Recombinant salmonella paratyphi A and application thereof

Abstract

The invention provides a salmonella paratyphi A strain for recombining and expressing Vi polysaccharide, which is characterized in that a viaB operon for expressing the Vi polysaccharide from salmonella paratyphi is cloned into a salmonella paratyphi A genome by a genetic engineering technology, so that the expression of the Vi polysaccharide in the salmonella paratyphi A is realized; furthermore, a constitutive strong promoter (Tac promoter) is used for replacing a viaB natural promoter to realize high expression of the Vi polysaccharide under an in-vitro culture condition; further, the msbB gene of the strain is knocked out to reduce the acylation level of the LPS and reduce the toxicity of the LPS; the vesicle vaccine prepared from the strain can induce immune protection effects on two serotypes.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine engineering, and relates to a vesicle vaccine capable of producing bivalent immunoprotective effect against typhoid and paratyphoid A. BACKGROUND

[0002] Typhoid and paratyphoid are intestinal infectious diseases caused by Salmonella typhi and four serotypes of Salmonella paratyphi A, B and C, collectively known as enteric fever, which is a global public health problem that seriously threatens human health.

[0003] In recent years, the epidemiological characteristics of enteric fever infection are highlighted in three aspects. First, the infection rate is high. Second, the incidence of paratyphoid A has increased significantly. In the past, the pathogen of enteric fever was mainly Salmonella typhi, accounting for more than 90%. However, recent surveys have shown that the composition of enteric fever pathogens has changed significantly worldwide: in the main epidemic area, paratyphoid A has increased significantly, accounting for as high as 35%, and in some areas, even more than the isolation rate of Salmonella typhi. Third, the problem of drug resistance is becoming increasingly serious. Due to the widespread prevalence of multi-drug resistant bacteria against first-line antibiotics (chloramphenicol, compound sulfamethoxazole and ampicillin), second-line fluoroquinolone antibiotics are currently used for treatment.

[0004] Vaccines are recognized as effective tools against infectious diseases and bacterial drug resistance. Although vaccines have almost no tolerance problems, some vaccines show selectivity to non-vaccine target serotypes, leading to serotype replacement. The development of typhoid vaccine has a long history, and currently three vaccines are mainly used: Salmonella typhi Ty21a oral attenuated live vaccine, Vi polysaccharide subunit vaccine and Vi polysaccharide conjugate vaccine. Vaccination is an effective means of preventing and controlling enteric fever, and the World Health Organization emphasizes the importance of typhoid vaccination in the latest position paper and recommends the vaccination of a new generation of conjugate vaccines. However, the current typhoid vaccine has a significant drawback: all three vaccines are prepared from Salmonella typhi, which has good preventive effect on typhoid caused by Salmonella typhi infection, and also has certain cross-protection on typhoid caused by Salmonella paratyphi B, but large-scale clinical trials and laboratory model experiments have shown that these vaccines lack effective cross-immunity against infection caused by Salmonella paratyphi A.

[0005] So far, there is no internationally recognized effective paratyphoid A vaccine on the market, and there is no bivalent vaccine covering typhoid and paratyphoid A on the market. In view of the significant increase in the incidence of paratyphoid A, the development of paratyphoid A vaccine, especially bivalent vaccine, has significant practical significance. SUMMARY

[0006] The application is based on a recombinant paratyphoid A Salmonella strain expressing Vi polysaccharide, in which a gene locus expressing Vi polysaccharide from Salmonella typhi is cloned into the genome of paratyphoid A Salmonella by genetic engineering technology to realize expression of Vi polysaccharide in paratyphoid A Salmonella; further, a strong constitutive promoter (Tac promoter) is used to replace the natural promoter to realize high expression of Vi polysaccharide under in vitro culture conditions; further, the gene of the strain is knocked out to reduce the acylation level of LPS and reduce the toxicity of LPS; and the vesicle vaccine prepared from the strain can induce immune protection against two serotypes. viaB viaB msbB

[0007] The application first provides a recombinant paratyphoid A Salmonella comprising a gene locus expressing Vi polysaccharide of Salmonella typhi, and the gene locus is constitutively expressed. viaB viaB

[0008] In an embodiment according to the application, the viaB gene locus is regulated for expression by a strong constitutive promoter; preferably, the promoter is a Tac promoter.

[0009] In an embodiment according to the application, the nucleotide sequence of the viaB gene locus is SEQ ID NO: 1.

[0010] In an embodiment according to the application, the nucleotide sequence of the Tac promoter is SEQ ID NO: 2.

[0011] In an embodiment according to the application, the gene locus is inserted into the rfb gene, and the rfb gene is inactivated; preferably, the recombinant paratyphoid A Salmonella is knocked out of the rfb gene. viaB phoN phoN msbB In an embodiment according to the application, the recombinant paratyphoid A Salmonella is knocked out of the rfb gene.

[0012] In an embodiment according to the application, the starting strain is paratyphoid A Salmonella CMCC50093.

[0013] In an embodiment according to the application, the preservation number of the recombinant paratyphoid A Salmonella is CCTCCM 20252795, the preservation unit is China Center for Type Culture Collection (CCTCC), the preservation address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, the classification name is paratyphoid A Salmonella SPA-VTM Salmonella enterica subsp. enterica serovar Paratyphi A SPA-VTM, and the preservation date is December 8, 2025. ​​​​​​​​

[0014] The application also provides use of the recombinant S. paratyphi A described above in the preparation of a vaccine for preventing typhoid fever; in an embodiment according to the application, the typhoid fever is an infection caused by S. typhi or S. paratyphi A infection.

[0015] The application further provides a S. paratyphi A vesicle prepared by a method comprising the following steps: The recombinant S. paratyphi A described above is inoculated into LB medium for culture; and then the recombinant S. paratyphi A vesicle is obtained by separation.

[0016] Another aspect of the application provides a pharmaceutical composition for preventing enteric fever, comprising the S. paratyphi A vesicle described above; preferably, further comprising an adjuvant, preferably, the adjuvant is Alhydrogel adjuvant 2% vaccine adjuvant; preferably, the volume ratio of the Alhydrogel adjuvant 2% vaccine adjuvant to the vesicle is 1:1.

[0017] The beneficial effects of the technical solutions of the application are as follows: The application constructs a S. paratyphi A vaccine strain with high expression of Vi polysaccharide and reduced endotoxin toxicity by genetic engineering technology, and the vesicle prepared by using the vaccine strain has dual immunoprotective efficacy and can effectively prevent typhoid fever caused by S. typhi or S. paratyphi A infection.

[0018] The strain SPA-VTM has a preservation number of CCTCC M 20252795, and is preserved by the China Center for Type Culture Collection (CCTCC) at 299 Bajiyilu, Wuchang District, Wuhan City, Hubei Province. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 For the construction of S. paratyphi A containing viaB (A) is a schematic diagram of the construction. Specifically, the phoN gene locus of S. typhi Ty2 strain is introduced into the chromosome of S. paratyphi A by homologous replacement of the S. paratyphi A neutral gene viaB (B) is a PCR identification diagram of the constructed strain. The relative positions of the primers are shown in the schematic diagram. viaB (C) is a result diagram of agglutination with Vi-specific antiserum caused by Vi production of the constructed strain SPAVi. (SPA is the abbreviation of S. paratyphi A; SPAVi is the abbreviation of S. paratyphi A containing

[0020] Figure 2 For the identification of the constructed strain by PCR phoPQ andhtrA The knockout results are shown in the figure. (A) shows the relative positions of the primers used in strain construction and identification. (B) shows... htrA The image shows the PCR identification results of the knockout, where lane 1: molecular weight marker; lanes 2 and 3: PCR products of primers Hk5 and Hk6; lanes 4 and 5: PCR products of primers Hk1 and Hk4. (C) is... phoPQ The image shows the PCR identification results of the knockout, where lane 1: molecular weight marker; lanes 2 and 3: PCR products of primers Pk5 and Pk6; lanes 4 and 5: PCR products of primers Pk1 and Pk4.

[0021] Figure 3 Replacement of Tac strong promoter tviA Original promoter schematic diagram.

[0022] Figure 4 Figure 1 shows the identification results of the Tac strong promoter replacement strains. (a) Agarose gel electrophoresis image of the SPA VPH-Ptac replacement strain. Lane M is the marker, lane 1 template is SPA VPH-Ptac genomic DNA, lane 2 template is SPAVPH genomic DNA, lane 3 template is PYG4-Ptac plasmid DNA, and the primer is Ptac identification primer F1R4. (b) Anti-Vi serum was diluted to 1 / 4, and 10 μl was mixed with 10 μl of bacterial culture to observe agglutination. (c) 10 μl of serum at different concentrations was mixed with 10 μl of SPA VPH, SPA VPH-Ptac, and Ty2 bacterial cultures, respectively, and the time and degree of agglutination were observed.

[0023] Figure 5 : msbB Construction of the knockout strain. (a) To identify the kanamycin resistance fragment in the integrated strain after PYG4-ΔmsbB was transformed into SPA VPH, the fragment size is 1234bp. Lane M is the marker, lanes 1-2 are the integrated strain, lane 3 is SPA VPH, and lane 4 is PYG4-ΔmsbB. (b) Agarose gel electrophoresis image of the SPA VTM strain. Knockout msbB After gene sequencing, the PCR band amplified by the ΔmsbB identification primers was 2433 bp in size, indicating that the gene was not knocked out. msbB The PCR band size of the gene amplification was 3372 bp. Lane M is the marker, lanes 1-2 are PCR products with SPA VTM strain as template, lane 3 is the PCR product with SPA VPH strain as template, and lane 4 is the PCR product with PYG4-ΔmsbB strain as template.

[0024] Figure 6 Evaluation of the immunogenicity of vesicle vaccines. (a) Intraperitoneal injection of 10 mg of vesicle vaccine into the immunized group and the non-immunized group, respectively.5 Survival rate of mice after CFU of SPA bacterial suspension (mixed with 5% mucin 1:1) (b) Survival rate of mice after CFU of Ty2 bacterial suspension (mixed with 5% mucin 1:1) (c, d, e) Levels of anti-S. paratyphi A LPS antibodies, anti-S. paratyphi A flagella antibodies, and anti-S. typhi Vi polysaccharide antibodies produced by mice on day 7, day 21, and day 35, respectively, after subcutaneous immunization with vesicles on day 0, day 14, and day 28, respectively. 5 Survival rate of mice after CFU of SPA bacterial suspension (mixed with 5% mucin 1:1) (b) Survival rate of mice after CFU of Ty2 bacterial suspension (mixed with 5% mucin 1:1) (c, d, e) Levels of anti-S. paratyphi A LPS antibodies, anti-S. paratyphi A flagella antibodies, and anti-S. typhi Vi polysaccharide antibodies produced by mice on day 7, day 21, and day 35, respectively, after subcutaneous immunization with vesicles on day 0, day 14, and day 28, respectively. DETAILED DESCRIPTION

[0025] To make the technical problems to be solved by the present application, technical solutions and advantages clearer, specific embodiments will be described in detail below with reference to the drawings.

[0026] Unless otherwise specified in the examples, the routine conditions or the conditions recommended by the manufacturer were used. Unless otherwise specified, the reagents or instruments used were conventional products that can be obtained by commercial purchase.

[0027] PrimeSTAR Max high-fidelity DNA polymerase (Takara, Dalian, China). 2x Taq master mix; ClonExpress II One Step Cloning Kit C115 (Vazyme), ApaL I; Nde I; Q5 High-Fidelity DNA polymerase; dNTP (New England Biolabs), bacterial competent cell kit (Shanghai Genechem Co., Ltd.), nalidixic acid (Macklin), kanamycin 100 mg / ml; 0.45 um cellulose acetate filter membrane; bacterial genomic DNA extraction kit; BCA protein quantification kit (Solarbio), Alhydrogel adjuvant 2% (InvivoGen).

[0028] Example 1 viaB Cloning of the locus and introduction into S. paratyphi A genome 1、 viaB Cloning of the locus PrimeSTAR Max high-fidelity DNA polymerase was used to PCR amplify from S. typhi Ty2 genomic DNA using primers viaB-F and viaB-R to obtain viaBLocus (about 13.9 kb) whose nucleotide sequence is SEQ ID NO: 1. The PCR reaction conditions are as follows: 98°C for 10 seconds, 55°C for 15 seconds, 72°C for 10 minutes, for a total of 5 cycles; 98°C for 10 seconds, 68°C for 10 minutes, for a total of 25 cycles. The PCR product is recovered by gel recovery. After Kpnl digestion, the amplified fragment is inserted into the low-copy vector pSTV28a (purchased from Takara Bio (Dalian) Co., Ltd.), transformed into E. coli HB101 by electroporation, and screened on agar plates containing 34 μg / ml chloramphenicol. After identification of the recombinant plasmid by Kpnl digestion, the sample is sent for DNA sequencing identification. Further detection of the recombinant plasmid by slide agglutination test using Vi diagnostic serum viaB Expression of the locus in E. coli. The correctly identified recombinant plasmid is named pSTV28-viaB.

[0029] 2. The viaB Locus is introduced into the genome of Salmonella paratyphi A Salmonella paratyphi A CMCC50093 purchased from the China Center for Type Culture Collection (Beijing, China) is used as the host strain, which is phoN replaced with the cloned viaB Locus. phoN The gene encodes an acid phosphatase unrelated to pathogenicity and is considered a neutral gene. The suicide plasmid pYG4 is used as a gene replacement vector, and its operation is as shown in Figure 1 Briefly, the overlapping PCR technique is used to amplify the upstream and downstream sequences of the Salmonella paratyphi A phoN gene using PNk1 / PNk2 and PNk3 / PNk4 as primers. By introducing Kpnl sites at the 5' ends of PNk2 and PNk3, a Kpnl site is formed in the center of the fusion fragment. Subsequently, the PCR product is digested with Bglll and Ndel endonucleases and ligated to the pYG4 vector to construct pYG4-phoNUD. The fragment containing viaB is obtained by Kpnl digestion from pSTV28-viaB, inserted into the Kpnl site between the upstream and downstream sequences of phoN in pYG4-phoNUD, and verified by Kpnl digestion and slide agglutination test. The final recombinant plasmid is named pYG4-viaB.

[0030] The recombinant plasmid pYG4-viaB was introduced into *Salmonella paratyphi A* CMCC50093 via electroporation. Chromosomal-integrated strains were screened using kanamycin resistance. After culturing kanamycin-resistant strains overnight in antibiotic-free liquid medium, reverse selection was performed on agar plates containing 5% sucrose. Successfully gene-substituted *Salmonella paratyphi A* strains were screened by PCR, and further verification was performed using a Vi antiserum slide agglutination assay. The final recombinant strain was named SPAVi.

[0031] 3. In the SPAVi strain htrA and phoPQ gene knockout Gene deletion is similar to gene replacement, except that a fusion DNA fragment consisting of upstream and downstream sequences of the target gene needs to be constructed in the pYG4 vector (primer sequences are shown in Table 1). The final attenuated strain producing Vi antigen was named SPA-VPH, and its identification is detailed below. Figure 2 .

[0032] Example 2 (Construction of recombinant plasmid containing Tac promoter sequence and promotion of promoter substitution) 1. The Tac promoter sequence was directly synthesized by Sangon Biotech (Shanghai) Co., Ltd. Primers for seamless cloning were designed; primer sequences are shown in Table 1. The designed primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd. The synthesized sequences are shown below: TTACAGAgacataagtctggtGGATCCGGAGCTTATCGACTGCACGGTGCACCAATGCTTCTGGCGTCAGGCAGCCATCGGAAGCTGTGGTATGGCTGTGCAGGTCGTAAATCACTGCATAATTCGTGTCGCTCAAGGCGCACTCCCGTTCTG GATAATGTTTTTTGCGCCGACATCATAACGGTTCTGGCAAATATTCTGAAATGAGCTGTTGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGAAttcATGAGGTTTCATCATTTCTGG (SEQ ID NO:2) 2. Extract SPA VPH genomic DNA and use it as a template for PCR amplification. viaB The first gene at the locus tviA Upstream and downstream fragments of the promoter. Amplification conditions: 98℃ for 30s; 35 cycles (98℃ for 10s, 70℃ for 30s, 72℃ for 30s); 72℃ for 2min.

[0033] PYG4 was linearized by double digestion with ApaLⅠ and NdeⅠ, and the tac promoter fragment was then extracted. viaB The upstream and downstream fragments of the promoter and linear PYG4 were used to construct the recombinant plasmid PYG4-Ptac via seamless cloning. Seamless cloning was performed using the ClonExpress II One Step Cloning Kit from Nanjing Novizan Biotechnology Co., Ltd. The recombinant plasmid was identified by sequencing. The identified recombinant plasmid was then transformed into the engineered strain S17-1 / λ Escherichia coli.

[0034] 3. Promoter Recombination and Replacement: Place a sterile 0.45 μm cellulose acetate filter membrane onto an LB agar plate. Add SPA VPH and S17-1 / PYG4-Ptac bacterial suspensions at a ratio of 4:1 or 9:1 onto the filter membrane. Incubate upright at 37°C for 12-16 hours until bacterial growth occurs. Scrape all bacterial growth onto 1 ml of sterile PBS using a sterile inoculation loop and mix thoroughly. After appropriate dilution, spread the bacterial suspension onto a plate containing 100 μg / ml kanamycin and 30 μg / ml nalidixic acid (NPA) and incubate at 37°C for at least 20 hours. Pick single colonies and perform PCR identification using Ptac identification primers. Inoculate the identified conjugating strains into LB liquid and incubate overnight at 37°C and 250 rpm for 12-16 hours. Spread 100 μl of the bacterial suspension onto a 5% sucrose plate for reverse selection. After single colonies grew on sucrose plates, PCR identification was performed using Ptac identification primers to screen for colonies that had been replaced with the tac promoter. The entire construction diagram is shown below. Figure 3 As shown, the identification results are as follows: Figure 4 As shown.

[0035] Example 3 ( msbB Gene knockout Gene knockout is similar to gene replacement, except that a fusion DNA fragment consisting of upstream and downstream sequences of the target gene needs to be constructed in the pYG4 vector (primer sequences are shown in Table 1). The resulting strain was named SPA-VTM, and the identification results are as follows: Figure 5 As shown.

[0036] Table 1 Primer Sequences

[0037]

[0038] Example 4 (Vessel preparation and evaluation of immunogenicity in a mouse model) 1. Vesicle preparation: Inoculate SPA-VTM into LB medium and incubate overnight at 37°C with shaking.

[0039] The vesicle extraction procedure was performed in accordance with the instructions for the bacterial vesicle isolation kit from Shanghai Bosheng Biotechnology Co., Ltd.

[0040] 2. The extracted vesicles were quantified for protein using the BCA Protein Assay Kit from Thermo Scientific to determine the concentration of the vesicles.

[0041] 3. 6-week-old female BALB / c mice were randomly divided into PBS and vesicle groups. Each mouse in the vesicle group was immunized with 50 μg of vesicles, and the concentration of the vesicles was ≥1.0 μg / μl. The vesicles were diluted with PBS to 50 μl, mixed with Alhydrogel adjuvant 2% vaccine adjuvant 1:1 (V:V) to form a vesicle mixture, and each mouse was subcutaneously immunized with 100 μl of the vesicle mixture on days 0, 14, and 28. The PBS group was subcutaneously injected with 100 μl of PBS.

[0042] ① On days 7, 21, 35, and 42, blood was collected from the orbital venous plexus of the mice, centrifuged at 3000 rpm for 20 min, and the supernatant was used to measure the antibody using an ELISA kit.

[0043] ② On day 35, the vesicle and PBS groups were subjected to bacterial challenge, and each mouse was challenged with 500 ul of Salmonella typhi or Salmonella paratyphi bacterial solution intraperitoneally. The survival of the mice was observed within 72 hours. As shown in Table 2 and Figure 6 , the vesicle vaccine immunization had a significant protective effect against challenge by both serotypes of Salmonella.

[0044] Table 2. Immune protection effect of subcutaneous inoculation of vesicles in a mouse model

[0045] The above describes preferred embodiments of the present application. It should be noted that, for those skilled in the art, without departing from the principles of the present application, several improvements and refinements can be made, and these improvements and refinements should also be considered within the scope of protection of the present application.

Claims

1. A recombinant Salmonella typhi that comprises a viaB locus that expresses a Salmonella typhi Vi polysaccharide, and said viaB locus is constitutively expressed.

2. The recombinant S. typhi of claim 1, wherein, The viaB The locus is regulated for expression by a constitutive strong promoter; preferably, the promoter is a Tac promoter.

3. The recombinant S. typhi of claim 1 or 2, wherein, The nucleotide sequence of the locus is SEQ ID NO:

1. viaB The nucleotide sequence of the locus is SEQ ID NO:

1.

4. The recombinant S. typhi of any one of claims 1-3, wherein, The nucleotide sequence of the Tac promoter is SEQ ID NO:

2.

5. The recombinant S. typhi of any one of claims 1-4, wherein, The viaB locus is inserted into phoN a gene, and the phoN gene is inactivated; preferably, the msbB gene knock-out of the recombinant S. typhi.

6. The recombinant S. typhi of any one of claims 1-5, wherein, The starting strain is Salmonella paratyphi A CMCC50093; preferably, the preservation number of the recombinant Salmonella paratyphi A is CCTCC M 20252795, and the preservation unit is China Center for Type Culture Collection (CCTCC), the preservation address is No. 299, Bajiyilu, Wuchang District, Wuhan City, Hubei Province.

7. Use of the recombinant Salmonella paratyphi A according to any one of claims 1-6 in the preparation of a vaccine for preventing enteric fever.

8. Use of the recombinant Salmonella paratyphi A according to any one of claims 1-7 in the preparation of a vaccine for preventing typhoid fever according to claim 7, wherein the enteric fever is typhoid fever caused by Salmonella typhi or Salmonella paratyphi A infection.

9. A Salmonella paratyphi A vesicle, which is prepared by a method comprising the following steps: Inoculating the recombinant Salmonella paratyphi A according to any one of claims 1-6 into LB medium for culture; and then isolating to obtain a Salmonella paratyphi A vesicle.

10. A pharmaceutical composition for preventing enteric fever, comprising the Salmonella paratyphi A vesicle according to claim 9; preferably, further comprising an adjuvant, preferably, the adjuvant is Alhydrogel adjuvant 2% vaccine adjuvant; preferably, the volume ratio of the Alhydrogel adjuvant 2% vaccine adjuvant to the vesicle is 1:1.

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