CN113151078A - Salmonella enteritidis outer membrane vesicle, preparation method and application thereof as avian salmonellosis subunit vaccine - Google Patents
Salmonella enteritidis outer membrane vesicle, preparation method and application thereof as avian salmonellosis subunit vaccine Download PDFInfo
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Abstract
The invention belongs to the field of microorganisms, and particularly relates to a salmonella enteritidis outer membrane vesicle, a preparation method and application thereof as a subunit vaccine for salmonellosis of fowl enteritis. The invention adopts LB liquid culture medium to culture salmonella enteritidis, and prepares outer membrane vesicles through centrifugation, filtration by a 0.22 mu m filter, ultrafiltration concentration and ultracentrifugation. The salmonella enteritidis outer membrane vesicle prepared by the invention is applied to chickens as a subunit vaccine to resist infection of salmonella enteritidis. The experimental result shows that the outer membrane vesicle has better immune protection effect and can be developed into subunit vaccine of avian salmonellosis.
Description
Technical Field
The invention relates to the field of microorganisms, and in particular relates to a salmonella enteritidis outer membrane vesicle, a preparation method and application thereof as a subunit vaccine for salmonellosis of fowl enteritis.
Background
Salmonella is a common zoonosis pathogen, and the serotypes are numerous and widely distributed, and can cause various diseases of human and animals. The salmonella enteritidis belongs to the serotype of the salmonella not typhosa, has a wide host spectrum, and is the serotype with the highest separation rate in poultry. Infection of poultry can lead to severe intestinal inflammation, and even death of some immunocompromised individuals. And the infection of adult chickens can cause the injury of the reproductive system. Human beings who eat poultry products contaminated with salmonella enteritidis can cause self-limiting gastroenteritis.
The prevention and control of salmonella is mainly realized by adopting antibiotics in poultry breeding enterprises in China. This is accompanied by an increase in resistance to salmonella and the emergence of multiple resistant strains. In recent years, vaccines have become one of the important tools against salmonellae. At present, commercial salmonella vaccines are mainly attenuated live vaccines and inactivated vaccines, and subunit vaccines are ideal vaccines due to good safety and no toxicity.
Disclosure of Invention
In order to solve the technical problems, the invention aims to provide an outer membrane vesicle of salmonella enteritidis, a preparation method and an application thereof as a subunit vaccine of salmonellosis enteritidis of poultry, wherein the outer membrane vesicle can effectively stimulate cellular immune response in chicken and can provide a very good immune protection effect for salmonella enteritidis, shows the potential of the outer membrane vesicle as the subunit vaccine and provides a foundation for the application of the outer membrane vesicle of salmonella enteritidis.
The technical scheme provided by the invention is as follows:
a Salmonella enteritidis outer membrane vesicle is prepared from in vitro cultured Salmonella enteritidis.
A preparation method of outer membrane vesicles of salmonella enteritidis comprises the following steps:
s1, culturing salmonella enteritidis in vitro to the logarithmic growth phase of bacteria, and then centrifuging, filtering and concentrating the concentrated solution of the culture supernatant without cells;
and S2, performing ultracentrifugation on the culture supernatant to obtain the salmonella enteritidis outer membrane vesicles.
In the preparation method as described above, preferably, in step S1, the culture medium for culturing salmonella enteritidis is LB liquid medium.
As in the above preparation method, preferably, in step S1, the centrifugation step includes: centrifuging at 4 deg.C and 1000 Xg for 10min, discarding precipitate, filtering with 0.22 μm disposable filter, and concentrating with ultrafiltration tube with molecular weight cutoff of 100 KDa.
In the preparation method as described above, preferably, in step S1, the ultrafiltration tube concentration step is: centrifuge at 3000 Xg for 5min at 4 deg.C, and concentrate to one sixth of the original volume.
As the preparation method, preferably, in step S2, the ultracentrifugation is to ultracentrifuge the cell-free culture supernatant at 4 ℃ and 39000 Xg for 1h, discard the supernatant, and resuspend the pellet in PBS buffer; and ultracentrifuging at 39000 Xg for 1h at 4 ℃ again as above, discarding supernatant, and resuspending the precipitate in PBS buffer solution to obtain the outer membrane vesicle for preparing salmonella enteritidis.
The outer membrane vesicle of the salmonella enteritidis obtained by the preparation method is applied to the preparation of subunit vaccine of salmonellosis of avian enteritis.
The salmonella enteritidis outer membrane vesicle obtained by the preparation method is used as a subunit vaccine of avian salmonellosis.
The invention further discloses a material for developing the outer membrane vesicle of the salmonella enteritidis into a subunit vaccine of the salmonellosis of avian enteritis.
The invention also provides a detection method of the subunit vaccine of salmonellosis of chicken enteritis, which adopts qRT-PCR to detect the relative expression quantity of the cell factors and compares the relative expression quantity with the relative expression quantity of the cell factors in the non-immunized PBS group.
Further, the qRT-PCR primers are as follows:
has the advantages that:
salmonella enteritidis is an important pathogenic bacterium for zoonosis, and can not only cause animal diseases, but also cause human acute gastroenteritis and other diseases by transferring polluted food to human. Vaccination is one of the effective measures for preventing and controlling salmonella infection at present, the outer membrane component contained in the outer membrane vesicle can stimulate the organism to generate adaptive immune memory, and the contained LPS can be used as a self-adjuvant and has certain safety when being used as a non-replicative vaccine. These factors have led to the potential of outer membrane vesicles for development as subunit vaccines. The outer membrane vesicles of the salmonella enteritidis are cultured by an LB (lysogeny broth) culture medium, centrifuged, filtered, ultrafiltered and ultracentrifuged, and are used as subunit vaccines to be inoculated to the Hailan white laying hens. The technical scheme of the invention provides a method for extracting outer membrane vesicles of salmonella, which is simple and easy to operate; the invention adopts the ultrafiltration tube concentration technology, the ultrafiltration tube concentration is more convenient to operate than the existing density gradient centrifugation method, and the purer outer membrane vesicle extract is obtained.
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FIG. 1 shows the SDS-PAGE results of Salmonella enteritidis C50041 OMV;
FIG. 2 is a scanning electron-projection microscope observation result of OMV of Salmonella enteritidis;
FIG. 3 is an analysis of cytokine expression in spleen after OMV immunization;
FIG. 4 is a survival curve of a flock after challenge;
FIG. 5 is the weight change of the chicken flocks after immunization;
FIG. 6 is the weight change of the chicken flock after challenge;
FIG. 7 shows the distribution and colonization of post challenge bacteria in chicken liver, spleen, ileum and cecum.
Detailed Description
Before the present embodiments are further described, it is to be understood that the scope of the invention is not limited to the particular embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments, and is not intended to limit the scope of the present invention; in the description and claims of the present application, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.
When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected unless the invention otherwise indicated. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention.
Example 1 preparation of outer Membrane vesicles of Salmonella enteritidis
1. Recovering the strain: streaking and inoculating a salmonella enteritidis C50041 strain stored at-70 ℃ to an LB (lysogeny broth) plate without antibiotics, culturing for 12h at 37 ℃, selecting a single colony on the LB plate, inoculating to an LB liquid culture medium containing 4mL, and performing shake culture at 37 ℃ and 180rpm for 8 h;
wherein the LB liquid culture medium is: weighing 5g of yeast extract, 10g of tryptone and 10g of NaCl, dissolving in 1000mL of distilled water, and autoclaving at 121 ℃ for 15 min; the LB solid culture medium is prepared by adding 15g agar powder into 1000ml LB liquid culture medium, autoclaving at 121 deg.C for 15min, and pouring into a plate when the temperature is reduced;
2. and (3) amplification culture: inoculating the Salmonella enteritidis bacterial liquid cultured for 8h into 400mL of LB liquid according to the proportion of 1:100, and performing shake culture at 37 ℃ and 180rpm until OD6000.4-0.6;
3. and (3) filtering and concentrating: centrifuging the Salmonella enteritidis culture solution at 4 deg.C and 1000 Xg for 10min, filtering the supernatant with 0.22 μm filter, concentrating the obtained cell-free supernatant with ultrafiltration tube with molecular weight cutoff of 100KDa, centrifuging at 4 deg.C and 4000rpm for 4min until the volume is concentrated to one sixth of the original volume;
collection of OMVs: ultracentrifugation is carried out on the concentrated liquid, the ultracentrifugation is carried out for 1h at the temperature of 4 ℃ and 39000 Xg, the supernatant is discarded, and the precipitate is resuspended by PBS buffer solution; and ultracentrifuging at 39000 Xg for 1h at 4 ℃ again as above, discarding supernatant, and resuspending the precipitate in PBS buffer solution to obtain the outer membrane vesicle for preparing salmonella enteritidis.
And (3) carrying out negative dyeing on the collected salmonella enteritidis outer membrane vesicles with uranyl acetate, and observing the outer membrane vesicle morphology by using a transmission electron microscope. The collected outer membrane vesicles were then subjected to SDS-PAGE validation and the BCA method was used to calculate the concentration of outer membrane vesicles from Salmonella enteritidis.
The OMV has a major band around 60kDa as shown in FIGS. 1 and 2, and the structure of OMV can be observed under transmission electron microscope, and the size is 20-200 nm.
Example 2: immunological effect analysis of outer membrane vesicle of salmonella enteritidis
2.1 Synthesis of Chicken cytokine primers
Synthesizing a probe method qRT-PCR primer for detecting the expression level of the chicken cytokine. The primers were synthesized by Bao bioengineering (Dalian) Co., Ltd.
TABLE 1 Chicken cytokine qRT-PCR primers
2.2 Total RNA extraction and cDNA Synthesis of Chicken spleen
100 helan white laying hens were randomly divided into 2 groups of 50 eggs. A blank control group was set up without immunization. Vaccinating 7-day-old Hainan white layer chicken with vaccine and PBS by intramuscular injection, performing booster immunization at 14-day-old, collecting spleen 7 and 14 days after immunization, respectively, soaking tissue sample in RNA protective agent, and storing in-70 deg.C refrigerator.
Tissue samples stored in a-70 ℃ freezer were removed and total spleen RNA was extracted as required by the QIAGEN Total RNA miniprep kit. 3 samples were run at each time point and total RNA concentration was measured using One-drop.
The extracted RNA was purified according to PrimeScriptTMRT reagent kit instructions, reverse transcription Synthesis of cDNA (tables 10-11), the reaction system is as follows:
table 2: gDNA removal system
The reaction conditions are as follows: 42 ℃ for 2min
TABLE 3 Synthesis System of cDNA
The reaction conditions were 37 ℃ for 15min and 85 ℃ for 5 s. The reverse transcribed cDNA was stored at-20 ℃ for further use.
2.3qRT-PCR detection of cytokines
And (3) diluting the cDNA into a proper concentration, and performing fluorescent quantitative PCR (polymerase chain reaction) by using the cDNA as a template to detect the expression level of each cytokine in the spleen of the chicken. The PCR reaction was as follows (20. mu.L):
TABLE 4qRT-PCR reaction System
The fluorescent quantitative PCR reaction system is as follows: 30s at 95 ℃; 5s at 95 ℃ and 34s at 60 ℃ for 40 cycles.
Spleen tissues of the chicken flocks in the immunized group and the chicken flocks in the non-immunized group are taken out 7 and 14 days after the immunization, and the relative expression amount of the cell factors is detected by qRT-PCR and is compared with the non-immunized PBS group. FIG. 3 shows that the IL-4 expression level was up-regulated within two weeks after immunization, and that the expression level in the immunized group was significantly higher than that in the control group (P < 0.01) after 14 days, indicating an enhanced humoral immune response. IL-6 has little difference in the initial stage of immunization (P > 0.05), and the expression level is obviously increased after 14 days of immunization (P < 0.001). IL-6 plays a role in sustaining infection resistance and can effectively promote antibody secretion. The IFN-gamma expression level of the immune group is obviously higher than that of the control group (P is less than 0.001), which shows that the cellular immune response is increased, and the intracellular parasitic bacteria such as salmonella are favorably eliminated. And IL-1 beta is high in expression amount in the early stage of immunity, and the expression amount is obviously reduced after 14 days.
Example 3: immunoprotection evaluation of Salmonella enteritidis outer membrane vesicles
Selecting single colony of CZ14-1, inoculating into LB liquid culture medium containing 4mL, shake culturing at 37 deg.C and 180rpm for 12h, inoculating into LB liquid containing 400mL at a ratio of 1:100, culturing at 37 deg.C and 180rpm for 8h, washing bacteria with sterile PBS for 3 times, adjusting bacteria concentration to 2 × 109CFU/mL；
100 helan white laying hens were randomly divided into 2 groups of 55 eggs. A blank control group was set up without immunization. Each group was further divided into 20 observation groups and 35 necropsy groups, and 7-day-old Hainan white layer chickens were inoculated with the vaccine and PBS by intramuscular injection, and booster immunization was carried out at 14-day-old, and the immunization dose was 60. mu.g/feather. Changes in body weight were measured in each group of chickens within 3 consecutive weeks after immunization. Challenge experiment was carried out on the chickens 1 week after the booster immunization, and wild strain CZ14-1 was inoculated by intramuscular injection at a dose of 2X 109CFU/mL. Dissecting the chickens in each group on days 1, 3, 7, 14 and 21 after challenge, taking the livers, spleens, ileums and cececa aseptically, weighing, adding 1mL of sterile PBS, thoroughly homogenizing, and taking 100 μ L of homogenized stock solution for continuous dilution in multiple proportion. Selecting 3 appropriate dilutions, collecting 10 μ L drop plate (BG solid plate) for each dilution, repeating for 3 times, standing in 37 deg.C constant temperature incubator for 12 hr, counting bacteria, and counting bacteriaBacterial load in the organs of the heart.
The survival curves of the chickens after challenge are shown in fig. 4, the PBS control group died and continuously died from day 2 of challenge, while the immune group died only from day 6 and the survival rate was 95%, and the mortality rate after the PBS group was 70%, indicating that OMV as a subunit vaccine provides good immune protection against challenge of salmonella enteritidis (table 5).
TABLE 5 immunoprotective efficacy of OMVs
The effect of OMV immunity on chicken growth capacity was assessed by measuring the change in body weight of the two groups. As shown in fig. 5 and 6, the growth of the immunized group chickens did not change significantly compared to the weight of the non-immunized group chickens, and the weight of the immunized group chickens after challenge was significantly higher than that of the non-immunized group at 7, 10, and 13 days.
The experimental results of in vivo colonization of bacteria after challenge are shown in fig. 7, the bacterial load in the liver of the PBS group is obviously greater than that of the OMV group on the first day after challenge, and reaches the maximum value after 3 days, but only one bacteria can be obtained in the OMV immune group on the third day, and the bacteria are completely eliminated in the liver after 7 days. The OMV immunization group had significantly lower numbers of bacteria in the spleen than the PBS group on challenge days 3 and 7. The bacterial numbers reached a maximum in the ileum in the PBS group on day three, whereas the bacterial load in the OMV immunized group was significantly lower than in the PBS group on days 3, 7, and the bacteria were completely cleared from the ileum on day 14. The OMV immunized group in the cecum had a small number of bacteria compared to the PBS group on day 7, and there was a significant difference.
The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.
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Claims (10)
1. The salmonella enteritidis outer membrane vesicle is characterized in that the salmonella enteritidis outer membrane vesicle is prepared by taking in-vitro cultured salmonella enteritidis as a raw material.
2. The method for preparing the salmonella enteritidis outer membrane vesicle of claim 1, comprising the following steps:
culturing salmonella enteritidis in vitro to logarithmic phase of bacteria, centrifuging, filtering, and concentrating cell-free culture supernatant concentrate;
and (4) performing ultracentrifugation on the culture supernatant to obtain the salmonella enteritidis outer membrane vesicles.
3. The method according to claim 2, wherein the culture medium for culturing Salmonella enteritidis is LB liquid medium.
4. The preparation method according to claim 2, wherein the centrifugation comprises the following specific steps: centrifuging at 4 deg.C for 10min at 1000 Xg, discarding precipitate, filtering with 0.22 μm disposable filter, and concentrating with ultrafiltration tube with molecular weight cutoff of 100 KDa.
5. The method of claim 4, wherein the step of concentrating the ultrafiltration tube comprises: centrifuge at 4 deg.C 3000 Xg for 5min, and concentrate to one sixth of the original volume.
6. The method of claim 1, wherein the ultracentrifugation is ultracentrifugation of cell-free culture supernatant at 39000 Xg for 1 hour at 4 ℃, discarding the supernatant, and resuspending the pellet in PBS buffer; and ultracentrifuging at 39000 Xg for 1h at 4 ℃ again as above, discarding supernatant, and resuspending the precipitate in PBS buffer solution to obtain the outer membrane vesicle for preparing salmonella enteritidis.
7. Use of the outer membrane vesicles of Salmonella enteritidis according to claim 1 for the preparation of a vaccine for salmonellosis enteritidis in avians.
8. A chicken salmonellosis enteritis subunit vaccine comprising the salmonella enteritidis outer membrane vesicle of claim 1.
9. The method for detecting the subunit vaccine of the chicken salmonellosis enteritis as claimed in claim 8, wherein qRT-PCR is adopted to detect the relative expression quantity of the cell factors, and the relative expression quantity is compared with the non-immunized PBS group.
10. The method for detecting the subunit vaccine of the salmonellosis enteritis in the chicken as claimed in claim 9, wherein the qRT-PCR primers are as follows:
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| CN114921398A (en) * | 2022-06-17 | 2022-08-19 | 南昌大学 | Method for preparing outer membrane vesicles of salmonella and application thereof |
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| CN114921398A (en) * | 2022-06-17 | 2022-08-19 | 南昌大学 | Method for preparing outer membrane vesicles of salmonella and application thereof |
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