Construction and application of salmonella choleraesuis three-gene deletion strain without resistance marker

Abstract

The invention discloses a salmonella choleraesuis three-gene deletion strain without a resistance marker, wherein the preservation number of the salmonella choleraesuis three-gene deletion strain is CCTCC (China Center For Type Culture Collection) M 20242059. The strain successively lacks a virulence regulating gene crp, an important nutrition metabolism gene asd for mediating diaminopimelic acid (DAP) synthesis, and an important carbon metabolism gene manA for mediating and coding mannose-6-phosphate isomerase. The salmonella choleraesuis gene deletion mutant strain is constructed by adopting a suicide vector and SacB reverse inheritance, and a non-resistant three-gene deletion mutant strain is obtained. The three-gene deletion mutant strain is clear in genetic background, free of resistance markers, good in safety and immunoprotectiveness to mice and capable of helping the mice to resist attack of high-lethal-dose salmonella choleraesuis, as the asd gene in the deletion strain is completely deleted in a traceless mode, a balanced lethal system plasmid pya3493-ApxIAN is electrically transformed into the deletion strain, and therefore the expression of the salmonella choleraesuis in the high-lethal-dose salmonella choleraesuis in the high-lethal-dose salmonella choleraesuis. The recombinant strain not only can grow in a DAP-free culture medium, but also can stably express foreign protein ApxIAN, and has a wide application prospect.

Description

Technical Field

[0001] The invention relates to the technical field of veterinary biological products, and in particular to the construction of a three-gene deletion strain of Salmonella choleraesuis without resistance markers and its application. Background Art

[0002] Salmonella enterica serovar Choleraesuis (S.Choleraesuis) is an important pathogen that causes paratyphoid fever in piglets, which manifests as septicemia and enterocolitis. Acute cases usually cause septicemia and lead to death. Chronic cases manifest as colitis, with a mortality rate of 25% to 50%. Surviving pigs grow slowly and carry pathogens for a long time, which seriously damages the economic interests of farmers.

[0003] The best strategy for preventing and controlling paratyphoid in piglets is vaccination. In the early years, inactivated vaccines were used, but they were eliminated due to severe side effects. The C500 strain is a weak strain obtained by chemically weakening the C78-1 strain by Fang Xiaowen and others in the 1960s. It has good immunogenicity, but the mechanism is unclear and there is still strong residual virulence. Therefore, many scholars use the strong strains of Salmonella choleraesuis C78-1, C78-2 or C78-3 as starting strains to develop genetically engineered attenuated live vaccines; among them, C78-2 is an isolated strain prevalent in Jiangsu Province, my country. After whole genome sequencing, the genetic background is clear. After genetic engineering attenuation, the pathogenicity to the host will be greatly reduced. It is more suitable to develop a attenuated live vaccine for use in my country.

[0004] Salmonella is an intracellular parasite that can adhere to the skin mucosa, respiratory tract, digestive tract and other parts through adhesins. Therefore, it can be used as an attenuated live vaccine to stimulate the body to produce efficient cellular and humoral immunity while also exerting its unique mucosal immunity. Attenuated Salmonella can also be used as a live vaccine carrier to carry various exogenous protective genes into the body, enabling the body to resist the attack of multiple pathogens at the same time. The Salmonella vector-exogenous protective gene recombinant bivalent vaccine constructed in this way has high immunogenicity, reduces costs, and reduces the number of vaccinations. In 1988, Nakayama et al. were the first to successfully construct a chromosome-plasmid balanced lethal system of mouse Salmonella, which can stably express exogenous antigens. Since then, many scholars have used C500 as the starting strain to construct a chromosome-plasmid balanced lethal system of suis Salmonella, and successfully constructed a batch of recombinant strains expressing exogenous proteins. Therefore, the use of the Salmonella balanced lethal system can realize the development of multi-linked and multivalent vaccines.

[0005] Salmonella choleraesuis has many virulence-related factors. Among them, the asd gene encodes aspartate β-galactose dehydrogenase, which is a key enzyme in the synthesis pathway of diaminopimelic acid (DAP). DAP is the basic component of the peptidoglycan of the Salmonella cell wall. The loss of the asd gene will cause the Salmonella to be unable to form a cell wall and to lyse and die. The lack of DAP in the body will cause the mutant strain to gradually degrade in the animal body. The virulence-related gene crp encodes adenylate cyclase receptor protein (cAMP receptor protein, crp). When the crp gene is knocked out, the strain lacks cAMP receptor protein, resulting in the inability of Salmonella to absorb cyclic adenosine monophosphate from the host, which will affect the metabolism of bacterial amino acids and carbon sources, as well as the expression of bacterial pili and flagella. The manA gene encodes mannose-6-phosphate isomerase, which can catalyze the isomerization of D-mannose and D-fructose in Escherichia coli and promote the metabolic absorption of carbon sources by bacteria. The deletion of the manA gene will affect the expression of related genes in the mannose, fructose and starch metabolic pathways of Escherichia coli, thereby affecting the uptake and utilization of carbon sources by Escherichia coli. Summary of the invention

[0006] The purpose of the present invention is to obtain a △crp / asd / manA three-gene deletion strain of Salmonella choleraesuis with clear genetic background, strong safety and no resistance marker.

[0007] The △crp / asd / manA (C78-2) triple gene deletion strain of the present invention can be used as a recipient bacterium containing an asd gene plasmid and can stably carry and express exogenous antigen genes.

[0008] To achieve the above-mentioned purpose, the present invention provides a three-gene deletion strain of Salmonella choleraesuis without a resistance marker, which is Salmonella choleraesuis △crp / asd / manA (C78-2), which was preserved in the China Center for Type Culture Collection (Wuhan University) on October 1, 2024, with the preservation number of CCTCC M 20242059.

[0009] The three-gene deletion strain of Salmonella choleraesuis without resistance markers successively lacks the virulence regulating gene crp of Salmonella choleraesuis, the important nutritional metabolism gene asd mediating the synthesis of diaminopimelic acid (DAP), and the important carbon metabolism gene manA mediating the encoding of mannose-6-phosphate isomerase. The △crp / asd / manA (C78-2) three-gene deletion strain has lower virulence than the parent strain C78-2 and the △crp / asd (C78-2) double gene deletion strain, better safety, and better immunogenicity.

[0010] The three-gene deletion strain of Salmonella choleraesuis without resistance markers △crp / asd / manA (C78-2) can form a complementary expression system with a plasmid expressing the asd gene. Further, the present invention provides an application of the three-gene deletion strain of Salmonella choleraesuis without resistance markers as an exogenous antigen expression bacterium or a carrier bacterium.

[0011] The three-gene deletion strain of Salmonella choleraesuis without resistance marker of the present invention is derived from Salmonella choleraesuis C78-2 (commercial strain purchased from the National Veterinary Microbiological Collection Center of the China Veterinary Drug Administration, number: CVCC79102). The PRE112 plasmid, the pya3493 plasmid containing asd gene expression, the χ6097 strain and the χ7213 strain used were all donated by Professor Zhao Zhanqin of Henan University of Science and Technology.

[0012] The three-gene deletion strain of Salmonella choleraesuis without resistance markers has successively deleted the ORF sequences of crp, asd, and manA genes, and the asd gene, crp gene, and manA gene are all traceless knockouts, and there is no risk of mutation complementation in the animal body. The knocked-out crp gene sequence is 627bp in its ORF sequence, and the ORF sequence is shown in SEQ ID No.1; the knocked-out asd gene sequence is 1101bp in its ORF sequence, and the ORF sequence is shown in SEQ ID No.2; the knocked-out manA gene sequence is 1170bp in its ORF sequence, and the ORF sequence is shown in SEQ ID No.3. The deletion strain cannot survive in the animal body, and cannot survive on a culture medium without DAP. After the plasmid expressing the asd gene is transferred, it can survive on a culture medium without DAP.

[0013] A large number of biological tests have shown that the triple-gene deletion strain of the present invention has reduced pathogenicity to animals, good safety, high immune protection, and can be used to carry and express exogenous antigen genes.

[0014] The construction of the three-gene deletion strain of Salmonella choleraesuis without resistance marker of the present invention comprises the following steps:

[0015] (1) Construction of a Salmonella choleraesuis △crp (C78-2) gene-deficient strain; primers were designed based on the genome sequence of Salmonella choleraesuis C78-2, and the left and right homologous arm fragments (1009 bp and 1148 bp) of the crp gene were amplified from the genome of Salmonella choleraesuis C78-2. The fragments were fused using tandem PCR to obtain the Crp-LR fragment, and the fragment and the pRE112 plasmid were digested with SphI and XbaI, respectively, to obtain the digested fragments, which were ligated overnight using T4 ligase and transformed into χ7213 competent medium. The strain was cultured on LA plates containing chloramphenicol, and a single colony was picked for PCR identification and sent to Bio-Tech for sequencing, thereby obtaining the pRE112-crpLR plasmid. The plasmid was electroporated into the C78-2 strain, and the crp gene deletion strain of C78-2 was screened out using chloramphenicol resistance screening and SacB reverse screening technology. After PCR identification and sequencing by Bioengineering, it was named △crp (C78-2).

[0016] (2) Construction of a Salmonella choleraesuis △crp / asd (C78-2) gene-deficient strain; primers were designed based on the genome sequence of Salmonella choleraesuis C78-2 to knock out primers, and the left and right homologous arm fragments (1145 bp and 983 bp) of the asd gene were amplified from the genome of Salmonella choleraesuis C78-2. They were fused using tandem PCR to obtain the asd-LR fragment, and the fragment and the pRE112 plasmid were digested with SphI and XbaI, respectively, to obtain the digested fragments, which were ligated overnight using T4 ligase and transformed into χ7213 competent cells. The cells were cultured on LA plates containing chloramphenicol, and single colonies were picked for PCR identification and sent to Bio-Tech for sequencing, thereby obtaining the pRE112-asdLR plasmid. The plasmid was electroporated into the △crp (C78-2) strain, and the asd gene deletion strain of C78-2 was screened out using chloramphenicol resistance screening and SacB reverse screening technology. After PCR identification and sequencing by Biotechnology, it was named △crp / asd (C78-2).

[0017] (3) Construction of a Salmonella choleraesuis △crp / asd / manA (C78-2) gene-deficient strain; primers were designed based on the genome sequence of Salmonella choleraesuis C78-2 to knock out primers, and the left and right homologous arm fragments of the manA gene (992 bp and 948 bp) were amplified from the genome of Salmonella choleraesuis C78-2. They were fused using tandem PCR to obtain the manA-LR fragment, and the fragment and the pRE112 plasmid were digested with SphI and XbaI, respectively, to obtain the digested fragments, which were ligated overnight using T4 ligase and transformed into χ7213 competent cells. The cells were cultured on LA plates containing chloramphenicol, and single colonies were picked for PCR identification and sent to Bio-Tech for sequencing, thereby obtaining the pRE112-manALR plasmid. The plasmid was electroporated into the △crp / asd (C78-2) strain, and the asd gene deletion strain of C78-2 was screened out using chloramphenicol resistance screening and SacB reverse screening technology. After PCR identification and sequencing by Bioengineering, it was named △crp / asd / manA (C78-2).

[0018] (4) Biological characteristics of Salmonella choleraesuis △crp / asd / manA (C78-2); its biological characteristics were studied by detecting its genetic stability and growth characteristics.

[0019] (5) Virulence determination of Salmonella choleraesuis △crp / asd / manA (C78-2); The triple gene deletion mutant has lost the ability to synthesize diaminopimelic acid (DAP) and therefore cannot grow in a DAP-negative environment. Transforming the plasmid containing the asd gene into the △crp / asd / manA (C78-2) strain can complement the asd gene in the plasmid with the triple gene deletion strain, thereby enabling the triple gene deletion strain to survive under DAP-negative conditions. Therefore, the pya3493 plasmid was electroporated into the △crp / asd (C78-2) and △crp / asd / manA (C78-2) strains to obtain the △crp / asd (pya3493) and △crp / asd / manA (pya3493) strains, which were then used to infect 6-8 week old Kunming mice by intraperitoneal injection and gavage, and their LD50 was calculated to evaluate the virulence of the △crp / asd / manA (C78-2) triple gene deletion strain to mice.

[0020] (6) Evaluation of the immune protection of Salmonella choleraesuis △crp / asd / manA (C78-2) on mice; After the pya3493 plasmid was electroporated into the triple-gene deletion strain, the △crp / asd / manA (pya3493) strain was obtained, and after expansion culture, the bacteria were collected, washed and resuspended in PBS, and diluted to a certain dose to prepare the △crp / asd / manA (pya3493) live vaccine; at the same time, the bacterial solution and aluminum hydroxide adjuvant were mixed in a ratio of 9:1 to prepare the △crp / asd / manA (pya3493) strain live vaccine for intramuscular injection; the C78-2 strain after expansion culture was added with formaldehyde at a final concentration of 0.2%, and inactivated in a refrigerator at 4 degrees for 48 hours, the bacteria were collected, washed and resuspended in PBS, and the bacterial solution and aluminum hydroxide adjuvant were mixed in a ratio of 9:1 to prepare the C78-2 strain inactivated vaccine for intramuscular injection. 50 mice were prepared and divided into five groups, including oral immunization with △crp / asd / manA (pya3493) live vaccine, intramuscular injection of △crp / asd / manA (pya3493) live vaccine, oral immunization with C500 (commercialized), intramuscular injection of C78-2 inactivated vaccine and PBS group. After the first immunization, booster immunization was performed 14 days later, and 6 mice were randomly selected for tail-cutting blood collection, serum was collected, and IgA and IgG levels against C78-2 were detected. Subsequently, a lethal dose of C78-2 strain was used for oral infection, and the survival of mice was observed for 3 consecutive weeks. Thus, the immune protection efficacy of Salmonella choleraesuis △crp / asd / manA (C78-2) on mice was evaluated.

[0021] (7) Detection of exogenous gene carrying and expression in Salmonella choleraesuis △crp / asd / manA (C78-2); The truncated sequence of the ApxI gene of porcine Actinobacillus pleuropneumoniae (ApxIAN) was sent to Wuhan Jinkairui Co., Ltd. for Salmonella codon optimization and synthesis, and carried a 6x His tag at the C-terminus. The sequence was constructed into the pya3493 plasmid by homologous recombination. After PCR identification and sequencing by Shenggong Co., Ltd., the pya3493-ApxIAN plasmid was obtained, which was electroporated into the △crp / asd / manA (C78-2) strain to obtain the △crp / asd / manA (pya3493-ApxIAN) recombinant strain, which was expanded and cultured in LB medium, the bacteria were collected, crushed, the supernatant and the precipitate were separated, and the expression of ApxIAN protein was detected by WB.

[0022] The △crp / asd / manA (C78-2) strain of the present invention lacks the asd gene, and a balanced lethal plasmid containing the asd gene and the exogenous protein can be transformed into the strain, and the asd gene on the plasmid can complement the triple-gene deletion mutant strain, thereby solving the problem of the mutant strain surviving in the animal body, and stably expressing the exogenous protein. At the same time, the crp and manA genes are missing, so that the strain further loses the ability to metabolize carbon sources, and the triple-gene deletion mutant strain is less pathogenic to mice than the double-gene deletion strain △crp / asd (C78-2), and has better safety.

[0023] Beneficial effects of the present invention:

[0024] 1. The triple-gene deletion mutant of Salmonella choleraesuis provided by the present invention weakens the virulence of wild-type Salmonella choleraesuis, is safe, has no resistance markers, has no adverse side effects on mice, and can provide mice with immune protection against a high lethal dose of Salmonella choleraesuis, and has the potential to be developed into an attenuated live vaccine of Salmonella choleraesuis.

[0025] 2. The three-gene deleted strain of Salmonella choleraesuis of the present invention can form a complementary strain with a plasmid containing the asd gene. The plasmid containing the asd gene can carry foreign genes and express corresponding proteins, and has the potential to be developed into a live vaccine for the prevention and control of multiple pathogens. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 It is the suicide plasmid map for knocking out related genes; a, b, and c are the plasmid maps of pRE112-crpLR, pRE112-asdLR, and pRE112-manALR, respectively.

[0027] Figure 2 These are the amplified fragments and restriction enzyme fragments of the left and right homology arms of crp; a, amplification of the left and right homology arms of crp; 1 / 2: crp-R fragment, 3 / 4: crp-L fragment; b, homology arm fusion fragment crp-LR; c, crp-LR restriction enzyme fragment; M: 2000DNAMaker, M1: 5000DNAMaker.

[0028] Figure 3 It is the restriction fragment of pRE112 plasmid; M1: 5000 DNAMaker, M2: 15000 DNAMarker.

[0029] Figure 4 PCR identification of recombinant plasmid pRE112-crpLR transformants; in the figure, 1-8: plasmid transformant identification; P: pRE112 plasmid control; H: ddH2O blank control; M1: 5000 DNA Marker.

[0030] Figure 5 Flow chart for obtaining the △crp / asd / manA (C78-2) strain.

[0031] Figure 6 PCR identification of crp gene deletion in C78-2 strain; in the figure, 1-7: identification of crp gene deletion transformants; 8: C78-2 control; H: ddH2O blank control; M1: 5000DNAMarker.

[0032] Figure 7 These are the amplified fragments and restriction enzyme-cut fragments of the left and right homologous arms of asd; a, amplified left and right homologous arms of asd, 1 / 2: asd-L fragment, 3 / 4: asd-R fragment; b, asd-LR homologous arm fusion fragment; c, asd-LR restriction enzyme-cut fragment; M: 2000DNAMaker, M1: 5000DNAMarker.

[0033] Figure 8 PCR identification of recombinant plasmid pRE112-asdLR transformants; in the figure, 1-10: plasmid transformant identification; P: pRE112 plasmid control; H: ddH2O blank control; M1: 5000 DNA Marker.

[0034] Fig. 9 PCR identification of asd gene deletion in △crp (C78-2) strain; in the figure, 1-12: identification of asd gene deletion transformants; 13: C78-2 control; H: ddH2O blank control; M1: 5000DNAMarker.

[0035] Fig.10 These are the amplified fragments and restriction enzyme fragments of the left and right homology arms of manA; a, amplification of the left and right homology arms of manA; 1 / 2: manA-R fragment, 3 / 4: manA-L fragment; b, manA-LR homology arm fusion fragment; c, manA-LR restriction enzyme fragment; M: 2000DNAMaker, M1: 5000DNAMarker.

[0036] Fig.11 PCR identification of recombinant plasmid pRE112-manALR transformants; in the figure, 1-10: plasmid transformant identification; P: pRE112 plasmid control; H: ddH2O blank control; M1: 5000 DNA Marker.

[0037] Fig.12 PCR identification of manA gene deletion in △crp / asd (C78-2) strain; in the figure, 1-12: identification of manA gene deletion transformants; 13: C78-2 control; H: ddH2O blank control; M1: 5000 DNA Marker.

[0038] Fig.13 PCR was used to detect the genetic stability of the △crp / asd / manA (C78-2) strain; a, PCR identification of crp gene deletion; b, PCR identification of asd gene deletion; c, PCR identification of manA gene deletion; 1: 1st generation strain; 2: 10th generation strain; 3: 20th generation strain; 4: 30th generation strain; 5: 40th generation strain; 6: 50th generation strain; 7: C78-2 strain; H: ddH2O blank control; M1: 5000DNAMarker.

[0039] Fig.14 Shown are the growth curves of the C78-2 triple / double deletion strain and the recombinant strain containing the asd gene plasmid.

[0040] Fig.15 Amplification of ApxIAN fusion fragment and pya3493 plasmid linearized fragment; a, Amplification of ApxIAN fragment; b, Amplification of pya3493 plasmid linearized fragment; M: 2000DNAMaker, M1: 5000DNAMarker.

[0041] Fig.16 PCR identification of the transformants of the recombinant plasmid pya3493-ApxIAN; in the figure, 1-9: transformant identification; H: ddH2O blank control; M: 2000DNAMarker.

[0042] Fig.17 The recombinant plasmid pya3493-ApxIAN was transformed into the △crp / asd / manA (C78-2) strain for PCR identification; in the figure, 1-6: transformant identification; 7: pya3493-ApxIAN plasmid control; H: ddH2O blank control; M: 2000DNAMarker.

[0043] Fig.18 WB detection of the expression of ApxIAN exogenous protein in the recombinant strain; △CAM+P is the control strain; P-ApxIAN: recombinant bacteria expressing ApxIAN protein. DETAILED DESCRIPTION

[0044] The present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can understand.

[0045] Example 1: Construction of a Salmonella choleraesuis Δcrp (C78-2) gene-deficient strain

[0046] 1. Construction of crp deletion plasmid

[0047] Salmonella choleraesuis C78-2 was purchased from the National Veterinary Microbiology Collection Center of the China Veterinary Drug Administration. Solid strain powder was streaked on LB solid plates for single colony culture. Single colonies were picked and expanded in LB liquid culture medium. Genome extraction was performed using a bacterial genome extraction kit (purchased from Beijing Tiangen Company) and sent to Tianjin Novogene Company for whole genome sequencing. The plasmids lacking crp, asd, and manA genes in the present invention all use pRE112 suicide vectors. The plasmid map is shown in Figure 1 As shown. Primers were designed based on the genome sequence of Salmonella choleraesuis C78-2 (not uploaded yet) and the genome of Salmonella choleraesuis C500 (NZ_CP007639), as shown in Table 1. The left homology arm fragment Crp-L (1009 bp, as shown in Table 1) of the crp gene was amplified from the genome of Salmonella choleraesuis C78-2 using primers CrpL-SphI-F / CrpL-RH-R. Figure 2 ), the right homology arm fragment Crp-R (1148 bp, as Figure 2 ), Crp-L and Crp-R were fused by tandem PCR to obtain the Crp-LR fragment (2157 bp, as Figure 2 ), at which point Sph I and Xba I restriction sites were introduced at both ends, and 1 μg of each fragment and pRE112 plasmid were digested with Sph I and Xba I (purchased from TaKaRa) at 37°C for 1 h, respectively, and a nucleic acid gel recovery kit (purchased from Beijing Tiangen Company) was used to recover the DNA fragment to obtain the Crp-LR restriction fragment (such as Figure 2 ) and pRE112 plasmid fragments (such as Figure 3 ); 100 ng of the Crp-LR restriction fragment and 100 ng of the pRE112 plasmid restriction fragment were connected overnight at 4°C using T4 ligase (purchased from TaKaRa), and the ligation product was transformed into the χ7213 competent state according to the Escherichia coli chemical transformation method, coated on an LB solid plate containing chloramphenicol, and placed in a 37°C incubator for 24 hours. A single colony was picked and PCR identified using pRE112-ID-F / R primers. The band size of 2615 bp was suspected to be the correct plasmid bacteria. The identification result was as follows Figure 4 As shown, the suspected correct single colony was inoculated into LB medium containing chloramphenicol resistance, and cultured in a shaker at 37°C and 180r / min for 12 hours. A small amount of plasmid extraction kit (purchased from Beijing Tiangen Company) was used, and the extracted plasmid was sent to Shanghai Shenggong Company for sequencing. The sequencing results were correctly aligned, thereby obtaining the pRE112-crpLR plasmid.

[0048] Table 1 Primers related to crp gene deletion plasmid and strain identification

[0049]

[0050] PCR amplification reaction system for Crp-L and Crp-R fragments:

[0051]

[0052] PCR reaction conditions:

[0053]

[0054] Tandem PCR system of Crp-L and Crp-R 1:

[0055]

[0056] Tandem PCR reaction conditions for Crp-L and Crp-R 1:

[0057]

[0058] Tandem PCR system 2 of Crp-L and Crp-R:

[0059]

[0060] Tandem PCR reaction conditions 2 for Crp-L and Crp-R:

[0061]

[0062]

[0063] PCR identification of pRE112-crpLR plasmid reaction system:

[0064]

[0065] PCR reaction conditions:

[0066]

[0067] 2. Construction of the crp gene-deficient strain of Salmonella choleraesuis Δcrp (C78-2)

[0068] The process of the crp gene deletion program for Salmonella choleraesuis is as follows Figure 5As shown; the pRE112-crpLR plasmid was first electroporated into the C78-2 strain. Pick a single colony of C78-2 and add it to 4ml LB medium. Cultivate it in a shaker at 37℃ and 180r / min overnight. Pipette 100μl of bacterial solution into 10ml LB medium. Cultivate it in a shaker at 37℃ and 180r / min until OD600 is about 0.6. Precool it on ice for 30min. Then centrifuge it at 5000r / min and 4℃ for 10min, discard the supernatant, resuspend the bacteria in precooled 10% glycerol water, centrifuge it at 5000r / min and 4℃ for 10min, discard the supernatant, repeat the washing of the bacteria 2-3 times, resuspend the bacteria in 80μl precooled glycerol water, add 20μl of pRE112-crpLR plasmid, mix and transfer to a 0.2cm electric transfer cup, adjust the voltage of the electric transfer instrument to 2500v, perform 1 electric shock, add LB medium and transfer to 2ml EP tube, shake and culture in a shaker at 37°C and 180r / min for 1h, spread on LB solid culture medium containing chloramphenicol, place in a 37°C incubator for 24-48h, pick a single colony and place it in LB culture medium containing 10% sucrose, shake and culture in a shaker at 37°C and 180r / min overnight, take 100μl of bacterial solution and add it to 900μl PBS, dilute it at a 10-fold ratio and spread it on LB solid culture medium containing 10% sucrose, place in a 37°C incubator for 24-48h, pick a single colony and use CrpLR-ID-F / CrpLR-ID-R primers for PCR identification. If the band size is 2908bp, it is determined to be a wild strain; if the band size is 2281bp, it is determined to be a crp gene deletion strain; the suspected correct one is purified again and then PCR identified. The identification result is as follows Figure 6 As shown, the suspected correct colony was sequenced, and after sequencing was correct, the C78-2 crp gene deletion strain was obtained and named △crp (C78-2).

[0069] PCR reaction system for identifying crp gene deletion:

[0070]

[0071] PCR reaction conditions:

[0072]

[0073] Example 2: Construction of a Salmonella choleraesuis Δcrp / asd (C78-2) gene-deficient strain

[0074] 1. Construction of asd deletion plasmid

[0075] The plasmid lacking the asd gene in the present invention adopts the pRE112 suicide vector, and the plasmid map is as follows: Figure 1As shown. According to the sequence of the Salmonella choleraesuis C78-2 genome (not uploaded yet) and the reference Salmonella choleraesuis C500 genome (NZ_CP007639), primers were designed to knock out primers, as shown in Table 2, and asdL-SphI-F / asdL-RH-R primers were used to amplify the left homologous arm fragment asd-L (1145 bp, as shown in Table 2) of the asd gene from the Salmonella choleraesuis C78-2 genome. Figure 7 ), the right homology arm fragment asd-R (983 bp, asdR-RH-F / asdR-XbaI-R) of the asd gene was amplified from the genome of Salmonella choleraesuis C78-2. Figure 7 ), asd-L and asd-R were fused by tandem PCR to obtain asd-LR fragment (2128 bp, as Figure 7 ), at which point Sph I and Xba I restriction sites were introduced at both ends, 1 μg of each fragment and pRE112 plasmid were digested with Sph I and Xba I (purchased from TaKaRa) at 37°C for 1 h, and a nucleic acid gel recovery kit (purchased from Beijing Tiangen Company) was used to recover the DNA fragment to obtain the asd-LR restriction fragment (such as Figure 7 ) and pRE112 plasmid fragments (such as Figure 3 ); 100 ng of asd-LR digested fragment and 100 ng of pRE112 plasmid digested fragment were connected by T4 ligase (purchased from TaKaRa) at 4°C overnight, and the ligation product was transformed into χ7213 competent cells according to the E. coli chemical transformation method, spread on LB solid plates containing chloramphenicol, and placed in a 37°C incubator for 24 hours. Single colonies were picked for PCR identification using pRE112-ID-F / R primers. The band size of 2586 bp was suspected to be the correct plasmid bacteria. The identification results were as follows Figure 8 As shown, the suspected correct single colony was inoculated into LB medium containing chloramphenicol resistance, and cultured in a shaker at 37°C and 180r / min for 12 hours. A plasmid small amount extraction kit (purchased from Beijing Tiangen Company) was used, and the extracted plasmid was sent to Shanghai Shenggong Company for sequencing. The sequencing results were correctly aligned, thereby obtaining the pRE112-asdLR plasmid.

[0076] Table 2 Primers related to asd gene deletion plasmid and strain identification

[0077]

[0078] PCR amplification of asd-L and asd-R fragments reaction system:

[0079]

[0080] PCR reaction conditions:

[0081]

[0082] Tandem PCR system of asd-L and asd-R 1:

[0083]

[0084]

[0085] Tandem PCR reaction conditions for asd-L and asd-R 1:

[0086]

[0087] Tandem PCR system 2 of asd-L and asd-R:

[0088]

[0089] Tandem PCR reaction conditions 2 for asd-L and asd-R:

[0090]

[0091] PCR identification of pRE112-asdLR plasmid reaction system:

[0092]

[0093]

[0094] PCR reaction conditions:

[0095]

[0096] 2. Construction of the crp and asd double gene deletion strain of Salmonella choleraesuis △crp / asd (C78-2)

[0097] The process of the asd gene deletion program for Salmonella choleraesuis is as follows Figure 5As shown; the pRE112-asdLR plasmid was first electroporated into the △crp (C78-2) strain. Pick a single colony of △crp (C78-2) and add it to 4ml LB medium. Cultivate it in a shaker at 37°C and 180r / min overnight. Pipette 100μl of bacterial solution into 10ml LB medium. Cultivate it in a shaker at 37°C and 180r / min until OD600 is about 0.6. Precool it on ice for 30min. Then centrifuge it at 5000r / min and 4°C for 10min, discard the supernatant, resuspend the bacteria with precooled 10% glycerol water, centrifuge it at 5000r / min and 4°C for 10min, discard the supernatant, repeat the washing of the bacteria 2-3 times, resuspend the bacteria with 80μl precooled glycerol water, add 20μl of pRE112-asdLR plasmid, mix and transfer to a 0.2cm electric transfer cup, adjust the voltage of the electric transfer instrument to 2500v, perform 1 electric shock, add LB medium and transfer to 2ml EP tube, shake and culture in a shaker at 37°C and 180r / min for 1h, spread on LB solid culture medium containing chloramphenicol, place in a 37°C incubator for 24-48h, pick a single colony and place it in LB+DAP culture medium containing 10% sucrose, shake and culture in a shaker at 37°C and 180r / min overnight, take 100μl of bacterial solution and add it to 900μl PBS, dilute it at a 10-fold ratio and spread on LB+DAP solid culture medium containing 10% sucrose, place in a 37°C incubator for 24-48h, pick a single colony and use asdLR-ID-F / asdLR-ID-R primers for PCR identification. If the band size is 3477bp, it is determined to be a wild strain; if the band size is 2376bp, it is determined to be an asd gene deletion strain; the suspected correct one is purified again and then PCR identified. The identification result is as follows Fig. 9 As shown, the suspected correct colonies were sequenced, and after sequencing was correct, the asd gene deletion strain in the △crp (C78-2) strain was obtained and named △crp / asd (C78-2).

[0098] PCR reaction system for identifying asd gene deletion:

[0099]

[0100]

[0101] PCR reaction conditions:

[0102]

[0103] Example 3: Construction of a Salmonella choleraesuis Δcrp / asd / manA (C78-2) gene-deficient strain

[0104] 1. Construction of manA-deficient plasmid

[0105] The plasmid lacking the manA gene in the present invention adopts the pRE112 suicide vector, and the plasmid map is as follows: Figure 1 As shown. Primers were designed based on the genome sequence of Salmonella choleraesuis C78-2 (not uploaded yet) and the genome of Salmonella choleraesuis C500 (NZ_CP007639), as shown in Table 2. The left homologous arm fragment manA-L (992 bp, as shown in Table 2) of the manA gene was amplified from the genome of Salmonella choleraesuis C78-2 using primers manAL-SphI-F / manAL-RH-R. Fig.10 ), the right homology arm fragment manA-R (948 bp, as shown in Figure 2) of the manA gene was amplified from the genome of Salmonella choleraesuis C78-2 using primers manAR-RH-F / manAR-XbaI-R. Fig.10 ), manA-L and manA-R were fused by tandem PCR to obtain the manA-LR fragment (1940 bp, as Fig.10 ), at which point Sph I and Xba I restriction sites were introduced at both ends, and 1 μg of each fragment and pRE112 plasmid were digested with Sph I and Xba I (purchased from TaKaRa) at 37°C for 1 h, respectively. A nucleic acid gel recovery kit (purchased from Beijing Tiangen Company) was used to recover the DNA fragment to obtain the manA-LR restriction fragment (such as Fig.10 ) and pRE112 plasmid fragments (such as Figure 3 ); 100 ng of manA-LR restriction fragment and 100 ng of pRE112 plasmid restriction fragment were connected by T4 ligase (purchased from TaKaRa) at 4°C overnight, and the ligation product was transformed into χ7213 competent cells according to the Escherichia coli chemical transformation method, coated on LB solid plates containing chloramphenicol, and placed in a 37°C incubator for 24 hours. Single colonies were picked for PCR identification using pRE112-ID-F / R primers. The band size of 2398 bp was suspected to be the correct plasmid bacteria. The identification results were as follows Fig.11 As shown, the suspected correct single colony was inoculated into LB medium containing chloramphenicol resistance, and cultured in a shaker at 37°C and 180r / min for 12h. A plasmid mini-extraction kit (purchased from Beijing Tiangen Company) was used, and the extracted plasmid was sent to Shanghai Shenggong Company for sequencing. The sequencing results were correctly aligned, thereby obtaining the pRE112-manALR plasmid.

[0106] Table 3 Primers related to manA gene deletion plasmid and strain identification

[0107]

[0108]

[0109] PCR amplification reaction system for manA-L and manA-R fragments:

[0110]

[0111] PCR reaction conditions:

[0112]

[0113] Tandem PCR system of manA-L and manA-R 1:

[0114]

[0115] Tandem PCR reaction conditions for manA-L and manA-R 1:

[0116]

[0117] Tandem PCR system 2 of manA-L and manA-R:

[0118]

[0119] Tandem PCR reaction conditions 2 for manA-L and manA-R:

[0120]

[0121] PCR identification of pRE112-manALR plasmid reaction system:

[0122]

[0123]

[0124] PCR reaction conditions:

[0125]

[0126] 2. Construction of the crp, asd, and manA triple-gene deletion strain of Salmonella choleraesuis △crp / asd / manA (C78-2)

[0127] The process of manA gene deletion in Salmonella choleraesuis is as follows Figure 5As shown; the pRE112-manALR plasmid was first electroporated into the △crp / asd (C78-2) strain. Pick a single colony of △crp / asd (C78-2) and add it to 4ml LB+DAP medium. Incubate it in a shaker at 37°C and 180r / min overnight. Pipette 100μl of bacterial solution into 10ml LB medium. Incubate it in a shaker at 37°C and 180r / min until OD600 is about 0.6. Precool it on ice for 30min. Centrifuge it at 5000r / min and 4°C for 10min. Discard the supernatant. Resuspend the bacteria in precooled 10% glycerol water. Centrifuge it at 5000r / min and 4°C for 10min. Discard the supernatant. Repeat the washing of the bacteria 2-3 times. Resuspend the bacteria in 80μl precooled glycerol water. Add 20μl of pRE112-manALR plasmid. Mix and transfer to a 0.2cm electric transfer cup. Adjust the voltage of the electric transfer instrument to 2500v. Perform one electric shock. Add LB+DAP medium and transfer to 2ml EP tube, shake and culture in a shaker at 37°C and 180r / min for 1h, spread on LB+DAP solid culture medium containing chloramphenicol, place in a 37°C incubator for 24-48h, pick a single colony and place it in LB+DAP culture medium containing 10% sucrose, shake and culture in a shaker at 37°C and 180r / min overnight, take 100μl of bacterial solution and add it to 900μl PBS, dilute it at a 10-fold ratio and spread on LB+DAP solid culture medium containing 10% sucrose, place in a 37°C incubator for 24-48h, pick a single colony and use manALR-ID-F / manALR-ID-R primers for PCR identification. If the band size is 3266bp, it is determined to be a wild strain; if the band size is 2096bp, it is determined to be a manA gene deletion strain; the suspected correct one is purified again and then PCR identified. The identification result is as follows Fig.12 As shown, the suspected correct colonies were sequenced, and after sequencing was correct, the manA gene deletion strain in the △crp / asd(C78-2) strain was obtained and named △crp / asd / manA(C78-2).

[0128] PCR reaction system for identifying manA gene deletion:

[0129]

[0130]

[0131] PCR reaction conditions:

[0132]

[0133] Example 4: Biological characteristics of Salmonella choleraesuis Δcrp / asd / manA (C78-2)

[0134] 1. Determination of genetic stability of Salmonella choleraesuis △crp / asd / manA (C78-2); streak the △crp / asd / manA (C78-2) strain on LB solid medium containing DAP, place it in a 37°C incubator for 24-48 hours, pick a single colony and transfer it to LB medium containing 50μg / ml DAP, and shake and culture it in a shaker at 37°C and 180r / min for 18 hours; transfer it to LB medium containing DAP at a ratio of 1:1000 and culture it for 16 hours, and the cultured bacterial liquid is the second generation; transfer culture again until 50 consecutive transfers are performed, and the cultured bacterial liquid is the 50th generation. The bacterial suspensions of the 1st, 10th, 20th, 30th, 40th and 50th generations were subjected to genome extraction according to the instructions of the bacterial whole genome extraction kit (purchased from Beijing Tiangen Company). The genome was used as a template for PCR identification of crp gene, asd gene and manA gene. The PCR amplification system and amplification conditions were the same as those in Examples 1, 2 and 3, respectively. The identification results are shown in FIG. Fig.13 As shown, the PCR bands of the three genes in each generation were consistent, indicating that the three genes in the △crp / asd / manA (C78-2) strain were very stable after deletion and no reverse mutation occurred.

[0135] 2. Growth characteristic test of Salmonella choleraesuis △crp / asd / manA (C78-2); first, the pya3493 plasmid containing the asd gene was electroporated into the △crp / asd (C78-2) and △crp / asd / manA (C78-2) strains to obtain strains △crp / asd (pya3493) and △crp / asd / manA (pya3493); C78-2, △crp / asd (pya3493) and △crp / asd / manA (pya3493) were inoculated into 5 ml LB medium, and △crp / asd (C78-2) and △crp / asd / manA (C78-2) strains were inoculated into 5 ml LB medium containing DAP, and cultured in a shaking incubator at 37°C and 180 r / min for 18 h; the strains were transferred to 50 ml at a ratio of 1:1000, respectively. LB medium or 50 ml LB medium containing DAP was cultured in a shaking incubator at 37°C and 180 rpm. 500 μl of bacterial solution was taken every hour to measure OD600 and a growth curve was drawn. The results were as follows: Fig.14As shown, the growth rate of △crp / asd (C78-2) and △crp / asd / manA (C78-2) strains was significantly slower than that of the wild strain C78-2. Even after the asd gene was complemented, the growth rate of △crp / asd (pya3493) and △crp / asd / manA (pya3493) was still significantly slower than that of the wild strain C78-2.

[0136] Example 5: Virulence determination of Salmonella choleraesuis Δcrp / asd / manA (C78-2)

[0137] The triple gene deletion mutant has lost the ability to synthesize diaminopimelic acid (DAP), so it cannot grow in a DAP-negative environment. The plasmid containing the asd gene is transformed into the △crp / asd / manA (C78-2) strain, which can complement the asd gene in the plasmid with the triple gene deletion strain, so that the triple gene deletion strain can survive under DAP-negative conditions. Therefore, the pya3493 plasmid was electroporated into the △crp / asd (C78-2) and △crp / asd / manA (C78-2) strains to obtain the △crp / asd (pya3493) and △crp / asd / manA (pya3493) strains.

[0138] 1. Evaluation of the toxicity of gene-deficient strains to mice by intraperitoneal injection

[0139] C78-2, △crp / asd (pya3493) and △crp / asd / manA (pya3493) were inoculated into 5 ml LB medium and cultured in a shaking incubator at 37°C and 180 r / min for 18 h; they were transferred into 20 ml LB medium at a ratio of 1:1000, cultured in a shaking incubator at 37°C and 180 r / min for 12 h, centrifuged at 5000 r / min and 4°C for 10 min, the supernatant was discarded, and the cells were washed with pre-cooled PBS buffer (NaCl 136.89 mM, KCl 2.67mM, Na2HPO48.1mM, KH2PO41.76mM) were used to resuspend the cells, centrifuged at 5000r / min and 4°C for 10min, the supernatant was discarded, the cells were washed 2-3 times, 1ml of pre-cooled PBS buffer was added to resuspend the cells, and the cells were diluted 10 times. 100μl of each dilution was applied to LB solid culture medium and placed in a 37°C incubator for 12h. The bacterial liquid volume was preliminarily calculated by counting the colonies on the plate. 40 female Kunming mice aged 6-8 weeks were purchased and divided into 4 large groups and 13 small groups. Three mice were randomly assigned to each group. C78-2, △crp / asd (pya3493) and △crp / asd / manA (pya3493) were diluted to 1.0×10 4cfu / ml, 1.0×10 5 cfu / ml, 1.0×10 6 cfu / ml, 1.0×10 7 cfu / ml, and infected each group of mice with 200μl by intraperitoneal injection. The death of mice was observed from the second day, and the observation was consistent until the 14th day. The data were statistically processed and shown in Table 4. All mice in the C78-2 group died; the △crp / asd (pya3493) group had mice die when infected with high doses; and there was no mouse death in the △crp / asd / manA (pya3493) group at all doses. It can be seen that the virulence of the △crp / asd / manA (C78-2) strain to mice is significantly lower than that of the C78-2 and △crp / asd (pya3493) strains.

[0140] Table 4 Determination of the toxicity of gene deletion strains to mice by intraperitoneal injection

[0141]

[0142] 2. Evaluate the toxicity of gene-deficient strains to mice by oral administration

[0143] C78-2, △crp / asd (pya3493) and △crp / asd / manA (pya3493) were inoculated into 5 ml LB medium and cultured in a shaker at 37°C and 180 r / min for 18 h; they were transferred to 50 ml LB medium at a ratio of 1:1000, cultured in a shaker at 37°C and 180 r / min for 12 h, centrifuged at 5000 r / min and 4°C for 10 min, the supernatant was discarded, the bacteria were resuspended in precooled PBS buffer, centrifuged at 5000 r / min and 4°C for 10 min, the supernatant was discarded, the bacteria were washed 2-3 times, 1 ml of precooled PBS buffer was added to resuspend the bacteria, diluted at a 10-fold ratio, 100 μl of each dilution was applied to LB solid culture medium, and cultured in a 37°C incubator for 12 h, and the bacterial liquid volume was preliminarily counted by counting the colonies on the plate. C78-2 was diluted to 2.0×10 4 cfu / 100μl, 2.0×10 5 cfu / 100μl, 2.0×10 6 cfu / 100μl, 2.0×10 7 cfu / 100μl, 2.0×10 8 cfu / 100μl, corresponding to 5 dose groups; △crp / asd (pya3493) was diluted to 2.0×10 7 cfu / 100μl, 2.0×10 8cfu / 100μl, 2.0×10 9 cfu / 100μl, 2.0×10 10 cfu / 100μl, corresponding to 4 dose groups; △crp / asd / manA(pya3493) was diluted to 2.0×10 7 cfu / 100μl, 2.0×10 8 cfu / 100μl, 2.0×10 9 cfu / 100μl, 2.0×10 10 cfu / 100μl, corresponding to 4 dose groups. 70 female Kunming mice aged 6-8 weeks were purchased and divided into 4 large groups and 18 small groups. Five mice were randomly assigned to each group. After the mice were bought back from the experimental animal center, they were raised for 2-3 days, deprived of food for 12 hours and water for 4 hours. The mice were first gavaged with 100μl 10% NaHCO3 buffer. After half an hour, 100μl bacterial solution and 100μl PBS were gavaged to each dose group. The death of mice was observed from the second day, and the observation was consistent until the 21st day. The data were statistically analyzed and processed. As shown in Table 4, mice in the C78-2 high-dose group died; a small number of mice died in the △crp / asd (pya3493) high-dose infection group; no mice died in all doses of the △crp / asd / manA (pya3493) group. It can be seen that the virulence of the △crp / asd / manA (C78-2) strain and the △crp / asd (pya3493) strain to mice is significantly lower than that of C78-2.

[0144] Table 5 Determination of virulence of gene deletion strains to mice by oral infection

[0145]

[0146] Example 6: Evaluation of the immune protection of Salmonella choleraesuis Δcrp / asd / manA (C78-2) in mice

[0147] 1. Preparation of △crp / asd / manA (C78-2) vaccine and immunization of mice

[0148] △crp / asd / manA (pya3493) was inoculated into 5 ml LB medium and cultured in a shaking incubator at 37°C and 180 r / min for 18 h; the culture was transferred into 100 ml LB medium at a ratio of 1:1000, and cultured in a shaking incubator at 37°C and 180 r / min for 12 h, centrifuged at 5000 r / min and 4°C for 10 min, the supernatant was discarded, the bacteria were resuspended in precooled PBS buffer, centrifuged at 5000 r / min and 4°C for 10 min, the supernatant was discarded, the bacteria were washed 2-3 times, 1 ml precooled PBS buffer was added to resuspend the bacteria, and the dilution was performed at a 10-fold ratio. 100 μl of each dilution was applied to LB solid medium, and cultured in a 37°C incubator for 12 h. The bacterial liquid volume was preliminarily calculated by counting the colonies on the plate. C78-2 was inoculated into 5ml LB medium and cultured in a shaker at 37℃ and 180r / min for 18h; it was transferred to 100ml LB medium at a ratio of 1:1000, and cultured in a shaker at 37℃ and 180r / min for 12h. 100μl of bacterial solution was diluted by 10 times, and 100μl of each dilution was coated with LB solid medium, and cultured in a 37℃ incubator for 12h. The amount of bacterial solution was preliminarily calculated by counting the colonies on the plate. At the same time, formaldehyde was added to the remaining C78-2 bacterial solution to a final concentration of 0.2%, mixed, and inactivated in a refrigerator at 4 degrees for 48h. Then centrifuge at 5000r / min and 4°C for 10min, discard the supernatant, resuspend the cells in pre-cooled PBS buffer, centrifuge at 5000r / min and 4°C for 10min, discard the supernatant, repeat the washing 2-3 times, and resuspend the cells in 1ml pre-cooled PBS buffer. Dilute the △crp / asd / manA (pya3493) bacterial solution to 1.67×10 7 CFU / 100μl, made into a live vaccine for the △CAM+P oral group; the △crp / asd / manA (pya3493) bacterial solution was diluted with PBS and mixed with aluminum hydroxide adjuvant at a ratio of 9:1, and the bacterial solution volume was also 1.67×10 7 CFU / 100μl, which is the live vaccine of △CAM+P intramuscular injection group; C500 (purchased from Shandong Huahong Company) live vaccine was diluted with PBS according to the instructions, and the dilution dose was 2.0×10 9 CFU / 100μl, for the C500 oral group, the live vaccine; the C78-2 was adjusted to 1.97×10 per mouse. 9 CFU, mixed with aluminum hydroxide adjuvant in a ratio of 9:1, is the inactivated vaccine for the C78-2 intramuscular injection group.

[0149] Fifty 3-week-old female Kunming mice were purchased and divided into 5 groups, with 10 mice in each group, namely △CAM+P oral group, △CAM+P intramuscular injection group, C500 oral group, C78-2 inactivated intramuscular injection group and PBS control group. The mice in the △CAM+P oral group, C500 oral group and PBS control group were deprived of food for 12 hours and water for 4 hours. They were first gavaged with 100μl 10% NaHCO3 buffer solution. After half an hour, they were gavaged with 100μl bacterial solution and 100μl PBS of each dose group. The mice in the △CAM+P intramuscular injection group and C78-2 inactivated intramuscular injection group did not need to be deprived of food and water. They only needed to be injected into the hind leg muscles. After immunization for 14 days, 100μl of blood was collected from the mice by tail cutting. The collected blood was placed in a 37℃ incubator for 1 hour, centrifuged at 3000r / min and 4℃ for 20 minutes, and the supernatant was aspirated into a new EP tube and stored in a -20℃ refrigerator for later use. At the same time, the mice in the corresponding groups were boosted with 1 immunization according to the corresponding immunization procedures.

[0150] 2. Detection of immune antibody levels in the serum of immunized mice

[0151] Blood was collected from mice before immunization. Blood was collected by tail cutting 14 and 28 days after the mice were immunized with the prepared vaccine to prepare serum. Salmonella serum antibodies were detected using C78-2 Salmonella bacteria as the coating antigen (refer to the ELISA method in the literature by Zhao Z, Xue Y, Wu B, Tang X, Hu R, Xu Y, Guo A, Chen H. Subcutaneous vaccination with attenuated Salmonella enterica serovar Choleraesuis C500 expressing recombinant filamentous hemagglutinin and pertactin antigens protects mice against fatal infections with both S. enterica serovar Choleraesuis and Bordetellabronchiseptica. Infect Immun. 2008 May; 76 (5): 2157-63). Equal volumes of mouse serum from each group at each time point were mixed and then anti-C78-2 serum antibodies were detected. The test results are shown in Table 6, which shows that the C78-2 inactivated intramuscular injection group and the △CAM+P intramuscular injection group of the present invention had higher immune levels in the second week after the first immunization; serum antibody test 2 weeks after the second immunization also showed that the C78-2 inactivated intramuscular injection group and the △CAM+P intramuscular injection group of the present invention had higher immune levels, especially the C78-2 inactivated intramuscular injection group had the highest humoral immunity level, while the △CAM+P oral group and the C500 oral group had significantly lower immune levels than the intramuscular injection group.

[0152] Table 6 IgG levels against C78-2 in mouse serum after various vaccinations

[0153]

[0154] 3. Determination of the survival rate of mice immunized after infection with the virulent strain C78-2

[0155] In the second week after the secondary immunization, 6 mice were randomly selected from each group, and all mice were orally challenged with a high lethal dose of C78-2 bacterial solution, and observed for 21 consecutive days. The protection rate after the challenge is shown in Table 7. The PBS control group began to show depression, messy fur, decreased appetite, and shrinking and gathering 5 days after the challenge; they began to die one after another until all died. The C78-2 inactivated vaccine group began to die one week after the challenge until all died. Mice in the C500 oral group and the △CAM+P oral group began to die one after another in the second week after the challenge. Only one mouse survived in the C500 oral group, and 3 mice survived in the △CAM+P oral group. The △CAM+P intramuscular injection group did not show any particularly obvious symptoms of the disease after the challenge, and all survived for 21 days. It can be seen that mice immunized with the △crp / asd / manA (pya3493) gene-deficient vaccine prepared by the present invention can resist the attack of a highly lethal dose of the virulent strain C78-2 of Salmonella choleraesuis, and the protection efficiency is better than that of C500 or inactivated vaccines regardless of oral or intramuscular immunization.

[0156] Table 7 Immunity protection rate after immunization of mice infected with lethal dose of C78-2

[0157]

[0158] Example 7: Detection of exogenous gene carrying and expression in Salmonella choleraesuis Δcrp / asd / manA (C78-2)

[0159] The truncated sequence of the ApxI gene of porcine Actinobacillus pleuropneumoniae (ApxIAN) was sent to Wuhan Jinkairui Company for Salmonella codon optimization and synthesis, and a 6x His tag was carried at the C-terminus. The sequence can be seen in SEQ ID No. 4. Using the synthetic sequence as a template, PCR amplification was performed using SApxI-RH-F / SApxI-RH-R. After electrophoresis, the band with a size of 1152 bp was the target band. Fig.15 It can be seen that after gel cutting, agarose gel electrophoresis was performed to recover the DNA fragment, which was the ApxIAN-RH fragment; pya3493 plasmid was used as a template and pyA3493-RH-F / pyA3493-RH-R primers were used for amplification. After electrophoresis, the band with a size of 3091 bp was the target band. Fig.15It can be seen that after gel cutting, agarose gel electrophoresis was performed to recover the DNA fragment, which was the pya3493-RH linearized fragment. 100 ng of the pya3493-RH fragment and 100 ng of the ApxIAN-RH fragment were subjected to a 37°C fusion reaction using a homologous recombination kit (purchased from Nanjing Novozymes). The reaction solution was added to the χ6097 competent medium, and heat-shocked for 90 seconds using the Escherichia coli chemical transformation method. LB culture medium was added and placed in a shaker at 37°C and 180 r / min for 1 hour of shaking culture. It was spread on LB solid culture medium and cultured in a 37°C incubator for 24 hours. A single colony was picked for PCR identification and amplified using pYA3493-trc-ID-F / pYA3493-trc-ID-R primers. After electrophoresis, the band size was about 1458 bp, which was suspected to be the correct band. Fig.16 It can be seen that the suspected correct plasmid transformed bacteria were transferred to LB culture medium, cultured in a shaker at 37°C and 180r / min for 12 hours, and the plasmid was extracted using a plasmid extraction kit. It was then sent to Shanghai Bioengineering Company for sequencing. After the sequence alignment was correct, it was the correct pya3493-ApxIAN plasmid. Pick the △crp / asd / manA (C78-2) colony and transfer it to LB+DAP medium for overnight culture. Transfer it to 30ml LB+DAP medium at a ratio of 1:1000, shake and culture in a shaker at 37°C and 180r / min until OD600 is about 0.6, place it on ice for precooling for 30min, then centrifuge it at 5000r / min and 4°C for 10min, discard the supernatant, resuspend the bacteria with precooled 10% glycerol water, centrifuge it at 5000r / min and 4°C for 10min, discard the supernatant, repeat the washing of the bacteria 2-3 times, resuspend the bacteria with 90μl precooled glycerol water, add 10μl of pya3493-ApxIAN plasmid, mix and transfer to a 0.2cm electric transfer cup, adjust the voltage of the electric transfer instrument to 2500v, perform 1 electric shock, add LB medium and transfer to 2ml EP tube, shake and culture in a shaker at 37°C, 180r / min for 1h, spread on LB solid medium, place in a 37°C incubator and culture for 24-48h, pick a single colony for PCR detection, the test results are as follows Fig.17As shown, it shows that the △crp / asd / manA (pya3493-ApxIAN) recombinant strain is obtained, a single colony is inoculated into 100 ml of LB medium, and cultured in a shaker at 37°C and 180 r / min for 16 hours, the bacteria are collected, centrifuged at 10000 r / min and 4°C for 10 min, the supernatant is discarded, the bacteria are washed twice with PBS, 5 ml of PBS is added to resuspend the bacteria, and a continuous flow high-pressure crusher is used for crushing, and then centrifuged at 12000 r / min and 4°C for 20 min, the supernatant and the precipitate are separated, and protein SDS loading buffer is added respectively, boiled in boiling water for 10 min, 10% DTT reagent is added, and SDS-PAGE electrophoresis samples are prepared. According to the new Saimei rapid preparation of SDS-PAGE method, a separation gel of 12% SDS-PAGE gel is prepared, and electrophoresis is performed at 80v for 30min and 120v for 1h, and then transfer Transfer to PVDF membrane, electrophoresis at 100V for 1h, block with 5% BSA for 2h, dilute anti-His mouse monoclonal antibody (purchased from Shanghai Shenggong Company) at 1:3000, incubate at room temperature for 1h; wash the membrane three times with TBST, 5min each time; dilute HRP-labeled goat anti-mouse secondary antibody (purchased from Thermo Fisher Scientific) at 1:10000, incubate at room temperature for 1h; wash the membrane three times with TBST, 5min each time; use high-sensitivity colorimetric solution (purchased from Thermo Fisher Scientific) and WB imager (purchased from Bio-Rad) for exposure, the results are as follows: Fig.18 As shown, it can be seen that the ApxIAN protein is expressed in the recombinant bacteria, indicating that the △crp / asd / manA (C78-2) strain and pya3493 complementation can carry and express exogenous genes.

[0160] Table 8 Primers for constructing related balanced lethal plasmids expressing ApxIAN protein

[0161]

[0162] PCR amplification of ApxIAN-RH fragment reaction system:

[0163]

[0164] PCR reaction conditions:

[0165]

[0166]

[0167] PCR amplification pya3493-RH fragment reaction system:

[0168]

[0169] PCR reaction conditions:

[0170]

[0171] PCR identification of pya3493-ApxIAN transformed bacteria reaction system:

[0172]

[0173] PCR reaction conditions:

[0174]

[0175] The present invention relates to a three-gene deleted strain of Salmonella choleraesuis △crp / asd / manA (C78-2) (whose deposit number is CCTCC M20242059). The strain successively lacks the virulence regulating gene crp, the important nutritional metabolism gene asd mediating the synthesis of diaminopimelic acid (DAP), and the important carbon metabolism gene manA mediating the encoding of mannose-6-phosphate isomerase. The present invention adopts suicide vector and SacB reverse genetics to construct a gene-deleted mutant strain of Salmonella choleraesuis, thereby obtaining a three-gene deleted mutant strain without resistance. The three-gene deleted mutant strain has a clear genetic background and no resistance marker, has good safety and immune protection for mice, can help mice resist the attack of high lethal doses of Salmonella choleraesuis, and because the asd gene in the deleted strain is completely tracelessly deleted, the balanced lethal system plasmid pya3493-ApxIAN is electroporated, and the recombinant strain can not only grow in a culture medium without DAP, but also can stably express the exogenous protein ApxIAN, and has broad application prospects.

Claims

1. A triple gene deletion strain of Salmonella choleraesuis without resistance marker, characterized in that: The asd gene, crp gene and manA gene in the attenuated Salmonella choleraesuis were completely deleted and named △crp / asd / manA (C78-2). It was deposited in the China Center for Type Culture Collection with the deposit number CCTCC M 20242059.

2. The three-gene deletion strain of Salmonella choleraesuis without resistance marker according to claim 1, characterized in that: 1) Its pathogenicity to mice is lower than that of wild strains or double-gene deletion strains; 2) Intramuscular injection of triple-gene deletion strains mixed with aluminum hydroxide adjuvant can produce better immune protection in mice.

3. Use of the three-gene deleted strain of Salmonella choleraesuis without resistance marker according to claim 1 or 2 in the preparation of a live vaccine of Salmonella choleraesuis without resistance marker.

4. A live vaccine of Salmonella choleraesuis without resistance marker, characterized in that: The invention comprises the three-gene deletion strain of Salmonella choleraesuis without resistance marker as claimed in claim 1 or 2 and an adjuvant.

5. The Salmonella choleraesuis live vaccine without resistance marker according to claim 4, characterized in that: The adjuvant is aluminum hydroxide adjuvant.

6. The method for constructing a three-gene deletion strain of Salmonella choleraesuis without resistance marker according to claim 1, characterized in that: 1) The asd gene, crp gene and manA gene are all seamlessly knocked out. The knocked-out crp gene sequence is 1170 bp in its ORF sequence, and the ORF sequence is shown in SEQ ID No. 1; the knocked-out asd gene sequence is 627 bp in its ORF sequence, and the ORF sequence is shown in SEQ ID No. 2; the knocked-out manA gene sequence is 1170 bp in its ORF sequence, and the ORF sequence is shown in SEQ ID No. 3; 2) The three-gene deletion strain of Salmonella choleraesuis without resistance marker can form a complementary expression system with the plasmid expressing the asd gene.

7. Use of a triple-gene deleted strain of Salmonella choleraesuis without resistance marker as an exogenous antigen expression bacterium or a carrier bacterium.

8. The use according to claim 7, characterized in that: A truncated version of ApxI of porcine Actinobacillus pleuropneumoniae (ApxIAN) can be expressed, the gene sequence of which is optimized according to the Salmonella codon and carries a 6x His tag at the C-terminus. The optimized sequence is shown in SEQ ID No.4.

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