CN115850404B - Recombinant erysipelothrix rhusiopathiae surface protection antigen A with tandem dominant epitope and application thereof - Google Patents
Recombinant erysipelothrix rhusiopathiae surface protection antigen A with tandem dominant epitope and application thereof Download PDFInfo
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
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Abstract
The invention belongs to the technical field of biology, and particularly relates to a recombinant erysipelothrix rhusiopathiae surface protection antigen A (surface protective antigen A, spaA) with tandem dominant epitopes and application thereof. The amino acid sequence of the recombinant erysipelas bacillus Sus surface protection antigen A of the tandem dominant epitope is shown as SEQ ID No.1, and the nucleotide sequence of the coded 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. And (3) transforming escherichia coli to obtain a recombinant expression strain. The recombinant erysipelothrix rhusiopathiae surface protection antigen A with the tandem dominant epitope is uniformly mixed with the adjuvant, so that an immune response can be induced by an organism after a mouse is immunized, and 100% immune protection effect for resisting 1000MLD virulent attack can be provided for the virus-challenged mouse; after immunization of pigs, 67% of immune protection against 2MLD virulent attack can be provided for the challenged pigs, and 100% of immune protection against 1MLD virulent attack can be provided for the challenged pigs.
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
Erysipelas in swine are caused by erysipelas bacillus suis (Erysipelothrix rhusiopathiae), and are also called "diamond skin diseases" due to the symptom of marked diamond-shaped skin injury, commonly called "fire marks". Acute type appears as septicemia accompanied by cutaneous diamond lesions, and chronic type appears as non-suppurative arthritis and proliferative endocarditis. Swine erysipelas are susceptible to Yu Xiaqiu in the period of high temperature and humidity, and swine of 2-6 months of age are most susceptible. Pig erysipelas have high morbidity and mortality and often take an aggregate outbreak situation, and pig death caused by pig erysipelas and pig ketone body devaluation caused by chronic symptoms cause great losses to farmers and breeding enterprises, and have negative effects on meat product supply and national economy.
Erysipelothrix rhusiopathiae is a gram-positive bacterium, has a slender or slightly curved shape, is round at two ends, has no capsule, does not form spores, and cannot move. The bacteria are sensitive to penicillin, streptomycin, tylosin, tetracycline and cephalosporins, but antibiotic therapy has poor effect on chronic infections. Erysipelothrix rhusiopathiae is sensitive to heat, but can survive in cured, smoked, frozen and dried meat stably, and can be transmitted through respiratory tract, digestive tract and blood. In addition, contaminated feed, drinking water, land, shelter, etc. are indirect vehicles for the bacteria. The erysipelothrix rhusiopathiae host is wide, and at present, separation reports are all available in mice, turkeys, chickens, ducks, cows, sheep, deer, moose and musk deer. Erysipelothrix rhusiopathiae can also infect people through skin wounds to cause local cellulitis, namely red algae like disease (euthapsilosis), and part of infectious bacteria in patients spread through blood diffusion, and finally cause joint and organ injury.
The main means for preventing and controlling swine erysipelas is vaccine immunization, and the commercial swine erysipelas vaccines in China at present comprise swine erysipelas inactivated vaccines and swine erysipelas live vaccines (G4T 10 strain and GC42 strain respectively). The immune effect of the swine erysipelas live vaccine is decisively influenced by antibiotics, and the immune process and the formulated implementation of medication are influenced to a certain extent, so that the raising end is more prone to selecting swine erysipelas inactivated vaccine. However, swine erysipelas inactivated vaccine also has some defects in the use process, such as a certain batch-to-batch difference in immune protection effect, because the content of key protective antigen components is different to a certain extent when swine erysipelas inactivated antigen is produced by using the traditional bacterial inactivated vaccine production process. Therefore, the development of subunit vaccines based on key protective antigen components would be an important development direction in the field of swine erysipelas prevention and control.
Surface protection antigen a (surface protective antigen A, spaA) is a key protective antigen of erysipelothrix rhusiopathiae. Different erysipelothrix rhusiopathiae strains may have different SpaA lengths, generally identical N-terminal lengths, and the main difference is the number of repetitions of the C-terminal repeat sequence. Most erysipelothrix rhusiopathiae strains SpaA full length 626aa,1-27aa are signal peptides, and extracellular secreted SpaA has a molecular weight of about 69kDa. Mice and pigs were immunized with the SpaA full-length protein and the N-terminus, which resulted in high levels of erysipelas protective antibodies, but not the C-terminus. The infection experiment is carried out by constructing swine erysipelas SpaA deletion strain by Borrathybay and the like, and the result shows that the pathogenicity and complement tolerance of the SpaA deletion strain to mice are obviously lower than those of wild strain, and the fact that the SpaA plays a critical role in swine erysipelas infection process is proved. These studies confirm that the erysipelas suis surface antigen SpaA has excellent immunogenicity, and establishes an important theoretical basis for the erysipelas suis surface antigen SpaA as a vaccine candidate antigen. In order to simplify the immunization program, reduce the stress response of frequent vaccination on pigs, reduce the labor cost of vaccination, and have market advantages compared with 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 critical protective antigen of erysipelothrix rhusiopathiae is compressed to the greatest 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 vaccines, the primary purpose of the invention is to provide a recombinant erysipelothrix rhusiopathiae surface protection antigen A with tandem dominant epitopes.
It is another object of the present invention to provide a nucleotide sequence encoding the recombinant erysipelothrix rhusiopathiae surface protection antigen a of the above tandem dominant epitope, which is artificially modified.
The third object of the invention is to provide a recombinant expression plasmid containing the nucleotide sequence of the recombinant erysipelothrix rhusiopathiae surface protection antigen A encoding the tandem dominant epitope.
The fourth object of the invention is to provide a recombinant E.coli capable of expressing the recombinant erysipelothrix rhusiopathiae surface protection antigen A of tandem dominant epitope.
A fifth object of the invention is to provide a swine erysipelas subunit vaccine.
A sixth object of the present invention is to provide the use of the recombinant erysipelothrix rhusiopathiae surface protection antigen a of the tandem dominant epitope described above.
The aim of the invention is achieved by the following technical scheme:
a recombinant erysipelothrix rhusiopathiae surface protection antigen A with tandem dominant epitope has an amino acid sequence shown as SEQ ID No. 1.
The nucleotide sequence of the recombinant erysipelothrix rhusiopathiae surface protection antigen A for encoding the tandem dominant epitope is shown as SEQ ID No.2.
A recombinant expression plasmid comprises the nucleotide sequence of the recombinant erysipelas bacillus suis surface protection antigen A for encoding the 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 which codes for the tandem dominant epitope is inserted into a multiple cloning site.
A recombinant E.coli is obtained by transforming E.coli BL21 (DE 3) strain 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 is named as follows: escherichia coli (Escherichia coli) which has been deposited at the China general microbiological culture Collection center (China Committee) for culture Collection of microorganisms at 10 and 12 days of 2022, having a deposit address of Beijing, chaoyang, national institute of microorganisms, national academy of sciences of China.
The preparation method of the recombinant erysipelothrix rhusiopathiae surface protection antigen A with tandem dominant epitope comprises the following steps:
(1) The nucleotide sequence SEQ ID No.2 of the recombinant erysipelas bacillus Sus surface protection antigen A of the coded tandem dominant epitope is subjected to optimization of an escherichia coli preference codon, the optimized nucleotide sequence is shown as SEQ ID No.6, and the optimized nucleotide sequence is artificially synthesized and inserted into 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 expression strain is subjected to fermentation culture, induced expression, thallus crushing, inclusion body denaturation, ion chromatography and dialysis renaturation to obtain a recombinant erysipelothrix rhusiopathiae surface protection antigen A with tandem dominant epitopes;
the recombinant expression plasmid is a nucleotide sequence for inserting a recombinant erysipelothrix rhusiopathiae surface protection antigen A which codes for a tandem dominant epitope into a pET-28b (+) vector cloning site.
The swine erysipelas subunit vaccine is prepared by fermenting and culturing the recombinant escherichia coli, inducing and expressing, crushing thalli, denaturing inclusion bodies, performing ion chromatography, dialyzing and renaturating, collecting target protein (recombinant swine erysipelas bacillus surface protection antigen A with tandem dominant epitope), and mixing the target protein with an immune adjuvant.
The immune Adjuvant is Manganese jely (MnJ) Adjuvant (gamma) colloidal Manganese Adjuvant.
The induction expression temperature is preferably 25 ℃.
The induction expression time is preferably 6h.
The IPTG induction expression concentration is preferably 0.5mmol/L.
The application of the recombinant erysipelas bacillus suis surface protection antigen A with the tandem dominant epitope in preparation of erysipelas suis vaccines.
Compared with the prior art, the invention has the following advantages and effects:
(1) According to the preference of the escherichia coli codon, the nucleotide sequence of the recombinant erysipelas bacillus suis surface protection antigen A containing a plurality of dominant antigen epitopes is artificially designed and synthesized, and the sequence is shown as SEQ ID No.2.
(2) The subunit vaccine prepared by the recombinant erysipelas Sus Domestica surface protection antigen A of the tandem dominant epitope expressed by the recombinant escherichia coli can induce organism to generate immune response after immunization of mice, and can provide 100% immune protection effect against 1000MLD virulent attack for the virus-challenged mice; after immunization of pigs, 67% of immune protection against 2MLD virulent attack can be provided for the challenged pigs, and 100% of immune protection against 1MLD virulent attack can be provided for the challenged pigs. 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 shows a graph of the results of rER-SpaAE protein purification.
1: a marker;2: not induced; 3: crushing the cell supernatant; 4: cleaning the supernatant by using the bacteria crushing and precipitation Triton-100; 5: washing the second supernatant by using the bacteria breaking and precipitating Trition-100; 6: the thallus is crushed and precipitated, PBS is washed to obtain supernatant; 7: washing to complete inclusion bodies (precipitation); 8: LB effluent; 9: WB effluent; 10: EB effluent; 11: supernatant of the dialysis completion liquid; 12: the dialysis was completed and the solution was precipitated.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but embodiments of the present invention are not limited thereto.
Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.
Reagents and materials used in the following examples are commercially available unless otherwise specified.
In the examples, the virulent virus-combating strain of erysipelas of swine is maintained by the supervision of Chinese veterinary medicine.
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 based on the amino acid sequence (AB 019124.1) of the erysipelas Sus domestica SpaA recorded in GenBank.
The SpaA amino acid sequence was analyzed using bioinformatics software to determine the position of the signal peptide (MKKKKHLFPKVSLMSCLLLTAMPLQTA) and the C-terminal repeat (EKSGGMATGWKKVADKWYYLDNTGAIVTGWKKVANKWYYLEKSGAMA TGWKKVSNKWYYLENSGAMATGWKKVSNKWYYLENSGAMATGWKKVA NKWYYLENSGAMATGWKKVSNKWYYLENSGAMATGWKKVANKWYYL), and the sequence between these 2 regions was subjected to epitope analysis to determine the dominant epitope (IGEQ, PVLPGTGVHAQEYNKMT, NQKVKP, EPKGYQS, EEIN, ELKNEGMS, IPELDEAY, VKYEGKVKGRA, DRIRS, PEAHE, LVSDSSEYNDKLN, RRNRQ, VYPNLER, SLKTIKDIKQRGKKLQ, QRSGDVRKPDV, KYQSVVDEEKNKLQDYLESDIFDSYSVDGEKIRNKEI, AQSISEIK, FQNEESDSKVEPESPVKVEKPVDEEKPKDQKKLVDQSKPESNSKEGWIKKDN K). The dominant epitope sequence is intercepted, the amino acid sequence of an open reading frame coding protein of a foreign protein is expressed by combining with a pET-28b (+) vector, and the amino acid sequence of a recombinant erysipelothrix rhusiopathiae surface protection antigen A with tandem dominant epitope is designed, wherein the specific sequence is shown as SEQ ID No. 1. IGEQPVLPGTGVHAQEYNKMT, NQKVKP, EPKGYQS, EEINELKNEGMS, IPELDEAY, VKYEGKVKGRADRIRS, PEAHELVSDSSEYNDKLN, RRNRQVYPNLER and SLKTIKDIKQRGKKLQQRSGDVRKPDVKYQSVVDEEKNKLQDYLESDIFDSY SVDGEKIRNKEIAQSISEIK,
FQNEESDSKVEPESPVKVEKPVDEEKPKDQKKLVDQSKPESNSKEGWIKKDN K is a dominant epitope of erysipelothrix rhusiopathiae surface protection antigen A, and corresponds to the dominant epitope of the natural protein. The nucleotide sequence of the coding sequence is matched for the amino acid sequences from the first dominant epitope to the last dominant epitope, the specific sequence is shown as SEQ ID No.5, the preferred codon optimization of escherichia coli is carried out on the nucleotide sequence, the sequence is shown as SEQ ID No.6, and the nucleotide sequence is synthesized by the Meter Mei and Biotechnology (Beijing) limited company by adopting a seamless cloning kit (Seamless Assembly Cloning Kit, product number C5891) according to the specification of the product.
(2) Construction of recombinant expression plasmids
(1) The nucleotide sequence of the artificially synthesized recombinant erysipelas bacillus suis surface protection antigen A encoding the tandem dominant epitope is inserted into the multiple cloning site of the pET-28b (+) expression vector, and positive clones are selected for plasmid full-length sequencing identification.
(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 encoding the tandem dominant epitope is arranged between the 5071 th and 5976 th bases, and consists of 906 bases, and the specific sequence is shown as SEQ ID No.2. In the nucleotide sequence SEQ ID No.2, a start sequence (atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat atggctagca tgactggtgg acagcaaatg ggtcgggac) and a terminal sequence (gcggccgcac tcgagcacca ccaccaccac cactga) are respectively open reading frame 5 'and 3' end sequences for expressing pET-28b (+) exogenous target proteins, and the sequences between the two are nucleotide sequences for encoding a tandem dominant epitope of the erysipelothrix rhusiopathiae surface protection antigen A. The terminal sequence gcggccgcac tcgagcacca ccaccaccac cactga has a 6×His coding sequence cacca ccaccaccac cac. The nucleotide sequence of the open reading frame of the recombinant expression plasmid for expressing the exogenous target protein is consistent with SEQ ID No.2 and is consistent with expectations. The recombinant expression plasmid was designated pET-28b (+) -spaAE.
EXAMPLE 2 recombinant E.coli acquisition and recombinant protein expression
(1) Obtaining recombinant E.coli
(1) The successfully identified recombinant expression plasmid pET-28b (+) -spaAE is transformed into BL21 (DE 3) competent cells and is gently beaten and mixed by a pipetting gun, and the mixture is kept stand on ice for 25min; (2) taking out competent cells from the ice bath, rapidly placing in a water bath at 42 ℃ for heat shock for 45s, taking out and incubating in the ice bath for 2min; (3) adding 900 mu L of SOC culture medium into the EP tube, and carrying out shaking culture for 4 hours at 37 ℃ on a bacterial culture table at 200 r/min; (4) gradient dilution of the transformant with SOC Medium (10) -1 、10 -2 、10 -3 ) 100. Mu.L of the above-mentioned dilution of the transformant was aspirated and spread on LB solid medium (30. Mu.g/mL kanamycin, 16. Mu.L of 50mg/mL isopropyl-. Beta. -D-thiogalactoside (IPTG) and 40. Mu.L of 20mg/mL X-gal were additionally added to the surface after solidification, and spread evenly using a sterile elbow L-type spreading bar; (5) the plate is placed in a bacteria incubator at 37 ℃ for culturing for 48 hours, and then the color of the bacterial colony is observed, wherein white bacterial colony is recombinantE.coli, designated as E.coli pET-28b (+) -spaAE/BL21 (DE 3) strain, is grown up, lyophilized and stored below 0 ℃. pET-28b (+) -spaAE BL21 (DE 3) has a preservation number of CGMCC No.25901, and the strain is preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) for 10 months and 12 days in 2022, and has a preservation address of North Xiyun No.1, 3 in the Korean region of Beijing, china academy of sciences of microorganisms.
(2) Recombinant protein expression
E.coli pET-28b (+) -spaAE/BL21 (DE 3) strain was inoculated in 8mL of LB liquid medium containing 30. Mu.g/mL kanamycin, and subjected to shaking culture at 37℃for 200r/min to OD 600 When the concentration is 0.6-0.8, adding IPTG with the final concentration of 0.5mmol/L respectively, inducing expression at 25 ℃ for 6 hours, centrifuging and collecting thalli after the culturing of the bacterial liquid is completed, and adding 10mL of lysis buffer [50mmol/L Tris-HCl,100mmol/L NaCl,5mmol/L EDTA (pH 8.5) according to the wet weight of each gram of thalli]The bacterial cells are resuspended in the proportion of (1), and crushed for 20min by a homogenizer in ice bath, wherein the crushing conditions are as follows: working for 30s, intermittent for 30s, and homogenizing rotating speed is 18000r/min. Centrifuging the crushed bacterial liquid at 4 ℃ and 8000r/min for 10min, and respectively collecting supernatant and precipitate. 40. Mu.L of the lysed sample was mixed with 10. Mu.L of 5 XSDS-PAGE loading buffer in a 1.5ml EP tube, boiled in water for 10min, centrifuged at 12000r/min for 5min, and subjected to 10% SDS-PAGE. The protein expression temperature was determined to be 25℃and the target protein was expressed in inclusion bodies.
And centrifuging the crushed bacterial liquid, and respectively collecting supernatant and sediment. And (3) uniformly mixing the cracked sample with SDS-PAGE loading buffer solution, boiling, denaturing and centrifuging, and performing SDS-PAGE electrophoresis detection to determine that the protein expression temperature is 25 ℃, wherein the target protein is expressed in inclusion bodies.
(3) Recombinant protein expression condition optimization
Further optimizing the concentration and time of the induced expression, inoculating the strain of E.coli pET-28b (+) -spaAE/BL21 (DE 3) with LB liquid medium containing kanamycin by the same method, and culturing at 37 ℃ under shaking at 200r/min until OD 600 When the concentration is 0.6-0.8, IPTG with the final concentration of 0.1, 0.5 and 1.0mmol/L is added, induced expression is respectively carried out at 25 ℃ and 37 ℃ for 6h and 18h, respectively sampling is carried out, and thallus treatment is carried out by adopting the same methodSDS-PAGE detection. The optimal condition for the final expression was 25℃and 0.5mmol/L IPTG was induced for 6 hours.
(4) Purification of recombinant proteins
Inoculating seed solution of Escherichia coli pET-28b (+) -spaAE/BL21 (DE 3) strain into 1L LB liquid medium containing kanamycin, shake culturing at 37deg.C to OD 600 When the expression level is 0.6 to 0.8, the expression is induced according to the determined optimal expression conditions, and bacterial cells are collected, crushed, washed, purified and renaturated and dialyzed to finally obtain the purified target protein rER-SpaAE, and the result is shown in figure 1.
EXAMPLE 3 preparation of swine erysipelas subunit vaccine Using recombinant proteins
(1) Preparation of recombinant protein immunogen of erysipelothrix rhusiopathiae rER-SpaAE
(1) First-stage seed propagation and identification: the strain for preparing the seedlings is escherichia coli BL21 (DE 3) [ pET-28b (+) -SpaAE ] strain of recombinant expression rER-SpaAE, the freeze-dried strain is re-dissolved by a small amount of LB liquid culture medium, streaked and inoculated on an LB solid flat plate containing kanamycin (25-50 mug/ml), the strain is placed at 37 ℃ for culturing for 16-20 hours, a typical single colony is selected, the LB liquid culture medium containing kanamycin is inoculated, 200r/min and 37 ℃ for culturing for 12-16 hours, sterilized glycerol (40% of final volume) is added, and the mixture is split, and the split-packed strain is used as first-stage seed for preparing the seedlings after pure inspection.
(2) And (3) secondary seed propagation and identification: taking first-stage seeds, inoculating the first-stage seeds into LB liquid medium containing kanamycin in an amount of 2% of the final volume, and culturing the first-stage seeds at 37 ℃ in a shaking way for 8-12 hours to obtain second-stage seeds.
(3) Preparation of antigen for seedling preparation: inoculating the second seed into LB liquid medium containing kanamycin at a final volume of 2%, and culturing at 37deg.C until OD of the culture 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 induction expression for 6 hours.
(4) And (3) breaking bacteria: the thalli are collected by centrifugation, the thalli are resuspended according to the proportion of adding 10mL bufferA per gram of the thalli wet weight, and the thalli are crushed for 20min by a homogenizer in ice bath, wherein the crushing conditions are as follows: working for 30s, intermittent for 30s, and homogenizing rotating speed is 18000r/min. Centrifuging the crushed bacterial liquid at 4 ℃ and 8000r/min for 10min, and collecting the precipitate.
(5) Cleaning inclusion bodies: the inclusion body pellet was washed with bufferA containing 1% Triton X-100 for 2 times, and with 1 XPBS for one time, 8000r/min was performed after each washing, and the inclusion body was collected by centrifugation for 10min, and the pellet was collected and washed continuously.
(6) Purifying: suspending the washed inclusion body according to the inclusion body Lysis buffer (containing 8mol/L urea) =1:10 (W/V), carrying out denaturation and dissolution, centrifuging (7000 r/min,10 min), and collecting the supernatant as a protein denaturation solution. And then assembling, balancing, loading, cleaning and eluting sequentially according to the Ni NTA Beads affinity chromatography instruction, and collecting the eluting target protein liquid.
(7) Renaturation: dialyzing the eluted target protein liquid, wherein the dialyzing solution comprises the following components in sequence: 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, 48 hours each time, after dialysis, 12000r/min, centrifuging at 4deg.C for 10min, collecting supernatant, sub-packaging, and storing at-80deg.C for use.
(8) Protein purity detection: the bands were detected by SDS-PAGE and gray scanned.
(9) Protein content detection: protein content was measured at 300. Mu.g/ml using BCA assay kit (Proriley, BCA assay microalbumin quantification kit, P1513).
Protein sterilization: purified swine erysipelas recombinant protein rER-SpaAE was sterilized by filtration through a 0.22 μm filter.
(2) Inspection of erysipelothrix rhusiopathiae rER-SpaAE recombinant protein immunogen
(1) Traits: the liquid was clear and colorless.
(2) And (3) sterile inspection: checking according to annex 3306 of Chinese animal pharmacopoeia, and aseptically growing.
(3) And (3) safety inspection: 5 mice weighing 16-18 g were subcutaneously injected with 0.3ml each, and the whole body was alive after observation for 10 days.
(3) Preparation of swine erysipelas subunit vaccine by recombinant protein
Every 400 mug of swine erysipelas recombinant protein rER-SpaAE is uniformly mixed with 1mL Manganese Jelly (MnJ) Adjuvant (gamma) colloidal manganese Adjuvant (2 mg/mL), and ultrapure water is added to supplement 12mL, and the swine erysipelas subunit vaccine is obtained after uniform mixing. The manganese adjuvant is a manganese nanoparticle water-based adjuvant with the manganese element content of 2 mg/mL. The core principle is that Mn2+ activates the cGAS-STING signal channel, efficiently and rapidly induces dendritic cells to mature and present antigen, thereby inducing adaptive immune response and generating immune memory. Manganese adjuvant advantages include: the antibody has the advantages of convenient use, quick antibody production, strong antibody affinity, no toxic or side effect, freezing storage or repeated freezing and thawing, and still can be immunized to obtain effective antibodies to antigens/hapten with weak immunogenicity or which cannot be acted by other adjuvants.
(4) Inspection of swine erysipelas subunit vaccine
(1) And (3) sterile inspection: the test is carried out according to annex 3306 of the current ' Chinese animal pharmacopoeia ' (Chinese animal pharmacopoeia committee, chinese people's republic of China, good two and year edition three, china agricultural press, 2021, hereinafter referred to as ' Chinese animal pharmacopoeia '), and the bacteria grow.
(2) And (3) safety inspection: 5 mice weighing 16-18 g were subcutaneously injected with 0.3ml each, and the whole body was alive after observation for 10 days.
Example 4 test of immune efficacy of swine erysipelas subunit vaccine against mice
(1) Grouping and immunization
Taking 4mL of subunit vaccine, adding 2.7mL Manganese Jelly (MnJ) Adjuvant (gamma) colloidal manganese Adjuvant (2 mg/mL), and uniformly mixing to obtain a diluted sample for testing the efficacy of the mice. 16 mice weighing 16-18 g were used, 10 of which were subcutaneously injected with 0.2ml of sample per mouse, and 6 of which were not vaccinated as controls.
(2) Attack toxin
After 21 days of inoculation, virulent mixed bacteria of erysipelothrix rhusiopathiae strain 1 (CVCC 43008) and type 2 (CVCC 43006) are used for virulent mixed bacteria, 10 immunized mice and 3 control mice are subcutaneously injected with 1000MLD virulent bacteria, and the other 3 control mice are subcutaneously injected with 1MLD virulent bacteria. The observation was carried out for 10 days.
(3) Results
Mice from the control group challenged with 1MLD (5 CFU) and 1000MLD (5000 CFU) died 3/3, and 10/10 protection (see Table 1) was given to immunized mice challenged with 1000MLD (5000 CFU), which was higher than the protection standard for mice for commercial swine erysipelas vaccine efficacy test.
TABLE 1 investigation of mouse immunogenicity and minimum immune dose by rER-SpaAE proteins
The result shows that the recombinant erysipelas bacillus suis surface protection antigen A with the tandem dominant epitope provided by the invention is uniformly mixed with an adjuvant, and can induce organisms to generate immune response after mice are immunized, so that 100% immune protection effect against 1000MLD virulent attack can be provided for the virus-challenged mice. The recombinant protein rER-SpaAE of erysipelas pig bacillus has good immunogenicity, can effectively protect mice from being attacked by type 1 and type 2 virulent of erysipelas pig bacillus, has an immune dose of 2 mug/mouse, and can achieve complete protection.
Example 5 test of immune efficacy of swine erysipelas subunit vaccine against swine
(1) Grouping and immunization
9 healthy and susceptible pigs of 4-6 months of age and weight of 23-26kg were used, 6 of which were subcutaneously injected with 3ml of vaccine and the other 3 were not vaccinated as controls.
(2) Attack toxin
After 21 days of inoculation, 3 immunized pigs were challenged with 2MLD swine erysipelas bacillus virulent ER24 strain broth, another 3 immunized pigs were challenged with 1MLD intramuscular injection, and control pigs were challenged with 1MLD intramuscular injection, and observed for 14 days.
(3) Results
Control pigs challenged with 1MLD (23 CFU) died, immunized pigs protected 3/3, and immunized pigs challenged with 2MLD (45 CFU) protected 2/3 (see Table 2) above the protection standard for commercial swine erysipelas vaccine efficacy tests with pigs.
TABLE 2 investigation of pig immunogenicity by rER-SpaAE proteins
The result shows that the recombinant erysipelas bacillus suis surface protection antigen A with the tandem dominant epitope provided by the invention is uniformly mixed with an adjuvant, and after immunization of pigs, the recombinant erysipelas bacillus suis surface protection antigen A can provide 67% of immune protection effect for the virulent attack of 2MLD for the virulent attack pigs, and can provide 100% of immune protection effect for the virulent attack of 1MLD for the virulent attack pigs. It can be seen that the recombinant protein rER-SpaAE of erysipelas suis has good immunogenicity, and can effectively protect pigs from being attacked by erysipelas suis virulent.
Claims (9)
1. A recombinant erysipelothrix rhusiopathiae surface protection antigen A with tandem dominant epitope has an amino acid sequence shown as SEQ ID No. 1.
2. The nucleotide sequence of the recombinant erysipelothrix rhusiopathiae surface protection antigen A encoding the tandem dominant epitope as set forth in claim 1 is shown in SEQ ID No.2.
3. A recombinant expression plasmid containing the nucleotide sequence of the recombinant erysipelas bacillus suis surface protection antigen A of the tandem dominant epitope coded by the claim 2, wherein the nucleotide sequence of the recombinant expression plasmid is shown as SEQ ID No. 3.
4. A recombinant E.coli obtained by transforming E.coli BL21 (DE 3) strain with the recombinant expression plasmid of claim 3.
5. A method for preparing the recombinant erysipelothrix rhusiopathiae surface protection antigen a of tandem dominant epitope of claim 1, comprising the steps of:
(1) Constructing a recombinant expression plasmid, wherein the nucleotide sequence of the recombinant expression plasmid is shown as SEQ ID No. 3;
(2) Transforming the recombinant expression plasmid into escherichia coli BL21 (DE 3 strain) to obtain a recombinant expression strain;
(3) The recombinant expression strain is subjected to fermentation culture, induced expression, thallus crushing, inclusion body denaturation, ion chromatography and dialysis renaturation to obtain the recombinant erysipelas bacillus Sus surface protection antigen A with tandem dominant epitopes.
6. The method of manufacturing according to claim 5, wherein: the temperature at which the expression is induced in the step (3) is 25 ℃; the induction expression time is 6h, and the IPTG induction expression concentration is 0.5mmol/L.
7. A erysipelothrix rhusiopathiae subunit vaccine, characterized in that the recombinant erysipelothrix rhusiopathiae surface protection antigen a of the tandem dominant epitope of claim 1 is mixed with an immunological adjuvant; the immune adjuvant is Manganese Jelly Adjuvant gamma colloid manganese adjuvant.
8. The swine erysipelas subunit vaccine of claim 7, wherein: 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.
9. Use of the recombinant erysipelothrix rhusiopathiae surface protection antigen a of tandem dominant epitope of claim 1 for preparing erysipelothrix rhusiopathiae vaccine.
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| 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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| 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 |
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