CN114990042A - Salmonella containing lipopolysaccharide capable of expressing shigella dysenteriae O antigen, preparation method and application thereof - Google Patents
Salmonella containing lipopolysaccharide capable of expressing shigella dysenteriae O antigen, preparation method and application thereof Download PDFInfo
- Publication number
- CN114990042A CN114990042A CN202210721437.9A CN202210721437A CN114990042A CN 114990042 A CN114990042 A CN 114990042A CN 202210721437 A CN202210721437 A CN 202210721437A CN 114990042 A CN114990042 A CN 114990042A
- Authority
- CN
- China
- Prior art keywords
- salmonella
- antigen
- plasmid
- outer membrane
- lipopolysaccharide
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/02—Bacterial antigens
- A61K39/025—Enterobacteriales, e.g. Enterobacter
- A61K39/0283—Shigella
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
- C07K14/25—Shigella (G)
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
- C07K14/255—Salmonella (G)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
- A61K2039/523—Bacterial cells; Fungal cells; Protozoal cells expressing foreign proteins
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/101—Plasmid DNA for bacteria
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/42—Salmonella
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses salmonella capable of expressing lipopolysaccharide of Shigella dysenteriae O antigen, which can express lipopolysaccharide of Shigella dysenteriae O antigen on outer membrane vesicles of recombinant salmonella, and salmonella removes the self O antigen of salmonella through gene modification; removing FliC and FljB flagellin which interfere the immune response of the outer membrane vesicle of the salmonella; removing the outer membrane proteins OmpA, OmpC and OmpD which are not necessary for the salmonella. The invention also discloses application of the bacterium in preparation of Shigella dysenteriae resistant vaccines.
Description
Technical Field
The invention relates to the field of physical technology, genetic engineering and microbial fermentation, in particular to salmonella containing lipopolysaccharide capable of expressing shigella dysenteriae O antigen, a preparation method and application thereof.
Background
Shigella spp is a gram-negative bacterium, commonly called Shigella dysenteriae, which is highly infectious and pathogenic and causes severe diarrhea and even death in humans. Dysentery is a high-incidence intestinal infectious disease worldwide, and according to conservative estimation, the number of infected people is more than 1.6 hundred million and the number of dead people is more than 110 million every year worldwide. Dysentery is particularly frequently caused in developing countries, nearly 2000 thousands of people are infected with dysentery every year in China, the annual accumulated number of diseases is always in the third place, and only after tuberculosis and hepatitis B, the prevention and treatment of the dysentery is a problem which cannot be ignored.
With the problem of antibiotic resistance becoming more and more serious, the trend has been toward the prevention and treatment of bacterial infections by vaccines. Although no satisfactory dysentery vaccine is found at present, researches have found some potential candidate vaccine targets, wherein the Lipopolysaccharide (Lipopolysaccharide) of Shigella can show better protective power, and researches prove that the complex of the Lipopolysaccharide and protein of Shigella can stimulate very high serum antibody titer of mice and can show certain protective power, and clinical experiments show that the O antigen polysaccharide of Fowles 2a (Shigella flexneri 2a) can cause specific immune protection, but the most key problem of the Lipopolysaccharide is that the O antigen polysaccharide enters a host body, cannot effectively reach the immune system of the host and is recognized by the immune system, so that the immune failure is caused with certain probability. If this problem can be solved, lipopolysaccharide has the potential to be developed into a highly effective vaccine target. Lipopolysaccharide is the main virulence factor of most gram-negative bacteria, is also an important component of the outer membrane of bacteria, and consists of an O-antigen (O antigen), core polysaccharide (core polysaccharide) and lactone A (lipid A). Through comparison of lipopolysaccharide synthesis gene clusters of salmonella and shigella, the O antigen gene clusters are slightly different in structure, but enzymes used for synthesizing and combining the O antigen on a core polysaccharide site are all RfaL, so that the O antigen of the shigella is synthesized on an outer membrane of the salmonella and is possibly combined on core oligosaccharide of the salmonella in a synthesis way. Similar strategies are utilized to express an O antigen gene cluster of Escherichia coli O78 in Salmonella enteritidis, and animal experiments show that the O antigen presented by the Salmonella can stimulate the body to generate specific immune response.
The outer membrane vesicles of the salmonella have proved to be a high-efficiency presentation carrier, and researches prove that PspA protein presented by the outer membrane vesicles of the salmonella can stimulate a host to generate a better immune response and provide certain protection. The outer membrane vesicle can wrap the presenting antigen due to the characteristic of the lipid bilayer of the outer membrane vesicle and is not cleared away by the organism too fast, and simultaneously, the outer membrane vesicle can stimulate the organism to cause immune protective reaction and avoid the safety risk of attenuated live vaccine and inactivated vaccine, and can present protein to the deep part of a host so as to better stimulate the organism to generate immune protection, so the outer membrane vesicle can be used as an efficient antigen presenting carrier. An ideal outer membrane vesicle presenting vector should have the following characteristics: 1) the outer membrane vesicle carrier can stimulate a host to generate good immune protection reaction, but avoids the interference of the presented foreign antigen by meaningless immune reaction as much as possible; 2) the purification steps of the outer membrane vesicles are simplified as much as possible, and the production of a large amount of outer membrane vesicles as much as possible can reduce the production cost; 3) the carrier itself may act as an adjuvant to help the presented antigen be recognized by the host immune system and thus exert greater efficacy.
Outer membrane vesicle vaccines have been the focus of research on vaccines for many pathogenic bacteria due to their many incomparable advantages. However, most of the outer membrane vesicle vaccines of pathogenic bacteria at present only stay on the outer membrane vesicles of wild strains to be evaluated, and the outer membrane vesicles serving as vaccine carriers also stay on nontoxic escherichia coli engineering bacteria to be researched. Outer membrane vesicle vaccines are in the hot spot of research in the ascending phase worldwide, and in China, the research is almost blank. If a vaccine presentation platform based on outer membrane vesicles can be successfully developed, the platform has great prospect in both basic research and transformation application. .
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides the defects of the prior method, and the problem to be solved by the invention is to provide a recombinant salmonella strain which can express extracellular polysaccharide of Shigella dysenteriae O antigen and can be used as a platform to prepare glycoprotein vaccine for resisting Shigella dysenteriae, aiming at solving part of the problems in the prior art or at least relieving part of the problems in the prior art.
A salmonella bacterium comprising lipopolysaccharide capable of expressing shigella dysenteriae O antigen, characterized by: the lipopolysaccharide is expressed on an outer membrane vesicle of the recombinant salmonella, and the salmonella removes a salmonella self-O antigen through gene modification; removing FliC and FljB flagellin which interfere the immune response of the outer membrane vesicle of the salmonella; the outer membrane proteins OmpA, OmpC and OmpD, which are not necessary for the salmonella itself, are removed.
Preferably, the salmonella further comprises: overexpression of the enzyme pagL (Δ pagL:: P) which controls deacylation of lipid A Synthesis trc pagL) that changes the wild-type lipid a structure to a lipid a monophosphate structure, reducing the ability of lipid a to be recognized by TLR 4.
The construction method of salmonella containing lipopolysaccharide capable of expressing shigella dysenteriae O antigen comprises the following steps:
(1) constructing an expression plasmid by taking pYA3337 as a template plasmid, wherein the plasmid contains a pSC101 replicon and an asd original copy, and the plasmid and the asd gene knocked out from the genome form a balanced lethal system;
(2) constructing a plasmid containing Shigella O antigen gene cluster by adopting a synthetic biology method: overlapping fragments (overlap) are designed through two strategies of a yeast recombination system or a kit Gibson assembly kit from NEB, a target fragment and a vector fragment are obtained by high-fidelity PCR amplification, and different assembly systems are utilized to assemble a complete plasmid. All plasmid construction procedures will use DH5 alpha and c6097 (asd deficient of DH5 alpha) as plasmid propagation hosts;
(3) constructing salmonella expressing lipopolysaccharide of shigella dysenteriae O antigen: the constructed plasmid is transferred into the constructed outer membrane vesicle mutant strain of the salmonella typhimurium, and the biological characteristics of the recombinant vaccine are measured to evaluate the influence of the expression of the heterologous polysaccharide on the outer membrane vesicle of the salmonella.
Salmonella containing lipopolysaccharide capable of expressing Shigella dysenteriae O antigen is used in preparation of Shigella dysenteriae resisting vaccine and in preparation of high-efficiency polysaccharide presentation platform.
Has the advantages that:
the technical scheme of the invention is based on the characteristic that the structures and the synthetic routes of Lipopolysaccharide (LPS) of Shigella dysenteriae and salmonella are similar, and the lipopolysaccharide derived from Shigella dysenteriae O antigen is expressed in the salmonella, and meanwhile, the self O antigen of the salmonella is removed through gene modification; removing FliC and FljB flagellin which interfere the immune response of the outer membrane vesicle of the salmonella; the outer membrane proteins OmpA, OmpC and OmpD, which are not necessary for the salmonella itself, are removed.
Drawings
FIG. 1 is a diagram of the concept of constructing Salmonella capable of expressing lipopolysaccharide of Shigella dysenteriae O antigen;
FIG. 2 is a technical roadmap for the construction of Salmonella;
FIG. 3 is the construction diagram of Shigella O antigen polysaccharide antigen expression plasmid;
FIG. 4 is a flow chart of an animal experiment for evaluating the presentation efficiency of outer membrane vesicle antigenic proteins;
FIG. 5 is a graph showing the result of analysis of IgA antibody content.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to examples, and the equipment and reagents used in the examples and test examples are commercially available without specific reference. The specific embodiments described herein are merely illustrative of the invention and are not intended to be limiting.
Various modifications to the precise description of the invention will be readily apparent to those skilled in the art from the information contained herein without departing from the spirit and scope of the appended claims. It is to be understood that the scope of the invention is not limited to the procedures, properties, or components defined, as these embodiments, as well as others described, are intended to be merely illustrative of particular aspects of the invention. Indeed, various modifications of the embodiments of the invention which are obvious to those skilled in the art or related fields are intended to be included within the scope of the following claims.
For a better understanding of the invention, and not as a limitation on the scope thereof, all numbers expressing quantities, percentages, and other numerical values used in this application are to be understood as being modified in all instances by the term "about". Accordingly, unless expressly indicated otherwise, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. In the present invention, "about" means within 10%, preferably within 5% of a given value or range.
In the following examples of the present invention, the temperature is not particularly limited, and all of the conditions are normal temperature conditions. The normal temperature refers to a natural room temperature condition in four seasons, no additional cooling or heating treatment is performed, and the normal temperature is generally controlled to be 10-30 ℃, and is preferably 15-25 ℃.
Example 1 construction of Salmonella outer Membrane vesicle vector
Construction method of Δ OmpA-deleted Strain: taking a Salmonella typhimurium UK-1 strain as a template, finding an OmpA gene and an upstream and downstream sequence thereof based on the principle of homologous recombination, obtaining an upstream homologous arm OmpA-L of a target gene through amplification of OmpA-A and OmpA-B, and obtaining a downstream homologous arm OmpA-R of the target gene through amplification of OmpA-C and OmpA-D; then carrying out fusion PCR by taking the OmpA-L, OmpA-R gene fragment as a template to amplify the OmpA-LR fragment deleted with the target gene, and then cloning the OmpA-LR to a suicide vector pRE112, namely pRE112: CmR-OmpA-LR; then transformed into 2, 6-diaminopimelic acid DAP-deficient strain chi 7213, and PCR shows a specific positive band which is consistent with the size of an expected fusion fragment; the double enzyme cutting result also shows that the suicide vector pRE112 is successfully constructed by CmR-OmpA-LR, and is frozen and stored after the enzyme cutting and the sequence identification are correct.
QS002 Salmonella comprising a Δ fliC Δ fljB Δ ompA Δ ompC Δ ompD gene deletion and QS003 Salmonella comprising a Δ fliC Δ fljB Δ ompA Δ ompC Δ ompD Δ pagL:PtrcpagLgene deletion and mutation can be prepared by the above method.
Example 2 construction of Shigella O antigen polysaccharide expression vector
pYA3337 is used as a template plasmid to construct an expression plasmid, the plasmid contains a pSC101 replicon, the replicon is a single copy plasmid, the synthesis and assembly of protein by bacteria can be best simulated, the synthesized Shigella O antigen polysaccharide is assembled on core oligosaccharide (core) of salmonella, in addition, an asd original is also contained, a balanced lethal system is formed by the synthesized asd O antigen polysaccharide and a genome knocked-out asd gene, and the use of antibiotics in the process of culturing bacteria is avoided.
As the Shigella O antigen gene cluster is large and is not easy to insert into an expression plasmid, the construction of the plasmid is completed by a synthetic biology method. We will use two different synthetic biology approaches to ensure the successful construction of expression plasmids: yeast recombination System or kit Gibson assembly kit from NEB. Both strategies are to use the overlapping fragment (overlap) design, obtain the target fragment and the vector fragment by high fidelity PCR amplification, and utilize different assembly systems to assemble into a complete plasmid (the specific operation steps are performed according to the instructions). All plasmid construction procedures will use DH5 α and c6097 (asd deficient in DH5 α) as plasmid propagation hosts. And transferring the constructed plasmid into the constructed outer membrane vesicle mutant strain of the salmonella typhimurium to construct a shigella O antigen polysaccharide expression vector.
Example 3 isolation and purification of outer Membrane vesicles from Salmonella
The method for separating and purifying the outer membrane vesicles of the salmonella is formed by slightly modifying the method disclosed by Muralnath et al. First, a single colony was picked and the bacteria were cultured, overnight with 1: 100 was inoculated into 2L of liquid LB medium, cultured overnight at 37 ℃ until the OD value became 1 (about 12 to 15 hours), centrifuged to remove the cells at 4 ℃ and 10,000 Xg. The collected supernatant was filtered through a 0.45 μm filter to remove the remaining cells, and then the outer membrane vesicles in the supernatant were collected by ultracentrifugation (centrifugation conditions 2h, 40,000 Xg, 4 ℃). The collected outer membrane vesicles were resuspended with DPBS and filtered again with 0.45 μm filter. The initially obtained outer membrane vesicles also need to be purified by density gradient centrifugation. Using 10mM HEPES buffer solution containing 0.85% NaCl as diluent, preparing density gradient centrifugation buffer solution into discontinuous density gradient centrifugation device from top to bottom according to the concentration of 20-45% by taking each layer as 2ml, and taking centrifugation at 4 ℃ overnight by adding the outer membrane vesicles collected in the last step on the uppermost layer of the density gradient centrifugation device, wherein the centrifugation conditions are 200,000 Xg. The density gradient layer containing a large amount of outer membrane vesicles was collected by ultracentrifugation (40,000 Xg, 4 ℃, 1 hour), washed once with DPBS to remove impurities therefrom, and stored in a refrigerator at-80 ℃ for later use.
Example 4 evaluation of immunoprotective efficacy of constructed Shigella polysaccharide recombinant vaccines using a mouse model
Non-immunized BALB/c mice of 6 weeks are purchased, unified into females, 5 mice are taken in each cage, the temperature is appropriate, the illumination is ensured for 12 hours, the mice are adapted to be cultured in cages to 7 weeks of age in a new environment, the mice are randomly grouped, and the mice are forbidden and fasted 6 hours before immunization.
1) Immunization procedure and sample collection:
immunization: two immunization routes, including nasal drip (intrasal) and intraperitoneal injection (intraepithelial), were used to evaluate the immunological potency of shigella recombinant polysaccharide vaccines to elicit body-generated immunity and the ability to protect against wild strain challenge. Newly purchased mice were bred for one week to adapt to a new environment, and the mice were divided into several groups according to 10 mice per group, and from the first immunization, 0 day and 30 days were used as a primary immunization and a booster immunization (secondary immunization), respectively. The immunization dose is shown in Table 1 (nasal drop immunization in 10. mu.l PBS; peritoneal immunization in 100. mu.l PBS). The control group was immunized with a corresponding volume of PBS buffer as a negative control.
TABLE 1 Immunometric assay
The experimental setup was as follows:
collecting samples: collecting blood from puncture by orbital sinus vein, collecting at 4 weeks after first-time immunization and 4 weeks after second-time immunization, standing the collected blood at 37 deg.C for one hour, standing at 4 deg.C overnight, separating out serum, and collecting serum in refrigerator at-80 deg.C for use. Collection of vaginal secretion samples: mu.l of PBS buffer was used to wash the vaginal opening and collect vaginal secretions and stored in a freezer at-80 ℃ until use. Three mice were sacrificed at 4 weeks post-primary and 4 weeks post-secondary, and alveolar lavage fluid was collected to determine their IgA antibody content. Results as shown in figure 5, mucosal IgA levels were lower in all mice except QS003 group 2 weeks after immunization (P < 0.05). Mucosal IgA levels were significantly elevated after booster immunization, and mucosal IgA levels were significantly higher in QS002 and QS003 groups than in wild type OMV immunized mice (P < 0.01).
2) And (3) immunodetection:
quantitative ELISA detection: antibodies specific for shigella LPS antigen, including IgG, IgG1, IgG2a, and IgA, were analyzed in serum, alveolar lavage fluid, and vaginal secretions. The coating antigen is shigella LPS antigen, and the coating concentration is 0.25 ug/ml. In the research, a method for drawing standard curves of different antibody specificities is adopted to quantify the content of the antibodies contained in different individuals, so that a purified mouse Ig subtype standard substance needs to be diluted by a multiple of 2 times from 0.5 mu g/mu l and coated according to the same method, and meanwhile, a sample to be detected is diluted by shigella lipopolysaccharide of 1 mu g/well in 100 mu l of coating buffer solution, added into a 96-hole ELISA plate and placed at 4 ℃ for overnight incubation. After overnight incubation, the ELISA plates were washed three times with PBST (PBS containing 0.1% Tween 20), then blocked with 2% BSA buffer and allowed to stand at room temperature for 2 hours. Different serum samples and vaginal secretion samples were diluted in 100. mu.l wells and each sample was run in triplicate and left at room temperature for 1 hour after sample addition. After the PBST is washed for three times, different biotin-labeled goat anti-mouse IgG, IgG1, IgG2a and IgA are added into different plates respectively, the content of different Ig subtypes is determined, and the plates are still placed at room temperature for 2 hours. After three PBST washes, alkaline phosphatase-labeled streptavidin was added for 1 hour. Finally, pNpp (p-nitrophenylphosphate) was used for color development. Color reaction (absorbance) the absorbance at a wavelength of 405nm was measured. Values greater than the PBS control were considered positive. Drawing the standard curve by using curve expert software, obtaining the standard curve by using a log-log regression curve function, substituting the measured sample numerical value to obtain the antibody content in the sample, and converting the final Ig antibody content into the dilution multiple of the sample to obtain the final antibody content. The antibodies in QS002 and QS003 groups were more than 5-fold higher than the wild type group.
3) Evaluation of efficacy of challenge protection: the method is implemented by evaluating the attack protection efficacy of the shigella polysaccharide recombinant vaccine on wild shigella by using a shigella mouse lung pathological model:
performing challenge on the fifth week after the second immunization, performing challenge by using Shigella with lethal measurement of 109CFU/10 mul DPBS in a nasal drip mode, dissecting 3 mice from an immune group and a control group respectively after 24 hours of challenge, taking the lung of the mice for pathological section preparation, sequentially soaking the tissues in ethanol and xylene, embedding the tissues with paraffin, dehydrating the tissues, cutting the paraffin-embedded samples into 5mm sections after dehydration, and performing hematoxylin-eosin staining. The results of the staining were analyzed by photographic observation under an electron microscope, reflecting the protective efficacy of the vaccine by the pathological section and the statistics of the pathological lesion scores. QS003 immunoprotection > QS002 > wild type was analyzed under an electron microscope.
The pathological lesion score is detailed as follows: score 0 indicates no significant inflammatory response or pathology; score 1 indicates a small or scattered lesion, affecting less than 10% of the tissues, with a small amount of perivascular viable peribronchial lymphocyte infiltration; 2 points indicate that the lymphocyte aggregation around mild blood vessels or bronchia, pneumonia or diffusion exist, and the total volume of inflammation does not exceed 10-20% of the whole tissue; the 3 points represent toxic lesions, rich lymphocyte infiltration is usually formed around blood vessels and bronchi, and the inflammation area is 20-30% of the total tissue area; score 4 indicates extensive pneumonia and significant inflammation area over 30% of the total tissue area; score 5 indicates that the lesion and inflammation area exceeds 50% of the total area.
b. And (3) taking lung lavage fluid of a part of mice 24 hours after the challenge to measure the cytokines, wherein the steps of collecting the lung lavage fluid are briefly as follows: after the mice are sacrificed, the neck is dissected, the trachea is exposed, a puncture needle or a 4.5-gauge needle is used for intubation, precooled PBS buffer solution 0.8 ml is used for lavage for 3-5 times, and the collected lavage solution is placed at minus 80 ℃ for standby. And finally detecting the content of IgA in the alveolar lavage fluid.
c. The remaining mice were then continued to observe their weight changes and whether they died, the number of dead mice per day was recorded, observations continued until 14 days after challenge, and final statistics were taken for protective efficacy analysis. Of these QS003 group mice were all alive, followed by QS002 group.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (4)
1. A salmonella bacterium comprising lipopolysaccharide capable of expressing shigella dysenteriae O antigen, characterized by: the lipopolysaccharide is expressed on outer membrane vesicles of recombinant salmonella, and the salmonella removes salmonella self-O antigen through gene modification; removing FliC and FljB flagellin which interfere the immune response of the outer membrane vesicle of the salmonella; removing the outer membrane proteins OmpA, OmpC and OmpD which are not necessary for the salmonella.
2. The salmonella of claim 1, wherein:
the salmonella comprises: overexpression of a mutation in pagL, an enzyme that controls deacylation of lipid a synthesis, changes the wild-type lipid a structure to a lipid a monophosphate structure, reducing the ability of lipid a to be recognized by TLR 4.
3. A process for preparing the salmonella bacterium containing lipopolysaccharide capable of expressing shigella dysenteriae O antigen of any one of claims 1 to 2, comprising the steps of:
(1) constructing an expression plasmid by taking pYA3337 as a template plasmid, wherein the plasmid comprises a pSC101 replicon and an asd original copy, and the plasmid and the asd gene knocked out from the genome form a balanced lethal system;
(2) constructing a plasmid containing Shigella O antigen gene cluster by adopting a synthetic biology method: overlapping fragments are designed through two strategies of a yeast recombination system or a reagent kit Gibson assembly kit from NEB, a target fragment and a vector fragment are obtained through high-fidelity PCR amplification, and different assembly systems are utilized to assemble a complete plasmid. All plasmid construction operations will use DH5 alpha and c6097 as plasmid propagation hosts;
(3) constructing salmonella expressing lipopolysaccharide of shigella dysenteriae O antigen: the constructed plasmid is transferred into the constructed outer membrane vesicle mutant strain of the salmonella typhimurium, and the biological characteristics of the recombinant vaccine are measured to evaluate the influence of the expression of the heterologous polysaccharide on the outer membrane vesicle of the salmonella.
4. The use of salmonella containing lipopolysaccharide capable of expressing shigella dysenteriae O antigen as claimed in any one of claims 1-2 in the preparation of a shigella dysenteriae vaccine and in the preparation of a high performance polysaccharide presentation platform.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202210721437.9A CN114990042A (en) | 2022-06-17 | 2022-06-17 | Salmonella containing lipopolysaccharide capable of expressing shigella dysenteriae O antigen, preparation method and application thereof |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202210721437.9A CN114990042A (en) | 2022-06-17 | 2022-06-17 | Salmonella containing lipopolysaccharide capable of expressing shigella dysenteriae O antigen, preparation method and application thereof |
Publications (1)
Publication Number | Publication Date |
---|---|
CN114990042A true CN114990042A (en) | 2022-09-02 |
Family
ID=83036310
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202210721437.9A Pending CN114990042A (en) | 2022-06-17 | 2022-06-17 | Salmonella containing lipopolysaccharide capable of expressing shigella dysenteriae O antigen, preparation method and application thereof |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN114990042A (en) |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN104797268A (en) * | 2012-09-06 | 2015-07-22 | 埃韦利库雷生物技术有限公司 | A novel live attenuated shigella vaccine |
CN105154377A (en) * | 2015-07-17 | 2015-12-16 | 河南科技大学 | Recombinant Salmonella enteria serovar Pullorum, (S. Pullorum), as well as preparation method and application thereof |
CN111793591A (en) * | 2020-06-22 | 2020-10-20 | 南昌大学 | Salmonella mutant strain capable of efficiently stimulating immune response and construction method and application thereof |
CN114214351A (en) * | 2022-01-05 | 2022-03-22 | 南昌大学 | Shigella polysaccharide expression plasmid and application thereof |
-
2022
- 2022-06-17 CN CN202210721437.9A patent/CN114990042A/en active Pending
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN104797268A (en) * | 2012-09-06 | 2015-07-22 | 埃韦利库雷生物技术有限公司 | A novel live attenuated shigella vaccine |
CN105154377A (en) * | 2015-07-17 | 2015-12-16 | 河南科技大学 | Recombinant Salmonella enteria serovar Pullorum, (S. Pullorum), as well as preparation method and application thereof |
CN111793591A (en) * | 2020-06-22 | 2020-10-20 | 南昌大学 | Salmonella mutant strain capable of efficiently stimulating immune response and construction method and application thereof |
CN114214351A (en) * | 2022-01-05 | 2022-03-22 | 南昌大学 | Shigella polysaccharide expression plasmid and application thereof |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN111793591B (en) | Salmonella mutant strain capable of efficiently stimulating immune response and construction method and application thereof | |
JP5451619B2 (en) | Attenuated Mycoplasma Gallicepticum strain | |
JP2000504726A (en) | Vaccine of Salmonella Typhimurium | |
Heidary et al. | Colonization and investigation of vibrio cholera recombination protein in e-coli | |
Cassell et al. | Immune response of pathogen-free mice inoculated intranasally with Mycoplasma pulmonis | |
JP5087604B2 (en) | Novel bacteria responsible for poultry disease and vaccines derived therefrom | |
CN106928373B (en) | Multi-epitope mucosal vaccine for mycoplasma hyopneumoniae | |
KR100628657B1 (en) | Bacteria attenuated by a non-reverting mutation in each of the AroC, OmpF and OmpC genes, useful as vaccines | |
US9597386B2 (en) | Outer membrane proteins of Histophilus somni and methods thereof | |
CN108774628B (en) | Escherichia coli engineering bacterium for synthesizing neonatal meningitis escherichia coli glycoprotein conjugate vaccine and application | |
CN113943714A (en) | Cat calicivirus strain and application thereof | |
CN105154377B (en) | Recombinant salmonella pullorum, preparation method and application | |
Jamal Hussaini et al. | Recombinant Clone ABA392 protects laboratory animals from Pasteurella multocida Serotype B | |
JPS6122A (en) | Chemically ruled vaccine for urinary tract infection | |
JP2012502898A (en) | Non-typeable Haemophilus influenzae vaccine and uses thereof | |
CN114990042A (en) | Salmonella containing lipopolysaccharide capable of expressing shigella dysenteriae O antigen, preparation method and application thereof | |
CN111100192A (en) | Porcine infectious actinobacillus pleuropneumoniae pilus ApfA antigen and immune characteristic and application thereof | |
CN101979504A (en) | Yersinia pestis ghost, preparation method thereof, and application of Yersinia pestis ghost serving as vaccine | |
Jaglic et al. | Characterisation of Pasteurella multocida isolated from rabbits in the Czech Republic | |
Zheng et al. | Construction of a novel Shigella live-vector strain co-expressing CS3 and LTB/STm of enterotoxigenic E. coli | |
KR102264989B1 (en) | Endotoxin-reduced Escherichia coli trivalent inactivated vaccine composition for preventing Avian colibacillosis | |
Han et al. | Evaluation of the protective efficacy of six major immunogenic proteins of Mycoplasma Synoviae | |
US5861163A (en) | Process for preparing a vaccine against pseudomonas aeruginosa using attenuated strains | |
Lu et al. | Hyperimmune serum from rabbits immunized with potassium thiocyanate extract of Pasteurella multocida protects against homologous challenge | |
CN116462743B (en) | Acinetobacter baumannii translation elongation factor recombinant protein, preparation method and application |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination |