CN116790456B - Bovine origin A type Pasteurella multocida strain, vaccine and application - Google Patents
Bovine origin A type Pasteurella multocida strain, vaccine and application Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/285—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Pasteurellaceae (F), e.g. Haemophilus influenza
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/02—Bacterial antigens
- A61K39/102—Pasteurellales, e.g. Actinobacillus, Pasteurella; Haemophilus
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
- A61K2039/552—Veterinary vaccine
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Abstract
The invention discloses a bovine origin A type Pasteurella multocida strain, a vaccine and application, wherein the strain is a bovine origin A type Pasteurella multocida cra gene deletion strain (delta cra), which is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of M20221451, and the strain is further prepared into an inactivated vaccine for immunization, so that the cross immune protection of mice and poultry against infection of different serotypes and different animal origin Pasteurella multocida can be promoted. The invention can promote the cross immune protection of animals against infection of different serotypes and different animal sources of Pasteurella multocida, has 100 percent of immune protection against infection of bovine-derived type A, B-derived type and swine-derived type A Pm in a mouse model, and has 70 percent, 90 percent and 80 percent of immune protection rate against bovine-derived type F, avian-derived type A and rabbit-derived type A Pm respectively; the immune protection rate of the poultry model against avian source A type Pasteurella multocida infection reaches 100 percent.
Description
Technical Field
The invention relates to the technical field of genetic engineering, in particular to a bovine origin A type Pasteurella multocida strain with cra gene deletion, a vaccine and application.
Background
Pasteurella multocida (Pasteurella multocida, pm) is a zoonotic pathogen that colonizes the upper respiratory tract of healthy animals as a symbiotic bacteria. Under various stress environmental conditions (such as long-distance transportation, environmental change, immunity reduction and the like), pm is greatly proliferated and spread to the lower respiratory tract to enter the lung or invade other parts through blood circulation, so that diseases such as pneumonia, hemorrhagic septicemia and the like are caused, animals die seriously, pm infects people, and symptoms such as shock, meningitis and the like can be caused.
Pm can be classified into A, B, D, E, F capsular serotypes according to differences in capsular antigens, and 1-16 LPS serotypes according to differences in Lipopolysaccharide (LPS) antigen components. The common vaccine may only have protection effect on the infection of strains of the same animal source or the same serotype, and the cross protection among the different serotypes of the pasteurellosis bacillus is weak, while the existing commercial vaccine only has protection effect on the infection of homotypic Pm, which brings great challenges to the prevention and control of the pasteurellosis bacillus. Thus, the development of a vaccine against pasteurellosis is particularly important.
The early-stage laboratory researches find that the deletion of quorum sensing regulatory genes qseC and luxS and a gene hyaD related to capsule synthesis endows the strain with cross protection property, and the cra is taken as a transcription regulatory gene for regulating and controlling carbon nutrition metabolism, and the influence of the cra on the aspects of carbon metabolism process, virulence effect, immune protection and the like of the Pasteurella multocida is unknown.
Disclosure of Invention
In view of the above-mentioned shortcomings of the prior art, it is an object of the present invention to provide a strain of pasteurella multocida with a deletion of the cra gene and further use it for immunization, promoting cross protection of animals against infection with pasteurella multocida of different serotypes and different animal origin.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
A bovine origin A type Pasteurella multocida strain is a cra gene deletion strain of bovine origin A type Pasteurella multocida PmCQ strain, which is preserved in China Center for Type Culture Collection (CCTCC) NO: M20221451.
Further, in the cra gene deletion strain, the amino acid sequence of the cra gene deletion part is SEQ ID NO.1.
Further, the bovine-derived pasteurella multocida strain has a cross-immune protection function and has the ability to cross-species infect poultry.
The invention also provides a Pasteurella multocida vaccine which is prepared by adopting the bovine origin A-type Pasteurella multocida strain.
Further, there is provided the use of the pasteurella vaccine for vaccination to promote cross-immune protection of animals against infection with pasteurella multocida of different serotypes, different animal sources.
Further, the cross protection is cross immune protection against bovine-derived type a, type B, type F pasteurella multocida infection.
Further, the cross-protection is cross-immune protection against swine and rabbit origin pasteurella multocida infection.
Further, the cross-protection is cross-immune protection against avian pasteurella multocida of type a, superior to avian pasteurella multocida vaccines.
Compared with the prior art, the invention has the following beneficial effects:
1. The invention discovers that the bovine-derived A-type Pasteurella multocida with the cra gene deleted has excellent cross protection characteristics, can be used as candidate strains for the development of Pm broad-spectrum vaccines, and lays a foundation for the commercialized application of the general vaccine of the Pasteurella multocida; the vaccine prepared from the extract has wide application prospect.
2. The deletion of the gene causes the change of host tropism of the bovine strain, and when the gene is not deleted, the strain cannot infect birds, but the deletion of the gene can infect birds, thereby providing a direction for researching cross-species infection of the pasteurella multocida.
3. From the perspective of vaccine, after cra gene of bovine strain is deleted, the invention has good cross protection effect on fowl-origin, pig-origin, rabbit-origin and bovine-origin pasteurella multocida in a mouse model; particularly in a model of poultry, the vaccine prepared by the strain also has good cross protection for the infection of the poultry-derived pasteurella multocida, and is better than the vaccine prepared by the poultry-derived pasteurella multocida.
4. The cattle source A type Pasteurella multocida vaccine with the cra gene deletion can promote the cross protection of animals on infection of different serotypes and different animal sources Pasteurella multocida, including the cross protection of cattle source A type, B type, F type and pig source, poultry source and rabbit source A type Pasteurella multocida infection, wherein the delta cra inactivated vaccine has 100 percent of immune protection on cattle source A type, B type and pig source A type Pm, and the protection rate on cattle source F type, poultry source A type and rabbit source A type Pm respectively reaches 70 percent, 90 percent and 80 percent.
Drawings
FIG. 1 is a schematic representation of the construction of a knockout strain and the associated primer binding sites of the present invention;
FIG. 2 is a graph showing survival of the detoxified mice after immunization with the inactivated vaccine of the present invention; wherein A. Attack toxin PmCQ1; B. toxin counteracting PmCQ; c, counteracting toxic substances PmCQ4; D. toxin counteracting PmCQ; e.pmb; F. counteracting toxic substances PmF; G. counteracting toxic substances Pmp; H. attack toxin PmQ; I. attack toxin PmR;
FIG. 3 is a view showing organ colonization according to the present invention; wherein, a, b, c, d, e, f, g, h, pmQ, i, pmR are both attack PmCQ, pmCQ, pmCQ, pmCQ;
FIG. 4 is a graph showing HE staining of lung tissue infection in immunized mice according to the invention;
FIG. 5 is a plot of the change in serum IgG antibody levels of delta cra inactivated vaccine immunized mice according to the invention; wherein, A. Anti PmCQ antibody dynamic change, B. Anti PmCQ2 antibody dynamic change, C. Anti PmCQ antibody dynamic change, D. Anti PmCQ antibody dynamic change, E. Anti PmB antibody dynamic change, F. Anti PmF antibody dynamic change, G. Anti Pmp antibody dynamic change, H. Anti PmQ antibody dynamic change, I. Anti PmR antibody dynamic change;
FIG. 6 is a graph showing the survival of the virus-combating avian Pasteurella multocida after the immunization of chickens with an inactivated vaccine according to the invention.
Detailed Description
In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described with reference to specific examples, but the embodiments of the present invention are not limited thereto.
1. Construction method of cattle-derived Pasteurella multocida PmCQ strain cra gene knockout strain delta cra and vaccine preparation method
EXAMPLE 1 construction of Deltacra by homologous recombination
1. Material
1.1 Strain
Bovine origin A type Pasteurella multocida PmCQ (A: L3) is isolated from the lungs of calf suffering from pneumonia in a certain cattle farm of Chongqing, and is temporarily stored in a refrigerator at-80 ℃ in a laboratory for controlling and controlling epidemic disease of beef cattle in the university of West south animal medical college.
Plasmid pUC19oriKan R used as a gene knockout was constructed and stored in the laboratory, and protein expression vector pET30a was temporarily stored in the laboratory. E.coli DH 5. Alpha. And E.coli BL21 (DE 3) competent cells were purchased from Bomaide Biotechnology Co.
1.2 Primers
Relevant primers (table 1) were designed for the construction of the PmCQ strain cra knockout strain and the construction of the anaplerotic strain using NCBI gene library, snapGene and BioXM software, and synthesized and purified by Shanghai biological engineering limited.
TABLE 1 related primers required for construction of knockout strains
2. Method of
2.1 Construction of the knockout plasmid vector pUC19oriKanR
The transformation of the homologous recombination knockout plasmid pUC19oriKan R is mainly completed by a laboratory, and a Kan expression nucleus, a temperature sensitive plasmid and a Pasteurella replication initiation site are inserted between two restriction sites of the original pUC19 plasmid KpnI and EcoRI.
2.2 Construction of recombinant plasmid pUC19 oriKanR-Delta craup +Down
2.2.1 Amplification, purification and ligation of the upstream and downstream homology arms of the cra Gene
(1) Amplification and purification of the upstream and downstream homology arms: primers 5'ARM-F/5' ARM-R and 3'ARM-F/3' ARM-R are used for amplifying an upstream homology arm and a downstream homology arm of the cra gene respectively, and a glue recovery kit (manufactured by Kangji Biotechnology Co., ltd.) is used for recovering the upstream homology arm and the downstream homology arm respectively.
(2) Upstream and downstream homology arm ligation: the purified upstream and downstream homology arms of cra were ligated and the upstream and downstream homology arms of cra Δcra up+down were recovered, and stored at-20℃after determining the DNA concentration.
2.2.2 Acquisition of recombinant plasmid pUC19oriKan R–Δcraup+down
(1) Extraction of pUC19oriKan R homologous recombinant plasmid: plasmids were extracted according to the instructions of the plasmid purification kit (Kangji Biotechnology Co., ltd.) and the concentration was measured by an enzyme-labeled instrument and stored at-20 ℃.
(2) Double-digested pUC19oriKan R plasmid: cleavage sites BamHI and HindIII; and (3) enzyme cutting: the enzyme is digested with BamHI at 30℃for 2h and then with HindIII at 37℃for 2h.
(3) Purification of the linear vector: the linearized vector after enzyme digestion is recovered according to the specification of a gel recovery kit (manufactured by Kangji Biotechnology Co., ltd.) and is stored at-20 ℃ after measuring the DNA concentration by an enzyme-labeled instrument.
(4) And (3) connection: the purified upstream and downstream homology arm Δcra up+down fragment of cra was ligated with the digested pUC19oriKan R linearized vector using Exnase TM II ligase. The reaction procedure: the tube wall reagent was centrifuged briefly and incubated at 37℃for 30min ℃and cooled in an ice-water mixture for storage at-20 ℃.
(5) Conversion of ligation products: the ligation product was transformed into E.coli DH 5. Alpha. Competent cells, and after 10min of resuscitation in a 30℃incubator, the cells were cultured for 2h on a shaking table at 30℃and 220r/min, and the bacterial solution was spread on LB plates containing 100. Mu.g/mL Amp, and cultured for 16h at 30 ℃.
(6) Screening of positive clones: several single clones on the plate were picked with a pipette, colony PCR amplification verification was performed, and positive clones were screened.
(7) Sequencing: the positive clones are picked, amplified and cultured, plasmids are extracted and sent to a worker for sequencing, and the universal primers M13-48 are adopted for sequencing.
2.3 PmCQ2 preparation of competent cells
(1) Streaking and activating the frozen PmCQ bacterial liquid on a Ma Dingshi broth solid culture medium, and culturing in an inversion way for 20 hours in a 37 ℃ incubator;
(2) 2-3 single colonies are selected and cultured in 5mL Ma Dingshi broth culture medium at 37 ℃ in a 220r/min culture box for 10 hours, transferred into a triangular flask containing 100mL Ma Dingshi broth culture medium according to 1% inoculum size, cultured again in 37 ℃ in a 220r/min culture box until OD 600 =0.2-0.4, added with 0.1% hyaluronidase, and continuously cultured until OD 600 =1.0-1.2, and subjected to ice bath for 30 minutes;
(3) Transferring the bacterial liquid after ice bath into a 50mL centrifuge tube, centrifuging at 5,000r/min and 5min and 4 ℃ and collecting bacterial bodies;
(4) Adding 30mL of 10% pre-cooled glycerol to resuspend the thalli, centrifuging at 5,000r/min and 5min and 4 ℃ to keep the thalli;
(5) Repeating the step (4) twice;
(6) Finally, 2mL of 10% pre-cooling glycerol is added to resuspend the thalli, 100 mu L/tube split charging of the thalli liquid is carried out, and the thalli are preserved at the temperature of minus 80 ℃ for standby after quick freezing by liquid nitrogen.
2.4 Electric transformation PmCQ2 of recombinant plasmid pUC19oriKan R–Δcraup+down
(1) Thawing the competent cells of recombinant plasmids pUC19oriKan R–Δcraup+down and PmCQ in an ice-water mixture;
(2) Transferring 10 mu L of recombinant plasmid into PmCQ competent cells, and uniformly mixing the light bullet wall;
(3) Transferring the mixed solution into a precooled electric shock cup, and carrying out electric shock for 5ms conversion under the conditions of voltage 2,500V and resistance 500 omega;
(4) Transferring the mixed solution to 900 mu L Ma Dingshi broth containing 10% fetal bovine serum after electric shock is over, standing at 30deg.C for 10min, recovering at 220r/min, and shake culturing at 30deg.C for 3 hr;
(5) After centrifugation of the mixture at 5,000r/min for 5min, 100. Mu.L of the supernatant was retained for resuspension of the cells, which were then plated on Martin plates containing 50. Mu.g/mL Kan resistance and incubated at 30 ℃.
The construction of the knockout strain and the associated primer binding sites are shown in FIG. 1.
2.5 Screening of Gene-deleted Strain Δcra
(1) Picking single colony growing on the 2.4 plate, and carrying out PCR amplification by using pUC19-F/pUC19-R primer to ensure that the recombinant plasmid is transferred into PmCQ competent cells;
(2) After verifying that the recombinant plasmid is successfully transferred into PmCQ < 2> competent cells, respectively streaking bacterial solutions on Ma Dingshi broth solid medium containing 50 mug/mL Kan resistance, and culturing at 30 ℃ for 24 hours to enable the recombinant plasmid insert to exchange with PmCQ genome;
(3) Selecting single colony growing on the culture medium (2) and carrying out PCR amplification by using mcra-F/mcra-R primer, and screening single colony with short band compared with wild type PmCQ genome (namely the target gene cra and delta cra up+down are exchanged);
(5) The single colony with the genome exchanged through PCR amplification verification is picked and diluted in normal saline, the diluted colony is coated in a martin-free culture medium, and after the culture is carried out for 24 hours at 30 ℃, the PCR amplification is carried out again by using mcra-F/mcra-R primers until the condition that the screened nucleic acid gel electrophoresis shows only short bands compared with PmCQ (namely, the delta cra up+down is completely exchanged with cra);
(6) Elimination of resistance: by utilizing the characteristics of a temperature-sensitive knockout vector (no replication at 42 ℃), single colonies with complete genome exchange are diluted by normal saline and then spread on Ma Dingshi broth agar culture medium without Kan resistance, cultured for 24h at 42 ℃, PCR amplification is carried out by pUC19-F/pUC19-R until single colonies with the pair of primers not amplified out a strip (namely plasmid loss) are screened out, the colonies are picked and streaked on Ma Dingping plates with Kan resistance, and sterile colony growth is carried out after culturing for 24 hours at 30 ℃, so that the elimination of Kan resistance is confirmed.
In the cra gene deletion strain, the amino acid sequence of the cra gene deletion part is SEQ ID NO.1.
The cra gene deletion strain of the bovine-derived A-type Pasteurella multocida PmCQ strain is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of M20221451.
2.6 Verification of genetic stability of Gene-deleted Strain Δcra
The knocked-out strain is continuously passaged for 30 times in vitro, then is subjected to PCR amplification verification by using a plurality of pairs of primers, then bacterial liquid is subjected to intraperitoneal toxicity attack to infect mice, lung homogenate is collected, streaks are carried out on Ma Dingshi broth culture medium, a plurality of single colonies are picked, and PCR amplification is performed by using a plurality of pairs of primers, so that the stable inheritance of delta cra in vivo and in vitro is ensured.
Example 2 preparation of vaccine against Cra Gene-deleted Strain of bovine Pasteurella multocida PmCQ Strain
1. Raw materials
1.1 Strain
Pasteurella multocida of bovine origin A PmCQ, pasteurella multocida of bovine origin A PmCQ (A: L3), pmCQ (A: L3), pmCQ (A: L3) were isolated from calf nasal swabs from a farm of Chongqing, pasteurella multocida of bovine origin B PmB (B: L2, CVCC 470) and Pasteurella multocida of porcine origin A Pmp (A: L1, CVCC 1662) were purchased from China veterinary microbiological strain collection center, pasteurella multocida of bovine origin F PmF (F: L3) was isolated from lung of calf having pneumonia from a farm of Mianyang, pasteurella multocida of avian origin A PmQ (A: L1) was isolated from death duck from a farm of Chongqing, and Pasteurella multocida of rabbit A PmR was isolated from rabbit liver of Chongqing. All the strains are temporarily stored in a refrigerator at-80 ℃ in a beef cattle epidemic prevention and control research laboratory of the university of southwest animal medical college.
1.2 Animals
Female Kunming mice (18-22 g), chicks (ROSS 308,7 days old).
2. Preparation of inactivated vaccine
(1) The PmCQ of the culture to logarithmic phase and the Deltacra seed solution screened in example 1 were transferred to a triangular flask at a ratio of 1:100, and the culture was expanded, and after pure examination and dilution counting, the bacterial solution was concentrated by centrifugation at 13,000r/min for 20 min.
(2) Re-suspending the thalli with a proper amount of culture solution, adding formaldehyde solution with the final concentration of 0.15 percent into an incubator at 37 ℃ for inactivation for 24 hours, and carrying out vortex oscillation once every 4-6 hours;
(3) The inactivated bacterial liquid and PBS are fully mixed with 15AVG mineral oil adjuvant according to the proportion of 4:1 respectively to prepare PBS emulsifier, 5.0X10 9 CFU/mL of deltacra inactivated vaccine and PmCQ inactivated vaccine.
(4) And culturing the inactivated Miao Tubu on a Ma Dingshi broth culture medium at 37 ℃ for 24 hours, detecting whether viable bacteria exist, simultaneously respectively inoculating the PmCQ inactivated vaccine, the deltacra inactivated vaccine and the PBS emulsifier under the back skin of 6 mice, evaluating the safety of the vaccine, and storing at 4 ℃ after the safety detection of the vaccine.
2. Determination and evaluation of cross-protection of cra Gene-deleted Strain Δcra
One mouse model
1. Cross-protective assay of Δcra inactivated vaccine in mice
(1) Immunization procedure: 270 female Kunming mice were randomly divided into 27 groups (n=10), the back of the mice was subcutaneously multi-vaccinated, while PBS emulsifier was injected as a control, boosted once after 14d prime, and the immunization procedure and challenge strains were as shown in Table 2:
TABLE 2 immunization procedure and detoxification
(2) Challenge of counteracting toxic substances: and after 21d of first time, the muscle toxicity attack detection and inactivation vaccine cross protection effect. The challenge amounts were 3.8×107CFU PmCQ1(LD50＝3.8×102CFU)、4.8×107CFU PmCQ2(LD50＝3.4×103CFU)、4.2×107CFU PmCQ4(LD50＝2.1×103CFU)、4.5×107CFU PmCQ5(LD50＝4.5×103CFU)、1.0×107CFU PmB (LD50＝5.0×103CFU)、6.5×108CFU PmF(LD50＝1.0×108CFU)、10CFU PmQ(LD50≈1CFU)、 10CFU PmP(LD50≈1CFU)、2.0×106CFU PmR(LD50＝1.0×104CFU), challenge times per day, 2 observations were made each day, the clinical symptoms of mice were recorded, euthanasia was performed when dying, the number of mice dying was recorded, and the recordings were continued for 1 week.
2. Determination of toxicity counteracting, lung colonisation and pathological injury after immunization
162 Female Kunming mice were randomly divided into 27 groups (n=6), 9 groups of Δcra inactivated vaccine, pmCQ inactivated vaccine and control group were each used to subcutaneously immunize the back of the mice at multiple points, and the mice were challenged 21d later. After 12h of infection, the mice are euthanized, the lungs, livers and spleens of the mice are collected, weighed, homogenized, diluted and plated, and the bacterial content of each organ is calculated; lungs of three mice in each group were randomly collected and fixed with 4% paraformaldehyde, and three groups (Δcra inactivated vaccine, pmCQ inactivated vaccine and control group) of uninfected groups (n=3) were simultaneously set, lungs were collected at the same time point, and the mice were sent to the inside organism company after being fixed with 4% paraformaldehyde to prepare pathological sections.
(II) chick model
1. Cross-protective assay of Δcra inactivated vaccine in chickens
(1) Immunization procedure: the 40 chicks (ROSS 308,7 days old) were randomly divided into 4 groups (n=10), the chick pectoral muscles were vaccinated with Δcra, pmQ and PmCQ inactivated vaccine, respectively, and PBS emulsifier was injected as a control, and were boosted once after 14d of priming, and the immunization procedure was as in table 2.
(2) Challenge of counteracting toxic substances: and detecting the cross protection effect of the inactivated vaccine by using 100CFU PmQ of chest muscle detoxification after 21 days of first immunization. After challenge, the chickens were observed 2 times daily, and the clinical symptoms and death time of the chickens were recorded for 1 week.
(III) analysis of results
1. Safety inspection of inactivated vaccine
The sterilization test is carried out after the inactivation of delta cra and PmCQ formaldehyde and the emulsification of the adjuvant, and the result shows that the bacteria liquid after the treatment is coated on the MD flat plate and no mixed bacteria grow out, thus proving that the sterilization test of the inactivated vaccine is qualified; after the mice are respectively inoculated with the inactivated vaccine subcutaneously, no obvious tumor is generated, the mental state of the mice is good, and the vaccine safety is proved to be good.
2. Results of cross protection test of inactivated vaccine in mice
To evaluate the cross-protective properties of the Δcra strain, the Δcra-inactivated vaccine had 100% immune protection rate against bovine-derived type a (CQ 1, CQ2, CQ4, CQ 5) (fig. 2A-D), bovine-derived type B (fig. 2E) and porcine-derived type a Pm (fig. 2G) and 70%, 90%, 80% protection rate against bovine-derived type F, avian-derived type a and rabbit-derived type a Pm (fig. 2F, h, i), respectively, after immunization of 21D against muscle challenge PmCQ, pmCQ2, pmCQ, pmCQ5, pmB, pmF, pmP, pmQ, pmR against Kunming mice, as shown in fig. 2, following one week of observation; whereas PmCQ inactivated vaccine has more than 70% immune protection rate to bovine type A Pm (FIG. 2A-D), and almost no cross protection to bovine type B, F and other animal sources Pm (FIG. 2E-I). The results show that the Deltacra strain has good cross immune protection characteristics.
3. After the inactivated vaccine is immunized, the organ colonization result of the infected mice
To evaluate the resistance of Δcra-and PmCQ-inactivated vaccine-immunized mice to pathogenic bacterial infection, pmCQ, pmCQ2, pmCQ, pmCQ, pmB, pmF, pmP, pmQ, pmR were infected respectively after 21d immunization of the inactivated vaccine, lung, liver, spleen organs were collected from mice 12h post infection, and diluted plating counts were performed after tissue homogenization. The results show that the mice immunized by the delta cra inactivated vaccine infected by each strain have almost no colonization of various organs (figures 3A-I) and can rapidly clear bacteria in the body; however, pmCQ inactivated mice had a number of colonization of each organ after infection, wherein the colonization of bovine-derived type a pasteurella multocida (PmCQ, pmCQ, pmCQ, pmCQ 5) was significantly lower in each organ than in the control group (fig. 3A-D), indicating that PmCQ inactivated mice had a certain clearance of bovine-derived type a pasteurella multocida infection. The above results indicate that mice immunized with Δcra inactivated vaccine have very strong resistance to infection with other strains, whereas PmCQ mice immunized with the inactivated vaccine have a certain clearance ability only against infection with bovine-derived pasteurella multocida a, which also verifies the cross-protection properties exhibited in fig. 3.
4. Results of lung pathological injury of infected mice after immunization with inactivated vaccine
To further evaluate the anti-pathogenic infection ability of Δcra-and PmCQ-inactivated mice, mice were immunized 21d later with the inactivated vaccine, pmCQ, pmCQ2, pmCQ, pmCQ, pmB, pmF, pmP, pmQ, pmR, respectively, lungs of mice 12H after the collection were fixed with 4% paraformaldehyde, and H & E stained. The results show that the lung pathological lesions are lighter after the Δcra inactivated vaccine immunized mice are infected, almost no change of cell structure or inflammatory cell infiltration is caused, but PmCQ mice in the immunized group and mice in the control group show obvious pathological lesions (figure 4).
5. Serum antibody level change results of delta cra inactivated vaccine immunized mice
After the mice are immunized for the first time, blood of tail veins of the mice in a delta cra immune group, a negative control group and a blank control group are collected every 7d, igG antibody levels in serum are measured, the serum is continuously detected for 119d, the dynamic change results of the antibodies are shown in figure 5, the IgG antibody levels in bodies of the mice immunized by delta cra inactivated vaccine reach the highest basically after 21d, the mice can be maintained for about 9 weeks, and the serum IgG antibodies of the mice in the blank control group are zero all the time.
6. Cross protection test of inactivated vaccine in chicken
In order to evaluate the cross protection property of the delta cra strain in the poultry body, pmQ muscle is selected for virus attack infection after the delta cra inactivated vaccine is immunized for 21d, and the survival condition is continuously observed for one week, as shown in figure 6, the delta cra inactivated vaccine has the protection function on infection of avian origin type A Pasteurella multocida.
Finally, it should be noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the technical solution, and those skilled in the art should understand that modifications and equivalents may be made to the technical solution of the present invention without departing from the spirit and scope of the present invention, and all such modifications and equivalents are included in the scope of the claims.
Biological material sample preservation information
Biological material naming: pasteurella multocida PmCQ. DELTA.cra (Pasteurella multocida PmCQ. DELTA.cra)
Preservation unit name: china center for type culture Collection
Deposit unit address: university of Chinese Wuhan
Preservation date: 2022 9/20
Preservation number: CCTCC NO: M20221451.
Claims (6)
1. The bovine origin A type Pasteurella multocida strain is characterized in that the strain is a cra gene deletion strain of bovine origin A type Pasteurella multocida PmCQ strain, which is preserved in China center for type culture collection, and the preservation number is CCTCC NO: M20221451.
2. The bovine origin a-type pasteurella multocida strain according to claim 1, wherein the amino acid sequence of the cra gene deleted part in the cra gene deleted strain is SEQ ID No.1.
3. The bovine origin pasteurella multocida strain of claim 1 or 2, characterized in that it has a cross-immune protective function and has the ability to cross-species infect poultry.
4. A pasteurella multocida vaccine, characterized in that it is obtainable with a pasteurella multocida strain of bovine origin a according to claim 1 or 2.
5. Use of a strain of pasteurella multocida of bovine origin a according to claim 1 for the preparation of a vaccine for cross-immune protection against multiple infections with pasteurella multocida of animal origin;
the cross immune protection is the cross immune protection to bovine type A, B and F type Pasteurella multocida infection or swine and rabbit type A Pasteurella multocida infection.
6. Use of a strain of pasteurella multocida of bovine origin a according to claim 5, characterized in that the cross-immune protection is of pasteurella multocida of avian origin a.
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| CN1325731A (en) * | 2000-06-09 | 2001-12-12 | 阿克佐诺贝尔公司 | Living toxic-reduced bacteria as vaccine |
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| CN113801812A (en) * | 2021-09-02 | 2021-12-17 | 华中农业大学 | Pasteurella multocida and application thereof |
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| CN1325731A (en) * | 2000-06-09 | 2001-12-12 | 阿克佐诺贝尔公司 | Living toxic-reduced bacteria as vaccine |
| KR20080060859A (en) * | 2006-12-27 | 2008-07-02 | 전남대학교산학협력단 | Avirulent attenuated strain of pasteurella multocida and live vaccine containing the same |
| CN113801812A (en) * | 2021-09-02 | 2021-12-17 | 华中农业大学 | Pasteurella multocida and application thereof |
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