Chicken salmonella strain stably expressing standard type o antigen and construction method and application thereof

Abstract

This invention discloses a strain of Salmonella pullorum stably expressing the standard O antigen, its construction method, and its application. By knocking out the SPUL_2404 gene in the standard Salmonella pullorum strain, the obtained SPUL_2404 gene-deleted strain stably expresses the standard O antigen. Agglutination tests showed that this strain exhibited a typical agglutination reaction with the standard O123 serum of Salmonella pullorum, but not with the variant O122 serum, and the strain showed good stability of the standard O antigen after multiple passages. Therefore, the SPUL_2404 gene-deleted Salmonella pullorum strain can be used to prepare the standard agglutination antigen component in the diagnostic agglutination antigen for Salmonella pullorum.

Description

Technical Field

[0001] This invention belongs to the field of avian salmonellosis prevention and control technology, specifically relating to a strain that stably expresses the standard O antigen of Salmonella pullorum, its construction method, and its application. Background Technology

[0002] Pullorum disease, caused by Salmonella pullorum, is a serious threat to the global poultry industry. It primarily affects chicks aged 2-3 weeks, causing pullorum disease and death. In adult chickens, infection leads to slowed growth and decreased egg production, resulting in significant economic losses for the poultry industry. The disease can spread both horizontally and vertically, with vertical transmission being the primary route. Purifying breeding stock for pullorum control is crucial. Effective purification relies on efficient detection technologies and reagents; therefore, developing efficient and stable diagnostic reagents is essential for pullorum eradication.

[0003] The primary method for detecting pullorum disease in chickens is through whole blood or serum agglutination tests using pullorum antigens. Due to differences in the O12 antigen of Salmonella Pullorum, two serotypes exist: a standard type and a variant. The standard type of Salmonella Pullorum contains O121 and O123 in its O12 antigen, while the variant type contains O121 and O122, leading to differences in their serum recognition capabilities. Therefore, pullorum antigens are typically prepared by combining the standard and variant strains of Salmonella Pullorum.

[0004] However, the O antigen of the strains used to prepare agglutinated antigens is unstable. For example, the standard serotype of *Salmonella pullorum* exhibits numerous variant subtypes in its progeny clones with changes in subculturing and culture conditions; that is, the O antigen transforms from the standard serotype to the variant serotype. This results in uncontrollable standard antigen components in the prepared agglutinated antigens, insufficient antigen stability, and affects detection accuracy. Summary of the Invention

[0005] Based on the identification of the key enzymes that transform the standard O antigen of Salmonella pullorum into a variant, this invention uses genetic engineering technology to construct a Salmonella pullorum strain that stably expresses the standard O antigen, and uses it for the preparation of the standard agglutination antigen of Salmonella pullorum.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A strain that stably expresses the O antigen of Salmonella pullorum standard type, wherein the strain is a Salmonella pullorum standard type strain with the SPUL_2404 gene knocked out, and the nucleotide sequence of the SPUL_2404 gene is shown in SEQ ID NO.1.

[0008] Preferably, the SPUL_2404 gene is knocked out by homologous recombination, and the method includes the following steps:

[0009] (1) Amplify the upstream and downstream homologous arms of the SPUL_2404 gene; preferably, the primer sequences for amplifying the upstream homologous arm of the SPUL_2404 gene are shown in SEQ ID NO.4 and 5, and the primer sequences for amplifying the downstream homologous arm are shown in SEQ ID NO.6 and 7.

[0010] (2) Connect the suicide plasmid pRE112 and the upstream and downstream homologous arms of the SPUL_2404 gene to construct the homologous recombinant suicide plasmid pRE112-ΔSPUL_2404.

[0011] (3) The Escherichia coli containing the homologous recombinant suicide plasmid pRE112-ΔSPUL_2404 was combined with the standard strain of Salmonella pullorum. The homologous recombinant suicide plasmid pRE112-ΔSPUL_2404 was transformed into the standard strain of Salmonella pullorum through binding transfer, and the SPUL_2404 gene deletion strain was obtained by screening.

[0012] Stability analysis of O antigen of SPUL_2404 gene-deleted Salmonella pullorum: The SPUL_2404 gene-deleted strain reacted with O123 serum but not with O122 serum, indicating that the SPUL_2404 gene-deleted strain is the standard type strain. After 40 generations of continuous subculturing of the SPUL_2404 gene-deleted strain, single colonies were selected and reacted with standard type serum O123 and variant type serum O122, respectively. The results showed that all 100 single colony subclones of the strain reacted only with standard type serum O123, exhibiting good stability of the standard type O antigen.

[0013] Application of SPUL_2404 gene-deleted Salmonella pullorum in the preparation of standard agglutination antigen for pullorum disease: Cultured Salmonella pullorum △SPUL_2404 was inactivated with formalin and stained with crystal violet to obtain a stable standard agglutination antigen. Plate agglutination test showed that the standard agglutination antigen prepared with △SPUL_2404 had a good agglutination effect on chicken serum infected with standard strain of Salmonella pullorum.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] Current methods for preparing pullorum antigens in chickens involve combining standard and variant strains of *Salmonella pullorum* in a fixed ratio. However, with variations in passage and culture conditions, the strains used to prepare pullorum antigens are unstable. The O antigen of the standard strain can transform into a variant, leading to changes in the composition of the prepared pullorum antigen, resulting in insufficient stability and affecting detection accuracy. This invention, based on the initial identification of the key enzyme causing the transformation of the standard O antigen into a variant, utilizes genetic engineering to knock out the key enzyme gene SPUL_2404, which leads to the transformation of the O antigen in the standard strain. This prevents the transformation of the O antigen in the standard strain into a variant, resulting in a stable standard strain of *Salmonella pullorum* expressing the standard O antigen. Therefore, the pullorum antigen prepared from this strain has a stable and controllable standard O antigen composition, effectively improving the stability of the agglutination antigen detection reagent. Attached Figure Description

[0016] Figure 1 Electrophoresis diagram of the amplified products of the homologous arm of SPUL_2404. M: DL2000 Marker; 1: Amplified product of the upstream homologous arm of SPUL_2404; 2: Amplified product of the downstream homologous arm of SPUL_2404.

[0017] Figure 2 Image of homologous recombination suicide plasmid pRE112-ΔSPUL_2404 after enzyme digestion. M: DL2000 Marker; 1: EcoRI / HindIII double digestion of pRE112-ΔSPUL_2404.

[0018] Figure 3 : PCR identification diagram of the SPUL_2404 gene deletion strain. M: DL2000 Marker; 1: Amplification product of the outer primer of the wild-type SPUL_2404 gene; 2: Amplification product of the outer primer of C79-3 / ΔSPUL_2404.

[0019] Figure 4 : Reaction diagram of gene deletion strain C79-3 / ΔSPUL_2404 with O123 serum and O122 serum.

[0020] O123 is the standard single-factor serum, and O122 is the variant single-factor serum. Detailed Implementation

[0021] Example 1: Construction of a Salmonella Pullorum SPUL_2404 gene-deleted strain

[0022] 1. Amplification of the homologous arm of the SPUL_2404 gene

[0023] Referring to the nucleotide sequence of the SPUL_2404 gene of Salmonella Pullorum (i.e., the SPUL_2404 gene with accession number CP003047.1), primers were designed based on the upstream and downstream homologous arms of the SPUL_2404 gene: upstream homologous arm forward primer 2404Up-F: 5′-ggagagctcgatatcgcatgcCCTATTTTCTATAAAACGGT TCGCG-3′, upstream homologous arm reverse primer 2404Up-R: 5′-gTCTTAATTATGTACTTAGGTC TTTTTTTGG-3′; downstream homologous arm forward primer 2404Down-F: 5′-cctaagtacataattaagaCATT CATGTATAATTAATGTTCCCCG-3′, downstream homologous arm reverse primer 2404Down-R: 5′-tcttc tagaggtaccgcatgcCACGCTTCAGTAGCTGGAAAAA-3′.

[0024] Genomic DNA was extracted from Salmonella pullorum strain C79-3 (standard strain). Using this genomic DNA as a template, the upstream and downstream homologous arms of the SPUL_2404 gene were amplified by PCR using the primers described above. The PCR reaction system was: 25 μL primerSTAR, 1.5 μL upstream primer, 1.5 μL downstream primer, 20 μL ddH2O, and 2 μL template. The PCR products were identified by 1% agarose gel electrophoresis. The results are shown below. Figure 1 As shown, two specific DNA bands with sizes of 859 bp and 848 bp (sequences shown in SEQ ID NO.2 and SEQ ID NO.3) were obtained, which correspond to the size of the target DNA fragment. The PCR product was then extracted using a DNA gel extraction kit.

[0025] 2. Preparation of Escherichia coli X7213 competent cells

[0026] Single colonies of *E. coli* X7213 were picked from LB agar plates and inoculated into 2 mL of LB liquid medium. The culture was incubated at 37°C with shaking at 200 rpm for 12 h. The activated bacterial culture was then transferred 1:1000 to 50 mL of LB liquid medium and incubated at 37°C with shaking at 200 rpm for 3-4 h (until OD). 600(Value approximately 0.6). Transfer the bacterial culture to sterile centrifuge tubes in a laminar flow hood and incubate on ice for 30 min. Centrifuge the bacterial culture at 4000 rpm for 10 min using a pre-chilled centrifuge and discard the supernatant. Resuspend the bacterial cells in 20 mL of ice-chilled sterile 0.1 mol / L CaCl2, incubate on ice for 30 min, and centrifuge at 4000 rpm for 10 min at 4°C. Discard the supernatant, add an appropriate amount of ice-chilled 0.1 mol / L CaCl2 to resuspend the bacterial cells again, and add sterile glycerol to a final concentration of 15%. Aliquot into 200 μL tubes and store at -80°C for later use.

[0027] 3. Construction of recombinant plasmids

[0028] Plasmid pRE112 was extracted and recovered by SphⅠ digestion. The purified upstream and downstream homologous arm fragments of the SPUL_2404 gene and the digested plasmid pRE112 were ligated using infusion. The ligation product was transformed into E. coli X7213 competent cells and plated on DAP (50 μg / ml) + chloramphenicol (40 μg / ml) antibiotic plates for screening positive clones. After successful colony PCR verification using 2404Up-F and 2404Down-R primers, the plasmid was extracted and identified by EcoRI / HindⅢ double digestion. The results are as follows. Figure 2 As shown, double digestion of the plasmid yielded two DNA fragments, one of which matched the size of the 1707 bp target DNA fragment. The correctly digested recombinant plasmid pRE112-ΔSPUL_2404 was sequenced, and the sequencing results showed that the recombinant plasmid was successfully constructed.

[0029] 4. Joining transfer

[0030] Donor strain X7213 containing the recombinant plasmid pRE112-ΔSPUL_2404 and recipient strain (Salmonella pullorum C79-3) were picked and inoculated separately into LB broth and cultured overnight at 37°C with shaking for 10 hours. The bacterial suspensions were then transferred at a 1:1000 volume ratio to LB broth containing 50 μg / ml chloramphenicol and antibiotic-free medium, respectively, and cultured at 37°C with shaking until OD. 600 Approximately 0.8, take 1 mL of bacterial culture from each, centrifuge at 4000 rpm for 5 min to collect the bacteria, wash twice with LB medium containing 0.01 mol / L MgSO4, mix the donor and recipient bacteria at a 1:10 volume ratio, and take 200 μL to spread on an NC membrane. Place on an LB plate containing DAP (50 μg / mL) and incubate at 37°C for 6-8 h. Wash off the bacteria from the membrane, wash twice with 1 mL of LB liquid medium containing Mg ions, dilute and spread on an LB plate containing chloramphenicol (40 μg / mL), and incubate overnight at 37°C.

[0031] 5. Recombinant identification

[0032] Single colonies were picked and PCR was performed using homologous primers 2404Up-F and 2404Down-R. If two bands of different sizes were amplified, with the difference between the large and small bands exactly equal to the gene size (1653 bp), it was identified as a single exchanger. The selected single exchanger cells were inoculated into NaCl-free LB broth and incubated at 37°C for 12-24 hours. They were then transferred to LB broth containing 15% sucrose and incubated at 37°C with shaking for 2 hours. The bacterial culture was then spread onto LB agar plates containing 15% sucrose using an inoculation loop and incubated at 37°C for 12 hours. Single colonies were picked using a sterilized pipette tip and spotted onto plates containing chloramphenicol (40 μg / ml) and antibiotic-free plates, and incubated at 37°C for 12 hours. Single colonies that did not grow on chloramphenicol plates but grew on antibiotic-free plates were picked and re-identified by PCR using primers 2404Up-F and 2404Down-R. The results were as follows. Figure 3 As shown, the amplified bacterial cell with a band size of 1707 bp is the SPUL_2404 gene deletion strain C79-3 / ΔSPUL_2404 of Salmonella pullorum C79-3.

[0033] 6. Serological analysis

[0034] Strawberry strain C79-3 / ΔSPUL_2404 was streaked onto Martin agar plates and incubated overnight. Single colonies were then picked and added to an appropriate amount of physiological saline to adjust the bacterial concentration to 1.0 × 10⁻⁶. 10 CFU / ml. Take a clean glass slide, and use a pipette to drop 25 μl of Salmonella O122 serum and O123 serum (purchased from the China Institute of Veterinary Drug Control) onto the slide. Then, add 25 μl of bacterial suspension to each of the O122 and O123 serums, and gently shake the slide to mix thoroughly. Determine the results after 2 minutes. Figure 4 As shown, the gene-deleted strain C79-3 / ΔSPUL_2404 reacted with O123 serum but not with O122 serum, indicating that this strain is the standard type.

[0035] Example 2: Antigen stability analysis of gene-deleted strain C79-3 / ΔSPUL_2404

[0036] After 40 consecutive passages of the parental strain C79-3 (standard strain) and the gene-deleted strain C79-3 / ΔSPUL_2404, the cultures were streaked onto Martin agar plates and incubated overnight. One hundred single colonies from each strain were then picked and added to an appropriate amount of physiological saline, with the bacterial concentration of each single colony adjusted to 1.0 × 10⁻⁶. 10CFU / ml. Take a clean glass slide, and use a pipette to drop 25 μl of Salmonella O122 serum and O123 serum (purchased from the China Institute of Veterinary Drug Control) onto the slide. Then, add 25 μl of bacterial suspension to each of the O122 and O123 serums, and gently shake the slide to mix thoroughly. Determine the results after 2 minutes. As shown in Table 1, among 100 single colony subclones of the parental strain C79-3, 12 subclones reacted with the variant serum (O122), indicating serotype variation. In contrast, all 100 single colony subclones of the gene-deleted strain C79-3 / ΔSPUL_2404 reacted only with the standard serum (O123), demonstrating good O antigen stability.

[0037] Table 1. Antigenic stability analysis of Salmonella pullorum gene-deleted strain C79-3 / SPUL_2404

[0038]

[0039] Note: O122+O123 indicates that it can undergo agglutination reactions with both O122 serum and O123 serum.

[0040] Example 3: Preparation and application of stable standard agglutination antigen

[0041] The constructed stable O antigen-expressing strain C79-3 / △SPUL_2404 was densely streaked onto Martin agar plates and incubated for 24 h. After washing with Martin broth, the cells were transferred to large Martin agar plates and incubated upright for 24 h, then inverted and incubated upside down for another 24 h. After 48 h, the cells were washed with PBS containing 1% formalin, inactivated at 37°C for 24 h, and then mixed thoroughly with twice the volume of anhydrous ethanol. The mixture was allowed to settle naturally at 4°C until complete bacterial precipitation. The precipitate was centrifuged, washed once with PBS containing 10% glycerol, resuspended, and the bacterial concentration adjusted to approximately 1.5 × 10⁻⁶. 10 A stable standard agglutination antigen is prepared by adding CFU / ml of ethanol solution of crystal violet to a final concentration of 0.01% and stirring at low speed to ensure thorough staining.

[0042] Standardized pullorum-positive serum prepared by immunizing SFP chickens with standard strains from different sources was serially diluted 2-fold. A self-made standard agglutination antigen and a commercial antigen were used to simultaneously detect the diluted serum, comparing the sensitivity of the self-made standard agglutination antigen and the commercial antigen to serum infected with the standard strains. The results are shown in Table 2. The agglutination antigen prepared with C79-3 / △SPUL_2404 showed better sensitivity than the commercial agglutination antigen, still producing agglutination even after high-fold dilution of the serum.

[0043] Table 2. Sensitivity analysis of standard agglutinated antigen and standard serum prepared by C79-3 / △SPUL_2404

[0044]

[0045] - The liquid is a uniform blue color with no particulate matter precipitated.

[0046] +: A small amount of fine particulate matter precipitates out, but the solution remains blue;

[0047] ++: Obvious blue particles are precipitated, and the liquid still shows a slight blue tint; +++: Large blue particles are precipitated, and the liquid becomes transparent.

Claims

1. Stable expression of Salmonella pullorum (Salmonella fowleri) Salmonella enterica subsp. enterica The strain of *Serovar Pullorum* with standard type O antigen is characterized by, The strain in question is a standard strain of Salmonella pullorum with the SPUL_2404 gene knocked out, and the nucleotide sequence of the SPUL_2404 gene is shown in SEQ ID NO.

1.

2. The strain according to claim 1, characterized in that, The SPUL_2404 gene was knocked out using homologous recombination.

3. The use of the strain described in claim 1 or 2 in the preparation of standard agglutination antigen of Salmonella pullorum.

4. A method for constructing a strain stably expressing the standard O antigen of Salmonella pullorum, characterized in that, The method involves constructing a homologous recombinant suicide plasmid pRE112-ΔSPUL_2404 carrying upstream and downstream homologous arms of the SPUL_2404 gene using the suicide plasmid pRE112, transforming the homologous recombinant suicide plasmid pRE112-ΔSPUL_2404 into a standard strain of Salmonella pullorum via binding transfer, and then screening to obtain strains with the SPUL_2404 gene deletion.

5. The construction method according to claim 4, characterized in that, The primer sequences for amplifying the upstream homologous arm of the SPUL_2404 gene are shown in SEQ ID NO.4 and 5, and the primer sequences for amplifying the downstream homologous arm of the SPUL_2404 gene are shown in SEQ ID NO.6 and 7.

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