid of Escherichia coli pET-28b (+) -spaAE/BL21 (DE 3) strain to 1L LB liquid culture medium containing kanamycin, and shake-culturing at 37 deg.C to OD 600 When the expression level is 0.6 to 0.8, induced expression is performed according to the determined optimal expression conditions, and the cells are collected and crushed, washed, purified, and renatured for dialysis to finally obtain the purified target protein rER-SpaAE, and the results are shown in FIG. 1.
EXAMPLE 3 preparation of Swine erysipelas subunit vaccine with recombinant proteins
(1) Preparation of erysipelothrix rhER-SpaAE recombinant protein immunogen
(1) First-order seed propagation and identification: the strain for preparing the vaccine is Escherichia coli BL21 (DE 3) [ pET-28b (+) -SpaAE ] strain for recombinant expression of rER-SpaAE, a freeze-dried strain is redissolved by a small amount of LB liquid culture medium, the redissolved strain is inoculated on an LB solid plate containing kanamycin (25-50 mu g/ml) in a streak manner, the culture is carried out for 16-20 hours at 37 ℃, a typical single colony is selected, the LB liquid culture medium containing kanamycin is inoculated, the culture is carried out for 200r/min at 37 ℃ for 12-16 hours, sterilized glycerol (40 percent of the final volume) is added, the mixture is subpackaged, and the qualified product is used as a first-grade seed for preparing the vaccine after pure inspection.
(2) Secondary seed propagation and identification: taking the first-stage seeds, inoculating the first-stage seeds to an LB liquid culture medium containing kanamycin in an amount of 2 percent of the final volume, and placing the first-stage seeds at 37 ℃ for shaking culture for 8 to 12 hours to obtain second-stage seeds.
(3) Preparing an antigen for preparing the vaccine: taking secondary seeds, inoculating to LB liquid culture medium containing kanamycin in an amount of 2% of the final volume, culturing at 37 deg.C, and allowing culture OD 600 When the value is 0.6-0.8, the temperature is reduced to 25 ℃, and IPTG with the final concentration of 0.5mM is added for inducing expression for 6h.
(4) Breaking the bacteria: centrifugally collecting thalli, re-suspending the thalli according to the proportion of adding 10mL of buffer A to the wet weight of each gram of thalli, and crushing the thalli for 20min in an ice bath homogenizer under the crushing conditions that: the operation is carried out for 30s, the intermittence is carried out for 30s, and the homogenizing rotating speed is 18000r/min. And centrifuging the crushed bacterial liquid at 4 ℃ and 8000r/min for 10min, and collecting the precipitate.
(5) Cleaning the inclusion bodies: washing the inclusion body precipitate with bufferA containing 1% Triton X-100 for 2 times, washing with 1 × PBS for one time, performing 8000r/min after each washing, centrifuging for 10min, collecting the inclusion body, and washing.
(6) And (3) purification: suspending the washed inclusion bodies according to an inclusion body Lysis buffer (containing 8mol/L urea) =1 (W/V), performing denaturation and dissolution, and then centrifuging (7000 r/min,10 min), and collecting the supernatant, namely the protein denaturation solution. Then assembling, balancing, sampling, washing and eluting according to the specification of Ni NTA Beads affinity chromatography in sequence, and collecting the eluted target protein solution.
(7) Renaturation: dialyzing the eluted target protein solution, wherein the dialyzed solution sequentially comprises: 0.01mol/L PBS containing 6mol/L urea, 0.01mol/L PBS containing 4mol/L urea, 0.01mol/L PBS containing 2mol/L urea and 0.01mol/L PBS are respectively added, 48 hours are carried out each time, after the dialysis is finished, 12000r/min is carried out, the centrifugation is carried out for 10min at 4 ℃, and after the supernatant is collected and subpackaged, the supernatant is preserved for standby at 80 ℃ below zero.
(8) Protein purity detection: the bands were examined by SDS-PAGE and grey-scanned.
(9) And (3) protein content detection: the protein content was measured using BCA assay kit (Prolate, BCA miniprotein quantitation kit, P1513) and was 300. Mu.g/ml.
And (3) bacterium removal of the protein R: the purified swine erysipelas recombinant protein rER-SpaAE was sterilized by filtration through a 0.22 μm filter.
(2) Test of Erer-SpaAE recombinant protein immunogen of erysipelothrix rhusiopathiae
(1) The characteristics are as follows: a colorless clear liquid.
(2) And (4) sterile inspection: the test was carried out according to appendix 3306 of Chinese veterinary pharmacopoeia, and the growth was carried out aseptically.
(3) And (4) safety inspection: 5 mice each weighing 16 to 18g were injected subcutaneously with 0.3ml, and the mice were observed for 10 days to be all alive.
(3) Preparation of swine erysipelas subunit vaccine by using recombinant protein
Mixing 400 mu g of swine erysipelas recombinant protein rER-SpaAE with 1mL of Manganese Jelly (MnJ) Adjuvant (gamma) colloidal Manganese Adjuvant (2 mg/mL), adding ultrapure water to supplement 12mL, and mixing uniformly to obtain the swine erysipelas subunit vaccine. The manganese adjuvant is a manganese nanoparticle aqueous adjuvant with the manganese element content of 2 mg/mL. The core principle is that Mn & lt 2+ & gt activates a cGAS-STING signal path, dendritic cells are efficiently and rapidly induced to mature and present antigens, and then adaptive immune response is induced and immune memory is generated. Manganese adjuvants have advantages including: the antibody has the advantages of convenient use, fast antibody generation, strong antibody affinity, no toxic or side effect, freezing storage or repeated freezing and thawing, and can still immunize antigens/haptens with weak immunogenicity or other adjuvants which cannot play a role to obtain effective antibodies.
(4) Testing of swine erysipelas subunit vaccine
(1) And (3) sterility test: the test was carried out according to the supplement 3306 of the present "Chinese veterinary pharmacopoeia" (Committee of Chinese veterinary pharmacopoeia, pharmacopoeia of people's republic of China, good quality, good year edition, three parts, china agricultural publishing house, 2021, hereinafter referred to as "Chinese veterinary pharmacopoeia") and the growth was carried out aseptically.
(2) And (4) safety inspection: 5 mice each weighing 16 to 18g were injected subcutaneously with 0.3ml, and the mice were observed for 10 days to be all alive.
Example 4 immunopotency test of porcine erysipelas subunit vaccine in mice
(1) Grouping and immunization
4mL of subunit vaccine is taken and added with 2.7mL of Manganese Jelly (MnJ) Adjuvant colloidal Manganese (2 mg/mL) and mixed evenly, thus obtaining diluted samples for testing the efficacy of the mice. 16 mice weighing 16-18 g, 10 of which were injected subcutaneously with 0.2 ml/mouse, and 6 were not inoculated as controls.
(2) Counteracting toxic substances
After 21 days of inoculation, the mixed bacterial liquid of erysipelothrix rhusiopathiae type 1C 43008 strain (CVCC 43008) and type 2C 43006 strain (CVCC 43006) is used for virus challenge, 1000MLD virulent bacterial liquid is injected subcutaneously into 10 immune mice and 3 control mice, and 1MLD virulent bacterial liquid is injected subcutaneously into the other 3 control mice. The observation period was 10 days.
(3) Results
3/3 of the mice from the control group challenged with 1MLD (5 CFU) and 1000MLD (5000 CFU) died, and 10/10 of the mice immunized with 1000MLD (5000 CFU) challenged (see Table 1) was higher than the protection criteria of mice tested for efficacy test of commercial swine erysipelas vaccine.
TABLE 1 study of rER-SpaAE proteins on immunogenicity and minimum immunization dose in mice
The result shows that the recombinant erysipelothrix rhusiopathiae surface protection antigen A with the tandem dominant epitope provided by the invention is uniformly mixed with an adjuvant, and can induce an organism to generate an immune response after a mouse is immunized, and can provide a 100% immune protection effect for resisting 1000MLD virulent attack for an attacking mouse. The erysipelothrix rhusiopathiae recombinant protein rER-SpaAE has good immunogenicity, can effectively protect mice from being attacked by erysipelothrix rhusiopathiae type 1 and type 2 virulent viruses, and has the immunizing dose of 2 mu g/mouse, so that complete protection can be achieved.
Example 5 immunopotency test of porcine erysipelas subunit vaccine in pigs
(1) Grouping and immunization
9 healthy susceptible pigs of 4-6 months of age and 23-26kg of body weight were injected 3ml under 6 scalps and 3 other pigs were not vaccinated as controls.
(2) Counteracting toxic substances
After 21 days of inoculation, 3 immune pigs are attacked by intramuscular injection of 2MLD erysipelothrix rhusiopathiae ER24 strain liquid, another 3 immune pigs are attacked by intramuscular injection of 1MLD, and control pigs are attacked by intramuscular injection of 1MLD, and observation is carried out for 14 days.
(3) As a result, the
3/3 deaths of control pigs challenged with 1MLD (23 CFU), 3/3 protection of immunized pigs, 2/3 protection of immunized pigs challenged with 2MLD (45 CFU) (see Table 2), are higher than the protection criteria for commercial swine erysipelas vaccine efficacy test with swine test.
TABLE 2 study of rER-SpaAE proteins on immunogenicity of pigs
The result shows that the recombinant erysipelothrix rhusiopathiae surface protection antigen A with the tandem dominant epitope provided by the invention is uniformly mixed with an adjuvant, and after a pig is immunized, 67% of immune protection effect for resisting 2MLD virulent attack can be provided for the attacking pig, and 100% of immune protection effect for resisting 1MLD virulent attack can be provided for the attacking pig. It can be seen that the erysipelothrix rhusiopathiae recombinant protein rER-SpaAE has good immunogenicity, and can effectively protect pigs from being attacked by virulent erysipelothrix rhusiopathiae.
Claims (10)
1. A recombinant erysipelothrix rhusiopathiae surface protection antigen A with tandem dominant epitopes has an amino acid sequence shown in SEQ ID No. 1.
2. The nucleotide sequence of the recombinant erysipelothrix rhusiopathiae surface protection antigen A for coding the tandem dominant epitope is shown as SEQ ID No.2.
3. A recombinant expression plasmid, which contains the nucleotide sequence of the recombinant erysipelothrix rhusiopathiae surface protection antigen A of the coding tandem dominant epitope of claim 2, and the nucleotide sequence of the recombinant expression plasmid is shown as SEQ ID No. 3.
4. The recombinant expression plasmid of claim 3, wherein: the starting vector is pET-28b (+), and the recombinant expression plasmid is pET-28b (+) -spaAE, namely, a nucleotide sequence SEQ ID No.2 of a recombinant erysipelothrix rhusiopathiae surface protection antigen A for coding a tandem dominant epitope is inserted into a multiple cloning site.
5. A recombinant Escherichia coli obtained by transforming the recombinant expression plasmid of claim 3 or 4 into Escherichia coli BL21 (DE 3).
6. A method for preparing recombinant erysipelothrix rhusiopathiae surface protection antigen a of the tandem dominant epitope as claimed in claim 1 or 2, characterized by comprising the steps of:
(1) Carrying out optimization of preferred codons of escherichia coli on a nucleotide sequence SEQ ID No.2 of a recombinant erysipelothrix rhusiopathiae surface protection antigen A for coding the tandem dominant epitope, wherein the optimized nucleotide sequence is shown as SEQ ID No.6, artificially synthesizing, and inserting a pET-28b (+) vector to obtain a recombinant expression plasmid;
(2) Transforming the recombinant expression plasmid into escherichia coli BL21 (DE 3 strain) to obtain a recombinant expression strain;
(3) The recombinant erysipelothrix rhusiopathiae surface protection antigen A with tandem dominant epitopes is obtained by carrying out fermentation culture, induction expression, thallus crushing, inclusion body denaturation, ion chromatography and dialysis renaturation on the recombinant expression strain;
the recombinant expression plasmid is a nucleotide sequence of a recombinant erysipelothrix rhusiopathiae surface protection antigen A which codes a tandem dominant epitope and is inserted into a pET-28b (+) carrier cloning site.
7. The method of manufacturing according to claim 6, characterized in that: the temperature for inducing expression in the step (3) is 25 ℃; the induced expression time is 6h, and the induced expression concentration of IPTG is 0.5mmol/L.
8. A swine erysipelas subunit vaccine, which is characterized by being obtained by mixing the recombinant erysipelas swine erysipelas bacillus surface protection antigen A of the tandem dominant epitope in claim 1 with an immunologic adjuvant; the immune Adjuvant is a Manganese Jelly (MnJ) Adjuvant colloidal Manganese Adjuvant.
9. The swine erysipelas subunit vaccine of claim 8, characterized in that: the concentration of the recombinant erysipelothrix rhusiopathiae surface protection antigen A of the tandem dominant epitope in the erysipelothrix rhusiopathiae subunit vaccine is not lower than 100 mug/head.
10. The use of the tandem dominant epitope recombinant erysipelothrix rhusiopathiae surface protection antigen A of claim 1 in the preparation of a swine erysipelas vaccine.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000047744A1 (en) * | 1999-02-10 | 2000-08-17 | The Rockefeller University | Antigen of erysipelothirx rhusiopathiae comprising an immuno-protective epitope |
| CN101031647A (en) * | 2004-02-27 | 2007-09-05 | 财团法人化学及血清疗法研究所 | Process for producing erysipelothrix rhusiopathiae surface protective antigen mutant in escherichia coli |
| JP2014000016A (en) * | 2012-06-15 | 2014-01-09 | National Agriculture & Food Research Organization | Novel antigenic protein of erysipelothrix rhusiopathiae, gene thereof, recombinant vector, and the use thereof |
| CN110183520A (en) * | 2019-05-25 | 2019-08-30 | 青岛易邦生物工程有限公司 | A kind of brickpox SpaA albumen and its preparing the application in vaccine |
| CN113388624A (en) * | 2020-09-21 | 2021-09-14 | 浙江理工大学 | Preparation method of swine erysipelas SpaA antigen protein and optimized clone thereof |
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000047744A1 (en) * | 1999-02-10 | 2000-08-17 | The Rockefeller University | Antigen of erysipelothirx rhusiopathiae comprising an immuno-protective epitope |
| CN101031647A (en) * | 2004-02-27 | 2007-09-05 | 财团法人化学及血清疗法研究所 | Process for producing erysipelothrix rhusiopathiae surface protective antigen mutant in escherichia coli |
| JP2014000016A (en) * | 2012-06-15 | 2014-01-09 | National Agriculture & Food Research Organization | Novel antigenic protein of erysipelothrix rhusiopathiae, gene thereof, recombinant vector, and the use thereof |
| CN110183520A (en) * | 2019-05-25 | 2019-08-30 | 青岛易邦生物工程有限公司 | A kind of brickpox SpaA albumen and its preparing the application in vaccine |
| CN113388624A (en) * | 2020-09-21 | 2021-09-14 | 浙江理工大学 | Preparation method of swine erysipelas SpaA antigen protein and optimized clone thereof |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116948043A (en) * | 2023-07-27 | 2023-10-27 | 广东光峰生物技术有限公司 | Swine erysipelas subunit vaccine and detection method and application thereof |
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