CN101962625A - Salmonella choleraesuis gene deletion mutant without resistant marker and vaccine thereof - Google Patents

Abstract

The present invention relates to a Salmonella choleraesuis gene deletion mutant strain without resistance marker SalmonellacholeraesuisC7821, and its preservation number is CCTCCNO: M2010102; it also relates to a Salmonella choleraesuis gene deletion vaccine prepared by the strain. The gene deletion mutant strain of Salmonella choleraesuis without resistance marker of the present invention is derived from Salmonella choleraesuis C78-2 strain (commercial strain purchased from National Veterinary Microorganism Collection Center of China Veterinary Drug Control Institute). The main virulence regulation gene crp of the said Salmonella choleraesuis gene deletion mutant strain has been deleted by 320bp, so that the bacterium cannot absorb cAMP from mammals, grows relatively slowly, and has greatly reduced virulence, so it has high safety. Animal experiments have confirmed that the gene deletion mutant strain has good immune protection. The gene deletion vaccine prepared by the invention can stimulate pigs to produce a protective immune response against the homologous wild strain of Salmonella choleraesuis, and effectively prevent the infection of Salmonella choleraesuis.

Description

A gene deletion mutant strain of Salmonella choleraesuis without resistance marker and its vaccine

technical field

The invention relates to a gene deletion mutant strain of Salmonella choleraesuis without resistance marker, and also relates to a gene deletion vaccine of Salmonella choleraesuis prepared by the strain, which belongs to the technical field of genetic engineering of animal bacteria.

Background technique

Salmonella choleraesuis ( Salmonella choleraesuis ) is the main pathogen that causes paratyphoid fever in piglets aged 2 to 4 months. Pigs can easily cause acute sepsis, chronic necrotic enteritis, intractable diarrhea and other typical symptoms after infection, and often cause a large number of weaned piglets to become ill. Such as concomitant or secondary infection of other diseases or untimely treatment, the mortality rate is relatively high, causing heavy losses. Animals infected with Salmonella or their products are contaminated, which can easily cause food poisoning after human consumption. Therefore, this pathogen is also of great significance in public health.

Salmonella choleraesuis belongs to the genus Salmonella ( Salmonellus ), is a Gram-negative bacillus, facultatively anaerobic, has flagella all over the body, can move, and does not form spores. Its optimum growth temperature is 37°C, and its optimum pH is 6.8-7.8. Salmonella choleraesuis has strong resistance to external factors such as drying, corruption, and sunlight, and can reproduce in the temperature range of 7-45°C, and can survive after being frozen or freeze-dried. The bacteria can survive for weeks, months or even years in suitable organic matter, but is not very resistant to disinfectants. Salmonella choleraesuis also exists latently in the intestinal tract or gallbladder of some healthy pigs to varying degrees. When the pigs' resistance is reduced due to poor feeding management and feeding environment, it often causes disease in pig herds.

People first used the inactivated whole-bacteria vaccine to prevent paratyphoid fever in piglets caused by Salmonella choleraesuis. Because the inactivated vaccine is easy to cause systemic and local reactions, it can only stimulate humoral immunity, cannot provide cross-protection, and needs multiple booster immunizations. , the application of such vaccines is limited. Subsequently, attenuated Salmonella were found to be more effective at inducing humoral and cellular immune responses than inactivated or subunit vaccines. Therefore, although traditional inactivated vaccines and subunit vaccines have been used all the time, they are gradually replaced by attenuated live vaccines due to poor immune effects and inconvenient immunization routes. Various Salmonella attenuated live vaccines have been widely used worldwide and have achieved good preventive effects (Curtiss R, III, Doggett T, Nayak A, Srinivasan J. 1996. Strategies for the use of live recombinant avirulent bacterial vaccines for mucosal immunization, p. 499–511. In H. Kiyono and M. F. Kagnoff (ed.), Essentials of mucosal immunology. Academic Press, San Diego, Calif; Sirard J C, Niedergang F, Kraehenbuhl J P. 1999. Live attenuated Salmonella : a paradigm of mucosal vaccines. Immunol. Rev. 171:5–26). In the early 1960s in my country, Fang Xiaowen et al. inoculated the highly virulent Salmonella choleraesuis strain C78-1 with good antigenicity into the medium containing thallium acetate and propagated it for hundreds of generations, and selected a weak strain with good immunogenicity. , was named C500 (Fang Xiaowen et al., Research on attenuated paratyphoid vaccine for piglets, Journal of Animal Husbandry and Veterinary Medicine, 1981(2):29–36). The C500 strain has been promoted and used throughout the country, which has effectively controlled piglet paratyphoid in my country (Huang Changbing et al., Research on Oral Immunization of Piglet Paratyphoid Attenuated Ventilated Freeze-dried Vaccine, Chinese Agricultural Sciences, 1981(6):89-94). However, the attenuated vaccine strain C500 is produced from a strong wild strain through chemical attenuation, and its genetic background is unclear, so there is a risk of virulence returning to strength. In addition, it has also been found in clinical application that the attenuated vaccine still has some residual virulence, which can cause strong side effects in some immunized pigs (Kang Kai, piglet paratyphoid live vaccine, Chinese Journal of Veterinary Medicine, 2003 (37) 37-49 ). Therefore, there is still a need to develop a novel Salmonella choleraesuis attenuated vaccine with clear genetic background, stable traits, and better biological safety.

With the development of modern biology and DNA recombination technology, people have conducted in-depth research and understanding on the virulence genetics of Salmonella. This provides the possibility to introduce multiple (Multiple), limited (Defined), attenuated (Attenuated) and irreversible (Irreversible) mutations in the pathogen genome, and this mutation is the genetic basis for the preparation of attenuated live vaccines. Compared with inactivated vaccines and subunit vaccines, attenuated live vaccines have the advantages of high efficiency, low cost, and convenient use. An ideal attenuated live vaccine should meet the following conditions: (1) complete attenuation: the residual toxicity of the mutant strain is generally not acceptable; (2) highly immunogenic: the live vaccine should be able to effectively stimulate the body to produce a specific immune response; ( 3) Unable to reverse mutation: prevent the occurrence of reverse mutation and reduce the risk of strong virulence; (4) No pollution to the environment: the vaccine should remain in the body for a specific period of time, and be cleared by a certain mechanism after the immune response is induced and does not remain (5) Harmless to future generations: the protective immunity induced by the vaccine should be passed on to the next generation, and the vaccine itself should not be transmitted vertically; (6) Economical and convenient to use: such as oral, intranasal, intramuscular injection, etc. Immunization method is relatively simple and convenient.

It has become a research hotspot in the world to use genetic engineering to attenuate Salmonella to screen and obtain attenuated vaccine strains. This is mainly because there is a clinical demand for new attenuated Salmonella vaccines, and Salmonella can be used as a vector for the development of other pathogenic bacteria vaccines (Sirard J C, Niedergang F, Kraehenbuhl J P. 1999. Live attenuated Salmonella : a paradigm of mucosal vaccines. Immunol . Rev. 171:5–26; Spreng S, Dietrich G. Weidinger G.. 2006. Rational design of Salmonella -based vaccination strategies. Methods 38:133–143). Salmonella virulence factors closely related to pathogenicity mainly include: lipopolysaccharide, enterotoxin, cytotoxin, invasion and virulence genes, and virulence regulation genes. People try to delete some of the virulence-related genes through various genetic engineering methods, so that the virulence of Salmonella is reduced, but its immunogenicity is still retained, and finally the Salmonella gene deletion attenuated vaccine strain is obtained through screening. For example, deletion of the aroA trophic synthesis-related gene ( aroA gene encodes 5-enolpyruvylshikimate-3-phosphate synthase, which catalyzes an intermediate reaction in the chorismate pathway of 2,4-dihydroxybenzoate, produces aromatic amino acids) (Hoiseth S K, Stocker B A. 1981. Aromatic-dependent Salmonella typhimurium are non-virulent and effective as live vaccines. Nature, 291:238–239; Dougan G, Maskell D, Pickard D, Hormaeche C. 1987. Isolation of stable aroA mutants of Salmonellatyphi Ty2: properties and preliminary characterization in mice. Mol Gen Genet, 207(2-3):402–405), cya and crp virulence regulation genes ( cya encodes cyclic adenylate synthase, crp encodes cAMP receptor protein, and mutants of cya and crp genes affect the expression of genes involved in carbohydrate and amino acid metabolism and the expression of pili and flagella) (Curtiss R Ⅲ, Kelly S M. 1987. Salmonella typhimurium deletion mutants lacking adenylate cyclase and cyclic AMP receptor protein are avirulent and immunogenic. Infect Immun, 55(12):3035–3043; Kelly S M, Bosecker B A, Curtiss R Ⅲ. 1992. Characterization and protective properties of attenuated mutants of Salmonella choleraesuis . Infect 60: Immun, 4881–4890), Dam and phoP / phoQ gene regulation related factors ( Dam gene encodes DNA adenylate methylase, regulates the expression of at least 40 genes activated by PhoP , while phoP / phoQ gene encodes transcriptional activator/sensor Kinase) (Galan J E, Curtiss Ⅲ R. 1989. Virule nce and vaccine potential of phoP mutants of Salmonellatyphimurium . Microb Pathogen, 6:433–443; Hohmann E L, Oletta C A, Miller S I. 1996. Evaluation of a phoP / phoQ -deleted, aroA -deleted live oral Salmonella typhi vaccine strain in human volunteers. Vaccine, 14(1):19–24) and so on. Among them, some Salmonella gene deletion strains (mainly Salmonella typhimurium, including a small amount of Salmonella choleraesuis gene deletion strains) showed a significant decrease in virulence and good immune efficacy to experimental animals.

Unlike inactivated vaccines and subunit vaccines, live Salmonella vaccines can invade the lymphatic system, more effectively stimulate the host's humoral and cellular immunity, and stimulate systemic and mucosal immune responses through natural infection routes such as oral or intranasal vaccination (Curtiss R, III, Doggett T, Nayak A, Srinivasan J. 1996. Strategies for the use of live recombinant avirulent bacterial vaccines for mucosal immunization, p. 499–511. In H. Kiyono and M. F. Kagnoff (ed.), Essentials of mucosal immunization Immunology. Academic Press, San Diego, Calif; Sirard J C, Niedergang F, Kraehenbuhl J P. 1999. Live attenuated Salmonella : a paradigm of mucosal vaccines. Immunol. Rev. 171:5–26). In this way, the attenuated live vaccine may be a feasible method to solve the shortage of current commercial vaccines. There are still many defects in the existing genetic engineering methods for constructing Salmonella gene-deleted attenuated vaccine strains. For example, there is randomness in the mutation method mediated by transposons, which is only suitable for the screening of mutants with obvious changes in phenotype, and mutant strains that do not cause obvious phenotypic changes cannot be obtained through these methods; Although the method of source recombination leading to target gene mutation is accurate, the obtained mutant strains often contain resistance markers in the end, and cannot be used as vaccine strains for vaccine production because they do not meet the biosafety requirements.

Contents of the invention

The purpose of the present invention is to provide a gene deletion mutant strain of Salmonella choleraesuis without resistance marker, which has better immunogenicity and strong safety.

In addition, the object of the present invention is to provide a Salmonella choleraesuis gene deletion vaccine prepared by using the gene deletion mutant strain of Salmonella choleraesuis.

In order to achieve the above object, the technical solution of the present invention adopts a Salmonella choleraesuis gene deletion mutant strain without resistance marker , Salmonella choleraesuis C7821, and its preservation number is CCTCC NO: M2010102.

The gene deletion mutant strain of Salmonella choleraesuis without the resistance marker has lost the main virulence regulation gene crp of Salmonella choleraesuis. After the deletion, the virulence of Salmonella choleraesuis strain was significantly weakened, but it still had good immunogenicity.

The technical solution of the present invention is also to adopt a Salmonella choleraesuis gene deletion vaccine comprising the Salmonella choleraesuis gene deletion mutant strain Salmonella choleraesuis C7821 without resistance marker.

The gene deletion mutant strain of Salmonella choleraesuis without the resistance marker has lost the main virulence regulation gene crp of Salmonella choleraesuis.

The gene deletion mutant strain of Salmonella choleraesuis without resistance marker of the present invention is derived from Salmonella choleraesuis C78-2 (commercial strain purchased from National Veterinary Microorganism Collection Center of China Veterinary Drug Control Institute). The main virulence regulatory gene crp of the said Salmonella choleraesuis gene deletion mutant strain has been deleted by 320 bp, resulting in that the bacterium cannot absorb cAMP from mammals, grows relatively slowly, and has greatly reduced virulence, so it has high safety . Animal experiments have confirmed that the gene deletion mutant strain has good immune protection.

The basic construction method of the present invention does not contain the resistance marker Salmonella choleraesuis gene deletion mutant strain: use genetic engineering technology to delete the virulence regulation gene of the wild virulent strain of Salmonella choleraesuis C78-2 (abbreviated as C78-2, the same below) crp to construct a new mutant strain △crp C78-2, which we named as C7821. A large amount of biological experiment data proves that the gene deletion strain C7821 prepared by the present invention can be used to prepare attenuated live vaccine against Salmonella choleraesuis infection.

The main advantages of the present invention are: (1) The gene-deleted mutant strain of Salmonella choleraesuis of the present invention has good immune protection, and has weak toxicity, and has no adverse side effects on immunized pigs; the vaccine prepared by the strain , which is safer than traditional vaccines. The vaccine made by the gene deletion mutant strain constructed by the parent bacteria can stimulate pigs to produce a protective immune response against the homologous wild strain of Salmonella choleraesuis, and effectively prevent the infection of Salmonella choleraesuis , has a broad market application prospect. (2) The gene deletion mutant strain of Salmonella choleraesuis of the present invention retains the immune protection effect against Salmonella choleraesuis infection, and has a clear genetic background and stable traits, thus having better biological safety. (3) The gene-deleted mutant strain of Salmonella choleraesuis of the present invention does not contain resistance markers, and fully meets the biological safety requirements of vaccines.

The Salmonella choleraesuis gene deletion mutant strain without resistance marker of the present invention , Salmonella choleraesuis C7821, was deposited on April 26, 2010 at the China Center for Type Culture Collection at Wuhan University, and its preservation number is CCTCC NO: M2010102.

Description of drawings

Fig. 1 is the physical map of transfer plasmid pRE△crp prepared by the present invention;

Fig. 2 is the flowchart that the transfer plasmid pREcrp12 that the present invention prepares constructs;

Fig. 3 is the PCR detection electrophoresis pattern of the Salmonella choleraesuis crp gene deletion bacterial strain Δcrp C78-2 that does not contain resistance marker in the present invention;

Figure 3A is the PCR identification map of △crp C78-2 mutant strain (Pr5/Pr6); among them, M in the figure is DNA marker (DL 2,000); 1 is H ₂ O control; 2 is parental strain C78-2 control (918 bp ); 3 is the screened △crp C78-2 mutant strain (599 bp);

Figure 3B: PCR identification map of △crp C78-2 mutant strain (Pr7/Pr6), M in the figure is DNA marker (DL 2,000); 1 is pRE112 plasmid control; 2 is H ₂ O control; 3 is parental strain C78-2 Control (1731 bp); 4 is the screened △crp C78-2 mutant (1412 bp);

Fig. 4 is the colony morphology of the Salmonella choleraesuis mutant strain Δcrp C78-2 without resistance marker in the present invention on MacConkey agar with 1% maltose added.

Figure 4A is the colony morphology of the Δcrp C78-2 mutant strain on LB culture;

Figure 4B is the colony morphology (colorless) of the △crp C78-2 mutant strain on MacConkey agar containing 1% maltose;

Figure 4C is the colony morphology (red) of the parental strain C78-2 on MacConkey agar containing 1% maltose;

Fig. 5 is the growth characteristic test result of the Salmonella choleraesuis mutant strain C7821 not containing the resistance marker in the present invention;

Fig. 6 is the result of the genetic stability experiment of the Salmonella choleraesuis mutant strain Δcrp C78-2 that does not contain a resistance marker in the present invention;

Figure 6A is the PCR identification diagram of primers Pr5/Pr6 on the deletion strain △crp C78-2, in which M: DNA marker (DL 2,000); 1 is H ₂ O control; Amplification results of the deletion strain templates of the 40th and 50th generations;

Figure 6B is the PCR identification diagram of primers Pr7/Pr6 on the deletion strain △crp C78-2, in which M: DNA marker (DL 2,000); 1 is H ₂ O control; 2-7 are 1, 10, 20, 30, Amplification results of deletion strain templates at passages 40 and 50.

Detailed ways

Example 1 Construction of Salmonella choleraesuis C78-2 crp gene deletion strain

1. Primer design (for gene cloning and molecular detection)

Refer to literature (Xu Yindi, et al. Construction and identification of a balanced lethal vector system for △crp △asd deletion strain of Salmonella choleraesuis C500 strain. Acta Biological Engineering, 2006, 22(3):366–372) and the reported Salmonella typhi strain Ty2 The crp gene sequence (GenBank No: AE016847) designed 2 pairs of primers (pr1/pr2 and pr3/pr4, see Table 1), from the virulent strain of Salmonella choleraesuis C78-2 (purchased from the National Veterinary Microorganism Collection of China Veterinary Drug Administration The upper and lower segments of the crp gene, crp1 (upper arm) and crp2 (lower arm), were respectively amplified in the genome of the Central Commercial Strain), and the sizes of the amplified fragments were 1048 bp and 1743 bp, respectively, and the two ends of the upper arm were respectively introduced with Xba I and Bam H I enzyme cutting sites point, Xho I and Kpn I restriction sites were introduced at the two ends of the lower arm respectively. In addition, 2 pairs of primers (pr5/pr6 and pr7/pr6, see Table 1) were designed to identify the C78-2 parental strain and crp- deficient strain. Primers were synthesized by Shanghai SANGON Company.

Table 1 PCR primers

2. Cloning of the upstream and downstream fragments crp1 (upper arm) and crp2 (lower arm) of the C78-2 crp gene of Salmonella choleraesuis

Streak the lyophilized Salmonella choleraesuis C78-2 on LB solid medium, the composition of LB solid medium is: 10 g tryptone, 5 g yeast extract, 5 g sodium chloride, 15 g agar powder, distilled water Dilute to 1000 mL, pressurize at 121°C for 25 minutes, and incubate at 37°C for 16 hours. Pick a single colony and inoculate it in LB liquid medium (10 g tryptone, 5 g yeast extract, 5 g sodium chloride, distilled water to 1000 mL, 121°C and high pressure for 25 min), 37°C, 200 r/min Incubate with shaking for 16 h. According to the instructions of the bacterial genome extraction kit (purchased from Beijing Tiangen Company), the genome was extracted as a PCR template.

Both crp1 and crp2 amplification reactions were carried out in a 25 μL system. The reaction system (PCR-related reagents were purchased from Dalian TaKaRa Company) was as follows: template DNA 1 μL, 10×PCR buffer 2.5 μL, 25 mmol/L MgCl2 2 μL , 1 μL of 10 μmol/L upstream and downstream primers, 1 μL of 2 mmol/L dNTPs, 0.5 μL of 2 U/μL Taqase, and 16 μL of ddH2O.

The amplification conditions of crp1 and crp2 were as follows: denaturation at 95°C for 5 minutes, followed by cycling, and the cycle parameters were 1 minute at 94°C, 1 minute at 57°C, and 2 minutes at 72°C. After 30 cycles, extend at 72°C for 10 min. The amplified PCR product was analyzed by 0.8% agarose gel electrophoresis, and the size of the amplified two fragments was comparable to the expected size, which were 1048 bp and 1743 bp, respectively. The obtained target gene was cloned into the pMD18-T vector (purchased from Dalian TaKaRa Company), and sent to Dalian TaKaRa Company for the determination of the exogenous gene sequence.

3. Construction of pREcrp12 transfer plasmid

The crp1 and pBluescriptSK(+) vectors (purchased from Stratagene, USA) were double-digested with Xba I and BamH I, recovered and purified, ligated with T4 DNA ligase (purchased from Dalian TaKaRa Company), and transformed into DH5α competent cells in a water bath at 16°C for 12 h Bacteria (purchased from Dalian TaKaRa Company) were cultured on a solid LB plate at 37°C for 12 h, picked into liquid LB medium, cultured at 37°C and shaken at 200 r/min for 12 h, and a plasmid extraction kit (purchased from Beijing TIANGEN Company) to prepare a small amount of plasmid to obtain plasmid pSKcrp1. Then use Xho I and Kpn I to double digest crp2 and plasmid pSKcrp1. After recovery, they were ligated with T4 DNA ligase, transformed into DH5α competent bacteria, cultured at 37°C for 12 hours, picked into liquid LB medium, shaken at 37°C and 200 r/min for 12 hours, and prepared a small amount of plasmids to obtain plasmid pSKcrp12. The transfer plasmid pSK△crp and the suicide plasmid pRE112 were double digested with Xba I and Kpn I (gifted by Dr. Roy Curtiss III, Washington University; Miller, V. L., Mekalanos J. J. 1988. A novel suicide vector and its use in construction on insertion mutations: osmoregulation of outer membrane proteins and virulence determinants in Vibrio cholerae requires toxR. J. Bacteriol. 170 : 2575–2583), recovered the crp1+crp2 fragment and plasmid pRE112, ligated with T4 DNA ligase, 16°C water bath for 12 h, electrophoresis Transformation (translated by Huang Peitang et al. Sambrook J, Russell D W. Molecular Cloning Experiment Guide (Third Edition). Beijing: Science Press, 2002) Escherichia coli χ7213 (gifted by Professor Dr. Roy Curtiss III, Washington University ; Edwards, R. A., L. H. Keller, and D. M. Schifferli. 1998. Improved allelic exchange vectors and their use to analyze 987P fimbria gene expression. Gene 207:149-157) construct recombinant strain χ7213 (pRE△crp), cultured at 37°C for 12h to pick Bacteria were transferred to liquid LB medium, shaken at 37°C and 200 r/min for 12 h, and a small amount of plasmid was prepared to obtain the pREcrp12 transfer plasmid. The physical map is shown in Figure 1. The results of restriction enzyme digestion confirmed that the constructed transfer plasmid pREcrp12 was correct. The construction process of transfer plasmid pREcrp12 is shown in Figure 2.

4. Construction of Salmonella choleraesuis C78-2 crp gene deletion strain △crpC78-2

The conjugative transfer was performed with χ7213 (pREcrp12) as the donor strain and C78-2 as the recipient strain. Donor bacteria and recipient bacteria were cultured overnight in LB medium, sterile phosphate buffer solution (PBS, NaCl 8.0 g, KCl 0.2 g, Na2HPO4.12H2O 2.9 g, KH2PO4 0.2 g, distilled water to 1000 mL, Autoclave at 121°C for 30 min), wash twice, adjust the bacterial concentration to OD600 of 0.8, take 100 μL bacterial suspension and mix. Paste the sterile nitrocellulose filter membrane on a solid LB plate, drop the mixed bacteria solution on the filter membrane, and incubate at 37°C for 12 hours, and simultaneously serve as donor and recipient controls. Wash the bacterial solution on the filter membrane, wash it twice with sterile PBS, coat a solid LB plate containing chloramphenicol (Cm, final concentration 30 μg/ml), incubate at 37°C for 12 h, and transfer the Cm-resistant colony to the plate containing Cm and 5% sucrose LB plates were used to screen Cm-resistant (Cmr) zygotes, the genome was extracted, and identified by amplification with primers pr5/pr6 (see Table 1) (see Figure 3A). Positive zygotes were cultured in LB liquid medium without resistance and without NaCl for 12 h, serially diluted 10 times, spread on a solid LB plate without NaCl containing 5% sucrose, picked a single colony and replicated in a medium containing Cm and 5% sucrose The solid plate, screening Cm sensitive (Cms) colonies. The genome was extracted, amplified again with primers pr5/pr6 and identified, and the crp-deficient strain was further identified with primers pr7/pr6 (Table 1) (see Figure 3B). The results showed that the constructed C78-2 crp gene deletion strain was correct, and we named it: Salmonella choleraesuis gene deletion mutant strain Salmonella choleraesuis C7821.

Example 2 Identification and biological characteristics of Salmonella choleraesuis C78-2 crp gene deletion mutant strain C7821

1. Phenotype identification of gene deletion strain C7821

The gene deletion strain C7821 (experiment number: △crpC78-2) and the parental strain C78-2 were streaked and inoculated on solid LB plates, and then transferred to glucose, maltose, lactose, sucrose, rhamnose, mannose, arabinose, xylose , dulcitol, urea and other carbon sources and H ₂ S and other biochemical identification tubes (purchased from Hangzhou Tianhe Company) for biochemical reactions. O and H antigens were identified according to the instructions of serum factors (purchased from China Veterinary Drug Administration). The results showed that the biochemical characteristics of the gene deletion strain C7821 were not completely consistent with the parent strain C78-2. The parent strain C78-2 can utilize maltose, and it shows red colonies on MacConkey agar with maltose (see Figure 4C), but after the crp gene is deleted, it cannot utilize maltose, so it still shows colorless colonies on MacConkey agar with maltose (See Figure 4B). Different from the parent strain C78-2, the gene deletion strain C7821 could no longer utilize maltose, glucose, rhamnose, mannose and xylose, but the utilization and phenotype of other carbon sources were consistent. The serotype of the gene-deleted strain C7821 was 6,7:C:1,5, which was consistent with the parental strain C78-2, and still conformed to the typical antigenic characteristics of Salmonella choleraesuis.

2. Analysis of the growth characteristics of gene deletion strain C7821

The gene deletion strain C7821 was cultured on LB plates at 37°C for 12 h, and the colony diameter was about 1.0 mm, which was significantly smaller than that of the parent strain C78-2 (2.4 mm). SHAPE \* MERGEFORMAT Parental strain C78-2 and gene deletion strain C7821 were cultured from 106 CFU/mL, samples were taken every 1 h, and viable counts were performed. The results showed that the growth rate of the gene deletion strain C7821 was significantly slower than that of the parent strain C78-2 (see Figure 5), and its mean generation interval (Mean generation time) was 37.2 min, which was 11.2 min longer than that of the parent strain (26.0 min).

3. Genetic stability of gene deletion strain C7821

Streak culture the gene-deleted strain C7821 prepared by the present invention on a solid LB plate, pick a single colony into the liquid LB medium, culture at 37°C, 200 r/min for 16 h, transfer at a volume ratio of 1:1000 Cultured in LB liquid medium for 12 h, then transferred to LB liquid medium again at a ratio of 1:1000 by volume, and performed 50 consecutive transfers. Another streak was inoculated on MacConkey agar with 1% maltose, and the color of the colony was observed. The results showed that the colony could not turn red and still conformed to the phenotypic characteristics of crp gene deletion. At the same time, primers pr5/pr6 and pr6/pr7 were used for PCR amplification, and the inheritance of the crp deletion gene in the gene deletion strain C7821 is shown in Figure 6. Figure 6 shows that there is no visible difference in the amplification results of the 1st, 10th, 20th, 30th, 40th and 50th generation templates of the deletion strain, indicating that the gene deletion strain C7821 prepared by the present invention can be inherited stably.

Example 3 Preparation of Salmonella choleraesuis Gene Deletion Vaccine

The obtained Salmonella choleraesuis gene deletion mutant strain C7821 (experiment number: △ crp C78-2) was identified, and each generation was inoculated on 1% maltose MacConkey agar medium to observe the colony color, and the crp of Salmonella choleraesuis was used to Genetic PCR detection was performed to identify the genetic stability of the missing bacteria. After 50 passages, it was found that the colonies of the deletion mutant strain C7821 could not turn red on MacConkey agar medium with 1% maltose, which still conformed to the phenotypic characteristics of crp gene deletion; Stablize. The Salmonella choleraesuis gene deletion mutant strain C7821 was cultured on LB solid medium, and a single colony was picked and cultured in LB liquid medium until the concentration of viable bacteria reached 1×10 ¹⁰ CFU/mL. Add gelatin protectant according to the ratio of bacterial solution:gelatin protectant (volume:volume) 7:1 (the gelatin protectant preparation method is: add 40 g of sucrose and 8 g of gelatin per 100 mL of deionized water, after fully melting, Sterilize at 121°C for 30 min and store for later use), put in sterilized freeze-dried bottles according to 2.0 mL/bottle, freeze-dry in a freeze-dryer at -50°C, freeze-dry for 36 h, press the cap, and use 10% aluminum gel normal saline was dissolved and carried out viable count (CFU), and confirmed that there was no contamination by miscellaneous bacteria, and stored at -20°C for future use as a vaccine strain for developing recombinant vaccines.

Example 4 Safety Evaluation of Salmonella Cholerasuis Gene Deletion Vaccine C7821

1. Toxicity determination of the gene deletion bacterial strain C7821 of the present invention

In order to test the toxicity of the constructed gene deletion strain C7821 (experiment number: △ crp C78-2) to BALB/c mice, 32 BALB/c mice were equally divided into 2 large groups, each with 4 group. The mice in the first group were orally infected with C7821, and each mouse in each group was injected with 2.1×10 ⁵ CFU, 2.1×10 ⁶ CFU, 2.1×10 ⁷ CFU, and 2.1×10 ⁸ CFU; the second group was orally infected with the parent bacteria For C78-2, each group was injected with 2.1×10 ⁵ CFU, 2.1×10 ⁶ CFU, 2.1×10 ⁷ CFU and 2.1×10 ⁸ CFU respectively. Record the death situation and calculate the median lethal dose (50% lethal dose, LD ₅₀ ). To evaluate the degree of virulence weakening of the gene deletion strain C7821 compared with the parent strain C78-2. The results are shown in Table 2: After injecting mice with 4 concentrations of the parental strain, the lowest concentration (2×10 ⁴ CFU) and the highest concentration (2×10 ⁷ CFU) all killed the mice; while the gene deletion vaccine strain of the present invention C7821 (experiment number: △ crp C78-2) orally infected mice, no mice died from the lowest concentration (2×10 ⁵ CFU) to the highest concentration (2×10 ⁷ CFU). The calculated results showed that the LD ₅₀ of the gene deletion strain C7821 and the parent strain C78-2 were 1.5×10 ⁷ CFU and 3.3×10 ⁶ CFU, respectively, and the virulence of C7821 was 1.325×10 ⁴ times lower than that of the parent strain C78-2. This shows that the virulence of the gene deletion mutant strain C7821 of the present invention is significantly lower than that of the parent strain C78-2.

the

Table 2 Gene deletion vaccine bacterial strain of the present invention and parent bacterial strain virulence comparative test

2. Safety evaluation of the gene deletion strain C7821 (experiment number: △ crp C78-2) of the present invention on pigs

Inoculate 10 4-week-old weaned piglets with 2 mL (containing 4.0×10 ¹⁰ CFU viable bacteria count) per pig orally with the C7821 gene deletion vaccine prepared by the present invention, and some of the inoculated piglets have mild transient fever and body temperature Elevated response, returned to normal after two days. During this period, the mental appetite of piglets injected with the gene deletion vaccine prepared by the present invention was normal, no abnormal changes were found, and Salmonella antibodies could be detected after 2 weeks of immunization. The parental strain C78-2 was orally inoculated with 2 mL per pig (containing 3.6×10 ¹⁰ CFU of live bacteria) to inoculate 4 weaned piglets at the age of 4 weeks. Abdominal cyanosis and other clinical symptoms; death began 3 days after infection, and all died within 7 days. The pathological autopsy of 4 piglets in this group showed typical pathological changes of acute piglet paratyphoid fever.

The gene deletion strain C7821 prepared by the present invention was injected into 10 pregnant sows by injecting 2 mL (containing 4.0×10 ¹⁰ CFU viable bacteria count) per pig. The spirit and appetite of all pregnant sows were normal, and no abnormal changes were found. Antibodies to Salmonella could be detected after one week. Compared with non-vaccinated pregnant sows, the litter size was basically the same, and there were no stillbirths or mummified fetuses. These two tests confirm that the gene deletion strain C7821 prepared by the present invention is safe for both weaned piglets and pregnant sows.

Example 5 Detection of the immune efficacy of the gene deletion strain C7821 of the present invention in mice

1. Immunization procedure for mice

Fifteen Salmonella-negative BALB/c mice aged 5 to 6 weeks were divided into three groups on average, namely the C7821 gene deletion vaccine prepared by the present invention (see Example 3 for the preparation method), and the C500 attenuated vaccine control group (see the implementation method for the preparation method) Example 3: The C500 strain was purchased from the National Veterinary Microorganism Collection Center of the China Veterinary Drug Administration) and the LB control group. The way of immunization was oral inoculation with 0.2 mL (containing 1.2×10 ⁷ CFU of live bacteria) bacterial liquid or LB medium, and a booster immunization 14 days later. 0 day before immunization, 14 days and 28 days after the first immunization, respectively, the C78-2 Salmonella bacterium was used as the coating antigen to detect Salmonella serum antibodies (standard ELISA method, refer to Zhao Z, Xue Y, Wu B, Tang X, Hu R , Xu Y, Guo A, Chen H. 2008. 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 Bordetella bronchiseptica. Infect Immun. 76(5 ):2157-63.).

2. Detection of humoral immune antibody level in immunized mice

The blood was collected before the mice were immunized, and the second and third blood samples were collected 14 and 28 days after the first immunization, respectively. The blood was collected from the tail vein under negative pressure, and the serum was separated and mixed in equal volumes to detect anti-Salmonella serum antibodies. The results are shown in Table 3. It can be seen from Table 3 that the Salmonella antibodies of the mice in the C7821 gene deletion vaccine group and the C500 attenuated vaccine group of the present invention were both 1:320 in the second week after the first immunization. Two weeks after the second immunization (that is, 4 weeks after the first immunization), the Salmonella antibody titers of the mice in the C7821 gene deletion vaccine group and the C500 attenuated vaccine group of the present invention rose to 1:5120 and 1:2560, respectively. The LB controls performed simultaneously were all negative (<1:10). The above test results show that the C7821 gene deletion vaccine prepared by the present invention can induce the body to produce a specific anti-Salmonella humoral immune response after immunizing mice, and the titer level is higher than that of the traditionally used C500 attenuated vaccine.

Table 3 Detection of serum antibody in mice immunized with C7821 gene deletion vaccine prepared by the present invention (ELISA method)

         

3. Protective test of BALB/c mice immunized with C7821 gene deletion vaccine

Thirty Salmonella-negative BALB/c mice aged 5 to 6 weeks were divided into three groups on average, namely the C7821 gene deletion vaccine prepared by the present invention (see Example 3 for the preparation method), and the C500 attenuated vaccine control group (see Example 3 for the preparation method) Example 3), LB control group. The C7821 gene deletion vaccine group prepared by the present invention and the C500 attenuated vaccine vaccine group control group were orally inoculated with 0.2 mL (viable bacteria content 1.2×10 ⁷ CFU) culture solution in BALB/c mice; each LB control group was orally inoculated with 0.2 mL LB Medium. After 14 days, booster immunization once.

In the second week after the second immunization (4 weeks after the first immunization), 10 mice were randomly selected from each group, and 10×LD ₅₀ (containing 2.6×10 ⁵ CFU) virulent strain C78-2 of Salmonella choleraesuis was used to treat the mice Oral challenge poison, observe 30 days. The mice in the LB control group began to show depression, non-eating, and crowding together 12 hours after challenge. Deaths began to appear on the 3rd day, the death peak appeared on the 5th day, and all died by the 8th day. The C7821 gene deletion vaccine group prepared by the present invention has no obvious symptoms and no death after challenge. The condition of the C500 attenuated vaccine control group was similar to that of the C7821 gene deletion vaccine group. This shows that the mice immunized with the C7821 gene deletion vaccine prepared by the present invention can resist the challenge of 10×LD ₅₀ Salmonella choleraesuis strength strain C78-2. The protection rate after challenge is shown in Table 4.

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Table 4 The C7821 gene deletion vaccine prepared by the present invention is against Salmonella choleraesuis C78-2 virulent strain

Protective evaluation of challenged BALB/c mice

Example 6 Detection of the immune efficacy of the C7821 gene deletion vaccine prepared in the present invention in piglets

1. Pig immunization procedure:

Select 20 piglets 20-25 days old and negative for Salmonella and Bordetella bronchiseptica. The test is divided into 3 groups. The first group is the LB control group, numbered 1-4, and the second group is the C500 attenuated vaccine control group, numbered 5-12, the third group is the C7821 gene deletion vaccine group of the present invention, numbered 13-20.

Each pig in the LB control group was injected with 2 mL of LB medium; each pig in the C7821 gene deletion vaccine group of the present invention was inoculated with 2 mL of the C7821 gene deletion vaccine by superficial neck intramuscular injection (see Example 3 for the preparation method, and the live bacteria content 4.0×10 ⁹ CFU); the C500 attenuated vaccine control group was inoculated with 2 mL of C500 attenuated vaccine by intramuscular injection in the neck (see Example 3 for the preparation method, and the live bacteria content was 4.0×10 ⁹ CFU). Blood was collected once at 2 weeks and 4 weeks after immunization, and the serum antibody of Salmonella was detected by ELISA method (standard ELISA method, the method was the same as in Example 5, referring to Zhao Z, et al . 2008. 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 Bordetella bronchiseptica. Infect Immun. 76(5):2157-63.).

2. ELISA antibody level detection of immunized piglets

Blood was collected from the anterior vena cava of 4 pigs in each group at 2 and 4 weeks after immunization, and the serum was separated. The sera of the 4 pigs in each group were mixed in equal proportions, and the serum antibodies to Salmonella were detected by indirect ELISA method. The results are shown in Table 5: 2 weeks after immunization, the Salmonella antibodies of the C7821 gene deletion vaccine group and the C500 attenuated vaccine group of the present invention were 1:320 and 1:160, respectively. Four weeks after immunization, the Salmonella antibody titers of the C7821 gene deletion vaccine group and the C500 attenuated vaccine group of the present invention rose to 1:2560 and 1:1280, respectively. The LB controls performed simultaneously were all negative (<1:10). The above results show that the C7821 gene deletion vaccine prepared by the present invention can induce the humoral immune response of the body to produce specific anti-Salmonella specific antibodies after immunizing piglets.

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Table 5 Detection of serum antibody of piglets immunized with C7821 gene deletion vaccine prepared by the present invention (ELISA method)

3. Protective test of piglets immunized with C7821 gene deletion vaccine prepared by the present invention

Twenty-one days after immunization, a total of 20 piglets in 3 groups were challenged, and each pig was intravenously injected with 1 mL of C78-2 virulent strain bacterial solution (containing about 5×LD ₅₀ , that is, 2.0×10 ⁹ CFU of live bacteria). Attack virus, observe 30 days. Piglets in the LB control group showed body temperature rising to 41-42°C after inoculation, lassitude, prostration, loss of appetite or exhaustion, dyspnea, walking shaking, vomiting and diarrhea. Cyanosis of extremities and abdomen. They began to die 4 days after the challenge, and all died within 7 days. The piglets in the C7821 gene deletion vaccine group and the C500 attenuated vaccine group of the present invention have no obvious symptoms after being challenged, and all survive. This shows that the C7821 gene deletion vaccine has the ability to protect immunized piglets against the infection of Salmonella choleraesuis, so that it can resist the challenge of 5×LD ₅₀ strain C78-2 of Salmonella choleraesuis strength. The protection rate after challenge is shown in Table 6.

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Table 6 The C7821 gene deletion vaccine prepared by the present invention is effective against Salmonella choleraesuis

Protection of piglets challenged by C78-2 wild-type virulent strain

The test results of the embodiments of the present invention show that the Salmonella choleraesuis gene deletion vaccine (C7821) of the present invention can stimulate the body to produce high-titer specific antibodies against Salmonella after immunizing piglets, has very low toxicity to pigs, and has good safety. In the immune protection test, it has a high challenge protection rate against Salmonella choleraesuis. In addition, the Salmonella choleraesuis gene deletion vaccine (C7821) of the present invention has a clear genetic background, does not contain resistance markers, has stable properties, and fully meets the biological safety requirements of veterinary vaccines.

Finally, it should be noted that the above embodiments are only used to illustrate rather than limit the technical solution of the present invention, although the present invention has been described in detail with reference to the above embodiments. Those skilled in the art should understand that the present invention can still be modified or equivalently replaced without departing from the spirit and scope of the present invention. Any modification or partial replacement should be covered by the claims of the present invention.

Claims (5)

1. A gene deletion mutant strain of Salmonella choleraesuis C7821 without a resistance marker, the preservation number of which is CCTCC NO: M2010102. 2. The Salmonella choleraesuis gene deletion mutant strain not containing the resistance marker according to claim 1, characterized in that: the Salmonella choleraesuis gene deletion mutant strain not containing the resistance marker has lost the main toxin of Salmonella choleraesuis. A 320 bp DNA fragment of the force-regulated gene crp . 3. A Salmonella choleraesuis gene deletion vaccine containing the Salmonella choleraesuis gene deletion mutant strain Salmonella choleraesuis C7821 without resistance marker. 4. The Salmonella choleraesuis gene-deleted vaccine according to claim 3, characterized in that: the Salmonella choleraesuis gene-deleted mutant strain that does not contain resistance markers has lost 320 of the main virulence regulator gene crp of Salmonella choleraesuis. bp DNA fragment. 5. Application of a Salmonella choleraesuis gene deletion mutant strain without resistance marker in the preparation of a Salmonella choleraesuis gene deletion vaccine. the

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