Construction method and application of auxotrophic riemerella anatipestifer strain

Abstract

The invention provides a construction method and application of an auxotroph riemerella anatipestifer strain, and belongs to the technical field of biology. The invention provides a mutant strain of riemerella anatipestifer. The mutant strain comprises a strain obtained after an alanine racemase Alr gene in the riemerella anatipestifer RA-LZ01 is knocked out. The mutant strain is obtained by knocking out the alanine racemase Alr gene in the riemerella anatipestifer RA-LZ01, the toxicity of the obtained mutant strain is greatly reduced compared with that of the riemerella anatipestifer RA-LZ01, and infection of wild type riemerella anatipestifer can be efficiently resisted. Results of the embodiment prove that healthy ducklings immunized by the riemerella anatipestifer mutant strain RA-LZ01 delta Alr prepared by the invention are used for preventing riemerella anatipestifer diseases, and the riemerella anatipestifer mutant strain RA-LZ01 delta Alr has the advantages of safety, high protection rate and the like. The mutant strain has a wide application prospect in the field of riemerella anatipestifer disease prevention and treatment.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a method for constructing an auxotrophic Riemerella anatipestifer strain and application thereof. Background Art

[0002] Riemerella anatipestifer is a rod-shaped or oval, spore-free, flagella-free Gram-negative bacterium that can form a capsule. It is bipolar after being stained with Wright's stain. It belongs to the family Flavobacteriaceae, the genus Riemerella, and is the main pathogen that causes duck infectious serositis and acute and chronic sepsis. The growth of this bacterium has relatively high nutritional requirements, and it does not grow on ordinary culture medium and MacConkey medium. Since this bacterium is facultatively anaerobic, when it is first isolated from the diseased material, it should be streaked on a whole blood tryptone soy agar plate and inverted in a 37°C incubator with 5% CO2 for 24 to 48 hours to grow a single colony that is colorless, round, slightly protruding, and smooth on the surface, with a diameter of about 1 to 2 mm.

[0003] Riemerella anatipestifer, also known as duck infectious serositis, is a type of disease that has a wide impact on the duck farming industry. Riemerella anatipestifer is a septicemic disease caused by Riemerella anatipestifer that infects a variety of birds such as domestic ducks, geese or turkeys. For a long time, it has caused serious economic losses to the duck farming industry. Under natural conditions, it can be transmitted horizontally through various channels, such as drinking water, feed, respiratory secretions, etc., and can also be transmitted vertically through breeding eggs. The disease mainly infects young ducks, and the typical symptoms are increased eye and nasal secretions and green diarrhea; in the late stage of infection, neurological symptoms such as head and neck tremors and lameness can be observed; surviving animals may have stunted development, and severely infected ducks can cause death. Symptoms such as fibrinous pericarditis, perihepatitis, and air sac inflammation can be found in autopsies of dead ducks.

[0004] In order to cope with the increasingly serious drug resistance of Riemerella anatipestifer, the development of vaccines is an effective way, but the current vaccines for the prevention and / or treatment of Riemerella anatipestifer often have poor preventive and therapeutic effects. Summary of the invention

[0005] In view of the defects in the prior art, the object of the present invention is to provide a mutant strain of Riemerella anatipestifer, which can be used as a live vaccine for preventing and treating Riemerella anatipestifer and has the characteristics of safety and high efficiency.

[0006] The objective of the present invention is achieved through the following technical solutions:

[0007] The invention provides a mutant strain of Riemerella anatipestifer. The mutant strain comprises a strain obtained by knocking out an alanine racemase Alr gene in Riemerella anatipestifer RA-LZ01.

[0008] The invention provides a mutant strain RA-LZ01ΔAlr of Riemerella anatipestifer. The deposit number of the mutant strain RA-LZ01ΔAlr is CCTCC NO: M 2024877.

[0009] The present invention provides a method for constructing Riemerella anatipestifer RA-LZ01ΔAlr according to the above technical solution, comprising:

[0010] Using Riemerella anatipestifer RA-LZ01 as the basic strain, the alanine racemase Alr gene was knocked out to obtain the mutant strain RA-LZ01ΔAlr.

[0011] Preferably, the method for knocking out the alanine racemase Alr gene comprises the following steps:

[0012] The targeting fragment is cloned into a suicide plasmid to obtain a recombinant suicide plasmid; the nucleotide sequence of the targeting fragment is shown in SEQ ID NO.11;

[0013] Transforming the recombinant suicide plasmid into competent Escherichia coli cells and culturing until a single clone is formed to obtain a donor strain;

[0014] The donor strain is combined with the recipient strain Riemerella anatipestifer RA-LZ01 and cultured until a single clone is formed to obtain Riemerella anatipestifer RA-LZ01ΔAlr.

[0015] Preferably, the method for preparing the targeting fragment comprises:

[0016] The upstream homology arm, the downstream homology arm of the Alr gene and the erythromycin gene ermF were amplified respectively to obtain the upstream homology arm gene fragment of the Alr gene, the downstream homology arm gene fragment of the Alr gene and the ermF gene fragment;

[0017] The upstream homologous arm gene fragment of the Alr gene, the ermF gene fragment and the downstream homologous arm gene fragment of the Alr gene are connected to obtain a targeting fragment.

[0018] Preferably, the primers for amplifying the upstream homology arm of the Alr gene include Alr-Up-F and Alr-Up-R; the nucleotide sequence of the Alr-Up-F is shown in SEQ ID NO.5; the nucleotide sequence of the Alr-Up-R is shown in SEQ ID NO.6;

[0019] The primers for amplifying the downstream homology arm of the Alr gene include Alr-Do-F and Alr-Do-R; the nucleotide sequence of the Alr-Do-F is shown in SEQ ID NO.9; the nucleotide sequence of the Alr-Do-R is shown in SEQ ID NO.10;

[0020] The primers for amplifying the ermF fragment of the erythromycin gene include Erm-F and Erm-R; the nucleotide sequence of the Erm-F is shown in SEQ ID NO.7; the nucleotide sequence of the Erm-R is shown in SEQ ID NO.8.

[0021] The present invention provides the use of the mutant strain described in the above technical scheme, the mutant strain RA-LZ01ΔAlr described in the above technical scheme, or the mutant strain RA-LZ01ΔAlr constructed by the construction method described in the above technical scheme in preparing products for preventing and / or treating diseases caused by Riemerella anatipestifer.

[0022] The present invention provides a live vaccine of Riemerella anatipestifer, comprising: the mutant strain described in the above technical solution, the mutant strain RA-LZ01ΔAlr described in the above technical solution, or the mutant strain RA-LZ01ΔAlr constructed by the construction method described in the above technical solution.

[0023] The present invention provides a method for preparing a Riemerella anatipestifer live vaccine, comprising the following steps:

[0024] The Riemerella anatipestifer RA-LZ01ΔAlr strain is cultured in a medium containing D-alanine to obtain a Riemerella anatipestifer RA-LZ01ΔAlr bacterial liquid;

[0025] After the Riemerella anatipestifer RA-LZ01ΔAlr bacterial liquid is separated to obtain bacterial bodies, the bacterial bodies are washed and resuspended to obtain a Riemerella anatipestifer live vaccine.

[0026] Preferably, the mass concentration of D-alanine in the culture medium is ≥50 mg / L.

[0027] Beneficial effects of the present invention:

[0028] The present invention provides a mutant strain of Riemerella anatipestifer, wherein the mutant strain includes a strain obtained by knocking out the alanine racemase Alr gene in Riemerella anatipestifer RA-LZ01. Alanine racemase (Alr; EC 5.1.1.1) is a homodimeric enzyme containing pyridoxal-5' phosphate (PLP), which catalyzes the mutual conversion of L-alanine into D-alanine. D-alanine is an important component of peptidoglycan in the cell wall of Gram-positive and Gram-negative bacteria. Peptidoglycan (PG) is the main component of bacterial cell walls, enabling bacteria to tolerate harsh environments. In addition, PG is also essential for maintaining cell morphology and normal growth and division. The present invention obtains a mutant strain by knocking out the alanine racemase Alr gene in Riemerella anatipestifer RA-LZ01. The obtained mutant strain can grow normally after adding D-alanine in the culture medium, and the toxicity is greatly reduced compared with Riemerella anatipestifer RA-LZ01, and can effectively resist the infection of wild-type Riemerella anatipestifer. The results of the embodiment show that the Riemerella anatipestifer mutant strain RA-LZ01ΔAlr prepared by the present invention is used to immunize healthy ducklings for the prevention of Riemerella anatipestifer disease, which has the advantages of safety and high protection rate. The mutant strain will have a wide range of application prospects in the field of prevention and treatment of Riemerella anatipestifer disease.

[0029] Biological deposit information

[0030] Riemerella anatipestifer, strain number RA-LZ01ΔAlr, was deposited in the China Center for Type Culture Collection (CCTCC) on May 8, 2024, Wuhan, China, with the deposit number CCTCCNO: M 2024877. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0032] Figure 1 This is the result of PCR identification of the donor bacteria;

[0033] Figure 2 This is the electrophoresis detection result of the PCR product amplified by the conservative primers OmpA-F / OmpA-R of Riemerella anatipestifer;

[0034] Figure 3This is the electrophoresis detection result of the PCR product amplified by the Alr gene primers Alr-F / Alr-R;

[0035] Figure 4 The results of blood bacterial load determination of ducklings infected with wild strain and RA-LZ01ΔAlr at different times;

[0036] Figure 5 This is the monitoring result of duckling survival rate after challenge with wild strain and RA-LZ01ΔAlr;

[0037] Figure 6 This is the weight monitoring result of ducklings after infection with wild strain and RA-LZ01ΔAlr;

[0038] Figure 7 This is the result of measuring the bacterial load in the blood of ducklings challenged with poison after immunization;

[0039] Figure 8 This is a graph showing the survival rate test results of ducklings challenged with poison after immunization. DETAILED DESCRIPTION

[0040] The invention provides a mutant strain of Riemerella anatipestifer. The mutant strain comprises a strain obtained by knocking out an alanine racemase Alr gene in Riemerella anatipestifer RA-LZ01.

[0041] Alanine racemase (Alr; EC 5.1.1.1) is a homodimeric enzyme containing pyridoxal-5' phosphate (PLP), which catalyzes the mutual conversion of L-alanine into D-alanine. D-alanine is an important component of peptidoglycan in the cell walls of Gram-positive and Gram-negative bacteria. Peptidoglycan (PG) is the main component of bacterial cell walls, enabling bacteria to tolerate harsh environments. In addition, PG is also essential for maintaining cell morphology and normal growth and division. The present invention obtains a mutant strain RA-LZ01ΔAlr by knocking out the alanine racemase Alr gene in Riemerella anatipestifer RA-LZ01. The obtained mutant strain RA-LZ01ΔAlr can grow normally after adding D-alanine to the culture medium, and its toxicity is greatly reduced compared with Riemerella anatipestifer RA-LZ01, and it can effectively resist infection by wild-type Riemerella anatipestifer.

[0042] The present invention provides a mutant strain RA-LZ01ΔAlr of Riemerella anatipestifer, and the deposit number of the mutant strain RA-LZ01ΔAlr is CCTCC NO: M 2024877. The present invention uses the mutant strain RA-LZ01ΔAlr of Riemerella anatipestifer to immunize healthy ducklings for preventing Riemerella anatipestifer disease, which has the advantages of safety, high protection rate, etc.

[0043] The present invention provides a method for constructing Riemerella anatipestifer RA-LZ01ΔAlr, comprising:

[0044] Using Riemerella anatipestifer RA-LZ01 as the basic strain, the alanine racemase Alr gene was knocked out to obtain the mutant strain RA-LZ01ΔAlr.

[0045] The present invention has no particular limitation on the method for knocking out the alanine racemase Alr gene, and any conventional method in the art may be used. In the present invention, the method for knocking out the alanine racemase Alr gene preferably comprises the following steps:

[0046] The targeting fragment is cloned into a suicide plasmid to obtain a recombinant suicide plasmid; the nucleotide sequence of the targeting fragment is shown in SEQ ID NO.11;

[0047] Transforming the recombinant suicide plasmid into competent Escherichia coli cells and culturing until a single clone is formed to obtain a donor strain;

[0048] The donor strain is combined with the recipient strain Riemerella anatipestifer RA-LZ01 and cultured until a single clone is formed to obtain Riemerella anatipestifer RA-LZ01ΔAlr.

[0049] The present invention has no special limitation on the preparation method of the targeting fragment, and the targeting fragment can be prepared by conventional methods in the art. In the present invention, the preparation method of the targeting fragment preferably includes: respectively amplifying the upstream homology arm, the downstream homology arm and the erythromycin gene ermF of the Alr gene to obtain the upstream homology arm gene fragment of the Alr gene, the downstream homology arm gene fragment of the Alr gene and the ermF gene fragment; connecting the upstream homology arm gene fragment of the Alr gene, the ermF gene fragment and the downstream homology arm gene fragment of the Alr gene to obtain the targeting fragment.

[0050] The present invention preferably uses the genome of Riemerella anatipestifer RA-LZ01 as a template to amplify the upstream homologous arm and the downstream homologous arm of the Alr gene respectively. The present invention preferably adopts a bacterial liquid PCR method to amplify the upstream homologous arm and the downstream homologous arm of the Alr gene respectively. The primers for amplifying the upstream homologous arm of the Alr gene of the present invention preferably include Alr-Up-F and Alr-Up-R; the nucleotide sequence of the Alr-Up-F is preferably as shown in SEQ ID NO.5; the nucleotide sequence of the Alr-Up-R is preferably as shown in SEQ ID NO.6. The primers for amplifying the downstream homologous arm of the Alr gene of the present invention preferably include Alr-Do-F and Alr-Do-R; the nucleotide sequence of the Alr-Do-F is preferably as shown in SEQ ID NO.9; the nucleotide sequence of the Alr-Do-R is preferably as shown in SEQ ID NO.10. As an practicable manner, the PCR amplification system for amplifying the upstream homologous arm and the downstream homologous arm of the Alr gene respectively in the present invention preferably includes: 25 μL of 2×Taq enzyme, 2 μL of upstream primer, 2 μL of downstream primer, 5 μL of RA-LZ01 inactivated bacterial solution, and ddH2O added to a final volume of 50 μL; the concentration of the upstream primer is preferably 10 μM; the concentration of the downstream primer is preferably 10 μM. As an practicable manner, the reaction procedure of the PCR preferably includes: 95°C 5min; 95°C 30s, 60°C 30s, 72°C 30s; 35 cycles; 72°C 10min. After the PCR reaction is completed, the present invention preferably sequences the PCR product to detect the accuracy of the obtained PCR product. After the PCR reaction is completed, the present invention preferably further includes purifying the obtained PCR products to obtain the upstream homologous arm gene fragment of the Alr gene and the downstream homologous arm gene fragment of the Alr gene. The present invention does not specifically limit the purification method, and conventional purification methods in the art can be used.

[0051] The present invention preferably uses the genome of the LJW-2 strain as a template to amplify the erythromycin gene ermF. The present invention preferably uses a bacterial solution PCR method to amplify the erythromycin gene ermF. The primers for amplifying the erythromycin gene ermF fragment of the present invention preferably include Erm-F and Erm-R; the nucleotide sequence of the Erm-F is preferably as shown in SEQ ID NO.7; the nucleotide sequence of the Erm-R is preferably as shown in SEQ ID NO.8. As an practicable manner, the PCR amplification system for amplifying the erythromycin gene ermF of the present invention preferably includes: 25 μL of 2×Taq enzyme, 2 μL of upstream primer, 2 μL of downstream primer, 5 μL of inactivated bacterial solution of the LJW-2 strain, and ddH2O added to a final volume of 50 μL; the concentration of the upstream primer is preferably 10 μM; the concentration of the downstream primer is preferably 10 μM. As an practicable manner, the reaction procedure of the PCR preferably includes: 95°C 5min; 95°C 30s, 58°C 30s, 72°C 30s; 35 cycles; 72°C 10min. After the PCR reaction is completed, the present invention preferably sequences the PCR product to detect the accuracy of the obtained PCR product. After the PCR reaction is completed, the present invention preferably further comprises purifying the obtained PCR product to obtain the ermF gene fragment. The present invention has no particular limitation on the purification method, and any conventional purification method in the art can be used.

[0052] After obtaining the upstream homology arm gene fragment of the Alr gene, the downstream homology arm gene fragment of the Alr gene and the ermF gene fragment, the present invention preferably connects the upstream homology arm gene fragment of the Alr gene, the ermF gene fragment and the downstream homology arm gene fragment of the Alr gene to obtain a targeting fragment. The present invention preferably first connects the upstream homology arm gene fragment of the Alr gene with the ermF gene fragment, and then connects it with the downstream homology arm gene fragment of the Alr gene. The present invention is not particularly limited to the method of connection, and conventional connection methods in the art can be used. The present invention preferably uses fusion reaction PCR to first connect the upstream homology arm gene fragment of the Alr gene with the ermF gene fragment; as an implementable method, the reaction system of the fusion reaction PCR preferably includes: 2.5 μL of the upstream homology arm gene fragment of the Alr gene, 2.5 μL of the ermF gene fragment, 25 μL of 2×Taq enzyme, and ddH2O added to a final volume of 50 μL; as an implementable method, the reaction procedure of the fusion reaction PCR preferably includes: 95°C for 5 min; 95°C for 30 s, 60°C for 30 s, 72°C for 1 min; 15 cycles; 72°C for 10 min. After the fusion reaction PCR is completed, a fusion reaction PCR product is obtained. After obtaining the fusion reaction PCR product, the present invention preferably performs PCR amplification on the fusion reaction PCR product; the PCR amplification preferably uses Alr-Up-F as the upstream primer and preferably uses Erm-R as the downstream primer; as an practicable manner, the reaction system of the PCR amplification preferably includes: 25 μL of 2×Taq enzyme, 2 μL of upstream primer, 2 μL of downstream primer, 5 μL of fusion reaction PCR product, and ddH2O added to a final volume of 50 μL; the concentration of the upstream primer is preferably 10 μM; the concentration of the downstream primer is preferably 10 μM. In the present invention, as an practicable manner, the reaction procedure of the PCR amplification preferably includes: 95°C for 5 min; 95°C for 30 s, 60°C for 30 s, 72°C for 1 min; 35 cycles; 72°C for 10 min. After the PCR amplification reaction is completed, the present invention preferably obtains a gene fragment of 1401 bp in size, which is the Alr-U-ermF fragment.

[0053] After obtaining the Alr-U-ermF fragment, the present invention preferably connects the Alr-U-ermF fragment with the downstream homology arm gene fragment of the Alr gene. In the present invention, the connection method is the same as above, specifically as follows: the present invention preferably uses fusion reaction PCR to first connect the Alr-U-ermF fragment with the downstream homology arm gene fragment of the Alr gene; as an implementable method, the reaction system of the fusion reaction PCR preferably includes: 2.5 μL of Alr-U-ermF fragment, 2.5 μL of downstream homology arm gene fragment of the Alr gene, 2×Taq enzyme 25 μL, ddH2O added to a final volume of 50 μL; as an implementable method, the reaction procedure of the fusion reaction PCR preferably includes: 95°C 5min; 95°C 30s, 60°C 30s, 72°C 1min; 15 cycles; 72°C 10min. After the fusion reaction PCR is completed, the fusion reaction PCR product is obtained. After obtaining the fusion reaction PCR product, the present invention preferably performs PCR amplification on the fusion reaction PCR product; the PCR amplification preferably uses Alr-Up-F as the upstream primer and Alr-Do-R as the downstream primer; as an practicable manner, the reaction system of the PCR amplification preferably includes: 25 μL of 2×Taq enzyme, 2 μL of upstream primer, 2 μL of downstream primer, 5 μL of fusion reaction PCR product, and ddH2O added to a final volume of 50 μL; the concentration of the upstream primer is preferably 10 μM; the concentration of the downstream primer is preferably 10 μM. In the present invention, as an practicable manner, the reaction procedure of the PCR amplification preferably includes: 95°C for 5 min; 95°C for 30 s, 60°C for 30 s, 72°C for 1 min; 35 cycles; 72°C for 10 min. After the PCR amplification reaction is completed, the present invention preferably obtains a gene fragment of 2001 bp in size, which is the Alr-U-ermF-Alr-D fragment. In the present invention, the Alr-U-ermF-Alr-D fragment is the targeting fragment. The present invention preferably performs electrophoresis detection and sequencing on the targeting fragment to confirm that the correct targeting fragment is obtained. In the present invention, the nucleotide sequence of the targeting fragment is shown as SEQ ID NO.11. The present invention preferably also includes purifying the targeting fragment. The present invention does not specifically limit the purification method, and any conventional purification method in the art can be used.

[0054] After obtaining the targeting fragment, the present invention clones the targeting fragment into a suicide plasmid. The present invention does not specifically limit the cloning method, and any conventional cloning method in the art can be used. In the present invention, the suicide plasmid preferably includes pRE112. In the present invention, the cloning method preferably includes: performing double enzyme digestion on the targeting fragment and the suicide plasmid, respectively. The double enzyme digestion described in the present invention is preferably performed using XbaⅠ and SphⅠ. The present invention does not specifically limit the double enzyme digestion method, and any conventional double enzyme digestion method in the art can be used. After performing double enzyme digestion, the present invention preferably obtains the target fragment after enzyme digestion and the suicide plasmid linear vector, respectively. After obtaining the target fragment after enzyme digestion and the suicide plasmid linear vector, the present invention preferably connects the target fragment after enzyme digestion and the suicide plasmid linear vector. The connection described in the present invention is preferably performed using T4 ligase. After the connection is completed, the present invention obtains a recombinant suicide plasmid.

[0055] After obtaining the recombinant suicide plasmid, the present invention preferably transforms the recombinant suicide plasmid into Escherichia coli competent cells, cultures until monoclonal formation, and obtains a donor strain. The present invention does not specifically limit the transformation method, and any conventional transformation method in the art can be used. In the present invention, the Escherichia coli competent cells preferably include X7213 strains. In the present invention, the transformation preferably includes: after mixing the recombinant suicide plasmid with the Escherichia coli competent cells, performing a first ice bath, a heat shock, and a second ice bath in sequence. As an practicable manner, the present invention preferably adds 2 μL of a recombinant suicide plasmid with a concentration of 200 ng / pL to 100 μL of competent cells of the X7213 strain for mixing. As an practicable manner, the time of the first ice bath in the present invention is preferably 30 min; the temperature of the heat shock is preferably 42° C.; the time of the heat shock is preferably 45 s; and the time of the second ice bath is preferably 3 min. After the second ice bath is completed, the present invention preferably resuscitates the obtained strain. In the present invention, as an practicable manner, the resuscitation culture is preferably carried out in a non-resistant LB liquid culture medium; the temperature of the resuscitation culture is preferably 37°C; the time of the resuscitation culture is preferably 1h; the resuscitation culture is preferably carried out at a speed of 200r / min. As an practicable manner, after the resuscitation culture is completed, the present invention preferably applies the strain after the resuscitation culture to an LB solid plate containing 50mg / L chloramphenicol and 50mg / LDPA for culture; after the culture is cultured to obtain a single colony, the present invention preferably picks a single colony for enrichment culture in an LB liquid culture medium containing 50mg / L chloramphenicol and 50mg / LDPA; the temperature of the enrichment culture is preferably 37°C; the time of the enrichment culture is preferably 4h. After the enrichment culture is completed, the present invention preferably performs PCR verification on the obtained bacterial solution. In the present invention, the PCR verification is preferably performed with Alr-Up-F as the upstream primer and preferably with Alr-Do-R as the downstream primer. The system and method for PCR verification of the present invention are the same as above, and will not be repeated here. The present invention confirms the donor strain through the PCR verification.

[0056] After obtaining the donor strain, the present invention combines the donor strain with the recipient strain Riemerella anatipestifer RA-LZ01, and cultures them until a single clone is formed to obtain Riemerella anatipestifer RA-LZ01ΔAlr.

[0057] The present invention has no particular limitation on the combining method, and any conventional combining method in the art may be used.

[0058] After obtaining the donor strain, as an practicable method, the present invention preferably cultured the donor strain in LB liquid medium containing 50 mg / L DPA and 50 mg / L chloramphenicol until OD 600 The donor bacterial solution was obtained at about 0.6. The OD600 After about 0.6 of the donor bacterial liquid, the present invention preferably takes the donor bacterial liquid for centrifugation, collects the bacteria, and obtains the donor strain bacteria; as an practicable manner, the centrifugal speed is preferably 6000r / min; the centrifugal time is preferably 5min; the centrifugal temperature is preferably 4°C. After obtaining the donor strain bacteria, the present invention preferably resuspends the donor strain bacteria in MgSO4 solution; as an practicable manner, the molar concentration of the MgSO4 solution is preferably 10mmoL / L; the volume ratio of the MgSO4 solution to the donor bacterial liquid is preferably 2:1. After resuspension, the present invention preferably centrifuges the obtained resuspended bacterial liquid; as an practicable manner, the centrifugal speed is preferably 6000r / min; the centrifugal time is preferably 10min; the centrifugal temperature is preferably 4°C. The present invention preferably repeats the centrifugation after resuspension with MgSO4 solution for 3 times, and finally mixes the obtained bacteria with the MgSO4 solution to obtain a resuspended donor bacteria; as an practicable method, the molar concentration of the MgSO4 solution is preferably 10mmoL / L; the volume of the MgSO4 solution is preferably 500μL. The present invention preferably combines the resuspended donor bacteria with the recipient strain.

[0059] In the present invention, as an practicable method, the recipient bacterial solution Riemerella anatipestifer RA-LZ01 is preferably cultured in a medium containing 2 mg / L polymyxin B in TSB until OD 600 The OD value is about 1.0, and the recipient bacterial solution is obtained. 600 After about 1.0 of the acceptor bacterial solution, the present invention preferably takes the acceptor bacterial solution for centrifugation, collects the bacterial cells, and obtains the acceptor strain bacterial cells; as an practicable manner, the centrifugal speed is preferably 6000r / min; the centrifugal time is preferably 5min; the centrifugal temperature is preferably 4°C. After obtaining the acceptor strain bacterial cells, the present invention preferably resuspends the acceptor strain bacterial cells with MgSO4 solution; as an practicable manner, the molar concentration of the MgSO4 solution is preferably 10mmoL / L; the volume ratio of the MgSO4 solution to the acceptor bacterial solution is preferably 4:1. After resuspension, the present invention preferably centrifuges the obtained resuspended bacterial solution; as an practicable manner, the centrifugal speed is preferably 6000r / min; the centrifugal time is preferably 10min; the centrifugal temperature is preferably 4°C. The present invention preferably repeats the centrifugation after resuspension with MgSO4 solution for 3 times, and finally mixes the obtained bacteria with the MgSO4 solution to obtain a resuspended recipient bacteria; as an practicable method, the molar concentration of the MgSO4 solution is preferably 10mmoL / L; the volume of the MgSO4 solution is preferably 500μL. The present invention preferably combines the resuspended recipient bacteria with the donor strain.

[0060] After obtaining the resuspended donor bacteria and the resuspended acceptor bacteria, the present invention preferably mixes the resuspended donor bacteria and the resuspended acceptor bacteria to obtain a mixed bacterial solution. After obtaining the mixed bacterial solution, the present invention preferably filters the mixed bacterial solution; as an practicable manner, the filtration is preferably performed using a 0.22 μm sterile nitrocellulose membrane. After the filtration is completed, as an practicable manner, the present invention preferably affixes the sterile side of the filter membrane to a TSA plate containing 50 mg / LDPA for culturing, and the culturing temperature is preferably 37°C; the culturing time is preferably 24h; the culturing is preferably performed in a 5% CO2 incubator. After the culturing is completed, as an practicable manner, the present invention preferably uses 2mL 10mmoL / L MgSO4 to wash off the bacterial moss on the filter membrane to obtain a coated bacterial solution. After obtaining the coated bacterial solution, as an practicable method, the present invention preferably applies the coated bacterial solution to a TSA plate containing 1 mg / L erythromycin, 2 mg / L polymyxin B, 100 mg / LD-alanine, and 5% fetal bovine serum for screening and culture; the temperature of the screening and culture is preferably 37°C; the time of the screening and culture is preferably 24 to 48 hours; the screening and culture is preferably carried out in a 5% CO2 incubator. After the screening and culture obtains a single colony, as an practicable method, the present invention preferably picks the obtained single colony and inoculates it in a TSA medium containing 1 mg / L erythromycin, 2 mg / L polymyxin B, 100 mg / LD-alanine, and 5% fetal bovine serum for enrichment culture; the temperature of the enrichment culture is preferably 37°C; the time of the enrichment culture is preferably 6 to 8 hours. After the enrichment culture is completed, the present invention preferably performs PCR verification on the obtained strain. As an practicable manner, the PCR verification of the present invention preferably uses conservative primers OmpA-F / OmpA-R of Riemerella anatipestifer and Alr gene primers Alr-F / Alr-R for PCR verification respectively; the nucleotide sequence of the OmpA-F is preferably as shown in SEQ ID NO.1; the nucleotide sequence of the OmpA-R is preferably as shown in SEQ ID NO.2; the nucleotide sequence of the Alr-F is preferably as shown in SEQ ID NO.3; the nucleotide sequence of the Alr-R is preferably as shown in SEQ ID NO.4. The present invention does not specifically limit the method of the PCR verification, and conventional methods in the art can be used. The present invention performs the PCR verification, and if the OmpA gene can amplify the target band but the Alr gene cannot amplify the target band, it means that the Alr gene is successfully knocked out, proving that the recombinant bacteria, i.e., Riemerella anatipestifer RA-LZ01ΔAlr, are obtained.

[0061] The Riemerella anatipestifer RA-LZ01ΔAlr constructed by the above technical scheme of the present invention is a mutant strain obtained by knocking out the alanine racemase Alr gene in Riemerella anatipestifer RA-LZ01. The obtained mutant strain can grow normally after adding D-alanine in the culture medium, and the toxicity is greatly reduced compared with Riemerella anatipestifer RA-LZ01, and can effectively resist the infection of wild-type Riemerella anatipestifer. The results of the embodiment show that the Riemerella anatipestifer mutant strain RA-LZ01ΔAlr prepared by the present invention is used to immunize healthy ducklings for the prevention of Riemerella anatipestifer disease, which has the advantages of safety and high protection rate. The mutant strain will have a wide range of application prospects in the field of prevention and treatment of Riemerella anatipestifer disease.

[0062] The present invention provides the use of the mutant strain described in the above technical scheme, the mutant strain RA-LZ01ΔAlr described in the above technical scheme, or the mutant strain RA-LZ01ΔAlr constructed by the construction method described in the above technical scheme in preparing products for preventing and / or treating diseases caused by Riemerella anatipestifer.

[0063] The present invention provides a live vaccine of Riemerella anatipestifer, comprising: the mutant strain described in the above technical solution, the mutant strain RA-LZ01ΔAlr described in the above technical solution, or the mutant strain RA-LZ01ΔAlr constructed by the construction method described in the above technical solution.

[0064] The present invention provides a method for preparing a Riemerella anatipestifer live vaccine, comprising the following steps:

[0065] The Riemerella anatipestifer RA-LZ01ΔAlr strain is cultured in a medium containing D-alanine to obtain a Riemerella anatipestifer RA-LZ01ΔAlr bacterial liquid;

[0066] After the Riemerella anatipestifer RA-LZ01ΔAlr bacterial liquid is separated to obtain bacterial bodies, the bacterial bodies are washed and resuspended to obtain the Riemerella anatipestifer live vaccine.

[0067] In the present invention, the mass concentration of D-alanine in the culture medium is ≥50 mg / L.

[0068] The present invention cultivates the Riemerella anatipestifer RA-LZ01ΔAlr strain in a culture medium containing D-alanine to obtain a Riemerella anatipestifer RA-LZ01ΔAlr bacterial solution. In the present invention, the culture medium preferably includes TSB culture medium; the mass concentration of D-alanine in the culture medium is preferably ≥50 mg / L, more preferably 50-100 mg / L. In a specific embodiment of the present invention, the mass concentration of D-alanine may be 50 mg / L, 60 mg / L, 70 mg / L, 80 mg / L, 90 mg / L or 100 mg / L. The culture temperature of the present invention may be 37°C; the culture time may be 12 hours; the culture is preferably accompanied by rotation; the rotation speed may be 200 r / min. The present invention preferably adopts the above-mentioned culture method to culture the Riemerella anatipestifer RA-LZ01ΔAlr strain twice, and then prepares a live vaccine with the obtained bacterial solution. In the present invention, the two cultures are the first culture and the second culture respectively; the first culture may also be referred to as recovery culture. After the second culture is completed, the present invention obtains the bacterial solution of Riemerella anatipestifer RA-LZ01ΔAlr. The OD of the bacterial solution of Riemerella anatipestifer RA-LZ01ΔAlr in the present invention is 600 Preferably about 1.0.

[0069] After obtaining the bacterial liquid of Riemerella anatipestifer RA-LZ01ΔAlr, the present invention preferably separates the bacterial liquid of Riemerella anatipestifer RA-LZ01ΔAlr to obtain bacterial bodies, washes and resuspends the bacterial bodies to obtain a live vaccine of Riemerella anatipestifer. The present invention does not specifically limit the method for isolating bacterial bodies, and any conventional separation method in the art can be used. In a specific embodiment of the present invention, the method for isolating bacterial bodies can be centrifugation; the speed of the centrifugation can be 3000r / min. After the bacterial bodies are separated, the present invention preferably washes the bacterial bodies; the washing is preferably performed using a PBS solution; the number of washes can be 3 times. After washing, the present invention preferably uses PBS to resuspend the bacterial bodies to obtain a bacterial suspension, and the bacterial suspension can be used as a live vaccine of Riemerella anatipestifer. The present invention preferably adjusts the number of live bacteria in the bacterial suspension to 1×10 9 CFU / mL.

[0070] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0071] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0072] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0073] The RA-LZ01 strain of Riemerella anatipestifer (GenBank accession number: NZ_CP045564.1) and LJW-2 are recorded in the literature (Construction of a Riemerella anatipestifer MFS efflux pump rant gene deletion strain and its mediated drug resistance, Trade-offs, Journal of Animal Husbandry and Veterinary Medicine, 2021); Escherichia coli X7213 and suicide plasmid pRE112 were purchased from NTCC National Type Culture Collection Center; the shuttle plasmid pCPRA is recorded in the literature (Construction of a Riemerella anatipestifer MFS efflux pump rant gene deletion strain and its mediated drug resistance, Trade-offs, Journal of Animal Husbandry and Veterinary Medicine, 2021); pMD-19T vector was purchased from TaKaRa (Dalian) Company; zero vector was purchased from Beijing Quanshijin Company. Tryptone soy broth (TSB) and tryptone soy agar (TSA) were purchased from difcoTM, USA; 2,6-diaminopimelate (DPA) was purchased from Tokyo Chemical Industry Co., Ltd.; LB broth and LB agar were purchased from Beijing Solarbio Company.

[0074] The preparation of the main reagents in the following Example 1:

[0075] 0.1 mol / L CaCl2 solution: Weigh 1.11 g CaCl2 and dissolve it in 100 mL deionized water. Filter and sterilize it with a 0.22 μm filter membrane and store it at 4°C for later use.

[0076] 10mmol / LMgSO4 solution: weigh 2.4647g MgSO4 and dissolve it in 1000mL deionized water, filter it with a 0.22μm filter membrane to sterilize it, and store it at 4℃ for later use.

[0077] 2 mg / mL erythromycin solution: Weigh 0.1 g erythromycin and dissolve it in 50 mL sterile water. Filter and sterilize with a 0.22 μm bacterial filter and store in aliquots at -20°C for later use.

[0078] 50 mg / mL 2,6-diaminopimelic acid (DPA) solution: weigh 0.5 g of 2,6-diaminopimelic acid and dissolve it in 10 mL of sterile water. Filter and sterilize it with a 0.22 μm bacterial filter and store it in aliquots at -20°C for later use.

[0079] 50 mg / mL chloramphenicol solution: Weigh 0.5 g chloramphenicol and dissolve it in 10 mL anhydrous ethanol. Filter and sterilize with a 0.22 μm bacterial filter and store in aliquots at -20°C for later use.

[0080] LB liquid culture medium: weigh 10 g of tryptone, 5 g of yeast extract, and 5 g of sodium chloride (NaCl), dissolve in deionized water to a volume of 1 L, and sterilize at 121° C. for 15 min.

[0081] LB solid medium: weigh 10 g of tryptone, 5 g of yeast extract, 5 g of sodium chloride (NaCl), and 10 g of agar, dissolve in deionized water to make up to 1 L, and sterilize by high pressure at 121°C for 15 min.

[0082] Tryptone soy broth (TSB): weigh 30 g of tryptone soy broth and dissolve it in deionized water to make up to 1 L. Autoclave at 121°C for 15 min.

[0083] Tryptone soy agar (TSA): weigh 30 g of tryptone soy agar and dissolve it in deionized water to 1 L, then sterilize it by autoclaving at 121°C for 15 min.

[0084] Example 1

[0085] Construction of Alr gene deletion strain of Riemerella anatipestifer RA-LZ01

[0086] In this study, suicide plasmid pRE112 was used as a vector to construct an insertional inactivation mutant of Riemerella anatipestifer. On this basis, a pRE112 plasmid with an erythromycin resistance gene was constructed, and the deletion mutant of Riemerella anatipestifer was constructed by replacing the resistance gene. This method avoids the influence of the vector, and only one resistance screening is required to obtain the mutant. The fusion PCR method was used to fuse the upstream and downstream homologous arms of the gene to be deleted with the erythromycin resistance gene to construct a targeting fragment, in which the resistance gene is located between the upstream and downstream homologous arms of the gene. The targeting fragment was then connected to the pRE112 vector to construct a recombinant mutation vector, which was transformed into the competent cells of Escherichia coli X7213 to obtain the donor bacteria, and then the deletion mutant of Riemerella anatipestifer was obtained.

[0087] The specific steps are as follows:

[0088] 1. Preparation of Escherichia coli X7213 competent cells

[0089] (1) Escherichia coli X7213 glycerol culture was inoculated into LB liquid medium containing 50 mg / L DPA at a volume ratio of 1:100, and cultured at 37°C, 200 r / min, overnight. The revived bacterial solution was then inoculated into 50 mL of fresh LB liquid medium containing 50 mg / L DPA at a volume ratio of 1:100, and cultured at 37°C, 200 r / min, until OD 600 About 0.6.

[0090] (2) The cultured bacterial suspension was placed in a sterile 50 mL centrifuge tube and placed in an ice bath for 30 min. The tube was then centrifuged at 5000 rpm at 4°C for 10 min to collect the bacterial cells.

[0091] (3) Add 15 mL of pre-cooled 0.1 mol / L CaCl2 solution to resuspend the cells, centrifuge at 5000 rpm for 10 min at 4°C, and repeat the wash three times (i.e., repeatedly use 0.1 mol / L CaCl2 solution to resuspend the cells).

[0092] (4) Discard the supernatant, add 0.1 mol / L CaCl2 containing 10% glycerol at a volume ratio of 1:100 to resuspend the cells, and dispense into 1.5 mL sterile centrifuge tubes, 100 μL per tube. Store in a -80°C refrigerator for later use.

[0093] 2. Primer Design and Synthesis

[0094] According to the whole genome sequence of Riemerella anatipestifer RA-LZ01 strain (GenBank accession number: NZ_CP045564.1) and the sequence of erythromycin gene (GenBank accession number: AP022313.1) published on NCBI, primers for amplifying the upstream and downstream homologous arms of Alr gene and erythromycin gene were designed. At the same time, specific primers for identifying Riemerella anatipestifer were cited, analyzed and designed (see Table 1). Primers were synthesized by Xi'an Qingke Jersey Biotechnology Co., Ltd.

[0095] Table 1 Upstream and downstream homology arm genes of target genes and identification primer sequences

[0096]

[0097]

[0098] 3. Construction of knockout vector and donor bacteria

[0099] The genome of the parent strain RA-LZ01 was used as a template, and the primers Alr-Up-F and Alr-Up-R were used to amplify the upstream homologous arm Alr-U of the Alr gene; the downstream homologous arm Alr-D of the Alr gene was amplified using Alr-Do-F and Alr-Do-R; the genome of the LJW-2 strain was used as a template, and the primers Erm-F and Erm-R were used to amplify the erythromycin gene ermF (the gene sequences of the upstream and downstream homologous arms and the erythromycin gene sequence are shown in SEQ ID NO.11). Among them, the PCR amplification system for the amplification of the upstream and downstream homologous arms: 2×Taq enzyme 25μL, 10μM upstream primer 2μL, 10μM downstream primer 2μL, RA-LZ01 inactivated bacterial solution 5μL, ddH2O was added to the final volume of 50μL. Reaction program: 95℃5min; 95℃30s, 60℃30s, 72℃30s, 35 cycles; 72℃10min. PCR amplification system of erythromycin resistance gene: 25 μL of 2×Taq enzyme, 2 μL of Erm-F / Erm-R (10 μM), 5 μL of inactivated bacterial solution of LJW-2 strain, ddH2O added to a final volume of 50 μL. Reaction procedure: 95°C for 5 min, (95°C for 30 s, 58°C for 30 s, 72°C for 30 s; 35 cycles) 72°C for 10 min.

[0100] The purified gene fragments of Alr-U, ermF and Alr-D were fused in sequence. First, the fusion reaction of Alr-U and ermF was carried out. The PCR reaction system was: 2.5 μL of purified products of Alr-U and ermF, 25 μL of 2×Taq enzyme, and ddH2O was added to a final volume of 50 μL. Reaction procedure: 95℃5min, (95℃30s, 60℃30s, 72℃1min; 15 cycles) 72℃10min. The obtained PCR reaction product was used as a template for PCR amplification. The reaction system was: 25 μL of 2×Taq enzyme, 2 μL of Alr-Up-F / Erm-R (10 μM), 5 μL of PCR reaction solution, and ddH2O was added to a final volume of 50 μL. Reaction procedure: 95℃5min, (95℃30s, 60℃30s, 72℃1min; 35 cycles) 72℃10min. The obtained gene fragment of 1401 bp in size was fused with the downstream homology arm Alr-D in the same way to obtain a 2001 bp PCR product. The PCR product was subjected to agarose gel electrophoresis and gel cutting and purification, and sent to Xi'an Qingke Jersey Biotechnology Co., Ltd. for sequencing verification, confirming that the correct targeting fragment was obtained, as shown in SEQ ID NO.11, specifically:

[0101]

[0102]

[0103] Among them, the black bold font in the upper part is the upstream homology arm 600bp (1-600bp), the non-bold font in the middle is the erythromycin gene 801bp (601-1401bp), and the black bold font in the lower part is the downstream homology arm 600bp (1402-2001bp).

[0104] The target fragment and suicide vector pRE112 were double-digested with XbaⅠ and SphⅠ. The digestion system was: 20μL of target fragment or pRE112 plasmid, 2μL of XbaⅠ, 2μL of SphⅠ, 4μL of H Buffer, and ddH2O added to a final volume of 40μL. Digestion was performed at 37℃ for 4h. The target fragment and pRE112 linear vector after digestion were recovered by gel and ligated overnight with T4 ligase. The ligation system was: 1μL of T4 ligase, 1μL of T4 ligase buffer, 6μL of target fragment, and 2μL of pRE112 linear vector. The recombinant suicide plasmid pRE112-ErmF was obtained.

[0105] Add 2 μL of recombinant plasmid ppRE112-ErmF (200 ng / pL) to 100 μL of competent cells of X7213 strain, place on ice for 30 min, heat shock at 42°C for 45 s, and place on ice for 3 min. Then add 500 μL of non-resistant LB liquid culture medium. Resuscitate at 37°C and 200 r / min for 1 h. Spread on LB solid plates containing 50 mg / L chloramphenicol and 50 mg / LDPA for culture. Pick a single colony and enrich it with the same resistance (inoculate a single colony in LB liquid culture medium containing 50 mg / L chloramphenicol and 50 mg / LDPA and culture it at 37°C for 6 h). After enrichment, use primers Alr-Up-F / Alr-Do-R for PCR verification. The electrophoresis detection results are as follows: Figure 1 As shown, Figure 1 Lanes 1 to 4 in the figure are different single colonies, and lane 5 is a negative control group. The PCR product was sequenced and compared, and the result is shown in SEQ ID NO. 11. The positive donor bacteria obtained were named X7213-pRE112-ErmF.

[0106] 4. Obtaining recombinant bacteria

[0107] The gene deletion strain was constructed by combining transfer and homologous recombination.

[0108] The donor bacteria X7213-pRE112-ErmF was cultured in LB liquid medium containing 50 mg / L L DPA and 50 mg / L chloramphenicol until OD 600 About 0.6.

[0109] The recipient strain RA-LZ01 was cultured in TSB medium containing 2 mg / L polymyxin B until OD 600About 1.0.

[0110] Take 2mL of donor bacteria and 4mL of recipient bacteria, centrifuge at 4℃6000r / min for 5min, and collect the bacteria separately. Resuspend with 1mL10mmoL / L MgSO4, centrifuge at 4℃6000r / min for 10min, and repeat this step three times. Then add 500μL10mmoL / L MgSO4 solution to each, resuspend the donor bacteria and recipient bacteria separately and mix them, filter with 0.22μm sterile nitrocellulose membrane, remove the filter membrane, and stick the sterile side of the body to a 50mg / L The plate was plated with DPA and cultured in a 37°C, 5% CO2 incubator for 24 h. Then, 2 mL of 10 mmoL / L MgSO4 was used to wash off the bacterial moss on the filter membrane. 100 μL was spread on a TSA plate containing 1 mg / L erythromycin, 2 mg / L polymyxin B, 100 mg / L D-alanine, and 5% fetal bovine serum. The plate was placed in a 37°C, 5% CO2 incubator for 24-48 h. A single colony was picked and enriched (enrichment was specifically performed by enriching the plate with TSB medium at 37°C and 200 r / min for 6-8 h). PCR verification was performed using conservative primers OmpA-F / OmpA-R for Riemerella anatipestifer and primers Alr-F / Alr-R for Alr gene. The electrophoresis detection results were as follows: Figure 2 and Figure 3 As shown. Figure 2 This is the electrophoresis detection result of the PCR product amplified by the conservative primers OmpA-F / OmpA-R of Riemerella anatipestifer; Figure 3 This is the electrophoresis detection result of the PCR product amplified by Alr gene primers Alr-F / Alr-R. Figure 2 and Figure 3 1 to 7 in the figure are the detection results of different single colonies. Figure 2 1 to 7 and Figure 3 Lanes 1 to 7 in the figure correspond to the same single colony, for example, Figure 2 1 and Figure 3 The 1 in the figure corresponds to a single colony.

[0111] If the OmpA gene can amplify the target band but the Alr gene cannot amplify the target band, it means that the Alr gene knockout is successful. Figure 2 and Figure 3 It can be seen that the Alr gene was successfully knocked out in the single colonies corresponding to 5, 6, and 7. The amplified products were sequenced and compared. The Alr gene deletion strain with successful Alr gene knockout, namely the Riemerella anatipestifer Alr gene deletion strain, was named RA-LZ01ΔAlr, which can also be called Riemerella anatipestifer RA-LZ01ΔAlr.

[0112] 5. Stability test of Riemerella anatipestifer RA-LZ01ΔAlr (recombinant strain ΔAlr)

[0113] The recombinant bacteria ΔAlr was streaked on a non-resistant TSA solid medium containing 100 mg / L D-alanine and cultured in a 37°C incubator for 24 hours. The single colonies that grew were randomly picked and cultured in a non-resistant TSB containing 100 mg / L D-alanine, and cultured at 37°C 200r / min for 18 hours. The shaken bacterial solution was cultured and subcultured 30 times in the same way as above, and each generation of bacterial solution was recorded and saved and stored in a -80°C refrigerator. The primers OmpA-F / OmpA-R and Erm-F / Erm-R were used to perform PCR verification on the bacterial solution of the 1st, 5th, 10th, 15th, 20th, 25th, and 30th generations, and the amplified products were sequenced and compared to verify the stability of the recombinant bacteria ΔAlr (RA-LZ01ΔAlr). The results showed that the resistance gene successfully replaced the target gene, and it was stably present even after continuous transmission to the 30th generation, proving that the recombinant bacteria (RA-LZ01ΔAlr) was stable.

[0114] 6. Growth curves of Riemerella anatipestifer RA-LZ01ΔAlr (Riemerella anatipestifer Alr gene deletion strain, or deletion strain ΔAlr) and Riemerella anatipestifer RA-LZ01 strain

[0115] The parent strain RA-LZ01 and the deletion strain ΔAlr (RA-LZ01ΔAlr) strain (stored in glycerol) were inoculated into 5 mL TSB liquid culture medium at a volume ratio of 1:100, and 100 mg / LD-alanine was added to the culture medium containing the deletion strain ΔAlr. The culture was cultured at 37°C and 200 r / min until OD 600 The colony-forming unit (CFU) of each strain was determined. The deletion strain ΔAlr was washed three times with sterile saline and then the CFU was determined. The initial concentration of each strain was adjusted to 10 using TSB liquid medium. 9 CFU / mL. Inoculate into 10mL TSB liquid medium at a ratio of 1:100, that is, the inoculation amount of each strain is 10 8 CFU / mL. D-alanine at final concentrations of 10 mg / L, 20 mg / L, 30 mg / L, 40 mg / L, 50 mg / L, and 100 mg / L was added to the culture medium of the deletion strain ΔAlr, and cultured at 37°C and 200 r / min. 150 μL of the bacterial solution was taken every 1 hour and its OD was measured using a UV spectrophotometer. 600The values ​​were measured for a total of 15 hours. Three parallel tests were performed for each strain. The experimental results showed that 10 mg / L D-alanine could not cause Riemerella anatipestifer RA-LZ01ΔAlr to show signs of growth. As the concentration of added D-alanine increased, the growth trend of Riemerella anatipestifer RA-LZ01ΔAlr became closer to the wild strain. Until the added concentration reached 50 mg / L and above, the growth trend of Riemerella anatipestifer RA-LZ01ΔAlr was not significantly different from that of the wild strain.

[0116] It can be seen that the growth of the Alr-deficient strain of Riemerella anatipestifer was severely dose-dependent with the concentration of D-alanine. With the increase of D-alanine concentration, the growth of the strain gradually returned to normal.

[0117] Example 2

[0118] Effects of Riemerella anatipestifer RA-LZ01ΔAlr (Alr gene deletion strain of Riemerella anatipestifer, or deletion strain ΔAlr) on bacterial virulence and immunogenicity

[0119] 1. Safety evaluation of the deletion strain ΔAlr

[0120] (1) To evaluate the safety of the deletion strain ΔAlr (i.e., the Alr gene deletion strain of Riemerella anatipestifer, RA-LZ01ΔAlr) as a vaccine, 1×10 9 Three-day-old ducklings (n=6, 6 biological replicates per experimental group) were infected with RA-LZ01 and RA-LZ01ΔAlr strains at 1×10 CFU / mL by thigh muscle injection. 8 The specific injection method is to take 1×10 9 CFU / mL of bacterial solution, 0.1mL was injected into each duckling.

[0121] The number of bacteria in the blood of infected ducklings was detected at different times. The test results are shown in Table 2 and Figure 4 As shown in the figure, RA-LZ01 corresponds to the number of bacteria in the ducklings injected with RA-LZ01; ΔAlr corresponds to the number of bacteria in the ducklings injected with RA-LZ01ΔAlr.

[0122] Table 2 Bacterial counts in the blood of infected ducklings in the RA-LZ01 and RA-LZ01ΔAlr strain injection groups at different times

[0123]

[0124] From Table 2 and Figure 4The wild-type strain multiplied in the blood and maintained high levels of bacteremia during the experiment. In contrast, the D-alanine auxotrophic strain of RA-LZ01 (i.e., the Riemerella anatipestifer Alr gene deletion strain) failed to replicate significantly and was cleared from the blood.

[0125] (2) To evaluate the effect of D-alanine malnutrition on the virulence and pathogenesis of R. anatipestifer, we evaluated the ability of the RA-LZ01ΔAlr mutant to infect ducklings (n = 10).

[0126] Use 1×10 9 CFU / mL Wild-type strain and deletion strain were injected into the thigh muscle to infect 3-day-old ducklings, with 0.1 mL injected into each duckling. Inject once, and then observe the survival every day. Monitor the body weight every 3 days.

[0127] At the same time as the above strains were injected, a negative control group and a blank control group were set up. The negative control group was injected with PBS equal to the above bacterial solution; the blank control group did not receive any treatment. Each experimental group was subjected to 10 biological replicates, i.e., n=10. The number of deaths of ducklings in the RA-LZ01 and RA-LZ01ΔAlr strain injection groups was observed every day, and the weight of ducklings in the RA-LZ01ΔAlr strain injection group, the negative control group, and the blank control group was monitored every 3 days during the infection period. The survival rate test results of ducklings in the RA-LZ01 and RA-LZ01ΔAlr strain injection groups are shown in Tables 3 and Figure 5 The weight monitoring results of ducklings in the RA-LZ01ΔAlr strain injection group, negative control group and blank control group are shown in Table 4 and Figure 6 In Table 3, RA-LZ01 corresponds to the number of ducklings that died in the RA-LZ01 injection test group; ΔAlr corresponds to the number of ducklings that died in the RA-LZ01ΔAlr injection test group.

[0128] Table 3 Number of ducklings killed at different times in the RA-LZ01 and RA-LZ01ΔAlr strain injection groups

[0129]

[0130] Table 4 Weight of ducklings in the RA-LZ01ΔAlr strain injection group at different times

[0131]

[0132] From Table 3 and Figure 5As shown in Table 4, all ducklings died within 72 hours after the wild-type strain was injected. In contrast, all animals inoculated with the same amount of the deletion strain showed no obvious signs of disease, appeared active and healthy, and survived the infection for 7 days. After that, all of them were still alive after 14 days of observation. This proves that the loss of Alr leads to a significant reduction in the virulence of Riemerella anatipestifer. From Table 4 and Figure 6 It was found that at 21 days, the ducklings used in the three groups of experiments were not dead and appeared active and healthy, proving that infection with the Alr gene deletion strain would not cause harm to the ducklings and would not cause changes in the weight of the ducklings.

[0133] 2. Study on the immunogenicity of the deletion strain ΔAlr

[0134] (1) Vaccines and virulent viruses

[0135] The Riemerella anatipestifer RA-LZ01ΔAlr (RA-LZ01ΔAlr) prepared in Example 1 was used as a live vaccine strain, and the virulent RA-LZ01 strain was used for challenge.

[0136] The preparation method of live vaccine is:

[0137] RA-LZ01ΔAlr stored in glycerol was cultured in TSB medium with a final concentration of 100 mg / L at 37°C and 200 rpm for 12 h to obtain the revived bacterial solution; the revived bacterial solution was then transferred and cultured under the same conditions until the OD 600 The cultured bacterial solution was then centrifuged at 3000 r / min to collect the cells and washed three times with sterile PBS. The cells were then resuspended in PBS and adjusted to OD 600 About 1.0, and record its CFU and adjust to 1×10 9 CFU / mL.

[0138] (2) Experimental animals

[0139] 3-day-old healthy susceptible ducklings.

[0140] (3) Evaluation of immune protection effect

[0141] Fifty healthy susceptible ducklings at 3 days old were randomly divided into 5 groups, 10 in each group. Group 5 was the non-immunized control group. Groups 1 to 4 were injected with 0.1 mL of bacterial solution of RA-LZ01ΔAlr vaccine strain, 0.1 mL of bacterial solution of RA-LZ01ΔAlr vaccine strain containing a final concentration of 20 mg / L D-alanine (D-Ala), 0.1 mL of bacterial solution of RA-LZ01ΔAlr vaccine strain containing a final concentration of 50 mg / LD-Ala, and 0.1 mL PBS as PBS immunized control groups. 14 days after immunization, the same dose was used for booster immunization. At the same time, the feeding, spirit, morbidity or death were recorded. 14 days after the booster immunization, groups 1 to 4 were injected with 0.1 mL (1×10 9 CFU / mL) RA-LZ01 strain was highly virulent. Six ducks were randomly selected from each experimental group at 24h and 48h after the challenge to test the blood bacterial load, and the blood bacterial load of the duckling group was recorded. The blood bacterial load of ducklings in each experimental group is shown in Table 5 and Figure 7 The ducklings were observed for 7 days and their feeding, spirit, morbidity or death were recorded. The survival rate of ducklings in each experimental group is shown in Table 6 and Figure 8 As shown. RA-LZ01 in Table 5 and Table 6 corresponds to the relevant test results of the PBS immunization control group; ΔAlr corresponds to the relevant test results of the RA-LZ01ΔAlr vaccine strain injected; 20+ΔAlr corresponds to the relevant test results of the RA-LZ01ΔAlr vaccine strain injected with 20mg / LD-Ala; 50+ΔAlr corresponds to the relevant test results of the RA-LZ01ΔAlr vaccine strain injected with 50mg / LD-Ala.

[0142] Table 5 Bacterial load in duckling blood of each experimental group

[0143]

[0144] Table 6 Death number of ducklings in each experimental group

[0145]

[0146]

[0147] The test results showed that all ducklings were in good spirits after immunization, and their food and water intake were normal, with no significant difference compared with the control group. Figures 7-8As shown in the figure, after the virus attack, the blood bacterial load in the control group increased sharply within 48 hours, while the blood bacterial load in the immunized group was gradually cleared. In terms of survival, the ducklings in the control group showed depression, head and neck tremors, and a significant reduction in food and water intake within 24 hours, and began to die. The number of duckling deaths increased sharply within 48 hours, until all died at 72 hours. The ducklings immunized with the RA-LZ01ΔAlr vaccine strain had no obvious clinical symptoms and survived during the observation period. In the immunized groups containing RA-LZ01ΔAlr with a final concentration of 20 mg / LD-Ala and RA-LZ01ΔAlr with a final concentration of 50 mg / L D-Ala, the ducklings also had no obvious clinical symptoms, and only 1-2 ducklings died during the observation period.

[0148] 3. Evaluation of the immune protection effect of Riemerella anatipestifer RA-LZ01ΔAlr (RA-LZ01ΔAlr) prepared in Example 1 as a live vaccine strain against other Riemerella anatipestifer serotype strains

[0149] 70 healthy susceptible ducklings at 3 days old were randomly divided into 7 groups, with 10 ducks in each group. Group 5 was the non-immunized control group. Groups 1 to 3 were injected with 0.1 mL of RA-LZ01ΔAlr vaccine strain into the leg muscles. Groups 4 to 6 were injected with 0.1 mL PBS as the PBS immunized control group. 14 days after immunization, the same dose was used for booster immunization. At the same time, the feeding, spirit, morbidity or death were recorded. 14 days after the booster immunization, each thigh muscle of Group 1 was injected with 0.1 mL (1×10 9 CFU / mL) Riemerella anatipestifer CH-1 (serotype 1), recorded as the ΔAlr immunized CH-1 challenge group; the second group was injected with 0.1 mL (1×10 9 CFU / mL) Riemerella anatipestifer Yb2 (serotype 2), recorded as the Yb2 challenge group after ΔAlr immunization; the third group was injected with 0.1 mL (1×10 9 CFU / mL) Riemerella anatipestifer HXb2 (serotype 10), recorded as the ΔAlr-immunized HXb2 challenge group; the fourth group was injected with 0.1 mL (1×10 9 CFU / mL) Riemerella anatipestifer CH-1 (serotype 1) was used as the type 1 control group and recorded as the CH-1 group. In the fifth group, 0.1 mL (1×10 9 CFU / mL) Riemerella anatipestifer Yb2 (serotype 2) was used as the type 2 control group and recorded as the Yb2 group. In group 6, 0.1 mL (1×109 CFU / mL) Riemerella anatipestifer HXb2 (serum type 10), as the type 10 control group, recorded as the HXb2 group. 24h and 48h after the challenge, 6 of them were randomly selected from each experimental group for blood bacterial load detection, and the blood bacterial load of the duckling group was recorded. The blood bacterial load of the ducklings in each experimental group is shown in Table 7. At the same time, the feeding, spirit, morbidity or death were recorded for 7 days. The mortality rate of the ducklings in each experimental group is shown in Table 8.

[0150] Table 7 Bacterial load in duckling blood of each experimental group

[0151]

[0152]

[0153] Table 8 Death number of ducklings in each experimental group

[0154]

[0155] As shown in Tables 7 and 8, compared with the unvaccinated RA-LZ01ΔAlr vaccine, the vaccinated ducklings have a protective effect against high-dose challenge of other serotypes of wild strains. In addition, the bacteria in the ducklings are easily cleared within 2-3 days and do not cause any clinical symptoms in the ducklings. It can be seen that the RA-LZ01ΔAlr vaccine not only has a protective effect on its wild strain RA-LZ01, but also has a good immune protection effect on other sera of Riemerella anatipestifer.

[0156] In summary, the vaccine strain provided by the present invention has an immune protection effect on other Riemerella anatipestifer serotype strains, such as Riemerella anatipestifer CH-1 (serotype 1), Riemerella anatipestifer Yb2 (serotype 2), and Riemerella anatipestifer HXb2 (serotype 10), in addition to the immune effect on the wild type RA-LZ01. The immune protection effect is consistent with the results of RA-LZ01.

[0157] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A mutant strain of Riemerella anatipestifer, characterized in that: The mutant strain includes a strain obtained by knocking out the alanine racemase Alr gene in Riemerella anatipestifer RA-LZ01.

2. A mutant strain RA-LZ01ΔAlr of Riemerella anatipestifer, characterized in that: The deposit number of the mutant strain RA-LZ01ΔAlr is CCTCC NO: M 2024877.

3. A method for constructing Riemerella anatipestifer RA-LZ01ΔAlr according to claim 2, characterized in that: include: Using Riemerella anatipestifer RA-LZ01 as the basic strain, the alanine racemase Alr gene was knocked out to obtain the mutant strain RA-LZ01ΔAlr.

4. The construction method according to claim 3, characterized in that: The method for knocking out the alanine racemase Alr gene comprises the following steps: The targeting fragment is cloned into a suicide plasmid to obtain a recombinant suicide plasmid; the nucleotide sequence of the targeting fragment is shown in SEQ ID NO.11; Transforming the recombinant suicide plasmid into competent Escherichia coli cells and culturing until a single clone is formed to obtain a donor strain; The donor strain is combined with the recipient strain Riemerella anatipestifer RA-LZ01 and cultured until a single clone is formed to obtain Riemerella anatipestifer RA-LZ01ΔAlr.

5. The construction method according to claim 4, characterized in that: The method for preparing the targeting fragment comprises: The upstream homology arm, the downstream homology arm of the Alr gene and the erythromycin gene ermF were amplified respectively to obtain the upstream homology arm gene fragment of the Alr gene, the downstream homology arm gene fragment of the Alr gene and the ermF gene fragment; The upstream homologous arm gene fragment of the Alr gene, the ermF gene fragment and the downstream homologous arm gene fragment of the Alr gene are connected to obtain a targeting fragment.

6. The construction method according to claim 5, characterized in that: The primers for amplifying the upstream homology arm of the Alr gene include Alr-Up-F and Alr-Up-R; the nucleotide sequence of the Alr-Up-F is shown in SEQ ID NO.5; the nucleotide sequence of the Alr-Up-R is shown in SEQ ID NO.6; The primers for amplifying the downstream homology arm of the Alr gene include Alr-Do-F and Alr-Do-R; the nucleotide sequence of the Alr-Do-F is shown in SEQ ID NO.9; the nucleotide sequence of the Alr-Do-R is shown in SEQ ID NO.10; The primers for amplifying the ermF fragment of the erythromycin gene include Erm-F and Erm-R; the nucleotide sequence of the Erm-F is shown in SEQ ID NO.7; the nucleotide sequence of the Erm-R is shown in SEQ ID NO.

8.

7. Use of the mutant strain according to claim 1, the mutant strain RA-LZ01ΔAlr according to claim 2, or the mutant strain RA-LZ01ΔAlr constructed by the construction method according to claims 3 to 6 in the preparation of products for preventing and / or treating diseases caused by Riemerella anatipestifer.

8. A Riemerella anatipestifer live vaccine, characterized in that: include: The mutant strain according to claim 1, the mutant strain RA-LZ01ΔAlr according to claim 2, or the mutant strain RA-LZ01ΔAlr constructed by the construction method according to claims 3 to 6.

9. A method for preparing a Riemerella anatipestifer live vaccine, characterized in that: The following steps are involved: The Riemerella anatipestifer RA-LZ01ΔAlr strain is cultured in a medium containing D-alanine to obtain a Riemerella anatipestifer RA-LZ01ΔAlr bacterial liquid; After the Riemerella anatipestifer RA-LZ01ΔAlr bacterial liquid is separated to obtain bacterial bodies, the bacterial bodies are washed and resuspended to obtain a Riemerella anatipestifer live vaccine.

10. The preparation method according to claim 9, characterized in that: The mass concentration of D-alanine in the culture medium is ≥50 mg / L.

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