A recombinant salmonella enteritidis and its construction method and application

Abstract

The application discloses a recombinant Salmonella enteritidis, a construction method and application thereof. The recombinant Salmonella enteritidis is obtained by simultaneously knocking out msbB genes and purL genes in Salmonella enteritidis, and is applied to preparation of attenuated live vaccine. The attenuated strain of Salmonella enteritidis with double-gene deletion disclosed by the application has a significantly reduced virulence compared with wild-type Salmonella enteritidis, a significantly shortened in-host colonization time, no adverse reaction after inoculation, a good protective effect on high-virulence Salmonella enteritidis, and can effectively eliminate high-virulence strain infection, and provides a solid immune protection effect for chicks.

Description

Technical Field

[0001] This invention relates to the field of biotechnology and genetically engineered vaccines, specifically to a recombinant Salmonella enteritidis, its construction method, and its application. Background Technology

[0002] Salmonella infection is a general term for acute and chronic diseases caused by Salmonella in animals and humans. Human infection with Salmonella mainly manifests as nausea, vomiting, and diarrhea; in animals, infection often presents with septicemia and enteritis, and can also cause abortion in pregnant animals (Jiao Xin'an. Salmonella Infection [M]. China Agriculture Press, 2015). Over the years, many countries worldwide have adopted measures such as biosafety, immunization, and antibiotic treatment to prevent and control different types of Salmonella infection. Although the prevalence of the disease has been controlled to some extent, due to various reasons, its prevention and control remains a long-term and arduous task.

[0003] Salmonella is a Gram-negative bacterium with a wide host spectrum and poses a serious threat. Among the many Salmonella serotypes, non-typhoidal Salmonella (NTS), mainly Salmonella enteritidis and Salmonella typhimurium, can endanger human health through various livestock and poultry products (Balasubramanian R, Im J, Lee JS, et al. The Global Burden and Epidemiology of Invasive Non-typhoidal Salmonella Infections[J]. Hum VaccinImmunother, 2019, 15: 1421-1426.). Salmonella enteritidis can cause latent infection in adult chickens and shed bacteria into the environment through feces, causing environmental pollution and making it difficult to eradicate the pathogen. Chicks infected with Salmonella enteritidis exhibit severe systemic infection and high mortality (Crump JA, Sjolund-Karlsson M, Gordon MA, et al. Epidemiology, Clinical Presentation, Laboratory Diagnosis, Antimicrobial Resistance, and Antimicrobial Management of Invasive Salmonella Infections[J]. Clin Microbiol Rev, 2015, 28: 901-937.). In recent years, reports have indicated that *Salmonella enteritidis* is gradually replacing *Salmonella typhimurium* as the most common foodborne Salmonella serotype. However, most scientific research still focuses on *Salmonella typhimurium*, with relatively little research on the pathogenic mechanisms and control of *Salmonella enteritidis* (Voetsch AC, Van G, Angulo FJ, et al. FoodNet Estimate of the Burden of Illness Caused by Nontyphoidal *Salmonella* Infections in the United States[J]. Clin Infect Dis, 2004: 127-134.). Therefore, strengthening basic research on the control of *Salmonella enteritidis* is imperative.

[0004] Salmonella vaccines are mainly classified into inactivated vaccines, subunit vaccines, and live attenuated vaccines. Among them, compared with inactivated vaccines and subunit vaccines, live attenuated vaccines have better immunogenicity. In addition to inducing humoral immunity, cellular immunity, and mucosal immunity, they can also serve as vaccine carriers, making them an important means of preventing Salmonella (Ma Xianghai, Yin Jiatong, Wang Juechu, et al. Research progress on avian Salmonella vaccines [J]. Animal Husbandry, 2021, 32: 8-9.).

[0005] In recent years, research on novel attenuated Salmonella vaccines constructed by knocking out virulence-related genes of virulent strains using genetic engineering technology has attracted much attention. These novel attenuated live vaccines have good safety and are not prone to reversion to virulence; they retain immunogenicity while being attenuated, and can multiply in immunized animals during the immune period, stimulating the body to produce a comprehensive systemic and local immune response, and can generate an immune response to multiple antigens of the pathogen; they provide robust immunity and have a long immune period, making them more advantageous than inactivated vaccines and subunit vaccines in preventing and controlling Salmonella infection in chickens, and therefore a more ideal vaccine.

[0006] Studies have shown that the msbB gene, which encodes an acyltransferase for lipid A, is a key regulator of the toxicity of Salmonella Typhimurium endotoxin (LPS). The absence of msbB leads to insufficient lipid A in LPS, reducing LPS endotoxin while maintaining structural integrity (Ling Yang, Zhuodong Chai, Jiaqian Qi, et al. Deficiency in themsbB Gene Reduced the Salmonella Typhimurium Virulence Through Mechanisms Beyond LPS Modification[J]. Microorganisms, 2025, 13(11): 2510.).

[0007] Studies have found that the purL gene encodes a key enzyme in the purine biosynthesis pathway. In the host tissue environment, the concentration of free purines is extremely low, which severely restricts the growth and reproduction of strains with this gene deletion, thereby achieving attenuation (D O'Callaghan, D Maskell, FY Liew, et al. Characterization of aromatic- andpurine-dependent Salmonella typhimurium: attention, persistence, and ability to induce protective immunity in BALB / c mice[J]. Infect Immun, 1988, 56(2):419-423.).

[0008] These genes may lead to a decrease in Salmonella virulence, but the effect of double knockout of the msbB and purL genes on the virulence of Salmonella enteritidis has not yet been studied and verified. Whether they can become targets for attenuated live vaccines or broad-spectrum antibacterial drugs requires further research. Summary of the Invention

[0009] Purpose of the invention: The primary objective of this invention is to provide a recombinant Salmonella enteritidis.

[0010] A second objective of this invention is to provide a method for constructing recombinant Salmonella enteritidis.

[0011] A third objective of this invention is to provide a method for reducing the virulence of Salmonella enteritidis.

[0012] The fourth objective of this invention is to provide the application of a method for constructing recombinant Salmonella or recombinant Salmonella enteritidis in the preparation of attenuated vaccines.

[0013] The final objective of this invention is to provide a vaccine to prevent Salmonella enteritidis.

[0014] Technical solution: In order to achieve the above objectives, the present invention provides a recombinant Salmonella enteritidis, wherein the recombinant Salmonella enteritidis simultaneously omits the msbB gene and the purL gene.

[0015] The nucleotide sequence of the msbB gene is shown in SEQ ID NO.13, and the nucleotide sequence of the purL gene is shown in SEQ ID NO.14.

[0016] The present invention also provides a method for constructing recombinant Salmonella enteritidis, the method comprising the following steps: knocking out the msbB gene and the purL gene in Salmonella enteritidis.

[0017] The method includes the following steps:

[0018] (1) Using the genomic DNA of Salmonella enteritidis as a template, the upstream and downstream homologous arms of the msbB gene and the purL gene were amplified by PCR. The amplification products were homologously recombined with the linear vectors to obtain plasmids with msbB gene deletion and plasmids with purL gene deletion. Then, they were transformed into Escherichia coli to obtain two donor bacteria.

[0019] (2) One of the donor bacteria obtained in step (1) is conjugated with the recipient bacteria Salmonella enterica for homologous single crossover recombination, and a strain with the corresponding gene deletion is obtained by homologous double crossover. The deleted strain is used as the recipient and conjugated with another donor bacteria obtained in step (1) for homologous single crossover recombination, and a strain with double deletion of msbB gene and purL gene is obtained by screening.

[0020] Among them, the screening method described in step (2) includes screening using chloramphenicol resistance and sucrose sensitivity.

[0021] The invention also provides a method for reducing the virulence of Salmonella enteritidis by knocking out the msbB gene with the nucleotide sequence shown in SEQ ID NO.13 and the purL gene with the nucleotide sequence shown in SEQ ID NO.14 in Salmonella enteritidis, or by constructing recombinant Salmonella enteritidis according to the method described.

[0022] The invention also provides the application of a method for constructing recombinant Salmonella or a recombinant Salmonella enteritidis in the preparation of attenuated vaccines.

[0023] The attenuated vaccine includes a vaccine to prevent Salmonella enteritidis infection in poultry.

[0024] The poultry mentioned therein includes one or more of the following: broiler chickens, breeding chickens, laying hens, meat ducks, laying ducks, breeding ducks, meat geese, breeding geese, pigeons, and quails.

[0025] The present invention also provides a vaccine for the prevention of Salmonella enteritidis, the vaccine containing the recombinant Salmonella enteritidis described above.

[0026] Beneficial effects: Compared with the prior art, the present invention has the following outstanding and significant advantages:

[0027] (1) The attenuated strain of Salmonella enteritidis with simultaneous deletion of two genes provided by the present invention has significantly reduced virulence compared with wild-type Salmonella enteritidis. The virulence of the attenuated strain was calculated to be 178 times lower than that of the wild strain using the Reed-Muench method. The colonization time in the host was significantly shortened. No adverse reactions were observed in chickens after inoculation. It provides good protection against highly virulent Salmonella enteritidis and can effectively eliminate infection by highly virulent strains.

[0028] (2) The vaccine candidate strain provided by this invention exhibits good immunogenicity when applied to inactivated Salmonella enteritidis vaccine, showing no significant difference compared to wild-type strain S; the vaccine candidate strain possesses DIVA characteristics, allowing for the differentiation between vaccine-immunized chickens and wild-type infected chickens by detecting flagellar antibodies; when the immunization dose is 1.0 × 10⁻⁶... 7 At CFU / feather, the protective efficacy of the vaccine candidate strain remained at 75%~100% under different challenge doses, providing solid immune protection for chicks. Attached Figure Description

[0029] Figure 1 This is a diagram showing the PCR verification of the inner and outer primers for the SE gene deletion strain.

[0030] Figure 2 Figure showing the bacterial load in organs of SPF chickens after inoculation with SE-MP and SE;

[0031] Figure 3 Figure showing the changes in average body weight of SPF chickens after inoculation with SE-MP and SE;

[0032] Figure 4 Figure showing the injection sites and organs of SPF chickens after inoculating them with SE-MP and SE;

[0033] Figure 5 Figure showing the results of serum antibody IgY detection in SPF chickens after inoculation with SE-MP and SE;

[0034] Figure 6 DIVA differential diagnosis results of SPF chickens inoculated with SE-MP and SE;

[0035] Figure 7 Organ phenotypes observed in SPF chickens inoculated with high doses of SE-MP;

[0036] Figure 8 Liver pathology images (200×) of SPF chickens inoculated with SE-MP and SE.

[0037] Figure 9 Pathological images of spleens in SPF chickens inoculated with SE-MP and SE (200×). Detailed Implementation

[0038] The technical solution provided by the present invention will be described in detail below with reference to the embodiments. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments were all purchased from conventional biochemical reagent stores.

[0039] In this example, Salmonella enteritidis C50041 was isolated and preserved from the Jiangsu Provincial Key Laboratory of Zoonoses. Bacterial genomic DNA extraction kit was purchased from Tiangen Biotech Co., Ltd., catalog number RK127-B; Prime STAR MaxPremix (2×) was purchased from Vazyme, catalog number P222-02; 2× CE Mix was purchased from Vazyme, catalog number C117. Rabbit anti-chicken HRP-labeled secondary antibody was purchased from Abcam, catalog number ab6721.

[0040] Example 1: Construction of Salmonella Enteritidis C50041 mutant strain

[0041] 1. Primer design

[0042] Based on the published Salmonella genome sequence (CP013097) in GeneBank, specific upstream and downstream homologous arm sequences were designed using Primer 5.0 software. The primers are shown in Table 1.

[0043] Table 1 Primer sequences used for PCR amplification

[0044]

[0045]

[0046] 2. Construction of recombinant suicide plasmid pGMB152-ΔmsbB

[0047] Single colonies of *Salmonella enteritidis* C50041 (SE) were picked and cultured in LB broth at 37°C and 180 rpm for 12–16 h until the logarithmic growth phase. After centrifugation, the bacterial cells were collected, and the bacterial genome was extracted using a bacterial genomic DNA extraction kit. Using the extracted SE genome as a DNA template, homologous fragments of the target gene (msbB-Up / Down) were amplified using specific primers msbB-Up-F / R (sequences shown in SEQ ID NO.1 and SEQ ID NO.2) and msbB-Down-F / R (sequences shown in SEQ ID NO.3 and SEQ ID NO.4). The PCR reaction system is shown in Table 2. The PCR products were identified by 1% agarose gel electrophoresis, recovered using an agarose gel extraction kit, and their concentration was determined. The products were stored at -20°C for later use.

[0048] Table 2 PCR reaction system and reaction conditions

[0049]

[0050] Homologous fragments msbB-Up and msbB-Down were ligated to the linearized pGMB152 vector (pGMB152 is published in "Geng, H.; Luo, L.; Zhang, J.; Gao, J.; Geng, S.; Barrow, P. Identification of a Novel Regulatory Gene, trmE, that Orchestrates Salmonella Flagellar Synthesis and Virulence. Microorganisms 2025, 13, 1455.") using a one-step ligation method to obtain the pGMB152-ΔmsbB recombinant plasmid. The ligation system is shown in Table 3.

[0051] Table 3 Connection of reaction system and reaction conditions

[0052]

[0053] 3. Transformation of recombinant suicide plasmid pGMB152-ΔmsbB

[0054] E. coli Spy372 and E. coli χ7213 competent cells were prepared using a supercompetent cell preparation kit (E. coli Spy372 and E. coli χ7213 competent cells are publicly available in "Geng, H.; Luo, L.; Zhang, J.; Gao, J.; Geng, S.; Barrow, P. Identification of a Novel Regulatory Gene,trmE, that Orchestrates Salmonella Flagellar Synthesis and Virulence. Microorganisms 2025, 13, 1455."). 10 μL of recombinant plasmid pGMB152-ΔmsbB was added to 100 μL of ice-cold E. coli Spy372 competent cells. The cells were gently tapped to mix, without pipetting, and incubated on ice for 30 min. After an ice bath, the cells were heat-shocked at 42°C for 90 sec, and immediately cooled on ice for 2–3 min. Add 900 μL of LB liquid medium to the tube and incubate at 37°C and 180 rpm for 2 h on a shaker. Spread an appropriate amount of bacterial culture onto LB plates containing IPTG (isopropyl-β-D-thiogalactopyranoside, 100 μM), X-Gal (5-bromo-4-chloro-3-indole-β-D-galactopyranoside, 40 μg / mL), Amp (ampicillin, 100 μg / mL), and Sm (streptomycin, 100 μg / mL), and incubate at 37°C in the dark for 12–16 h. Pick single blue colonies and perform PCR identification using the specific primers msbB-UP-F (sequence shown in SEQ ID NO.1) / msbB-Down-R (sequence shown in SEQ ID NO.4). Positive clones are E. coli Spy372 (pGMB152-ΔmsbB) containing the recombinant plasmid. The PCR reaction system is shown in Table 4.

[0055] Table 4 PCR reaction system and reaction conditions

[0056]

[0057] The recombinant plasmid of the verified E. coli Spy372 (pGMB152-ΔmsbB) strain was extracted and transformed into competent E. coli χ7213 cells using the same method as described above. However, DAP (diaminopimelic acid, 100 μg / mL) was added during the culture of E. coli χ7213 cells. The specific primers msbB-UP-F / msbB-Down-R were used to identify E. coli χ7213 (pGMB152-ΔmsbB). The PCR reaction system is shown in Table 4.

[0058] 4. Construction of recombinant Salmonella enteritidis C50041ΔmsbB

[0059] Conjugation transfer: Verified donor strain *E. coli* χ7213 (pGMB152-ΔmsbB) and recipient strain C50041 wild-type strain were inoculated into LB broth containing DAP (100 μg / mL) and LB broth without DAP, respectively, and cultured at 37°C and 180 rpm to the logarithmic phase. 4 mL of donor bacterial suspension and 1 mL of recipient bacterial suspension were collected by centrifugation at 5,000 rpm for 5 min. After washing once with sterile PBS, the cells were resuspended in 200 μL PBS and mixed. The mixed suspension was added dropwise to the center of an LB agar plate (containing DAP) lined with a 0.22 μm aqueous microporous filter membrane, membrane face up, and co-cultured at 30°C for 36 h. The filter membrane was removed, and the bacterial growth on the membrane was washed off with 1 mL of sterile PBS and transferred to a 1.5 mL centrifuge tube. After serial dilution, 100 μL was plated onto LB agar plates containing IPTG, X-Gal, Amp, and Sm, and incubated upside down at 37°C in the dark for 12–16 h. Blue single colonies were picked and verified by PCR using the msbB-UP-F / msbB-Down-R primers. Positive clones were identified as recombinant Salmonella enteritidis C50041 (pGMB152-ΔmsbB). The PCR reaction system is shown in Table 4.

[0060] Removal of the suicide plasmid: Select a single blue colony that has been verified correctly in the previous step and transfer it to LB broth containing 15% sucrose. Incubate at 37°C with shaking at 180 rpm for 12-16 h until the logarithmic growth phase. Take 1 mL of culture, wash once with sterile PBS, and then serially dilute. Spread 100 μL of the appropriately diluted bacterial suspension onto LB agar plates containing IPTG and X-Gal. After incubation at 37°C, select a single white colony and perform PCR amplification and identification using msbB-UP-F / msbB-Down-R to obtain recombinant Salmonella enteritidis C50041ΔmsbB without the suicide plasmid. The PCR reaction system is shown in Table 4.

[0061] 4. Construction of recombinant Salmonella enteritidis C50041ΔpurL

[0062] The construction method of C50041ΔpurL is the same as that of C50041ΔmsbB. Using the extracted SE genome as a DNA template, homologous fragments upstream and downstream of the target gene were amplified using specific primers purL-UP-F (SEQ ID NO.5) / purL-UP-R (SEQ ID NO.6) and purL-Down-F (SEQ ID NO.7) / purL-Down-R (SEQ ID NO.8), respectively. The homologous fragments were then ligated to the pGMB152 linearized vector using a one-step ligation method to obtain the pGMB152-ΔpurL recombinant plasmid.

[0063] During conjugation transfer, C50041 was used as the recipient bacterium and E. coli χ7213 (pGMB152-ΔpurL) was used as the donor bacterium. After co-culturing, recombinant bacteria were obtained. The suicide plasmid was then removed using 15% sucrose LB liquid medium to obtain the target deletion strain C50041ΔpurL.

[0064] 5. Construction of recombinant Salmonella enteritidis C50041ΔmsbBΔpurL

[0065] Using the C50041ΔmsbB obtained above as the recipient and the E. coli χ7213 (pGMB152-ΔpurL) obtained above as the donor, conjugation transfer was performed (the steps are the same as above). After co-culturing, recombinant bacteria were obtained. The suicide plasmid was then removed with 15% sucrose LB liquid medium to obtain recombinant Salmonella enteritidis C50041ΔmsbBΔpurL (SE-MP).

[0066] 6. PCR and sequencing verification of recombinant Salmonella enteritidis C50041ΔmsbBΔpurL (SE-MP)

[0067] PCR verification was performed using two inner and outer primers for the gene. PCR was performed using the outer primers msbB-UP-F / msbB-Down-R and purL-UP-F / purL-Down-R, and the results are as follows: Figure 1 As shown, the amplified bands of the gene deletion strains represented by lanes 1 and 5 are smaller than those of the parental strains represented by lanes 2 and 6. However, when using the inner primers msbB-in-F (SEQ ID NO. 9) / msbB-in-R (SEQ ID NO. 10) and purL-in-F (SEQ ID NO. 11) / purL-in-R (SEQ ID NO. 12), the deletion strains in lanes 3 and 7 did not amplify any bands, while the parental strains in lanes 4 and 8 did amplify any bands.

[0068] Simultaneously, C50041ΔmsbBΔpurL sequencing was performed to amplify and sequence the upstream and downstream homologous arms (UP-F / Down-R) of the msbB and purL genes. The sequencing results showed that the msbB and purL genes were missing in the vaccine strain compared with the SE sequence of the wild strain.

[0069] The successful construction of C50041ΔmsbBΔpurL was confirmed by PCR inner and outer primers and sequencing verification.

[0070] Example 2: Comparison of virulence between vaccine candidate strain SE-MP and wild-type strain SE

[0071] The vaccine candidate strain SE-MP and the wild-type strain SE were inoculated into LB liquid medium and cultured at 37°C for 12 h. Then, the bacterial culture was transferred to fresh LB liquid medium at a volume ratio of 1:50 and cultured for 6 h before collecting the bacterial cells. The bacterial cells were washed three times with sterile PBS and the bacterial concentration was adjusted using a spectrophotometer.

[0072] Seven-day-old SPF chicks were randomly divided into two groups and injected intramuscularly with the candidate vaccine strain SE-MP and the wild-type strain SE, respectively. The immunization dose was set at 1.0 × 10⁻⁶. 5 CFU / feather, 1.0×10 6 CFU / feather, 1.0×10 7 CFU / feather, 1.0×10 8 CFU / feather, 1.0×10 9 CFU / Yuhe 1.0×10 10 CFU / bird, 5 chicks per dose group, with a blank control group (SPF chicks received no treatment). Chicks were observed for 14 consecutive days post-immunization, and the survival and mortality rates were recorded. LD50 was calculated based on these results. 50 This study aimed to evaluate the virulence of vaccine candidate strains and wild-type strains to SPF chickens. The Reed-Muench method was used to calculate the LD50. 50 The logarithm of the dose in the maximum vaccination group is denoted as X. m The logarithm of the ratio of the high dose to the low dose in two adjacent groups is denoted as i, and the total mortality rate of animals in each group is represented by ΣP.

[0073] LD 50 The formula is as follows:

[0074] SPF Chicken LD 50 The analysis is shown in Table 5, SE-MP's LD 50 >1.0×10 10 CFU / feather. And the LD of wild-type SE. 50 It is 5.6 × 10 7CFU / feather. Calculations showed that the toxicity of SE-MP was 178-fold lower than that of the wild-type strain, indicating an order-of-magnitude reduction in SE-MP toxicity. The experiment strictly followed a dose gradient setting (10... 6 -10 10 (CFU / feather), which meets the safety design requirements for attenuated live vaccine strains.

[0075] Table 5. Effects of SE-MP vaccine candidate strain on LD50 in SPF chickens 50 Measurement

[0076]

[0077] Example 3: Colonization assay of vaccine candidate strain SE-MP in the host.

[0078] 1. The ability of SE-MP to be reduced in SPF chicks

[0079] Seven-day-old SPF chicks were randomly divided into 5 groups, including: a low-dose group of the SE-MP vaccine candidate strain (1.0 × 10⁻⁶). 7 CFU / feather), high-dose group of SE-MP vaccine candidate strain (1.0×10⁻⁶) 8 CFU / feather), SE wild-type low-dose group (1.0×10⁻⁶) 7 CFU / feather), SE wild-type high-dose group (1.0×10⁻⁶) 8 (CFU / bird) and a blank control group (SPF chicks received no treatment). On days 3, 7, 14, and 21 post-immunization, three chicks from each group were randomly selected and euthanized; liver and spleen were aseptically collected. Appropriate tissue samples were weighed and placed in sterile homogenization tubes, with 1 mL of sterile PBS added, and thoroughly homogenized in a tissue homogenizer. The tissue homogenates were serially diluted 10-fold, with appropriate dilutions (three replicates per dilution) and plated on XLT4 plates. After incubation at 37°C for 12–18 h, colony counting was performed. Based on the colony count results, the Salmonella load (CFU / g) in each tissue was calculated. Reduction dynamic curves were plotted using the logarithmic values ​​of Salmonella load at each time point to analyze the reduction dynamics of the vaccine candidate strain and the wild-type strain in the liver and spleen.

[0080] The results are as follows Figure 2 As shown, bacterial load was measured by necropsy starting from the third day after vaccination. In both the spleen and liver, the bacterial load of the SE wild-type strain was higher than that of the SE-MP vaccine candidate strain 14 days after immunization. After 21 days of immunization, the strains in both the liver and spleen were completely cleared. This indicates that SE-MP has good clearance in the host, significantly superior to the wild-type SE strain. Furthermore, the colonization level of SE-MP in organs showed a dose-dependent effect, with the bacterial load in the high-dose immunization group being significantly higher than that in the low-dose group.

[0081] 2. Weight difference analysis

[0082] To evaluate the effect of the vaccine candidate strain SE-MP on the production performance of SPF chickens, SE-MP and SE were respectively used at 1.0 × 10⁻⁶. 7 Seven-day-old SPF chickens were immunized with a CFU / feather dose. On days 0, 3, 7, 14, and 21 post-immunization, the same number of SPF chickens were randomly selected from each group for weighing. The results are as follows: Figure 3 As shown, compared with the control group (SPF chicks without any treatment), there was no significant difference in the body weight of SPF chicks in the SE-MP group; while the body weight of chicks inoculated with wild-type strains was significantly reduced, indicating that SE-MP does not affect the production performance of chickens (*P<0.05, ***P<0.001).

[0083] 3. Safety analysis of injection sites and organs

[0084] Seven-day-old SPF chicks were randomly divided into three groups: two immunization groups, one challenge group, and one blank control group. Chicks in the two immunization groups were immunized via intramuscular injection in the leg with 1.0 × 10⁻⁶ ppm. 6 CFU / feather, 1.0×10 7 The candidate vaccine strain was administered at a CFU / feather dose, and the challenge and blank control groups were injected with an equal volume of sterile PBS. On day 21 post-immunization, chicks in both the immunized and challenge groups were injected intramuscularly with the virulent strain Z11 (preserved in our laboratory, strain sequence available in GenBank: NC_003197.2) at a challenge dose of 2.0 × 10⁻⁶. 9 CFU / bird, and the blank control group was injected with an equal volume of sterile PBS. After 14 days of continuous observation after challenge, the chickens were euthanized and dissected to observe whether lesions occurred in the local tissues and organs at the immunization and challenge injection sites, including congestion, edema, necrosis and nodules.

[0085] The results are as follows Figure 4 As shown, no bacteria were isolated from the organs and tissues of the immunized group two weeks after challenge. Compared with the control group, no obvious hyperemia, fibrosis, or other abnormal changes were observed in the skin and subcutaneous tissue at the injection site of the immunized group. The local muscle fibers were neatly arranged without lysis or necrosis. The liver was uniform in color, smooth in surface, and had an intact capsule without abnormalities. The liver and spleen were of normal size, smooth in surface, and without nodules or necrotic spots. Chickens in the challenge group showed obvious lesions: obvious necrosis was visible in the local muscle at the injection site; black spot-like necrosis appeared on the surface of the spleen; and multiple grayish-yellow necrotic foci were also visible in the liver. The results indicate that SE-MP is non-pathogenic to SPF chickens and has a high safety profile.

[0086] Example 4 Immunoprotective assay of vaccine candidate strain SE-MP

[0087] 1. Immunogenicity test: IgY antibody detection

[0088] To evaluate the effect of the SE-MP vaccine candidate strain on the humoral immune response of SPF chicks, the serum specific IgY antibody titer at different time points after immunization with the vaccine candidate strain was detected by whole-cell coated ELISA.

[0089] Seven-day-old SPF chicks were randomly divided into five groups of five chicks each: two immunization groups, two challenge groups, and a control group. The immunization groups received an intramuscular injection of the candidate vaccine strain SE-MP via the leg, while the challenge groups received the wild-type strain SE via the same route. The dosage for both groups was 1.0 × 10⁻⁶. 7 CFU / Yuhe 1.0×10 8 CFU / bird, and the blank control group was injected with an equal volume of sterile PBS. On days 7, 14, and 21 post-immunization, three chicks from each group were randomly selected, blood was collected from the wing vein, serum was separated, and the titer of specific IgY antibodies in the chicken serum was detected by whole-cell coated ELISA.

[0090] The ELISA testing steps are as follows:

[0091] (1) Pretreatment of ELISA strips: Add 100 μL of 5% glutaraldehyde solution (prepared by mixing 80 mL of 0.1 mol / L NaHCO3 solution with 20 mL of 25% glutaraldehyde solution) to each well, incubate in a water bath at 37℃ for 2 h, and wash 5 times with sterile distilled water for 3 min each time.

[0092] (2) Antigen coating: The overnight cultured Salmonella enteritidis SE-MP and SE bacterial cultures were centrifuged to collect the bacterial cells, washed with sterile PBS and resuspended, and the bacterial concentration was adjusted to 1×10⁻⁶. 8 CFU / mL. Add 100 μL of bacterial culture to each well and dry at 56°C overnight (22-24h).

[0093] (3) Fixation: The next day, take out the strips, add 100 μL of pre-cooled anhydrous methanol to each well, let stand at room temperature for 15 min, wash with sterile distilled water 3 times for 3 min each time to remove unbound antigens.

[0094] (4) Blocking: Add 200 μL of blocking solution (1% BSA prepared with PBST) to each well and block at 37℃ for 2 h. After blocking, wash with PBST 5 times for 3 min each time.

[0095] (5) Serum incubation: Serum samples from each group were serially diluted with 1% BSA prepared with PBST at a dilution of 1:200. 100 μL of diluted serum was added to each well. A negative control group and a blank control group were also set up. The samples were incubated at 37°C for 2 h. Then, the samples were washed three times with PBST for 3 min each time and patted dry on absorbent paper.

[0096] (6) Secondary antibody incubation: Add 100 μL of rabbit anti-chicken HRP-labeled secondary antibody diluted 1:1000 to each well and incubate in a water bath at 37℃ for 1 h. Wash 7 times with PBST for 3 min each time and pat dry.

[0097] (7) Color development and termination: Add 100 μL of TMB substrate to each well, develop color at 37℃ in the dark for 10 min, and then add 50 μL of 2 mol / L H2SO4 to terminate the reaction.

[0098] (8) Result determination: The OD of each well was measured using a microplate reader. 450 Value. Based on the OD of the sample well. 450 The value is greater than the OD of the negative control well. 450 The highest dilution factor of 2.1 times is used as the serum antibody titer.

[0099] The results are as follows Figure 5 As shown, SE-MP induced specific IgY antibodies in SPF chickens at both doses, and the titer increased with prolonged immunization time, indicating good immunogenicity. Compared with the wild-type SE strain, the antibody titer of the vaccine candidate strain showed no significant difference, indicating that gene deletion did not significantly weaken the immunogenicity of SE-MP.

[0100] 2. SE-MP's DIVA differential diagnostic capability test

[0101] To evaluate the DIVA differential diagnostic ability of the vaccine candidate strain SE-MP, the flagella-deficient characteristic of this vaccine candidate strain was utilized. Indirect ELISA was used to detect flagellar protein-specific antibodies in the serum of immunized chickens to verify whether it could differentiate from wild-type strain infection. Seven-day-old SPF chicks were randomly divided into 5 groups, including: a low-dose group of the vaccine candidate strain (1.0 × 10⁻⁶). 7 CFU / feather), high-dose group of vaccine candidate strain (1.0×10⁻⁶) 8 CFU / feather), low-dose wild-type strain (1.0×10⁻⁶) 7 CFU / feather), high-dose wild-type plant group (1.0×10⁻⁶) 8 (CFU / bird) and a blank control group. On day 21 post-immunization, blood was collected from chicks in each group to separate serum, which was stored at -20℃ for later use.

[0102] The steps for indirect ELISA detection of flagellin-specific antibodies are as follows:

[0103] (1) Antigen coating: Salmonella enteritidis flagellin was diluted to 1 μg / mL using CBS solution (carbonate buffer), and 100 μL was added to each well of a 96-well ELISA plate. The plate was then coated overnight at 4°C (12 h).

[0104] (2) Washing: Discard the coating solution, add 200 μL PBST (PBS containing 0.05% Tween-20) to each well and wash 3 times for 3 min each time, then pat dry on absorbent paper.

[0105] (3) Blocking: Add 200 μL of blocking solution (PBS containing 5% BSA) to each well and incubate at 37°C for 3 h.

[0106] (4) Washing: Same as step (2).

[0107] (5) Serum incubation: The serum samples of each group were serially diluted with PBS (initial dilution of 1:1000), and 100 μL of diluted serum was added to each well. The samples were incubated at 37°C for 2 h. A negative control was also set up.

[0108] (6) Washing: Same as step (2).

[0109] (7) Secondary antibody incubation: Add 100 μL of rabbit anti-chicken HRP-labeled secondary antibody diluted 1:1000 to each well (dilution solution is PBS), and incubate at 37℃ for 1 h.

[0110] (8) Washing: Add 200 μL PBST to each well and wash 7 times, 3 min each time, and pat dry.

[0111] (9) Color development and termination: Add 100 μL of TMB substrate to each well, develop color at 37℃ in the dark for 10 min, and then add 50 μL of 2 mol / L H2SO4 to terminate the reaction.

[0112] (10) Result determination: OD was measured using an enzyme-linked immunosorbent assay (ELISA) reader. 450 Value. Based on the OD of the sample well. 450 The value is greater than the OD of the negative control well. 450 The highest dilution factor of 2.1 times is used as the serum antibody titer.

[0113] If the titer of flagellin-specific antibodies in the serum of the vaccine candidate strain immunization group is significantly lower than that in the wild strain immunization group, and there is no significant difference from the blank control group, it indicates that the vaccine strain has good DIVA ability and can achieve serological differentiation between immunized animals and wild-type infected animals.

[0114] The results are as follows Figure 6 As shown, compared with the control group, no flagellated antibodies were detected in the vaccine candidate strain SE-MP (P / N<2.1), while high-titer flagellated antibodies were detected in wild-type SE-infected chickens (P / N>2.1). This allows for the differentiation between vaccine-immunized chickens and wild-type infected chickens by detecting flagellated antibodies. Therefore, this vaccine candidate strain has DIVA characteristics (***P<0.001).

[0115] 3. SE-MP immune protection efficiency

[0116] To evaluate the immunoprotective efficacy of the vaccine candidate strain SE-MP against SPF chicks, its immunoprotective efficacy against the virulent Salmonella enteritidis strain Z11 was determined by intramuscular injection followed by challenge.

[0117] The vaccine candidate strain SE-MP was administered intramuscularly to 7-day-old SPF chicks at a dose of 1.0 × 10⁻⁶. 5 CFU / feather, 1.0×10 6 CFU / feather, 1.0×10 7 CFU / feather concentration. On day 14 or 21 post-immunization, immunized chickens were challenged with a virulent Salmonella enteritidis strain Z11 via intramuscular injection at a dose of 1.0 × 10⁻⁶. 9 CFU / feather or 2.0×10 9 CFU / feather or 3.5×10 9 CFU / feather was used to challenge the vaccine, and its survival / death status was recorded for two weeks. Immunoprotective efficacy was calculated to evaluate the immunoprotective effect of the vaccine candidate strain. The formula for calculating vaccine protective efficacy is as follows:

[0118] Vaccine protection efficiency = (mortality rate in the control group - mortality rate in the immunized group) / mortality rate in the control group × 100%

[0119] The results of SE-MP immunoprotective efficacy are shown in Table 6. When the immunization dose is 1.0 × 10⁻⁶, the efficacy is as follows: 7 At CFU / feather, SE-MP showed the best immunoprotective effect on chicks at different challenge doses, maintaining a range of 75%–100%. The experiment was independently repeated three times, confirming that the vaccine candidate strain SE-MP has good immunoprotective efficiency and stability in chicks.

[0120] Table 6. Determination of the immunoprotective efficacy of the vaccine candidate strain SE-MP against SPF chicks.

[0121]

[0122] 4. Safety dose evaluation experiments of SE-MP at 10-100 times the standard dose.

[0123] To evaluate the safety of the high-dose vaccine candidate strain SE-MP in 7-day-old SPF chickens, 1.0 × 10⁻⁶ doses were administered. 7 10 times the CFU / feather (1.0×10 8 CFU / feather) and 100x (1.0×10 9 SPF chicks were immunized intramuscularly with a dose of CFU / bird. Fourteen days after immunization, the chicks were dissected to observe the liver and spleen phenotypes and perform pathological tissue analysis. Results are as follows: Figure 7 , Figure 8 , Figure 9As shown, compared with the blank group, 1.0×10 7 CFU / feather and its 10-fold and 100-fold doses of SE-MP did not cause death or lesions in SPF chickens, and no obvious abnormal lesions or necrosis were observed in the liver and spleen.

Claims

1. A recombinant Salmonella enteritidis, characterized in that, The recombinant Salmonella enterica contains both the msbB and purL genes absent in the Salmonella enterica.

2. The recombinant Salmonella enteritidis according to claim 1, characterized in that, The nucleotide sequence of the msbB gene is shown in SEQ ID NO.13, and the nucleotide sequence of the purL gene is shown in SEQ ID NO.

14.

3. A method for constructing recombinant Salmonella enteritidis according to claim 1, characterized in that, The method includes the following steps: knocking out the msbB and purL genes in Salmonella enteritidis.

4. The method for constructing recombinant Salmonella enteritidis according to claim 3, characterized in that, The method includes the following steps: (1) Using the genomic DNA of Salmonella enteritidis as a template, the upstream and downstream homologous arms of the msbB gene and the purL gene were amplified by PCR. The amplification products were homologously recombined with the linear vectors to obtain plasmids with msbB gene deletion and plasmids with purL gene deletion. Then, they were transformed into Escherichia coli to obtain two donor bacteria. (2) One of the donor bacteria obtained in step (1) is conjugated with the recipient bacteria Salmonella enterica for homologous single crossover recombination, and a strain with the corresponding gene deletion is obtained by homologous double crossover. The deleted strain is used as the recipient and conjugated with another donor bacteria obtained in step (1) for homologous single crossover recombination, and a strain with double deletion of msbB gene and purL gene is obtained by screening.

5. The method according to claim 4, characterized in that, The screening method described in step (2) includes screening using chloramphenicol resistance and sucrose sensitivity.

6. A method for reducing the virulence of Salmonella enteritidis, characterized in that, Recombinant Salmonella enterica was constructed by knocking out the msbB gene (nucleotide sequence shown in SEQ ID NO. 13) and the purL gene (nucleotide sequence shown in SEQ ID NO. 14) from Salmonella enterica, or by constructing recombinant Salmonella enterica according to the method described in claim 3.

7. The use of the recombinant Salmonella of claim 1 or 2, or the method of claim 3, in the preparation of an attenuated vaccine.

8. The application according to claim 7, characterized in that, The attenuated vaccine includes a vaccine to prevent Salmonella enteritidis infection in poultry.

9. The application according to claim 8, characterized in that, The poultry includes one or more of the following: broiler chickens, breeding chickens, laying hens, meat ducks, laying ducks, breeding ducks, meat geese, breeding geese, pigeons, and quails.

10. A vaccine for the prevention of Salmonella enteritidis, characterized in that, The vaccine contains the recombinant Salmonella enteritidis as described in claim 1.

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