CN116059333A - Bovine origin A type Pasteurella multocida gene deletion strain and application thereof - Google Patents
Bovine origin A type Pasteurella multocida gene deletion strain and application thereof Download PDFInfo
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- CN116059333A CN116059333A CN202211216971.0A CN202211216971A CN116059333A CN 116059333 A CN116059333 A CN 116059333A CN 202211216971 A CN202211216971 A CN 202211216971A CN 116059333 A CN116059333 A CN 116059333A
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- 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
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Abstract
The invention provides a bovine origin A type pasteurella multocida gene deletion strain and application thereof, wherein the bovine origin A type pasteurella multocida gene deletion strain is used for immunization to promote cross protection of animals on infection of different serotypes and different animal sources by pasteurella multocida; the cross protection is to bovine type A, B type, F type, swine source, fowl source and rabbit source ACross protection of type pasteurella multocida infection. The gene deletion strain ishyaDDeletion of the gene; or is as followsqseCDeletion of genes. The gene deletion strain is a cattle source Pasteurella multocida CQ2 strain (PmCQ 2) constructed by a homologous recombination methodhyaDGene-deleted strain, pmCQ2ΔhyaDOr (b)qseCGene-deleted strain PmCQ2ΔqseC. The saidhyaDGene and geneqseCThe gene deletion can obviously reduce the toxicity of wild strains, and has stronger cross protection effect on different serotypes and different host sources of Pasteurella multocida. Provides a candidate target for the research and development of the pasteurella multocida general vaccine.
Description
The present invention is based on the application filed as priority under application number 202111216800.3. Specific statement: the present invention relates to the preservation of biological materials for which priority is not required.
Technical Field
The invention relates to the technical field of genetic engineering, in particular to bovine-derived A-type Pasteurella multocida hyaD and qseC gene deletion strains, application thereof and construction of the gene deletion strains.
Background
Pasteurella multocida is a gram-negative and two-stage concentrated bacillus brevis, can infect various livestock and wild animals and also can infect people, and brings great economic loss to animal husbandry every year. According to the differences of capsular polysaccharides, the capsular polysaccharides can be divided into A, B, D, E, F capsular serotypes, and according to the LPS differences, the capsular polysaccharides can be divided into 16 serotypes, and the types of diseases caused by the different serotypes of main infection of animals are also obviously different. The pasteurella multocida can be used as symbiotic bacteria for field planting in the nasopharynx part, the respiratory tract and other parts of animals, but under the conditions of transportation stress, body resistance reduction and the like, the pasteurella multocida is propagated in a large quantity and spread to organs such as lung, liver and spleen and the like to cause epidemic diseases such as bovine hemorrhagic septicemia, swine plague, fowl cholera, rabbit pasteurellosis and the like.
At present, the prevention and control of the Pasteurella multocida mainly adopts the combination of vaccine prevention and antibiotic treatment. However, there are only a few commercial vaccines against Pasteurella multocida currently available, and each has a better immunoprotection against only certain serotypes of Pasteurella multocida of a single animal origin, with little or no cross-immunoprotection. The existing pasteurella multocida vaccines can not achieve the aim of effectively preventing and controlling the pasteurella multocida infection from the aspect of serotypes of the pasteurella multocida and the infectious animals. In the antibiotic treatment process, the long-term and large-scale use of antibiotics also causes more and more drug-resistant strains of the pasteurella multocida to appear, which also brings difficulty to the effective treatment of the pasteurella multocida infection. Therefore, the development of broad-spectrum pasteurella multocida vaccines and drugs which are not easy to generate drug resistance are key to effectively preventing and controlling pasteurella multocida infection. At present, although different types of pasteurella multocida vaccines, such as inactivated vaccine, attenuated vaccine, subunit vaccine, gene recombinant vaccine, DNA vaccine and the like are studied, the cross immunity protection effect is not ideal, and the universal cross protection vaccine with better effect is still lacking. The development of the related general vaccine has important significance in preventing the pasteurellosis, improving animal welfare and blocking zoonosis transmission.
Although the previous study by Homchampa et al found that aroA gene deletion enhanced the cross-immunoprotection of Pasteurella multocida of capsular type A, only aroA gene deletion strains were found to have some cross-protection against other LPS serotype strains of capsular type A, with a narrow cross-protection range; meanwhile, after the gene is deleted in different strains, the cross immunity protection of the strains is endowed with obvious difference.
Disclosure of Invention
Aiming at the defects existing in the prior art, the invention aims to provide a bovine-derived A-type Pasteurella multocida hyaD gene deletion strain, a qseC gene deletion strain and a construction method thereof.
Further, the application of the bovine-derived type A Pasteurella multocida gene deletion strain is also provided.
The invention adopts the following technical scheme:
a bovine origin type-a pasteurella multocida gene deletion strain, wherein the strain is a hyaD gene deletion strain of a PmCQ2 strain, the gene deletion strain pmcq2Δhyad is preserved in China center for type culture collection (CCTCC M2022067), and the preservation date is: 2022, 1 and 13.
A bovine origin type-a pasteurella multocida gene deletion strain, wherein the strain is a qseC gene deletion strain of a PmCQ2 strain, the gene deletion strain pmcq2Δqsec is preserved in China center for type culture collection (CCTCC M2022066), and the preservation number is CCTCC M2022066, and the preservation date is as follows: 2022, 1 and 13.
Further, the nucleotide sequence of the hyaD gene deletion part is SEQ ID NO.1, and the deletion of the fragment leads to inactivation of the hyaD gene function.
Further, the nucleotide sequence of the qseC gene deletion part is SEQ ID NO.2, and the fragment deletion leads to inactivation of the qseC gene function.
Further, the gene deletion strain PmCQ2ΔhyaD is constructed by a homologous recombination method, and comprises the following steps:
(1) Extracting the genomic DNA of a wild strain PmCQ2 by using a kit (DP 302-02, purchased from Tiangen biotechnology Co., ltd.) and respectively carrying out PCR amplification by using the PmCQ2 genomic DNA as a template and adopting upstream and downstream homology arm amplification primers 5'hyaDARM-F/R and 3' hyaDARM-F/R, and after nucleic acid electrophoresis of amplified products, cutting out target fragments, recovering the target fragments by using a glue recovery kit (CW 2302M, purchased from Kangji biotechnology Co., ltd.) to obtain purified 226bp upstream homology arm fragments and 251bp downstream homology arm fragments respectively;
(2) Using the upstream and downstream homology arm fragments as templates, adopting 5'hyaDARM-F and 3' hyaDARM-R primers to continue PCR amplification, and obtaining purified upstream and downstream homology arm fusion fragments after nucleic acid electrophoresis and gel recovery;
(3) pUC19oriKan containing temperature sensitive plasmid R After activation, overnight culture, plasmid extraction using plasmid extraction kit (CW 0500M, available from Kagaku, century Biotechnology Co., ltd.) was used to extract plasmid pUC19oriKan R Through BamH I (1605, available from TaKaRa) and Hind III (16)15, purchased from TaKaRa) after double digestion at 37℃for 2 hours (hor, h), the linearized vector fragment was recovered with a gel recovery kit;
(4) By using
HD Cloning Kit (PT 5162-1, purchased from Clontech) the double digested linearized vector and homologous arm fragments were ligated in a 37℃thermostat water bath for 30 minutes (minute, min), the ligation products were transformed into E.coli DH 5. Alpha. Competent cells, spread on LB plates containing 50. Mu.g/mL Kan resistance, incubated overnight at 37℃constant temperature, colonies were picked, colony PCR was performed using primers pUC19-F/pUC19-R, positive clones were screened, and sequencing verification was performed;
(5) Preparing PmCQ2 electrotransformation competence, coating the recombinant plasmid on a broth medium containing 100 mug/mL Kan Ma Dingshi after electrotransformation of PmCQ2 competence, screening a hyaD gene deletion strain through PCR amplification, and ensuring Kan resistance elimination and genetic stability through continuous passage for 30 times.
Further, the gene deletion strain pmcq2Δqsec is constructed by a homologous recombination method, and includes the steps of:
(1) Extracting the genomic DNA of a wild strain PmCQ2 by using a kit (DP 302-02, purchased from Tiangen biotechnology Co., ltd.) and respectively carrying out PCR amplification by using the PmCQ2 genomic DNA as a template and adopting an upstream and downstream homology arm amplification primer 5'qseCARM-F/R and a 3' qseCARM-F/R, and after nucleic acid electrophoresis, cutting a target fragment, and recovering by using a glue recovery kit (CW 2302M, purchased from Shi biological technology Co., ltd.) to obtain a purified 350bp upstream homology arm fragment and a 350bp downstream homology arm fragment respectively;
(2) Using the upstream and downstream homology arm fragments as templates, adopting 5'qseCARM-F and 3' qseCARM-R primers to continue PCR amplification, and obtaining purified upstream and downstream homology arm fusion fragments after nucleic acid electrophoresis and gel recovery;
(3) pUC19oriKan containing temperature sensitive plasmid R After activation, overnight culture, plasmid extraction using plasmid extraction kit (CW 0500M, available from Kagaku, century Biotechnology Co., ltd.) was used to extract plasmid pUC19oriKan R Through BamH I (1605, available from TaKaRa) and Hind III (16)15, purchased from TaKaRa) after double digestion at 37℃for 2 hours (hor, h), the linearized vector fragment was recovered with a gel recovery kit;
(4) By using
HD Cloning Kit (PT 5162-1, purchased from Clontech) the double digested linearized vector and homologous arm fragments were ligated in a 37℃thermostat water bath for 30 minutes (minute, min), the ligation products were transformed into E.coli DH 5. Alpha. Competent cells, spread on LB plates containing 50. Mu.g/mL Kan resistance, incubated overnight at 37℃constant temperature, colonies were picked, colony PCR was performed using primers pUC19-F/pUC19-R, positive clones were screened, and sequencing verification was performed;
(5) Preparing PmCQ2 electrotransformation competence, coating recombinant plasmid on a broth culture medium containing 100 mug/mL Kan Ma Dingshi after electrotransformation of PmCQ2 competence, screening out qseC gene deletion strain PmCQ2 delta qseC by PCR amplification, and ensuring Kan resistance elimination and genetic stability by continuous passage for 30 times.
The invention also provides application of the bovine-derived type A Pasteurella multocida gene deletion strain, which is used for preparing vaccines for immunization and promoting cross immune protection of animals to infection of different serotypes and different animal-derived Pasteurella multocida.
Further, the cross protection is cross immune protection against bovine-derived type a, type B, type F pasteurella multocida infection. And the cross protection is cross immune protection against infection of swine, avian, rabbit, and pasteurella multocida.
Compared with the prior art, the invention has the following beneficial effects:
1. the gene-deleted strains PmCQ2Δhyad and PmCQ2Δqsec of the Pasteurella multocida constructed by the invention are all non-resistance marker strains, compared with wild strains, the toxicity of the PmCQ2Δhyad is reduced by nearly ten thousands times, the toxicity of the PmCQ2Δqsec is reduced by nearly ten thousands times, the colonization amount of non-lethal dose infection in animal organs is low, and pathological damage is slight, so that the biological safety risk is avoided for the clinical application of the strain.
2. The invention constructs the deletion strains PmCQ2Δhyad and PmCQ2Δqsec, has simple in vitro culture conditions, has no difference between the growth rate and the wild strain, obviously reduces the capsule production, and compared with the wild strain, the invention has the advantages that thalli are easier to centrifuge and precipitate, thereby providing more favorable conditions for thalli culture and concentration in the vaccine preparation process.
3. The invention researches the application of the bovine-derived type A multi-killing Pasteurella multocida gene deletion strain, and the immunization promotes the cross protection of animals on infection of different serotypes and different animal-derived multi-killing Pasteurella multocida based on the bovine-derived type A multi-killing Pasteurella multocida gene deletion strain; the cross protection is the cross protection of type A, type B and type F of bovine, type A and type A pasteurella multocida infection of pig, poultry and rabbit. The bovine-derived A type Pasteurella multocida CQ2 strain is selected, and the hyaD gene and the quorum sensing gene qseC gene which are related to capsular polysaccharide synthesis and are screened out are deleted by deleting a plurality of genes, so that the deletion strain can be endowed with stronger and broad-spectrum cross immunity protection characteristics, and a foundation is laid for commercialized application of the Pasteurella multocida universal vaccine.
4. According to the invention, the PmCQ2Δqsec attenuated vaccine and the formaldehyde inactivation mode are selected and mixed with the mineral oil adjuvant to prepare the PmCQ2ΔhyaD inactivated vaccine, and the two vaccines are found to have good cross immune protection effects on bovine type A, B type, F type, swine source, avian source and rabbit source type A Pasteurella multocida, especially for infection of bovine type A, B type, avian source and rabbit source type A Pasteurella multocida, the protection effect is 100%, the immunized mice generate higher antibody level, the highest antibody level is maintained for more than 8 weeks, and the lung of the mice is not obviously pathologically damaged after immune attack, so that the PmCQ2Δqsec attenuated vaccine PmCQ2ΔhyaD inactivated vaccine can be used as a good vaccine development candidate target.
Drawings
FIG. 1 shows the construction and biological properties of the hyaD gene-deleted strain and the hyaD gene-complemented strain. Schematic construction of the gene-deleted strain. (B) PCR amplification verifies the construction conditions of the knocked-out strain and the anaplerotic strain, wherein 1, 4, 7, 10 and 13 take PmCQ2 genome as a template, 2, 5, 8, 11 and 14 take PmCQ2 delta hyaD genome as a template, and 3, 6, 9, 12 and 15 take PmCQ2 delta hyaD/phyaD genome as a template; 1. the primers 2, 3 are KMT1-F/R, the primers 4, 5, 6 are 5'ARM-F/3' ARM-R, the primers 7, 8, 9 are dhiaD-F/R, the primers 10, 11, 12 are pUC19-F/pUC19-R, and the primers 13, 14, 15 are mhiaD-F/mhiaD-R. (C) And (3) quantitatively verifying the construction conditions of the knocked-out strain and the anaplerotic strain by hyaD fluorescence. And (D) verifying the construction conditions of the knocked-out strain and the anaplerotic strain by using Westernblot. (E) growth curve. (F) colony morphology. (G) capsular quantification. (H) And standing at 4 ℃ for 2 hours, and quantifying the biological membrane by low-speed centrifugation (I) at 3,000r/min for 5 min.
FIG. 2 is a diagram showing the virulence analysis of the hyaD gene-deleted strain. (A) post-challenge death time profile. (B, C) lung, liver and spleen organ colonization 8h and 24h after toxin attack. (D-F) 24H after challenge, staining of lung H & E. (G) number of adherent macrophages after infection. (H) the amount of phagocytized macrophages after infection. (I-M) expression of inflammatory genes 8h after macrophage infection. (N-R) secretion of inflammatory factors 8h after macrophage infection.
Fig. 3 is a Δhyad cross-immunoprotection assay. After immunization of ΔhyaD inactivated vaccine for 21D, the survival rate of immunized mice was determined by challenge using the muscle pathways of (A) PmCQ1, (B) PmCQ2, (C) PmCQ4, (D) PmCQ5, (E) PmB, (F) PmF, (G) Pmp, (H) PmQ and (I) PmR, respectively.
Figure 4 shows antibody dynamics after Δhyad immunization and challenge lung H & E staining after immunization. (A) anti-PmCQ1 antibody dynamic change. (B) anti-PmCQ2 antibody dynamic change. (C) anti-PmCQ4 antibody dynamic change. (D) anti-PmCQ5 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 changes. (I) anti-PmR antibody dynamic changes. (J) comparison of antibodies after 21d immunization. (K) PmCQ1 infection after immunization. (L) PmCQ2 infection after immunization. (M) infection with PmCQ4 after immunization. (N) PmCQ5 infection after immunization. (O) PmB infection after immunization. (P) PmF infection after immunization. (Q) PmP infection after immunization. (R) infection with PmQ after immunization. (S) infection with PmR after immunization. (T) non-infection after immunization.
FIG. 5qRT-PCR verifies that ΔhyaD affects other gene expression. (A-D) genes involved in capsular synthesis. (E-F) biofilm formation-related genes. (G-H) iron transport-related genes. (I-L) LPS synthesis and transport related genes. (M) virulence gene Pm0442. (N) virulence gene ompA. (O) outer membrane protein-related genes.
FIG. 6 shows the construction and biological properties of qseC gene-deleted strain (ΔqseC) and qseC gene-complemented strain (C-qseC). PCR confirmation of mutant and complement strains. M: labeling DNA; lanes1-3: pmCQ2; lanes 4-6: Δqsec; lanes 7-9: C-qseC. Lanes1, 4, 7: detecting Pasteurella multocida (positive control) using the species-specific primer KMT 1-F/R; lanes 2, 5, 8, detect the qseC gene using primer comqseC-F/R; lanes 3, 6 and 9. The qseC gene was detected using the primer qseC5' ARM-F/R. (B) qseC gene transcripts were detected using qRT-PCR. (C) Growth curve of strain cultured in shake flask at 37℃and 200 r/min. (D) Colony morphology of PmCQ2, Δqsec and C-qseC was cultured on a martin broth agar plate. (E) production of capsular polysaccharides. (F) The bacterial cells were in a centrifugally packed state after centrifugation at 10,000r/min for 5 min. (G) quantification of biofilm formation. (H) With 5mM, 10mM or 20mM H, respectively 2 O 2 The treatment was carried out at 37℃for 1 hour. (I) Bacterial cells were exposed to 100mM, 200mM and 300mM NaCl at 37℃for 1h, respectively.
FIG. 7 is a qseC gene deleted strain virulence analysis. (A) Survival of mice infected with wild strains PmCQ2, Δqsec and C-qseC, respectively, n=10 mice/group. (B-D) pulmonary histopathological changes 24h after PmCQ2, Δqsec and C-qseC infection. (B) Venous and alveolar wall capillaries severely dilate and engorged, inflammatory cell infiltration; (C) The pulmonary alveoli wall capillary blood vessel expands slightly to be hyperemic, and part of pulmonary alveoli cavity is internally provided with a small amount of red blood cells; (D) Congestion bleeding, and a large amount of red blood cells and cellulose exude from the alveolar space.
Figure 8 is a delta qseC post immunization antibody assay. Mice were immunized with inactivated PmCQ2, inactivated Δqsec, and live Δqsec, respectively. Blood was collected from the tail vein 21 days after immunization for serum separation. Serum antibodies were detected against (a) PmCQ1, (B) PmCQ2, (C) PmCQ4, (D) PmCQ5, (E) PmB, (F) PmF, (G) PmP, (H) PmQ, (I) PmR. Mice were immunized with (J) PmCQ2 inactivated vaccine, (K) Δqsec inactivated vaccine, (L) Δqsec attenuated vaccine, and analyzed for IgG antibody titer.
FIG. 9 is a qseC gene deleted strain cross-protection assay. After 21 days of immunization of mice with Δqsec inactivated vaccine and PBS subcutaneous route, mice were muscle-infected with survival rates of (a) PmCQ1, (B) PmCQ2, (C) PmCQ4, (D) PmCQ5, (E) PmB, (F) PmF, (G) PmP, (H) PmQ, and (I) PmR, respectively. After immunization of mice with Δqsec attenuated vaccine and PBS muscle pathway for 21d, mice were then muscle infected with survival rates of (J) PmCQ1, (K) PmCQ2, (L) PmCQ4, (M) PmCQ5, (N) PmB, (O) PmF, (P) PmP, (Q) PmQ, and (R) PmR, respectively.
Detailed Description
The invention will be described in further detail with reference to specific embodiments and drawings.
1. Examples
1 materials and methods
1.1 Strain, plasmid and culture conditions
Pasteurella multocida of bovine origin A (PmCQ 1, pmCQ2, pmCQ4, pmCQ 5), pasteurella multocida of bovine origin F (PmF), pasteurella multocida of avian origin A (PmQ) and Pasteurella multocida of rabbit origin A (PmR) are all isolated and stored in a preventive veterinary laboratory at the university of West south; pasteurella multocida of bovine origin B (PmB, CVCC 470) and Pasteurella multocida of porcine origin A (Pmp, CVCC 1662) were purchased from the China center for type-B collection of veterinary microorganisms; competent cells of E.coli DH 5. Alpha. And E.coli BL21 (DE 3) were purchased from Beijing Bomaide Biotechnology Co. Plasmid pUC19oriKan for gene deletion R For laboratory early construction and preservation, plasmid pMc-Express was given away by the teaching of Sichuan university of agriculture Cao Sanjie. The Pasteurella multocida is streaked and activated by adopting Ma Dingshi broth agar culture medium (purchased from Qingdao sea Bo biotechnology Co., ltd.) and cultured at the constant temperature of 37 ℃ for 24 hours, and then single bacterial colonies are picked up and put into 5mL Ma Dingshi broth liquid culture medium and cultured in a shaking table at the temperature of 37 ℃ for 12 hours for standby. Coli strains containing different resistant plasmids were inoculated with 50. Mu.g/mL kanamycin (Kan, available from Bio-engineering Co., ltd.), 100. Mu.g/mL ampicillin (Amp, available from Bio-engineering Co., ltd.), and 50. Mu.g/mL chloramphenicol (Cl, available from Bio-engineering Co., ltd.) respectively, resistant Luria-Bertani (LB, available from Beijing obucing Biotechnology Co., ltd.) on agar medium and cultured in an incubator at 37℃for 24 hours for activation.
1.2 test animals and ethical statements
Animals used in this test were 6-8 weeks old, 18-22g Kunming mice and C57/BL6 female mice, purchased from Ensifer. All feeding and test modes adopted for the test animals meet the requirements of relevant documents of welfare and ethical committee of animals at the university of southwest and are supervised by ethical examination committee of the test animals at the university of southwest (permission Number: IACUC-20200803-01).
1.3 construction of Gene-deleted and anaplerotic Strain
Primers used for constructing hyaD and qseC gene deletion strains and the anaplerotic strain are shown in tables 1 and 2, respectively.
Extracting a wild strain PmCQ2 genome by using a kit (DP 302-02, purchased from Tiangen biotechnology Co., ltd.) and respectively amplifying homologous sequences of the PmCQ2 genome by using the PmCQ2 genome as a template and upstream and downstream homology arm 5'ARM-F/R and 3' ARM-F/-R primers, and respectively obtaining an upstream homology arm fragment and a downstream homology arm fragment by using a gel recovery kit (CW 2302M, purchased from Kangji biotechnology Co., ltd.) after nucleic acid electrophoresis; and (3) carrying out PCR amplification by using the upstream and downstream homology arms as templates and using 5'ARM-F and 3' ARM-R primers, and obtaining the fusion fragments of the upstream and downstream homology arms after nucleic acid electrophoresis and gel recovery. Plasmid pUC19oriKan R After overnight incubation after activation, plasmids were extracted with a kit (CW 0500M, available from Kagaku Biotechnology Co., ltd.) and linearized vectors were recovered with a gel recovery kit after double digestion at 37℃for 2 hours (hours, h) via BamH I (1605, available from TaKaRa) and Hind III (1615, available from TaKaRa). By using
HD Cloning Kit (PT 5162-1, available from Clontech)
After ligation of the double digested linearized vector and the homology arm fragment in a thermostatic waterbath at 37℃for 30 minutes (minute, min), the ligation product was transformed into E.coli DH 5. Alpha. Competent cells and spread on LB plates containing 50. Mu.g/mLKan resistance overnight, and the positive clone pUC19oriKan was verified using the primers pUC19-F/R R -a hyad upstream + downstream homology arm. The recombinant plasmid is coated on a broth medium containing 100 mug/ml Kan Ma Dingshi after electrotransformation of PmCQ2, the knockout strain is screened out through PCR amplification, and Kan resistance elimination and genetic stability are ensured through continuous passage for 30 times. pMc mass is utilized in construction of anaplerotic plantsThe full length of hyaD gene and qseC gene are amplified by using PmCQ2 as template and primers comhyaD-F/R and comqseC-F/R, respectively, and the fragment is amplified by using plasmid pMc as template and primers egfp-F and egfp-R and digested with BamHI and HindIII to obtain pUC19oriKan R Linearized vector ligation to obtain recombinant plasmid pUC19oriKan R After double digestion of the product with Sal I (F301 DA0001, available from Biotechnology Co., ltd.) and Xba I (F927 DA0002, available from Biotechnology Co., ltd.), the product was ligated with the recovered hyaD gene fragment with ligase, transformed, and screened to give recombinant plasmid pUC19oriKan R -communications D and pUC19oriKan R -combsec. The recombinant plasmid was also electrotransferred into the knockout strain and the anaplerotic strain was screened by PCR amplification on a broth medium containing 100. Mu.g/mLKan Ma Dingshi and passaged 30 consecutive times to ensure genetic stability.
TABLE 1hyaD Gene deleted Strain and the primers for constructing the same
TABLE 2 qseC Gene deleted Strain and the primers required for construction of the anaplerotic Strain
1.4 Western blot verification of HyaD protein expression
And (3) taking the PmCQ2 genome as a template, amplifying an upstream primer wbhyaD-F/R, and obtaining the hyaD gene truncated expression fragment after glue recovery. Meanwhile, pET32a (+) plasmid is activated on an Amp resistance plate by the method, the plasmid and BamH I and Hind III are extracted by a kit, and are connected with truncated hyaD fragments after double enzyme digestion, and then the plasmid is transformed into E.coli DH5 alpha competent cells to screen positive clones, and the plasmid is extracted again and transformed into E.coli BL21 (DE 3) competent cells. Protein-induced expression and purification were performed according to the laboratory pre-procedure. Simultaneously, the method of ultrasonic crushing is adopted to prepare PmCQ2, pmCQ2 delta hyaD and PmCQ2 delta hyaD/phyaD whole mycoprotein.
First purified protein and Freund's complete adjuvant (purchased from Sigma Co.) were combined according to 1: mixing in proportion 1 to make the final concentration of protein in the mixed solution be 50 mug/mu L, fully emulsifying by a vortex oscillator, and performing subcutaneous multipoint immunization on the back of the mouse by 100 mu L of the mixed solution; purified protein and Freund's incomplete adjuvant (purchased from Sigma) were mixed in the same volume to prepare the same concentration mixture, and 100. Mu.L of the mixture was subjected to subcutaneous multipoint immunization at the back after 14 days of first immunization to boost, and tail venous blood was collected after 14 days of second immunization. The collected tail venous blood is placed in an incubator at 37 ℃ for 1h, and placed at 4 ℃ for overnight precipitation of serum.
Three whole bacterial proteins of PmCQ2, pmCQ2ΔhyaD/phyaD are respectively used as antigens, hyaD truncated expression protein immune mouse serum is used as an antibody, sheep anti-mouse IgG-HRP (purchased from Thermo scientific company) is used as an enzyme-labeled secondary antibody, and the expression condition of the protein in wild strains, knocked-out strains and anaplerosis strains is verified according to a western blot of a laboratory early-stage method.
Growth curve
The wild strain, the gene deletion strain and the anaplerotic strain are selected, and the single colonies are placed into 5mL Ma Dingshi broth culture medium and cultured for 10 hours at the temperature of 37 ℃ at 220r/min in an incubator. Diluting and counting the bacterial liquid, and diluting to 2×10 with Ma Dingshi broth 8 CFU/mL followed by 1:100 inoculum sizes were transferred to fresh Ma Dingshi broth medium and incubated at 37℃at 220r/min and absorbance at OD600nm was measured every two hours.
Capsule quantification
Respectively sucking three bacterial solutions of a wild strain, a gene deletion strain and a anaplerotic strain which are cultivated to a logarithmic growth phase into a centrifuge tube with 1mL to 1.5mL, centrifuging at 13,000r/min for 10min, discarding the supernatant, washing twice with phosphate buffer (PBS, purchased from Soy Corp.) and then adding 1mLPBS solution to resuspend the bacterial solution, placing in a metal bath at 42 ℃ for 1h, and respectively counting the bacterial solutions before and after the metal bath. After that, the bacterial solutions of each tube were centrifuged again at 13,000r/min for 10min, and the supernatant was retained. 100. Mu.L of the supernatant was added to 900. Mu.L of capsule staining solution prepared from 0.2mg/mL of stains all powder (7423-31-6, available from Biotechnology Co., ltd.), 50% formamide (75-12-7, available from Colon chemical Co., ltd.) and 0.06% glacial acetic acid solution (available from Colon chemical reagent Co.) respectively, mixed by shaking, while 0, 0.5, 1, 2, 3, 4, 5. Mu.g/100. Mu.L of standard hyaluronic acid solution samples of different concentration gradients were added to 900. Mu.L of the volume of capsule staining solution, while absorbance at OD640 nm was measured, and the amounts of hyaluronic acid released from the three samples to be measured were calculated using the standard samples as standard curves.
Biofilm quantification
Counting three bacterial liquids of wild strain, gene deletion strain and anaplerotic strain cultured to logarithmic growth phase, and diluting with brain heart infusion broth (BHI, purchased from Qingdao sea Bo Biotechnology Co., ltd.) culture medium to bacterial content of 1×10 8 CFU/mL, 400. Mu.L of bacterial dilutions were transferred to 48 well cell culture plates, respectively, while negative control was 400. Mu.LBHI broth, mixed with horizontal shaking and incubated in an incubator at 37℃for 48h. The bacterial culture solution was aspirated and washed twice with 200. Mu.L of PBS solution to remove impurities and non-adherent bacteria, dried at room temperature, then added with 200. Mu.L of methanol (67-56-1, from Colon chemical Co., ltd.) fixing solution and gently washed three times at room temperature and dried at room temperature, then added with 1% crystal violet (548-62-9, from Biotechnology Co., ltd.) per well at 200. Mu.L/well and stained at 37℃for 30min, and likewise added with 200. Mu.L/well of PBS solution and gently washed three times and dried at room temperature, finally added with 200. Mu.L/well of 33% glacial acetic acid solution and dissolved at 37℃for 30min, the absorbance at OD630nm was read with an ELISA, and the bacterial solution was not added as a blank.
LD50 determination
Counting the gene deletion strain and the anaplerotic strain which are cultured to the logarithmic growth phase, carrying out muscle toxicity attack according to different concentration gradients, continuously observing for one week, and treating the mice as death and timely euthanasia when obvious hair frying and mental depression states appear. The death of mice was counted and the median lethal dose of the gene-deleted and complemented strains was calculated using the bliss method.
Organ colonization and H & E staining
Counting three bacterial liquids of a wild strain, a gene deletion strain and a anaplerotic strain which are cultivated to logarithmic growth phase, and then adding the bacterial liquids to the culture medium at a ratio of 2.0x10 6 CFU was intramuscular injected into mice, 6 Kunming mice per group. Mice 8 and 24 hours after the toxicity attack are euthanized, three organs of lung, liver and spleen are collected for homogenate, diluted and coated, the bacteria content is detected, and meanwhile, the lung of the mice after 24 hours of infection is collected and sent to Rijia Nuo biotechnology limited company to prepare pathological sections.
Infection of macrophages
The mouse peritoneal macrophages were collected and cultured in a 5% CQ2 incubator at 37℃according to the method established earlier in the laboratory. Inoculating 2X 10 cells per well in 48-well cell plates 5 After attaching to the surface of the macrophages, the macrophages are infected with PmCQ2, pmCQ2ΔhyaD and PmCQ2ΔhyaD/phyaD according to 1MOI respectively, a group of culture solution is discarded after 8 hours, PBS is added to wash for three times, 500 mu L of ciprofloxacin with the final concentration of 100 mu g/mL is added to each hole to treat for 30 minutes to kill extracellular bacteria, 500 mu LPBS is added to each hole to wash for three times, PBS solution containing 0.1% TritonX-100 (purchased from Solarbio) is added to each hole to fully lyse the cells, and the number of the adhered macrophages of various bacteria and the number of the phagocytized macrophages are respectively counted.
Attenuated vaccine and inactivated vaccine preparation
The preparation of the PmCQ2Δqsec attenuated vaccine is to directly culture the PmCQ2Δqsec for a stable period, count and directly immunize the live bacteria according to a certain dosage. And the inactivated vaccine of the two gene deletion strains is prepared as follows: the wild strain and the gene deletion strain cultured to the logarithmic growth phase are respectively cultured according to the following ratio of 1:100 transferCulturing in triangular flask until OD600 = 1.4-1.5, performing purity test and counting, concentrating, adding formaldehyde solution (commercially available from Chenopodium chemical Co., ltd.) with final concentration of 0.15%, inactivating in incubator at 37deg.C for 24 hr, wherein each interval for 3 hr is properly vortex-oscillated to inactivate thoroughly, mixing with 15AVG mineral oil emulsifier (supplied by Chongqing Australian biological Co., ltd.) at a volume ratio of 4:1 to obtain inactivated vaccine with final concentration of 5×10 9 CFU/mL. 100 mu L of inactivated vaccine is coated on a Martin plate, and 6 mice are selected to be respectively inoculated with 100 mu L of inactivated vaccine to the back subcutaneous for vaccine safety evaluation.
Cross protection assay
The Kunming mice were randomly grouped, 10 mice per group, and the prepared vaccination procedure was performed according to tables 3 and 4, respectively, wherein secondary immunization was performed after 7d interval from the priming in order to enhance the inactivated vaccine immunization effect. After 21d of priming, the hyaD gene deleted vaccine groups were each inactivated at 3.6X10 7 CFU PmCQ1 (muscle route: ld50=3.8x10) 2 CFU)、3.2×10 7 CFU PmCQ2 (muscle route: ld50=3.4×10) 3 CFU)、4.6×10 7 CFU PmCQ4 (muscle route: ld50=2.1×10) 3 CFU)、2.9×10 7 CFU PmCQ5 (muscle route: ld50=4.5×10) 3 CFU)、1.2×10 7 CFU PmB (muscle route: ld50=5.0×10 3 CFU)、4.4×10 8 CFU PmF (muscle pathway: 1.0X10) 8 CFU), 10CFU PmQ (muscle pathway: LD50≡1 CFU), 10CFU PmP (muscle pathway: LD50≡1 CFU), 2.2X10 6 CFU PmR (muscle pathway: 1.0X10) 4 CFU) muscle challenge and continuous observation for one week, while the qseC gene deleted vaccine groups were 3.8x10, respectively 7 CFU PmCQ1、4.8×10 7 CFU PmCQ2、3.6×10 7 CFU PmCQ4、4.5×10 7 CFU PmCQ5、1.0×10 7 CFU PmB、2.0×10 8 CFU PmF、10CFU PmP、10CFU PmQ、1.0×10 6 CFU PmR muscle attacks toxin and continuously observes for one week, and the mice have obvious phenomena of hair frying, mental depression and the like to judge death and euthanize in time. Immunization procedures and challenge are shown in tables 3 and 4.
Table 3hyaD Gene deleted vaccine immunization program and challenge
Table 4qseC Gene deleted vaccine immunization program and challenge
Antibody titer analysis
After primary immunization of the vaccine, blood serum of tail veins of mice is collected at intervals of 7, 14, 21, 28, 42, 56, 70, 84, 98 and 105 days respectively, and antibody titer determination is carried out according to an indirect ELISA method established in the earlier stage of a laboratory. PmCQ1, pmCQ2, pmCQ4, pmCQ5, pmB, pmF, pmQ, pmP and PmR whole mycoproteins were obtained by sonication, and the whole mycoproteins were coated overnight at 4℃with a coating solution (0.05M carbonate buffer, pH 9.6) at 1. Mu.g/well dilution. The next day PBST (PBS solution with 0.05% Tween 20, tween 20 from Biotechnology Co., ltd.) was washed 3 times for 5min each time, and then each well was blocked at 37deg.C for 1h with 5% skimmed milk powder (from Saguo Biotech Co.). The primary antibody is vaccine immunized mouse serum, the first hole is diluted 1:100, namely 2 mu L serum is added into 198 mu LPBST, the double dilution is uniformly mixed by blowing up and down with a 100 mu L row gun for 5 times, 100 mu L is sucked into the second hole, the same operation is repeated until the last hole is discarded 100 mu L liquid, and PBST is washed 3 times for 5min each time after incubation at 37 ℃ for 1h. HRP-labeled goat anti-mouse IgG (h+l) secondary antibody (purchased from Sigma) was diluted with PBST 1:10,000, 100 μl was added to each well, incubated for 1H at 37 ℃ and then washed again with PBST 3 times for 5min each, and the remaining liquid in the wells was dried. 100. Mu.L of TMB chromogenic solution (3, 3', 5' -tetramethylbenzidine, available from Beyotime) was added to each well, developed for 10min at room temperature in the dark, and 100. Mu.L of 1M sulfuric acid (available from Chongqing Chuan Dong chemical) stop solution was added to the mixture, and the OD450 value was read by an ELISA reader.
Post-immunization lung H & E staining
After the PmCQ2 delta hyaD inactivated vaccine is immunized, the lung of the mice after 12 hours of toxin attack and the lung of the mice without toxin attack are respectively collected and sent to Lijia NOO biological technology Co., ltd for preparing pathological sections.
qRT-PCR fluorescent quantitative analysis
And (3) centrifugally collecting bacterial solutions of the wild strain, the deletion strain and the anaplerotic strain which are cultured to the logarithmic growth phase, respectively extracting total RNA according to the specification of a bacterial RNA extraction kit (TR 214-50, purchased from the anabrosis organism), measuring the concentration by an enzyme-labeling instrument, regulating the concentration of the total RNA of the wild strain, the deletion strain and the anaplerotic strain to be the same concentration, and then synthesizing cDNA by using a reverse transcription kit (KR 118-02, purchased from the anabrosis organism). qRT-PCR fluorescence quantification was performed using cDNA as a template and the related primers (Table 5, table 6).
TABLE 5 qRT-PCR fluorescent quantitation of hyaD Gene deleted strains
Table 6qseC Gene deleted Strain qRT-PCR fluorescent quantitative required primer
Statistical analysis of data
Adopting Graphpad Prism 6 software to carry out experimental data analysis treatment, and carrying out t-test analysis, wherein p >0.05 is no significant difference (ns); p <0.05 (, p <0.01 (, p <0.001 (, p <0.0001 (, p) are significant differences).
2. Example verification results (one) hyaD Gene-deleted Strain-related verification results
1. hyaD gene deletion construction and biological characteristics thereof
The invention researches the effect of the hyaD gene in bovine source A type Pasteurella multocida PmCQ2, constructs a PmCQ2 hyaD gene deletion strain, the related primer binding site is shown in figure 1A, and verifies that the gene is not expressed in the knockout strain, the wild strain and the anaplerotic strain through PCR amplification, qRT-PCR fluorescent quantitation and Western blot on the DNA, RNA and protein levels respectively (figures 1B-D). After 30 times of in vitro passage, the knocked-out strain and the anaplerotic strain can still be inherited stably. When the knocked-out strain grows in the Martin broth liquid culture medium, the 6 th hour is obviously faster than that of the wild strain and the anaplerotic strain, and the growth speeds of the anaplerotic strain and the wild strain tend to be consistent (figure 1E). After 24h growth on Ma Dingshi broth plates, the morphology of the PmCQ2ΔhyaD colonies was significantly reduced compared to the wild strain, with the morphology size of the PmCQ2ΔhyaD/phyaD colonies being intermediate to the two (FIG. 1F). The knockout strain capsule content was significantly reduced, while the anaplerotic strain capsule content was higher than the knockout strain but still less than the wild strain (fig. 1G), and the knockout strain was extremely prone to centrifugation and sedimentation (fig. 1H). Meanwhile, compared with the PmCQ2 and the PmCQ2 delta hyaD/phyaD, the biological film content of the PmCQ2 delta hyaD is obviously improved, and the anaplerotic strain is between the two (figure 1I).
2. hyaD affects PmCQ2 virulence
In order to explore the influence of hyaD genes on the virulence of Pasteurella multocida PmCQ2, pmCQ2ΔhyaD and PmCQ2ΔhyaD/phyaD half-lethal dose are measured by different concentration gradient muscle toxicity, and the knockout strain PmCQ2ΔhyaDd50=2.30X10 is calculated by a bliss method 7 CFU, approximately half the lethal dose of wild strain PmCQ2 (ld50=3.43×10 3 CFU) 6700-fold, whereas the anaplerotic strain pmcq2Δhyad/phyadld50=1.43×10 4 CFU was about 4-fold that of the wild strain (table 7). At 2.0X10 6 The wild strain was found to die quickly within one day after CFU PmCQ2, pmcq2Δhyad and pmcq2Δhyad/phyaD muscle challenge, the knockout strain died only one within 5 days, the complemented strain died all within 7 days but the death time was delayed compared to the wild strain (fig. 2A). At the same time respectively 2.0X10 6 The CFU PmCQ2, pmcq2Δhyad and pmcq2Δhyad/phyaD muscle challenge were collected for 8h, 24h of lung, liver, spleen, respectively, and after homogenization, dilution plating and counting, it was found that the wild plants had the highest colonization amount in each organ, the knocked-out plants had the lowest colonization amount, the colonization amounts of the anaplerotic plants in the different organs were between the two, and the colonization amounts were all increased with the increase of time (fig. 2B-C).Mice were infected at the same dose, and the knockout strain caused less pathological damage to the lungs (FIGS. 2D-F)
TABLE 7PmCQ2ΔhyaD and PmCQ2ΔhyaD/phyaD LD50
3. Results of toxic effects of infected macrophages
To further verify the virulence of the knockdown strain, it was further verified at the cellular level that, after PmCQ2, pmCQ2ΔhyaD and PmCQ2ΔhyaD/phyaD respectively infected cells, the knockdown strain was found to adhere more easily to the surface of macrophages and be phagocytosed easily, the number of adhesion of the anaplerotic strain and phagocytosis by macrophages was intermediate between that of the wild strain and the knockdown strain (FIG. 2G-H), and the expression of inflammatory genes of macrophages TNF- α, IL-1β, IL-6, IL-12p40 and IL-17A and secretion of inflammatory factors of TNF- α, IL-1β, IL-6, IL-12p40 and IL-17A were promoted more, whereas the anaplerotic strain was not significantly different from the wild strain (FIG. 2I-R).
4. Detection of inactivated vaccine
After the inactivated vaccine is coated on a Ma Dingshi broth culture medium and is cultured for 48 hours at 37 ℃, no colony grows on a flat plate, which indicates that the vaccine inactivation effect is good. The inactivated vaccine is 3,000r/min, and no layering exists after the centrifugation for 3min, which indicates that the vaccine has better emulsification effect. After the inactivated vaccine is pre-inoculated into the mice, the mice have good mental state and appetite, and no obvious tumor exists under the back skin, so that the vaccine has good safety.
5. Cross protection effect
In order to evaluate the cross protection property of the PmCQ2 delta hyaD, the PmCQ2 delta hyaD is prepared into an inactivated vaccine, and after 21 days of immunization, the inactivated vaccine is subjected to muscle detoxification, so that 100% immunity protection is realized on bovine type A Pasteurella multocida (PmCQ 1, pmCQ2, pmCQ4, pmCQ 5) and bovine type B Pasteurella multocida PmB, and 80% protection rate is realized on bovine type F Pasteurella multocida PmF; the protection rates of 90%, 100% and 100% for rabbit, avian and swine origin type a pasteurella multocida PmR, pmQ, pmP (fig. 3A-I), respectively, demonstrate that PmCQ2 Δhyad can be used as an inactivated vaccine against infection by pasteurella multocida, in particular, infection by bovine origin type B pasteurella multocida.
6. Antibody dynamic changes
In order to monitor the dynamic change of the antibody level in the serum of the PmCQ2 delta hyaD inactivated vaccine immunized mice, an indirect ELISA method is adopted for judging the antibody titer. The results show that antisera had higher antibody levels against various pathogens and reached the highest antibody level substantially after 21d priming and maintained for about 8 weeks (fig. 4A-I), and that serum antibody levels after 21d immunization of mice with inactivated vaccine were compared to find that there was no significant difference between anti-bovine type a, type B pasteurella multocida IgG antibody levels and anti-PmCQ 2 antibody levels, while anti-other serotypes and other host-derived pasteurella multocida antibody titers were lower than PmCQ2 (fig. 4J).
7. Evaluation of pathological injury results after immune detoxification
To further verify the immune effect of the inactivated vaccine, lungs of immunized mice were collected after 12H of challenge, and H & E staining showed no obvious pathological damage to lungs of immunized mice compared with lungs of non-immunized mice (fig. 4T), and slight pathological damage to parts of lungs of immunized mice (fig. 4K-S).
8. hyaD affects PmCQ2 Gene expression
Based on the change of the related biological characteristics of the PmCQ2 delta hyaD, the expression quantity of the related genes of the PmCQ2, the PmCQ2 delta hyaD and the PmCQ2 delta hyaD/phyaD is quantitatively compared through qRT-PCR fluorescence, and the knockdown strain is compared with a wild strain and a anaplerotic strain, and the related capsular synthetic gene hexA, hexC, hexD, phyB is down-regulated; up-regulating expression of biomembrane synthesis related gene cya and oxyR; lipopolysaccharide LPS synthesis and transport related gene lpxD, wzzE, kdsA, lptG expression is down-regulated; the expression of iron transport related genes fecC and fecE is down-regulated, and the expression of other virulence genes Pm0442 and ompA is down-regulated; the outer membrane protein related genes metQ, plp4, plpE, lolA, lolB, lppB, pal, bamA, bamD, bamE, fbpB, fbpC, ompW, ompH, pcp, skp, hemR, fhaB2 were up-regulated.
As a result, it was found that ΔhyaD capsule was significantly reduced, biofilm was increased, virulence was significantly reduced, and LD was significantly reduced, as compared with the wild strain 50 Rising by about 6700 times, pathological injury caused by infection and colonization in visceraThe amount is obviously reduced, the macrophage is infected, the cell adhesion amount is increased and the macrophage is easy to phagocytose, and further the obvious up-regulation of the expression of the cell inflammatory factor is promoted; the prepared delta hyaD inactivated vaccine is used for immunizing mice (1 time of inactivated vaccine boosting), serum antibody detection is carried out after blood sampling and separation of tail veins every 5 days, different types of animal-derived Pasteurella multocida are respectively attacked 21 days after immunization, the protection rates of the delta hyaD inactivated vaccine on bovine-derived A-type, B-type and F-type Pasteurella multocida respectively reach 100%, 100% and 80%, and the immune protection rates of the delta hyaD inactivated vaccine on rabbit-derived, pig-derived and avian-derived A-type Pasteurella multocida respectively reach 90%, 100% and 100%. After immune attack, lung is not obviously pathologically damaged, and the antibody level reaches the highest level at 21d and is maintained for about 8 weeks. Therefore, the hyaD gene can obviously reduce the toxicity of wild strains after knocking out, has cross protection effect on the pasteurella multocida with different serotypes and different host sources, and provides a candidate target for the research and development of the pasteurella multocida vaccine.
(II) qseC Gene deletion Strain-related verification results
qseC gene deletion construction and biological Properties thereof
The invention researches the effect of qseC genes in bovine origin A type Pasteurella multocida PmCQ2, constructs a PmCQ2 qseC gene deletion strain, and in order to research the effect of QseC in Pasteurella multocida, we construct a qseC gene deletion strain (delta qseC) without a resistance marker and a qseC gene complementation strain C-qseC from the qseC gene deletion strain in bovine origin A type Pasteurella multocida capsular strain CQ 2. PCR amplification indicated that the qseC gene was not in the chromosomes of Δqsec and C-qseC, but rather was present in the C-qseC plasmid (FIG. 6A). To further confirm the mutant and the complementing strain, RT-PCR was performed, and the results indicated that the qseC gene transcript was only present in the wild-type and complementing strain, but not in the mutant (fig. 6B). The Δqsec was stable for more than 30 generations (data not shown), and the growth curve was similar to WT strain (fig. 6C). The colony morphology of Δqsec was much smaller than that of PmCQ2 and C-qseC (fig. 6D). Considering that the three strains grow at similar rates and that the capsular polysaccharide in PmCQ2 is abundant, the colony size of the mutant may be related to the decrease in capsular polysaccharide content in the cells. Capsular polysaccharides, Δqsec and C-qseC extracted from PmCQ2 were detected. The capsular polysaccharide production of Δqsec was significantly reduced compared to wild type, followed by C-qseC (fig. 6E). Since capsular polysaccharide content also affects the centrifugation status of bacteria, Δqsec cells were more easily centrifuged to the bottom of the tube, and the supernatant was clear and transparent (fig. 6F). The trend of capsular polysaccharide production in multocida capsular serotype a was opposite to that of the strain producing biofilm. The biofilm assay showed a significant increase in biofilm formation in Δqsec compared to PmCQ2 and C-qseC (fig. 6G), with qseC upregulating oxidative stress, osmotic pressure and heat shock resistance in haemophilus parasuis. However, qseC in Pasteurella multocida is involved in the negative regulation of oxidative stress and osmotic pressure resistance (FIG. 6H-I), in contrast to Haemophilus parasuis.
Effect of QseC on Pasteurella multocida virulence
QseC can regulate virulence of many bacterial pathogens. To investigate its effect on pasteurella multocida virulence, mice were injected with PmCQ2, Δqsec and C-qseC, respectively (3.48×10 5 CFU). Mice infected with Δqsec had significantly higher survival than those infected with PmCQ2 or C-qseC (fig. 7A). Compared to WT and C-qseC, qseC mutants induced a weaker inflammatory response in the lungs of mice (fig. 7B-C), and the bacterial load in the lungs of qseC mutant infected mice was significantly lower than PmCQ2 and C-qseC infected mice (fig. 7D-F). To further quantify the decline in virulence of the qseC mutant, a 50% lethal dosimetry was performed. The LD50 of the Δqsec trans-abdominal route is 5.28X10 7 CFU, 5.28x10 higher than PmCQ2 (celiac pathway LD50≡1 CFU) 7 Double, C-qseC (abdominal route ld50=2.48×10 4 CFU) 2.1×10 higher 3 Multiple (Table 8).
TABLE 8LD50 determination
3. Delta qseC can trigger stronger antibody reaction
To investigate the effect of qseC gene deletion on antibody production, ELISA was used to determine serum IgG titers of inactivated PmCQ2, inactivated Δqsec and live-inactivated Δqsec immunized mice. The antibody titer of Δqsec was significantly higher than PmCQ2 (fig. 8A-I), and the level of antibodies against the broad-spectrum pasteurella multocida strain generated by live immunization of Δqsec was much higher compared to the inactivated PmCQ2 and the inactivated Δqsec (fig. 8A-I). The serum antibody titers against bovine Pasteurella multocida A in immunized mice were significantly higher than those against Pasteurella multocida B, F and other animals (FIGS. 8J-L). The results indicate that live Δqsec induces more cross-reactive antibodies than inactivated PmCQ2 and inactivated Δqsec.
4. Delta qseC cross-protective assay
Cross-protection of Δqsec against other pasteurella multocida serotypes was investigated. The Kunming mice were vaccinated intramuscularly with live Δqsec, inactivated Δqsec, and inactivated PmCQ2, respectively. On day 21 after the first immunization, mice were challenged with 9 strains of Pasteurella multocida, respectively (Table 4). Both live Δqsec and inactivated Δqsec have strong cross-protection against pasteurella multocida in mice (fig. 9A-R). However, deactivated PmCQ2 exhibited very weak cross-protection. Immunization of mice with the Δqsec attenuated vaccine had 100% protection against infection by bovine pasteurella multocida capsular types a (fig. 9J-M) and B (fig. 9N), swine pasteurella multocida capsular type a (fig. 9P), and rabbit pasteurella multocida capsular type a (fig. 9R). Whereas live Δqsec has only 30% protection against seroinfection by bovine pasteurella multocida capsule type F (fig. 9O) and 80% protection against seroinfection by poultry pasteurella multocida capsule type a (fig. 9Q). While inactivated Δqsec also exhibited good cross-protection, live Δqsec performed better (fig. 9A-R). The results show that live delta qseC has potential as an attenuated vaccine against infection by homologous and heterologous strains of pasteurella multocida.
In conclusion, the invention discovers that the hyaD and qseC genes can influence the synthesis of PmCQ2 capsules, the formation of biological films, the growth speed and the virulence through constructing PmCQ2ΔhyaD knockout strain and PmCQ2ΔqseC knockout strain, and the knockout strain can provide cross protection for bovine type A, B and F serotypes and multi-killing Pasteurella strain of different host sources of avian sources, porcine sources and rabbit sources, so that the PmCQ2ΔhyaD and PmCQ2ΔqseC inactivated vaccine is possibly a novel vaccine for preventing and controlling the diseases.
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.
Claims (9)
1. A bovine origin type A Pasteurella multocida gene deletion strain, characterized in that the strain is a PmCQ2 strainhyaDDeletion strain of gene, said deletion strain of gene PmCQ2ΔhyaDThe culture medium is preserved in China Center for Type Culture Collection (CCTCC) M2022067.
2. A bovine origin type A Pasteurella multocida gene deletion strain, characterized in that the strain is a PmCQ2 strain qseCDeleted strain of gene, said deleted strain PmCQ2ΔqseCThe culture medium is preserved in China Center for Type Culture Collection (CCTCC) M2022066.
3. The bovine-derived type a gene-deleted strain of pasteurella multocida according to claim 1, characterized in that the strainhyaDThe nucleotide sequence of the gene deletion part is SEQ ID NO.1, and the deletion of the fragment leads to the inactivation of the hyaD gene function.
4. The bovine origin a-type pasteurella multocida gene deletion strain as claimed in claim 2, wherein the nucleotide sequence of the qseC gene deletion portion is SEQ ID No.2, the fragment deletion resulting inqseCInactivation of gene function.
5. The bovine-derived pasteurella multocida gene-deleted strain according to claim 1, characterized in that the gene-deleted strain pmcq2ΔhyaDConstructed by a homologous recombination method comprising the steps of:
(1) Extracting genomic DNA of wild strain PmCQ2 with kit (DP 302-02, available from Tiangen Biotechnology Co., ltd.) using PmCQ2 genomic DNA as templateAmplification of primer 5 'with upstream and downstream homology arms'hyaDARM-F/R and 3'hyaDARM-F/R carries out PCR amplification respectively, after the amplified products are subjected to nucleic acid electrophoresis, target fragments are cut off, and recovered by adopting a gel recovery kit (CW 2302M, purchased from century biotechnology Co., ltd.) to obtain purified 226bp upstream homology ARM fragments and 251bp downstream homology ARM fragments respectively;
(2) The upstream and downstream homologous arm fragments are used as templates, 5 'is adopted'hyaDARM-F and 3'hyaDThe ARM-R primer continues to carry out PCR amplification, and purified upstream and downstream homology ARM fusion fragments are obtained after nucleic acid electrophoresis and gel recovery;
(3) pUC19oriKan containing temperature sensitive plasmid R After activation, overnight culture, plasmid extraction using plasmid extraction kit (CW 0500M, available from Kagaku, century Biotechnology Co., ltd.) was used to extract plasmid pUC19oriKan R Warp yarnBamHI (1605, from TaKaRa) andHindIII (1615, from TaKaRa) was double digested at 37℃for 2 hours (hor, h) and the linearized vector fragment was recovered using a gel recovery kit;
(4) The double digested linearized vector and homology arm fragments were ligated In a 37℃thermostat water bath for 30 minutes (minute, min) using an In-Fusion library HD Cloning Kit (PT 5162-1, available from Clontech) and the ligation product was transformed intoE. coliIn DH5 alpha competent cells, coating on a LB plate containing 50 mug/mL Kan resistance, culturing overnight at a constant temperature of 37 ℃, picking up colonies, adopting a primer pUC19-F/pUC19-R to perform colony PCR, screening out positive clones, and then performing sequencing verification;
(5) Preparation of PmCQ2 electric conversion competence, and after electric conversion of the recombinant plasmid to PmCQ2 competence, the recombinant plasmid is coated on broth medium containing 100 mug/mL Kan Ma Dingshi, amplified by PCR, and screened out hyaDThe gene deletion strain is continuously passed for 30 times to ensure the elimination of Kan resistance and genetic stability.
6. The bovine-derived pasteurella multocida gene-deleted strain according to claim 2, characterized in that the gene-deleted strain pmcq2Δqsec is constructed by a homologous recombination method, comprising the steps of:
（1) Extracting the genomic DNA of the wild strain PmCQ2 with a kit (DP 302-02, purchased from Tiangen Biotechnology Co., ltd.) and amplifying the primer 5 'with the PmCQ2 genomic DNA as a template and an upstream and downstream homology arm'qseCARM-F/R and 3'qseCPerforming PCR amplification on ARM-F/R respectively, performing nucleic acid electrophoresis on an amplified product, cutting a target fragment, and recovering the target fragment by using a gel recovery kit (CW 2302M, purchased from century biotechnology Co., ltd.) to obtain a purified 350 bp upstream homology ARM fragment and a purified 350 bp downstream homology ARM fragment respectively;
(2) The upstream and downstream homologous arm fragments are used as templates, 5 'is adopted'qseCARM-F and 3'qseCThe ARM-R primer continues to carry out PCR amplification, and purified upstream and downstream homology ARM fusion fragments are obtained after nucleic acid electrophoresis and gel recovery;
(3) pUC19oriKan containing temperature sensitive plasmid R After activation, overnight culture, plasmid extraction using plasmid extraction kit (CW 0500M, available from Kagaku, century Biotechnology Co., ltd.) was used to extract plasmid pUC19oriKan R Warp yarnBamHI (1605, from TaKaRa) andHindIII (1615, from TaKaRa) was double digested at 37℃for 2 hours (hor, h) and the linearized vector fragment was recovered using a gel recovery kit;
(4) The double digested linearized vector and homology arm fragments were ligated In a 37℃thermostat water bath for 30 minutes (minute, min) using an In-Fusion library HD Cloning Kit (PT 5162-1, available from Clontech) and the ligation product was transformed intoE. coliIn DH5 alpha competent cells, coating on a LB plate containing 50 mug/mL Kan resistance, culturing overnight at a constant temperature of 37 ℃, picking up colonies, adopting a primer pUC19-F/pUC19-R to perform colony PCR, screening out positive clones, and then performing sequencing verification;
(5) Preparation of PmCQ2 electric conversion competence, and after electric conversion of the recombinant plasmid to PmCQ2 competence, the recombinant plasmid is coated on broth medium containing 100 mug/mL Kan Ma Dingshi, amplified by PCR, and screened outqseCGene-deleted strain PmCQ2ΔqseCAnd the elimination of Kan resistance and genetic stability are ensured through 30 times of continuous passages.
7. The application of the bovine-derived type A Pasteurella multocida gene deletion strain, which is characterized in that the bovine-derived type A Pasteurella multocida gene deletion strain as set forth in claim 1 or 2 is adopted to prepare a vaccine for immunization, so as to promote the cross immune protection of animals against infection of different serotypes and different animal-derived Pasteurella multocida.
8. The use of a gene-deleted strain of pasteurella multocida of bovine origin according to claim 7, characterized in that the cross-protection is a cross-immune protection against infection by pasteurella multocida of bovine origin type a, type B, type F.
9. The use of a gene-deleted strain of pasteurella multocida of bovine origin according to claim 7, characterized in that the cross-protection is a cross-immune protection against infection with pasteurella multocida of porcine origin, avian origin, rabbit origin.
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