A candidate strain of *Riebelella anatipestifer* with double gene deletion attenuated vaccine, its construction method and application

Abstract

This invention discloses a candidate strain of *R. anatipestifer* double-gene deletion attenuated vaccine, its construction method, and its application. The candidate strain of *R. anatipestifer* double-gene deletion attenuated vaccine is *R. anatipestifer* strain CH-1. B739_RS01935-B739_RS07625 A candidate attenuated live vaccine strain with double gene deletion, deposited at the China Center for Type Culture Collection (CCTCC) on May 22, 2025, with accession number CCTCC NO: M20251080, at Wuhan University, Wuhan, China, has been deposited on that date. The attenuated live strain constructed in this invention showed no significant impact on the weight gain of ducklings after immunization, demonstrating good safety. Furthermore, the protection rate against challenge with the wild-type virulent RACH-1 strain reached 83.3% after immunization, indicating excellent immunoprotective efficacy.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, specifically to a candidate strain of attenuated live vaccine with double gene deletion of Riekelia anatipestifera, its construction method, and its application. Background Technology

[0002] duck plague bacillus ( Riemerellaanatipestifer *Rhizoctonia solani* (RA) is the main pathogen causing duck serositis, infecting ducks, geese, and other poultry, and currently showing a trend of spreading to chickens. This disease has a wide distribution and has become one of the most serious bacterial infectious diseases threatening my country's duck farming industry. Currently, control of this disease mainly relies on inactivated vaccines and drug treatment. Inactivated vaccines have a complex preparation process; drug treatment easily leads to drug resistance in bacteria and causes environmental pollution, and under the current trend of "reducing and limiting antibiotic use," the use of antibacterial drugs is strictly restricted. Therefore, there is an urgent need to develop a genetically engineered attenuated vaccine that can be administered with a single injection and has a good immunogenic effect.

[0003] Therefore, this patent application is filed. Summary of the Invention

[0004] To address the above problems, this invention provides a candidate strain of attenuated live vaccine with double gene deletion of Riegeria anatipestifer, a construction method and application, and also provides an attenuated live vaccine for Riegeria anatipestifer disease.

[0005] The following technical solution is specifically adopted:

[0006] The first objective of this invention is to provide a candidate strain of *Riegeria anatipestifer* double-gene deletion attenuated vaccine, namely *Riegeria anatipestifer* strain CH-1. B739_RS01935-B739_RS07625 A candidate strain for a double-gene deletion attenuated live vaccine, namely *Riegeria anatipestifer* CH-1Δ. B739_RS01935 Δ B739_RS07625 (Classified and named Riemerella anatipestifer RACH-1ΔB739_RS01935ΔB739_RS07625), accession number CCTCC NO: M20251080, accession date May 15, 2025, deposited at China Center for Type Culture Collection, Wuhan University, Wuhan, China.

[0007] The second objective of this invention is to provide a method for constructing the above-mentioned candidate strains of *Riegeria anatipestifer* double-gene deletion attenuated vaccine, including the *Riegeria anatipestifer* CH-1 strain. B739_RS01935 Construction of gene deletion-free strains and Riegeria anatipestifer CH-1 strain B739_RS01935-B739_RS07625 Construction of a double gene deletion-free strain;

[0008] The duck plague rieger strain CH-1 B739_RS01935 The construction of gene deletion-free strains includes:

[0009] (1) B739_RS01935 upstream gene fragments B739_RS01935 Up、 B739_RS01935 Downstream gene fragments B739_RS01935 Amplification of Down;

[0010] (2) pBAD24:: B739_RS01935 Up- Cfx - SacB Construction of recombinant plasmids;

[0011] (3) pBAD24:: B739_RS01935 Up -Cfx - SacB-B739_RS01935 Construction of Down recombinant plasmid;

[0012] (4) B739_RS01935 Up- Cfx - SacB - B739_RS01935 Amplification of the Down fragment: using pBAD24:: B739_ RS01935 Up -Cfx - SacB-B739_RS01935 The recombinant plasmid was used as a template for the procedure.

[0013] (5) B739_RS01935 Up- Cfx - SacB - B739_RS01935 The Down fragment was naturally transformed into RA CH-1, and positive transformants were identified, yielding strain RA CH-1Δ. B739_RS01935 ::Up -Cfx-SacB- Down;

[0014] (6) B739_RS01935 Up-down fragment construction;

[0015] (7) B739_RS01935 Natural transformation of UP-DOWN fragments and identification of positive transformants: for strain RA CH-1Δ B739_RS01935 ::Up -Cfx-SacB- After Down was cultured, it was added B739_RS01935 The UP-DOWN fragment was cultured and identified by PCR to obtain strain RA CH-1Δ B739_RS01935 .

[0016] Preferably, in step (1), specific primers containing enzyme cleavage sites are designed. B739_RS01935 Up P1 (inclusive) EcoR I. Enzyme cleavage site) B739_RS01935 Up P2 (inclusive) Kpn I) Enzyme cleavage site B739_RS01935Up gene fragments were amplified, and specific primers were designed for each. B739_RS01935 Down P1 (inclusive) Pst I. Enzyme cleavage site) B739_RS01935 DownP2 (included) Hind III restriction enzyme sites) B739_RS01935 Amplification of the Down gene fragment;

[0017] The specific primers containing the enzyme cleavage site B739_RS01935 Up P1、 B739_RS01935 Up P2, B739_ RS01935 Down P1、 B739_RS01935 The sequences of Down P2 are shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO.4, respectively;

[0018] Step (2) includes using EcoR I enzymes and Kpn I enzymes respectively for B739_RS01935 Up DNA fragments and pBAD24:: Cfx - SacB The plasmid was double-digested with enzymes;

[0019] pBAD24:: Cfx - SacB The plasmid sequence is shown in SEQ ID NO.5;

[0020] Step (3) includes using Pst I enzymes and Hind Enzymes III respectively acted on B739_RS01935 Down DNA fragments and pBAD24:: B739_RS01935 Up- Cfx - SacB The recombinant plasmid was subjected to double enzyme digestion.

[0021] Preferably, in step (4), B739_RS01935 Up- Cfx - SacB - B739_RS01935 The sequence of the Down fragment is shown in SEQ ID NO.6;

[0022] In step (6), specific fusion primer fragments are designed. B739_RS01935 UP P1、 B739_RS01935 UPP2 amplification B739_RS01935 UP DNA fragment, design specific fusion primer fragment B739_RS01935 DOWN P1、 B739_ RS01935 DOWN P2 amplification B739_RS01935DOWN DNA fragments; then amplify the DNA fragments. B739_RS01935 UPDNA fragments and B739_RS01935 DOWN DNA fragments were obtained by overlap PCR. B739_RS01935 Up-down segment;

[0023] B739_RS01935 UP P1、 B739_RS01935 UPP2, B739_RS01935 DOWN P1、 B739_ RS01935 The sequences of DOWN P2 are shown in SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, and SEQ ID NO.12, respectively.

[0024] Preferably, in step (5), two pairs of identification primers are used for the identification of positive transformants, and the sequences of the two pairs of identification primers are shown in SEQ ID NO.1 and SEQ ID NO.8; and in SEQ ID NO.4 and SEQ ID NO.7;

[0025] In step (7), the positive transformant is identified using three pairs of primers. The sequences of the three pairs of primers are shown in SEQ ID NO.9 and SEQ ID NO.12; SEQ ID NO.7 and SEQ ID NO.13; and SEQ ID NO.8 and 14.

[0026] The sequences are as follows:

[0027] B739_RS01935 Up P1 (inclusive) EcoR I. Enzyme cleavage site):

[0028] SEQ ID NO.1: CG GAATTC GATATTAAATCACTACAAAC.

[0029] B739_RS01935 Up P2 (inclusive) Kpn I. Enzyme cleavage site:

[0030] SEQ ID NO.2: CGG GGTACC AAAATAGATTTAAAGTG.

[0031] B739_RS01935 Down P1 (inclusive) Pst I. Enzyme cleavage site:

[0032] SEQ ID NO.3: AA CTGCAG TATAAAGCCACACTAGAGGG.

[0033] B739_RS01935 Down P2 (inclusive) Hind III restriction enzyme sites):

[0034] SEQ ID NO.4: CCC AAGCTT GTTCATAATTAAAGGTGC.

[0035] pBAD24:: Cfx - SacB plasmid sequence:

[0036] SEQ ID NO.5:

[0037]

[0038] B739_RS01935 Up- Cfx - SacB - B739_RS01935 Down fragment sequence:

[0039] SEQ ID NO.6:

[0040]

[0041] Identification primers Cfx P1:

[0042] SEQ ID NO. 7: TTTCATGTTCCATAAATCAGC.

[0043] Identification primers SacB P2:

[0044] SEQ ID NO. 8: ACGCGTCGACGTCGGTTATTTGTTAACTGTTAATTGTCCT.

[0045] B739_RS01935 UP P1:

[0046] SEQ ID NO.9: GACCCTGTAACGCTTTCTAAAGACTATACTTTAAGAGAAG.

[0047] B739_RS01935 UP P2:

[0048] SEQ ID NO. 10: CCCTCTAGTGTGGCTTTATACATAAAATAGATTTAAAGTG.

[0049] B739_RS01935 DOWN P1:

[0050] SEQ ID NO. 11: CACTTTAAATCTATTTTATGTATAAAGCCACACTAGAGGG.

[0051] B739_RS01935 DOWN P2:

[0052] SEQ ID NO. 12: AGGTAGCTAAAGCGGTAGAAATATTACATAAGAAATATCC.

[0053] Identification primers Cfx P2:

[0054] SEQ ID NO. 13: TACACCTGTTTTTGCATTCTTTT.

[0055] Identification primers SacB P1:

[0056] SEQ ID NO. 14: CTAGTCTAGACTAGTTTTTTTTTAACATTTGATTTTGTAT.

[0057] Preferably, the duck plague rieger bacteria CH-1 strain B739_RS01935-B739_RS07625 The construction of a double-gene deletion-free strain includes:

[0058] (1) B739_RS07625 upstream gene fragments B739_RS07625 Up、 B739_RS07625 Downstream gene fragments B739_RS07625 Amplification of Down;

[0059] (2) pBAD24:: B739_RS07625 Up- Cfx - SacB Construction of recombinant plasmids;

[0060] (3) pBAD24:: B739_RS07625 Up -Cfx - SacB - B739_RS07625 Construction of Down recombinant plasmid;

[0061] (4) B739_RS07625 Up- Cfx - SacB - B739_RS07625 Amplification of the Down fragment: using pBAD24:: B739_ RS07625 Up -Cfx - SacB - B739_RS07625 The recombinant plasmid was used as a template for the procedure.

[0062] (5) B739_RS07625 Up- Cfx - SacB - B739_RS07625 Natural transformation of the Down fragment and identification of positive transformants were performed to obtain strain RA CH-1Δ B739_RS01935 Δ B739_RS07625 ::Up- Cfx - SacB -Down;

[0063] (6) B739_RS07625 Up-down fragment construction and fusion;

[0064] (7) B739_RS07625 Natural transformation of UP-DOWN fusion fragments and identification of positive transformants: RA CH-1Δ B739_RS01935 Δ B739_RS07625 ::Up- Cfx - SacB -Down was cultured and then added B739_RS07625 The UP-DOWN fusion fragment was cultured and identified by PCR to obtain strain RA CH-1Δ B739_RS01935 Δ B739_RS07625(i.e., Riegeria anatipestifer CH-1Δ) B739_RS01935 Δ B739_RS07625 ).

[0065] Preferably, in step (1), specific primers containing enzyme cleavage sites are designed. B739_RS07625 Up P1 (inclusive) EcoR I enzyme cleavage site), B739_RS07625 Up P2 (inclusive) Kpn I restriction site) B739_RS07625 Up gene fragments were amplified, and specific primers containing restriction enzyme sites were designed. B739_RS07625 Down P1 (inclusive) Sal I enzyme cleavage site), B739_ RS07625 Down P2 (inclusive) Pst I restriction site) B739_RS07625 Amplification of the Down gene fragment;

[0066] Specific primers containing enzyme cleavage sites B739_RS07625 Up P1、 B739_RS07625 Up P2, B739_ RS07625 Down P1、 B739_RS07625 The sequences of Down P2 are shown in SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17, and SEQ ID NO.18, respectively.

[0067] Step (2) includes using EcoR I enzyme and Kpn I enzymes respectively B739_RS07625 Up DNA fragments and pBAD24:: Cfx - SacB The plasmid was double-digested with enzymes;

[0068] Step (3) includes using Sal I enzyme and Pst I enzymes respectively B739_RS07625 Down clip and pBAD24:: B739_RS07625 Up- Cfx - SacB The recombinant plasmid was subjected to double enzyme digestion.

[0069] Preferably, in step (4), B739_RS07625 Up- Cfx - SacB - B739_RS07625 The sequence of the Down fragment is shown in SEQ ID NO.19;

[0070] In step (6), specific fusion primers are designed. B739_RS07625 UP P1、 B739_RS07625UP and P2 were amplified separately. B739_RS07625 UPDNA fragment, design specific fusion primers B739_RS07625 DownP1、 B739_ RS07625 DownP2 amplification B739_RS07625 DOWN DNA fragments; then amplify the DNA fragments. B739_RS07625 UPDNA fragments and B739_RS07625 DOWN DNA fragments were obtained by overlap PCR. B739_RS07625 Up-down segment;

[0071] Specific fusion primers B739_RS07625 UP P1、 B739_RS07625 UP P2, B739_RS07625 DownP1、 B739_RS07625 The sequences of DownP2 are shown in SEQ ID NO.20, SEQ ID NO.21, SEQ ID NO.22, and SEQ ID NO.23.

[0072] Preferably, in step (5), the identification of positive transformants uses two pairs of identification primers, the sequences of which are shown in SEQ ID NO.15 and SEQ ID NO.8; and SEQ ID NO.18 and SEQ ID NO.7.

[0073] In step (7), positive transformant identification uses three pairs of identification primers. The sequences of the three pairs of identification primers are shown in SEQ ID NO.7 and SEQ ID NO.13; SEQ ID NO.8 and SEQ ID NO.14; and SEQ ID NO.20 and SEQ ID NO.23.

[0074] The sequences are as follows:

[0075] B739_RS07625 Up P1 (inclusive) EcoR I restriction site):

[0076] SEQ ID NO.15: CG GAATTC CAATAATTTTGATATTAGGG.

[0077] B739_RS07625 Up P2 (inclusive) Kpn I (restriction site):

[0078] SEQ ID NO.16: GG GGTACC AGAATTAAAAATAGAACCGC.

[0079] B739_RS07625 Down P1 (inclusive)Sal I (restriction site):

[0080] SEQ ID NO.17: ACGC GTCGAC CATTAAAGACTAAATCTGAT.

[0081] B739_RS07625 Down P2 (inclusive) Pst I (restriction site):

[0082] As shown in SEQ ID NO.18: AA CTGCAG GGCAGATTTGGAAGCACAAC.

[0083] B739_RS07625 Up- Cfx - SacB - B739_RS07625 Down clip:

[0084] SEQ ID NO.19:

[0085]

[0086] B739_RS07625 UP P1:

[0087] SEQ ID NO. 20: ATTTTAGAAGGTATAGAAAAGTCTTCTTTAAAAATAGGAG.

[0088] B739_RS07625 UP P2:

[0089] SEQ ID NO. 21: CAATCAGATTTAGTCTTTAAATAGAATTAAAAATAGAACC.

[0090] B739_RS07625 DOWN P1:

[0091] SEQ ID NO. 22: GGTTCTATTTTTAATTCTATTTAAAGACTAAATCTGATTG.

[0092] B739_RS07625 DOWN P2:

[0093] SEQ ID NO. 23: GAGATTGTGTATTTCGTTAATCAAACAAGTAATAATAG.

[0094] The third objective of this invention is to provide the application of the above-mentioned candidate strain of *Riegeria anatipestifer* double gene deletion attenuated vaccine, specifically its application in the preparation of attenuated vaccines against *Riegeria anatipestifer* disease or in the preparation of drugs for the prevention and treatment of *Riegeria anatipestifer* infection.

[0095] The fourth objective of this invention is to provide an attenuated live vaccine against duck plague Riesella disease, containing the duck plague Riesella double-gene deletion attenuated live vaccine candidate strain as described above.

[0096] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0097] The attenuated strain RA CH-1Δ constructed in this invention B739_RS01935 Δ B739_RS07625 Immunizing ducklings had no significant impact on their weight gain and demonstrated good safety. RA CH-1Δ B739_RS01935 Δ B739_RS07625 After immunizing ducklings, the protection rate against challenge with the wild-type virulent strain RACH-1 reached 83.3%, indicating a good immunization protection effect. Attached Figure Description

[0098] To more clearly illustrate the technical solutions of the exemplary embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly described below. It should be understood that the following drawings only show some embodiments of the present invention and should not be considered as a limitation of the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort. In the drawings:

[0099] Figure 1 : B739_RS01935 Up、 B739_RS01935 PCR amplification results of Down homologous fragments: M: Marker; Lane 1: B739_RS01935 Up amplified fragment electrophoresis results; Lane 2: B739_RS01935 Down amplified fragment electrophoresis results.

[0100] Figure 2 pBAD24:: B739_RS01935 Up- Cfx - SacB Construction of recombinant plasmid: M: Marker; Lane 1: B739_RS01935 Up fragment identification electrophoresis results.

[0101] Figure 3 pBAD24:: B739_RS01935 Up- Cfx - SacB - B739_RS01935 Construction of the Down recombinant plasmid: M: Marker; Lane 1: B739_RS01935 Down fragment identification of electrophoresis results.

[0102] Figure 4 : B739_RS01935 Up- Cfx - SacB - B739_RS01935 Amplification of the Down fragment: M: Marker; Lane 1: B739_RS01935 Up- Cfx - SacB - B739_RS01935 Down fragment amplification electrophoresis results.

[0103] Figure 5 :RA CH-1Δ B739_RS01935 ::Up- Cfx - SacB -Down PCR identification: M: Marker; Lane 1: B739_RS01935 Up- Cfx - SacB Fragment amplification electrophoresis results; Lane 2: Cfx - SacB - B739_RS01935 Down fragment amplification electrophoresis results.

[0104] Figure 6 : B739_RS01935 Up-down fragment construction: M: Marker; swimlane 1: B739_RS01935 UP fragment amplification electrophoresis results; Lane 2: B739_RS01935 DOWN fragment amplification electrophoresis results; Lane 3: B739_RS01935 Electrophoresis results of UP-DOWN fusion fragments.

[0105] Figure 7 :RA CH-1 Δ B739_RS01935 PCR identification: M: Marker; Lane 1: RA CH-1Δ B739_ RS01935 template Cfx Gene identification electrophoresis results; Lane 2: RA CH-1Δ B739_RS01935 template SacB Gene identification electrophoresis results; Lane 3: RA CH-1Δ B739_RS01935 template B739_RS01935 Identifying electrophoresis results; Lane 4: RACH-1 as template B739_RS01935 Identify the electrophoresis results.

[0106] Figure 8 : B739_RS07625 Up、 B739_RS07625 PCR amplification results of Down homologous fragments: M: Marker; Lane 1: B739_RS07625 Up amplified fragment electrophoresis results; Lane 2: B739_RS07625 Down amplified fragment electrophoresis results.

[0107] Figure 9 pBAD24 ::B739_RS07625 Up- Cfx - SacB Construction of recombinant plasmid: M: Marker; Lane 1: B739_RS07625 Up fragment identification electrophoresis results.

[0108] Figure 10 pBAD24 ::B739_RS07625 Up- Cfx - SacB - B739_RS07625 Construction of the Down recombinant plasmid: M: Marker; Lane 1: B739_RS07625 Down fragment identification of electrophoresis results.

[0109] Figure 11 : B739_RS07625 Up- Cfx - SacB - B739_RS07625 Amplification of the Down fragment: M: Marker; Lane 1: B739_RS07625 Up- Cfx - SacB - B739_RS07625 Down fragment amplification electrophoresis results.

[0110] Figure 12 :RA CH-1Δ B739_RS01935 Δ B739_RS07625 ::Up- Cfx - SacB -Down PCR identification: M: Marker; Lane 1: B739_RS07625 Up- Cfx - SacB Fragment amplification electrophoresis results; Lane 2: Cfx - SacB - B739_ RS07625 Down fragment amplification electrophoresis results.

[0111] Figure 13 : B739_RS07625 Up-down fragment construction: M: Marker; swimlane 1: B739_RS07625 UP fragment amplification electrophoresis results; Lane 2: B739_RS07625 DOWN fragment amplification electrophoresis results; Lane 3: B739_RS07625 Electrophoresis results of UP-DOWN fusion fragments.

[0112] Figure 14 :RA CH-1 Δ B739_RS01935 Δ B739_RS07625 PCR identification: M: Marker; Lane 1: RACH-1Δ B739_RS01935 Δ B739_RS07625 template Cfx Gene identification electrophoresis results; Lane 2: RA CH-1 Δ B739_RS01935 Δ B739_RS07625 template SacB Gene identification electrophoresis results; Lane 3: RA CH-1 Δ B739_ RS01935 Δ B739_RS07625 template B739_RS07625 Gene identification electrophoresis results; Lane 4: RA CH-1 Δ B739_ RS01935 Δ B739_RS07625 template B739_RS07625 Gene identification electrophoresis results.

[0113] Figure 15 Experimental group (immune RA CH-1) B739_RS01935-B739_RS07625 Line graph of 15-day body weight of gene-deleted attenuated strain and control group. Detailed Implementation

[0114] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the embodiments and accompanying drawings. The illustrative embodiments and descriptions of this invention are only for explaining this invention and are not intended to limit this invention.

[0115] This invention employs the following method to construct a candidate strain of *Riekelium anatipestifer* double-gene deletion attenuated vaccine:

[0116] I. Construction of a strain of *Riebelella anatipestifer* CH-1 B739_RS01935 without scarring

[0117] 1. B739_RS01935 upstream gene fragments ( B739_RS01935 Up) B739_RS01935 Downstream gene fragments ( B739_RS01935 Amplification of Down: Specific primers were designed using the RA CH-1 genome as a reference. B739_ RS01935 Up P1 (inclusive) EcoR I. Enzyme cleavage site B739_RS01935 Up P2 (inclusive) Kpn I. Enzyme cleavage site B739_ RS01935 Down P1 (included) Pst I. Enzyme cleavage site B739_RS01935 Down P2 (inclusive) Hind (III restriction site), primer sequences are shown in SEQ ID NO.1–4. The primers were synthesized by Beijing Qingke Biotechnology Co., Ltd., and amplified using the RA CH-1 genome as a template. B739_RS01935 Up DNA fragments and B739_RS01935 Down DNA fragments. After determining the length of the PCR amplification product by agarose gel electrophoresis, the relevant DNA fragments were recovered using a DNA purification and recovery kit. Subsequently, the DNA fragment concentration was determined and the fragments were stored at -20°C.

[0118] 2. pBAD24:: B739_RS01935 Up- Cfx - SacB Construction of recombinant plasmids: First, using EcoR I enzymes and Kpn I enzymes were used to treat 1 μg of enzymes. B739_RS01935 Up DNA fragment and 1μg pBAD24- Cfx - SacBThe plasmid was double-digested with enzymes. Next, the digested products were recovered using a DNA purification and recovery kit, and their concentrations were determined. Then, a ligation reaction was performed, with the plasmid:fragment ratio required to be 1:3 (molar ratio), and ligation was carried out at 16℃ for 16 h. After ligation, a heat shock transformation experiment was performed. Using the transformed colonies as templates, colony PCR was performed for identification, and the recombinant plasmid was extracted. The identification primer sequences are shown in SEQ ID NO. 1~2, pBAD24- Cfx - SacB The plasmid sequence is shown in SEQ ID NO.5.

[0119] 3. pBAD24:: B739_RS01935 Up -Cfx - SacB-B739_RS01935 Construction of Down recombinant plasmid: First, using Pst I enzymes and Hind Enzyme III was used on 1 μg of [a substance / particle / concentrate]. B739_RS01935 Down DNA fragment and 1μg pBAD24:: B739_ RS01935 Up- Cfx - SacB The recombinant plasmid was double-digested with enzymes, and its concentration was determined. After ligation, heat shock transformation, and PCR identification, the recombinant plasmid pBAD24:: was extracted. B739_RS01935 Up -Cfx - SacB-B739_RS01935 Down. The primer sequences for identification are shown in SEQ ID NO. 3–4.

[0120] 4. B739_RS01935 Up- Cfx - SacB - B739_RS01935 Amplification of the Down fragment: using pBAD24:: B739_ RS01935 Up -Cfx - SacB - B739_RS01935 PCR amplification was performed using the Down recombinant plasmid as a template. The amplification primer sequences were SEQ ID NO.1 and SEQ ID NO.4. The plasmid was then purified using a DNA purification and recovery kit to obtain the desired DNA. B739_ RS01935 Up- Cfx - SacB - B739_RS01935 The down fragment, the fragment sequence is shown in SEQ ID NO.6.

[0121] 5. B739_RS01935 Up- Cfx - SacB - B739_RS01935 The Down fragment was naturally transformed into RA CH-1, and the identification of positive transformants (first homologous recombination) was performed as follows: The RA CH-1 parental strain was cultured in liquid medium to the logarithmic growth phase, and the bacterial culture was adjusted to 1 OD / mL. Subsequently, 300 μL of bacterial culture was taken and 2 μg of purified [fragment / material] was added. B739_RS01935 Up- Cfx - SacB - B739_RS01935 The fragment was removed, mixed well, and incubated at 37°C for 1 h. 100 μL of the bacterial suspension was then plated onto 5% sterile defibrinated sheep blood agar plates containing 1 μg / mL cefoxitin (Cfx) and incubated at 37°C for 24 h. Single colonies were identified by PCR, yielding strain RA CH-1Δ. B739_RS01935 ::Up -Cfx-SacB- Down, the sequences of the two pairs of identification primers are shown in SEQ ID NO.1 and SEQ ID NO.8; SEQ ID NO.4 and SEQ ID NO.7.

[0122] 6. B739_RS01935 UP-DOWN fragment construction: Using the RA CH-1 genome as a reference, specific fusion primer fragments were designed. B739_RS01935 UP P1 / P2 and DOWN P1 / P2. Amplification was performed using the RA CH-1 genome as a template. B739_ RS01935 UP DNA fragments and B739_RS01935 DOWN DNA fragment. Subsequently, [the study / analysis]... B739_RS01935 UP DNA fragments and B739_RS01935 DOWN DNA fragments were obtained by overlap PCR. B739_RS01935 The UP-DOWN fragment, primer sequences are shown in SEQ ID NO.9~12.

[0123] 7. B739_RS01935 Natural transformation of the UP-DOWN fusion fragment and identification of positive transformants (second homologous recombination): RA CH-1Δ B739_RS01935 ::Up- Cfx - SacB - The cells were cultured in liquid medium until the logarithmic growth phase, and the bacterial concentration was adjusted to 1 OD / mL. Then, 300 μL of the bacterial culture was taken and 2 μg of purified [agent / material] was added. B739_RS01935 The UP-DOWN fragment was incubated at 37℃ for 1 h. 100 μL of the bacterial suspension was then plated onto a 5% sterile defibrinated sheep blood agar plate containing 15% sucrose and incubated at 37℃ for 24 h. Single colonies that grew well on the sucrose plate were selected and subcultured on both antibiotic-free and cefoxitin-containing blood agar plates for 24 h. Colonies that grew on antibiotic-free blood agar plates but not on cefoxitin-containing blood agar plates were selected as candidate strains for PCR identification, yielding strain RA CH-1Δ. B739_RS01935 The sequences of the three pairs of identification primers are shown in SEQ ID NO.9 and SEQ ID NO.12; SEQ ID NO.7 and SEQ ID NO.13; SEQ ID NO.8 and 14.

[0124] II. Riegeria anatipestifer CH-1 strain B739_RS01935-B739_RS07625 Construction of double gene deletion-free strains

[0125] 1. B739_RS07625 upstream gene fragments ( B739_RS07625 Up) B739_RS07625 Downstream gene fragments ( B739_RS07625 Amplification of Down: Specific primers were designed using the RA CH-1 genome as a reference. B739_ RS07625 Up P1 (inclusive) EcoR I restriction site). B739_RS07625 Up P2 (inclusive) Kpn I restriction site). B739_ RS07625 Down P1 (included) Sal I restriction site). B739_RS07625 Down P2 (inclusive) Pst I restriction site), primer sequences are shown in SEQ ID NO.15–18. Primers were synthesized by Beijing Qingke Biotechnology Co., Ltd. and amplified using the RA CH-1 genome as a template. B739_RS07625 Up DNA fragments and B739_RS07625 Down DNA fragments were extracted. The length of the PCR amplification product was determined by agarose gel electrophoresis, and the relevant DNA fragments were recovered using a DNA purification and recovery kit. Subsequently, the DNA fragment concentration was determined, and the fragments were stored at -20°C.

[0126] 2. pBAD24:: B739_RS07625 Up- Cfx - SacB Construction of recombinant plasmids: First, using EcoR I enzyme and Kpn I enzyme at 1 μg B739_RS07625 Up DNA fragment and 1 μg pBAD24- Cfx - SacB The plasmid was double-digested with enzymes. Next, the digested products were recovered using a DNA purification and recovery kit, and their concentrations were determined. Then, a ligation reaction was performed, with the plasmid:fragment ratio required to be 1:3 (molar ratio), and ligation was carried out at 16℃ for 16 h. After ligation, a heat shock transformation experiment was performed. Using the transformed colonies as templates, colony PCR was performed for identification, and the recombinant plasmid was extracted. The identification primer sequences are shown in SEQ ID NO. 15-16, pBAD24- Cfx - SacB The plasmid sequence is shown in SEQ ID NO.5.

[0127] 3. pBAD24:: B739_RS07625 Up -Cfx - SacB - B739_RS07625 Construction of Down recombinant plasmid: First, using Sal I enzyme and Pst I enzyme was used to treat 1 μg of... B739_RS07625 Down fragment and 1 μg pBAD24 ::B739_ RS07625 Up- Cfx - SacB The recombinant plasmid was double-digested with enzymes, and its concentration was determined. After ligation, heat shock transformation, and PCR identification, the recombinant plasmid pBAD24:: was extracted. B739_RS07625 Up -Cfx - SacB-B739_RS07625 Down. The primer sequences for identification are shown in SEQ ID NO. 17-18.

[0128] 4. B739_RS07625 Up- Cfx - SacB - B739_RS07625 Amplification of the Down fragment: using pBAD24:: B739_ RS07625 Up- Cfx - SacB - B739_RS07625 PCR amplification was performed using the Down recombinant plasmid as a template. The amplification primer sequences were SEQ ID NO.17 and SEQ ID NO.20. The plasmid was then recovered using a DNA purification and recovery kit to obtain the desired DNA. B739_ RS07625 Up- Cfx - SacB - B739_RS07625 The down fragment, the fragment sequence is shown in SEQ ID NO.19.

[0129] 5. B739_RS07625 Up- Cfx - SacB - B739_RS07625 Down's spontaneous transformation and identification of positive transformants (first homologous recombination): RA CH-1Δ B739_RS01935 The deletion-free strain was cultured in liquid medium until the logarithmic growth phase, and the bacterial concentration was adjusted to 1 OD / mL. Then, 300 μL of the bacterial culture was taken and 2 μg of purified [agent / material] was added. B739_RS07625 Up- Cfx - SacB - B739_RS07625 The fragment was removed, mixed well, and incubated at 37°C for 1 hour. 100 μL of the bacterial suspension was then plated onto a 5% sterile defibrinated sheep blood agar plate containing 1 μg / mL cefoxitin and incubated at 37°C for 24 hours. Single colonies were identified by PCR, yielding strain RA CH-1Δ. B739_RS01935 Δ B739_RS07625 ::Up- Cfx - SacB -Down, the sequences of the two pairs of identification primers are shown in SEQ ID NO.15 and SEQ ID NO.8; SEQ ID NO.18 and SEQ ID NO.7.

[0130] 6. B739_RS07625 UP-DOWN Fragment Construction and Fusion: Specific fusion primers were designed using the RA CH-1 genome as a reference. B739_RS07625 UP P1 / P2 and DOWN P1 / P2. Amplification was performed using the RA CH-1 genome as a template. B739_RS07625 UPDNA fragments and B739_RS07625 DOWN DNA fragment. Subsequently, [the study / analysis]... B739_RS07625 UPDNA fragments and B739_RS07625 DOWN DNA fragments were obtained by overlap PCR. B739_RS07625 The UP-DOWN fragment, primer sequences are shown in SEQ ID NO.20~23.

[0131] 7. B739_RS07625 Natural transformation of the UP-DOWN fusion fragment and identification of positive transformants (second homologous recombination): RA CH-1Δ B739_RS01935 Δ B739_RS07625 ::Up- Cfx - SacB - The cells were cultured in liquid medium until the logarithmic growth phase, and the bacterial concentration was adjusted to 1 OD / mL. Then, 300 μL of the bacterial culture was taken and 2 μg of purified [agent / material] was added. B739_ RS07625 The UP-DOWN fusion fragment was incubated at 37°C for 1 hour. 100 μL of the bacterial suspension was then plated onto a 5% sterile defibrinated sheep blood agar plate containing 15% sucrose and incubated at 37°C for 24 hours. Single colonies that grew well on the sucrose plate were selected and subcultured on both antibiotic-free and cefoxitin-containing blood agar plates for 24 hours. Colonies that grew on antibiotic-free blood agar plates but not on cefoxitin-containing blood agar plates were selected as candidate strains for PCR identification, yielding strain RA CH-1 Δ. B739_ RS01935 Δ B739_RS07625 The sequences of the three identification primer pairs are shown in SEQ ID NO.7 and SEQ ID NO.13; SEQ ID NO.8 and 14; SEQ ID NO.20 and SEQ ID NO.23.

[0132] The present invention will be described in detail below through specific embodiments.

[0133] 1. RA CH-1 B739_RS01935 - B739_RS07625 Methods for constructing gene deletion strains

[0134] 1.1, RA CH-1 B739_RS01935 Methods for constructing gene deletion strains

[0135] 1.1.1 B739_RS01935 Up、 B739_RS01935 PCR amplification of Down homologous fragments

[0136] Using the RA CH-1 genome as a reference, two pairs of specific primers were designed: B739_RS01935 Up P1 / P2 (i.e.) B739_RS01935 Up P1 and B739_RS01935 Up P2) and Down P1 / P2 (i.e.) B739_RS01935 Down P1 and B739_RS01935 Down P2). Using the RA CH-1 genome as a template, PCR amplification was performed. The system and procedure are as follows:

[0137] B739_RS01935 PCR amplification system of Up fragment

[0138]

[0139] B739_RS01935 PCR amplification system of down fragment

[0140]

[0141] The reaction procedure is as follows:

[0142]

[0143] Note: The denaturation-annealing-extension cycle needs to be repeated 30 times throughout the PCR procedure.

[0144] After the PCR reaction was completed, 5 μL of the reaction solution was taken, and 1 μL of DNA Loading Buffer was added for sample loading. The agarose gel electrophoresis results are shown in the figure. Figure 1 The DNA fragments were recovered and their concentration was determined using NanoDrop, and stored at -20°C.

[0145] B739_RS01935 Up P1 (inclusive) EcoR I. Restriction site: CG GAATTC GATATTAAATCACTACAAAC.

[0146] B739_RS01935 Up P2 (inclusive) Kpn I. Restriction site: CGG GGTACC AAAATAGATTTAAAGTG.

[0147] B739_RS01935 Down P1 (inclusive) Pst I. Restriction site: AA CTGCAG TATAAAGCCACACTAGAGGG.

[0148] B739_RS01935 Down P2 (inclusive) Hind III restriction enzyme site): CCC AAGCTT GTTCATAATTAAAGGTGC.

[0149] 1.1.2, pBAD24:: B739_RS01935 Up- Cfx - SacB Construction of recombinant plasmids

[0150] Pick B739_RS01935 Up and pBAD24- Cfx - SacB The plasmid underwent double enzyme digestion, and the system is as follows:

[0151]

[0152] After 1 h of reaction, the enzyme digestion product was recovered and its concentration was determined using NanoDrop. Subsequently, a ligation reaction was performed, requiring a plasmid:fragment ratio of n:3 (molar ratio), as shown below:

[0153]

[0154] The ligation reaction was carried out in a metal bath at 16°C for 16 hours. After ligation, a transformation operation was performed, and the transformation of each component and its purpose are as follows:

[0155] (1) Competent cell group: 200 μL of competent Escherichia coli DH5α (to observe whether the competent cells are contaminated);

[0156] (2) Double digestion group: 15 μL plasmid digestion product (untreated with ligase) + 200 μL competent Escherichia coli DH5α (to observe whether the plasmid was completely digested by the restriction enzyme);

[0157] (3) Self-ligation group: 7.5 μL plasmid digestion product + 7.5 μL Solution I ligase + 200 μL competent Escherichia coli DH5α (to observe whether the plasmid self-ligates after digestion);

[0158] (4) Experimental group: 15 μL ligation product + 200 μL competent Escherichia coli DH5α (to observe whether the ligation was successful).

[0159] After incubating each component on ice for 10 min, immediately place it in a 37℃ water bath for 5 min. Then, add 1 mL of antibiotic-free LB liquid medium to each component and incubate at 37℃ for 1 h. After incubation, spread 200 μL of each component's bacterial suspension onto an LB resistant plate (containing 100 μg / mL Amp). Centrifuge the remaining bacterial suspension at 5500 rpm for 5 min, discard 600 μL of supernatant, resuspend the remaining bacterial suspension, and spread 200 μL onto an LB resistant plate (containing 100 μg / mL Amp). Incubate at 37℃ for 12 h. Inoculate the resulting single colonies into a new LB resistant plate (containing 100 μg / mL Amp) for subculturing and then perform colony PCR identification. The reaction system is as follows:

[0160]

[0161] The reaction procedure is as follows:

[0162]

[0163] Note: The denaturation-annealing-extension cycle needs to be repeated 30 times throughout the PCR procedure.

[0164] After the PCR reaction was completed, 5 μL of the reaction solution was taken, and 1 μL of DNA Loading Buffer was added for sample loading. The agarose gel electrophoresis results are shown in the figure. Figure 2 Preserve colonies that show positive bands.

[0165] B739_RS01935 Up P1 (inclusive) EcoR I. Enzyme cleavage site): CG GAATTCGATATTAAATCACTACAAAC.

[0166] B739_RS01935 Up P2 (inclusive) Kpn I. Restriction site: CGG GGTACC AAAATAGATTTAAAGTG.

[0167] 1.1.3、pBAD24:: B739_RS01935 Up -Cfx - SacB - B739_RS01935 Construction of Down recombinant plasmid

[0168] Pick Down and pBAD24:: B739_RS01935 Up B739_RS01935 The recombinant plasmid underwent double enzyme digestion, as shown in the following system:

[0169]

[0170] After 1 h of reaction, the enzyme digestion product was recovered and its concentration was determined using NanoDrop. Subsequently, a ligation reaction was performed, requiring a plasmid:fragment ratio of n:3 (molar ratio), as shown below:

[0171]

[0172] The ligation reaction was carried out in a 16℃ metal bath for 16 hours. After ligation, transformation was performed using the same method as in 1.1.2. The PCR reaction system is as follows:

[0173]

[0174] The reaction procedure is as follows:

[0175]

[0176] Note: The denaturation-annealing-extension cycle needs to be repeated 30 times throughout the PCR procedure.

[0177] After the PCR reaction was completed, 5 μL of the reaction solution was taken, and 1 μL of DNA Loading Buffer was added for sample loading. The agarose gel electrophoresis results are shown in the figure. -Cfx - SacB Preserve colonies that show positive bands.

[0178] Figure 3 Down P1 (inclusive) B739_RS01935 I. Restriction site: AA Pst TATAAAGCCACACTAGAGGG.

[0179] CTGCAG Down P2 (inclusive)B739_RS01935 III restriction enzyme site): CCC Hind GTTCATAATTAAAGGTGC.

[0180] 1.1.4 AAGCTT Up- B739_RS01935 - Cfx - SacB Amplification of the Down Fragment

[0181] pBAD24 was extracted using a plasmid miniprep kit. B739_RS01935 Up B739_RS01935 - -Cfx - B739_ SacB Down the recombinant plasmid. Use this plasmid as a template and dilute it to 10-20 ng / μL for PCR amplification. The reaction system is as follows:

[0182]

[0183] The reaction procedure is as follows:

[0184]

[0185] Note: The denaturation-annealing-extension cycle needs to be repeated 30 times throughout the PCR procedure.

[0186] After the PCR reaction was completed, 5 μL of the reaction solution was taken, and 1 μL of DNA Loading Buffer was added for sample loading. The agarose gel electrophoresis results are shown in the figure. RS01935 Recycled and measured using NanoDrop. Figure 4 Up- B739_RS~ - Cfx - B739_ SacB The concentration of the Down fragment was stored at -20°C.

[0187] RS01935 Up P1 (inclusive) B739_RS01935 I. Enzyme cleavage site): CG EcoR GATATTAAATCACTACAAAC.

[0188] GAATTC Down P2 (inclusive) B739_RS01935 III restriction enzyme site): CCC Hind GTTCATAATTAAAGGTGC.

[0189] 1.1.5 AAGCTT Up- B739_RS01935 - Cfx - SacBNatural transformation of Down fragments and identification of positive transformants (first homologous recombination)

[0190] RA CH-1 was inoculated into TSB medium and cultured at 37°C and 180 rpm until the logarithmic growth phase (OD200). 600 =1.0-1.5). After adjusting the bacterial suspension to 1 OD / mL, take 300 μL of bacterial suspension and add 2 μg of [unspecified ingredient]. B739_RS01935 Up- B739_RS01935 - Cfx - SacB Down the fragment, mix well, and incubate at 37℃ for 1 hour. After incubation, take 100 μL of bacterial suspension and spread it on a 5% sterile defibrinated sheep blood agar plate containing 1 μg / mL cefoxitin. Incubate at 37℃ for 24 hours. Pick grayish-white single colonies and subculture them on a fresh 5% sterile defibrinated sheep blood agar plate containing 1 μg / mL cefoxitin. Perform PCR identification on the grown colonies. The system and procedure are as follows:

[0191] System (1) (Up- B739_RS01935 )

[0192]

[0193] System (2) SacB -Down)

[0194]

[0195] The PCR procedure is as follows:

[0196]

[0197] Note: The denaturation-annealing-extension cycle needs to be repeated 30 times throughout the PCR procedure.

[0198] After the PCR reaction was completed, 5 μL of the reaction solution was taken, and 1 μL of DNA Loading Buffer was added for sample loading. The agarose gel electrophoresis results are shown in the figure. Cfx Positive colonies were streaked and passaged onto new 5% sterile defibrinated sheep blood agar plates containing 1 μg / mL cefoxitin (Cfx). The plates were incubated at 37°C for 24 h. The bacterial growth was then scraped into 1 mL of sterile defibrinated sheep blood using an inoculation loop and stored at -80°C to obtain RA CH-1Δ. Figure 5 ::Up- B739_RS01935 - Cfx -Down.

[0199] SacB Up P1 (inclusive) B739_RS01935 I. Restriction site: CG EcoR GATATTAAATCACTACAAAC.

[0200] GAATTC P2: ACGCGTCGACGTCGGTTATTTGTTAACTGTTAATTGTCCT.

[0201] SacB P1:TTTCATTGTTCCATAAATCAGC.

[0202] Cfx Down P2 (inclusive) B739_RS01935 III restriction enzyme site): CCC Hind GTTCATAATTAAAGGTGC.

[0203] 1.1.6、 AAGCTT Up-down fragment construction

[0204] Using the RA CH-1 genome as a reference, specific primer fragments were designed. B739_RS01935 UP P1 / P2 and DOWN P1 / P2, and amplify them respectively using the RA CH-1 genome as a template. B739_RS01935 UP clips and B739_ B739_RS01935 DOWN fragment. Obtained by overlapping PCR of the two. RS01935 The UP-DOWN fragment, primer sequences are shown in SEQ ID NO. 9-12.

[0205] (1) B739_RS01935 PCR amplification system of UP fragment

[0206]

[0207] (2) B739_RS01935 PCR amplification system for DOWN fragments

[0208]

[0209] Reaction Procedure

[0210]

[0211] Note: The denaturation-annealing-extension cycle needs to be repeated 30 times throughout the PCR procedure.

[0212] (3) B739_RS01935 UP clips and B739_RS01935 PCR fusion system of DOWN fragments

[0213]

[0214] The reaction procedure is as follows:

[0215]

[0216] Note: The process requires 5 cycles of "denaturation-annealing-extension".

[0217] After the reaction is complete, add to the reaction solution B739_RS01935 UP P1 and B739_RS01935 DOWN P2 2.5 μL each. Continue the reaction as follows:

[0218]

[0219] Note: The process requires 25 cycles of "modification-annealing-extension".

[0220] After the PCR reaction was completed, 5 μL of the reaction solution was taken, and 1 μL of DNA Loading Buffer was added for sample loading. The agarose gel electrophoresis results are shown in the figure. B739_RS01935 Recycled and measured using NanoDrop. Figure 6 The concentration of the Up-Down fragment was stored at -20°C.

[0221] B739_RS01935 UP P1:GACCCTGTAACGCTTTCTAAAGACTATACTTTAAGAGAAG.

[0222] B739_RS01935 UP P2:CCCTCTAGTGTGGCTTTATACATAAAATAGATTTAAAGTG.

[0223] B739_RS01935 DOWN P1: CACTTTAAATCTATTTTATGTATAAAGCCACACTAGAGGG.

[0224] B739_RS01935 DOWN P2: AGGTAGCTAAAGCGGTAGAAATATTACATAAGAAATATCC.

[0225] 1.1.7 B739_RS01935 Natural transformation of UP-DOWN fusion fragments and identification of positive transformants (second homologous recombination)

[0226] RA CH-1Δ B739_RS01935 ::Up- B739_RS01935 - Cfx -Down inoculate into TSB medium and incubate at 37°C and 180 rpm until the logarithmic growth phase (OD50). 600 =1.0-1.5). After adjusting the bacterial suspension to 1 OD / mL, take 300 μL of bacterial suspension and add 2 μg of [unspecified ingredient]. SacBUP-DOWN fragment, mix well, and incubate at 37℃ for 1 h. After incubation, take 100 μL of bacterial suspension and spread it on a 5% sterile defibrinated sheep blood agar plate containing 15% sucrose, and incubate at 37℃ for 24 h. Pick single colonies that grow well on the sucrose plate and subculture them on blood agar plates without antibiotics and blood agar plates containing 1 μg / mL cefoxitin for 24 h. Colonies that grow on blood agar plates without antibiotics but cannot grow on blood agar plates containing 1 μg / mL cefoxitin are selected as candidate strains for PCR identification. The system and procedure are as follows:

[0227] System (1) — B739_RS01935 :

[0228]

[0229] System (2) — Cfx :

[0230]

[0231] The reaction procedure is as follows:

[0232]

[0233] Note: The denaturation-annealing-extension cycle needs to be repeated 30 times throughout the PCR procedure.

[0234] System (3) — Target Gene

[0235]

[0236] The reaction procedure is as follows:

[0237]

[0238] Note: The denaturation-annealing-extension cycle needs to be repeated 30 times throughout the PCR procedure.

[0239] System (4) — Positive control RA CH-1

[0240]

[0241] The reaction procedure is as follows:

[0242]

[0243] Note: The denaturation-annealing-extension cycle needs to be repeated 30 times throughout the PCR procedure.

[0244] After the PCR reaction was completed, 5 μL of the reaction solution was taken, and 1 μL of DNA Loading Buffer was added for sample loading. The agarose gel electrophoresis results are shown in the figure. SacBPositive colonies were streaked onto new 5% sterile defibrinated sheep blood agar plates and incubated at 37°C for 24 hours. The bacterial growth was then scraped into 1 mL of sterile defibrinated sheep blood using an inoculation loop and stored at -80°C to obtain RA CH-1Δ. B739_ Figure 7 .

[0245] RS01935 P1:TTTCATTGTTCCATAAATCAGC.

[0246] Cfx P2:TACACCTGTTTTTGCATTCTTTT.

[0247] Cfx P1: CTAGTCTAGACTAGTTTTTTTTTAACATTTGATTTTGTAT.

[0248] SacB P2: ACGCGTCGACGTCGGTTATTTGTTAACTGTTAATTGTCCT.

[0249] SacB UP P1:GACCCTGTAACGCTTTCTAAAGACTATACTTTAAGAGAAG.

[0250] B739_RS01935 B739_RS01935 DOWN P2: AGGTAGCTAAAGCGGTAGAAATATTACATAAGAAATATCC.

[0251] 1.2, RA CH-1 B739_RS01935 - B739_RS07625 Methods for constructing gene deletion strains

[0252] 1.2.1、 B739_RS07625 Up、 B739_RS07625 PCR amplification of Down homologous fragments

[0253] Using the RA CH-1 genome as a reference, two pairs of specific primers were designed: B739_RS07625 Up P1 / P2 and Down P1 / P2. Using the RA CH-1 genome as a template, PCR amplification was performed. The system and procedure are as follows:

[0254] (1) B739_RS07625 PCR amplification system of homologous fragments of Up

[0255]

[0256] (2) B739_RS07625 PCR amplification system of down fragment

[0257]

[0258] The reaction procedure is as follows:

[0259]

[0260] Note: The denaturation-annealing-extension cycle needs to be repeated 30 times throughout the PCR procedure.

[0261] After the PCR reaction was completed, 5 μL of the reaction solution was taken, and 1 μL of DNA Loading Buffer was added for sample loading. The agarose gel electrophoresis results are shown in the figure. Figure 8 The DNA fragments were recovered and their concentration was determined using NanoDrop, and stored at -20°C.

[0262] B739_RS07625 Up P1 (inclusive) EcoR I restriction site): CG GAATTC CAATAATTTTGATATTAGGG.

[0263] B739_RS07625 Up P2 (inclusive) Kpn I restriction site): GG GGTACC AGAATTAAAAATAGAACCGC.

[0264] B739_RS07625 Down P1 (inclusive) Sal I restriction site): ACGC GTCGAC CATTAAAGACTAAATCTGAT.

[0265] B739_RS07625 Down P2 (inclusive) Pst I restriction site): AA CTGCAG GGCAGATTTGGAAGCACAAC.

[0266] 1.2.2、pBAD24:: B739_RS07625 Up- Cfx - SacB Construction of recombinant plasmids

[0267] Pick B739_RS07625 Up and pBAD24- Cfx - SacB The plasmid underwent double enzyme digestion, and the system is as follows:

[0268]

[0269] After 1 h of reaction, the enzyme digestion product was recovered and its concentration was determined using NanoDrop. Subsequently, a ligation reaction was performed, requiring a plasmid:fragment ratio of n:3 (molar ratio), as shown below:

[0270]

[0271] The ligation reaction was carried out in a metal bath at 16°C for 16 hours. After ligation, a transformation operation was performed, and the transformation of each component and its purpose are as follows:

[0272] (1) Competent cell group: 200 μL of competent Escherichia coli DH5α (to observe whether the competent cells are contaminated);

[0273] (2) Double digestion group: 15 μL plasmid digestion product (untreated with ligase) + 200 μL competent Escherichia coli DH5α (to observe whether the plasmid was completely digested by the restriction enzyme);

[0274] (3) Self-ligation group: 7.5 μL plasmid digestion product + 7.5 μL Solution I ligase + 200 μL competent Escherichia coli DH5α (to observe whether the plasmid self-ligates after digestion);

[0275] (4) Experimental group: 15 μL ligation product + 200 μL competent Escherichia coli DH5α (to observe whether the ligation was successful).

[0276] After incubating each component on ice for 10 min, immediately place it in a 37℃ water bath for 5 min. Then, add 1 mL of antibiotic-free LB liquid medium to each component and incubate at 37℃ for 1 h. After incubation, spread 200 μL of each component's bacterial suspension onto an LB resistant plate (containing 100 μg / mL Amp). Centrifuge the remaining bacterial suspension at 5500 rpm for 5 min, discard 600 μL of supernatant, resuspend the remaining bacterial suspension, and spread 200 μL onto an LB resistant plate (containing 100 μg / mL Amp). Incubate at 37℃ for 12 h. Inoculate the resulting single colonies into a new LB resistant plate (containing 100 μg / mL Amp) for subculturing and then perform colony PCR identification. The reaction system is as follows:

[0277]

[0278] The reaction procedure is as follows:

[0279]

[0280] Note: The denaturation-annealing-extension cycle needs to be repeated 30 times throughout the PCR procedure.

[0281] After the PCR reaction was completed, 5 μL of the reaction solution was taken, and 1 μL of DNA Loading Buffer was added for sample loading. The agarose gel electrophoresis results are shown in the figure. Figure 9 Preserve colonies that show positive bands.

[0282] B739_RS07625 Up P1 (inclusive) EcoR I restriction site): CG GAATTC CAATAATTTTGATATTAGGG.

[0283] B739_RS07625 Up P2 (inclusive) Kpn I restriction site): GG GGTACC AGAATTAAAAATAGAACCGC.

[0284] 1.2.3、pBAD24:: B739_RS07625 Up -Cfx - SacB - B739_RS07625 Construction of Down recombinant plasmid

[0285] Pick B739_RS07625 Down and pBAD24:: B739_RS07625 Up -Cfx - SacB The recombinant plasmid underwent double enzyme digestion, as shown in the following system:

[0286]

[0287] After 1 h of reaction, the enzyme digestion product was recovered and its concentration was determined using NanoDrop. Subsequently, a ligation reaction was performed, requiring a plasmid:fragment ratio of n:3 (molar ratio), as shown below:

[0288]

[0289] The connection reaction was carried out in a metal bath at 16℃ for 16 hours. After connection, a conversion operation was performed, using the same method as in 1.2.2. The reaction system is as follows:

[0290]

[0291] The reaction procedure is as follows:

[0292]

[0293] Note: The denaturation-annealing-extension cycle needs to be repeated 30 times throughout the PCR procedure.

[0294] After the PCR reaction was completed, 5 μL of the reaction solution was taken, and 1 μL of DNA Loading Buffer was added for sample loading. The agarose gel electrophoresis results are shown in the figure. Figure 10Preserve colonies that show positive bands.

[0295] B739_RS07625 Down P1 (inclusive) Sal I restriction site): ACGC GTCGAC CATTAAAGACTAAATCTGAT.

[0296] B739_RS07625 Down P2 (inclusive) Pst I restriction site): AA CTGCAG GGCAGATTTGGAAGCACAAC.

[0297] 1.2.4、 B739_RS07625 Up- Cfx - SacB - B739_RS07625 Amplification of Down homologous fragments

[0298] pBAD24 was extracted using a plasmid miniprep kit. B739_RS07625 Up -Cfx - SacB - B739_ RS07625 Down the recombinant plasmid. Use this plasmid as a template and dilute it to 10-20 ng / μL for PCR amplification. The reaction system is as follows:

[0299]

[0300] The reaction procedure is as follows:

[0301]

[0302] Note: The denaturation-annealing-extension cycle needs to be repeated 30 times throughout the PCR procedure.

[0303] After the PCR reaction was completed, 5 μL of the reaction solution was taken, and 1 μL of DNA Loading Buffer was added for sample loading. The agarose gel electrophoresis results are shown in the figure. Figure 11 Recycled and measured using NanoDrop. B739_RS07625 Up- Cfx - SacB - B739_ RS07625 The concentration of the Down fragment was stored at -20°C.

[0304] B739_RS07625 Up P1 (inclusive) EcoR I restriction site): CG GAATTC CAATAATTTTGATATTAGGG.

[0305] B739_RS07625Down P2 (inclusive) Pst I restriction site): AA CTGCAG GGCAGATTTGGAAGCACAAC.

[0306] 1.2.5 B739_RS07625 Up- Cfx - SacB - B739_RS07625 Natural transformation of Down fragments and identification of positive transformants (first homologous recombination)

[0307] RA CH-1Δ B739_RS01935 Inoculate into TSB medium and incubate at 37°C and 180 rpm until the logarithmic growth phase (OD200). 600 =1.0-1.5). After adjusting the bacterial suspension to 1 OD / mL, take 300 μL of bacterial suspension and add 2 μg of [unspecified ingredient]. B739_RS07625 Up- Cfx - SacB - B739_RS07625 Down the fragment, mix well, and incubate at 37℃ for 1 hour. After incubation, take 100 μL of bacterial suspension and spread it on a 5% sterile defibrinated sheep blood agar plate containing 1 μg / mL cefoxitin. Incubate at 37℃ for 24 hours. Pick grayish-white single colonies and subculture them on a fresh 5% sterile defibrinated sheep blood agar plate containing 1 μg / mL cefoxitin. Perform PCR identification on the grown colonies. The system and procedure are as follows:

[0308] System (1) (Up- SacB )

[0309]

[0310] System (2) Cfx -Down)

[0311]

[0312] PCR program

[0313]

[0314] Note: The denaturation-annealing-extension cycle needs to be repeated 30 times throughout the PCR procedure.

[0315] After the PCR reaction was completed, 5 μL of the reaction solution was taken, and 1 μL of DNA Loading Buffer was added for sample loading. The agarose gel electrophoresis results are shown in the figure. Figure 12Positive colonies were streaked and passaged onto new 5% sterile defibrinated sheep blood agar plates containing 1 μg / mL cefoxitin (Cfx). The plates were incubated at 37°C for 24 h. The bacterial growth was then scraped into 1 mL of sterile defibrinated sheep blood using an inoculation loop and stored at -80°C to obtain RA CH-1Δ. B739_RS01935 Δ B739_RS07625 ::Up- Cfx - SacB -Down.

[0316] B739_RS07625 Up P1 (inclusive) EcoR I restriction site): CG GAATTC CAATAATTTTGATATTAGGG.

[0317] SacB P2: ACGCGTCGACGTCGGTTATTTGTTAACTGTTAATTGTCCT.

[0318] Cfx P1:TTTCATTGTTCCATAAATCAGC.

[0319] B739_RS07625 Down P2 (inclusive) Pst I restriction site): AA CTGCAG GGCAGATTTGGAAGCACAAC.

[0320] 1.2.6、 B739_RS07625 Up-down fragment construction and fusion

[0321] Using the RA CH-1 genome as a reference, specific primer fragments were designed. B739_RS07625 UP P1 / P2 and DOWN P1 / P2, and amplify them respectively using the RA CH-1 genome as a template. B739_RS07625 UP clips and B739_ RS07625 DOWN fragment. Obtained by overlapping PCR of the two. B739_RS07625 The UP-DOWN fragment, primer sequences are shown in SEQ ID NO. 20-23.

[0322] (1) B739_RS07625 PCR amplification system of UP fragment

[0323]

[0324] (2) B739_RS07625 PCR amplification system for DOWN fragments

[0325]

[0326] The reaction procedure is as follows:

[0327]

[0328] Note: The denaturation-annealing-extension cycle needs to be repeated 30 times throughout the PCR procedure.

[0329] (3) B739_RS07625 UP clips and B739_RS07625 PCR fusion system of DOWN fragments

[0330]

[0331] The reaction procedure is as follows:

[0332]

[0333] Note: The process requires 5 cycles of "denaturation-annealing-extension".

[0334] After the reaction is complete, add to the reaction solution B739_RS07625 UP P1 and B739_RS07625 DOWN P2 2.5 μL each. Continue the reaction as follows:

[0335]

[0336] Note: The process requires 25 cycles of "modification-annealing-extension".

[0337] After the PCR reaction was completed, 5 μL of the reaction solution was taken, and 1 μL of DNA Loading Buffer was added for sample loading. The agarose gel electrophoresis results are shown in the figure. Figure 13 Recycled and measured using NanoDrop. B739_RS07625 The concentration of the Up-Down fragment was stored at -20°C.

[0338] B739_RS07625 UP P1:ATTTTAGAAGGTATAGAAAAGTCTTCTTTAAAAATAGGAG.

[0339] B739_RS07625 UP P2:CAATCAGATTTAGTCTTTAAATAGAATTAAAAATAGAACC.

[0340] B739_RS07625 DOWN P1: GGTTCTATTTTTAATTCTATTTAAAGACTAAATCTGATTG.

[0341] B739_RS07625 DOWN P2: GAGATTGTGTATTTCGTTAATCAAACAAGTAATAATAG.

[0342] 1.2.7 B739_RS07625 Natural transformation of UP-DOWN fusion fragments and identification of positive transformants (second homologous recombination)

[0343] Δ B739_RS07625 Δ B739_RS07625 ::Up- Cfx - SacB -Down inoculate into TSB medium and incubate at 37°C and 180 rpm until the logarithmic growth phase (OD50). 600 =1.0-1.5). After adjusting the bacterial suspension to 1 OD / mL, take 300 μL of bacterial suspension and add 2 μg of [unspecified ingredient]. B739_RS07625 The up-down fragments were mixed and incubated at 37°C for 1 hour. After incubation, 100 μL of the bacterial suspension was plated onto a 5% sterile defibrinated sheep blood agar plate containing 15% sucrose and incubated at 37°C for 24 hours. Single colonies that grew well on the sucrose plate were selected and passaged on both antibiotic-free and cefoxitin-containing blood agar plates for 24 hours. Colonies that grew on antibiotic-free blood agar plates but failed to grow on cefoxitin-containing blood agar plates were selected as candidate strains for PCR identification. The system and procedure are as follows:

[0344] System (1) — Cfx :

[0345]

[0346] System (2) — SacB :

[0347]

[0348] The reaction procedure is as follows:

[0349]

[0350] Note: The denaturation-annealing-extension cycle needs to be repeated 30 times throughout the PCR procedure.

[0351] System (3) — Target Gene

[0352]

[0353] The reaction procedure is as follows:

[0354]

[0355] Note: The denaturation-annealing-extension cycle needs to be repeated 30 times throughout the PCR procedure.

[0356] System (4) – Positive Control RA CH-1

[0357]

[0358] The reaction procedure is as follows:

[0359]

[0360] Note: The denaturation-annealing-extension cycle needs to be repeated 30 times throughout the PCR procedure.

[0361] After the PCR reaction was completed, 5 μL of the reaction solution was taken, and 1 μL of DNA Loading Buffer was added for sample loading. The agarose gel electrophoresis results are shown in the figure. Figure 14 Positive colonies were streaked onto new 5% sterile defibrinated sheep blood agar plates and incubated at 37°C for 24 hours. The bacterial growth was then scraped into 1 mL of sterile defibrinated sheep blood using an inoculation loop and stored at -80°C to obtain RA CH-1Δ. B739_ RS01935 Δ B739_RS07625 .

[0362] Cfx P1:TTTCATTGTTCCATAAATCAGC.

[0363] Cfx P2:TACACCTGTTTTTGCATTCTTTT.

[0364] SacB P1: CTAGTCTAGACTAGTTTTTTTTTAACATTTGATTTTGTAT.

[0365] SacB P2: ACGCGTCGACGTCGGTTATTTGTTAACTGTTAATTGTCCT.

[0366] B739_RS07625 UP P1:ATTTTAGAAGGTATAGAAAAGTCTTCTTTAAAAATAGGAG.

[0367] B739_RS07625 DOWN P2: GAGATTGTGTATTTCGTTAATCAAACAAGTAATAATAG.

[0368] 2. RA CH-1 B739_RS01935 - B7 Safety evaluation of double gene deletion strains as gene deletion candidate vaccines (impact on weight gain in ducklings).

[0369] Three-day-old ducklings were divided into two groups: Group 1, 12 ducklings were immunized with RA CH-1Δ B739_RS01935 Δ B739_ RS07625 Group 2 consisted of 12 ducklings that received no treatment. They were provided with ample antibiotic-free feed and water, and their weight was measured daily for 14 consecutive days. The results showed that, compared to the control group, the use of RA CH-1Δ B739_ RS01935 Δ B739_RS07625 Immunizing ducklings with double gene deletion strains does not significantly affect their normal growth. Figure 15 ).

[0370] 3. RA CH-1 B739_RS01935-B739_RS07625 Evaluation of the immunoprotective efficacy of double gene deletion strains as gene deletion candidate vaccines

[0371] Three-day-old ducklings were divided into two groups: Group 1 was treated with 1×10 9 CFU's RA CH-1Δ B739_RS01935 Δ B739_ RS07625 Vaccinate 3-day-old ducklings and challenge them with 100×LD virus 7 days after vaccination. 50 Group 1: RA CH-1 wild strain; Group 2: 3-day-old ducklings without any treatment, challenged with 100×LD50 after 7 days. 50 The LD50 of the wild-type RA CH-1 strain (as previously determined in the laboratory) was 2.24 × 10⁻⁶. 8 The morbidity and mortality rates of ducks within 10 days after challenge were calculated to assess the protective efficacy of the attenuated candidate vaccine. Results showed that RA CH-1Δ B739_RS01935 Δ B739_RS07625 The protection rate against wild-type RA CH-1 after immunization was 83.3% (Table 2).

[0372] Table 1. RA CH-1 B739_RS01935-B739_RS07625 Determination of the immune protection of double gene deletion strains against ducklings

[0373]

[0374] The above results indicate that this attenuated strain is safe and reliable in application, has a high protective effect against viral challenge, and can be used as a candidate strain for the development of novel gene-deleted vaccines.

[0375] The above specific embodiments further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above are merely specific embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A type of duck plague bacillus (Riebelella anatipestifer) Riemerella anatipestifer A candidate strain of a double-gene deletion attenuated live vaccine, characterized in that, It is the CH-1 strain of *Riebelella anatipestifer*. B739_RS01935-B739_RS07625 A candidate strain of attenuated live vaccine with double gene deletion, accession number CCTCC NO: M20251080, accession date May 15, 2025, is deposited at the China Center for Type Culture Collection, Wuhan University, Wuhan, China.

2. The duck plague bacillus (Riebelella anatipestifer) as described in claim 1. Riemerella anatipestifer Application of double-gene deletion attenuated vaccine candidate strains in the preparation of attenuated vaccines against duck plague or in the preparation of drugs for the prevention and treatment of duck plague infection.

3. A live attenuated vaccine against duck plague Riesella dysenteriae, characterized in that, Contains the duck plague bacillus as described in claim 1 ( Riemerella anatipestifer Candidate strains for attenuated live vaccine with double gene deletion.

Images