CN116284274A - Recombinant erysipelothrix rhusiopathiae surface antigen SpaA protein and application thereof - Google Patents
Recombinant erysipelothrix rhusiopathiae surface antigen SpaA protein and application thereof Download PDFInfo
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- CN116284274A CN116284274A CN202211616614.3A CN202211616614A CN116284274A CN 116284274 A CN116284274 A CN 116284274A CN 202211616614 A CN202211616614 A CN 202211616614A CN 116284274 A CN116284274 A CN 116284274A
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- recombinant
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- surface antigen
- erysipelothrix rhusiopathiae
- spaa protein
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Abstract
The invention discloses a recombinant erysipelas surface antigen SpaA protein and application thereof, wherein a wild type sequence with high conservation is adopted as a coding gene of the recombinant erysipelas surface antigen SpaA protein, so that the recombinant erysipelas surface antigen SpaA protein has good safety, immunogenicity and cross-serotype protection effects, can generate antibodies with enough immune protection, and has good application value for the construction of erysipelas related antigens and antibody diagnostic methods. Meanwhile, the protein has good immune compatibility when being mixed with other antigens, and can provide sufficient dosage space for other components.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a recombinant erysipelas bacillus surface antigen SpaA protein and application thereof.
Background
Erysipelas in pigs is caused by erysipelas bacillus (erysipelothrix rhusiopathiae) and is also called "diamond skin disease" due to the symptom of marked diamond skin injury, commonly called "fire mark" [1] . Acute type is manifested by septicemia accompanied by skin diamond damage, and chronic type is manifested by non-suppurative arthritis and proliferative endocarditis [2] . Swine erysipelas is susceptible to Yu Xiaqiu in high-temperature humid seasons, and swine of 2-6 months of age are most susceptible. Pig erysipelas have high morbidity, high mortality and frequent aggregate outbreak situation, and pig death caused by pig erysipelas and pig ketone body devaluation caused by chronic symptoms cause great loss to farmers and breeding enterprises, and have negative effects on meat product supply and national economy [3] 。
Erysipelothrix rhusiopathiae is a gram positive bacterium, and is shapedSlender or slightly curved, rounded at both ends, without capsule, without sporulation, unable to move [4] . The bacteria are sensitive to penicillin, streptomycin, tylosin, tetracycline and cephalosporins, but antibiotic therapy has poor effect on chronic infections [5] . Erysipelothrix rhusiopathiae is sensitive to heat but can survive in curing, smoking, freezing and drying meat [6] Can be transmitted through the respiratory tract, digestive tract and blood. In addition, the contaminated feed, drinking water, land, shelter and the like are indirect transmission media of the bacteria [7] . The erysipelothrix rhusiopathiae host is wide, and at present, separation reports are all carried out in mice, turkeys, chickens, ducks, cattle, sheep, deer, moose and musk deer [8-10] . Erysipelothrix rhusiopathiae can also infect humans through skin wounds, causing localized cellulitis, i.e. rhodophyta-like disease (euthapsigarid) [8] Part of the infectious bacteria in the patient spreads via blood diffusion, and finally causes joint and organ injury [10] 。
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 attenuated live vaccines (G4T 10 strain and GC42 strain respectively). The attenuated live vaccine has a certain immune side reaction, has relatively strict requirements on the body condition and health state of pigs, has decisive influence on the immune effect of the attenuated live vaccine by antibiotics, has a certain influence on the immune program and the preparation and implementation of medication, and therefore, the raising end is more prone to selecting swine erysipelas inactivated vaccine. However, the swine erysipelas inactivated vaccine also has some defects in the use process, for example, the vaccine is prepared after full-bacteria inactivation, local inflammation and toxic reaction of animals are easy to cause after immunization, and biological potential safety hazards such as exotoxin leakage or incomplete inactivation exist in the preparation process. Therefore, development of subunit vaccines with definite single component, good safety and strong immunogenicity will be a future development direction.
Erysipelothrix rhusiopathiae surface antigen SpaA (surface protective antigen A) was found to be the main protective antigen of erysipelothrix rhusiopathiae [4,11] . SpaA is about 64kDa, and immunization of mice and pigs with the protein produces high levels of erysipelas in pigsProtective antibodies against bacilli [12-15] . Structural sequence analysis of SpaA revealed that the C-terminal region of SpaA is very similar to the corresponding region of Streptococcus pneumoniae, both of which bind to bacterial surfaces by interaction with choline residues of teichoic acid [16] . Infection experiments are carried out on construction of swine erysipelas SpaA deletion strains by Borrathybay and the like, and the results show that the pathogenicity and complement tolerance of the SpaA deletion strains to mice are obviously lower than those of wild strains, and the results prove that the SpaA plays a critical role in swine erysipelas bacillus infection process [17] . These studies confirm that the erysipelothrix rhusiopathiae surface antigen SpaA has excellent immunogenicity. At present, the extraction of the natural SpaA surface protein has the problems of complex process, low yield, low purity and the like, which indirectly influences the reactivity, immunogenicity and related applications of the natural SpaA.
Disclosure of Invention
The invention aims to provide a erysipelas surface antigen SpaA protein of swine, which has good safety and immunogenicity, has good immune compatibility when mixed with other antigens and can provide sufficient dosage space, and a genetic engineering strain for expressing the protein.
The amino acid sequence of the recombinant erysipelas Sus Domestica surface antigen SpaA protein is shown in SEQ No. 1.
The nucleotide sequence of the encoding gene A of the recombinant erysipelas bacillus surface antigen SpaA protein is shown as SEQ No. 2.
The recombinant erysipelothrix rhusiopathiae surface antigen SpaA protein contains a tag which facilitates purification of the protein, preferably a C-terminal 6 histidine (6 xHis) tag.
The recombinant erysipelothrix rhusiopathiae surface antigen SpaA protein plasmid constructed by the invention is constructed by connecting the coding gene A between BamHI and HindIII in a cloning/expression region of a plasmid vector pET28 a.
The strain for secretory expression of recombinant erysipelas suis surface antigen SpaA protein constructed by the invention is Escherichia coli BL/S3 strain (BL 21 (DE 3) -S3 strain), the preservation number is CGMCC No.25902, the preservation classification name is Escherichia coli, the strain is preserved in China general microbiological culture Collection center (China Committee) for 10 months 12 in 2022, and the preservation address is North Chen West road No.1 and 3 in the Beijing area of the Chaetomium, and the China academy of sciences microbiological study.
When the recombinant erysipelas bacillus suis surface antigen SpaA protein (rER-SpaA) is expressed by utilizing the escherichia coli BL/S3 strain, the escherichia coli BL/S3 strain is inoculated in an LB liquid culture medium of kanamycin and is subjected to shake culture until OD 600 When the concentration is 0.6-0.8, IPTG with the final concentration of 0.5M is added, and the induction expression is carried out at 25 ℃ for 6 hours.
The recombinant erysipelothrix rhusiopathiae surface antigen SpaA protein can be applied to preparation of vaccines for preventing erysipelothrix rhusiopathiae and an evaluation method of erysipelothrix rhusiopathiae vaccines.
The recombinant erysipelothrix rhusiopathiae surface antigen SpaA protein can be used for preparing a diluent and combining with live vaccines, inactivated vaccines, effective protective antigens and the like.
The live vaccines comprise bacterial live vaccines and virus live vaccines, and the protective antigen comprises subunit vaccines such as outer membrane proteins, capsular polysaccharide, toxins and the like.
The recombinant erysipelas bacillus surface antigen SpaA protein is applied to preparing a diluent and is combined with a type A or type B pasteurellosis multi-virus live vaccine.
More preferably, the recombinant erysipelas bacillus surface antigen SpaA protein is added into aluminum hydroxide gum salt water as diluent, and is combined with the type A or type B live vaccine of the pasteurellosis.
The invention has the beneficial effects that:
1. the recombinant erysipelothrix rhusiopathiae surface antigen SpaA protein expresses a whole gene fragment of erysipelothrix rhusiopathiae SpaA protein, the coding gene adopts a wild type sequence with high conservation, and standard virus-attacking strains are used for proving that the recombinant erysipelothrix rhusiopathiae surface antigen SpaA protein has excellent immunogenicity, and can be used for preventing erysipelothrix rhusiopathiae.
2. According to the recombinant erysipelas bacillus surface antigen SpaA protein, a purification tag is added, so that a large amount of recombinant SpaA protein can be efficiently obtained by a classical purification method, and the preparation time and the production cost for separating and purifying target protein are reduced.
3. The recombinant erysipelothrix rhusiopathiae surface antigen SpaA protein prepared by adopting a genetic engineering method can be used as subunit vaccine effective antigen to replace the traditional method for culturing pathogenic erysipelothrix rhusiopathiae and inactivating and detoxication, so that the biosafety risk in the seed conservation and production process is greatly reduced.
4. The recombinant erysipelas bacillus surface antigen SpaA protein has good immune compatibility with other antigens, and can provide sufficient dosage space.
5. The recombinant erysipelas bacillus surface antigen SpaA protein can be used as an effective component of a vaccine for preventing erysipelas bacillosis of pigs, a solubilizer, a denaturant and the like are not needed to be added, the preparation process is simplified, and meanwhile, the safety of the vaccine is improved.
6. The recombinant erysipelothrix rhusiopathiae surface antigen SpaA protein can be used for preparing vaccine diluent and combining live vaccine.
Drawings
The SDS-PAGE identification of the recombinant strain BL/S3 in the genetic engineering of FIG. 1 determines the expression temperature.
1, protein marker;2, depositing after the empty vector bacteria are cracked; inducing BL/S3 bacteria to crack at the temperature of 3:25 ℃ and then precipitating; inducing BL/S3 bacteria to crack at the temperature of 4:25 ℃; inducing BL/S3 bacteria to crack at the temperature of 5:37 ℃; and 6, inducing BL/S3 bacteria to be lysed and then precipitating at 37 ℃.
FIG. 2 SDS-PAGE identification of genetically engineered recombinant BL/S3 strain to determine the induction dose and time.
1, protein marker;2, sedimentation after BL/S3 bacteria are not induced to crack; 3:0.1M IPTG induces BL/S3 bacteria to be decomposed for 6 hours and then deposited; 4:0.5M IPTG induces BL/S3 bacteria to be precipitated after 6h of lysis; 5:1.0M IPTG induces BL/S3 bacteria to be precipitated after 6h of lysis; 6:1.0M IPTG induces BL/S3 bacteria to be precipitated after 18h of lysis; 7:0.1M IPTG induces BL/S3 bacteria to crack for 6 hours, and supernatant is obtained; 8:0.5M IPTG induces BL/S3 bacteria to crack for 6 hours, and supernatant is obtained; 9:1.0M IPTG induced BL/S3 bacteria after 6h lysis; 10:1.0M IPTG induced BL/S3 bacteria 18h lysis of the supernatant.
FIG. 3 shows the identification result of Western blot (adopting swine erysipelas immune serum of murine origin) of recombinant BL/S3 strain.
1, protein Marker;2, extracting surface protein from erysipelas Sus Domestica bacillus; 3, sedimentation after BL/S3 bacteria are not induced to crack; 4, BL/S3 bacteria induce cracking and then precipitate; BL/S3 bacteria induced lysis of the supernatant.
FIG. 4rER-SpaA protein purification.
1, protein Marker; BL/S3 bacteria do not induce lysis precipitation; 3, BL/S3 bacteria induce lysis supernatant; 4, BL/S3 bacteria induce lysis Trition X-100 to wash supernatant; 5, BL/S3 bacteria induce lysis Trition X-100 to wash the supernatant of the second time; BL/S3 bacteria induce lysis PBS to clean the second supernatant; 7, BL/S3 bacteria induce lysis PBS to clean and finish precipitation; 8, loading effluent LB;9, eluting effluent LB; eluting effluent EB;11, dialyzing the supernatant; 12, precipitation of the dialysis completion solution.
Detailed Description
The technical solutions of the present invention will be clearly and completely described in connection with the embodiments, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention. In the following examples, conventional methods are used unless otherwise specified. The reagents, carriers, cells, and cells used, unless otherwise specified, are commercially available products well known in the art.
EXAMPLE 1 construction and identification of E.coli BL/S3 strain
1. Amplification of the Gene fragment of interest
After sequence hydrophilicity and hydrophobicity, signal peptide, transmembrane domain and basic structure theory evaluation are carried out on the SpaA gene sequence of the erysipelas Sus Domestica surface protein, the nucleotide sequence SEQ No.2 is selected as a target gene fragment. The target gene fragment was synthesized by Zhongmeitai and Biotechnology (Beijing) limited company using a seamless cloning kit (Seamless Assembly Cloning Kit, product number C5891) according to the product specification.
2. Recombinant protein expression vector construction
The enzyme cutting site is designed according to the target gene and the vector: and (3) respectively carrying out enzyme digestion on the PCR product and the plasmid vector pET28a by BamHI and HindIII, and then connecting the digested PCR product with the plasmid vector pET28a by using ligase to obtain the pET28a prokaryotic expression vector inserted with the target gene fragment. The well-connected plasmid is transformed into TOP10 competent cells, positive clones are detected and screened out for sequencing verification, amplification is carried out in LB liquid medium containing kanamycin after confirming that the positive clones are correct, the plasmid is extracted, and the plasmid is preserved below minus 15 ℃.
3. Construction of genetically engineered Strain expressing rER-SpaA
The plasmid obtained by extraction was transformed into competent cells of E.coli BL21 (DE 3), single colonies were picked up on a kanamycin LB plate (final concentration: 50. Mu.g/ml) to LB liquid medium containing kanamycin (final concentration: 50. Mu.g/ml), shake-cultured overnight at 37℃and the presence of the target DNA fragment was confirmed by PCR identification, which was designated as E.coli BL/S3 strain. Adding glycerol (final concentration of 20%) into the obtained BL/S3 strain, and performing gradient freezing and storage, and finally performing long-term freezing and storage at-70 ℃.
Example 2 expression and characterization of rER-SpaA
Expression of rER-SpaA
Inoculating Escherichia coli (E.coli) BL/S3 strain into LB liquid medium containing kanamycin, and shaking culturing at 37deg.C and 200rpm to OD 600 When the concentration is 0.6-0.8, adding IPTG with the final concentration of 0.5M, respectively inducing and expressing at 25 ℃ and 37 ℃ for 6 hours, centrifuging to collect thalli after the bacterial liquid culture is finished, and adding 10mL bufferA[50mM Tris-HCl,100mM NaCl,5mM 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: the operation is carried out for 30s, the interval is 30s, and the homogenizing rotating speed is 18000rpm. The crushed bacterial liquid is centrifuged at 8000rpm for 10min at 4 ℃, and the supernatant and the precipitate are collected respectively. 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 12000rpm for 5min, and subjected to 10% SDS-PAGE (FIG. 1). The protein expression temperature was determined to be 25℃and the target protein was expressed in inclusion bodies. Then BL/S3 strain is inoculated with LB liquid medium containing kanamycin by the same method, placed at 37 ℃ and shake-cultured at 200rpm until OD 600 At 0.6 to 0.8, IPTG was added at a final concentration of 0.1, 0.5 and 1.0M, and the cells were induced to express at 25℃for 6 hours and 18 hours, and then sampled, and treated with bacterial cells and SDS-PAGE was performed in the same manner (FIG. 2). The optimal conditions for the final determination of expression were 25℃and 0.5MIPTG inductionExpression was carried out for 6 hours.
Identification of rER-SpaA
And (3) performing Western blot identification on the bacterial liquid protein sample obtained by adopting BL/S3 strain optimal expression conditions and a method. And (3) loading 20 μl/hole, carrying out electrophoresis for 100min at constant pressure of 80V, taking the gel after finishing, cutting off the concentrated gel after simple flushing, soaking the gel in clear water, soaking the prepared PVDF membrane in methanol for 1min, and assembling a transfer membrane box according to the sequence of blackboard-sponge-filter paper-gel-membrane-filter paper-sponge-red plate, wherein the assembling process is carried out in pre-cooled transfer membrane liquid, and the transfer membrane adopts constant current of 300mA for 90min. And taking out the PVDF membrane from the incubation box after membrane transfer, and adding clean water for 3min. Blocking was then performed for 1h (TBST with 5% skim milk), primary antibody incubation for 1h (1:1000 swine erysipelas immune serum), secondary antibody incubation for 1h (1:3000 hrp-labeled goat anti-mouse IgG), 3-4 washes with TBST after each incubation was completed, 5min each time. After incubation, the following incubation was performed with developer a: developer b=2 ml: the chemiluminescent reagent was added dropwise to the membrane in a proportion of 2ml, and the membrane was imaged after soaking for 2min, the results are shown in FIG. 3. As can be seen from FIG. 3, rER-SpaA expressed by the strain after induction at 25 ℃ for 6 hours mainly exists in inclusion bodies, and rER-SpaA and surface proteins extracted from wild erysipelothrix rhusiopathiae can react with immune serum of erysipelothrix rhusiopathiae, so that rER-SpaA expressed by BL/S3 strain has antigen immunogenicity and spatial structure of erysipelothrix rhusiopathiae SpaA.
Purification of rER-SpaA
Inoculating Escherichia coli BL/S3 strain seed solution into 1.5L LB liquid medium containing kanamycin, shake culturing at 37deg.C to OD 600 When the expression level is 0.6 to 0.8, the protein is induced and expressed according to the determined optimal expression conditions, and the thalli are collected, crushed, washed, purified and renaturated and dialyzed to finally obtain the purified target protein rER-SpaA, and the protein content is measured by using a protein quantification kit of the BCA method as shown in FIG. 4. The theoretical molecular weight of the protein of interest rER-SpaA is about 77kDa.
Example 3 preparation and testing of erysipelothrix rhusiopathiae rER-SpaA recombinant protein immunogens
1. Preparation of erysipelothrix rhusiopathiae rER-SpaA recombinant protein immunogen
(1) First-stage seed propagation and identification: the strain for preparing the seedlings is escherichia coli BL/S3 strain for recombining and expressing rER-SpaA, the freeze-dried strain is thawed by a small amount of LB liquid culture medium, streaked and inoculated on an LB solid 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, the strain is cultured at 200rpm and 37 ℃ for 12-16 hours, sterilized glycerol (40% of final volume) is added, mixed and split charging is carried out, and the strain is used as first-stage seed for preparing the seedlings after pure inspection is qualified.
(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: the operation is carried out for 30s, the interval is 30s, and the homogenizing rotating speed is 18000rpm. The crushed bacterial liquid is centrifuged at 8000rpm for 10min at 4 ℃ and the precipitate is collected.
(5) Cleaning inclusion bodies: the inclusion body pellet was washed with bufferA containing 1% Triton X-100 2 times, and then with 1 XPBS once, and at each washing completion, the inclusion body was collected by centrifugation at 8000rpm for 10min, and the pellet was collected and washed continuously. The two-step cleaning method of bufferA and PBS can remove a large amount of impurity proteins, is beneficial to removing endotoxin, and improves the purity of the prepared erysipelothrix rhusiopathiae rER-SpaA recombinant protein immunogen.
(6) Purifying: and (3) suspending the washed inclusion body according to the inclusion body Lysis buffer (containing 8M urea) =1:10 (W/V), carrying out denaturation and dissolution, centrifuging (7000 rpm,10 min), and collecting the supernatant to obtain 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: 0.01M PBS containing 6M urea, 0.01M PBS containing 4M urea, 0.01M PBS containing 2M urea, and 0.01M PBS, 48 hr each time, centrifuging at 12000rpm at 4deg.C for 10min after dialysis, collecting supernatant, sub-packaging, and storing at-80deg.C for use.
(8) Protein purity detection: the protein purity was 85% as detected by SDS-PAGE and the bands were gray scanned.
(9) Protein content detection: protein content was measured at 33. Mu.g/ml using BCA assay kit (Proriley, BCA assay microalbumin quantification kit, P1513).
(10) Antigen preparation: preparing 40% aluminum gel physiological saline according to the requirements of annex of Chinese animal pharmacopoeia, diluting the qualified purified protein to 20 mug/ml with 40% aluminum gel physiological saline according to the protein content measurement result, fully mixing, and preserving at 2-8 ℃ for detection.
2. Inspection of erysipelothrix rhusiopathiae rER-SpaA recombinant protein immunogen
(1) Traits: after standing, the upper layer is transparent clear liquid, the lower layer is precipitated by a small amount, and the lower layer is uniformly mixed with suspension after shaking.
(2) The sterility test is carried out according to annex 3306 of Chinese animal pharmacopoeia, and the sterility growth is carried out.
(3) The weight of the safety test is 16-18 g, 5 mice are injected with 0.3ml of antigen subcutaneously, and all the mice are healthy and alive after observation for 10 days.
EXAMPLE 4 determination of immunogenicity of recombinant protein rER-SpaA
1. Test with mice
(1) Preparing immunogen: purified erysipelas recombinant protein rER-SpaA was mixed in equal volumes using 40% saline, after which the vaccine was diluted to the indicated concentration using 40% saline according to the protocol (see table 1 for details).
(2) Immunization: mice were used for the assay and immunized subcutaneously with different concentrations of rER-SpaA protein, 0.1 ml/mouse.
(3) Toxin counteracting: the results of challenge control and immunization of each group of mice with 1000MLD (6000 CFU) of CVCC43006 and CVCC43008 strain virulent bacteria are shown in table 2. The 1000MLD control group is 5/5 dead, the 1MLD control group is 4/5 dead, the swine erysipelas recombinant protein rER-SpaA has good immunogenicity, can effectively protect mice from being attacked by swine erysipelas bacillus virulence, and has a minimum immune dose of 2.0 mug/mouse, thus achieving complete protection.
TABLE 1 investigation of mouse immunogenicity and minimum immune dose by rER-SpaA protein
2. Inspection with pigs
(1) Preparing immunogen: purified erysipelas recombinant protein rER-SpaA was mixed in equal volumes using 40% saline, after which the vaccine was diluted to 20 μg/ml using 40% saline following the protocol (see table 2 for details).
(2) Immunization: the pig is tested by using healthy and susceptible pigs of 56-63 days old, and the subcutaneous immunity of the pig is realized by rER-SpaA protein, 5 ml/pig.
(3) Toxin counteracting: control and immunized pigs were challenged with 1MLD (30 CFU) swine erysipelas ER24 strain virulent bacteria solution and the results are shown in table 3. Control pigs 5/5 died and immunized pigs 4/5 protected. It can be seen that the swine erysipelas recombinant protein rER-SpaA has good immunogenicity, and can effectively protect swine from being attacked by swine erysipelas bacillus virulence.
TABLE 2 investigation of pig immunogenicity by rER-SpaA proteins
EXAMPLE 5 application of rER-SpaA protein to preparation of Combined vaccine for live vaccine against porcine Pasteurella multocida (type A, CA Strain)
Mixed immunized mice with rER-SpaA protein and live vaccine against porcine Pasteurella multocida (A, CA strain)
Purified recombinant protein rER-SpaA qualified by inspection is diluted to 20 mug/ml by using 20% aluminum hydroxide gelatin physiological saline, and live vaccine of swine pasteurellosis (A type, CA strain) is diluted to 1/10 head/ml by using 20% aluminum hydroxide gelatin physiological saline. The two components are uniformly mixed in equal quantity of 1:1. 55 mice weighing 18-22 g were used, 30 of which were divided into 3 groups of 10 and 25 of which were not vaccinated as controls. Each of groups 1, 2 and 3 was subcutaneously injected with 0.2 ml/dose of rER-SpaA protein-CA vaccine mixture (containing 2. Mu.g of rER-SpaA protein, 1/100 parts of CA vaccine). After 21 days of inoculation, challenge with erysipelothrix rhusiopathiae strain 1 type C43-8 (CVCC 43008), strain 2 type C43-6 (CVCC 43006) and strain a pasteurella multocida P71: group 1, 10 immunized mice and 5 control mice were injected subcutaneously with 1000MLD CVCC43008 strain of challenge bacteria solution respectively, and the other 5 control mice were injected subcutaneously with 1mld cvcc43008 strain of challenge bacteria solution respectively; group 2, 10 immunized mice and 5 control mice were injected subcutaneously with 1000MLD CVCC43006 strain of challenge bacteria solution respectively, and the other 5 control mice were injected subcutaneously with 1mld cvcc43006 strain of challenge bacteria solution respectively; group 3, 10 immunized mice and 5 control mice were injected subcutaneously with 1MLD P71 strain of challenge bacteria solution, and the mice were observed for 10 days and recorded for death, as shown in Table 4. The result shows that the swine erysipelas recombinant protein rER-SpaA has antigen compatibility with the A type Pasteurella multocida, provides immune protection for mice and does not influence the immune effect of the A type Pasteurella multocida.
TABLE 3 evaluation of Mixed immunoprotection Effect of swine erysipelas rER-SpaA protein and A-type Pasteurella multocida
Application of rER-SpaA protein and live vaccine (type B, EO630 strain) for swine pasteurellosis mixed immunization of mice
Purified recombinant protein rER-SpaA qualified by inspection is diluted to 20 mug/ml by using 20% aluminium hydroxide gelatin physiological saline, and live vaccine of swine pasteurellosis (type B, EO630 strain) is diluted to 1/3 head/ml by using 20% aluminium hydroxide gelatin physiological saline. The two components are uniformly mixed in equal quantity of 1:1. 60 mice weighing 16-18 g were used, 30 of which were divided into 3 groups of 10 animals each, and another 30 were not vaccinated as controls. Each of groups 1, 2 and 3 was subcutaneously injected with 0.2 ml/dose of the rER-SpaA protein-EO 630 vaccine mixture (containing 2. Mu.g of rER-SpaA protein, 1/30 parts of EO630 vaccine). After 14 days of inoculation, challenge with Pasteurella multocida C44-8 strain B (CVCC 44408): group 1, 10 immunized mice and 5 control mice were respectively subcutaneously injected with 2MLD CVCC44408 strain of challenge bacteria solution, and the other 5 control mice were respectively subcutaneously injected with 1MLD CVCC44408 strain of challenge bacteria solution; after 21 days of inoculation, challenge with erysipelothrix rhusiopathiae strain 1 type C43-8 (CVCC 43008) and strain 2 type C43-6 (CVCC 43006): group 2, 10 immunized mice and 5 control mice were injected subcutaneously with 1000MLD CVCC43008 strain of challenge bacteria solution respectively, and the other 5 control mice were injected subcutaneously with 1mld cvcc43008 strain of challenge bacteria solution respectively; group 3, 10 immunized mice and 5 control mice were injected subcutaneously with 1000MLD CVCC43006 strain of challenge bacteria solution respectively, and the other 5 control mice were injected subcutaneously with 1MLD CVCC43006 strain of challenge bacteria solution respectively, and the death of the mice was recorded after 10 days of observation, as shown in Table 4. The result shows that the swine erysipelas recombinant protein rER-SpaA has antigen compatibility with the B type Pasteurella multocida, provides immune protection for mice and does not influence the immune effect of the B type Pasteurella multocida.
TABLE 4 evaluation of Mixed immunoprotection Effect of swine erysipelas rER-SpaA protein and B-type swine Pasteurella multocida
At present, in the prevention and control of swine erysipelas in China, the pain point is lack of antigens with low biological safety risk, strong safety and stable immunogenicity, and the swine erysipelas surface antigen recombinant protein rER-SpaA has good safety, immunogenicity and cross-serotype protection effects, can generate antibodies with enough immune protection, and has good application value for the construction of swine erysipelas related antigens and antibody diagnostic methods. Meanwhile, the protein has good immune compatibility when being mixed with other antigens, and can provide sufficient dosage space for other components.
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Claims (10)
1. The recombinant erysipelothrix rhusiopathiae surface antigen SpaA protein is characterized in that the amino acid sequence of the SpaA protein is shown as SEQ No. 1.
2. The recombinant erysipelothrix rhusiopathiae surface antigen SpaA protein of claim 1, wherein: the SpaA protein contains a tag that facilitates purification of the protein, and the C-terminus contains a 6 histidine tag.
3. The recombinant erysipelothrix rhusiopathiae surface antigen SpaA protein is characterized in that: the nucleotide sequence of the encoding gene A of the SpaA protein is shown as SEQ No. 2.
4. A recombinant erysipelothrix rhusiopathiae surface antigen SpaA protein plasmid comprising the coding gene a of claim 3, wherein: constructed by ligating the coding gene A between BamHI and HindIII in the cloning/expression region of plasmid vector pET28 a.
5. The expression strain of the recombinant erysipelas bacillus surface antigen SpaA protein is escherichia coli BL/S3 strain, and the preservation number is CGMCC No.25902.
6. The recombinant erysipelothrix rhusiopathiae surface antigen SpaA protein-expressing strain of claim 5, wherein: when the escherichia coli BL/S3 strain is used for expressing the recombinant erysipelas suis surface antigen SpaA protein, the escherichia coli BL/S3 strain is inoculated in an LB liquid culture medium of kanamycin and is subjected to shake culture until OD 600 When the concentration is 0.6-0.8, IPTG with the final concentration of 0.5M is added, and the induction expression is carried out at 25 ℃ for 6 hours.
7. The use of a recombinant erysipelothrix rhusiopathiae surface antigen SpaA protein according to claim 1 or 2 for preparing a vaccine for preventing erysipelothrix rhusiopathiae and evaluating the erysipelothrix rhusiopathiae vaccine.
8. The recombinant erysipelothrix rhusiopathiae surface antigen SpaA protein of claim 1 or 2, which is used for preparing a diluent and combining with a live vaccine, an inactivated vaccine, an effective protective antigen and the like.
9. Use of a recombinant erysipelothrix rhusiopathiae surface antigen SpaA protein according to claim 1 or 2 for preparing a diluent for use in combination with a live vaccine of type a or type B pasteurellosis.
10. The use of the recombinant erysipelothrix rhusiopathiae surface antigen SpaA protein of claim 9, wherein: the recombinant erysipelothrix rhusiopathiae surface antigen SpaA protein is added into aluminum hydroxide gel saline as a diluent.
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| 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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