CN112546210A - Preparation method and application of salmonella inactivated vaccine - Google Patents
Preparation method and application of salmonella inactivated vaccine Download PDFInfo
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Abstract
The invention discloses a preparation method and application of an inactivated vaccine of a salmonella gene mutant strain, wherein a salmonella pullorum pagC gene deletion mutant strain delta PagC is prepared and obtained by a lambda-Red system. Inoculating overnight cultured delta PagC bacterial liquid into 50ml of liquid LB culture medium at a ratio of 1:100, and performing shaking culture at 37 ℃ and 180rpm until the OD value is about 0.8; adding formaldehyde solution with final concentration of 0.4%, further performing shaking culture for 16h, centrifuging at 4 deg.C and 5000rpm for 10min, collecting thallus, washing with sterile PBS buffer solution, and resuspending for 3 times to obtain stock solution. The adjusted dose is mixed with adjuvant 1:1, and then the mixture is fully emulsified and inoculated to animals. By preparing the salmonella inactivated vaccine, the vaccine strain has no adverse reaction after being inoculated to a mouse, and the mouse can be protected from infection of salmonella pullorum CVCC1800, salmonella enteritidis ATCC19585 and salmonella typhimurium CVCC 542. The vaccine can be used together with a PagC protein-based salmonella antibody indirect ELISA detection kit, so that the artificial immunity and the natural infection differential diagnosis of salmonella are realized, and good DIVA characteristics are shown.
Description
Technical Field
The invention relates to the technical field of veterinary biological products, in particular to a preparation method of a salmonella inactivated vaccine and application of the salmonella inactivated vaccine.
Background
Salmonella is an important food-borne pathogenic microorganism, and the pathogenic serotypes are more than 2500, most of the pathogenic serotypes have pathogenicity, cause diarrhea, enteritis, septicemia and abortion of dams, and have great significance for livestock and poultry breeding. Diseased and bacteria-bearing animals are the main infectious source of the disease, and can not only continuously expel toxin in the population, but also be vertically transmitted to the next generation. The traditional method for positively purifying the population according to clinical symptoms cannot eliminate the recessive infection population in time, and the purification work of salmonella needs to adopt a scheme of matching vaccine immunization and serological detection, so that the development of a DIVA (differentiation of Infected and preserved animals) vaccine is urgently needed, and clinical infection and artificial immunization can be distinguished in the serological detection.
The PagC protein is a highly conserved specific protein in the Salmonella, and the homology in the genus is as high as more than 95%. The pagC gene for coding the protein is widely distributed in various avian and murine salmonella serotypes, and is an ideal differential diagnosis target point of a marker vaccine.
Based on the consideration, the inventor develops a salmonella inactivated vaccine on the basis of a salmonella pullorum delta PagC mutant strain obtained by a lambda-Red homologous recombination method in a previous laboratory, and evaluates the immune effect and DIVA characteristics of the salmonella inactivated vaccine.
Disclosure of Invention
The invention aims to provide a preparation method and application of a salmonella gene mutant inactivated vaccine, wherein a vaccine strain has no adverse reaction after being inoculated into a mouse, and can protect the mouse from being infected by salmonella pullorum CVCC1800, salmonella enteritidis ATCC19585 and salmonella typhimurium CVCC 542.
In order to achieve the purpose, the invention provides the following technical scheme: a preparation method of a gene mutant strain salmonella inactivated vaccine comprises the following steps:
step S1, preparing and obtaining a salmonella pullorum genetic deletion mutant strain delta PagC by using a lambda-Red system;
step S2, streaking the LB plate of the deletion strain, selecting a single colony, transferring the single colony to a fresh LB culture medium for overnight culture;
step S3, inoculating overnight-cultured Salmonella pullorum delta PagC bacterial liquid into 50ml of liquid LB culture medium at a ratio of 1:100, and carrying out shaking culture at 37 ℃ and 180rpm until the OD value is about 0.8;
step S4, adding a formaldehyde solution with the final concentration of 0.4%, continuing to shake and culture for 16h, centrifuging at 4 ℃ and 5000rpm for 10min, collecting thalli, washing with sterile PBS buffer solution, and resuspending for 3 times to obtain a stock solution;
step S5, adjusting the stock solution for multiple times to dilute 10 times, and measuring OD value to 1 (the concentration of the stock solution is about 10 at this time)9CFU/ml)；
Step S6, concentrating the stock solution by one time, mixing the concentrated stock solution with an adjuvant in a ratio of 1:1, adding the mixture into a 2ml sterilized homogenate tube (containing steel balls), emulsifying the mixture by using a tissue homogenate instrument, taking 100 mu l of the mixture after complete emulsification, uniformly coating the mixture on an LB solid culture medium, culturing the mixture at 37 ℃ for 16 hours, and observing aseptic colonies growing out, thereby successfully preparing the salmonella pullorum CVCC1800 delta PagC formaldehyde inactivated vaccine.
An application of a salmonella inactivated vaccine comprises an immune protection test of a salmonella pullorum CVCC1800 delta PagC formaldehyde inactivated vaccine and DIVA characteristic evaluation of the salmonella pullorum CVCC1800 delta PagC formaldehyde inactivated vaccine.
Further, the source of the wild salmonella pullorum CVCC1800 in step S1 is the institute of veterinary medicine in china.
Further, the adjuvant type in step S6 is ISA 206.
Further, an immune protection test of the salmonella pullorum CVCC1800 delta PagC formaldehyde inactivated vaccine;
5.1 immunogenicity assay: serum potency detection
The prepared CVCC1800 delta PagC vaccine strain is injected into ICR mice of 3-4 weeks at multiple subcutaneous points, and the injection dose is 1x108CFU/one, 4 per group, and simultaneously setting up a PBS-injected negative control group, wherein the injection dose is 100 μ l each; the immunization is carried out for 3 times, and the immunization interval is 14 days each time; collecting blood from inferior jaw vein before first immunization and 13 days after each immunization, separating serum for next day, performing indirect ELISA test, and collecting each serumThe serum titer of the sample is detected after the sample is diluted by 1: 400;
along with the increase of the immunization times, the antibody titer of the vaccine group mice gradually rises, reaches a peak after the three-immunization, and has very obvious difference with a PBS control group;
5.2 protective efficacy test
Carrying out abdominal cavity challenge on the mice 14 days after the third immunization, observing survival states every day after the challenge, drawing survival curves and calculating immune protection rate;
5.3 pathological tissue changes of the cecum
On 14 days after the third immunization of the vaccine, the three strains of salmonella with different serotypes are used for virus challenge; the mice are killed in sequence according to the clinical symptoms, and the pathological changes of caecum tissues are observed;
pathological changes of the caecum: after three different serovars of salmonella infect mice, the cells of the caecal mucosa of the immune group of the mice are slightly necrotic and shed; the caecal mucosa of the corresponding PBS negative control group mice is seriously damaged, and the structure can not be clearly distinguished.
Furthermore, the sources of wild type salmonella enteritidis CVCC1800 and salmonella typhimurium CVCC542 are China veterinary microbial strain preservation management center.
Further, evaluating the DIVA characteristics of the salmonella pullorum CVCC1800 delta PagC formaldehyde inactivated vaccine;
and (3) coating an ELISA (enzyme-linked immunosorbent assay) ELISA plate with PagC antigen, and evaluating the differential diagnosis effect of the salmonella delta PagC vaccine. Antigen coating concentration was 2 μ g/ml, serum dilution fold was 1: 400. the positive serum is obtained by separating a salmonella pullorum CVCC1800 wild strain after a plurality of times of abdominal cavity infection of mice.
Compared with the prior art, the invention has the beneficial effects that:
according to the invention, by preparing the salmonella inactivated vaccine, no adverse reaction is generated after the vaccine strain is inoculated to a mouse, the mouse can be protected from infection of salmonella pullorum CVCC1800, salmonella enteritidis ATCC19585 and salmonella typhimurium CVCC542, the vaccine can be combined with a salmonella antibody indirect ELISA detection kit based on PagC protein, the artificial immunity and natural infection differential diagnosis of salmonella are realized, and good DIVA characteristics are shown.
Drawings
FIG. 1 is a schematic diagram showing the toxicity-counteracting dose and immunoprotection rate of three different serotypes of Salmonella of the present invention;
FIG. 2 is a diagram showing the results of the antibody titer detection according to the present invention;
FIG. 3 is a schematic illustration of the survival curve of mice infected with Salmonella pullorum CVCC1800 after immunization with the vaccine of the present invention;
FIG. 4 is a graph showing the survival curves of mice infected with Salmonella enteritidis ATCC19585 after immunization with the vaccine of the present invention;
FIG. 5 is a graphical representation of the survival curve of mice infected with Salmonella typhimurium CVCC542 after immunization with the vaccine of the present invention;
fig. 6 is a diagram showing pathological changes of the cecum tissue according to the present invention.
FIG. 7 is a graphical representation of the differential diagnostic efficacy of the vaccine evaluated based on the PagC antigen of the present invention;
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Referring to fig. 1-7, the present invention provides a technical solution: a preparation method of a salmonella inactivated vaccine comprises the following steps:
step S1, preparing and obtaining a salmonella pullorum genetic deletion mutant strain delta PagC by using a lambda-Red system;
step S2, streaking the LB plate of the deletion strain, selecting a single colony, transferring the single colony to a fresh LB culture medium for overnight culture;
step S3, inoculating overnight-cultured Salmonella pullorum delta PagC bacterial liquid into 50ml of liquid LB culture medium at a ratio of 1:100, and carrying out shaking culture at 37 ℃ and 180rpm until the OD value is about 0.8;
step S4, adding a formaldehyde solution with the final concentration of 0.4%, continuing to shake and culture for 16h, centrifuging at 4 ℃ and 5000rpm for 10min, collecting thalli, washing with sterile PBS buffer solution, and resuspending for 3 times to obtain a stock solution;
step S5, adjusting the stock solution for multiple times to dilute 10 times, and measuring OD value to 1 (the concentration of the stock solution is about 10 at this time)9CFU/ml)；
Step S6, concentrating the stock solution by one time, mixing the concentrated stock solution with an adjuvant in a ratio of 1:1, adding the mixture into a 2ml sterilized homogenate tube (containing steel balls), emulsifying the mixture by using a tissue homogenate instrument, taking 100 mu l of the mixture after complete emulsification, uniformly coating the mixture on an LB solid culture medium, culturing the mixture at 37 ℃ for 16 hours, and observing aseptic colonies growing out, thereby successfully preparing the salmonella pullorum CVCC1800 delta PagC formaldehyde inactivated vaccine.
An application of a salmonella inactivated vaccine comprises an immune protection test of a salmonella pullorum CVCC1800 delta PagC formaldehyde inactivated vaccine and DIVA characteristic evaluation of the salmonella pullorum CVCC1800 delta PagC formaldehyde inactivated vaccine.
The source of the wild salmonella pullorum CVCC1800 in the step S1 is the China institute of veterinary medicine;
the adjuvant type in step S6 in the present invention is ISA 206;
the invention relates to an immune protection test of salmonella pullorum CVCC1800 delta PagC formaldehyde inactivated vaccine;
5.1 immunogenicity assay: serum potency detection
The CVCC 1800. delta. PagC vaccine strain prepared in example 1 was injected subcutaneously at multiple sites into 3-4 week-old ICR mice at a dose of 1X108CFU/mouse, 4 mice per group, and a negative control group injected with PBS at a dose of 100. mu.l each was established. The immunization is carried out for 3 times, and the immunization interval is 14 days each time; collecting blood from inferior jaw vein before first immunization and 13 days after each immunization, separating serum for the next day, performing indirect ELISA test, diluting each serum sample by 1:400, and detecting the serum titer. Specific methods can be found in the literature: xutuo, gaogongensis, Gongsen, Luxiajuan, Spodoptera glauca and Liuzhou, and research on detection of serum antibody of fowl cholera by ELISA (enzyme-Linked immunosorbent assay) using holomycete antigen dry coating method [ J]Chinese poultry, 2009,31(5): 31-33.
The results are shown in FIG. 2: with the increase of the immunization times, the antibody titer of the mice in the vaccine group gradually rises, reaches a peak after the third immunization, and is very obviously different from that of the PBS control group.
5.2 protective efficacy test
The mice were subjected to abdominal cavity challenge on day 14 after the third immunization (the challenge dose is shown in fig. 1), and the survival status was observed every day after challenge, and a survival curve was plotted and the immune protection rate was calculated. (the survival curves are shown in FIG. 3, FIG. 4 and FIG. 5)
5.3 pathological tissue changes of the cecum
On day 14 after the third immunization of the vaccine, the three strains of salmonella of different serotypes are used for virus challenge. Mice were sacrificed in succession according to their clinical symptoms and observed for pathological changes in the cecal tissues.
The pathological changes of the caecum are shown in FIG. 6 (the pictures A, C and E are pathological sections of caecum after the immunized mice are infected with Salmonella pullorum, Salmonella enteritidis and Salmonella typhimurium, respectively; B, D and F are corresponding PBS control groups): after three different serovars of salmonella infect mice, the cells of the caecal mucosa of the immune group of the mice are slightly necrotic and shed; the caecal mucosa of the corresponding PBS negative control group mouse is seriously damaged, and the structure can not be clearly distinguished;
the sources of wild type salmonella enteritidis CVCC1800 and salmonella typhimurium CVCC542 are China veterinary microorganism strain preservation management center;
according to the invention, the ELISA (enzyme-linked immunosorbent assay) ELISA plate is coated with PagC antigen, and the differential diagnosis effect of the salmonella delta PagC vaccine is evaluated. Antigen coating concentration was 2 μ g/ml, serum dilution fold was 1: 400. the positive serum is obtained by separating a salmonella pullorum CVCC1800 wild strain after a plurality of times of abdominal cavity infection of mice. Specific process references: establishment of indirect ELISA antibody detection method for salmonella and development of recombinant suipoxvirus live vector vaccine [ D ]](ii) a 2016 (differential diagnosis effect is shown in FIG. 7, solid line corresponds to positive serum OD of wild type Salmonella pullorum CVCC1800450nmReading, dotted line corresponds to negative serum OD450nmReading the values and attaching a specific value table).
It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (7)
1. The preparation method of the inactivated vaccine for salmonella is characterized by comprising the following steps:
step S1, preparing and obtaining a salmonella pullorum genetic deletion mutant strain delta PagC by using a lambda-Red system;
step S2, streaking the LB plate of the deletion strain, selecting a single colony, transferring the single colony to a fresh LB culture medium for overnight culture;
step S3, inoculating overnight-cultured Salmonella pullorum delta PagC bacterial liquid into 50ml of liquid LB culture medium at a ratio of 1:100, and carrying out shaking culture at 37 ℃ and 180rpm until the OD value is about 0.8;
step S4, adding a formaldehyde solution with the final concentration of 0.4%, continuing to shake and culture for 16h, centrifuging at 4 ℃ and 5000rpm for 10min, collecting thalli, washing with sterile PBS buffer solution, and resuspending for 3 times to obtain a stock solution;
step S5, adjusting the stock solution for multiple times to dilute 10 times, and measuring OD value to 1 (the concentration of the stock solution is about 10 at this time)9CFU/ml)；
Step S6, concentrating the stock solution by one time, mixing the concentrated stock solution with an adjuvant in a ratio of 1:1, adding the mixture into a 2ml sterilized homogenate tube (containing steel balls), emulsifying the mixture by using a tissue homogenate instrument, taking 100 mu l of the mixture after complete emulsification, uniformly coating the mixture on an LB solid culture medium, culturing the mixture at 37 ℃ for 16 hours, and observing aseptic colonies growing out, thereby successfully preparing the salmonella pullorum CVCC1800 delta PagC formaldehyde inactivated vaccine.
2. The method for preparing the inactivated vaccine for salmonella as claimed in claim 1, wherein the inactivated vaccine for salmonella comprises: the source of the wild salmonella pullorum CVCC1800 in the step S1 is the institute of veterinary medicine in China.
3. The method for preparing the inactivated vaccine for salmonella as claimed in claim 1, wherein the inactivated vaccine for salmonella comprises: the adjuvant type in step S6 is ISA 206.
4. The application of the inactivated vaccine for the salmonella is characterized in that: comprises an immune protection test of the salmonella pullorum CVCC1800 delta PagC formaldehyde inactivated vaccine and DIVA characteristic evaluation of the salmonella pullorum CVCC1800 delta PagC formaldehyde inactivated vaccine.
5. The inactivated vaccine for Salmonella according to claim 4, wherein:
immunoprotection test of salmonella pullorum CVCC1800 delta PagC formaldehyde inactivated vaccine;
5.1 immunogenicity assay: serum potency detection
The prepared CVCC1800 delta PagC vaccine strain is injected into ICR mice of 3-4 weeks at multiple subcutaneous points, and the injection dose is 1x108CFU/one, 4 per group, and simultaneously setting up a PBS-injected negative control group, wherein the injection dose is 100 μ l each; the immunization is carried out for 3 times, and the immunization interval is 14 days each time; collecting blood from inferior jaw vein before first immunization and 13 days after each immunization, separating serum for the next day, performing indirect ELISA test, diluting each serum sample by 1:400, and detecting the serum titer;
along with the increase of the immunization times, the antibody titer of the vaccine group mice gradually rises, reaches a peak after the three-immunization, and has very obvious difference with a PBS control group;
5.2 protective efficacy test
Carrying out abdominal cavity challenge on the mice 14 days after the third immunization, observing survival states every day after the challenge, drawing survival curves and calculating immune protection rate;
5.3 pathological tissue changes of the cecum
On 14 days after the third immunization of the vaccine, the three strains of salmonella with different serotypes are used for virus challenge; the mice are killed in sequence according to the clinical symptoms, and the pathological changes of caecum tissues are observed;
pathological changes of the caecum: after three different serovars of salmonella infect mice, the cells of the caecal mucosa of the immune group of the mice are slightly necrotic and shed; the caecal mucosa of the corresponding PBS negative control group mice is seriously damaged, and the structure can not be clearly distinguished.
6. The inactivated vaccine for Salmonella according to claim 5, wherein: the sources of wild type salmonella enteritidis CVCC1800 and salmonella typhimurium CVCC542 are China veterinary culture collection and management center.
7. The inactivated vaccine for Salmonella according to claim 4, wherein:
evaluating DIVA characteristics of salmonella pullorum CVCC1800 delta PagC formaldehyde inactivated vaccine;
and (3) coating an ELISA (enzyme-linked immunosorbent assay) ELISA plate with PagC antigen, and evaluating the differential diagnosis effect of the salmonella delta PagC vaccine. Antigen coating concentration was 2 μ g/ml, serum dilution fold was 1: 400. the positive serum is obtained by separating a salmonella pullorum CVCC1800 wild strain after a plurality of times of abdominal cavity infection of mice.
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