Chicken salmonella strains stably expressing variant o antigens and methods of construction and use thereof

Abstract

The application discloses a chicken salmonella pullorum strain stably expressing variant O antigen and a construction method and application thereof, and constructs an expression plasmid containing a J23119 promoter and a SPUL_2404 gene, and then the expression plasmid is transferred into chicken salmonella pullorum, so that a chicken salmonella pullorum strain overexpressing the SPUL_2404 gene is constructed to stably express the variant O antigen, and agglutination test shows that the strain only has a typical agglutination reaction with chicken salmonella pullorum variant serums, and does not have a typical agglutination reaction with chicken salmonella pullorum standard serums, and after subculture, O antigens of all subclone colonies are stable, and only have a typical agglutination reaction with the variant serums, so that the strain can be used for preparing chicken salmonella pullorum variant agglutination antigens.

Description

Technical Field

[0001] This invention belongs to the field of avian salmonellosis prevention and control technology, specifically relating to the method for constructing O antigen-stable Salmonella pullorum variant strains and their application in the preparation of pullorum agglutination antigens. Background Technology

[0002] Pullorum disease, caused by Salmonella pullorum, is a serious threat to the global poultry industry. It can spread both horizontally and vertically, with vertical transmission being the primary route. Cure breeding stock of pullorum and cull positive cases to interrupt the vertical transmission route are crucial for controlling the disease. Cure relies on efficient detection technologies and reagents; therefore, developing efficient, stable, and highly specific detection 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, studies have found that the O antigen of variant strains of Salmonella pullorum is unstable. With changes in subculturing and culture conditions, a certain number of strains in the progeny clones of the variant subtype exhibit the O antigen of the standard subtype. This leads to insufficient stability of the agglutination antigen prepared from this strain, affecting the accuracy of detection. Therefore, this invention, based on the identification of the key enzyme determining the interconversion between the standard and variant O antigens of Salmonella pullorum, uses gene overexpression technology to stably and efficiently express this enzyme, constructs a variant strain of Salmonella pullorum with stable O antigen, and uses it for the preparation of the variant O antigen component in Salmonella pullorum agglutination antigen. Summary of the Invention

[0005] The purpose of this invention is to provide a Salmonella pullorum variant strain with stable O antigen and its construction method. The principle is to overexpress the key enzyme gene for the formation of variant O antigen in Salmonella pullorum, thereby obtaining a Salmonella pullorum strain that stably expresses variant O antigen. The agglutination antigen prepared from this strain also maintains a stable and controllable variant O antigen.

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

[0007] A strain that stably expresses the variant O antigen of Salmonella pullorum, wherein the strain overexpresses the SPUL_2404 gene in Salmonella pullorum, and the nucleotide sequence of the SPUL_2404 gene is shown in SEQ ID NO.1.

[0008] Preferably, the expression of the SPUL_2404 gene is driven by the J23119 promoter, the nucleotide sequence of which is shown in SEQ ID NO.2. The construction method includes the following steps: constructing an expression plasmid containing the J23119 promoter and the SPUL_2404 gene, and transforming the expression plasmid into *Salmonella pullorum*.

[0009] Stability analysis of O antigen of Salmonella pullorum overexpressing the SPUL_2404 gene: The SPUL_2404 gene overexpressing strain reacted with serum O122 but not with serum O123, indicating that the SPUL_2404 gene overexpressing strain is a variant strain. After 40 generations of continuous passage of the SPUL_2404 gene overexpressing strain, single colonies were selected and reacted with standard serum O123 and variant serum O122, respectively. The results showed that 100 single colony subclones of the SPUL_2404 gene overexpressing strain reacted only with variant serum O122, exhibiting good stability of the variant antigen.

[0010] Application of SPUL_2404 gene overexpression of Salmonella pullorum in the preparation of variant agglutination antigen of Salmonella pullorum: The cultured Salmonella pullorum strain overexpressing SPUL_2404 gene was inactivated by formalin and stained with crystal violet to obtain a stable variant agglutination antigen. Plate agglutination test showed that the variant agglutination antigen prepared by this strain had a good agglutination effect on chicken serum infected with variant strain of Salmonella pullorum.

[0011] Compared with the prior art, the present invention has the following beneficial effects:

[0012] Currently, chicken pullorum agglutination antigens are prepared by combining standard and variant strains of *Salmonella pullorum* in a fixed ratio. However, the strains used to prepare *Salmonella pullorum* agglutination antigens are not stable. During passage and culture, the O antigen of the variant strains of *Salmonella pullorum* undergoes conversion to the standard type, leading to changes in the antigenic components of the chicken pullorum agglutination antigen prepared from this bacterium, resulting in insufficient stability and affecting detection accuracy. This invention, based on the initial identification of the key enzyme expressing the variant O antigen in *Salmonella pullorum*, utilizes genetic engineering technology to overexpress the key enzyme gene for forming the variant O antigen of *Salmonella pullorum* by replacing the constitutive promoter. This yields a variant strain of *Salmonella pullorum* that stably expresses the variant O antigen. Therefore, the chicken pullorum agglutination antigen prepared from this strain has a stable variant O antigen component, effectively improving detection stability. Attached Figure Description

[0013] Figure 1 Electrophoresis images of the amplified products of the pCDF-J23 vector and the SPUL_2404 fragment.

[0014] Figure 2 Identification of overexpression plasmid pCDF-J23-SPUL_2404. M: DL5000 Marker; 1: pCDF-J23 plasmid control; 2: negative control; 3-4: overexpression plasmid pCDF-J23-SPUL_2404.

[0015] Figure 3 : Reaction diagram of overexpression strain C79-3 / pCDF-J23-SPUL-O with serum of O123 and O122.

[0016] O123 is the standard single-factor serum, and O122 is the variant single-factor serum. Detailed Implementation

[0017] Example 1: Construction of a strain overexpressing the SPUL_2404 gene

[0018] 1. Cloning of the SPUL_2404 gene

[0019] The nucleotide sequence of the Salmonella Pullorum SPUL_2404 gene is shown in SEQ ID NO: 1 (accession number CP003047.1). Primers were designed based on the SPUL_2404 gene sequence: upstream primer SPUL_2404-F: 5′-agaaagaggagaaatactagATGAGAAGAAAAATGGTTAACAATAGATT-3′; downstream primer SPUL_2404-R: 5′-ctcagctaattaagcttttaTTATTTAATTATTTCCGTAATATTCTCAT TTG-3′. The lowercase part is homologous to the upstream and downstream of the vector insertion site, and the uppercase part is the primer for amplifying the SPUL_2404 gene.

[0020] Genomic DNA was extracted from Salmonella pullorum strain C79-3 (standard serotype subtype). Using this genomic DNA as a template, the SPUL_2404 gene was amplified by PCR using the primers described above. The PCR reaction mixture consisted of: 25 μL primer STAR mix, 2 μL upstream primer, 2 μL downstream primer, 19 μL ddH2O, and 2 μL template. The PCR products were identified by 1% agarose gel electrophoresis, and the results are as follows: Figure 1 As shown, a specific DNA band of approximately 1653 bp was obtained, which matches the size of the target DNA fragment. The PCR product was recovered by gel extraction using a DNA gel extraction kit.

[0021] 2. Construction of overexpression plasmids

[0022] The pCDF-J23-GFP plasmid was extracted, diluted, and used as a template. Primers were designed based on the location of the GFP fragment to linearize the plasmid for PCR amplification. The upstream primer pCDF-F was 5′-TAAAAGCTTAATTAG CTGAGCTTGGA-3′, and the downstream primer pCDF-R was 5′-CTAGTATTTCTCCTCTTTCTCTAG TGCTAGT-3′. The PCR reaction mixture consisted of: 25 μL primer STAR mix, 2 μL upstream primer, 2 μL downstream primer, 19 μL ddH2O, and 2 μL template. The PCR products were identified and recovered by 1% agarose gel electrophoresis. The results are shown below. Figure 1 As shown, a specific DNA band of 2420 bp was obtained, consistent with the size of the target DNA fragment. The recovered product was ligated with the SPUL_2404 gene containing the homologous arm fragment using infusion, transformed into DH5α, plated on streptomycin-resistant plates, and positive clones were picked and identified by PCR using universal plasmid primers S-J23-F: 5′-TAGGCGTATCACGAGGCA-3′ and S-J23-R: 5′-TCGGTTCA GGGCAGGGT-3′. The results are shown in the figure. Figure 2 As shown, the recombinant amplified a band of approximately 2057 bp. Sequencing of the PCR product showed that the overexpression plasmid pCDF-J23-SPUL_2404 was successfully constructed.

[0023] 3. Transformation of overexpression plasmids

[0024] Single colonies of Salmonella pullorum C79-3 were picked and incubated overnight at 37°C and 200 rpm. The next day, the culture was diluted 1:100 and transferred to 50 ml of LB liquid medium. Incubation was allowed for approximately 4 hours until OD (occurrence depth) was observed. 600Once the bacterial concentration reaches 0.5-0.6, 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. Add 20 mL of pre-chilled 10% glycerol to resuspend and wash the bacterial cells. Centrifuge the bacterial culture at 4000 rpm for 10 min and discard the supernatant. Repeat the washing process three times. After the final centrifugation to remove the supernatant, add an appropriate amount of pre-chilled 10% glycerol to resuspend the bacterial cells. Aliquot 100 μL / tube. Store any excess competent cells at -80°C for later use. Add 10 ng of the correctly identified plasmid pCDF-J23-SPUL_2404, place on ice for 20 min, then transfer to a pre-chilled 0.1 mm electroporation cuvette. Perform electroporation transformation at 1800 V and 200 Ω. Immediately after electroporation, add 800 μl of pre-chilled LB broth and shake at 37 °C and 200 rpm for 1 h to rejuvenate. Spread on streptomycin-resistant LB plates and incubate overnight. The resulting positive clones are the chicken pullorum strain C79-3 / SPUL_2404-O overexpressing the SPUL_2404 gene.

[0025] 4. Serological analysis

[0026] The parental strain C79-3 and the overexpression strain C79-3 / SPUL_2404-O were streaked onto Martin agar plates, incubated overnight, and single colonies were 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, pipette 25 μl of bacterial suspension into each of the O122 and O123 serums, and gently shake the slide to mix thoroughly. Determine the results after 2 minutes. Figure 3 As shown, strain C79-3 reacted with serum O123 but not with serum O122, indicating it is the standard strain. Strain C79-3 / SPUL_2404-O reacted with serum O122 but not with serum O123, suggesting that strain C79-3 / SPUL_2404-O has transformed into a variant strain.

[0027] Example 2: Antigen stability analysis of overexpression strain C79-3 / SPUL_2404-O

[0028] After 40 consecutive passages of parental strains C79-3 (standard strain), CVCC530 (variant strain), and C79-3 / SPUL_2404-O, 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. The bacterial concentration of each single colony was 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 serum, 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 parental strain C79-3, 12 clones reacted with the variant serum (O122). Among 100 single-colony subclones of strain CVCC530, 10 clones reacted with the standard serum (O123), indicating serotype variation. However, all 100 single-colony subclones of strain C79-3 / SPUL_2404-O reacted only with the variant serum (O122), demonstrating good O antigen stability.

[0029] Table 1. Antigenic stability analysis of Salmonella pullorum C79-3 / SPUL_2404-O strain.

[0030]

[0031] Note: O122+O123 indicates that it can undergo agglutination reactions with both O122 serum and O123 serum.

[0032] Example 3: Preparation and application of stable variant agglutinating antigens

[0033] The constructed overexpression strain C79-3 / SPUL_2404-O was densely streaked onto Martin agar plates and cultured for 24 h. After washing with Martin broth, the cells were transferred to large Martin agar plates and cultured upright for 24 h. The plates were then inverted and cultured for another 24 h. After 48 h, the cells were washed with PBS containing 1% formalin and inactivated at 37°C for 24 h. After 24 h, two volumes of anhydrous ethanol were added and thoroughly mixed. 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, and resuspended. The bacterial concentration was adjusted to approximately 1.5 × 10⁻⁶. 10 Stable variant agglutinating antigen is prepared by adding CFU / ml of ethanol solution containing crystal violet to a final concentration of 0.01% and stirring at low speed to ensure thorough staining.

[0034] Chicken white pullorum-positive sera prepared by immunizing SFP chickens with variant strains from different sources were serially diluted 2-fold. A self-prepared variant agglutination antigen and a commercial antigen were used to simultaneously detect the diluted sera, comparing their sensitivity. The results are shown in Table 2. The agglutination antigen prepared using strain C79-3 / SPUL_2404-O showed better sensitivity than the commercial agglutination antigen, still producing agglutination even after high-fold dilution of the serum.

[0035] Table 2. Application and sensitivity analysis of the variant agglutination antigen prepared from strain C79-3 / SPUL_2404-O.

[0036]

[0037]

[0038] - The liquid is a uniform blue color with no particulate matter precipitated.

[0039] +: A small amount of fine particulate matter precipitates out, but the solution remains blue;

[0040] ++: 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. A strain stably expressing the variant O antigen of Salmonella pullorum, characterized in that, The strain overexpresses the SPUL_2404 gene in a standard strain of Salmonella pullorum, 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 expression of the SPUL_2404 gene was driven by the J23119 promoter, the nucleotide sequence of which is shown in SEQ ID NO.

2.

3. The method for constructing the strain according to claim 1, characterized in that, Includes the following steps: An expression plasmid containing the J23119 promoter and the SPUL_2404 gene was constructed, and the expression plasmid was transformed into a standard strain of Salmonella pullorum.

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

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