CN113354742A

CN113354742A - Brucella gene engineering subunit vaccine and preparation method and application thereof

Info

Publication number: CN113354742A
Application number: CN202110505138.7A
Authority: CN
Inventors: 曹政; 付文贵; 付利芝; 何丹; 鲍伟; 穆德龙; 张月; 龙小玲
Original assignee: Chongqing Tangcheng Animal Husbandry Technology Co ltd; Chongqing Academy of Animal Sciences
Current assignee: Chongqing Tangcheng Animal Husbandry Technology Co ltd; Chongqing Academy of Animal Sciences
Priority date: 2021-05-10
Filing date: 2021-05-10
Publication date: 2021-09-07

Abstract

The invention relates to a brucella gene engineering subunit vaccine and a preparation method and application thereof, in particular to a tandem epitope protein of outer membrane protein of procaryotic expression brucella, which comprises antigen epitopes of a second group of outer membrane protein OMP2 protein of bovine brucella (B.abortus), a sheep brucella (B.melitensis), a pig brucella (B.suis) and a dog brucella (B.canis) and OMP25, OMP31, OMP28 and OMP22 protein in a third group of outer membrane protein. The vaccine has good safety and effectiveness, and cattle and sheep can stimulate good antibody level after intramuscular or subcutaneous injection.

Description

Brucella gene engineering subunit vaccine and preparation method and application thereof

Technical Field

The invention relates to the technical field of biological pharmacy, and relates to a Brucella genetic engineering subunit vaccine, and a preparation method and application thereof.

Background

Brucella is a gram-negative intracellular parasite, and there are 9 currently identified brucella species, 7 of which are terrestrial animals as hosts, namely, bovine species (b.abortus), ovine species (b.melitensis), porcine species (b.suis), ovine testis species (b.ovis), canine species (b.canis), gerbil species (b.neotomae), and hamster species (b.micoti). The hosts are 2 species of marine animals, namely whale species (B.ceti) and seal species (B.pinipedia). The Brucella melitensis is a pathogenic bacterium with extremely strong transmission and wide host, particularly, the Brucella melitensis has the strongest pathogenicity and can enter animal bodies through skin, oral cavity, mucous membrane and mating to cause infection, and diseased individuals can be transmitted to the outside of the body through various ways such as emulsion, semen, pus, vaginal secretion and the like.

Brucellosis belongs to a serious zoonosis infectious disease, has great harm to people and animals, and belongs to a type B infectious disease together with rabies, atypical pneumonia, highly pathogenic avian influenza and the like. The existing Brucella S19 strain live vaccine, Brucella S2 strain live vaccine and Brucella M5 strain live vaccine play an important role in the prevention and control work of Brucella disease. However, the application of the three vaccines is not enough: (1) the safety is poor, and the feed can not be inoculated to pregnant animals, breeding animals and dairy animals; (2) the vaccinated animals and the wild virus infected animals can not be identified; (3) the vaccine has infectivity on human and animals, and harms public health and safety. The above disadvantages make the vaccine use limited and not applicable to breeding stock and dairy animals. This has led to the prevalence of brucellosis being controlled only by monitoring and killing in most areas, continuing with a safe and effective brucella vaccine.

The outer membrane protein becomes a research hotspot of brucella, and has been found to be related to important propagation and growth processes of brucella such as iron uptake in host endosome, and has important immunogenicity and antigen protection effects. 7 outer membrane proteins have been discovered and can be divided into three groups of outer membrane proteins according to molecular weight. Wherein the first group of outer membrane proteins comprises OMP10, OMP19, etc., which are involved in the structural stability of the bacterium; the second group of outer membrane proteins is OMP2, which is involved in transport of bacterial nutrients; a third group of exoproteins, including OMP25, OMP31, and the like, are involved in maintaining the stability of the bacterial exo-modular functional structure. Wherein OMP25 plays an important role in the process of brucella infection and pathogenesis, and OMP31 is closely related to the iron intake of brucella in the process of endoparasitic. Therefore, a safe and effective novel vaccine is developed based on the brucella outer membrane protein, and the vaccine has important significance on the prevention, control and purification of brucella and public health safety.

Disclosure of Invention

In order to solve the problems, the invention provides the brucella outer membrane protein tandem epitope protein, and the vaccine prepared by using the brucella outer membrane protein tandem epitope protein can be used for animals such as cattle, sheep and the like, and can safely and effectively prevent brucella infection.

The invention aims to provide a brucella outer membrane protein tandem epitope protein, which is used for performing bioinformatics analysis on a second group of outer membrane proteins OMP2 of Brucella bovis (B.abortus), Brucella melitensis sheep (B.melitensis), Brucella suis swine (B.suis) and Brucella canis (B.canis), and OMP25, OMP31, OMP28 and OMP22 proteins in a third group of outer membrane proteins to obtain the epitope of the outer membrane proteins. Preparing a tandem epitope DNA fragment containing the outer membrane protein epitope through artificial synthesis, connecting the DNA fragment to a pET28a prokaryotic expression vector, and constructing a tandem epitope protein recombinant expression vector (pET28a-Mutie), wherein the specific technical scheme is as follows:

the brucella outer membrane protein tandem epitope protein has an amino acid sequence consisting of the amino acid sequences of sheep species Omp2a protein, sheep species Omp2b protein, cattle species Omp2b protein, pig species Omp2b protein, cattle species Omp25 protein, dog species Omp25 protein, pig species Omp25 protein, sheep testis species Omp25 protein, sheep species Omp31 protein, dog species Omp31 protein, sheep testis species Omp31 protein, pig species Omp31 protein, sheep testis species Omp22 protein, cattle species Omp22 protein, sheep species Omp28 protein and cattle species Omp28 protein.

Specifically, the amino acid sequence of the brucella outer membrane protein tandem epitope protein consists of 22-51 site and 144-158 site of the sheep Omp2a protein; 233-245 site and 314-336 site of the Omp2b protein of the sheep species; 4-29 of the bovine Omp2b protein; 41-53 sites of the pig strain Omp2b protein; 4-46, 187-197 and 143-154 of the Omp25 protein of the cattle species; 87-94 of canine Omp25 protein; the 120-137 site of the Omp25 protein of the pig strain; 10-27 of the sheep testis species Omp25 protein; 19-27 of the sheep seed Omp31 protein; 4-14 of the canine Omp31 protein; 95-102 site, 224-237 site of Omp31 protein of sheep testis species; 72-89 of the pig strain Omp31 protein; 157-177 of Omp22 protein of sheep testis species; 74-80 of the bovine Omp22 protein; the 240-position 247 of the sheep Omp28 protein and the 103-position 111 of the cattle Omp28 protein.

Specifically, the amino acid sequence of the brucella outer membrane protein tandem epitope protein is shown in SEQ ID No. 2.

The second purpose of the invention is to provide a Brucella outer membrane protein tandem epitope protein, and specifically, the nucleotide sequence of the Brucella outer membrane protein tandem epitope protein is shown as SEQ ID No. 1.

The invention also aims to provide a recombinant plasmid for expressing the Brucella outer membrane protein tandem epitope protein, and the specific technical scheme is as follows:

a recombinant plasmid for expressing the Brucella outer membrane protein tandem epitope protein comprises the nucleotide sequence of the Brucella outer membrane protein tandem epitope protein in the technical scheme.

The fourth purpose of the present invention is to protect a genetic engineering strain prepared by the recombinant plasmid in the technical scheme.

The specific method comprises the steps of transforming the recombinant plasmid in the scheme into host bacteria to construct a genetic engineering strain, identifying the genetic engineering strain to prepare a brucella outer membrane protein tandem epitope protein genetic engineering strain seed strain, and obtaining the host bacteria which are escherichia coli BL21 strains.

Further, the identification method of the genetic engineering strain comprises the steps of shake culturing the genetic engineering expression strain to a logarithmic phase by using an LB culture medium at 37 ℃, then adding IPTG with the final concentration of 1mM into the LB culture medium and continuing culturing for 2-3 hours. Centrifuging to collect thallus precipitate, re-suspending thallus with ice-cold physiological saline at a ratio of 10:1(W/V), centrifuging to collect thallus precipitate, repeating for 2-3 times, and removing culture medium components. The thalli is detected by SDS-PAGE electrophoresis, and compared with the thalli which is a host bacterium of the untransformed escherichia coli BL21, a band is obvious at 34 kD.

Further, the preparation method of the genetic engineering strain comprises the steps of shake culturing the identified genetic engineering strain to a logarithmic phase by using an LB culture medium at 37 ℃, collecting bacterial liquid under a clean condition, adding an ice-cold freeze-drying protective agent according to a ratio of 1:1, shake mixing uniformly, subpackaging and freeze-drying.

The fifth purpose of the invention is to request protection of the brucella subunit vaccine prepared by using the genetic engineering strain.

The sixth purpose of the invention is to provide a method for preparing a brucella subunit vaccine by using the genetic engineering strain in the technical scheme, which comprises the following specific technical scheme:

the method for preparing the brucella subunit vaccine by using the genetic engineering strain in the technical scheme comprises the following steps:

(1) fermenting and culturing the genetic engineering bacteria;

(2) adding a vaccine preparation buffer solution for resuspension, crushing and centrifuging to obtain a supernatant;

(3) purifying and filtering the supernatant to obtain an antigen solution for vaccine preparation;

(4) adding a freeze-drying protective agent, and freeze-drying to obtain the brucella subunit vaccine.

Further, the fermentation culture of the step (1) is specifically to inoculate the genetically engineered bacteria in a fermentation culture medium according to the proportion of 1:1000 for fermentation culture.

Specifically, the fermentation temperature is 35-38 ℃, when the OD value is more than 20, IPTG is added to the final concentration of 1mM, and the fermentation is continued for 2-3 hours.

Further, the fermentation medium comprises peptone 5g/L, yeast extract 2.5g/L, and glycerol 8g/L, KH₂PO₄10.5g/L、(NH₄)₂HPO₄6g/L, citric acid 1.7g/L, MgSO₄1.68g/L、ZnSO₄·7H₂O 5.25g/L、MnSO₄·4H₂O 0.5g/L、CaCl₂ 2.0g/L、FeSO₄·7H₂O10 g/L、Na₂B₄O₇·10H₂O 0.23g/L、(NH₄)₆Mo₇O₂₄0.1 g/L.

Further, the antigen solution for vaccine preparation described in step (3) is diluted with a vaccine preparation buffer solution until the target protein content is 10mg/mL, and then a lyoprotectant is added at a ratio of 1:1 to perform freeze-drying in step (4).

Specifically, the vaccine preparation buffer solution consists of Tris 6.057g/L, EDTA 0.146.146 g/L, NaCl 2.922g/L, DTT 0.77.77 g/L, urea 180.18g/L and water.

Further, the freeze-drying protective agent consists of Tris 6.057g/L, EDTA 0.146.146 g/L, NaCl 2.922g/L, sucrose 100g/L, skimmed milk powder 200g/L and water.

The seventh purpose of the present invention is to protect the application of the brucella subunit vaccine in the above technical scheme in the preparation of anti-brucella antibody.

The invention has the advantages that: the brucella gene engineering subunit vaccine provided by the invention has the main component of the prokaryotic expression brucella outer membrane protein tandem epitope protein, can stimulate good cellular immunity and humoral immunity after immunizing animals, is used for animals such as cattle and sheep, and can safely and effectively prevent brucella infection.

Moreover, safety and immunogenicity experiments are carried out, and cattle and sheep are immunized by subcutaneous or intramuscular injection, so that no immune injury or disease signs caused by vaccination are found, and the safety is realized. Cattle and sheep are immunized subcutaneously or intramuscularly twice in 0 day and 21 days, and the titer of the detected antibody can reach 1:51200 in 14 days after the second immunization, so the vaccine has good immunogenicity.

Drawings

FIG. 1 shows PCR detection of Mutie protein DNA

FIG. 2 is SDS-PAGE electrophoretic detection of Mutie protein

FIG. 3 shows the ELISA antibody level of Brucella gene engineering subunit vaccine of the present invention after immunization of sheep

FIG. 4 shows ELISA antibody levels of Brucella genetic engineering subunit vaccines of the present invention after immunization of cattle

Detailed Description

The present invention is further described in detail by the following examples, which should be understood that the present invention is not limited to the particular examples described herein, but is intended to cover modifications within the spirit and scope of the present invention.

The experimental procedures, for which specific conditions are not indicated in the following examples, are generally carried out according to conventional conditions, for example as described in the molecular cloning protocols (third edition, sambrook et al), or according to the conditions recommended by the manufacturers.

EXAMPLE 1 construction of recombinant expression vectors

The amino acid sequences of outer membrane proteins OMP2, OMP25, OMP31, OMP28 and OMP22 of Brucella from Genebank, sheep testis, pig and dog are subjected to bioinformatics analysis, and the following epitopes are screened from the outer membrane proteins of Brucella: 22-51 site and 144-158 site of sheep Omp2a protein (GenBank: AMM 72580.1); 233-245 site and 314-336 site of the sheep Omp2b protein (GenBank: AMM 72579.1); 4-29 of bovine Omp2b protein (GenBank: AAX 94577.1); 41-53 sites of pig Omp2b protein (GenBank: AAX 94574.1); 4-46, 187-197 and 143-154 of the cattle Omp25 protein (GenBank: AFJ 79953.1); 87-94 sites of canine Omp25 protein (GenBank: CDL 76104.1); 120-137 site of pig Omp25 protein (GenBank: AHN 46339.1); 19-27 sites of 10-27 sites of sheep testis Omp31 protein (GenBank: ACS50328.1) of sheep testis Omp25 protein (GenBank: ABU 93464.1); 4-14 sites of canine Omp31 protein (GenBank: AAL 27296.1); 95-102 site and 224-237 site of sheep testis Omp31 protein (GenBank: AAL 27294.1); 72-89 sites of pig Omp31 protein (GenBank: AAL 27286.1); 157-177 of Omp22 protein (GenBank: AAS84602.1) of sheep testis species; 74-80 sites of bovine Omp22 protein (GenBank: AAS 84601.1); 240-247 position of sheep seed Omp28 protein (GenBank: AAB 17713.1); 103-111 position of the bovine Omp28 protein (GenBank: AAX 74798.1). The amino acid sequences of the epitope proteins screened are shown in table 1.

TABLE 1 statistical table of amino acid sequences of epitope proteins

Sequencing the amino acid sequence of the epitope protein in a certain sequence to obtain the amino acid sequence of the Brucella tandem epitope protein Mutie, which is shown as SEQ: ID: 2, respectively. The nucleotide sequence of the Brucella tandem epitope protein Mutie is obtained by translating the amino acid sequence through bioinformatics software and optimizing codons, and is shown as SEQ: ID: 1 is shown. Artificially synthesizing a DNA fragment of the MutiE protein, introducing an Xba I enzyme cutting site at the 5 'end of the DNA fragment, and introducing an Xho I enzyme cutting site at the 3' end of the DNA fragment. The DNA fragment is connected to a multiple cloning site of a pET28a prokaryotic expression vector to construct a recombinant expression vector pET28a-MutiE, as shown in figure 1 (wherein M is a DNA Marker, 1 is a pET28a-MutiE vector sample, 2 is an engineering strain original seed sample, 3 is an engineering strain production seed sample, and NC is a blank Escherichia coli BL21 sample of an untransformed expression vector).

Example 2 plasmid stability characterization

Escherichia coli Bl21 cells are selected as host bacteria, the recombinant pET28a-MuTIE expression vector constructed in the embodiment 1 is transformed into Escherichia coli Bl21 competent cells by a heat shock method, and then genetic engineering strains for stably and highly expressing MuTIE protein are screened. The method comprises the following specific steps: firstly, unfreezing 100 mu L of prepared competent escherichia coli Bl21 cells on ice, and then adding 1.5 mu g of prepared pET28a-Mutie vector; ② after mixing, placing on ice for 30min, water bath at 45 ℃ for 90 s; ③ placing on ice for 3min, adding 700 microliter LB culture medium, activating for 1h at 37 ℃; fourthly, the activated bacterial liquid is coated on an LB solid plate with kanamycin concentration of 100 mu g/mL and is inversely cultured at 37 ℃ for overnight; fifthly, randomly selecting full single colonies, numbering the colonies, inoculating the colonies into 5mL LB liquid culture medium with kanamycin concentration of 100 mu g/mL, carrying out shake culture at 37 ℃, and adding IPTG (isopropyl-beta-thiogalactoside) after 6 hours until the final concentration is 1mM for induction expression for 2-3 hours. And then, carrying out streak subculture on the strain for expressing the MuTIE protein for 40 generations, and identifying that strains of 10 th generation, 20 th generation, 30 th generation and 40 th generation can stably express the MuTIE protein, and the growth condition of the strains is not different from the protein expression condition.

Example 3 seed library establishment

Establishing an original seed library: the strain prepared in example 2 was inoculated into 1.5mL LB medium containing 100. mu.g/mL kanamycin at 1% concentration, and shake-cultured at 37 ℃ until OD₆₀₀Reaching about 0.8. The culture broth was then streaked onto LB solid plates with a kanamycin concentration of 100. mu.g/mL, and cultured overnight at 37 ℃ in an inverted state. The single colony was randomly selected, numbered, inoculated into 5mL LB medium containing 100. mu.g/mL kanamycin, and shake-cultured at 37 ℃. Mixing the bacterial liquid cultured to logarithmic phase with a freeze-drying protective agent according to the proportion of 1:1, taking 3mL of the bacterial liquid in an aseptic 10mL penicillin bottle under aseptic condition, freeze-drying, and naming the bacterial liquid as the original seed of the Brucella genetic engineering subunit vaccine, wherein the PCR identification result is shown in figure 1.

Establishing a production seed library: re-dissolving the original seeds by using an LB culture medium, screening single colonies by using a scribing method, and randomly selecting full single colonies for amplification culture. Mixing the bacterial liquid cultured to the logarithmic phase with a freeze-drying protective agent according to the proportion of 1:1, taking 3mL of the bacterial liquid in an aseptic 10mL penicillin bottle under the aseptic condition, and freeze-drying the mixture to obtain the brucella genetic engineering subunit vaccine production seed. In order to ensure the stability of production batches, one production seed is taken each time during production, and is used for production after being directly activated, and the PCR identification result is shown in figure 1.

EXAMPLE 4 fermentation of the engineered Strain

Taking the production seeds which are freeze-dried and preserved, inoculating the production seeds into LB culture medium with 100 mu g/mL kanamycin according to the inoculation amount of 0.1 percent, carrying out amplification culture, and carrying out shake culture at 37 ℃ until the logarithmic phase. Adding fermentation culture medium and on-line mould into the fermentation tank in advance, adding the production seeds subjected to expanded culture into the fermentation tank according to the proportion of 1:1000 when the temperature of the culture medium is reduced to 35-38 ℃, and ventilating, stirring and fermenting at 37 ℃. Recording the OD value once per hour, adding IPTG with the final concentration of 1mM to induce the expression of the foreign protein when the OD value is more than 20, and continuing to ferment for 2-3 hours. The SDS-PAGE results are shown in FIG. 2 (the "M" is protein Marker, "NC" is blank E.coli BL21 sample of untransformed expression vector, "fermentation" is sample after fermentation of engineered bacteria, "purification" is antigen sample for vaccine preparation).

EXAMPLE 5 preparation of vaccine antigen solution

The fermented cells were collected by centrifugation, resuspended at a ratio of 1:10(W/V) with ice-cold vaccine formulation buffer, and then collected by centrifugation again, repeated 2-3 times, and the medium components were removed. The cells were resuspended at 1:10(W/V) in an ice-cold buffer prepared with a vaccine, disrupted by a high-pressure homogenizer, and the disrupted supernatant was collected by centrifugation. The supernatant was ultrafiltered with a hollow fiber column having a pore size of 0.45 μm to remove impurities. The filtrate was collected and concentrated by ultrafiltration using a hollow fiber column with a molecular weight cut off of 10kD to 20-30% of the original volume. Collecting the concentrated reflux, quantitatively detecting with BSA method to obtain a total protein content of not less than 30mg/mL, and detecting with SDS-PAGE electrophoresis to obtain a Mutie protein band content of not less than 80%. And sterilizing and filtering the reflux liquid to obtain the antigen solution for vaccine preparation. The antigen solution is light yellow clear liquid in appearance, the pH value is 7.0-7.2, the result of identifying the mixed bacteria through a blood plate is negative, the result of identifying the mycoplasma through semi-fluid and broth culture is negative, and the neutralizing titer of the antiserum is calculated to be higher than 1:2800 by adopting a Reed & Muench method.

Example 6 preparation and application of a Brucella genetic engineering subunit vaccine

The antigen solution for vaccine preparation in example 5 is diluted with a vaccine preparation buffer solution to ensure that the Mutie protein content is not less than 10mg/mL, and then a freeze-drying protective agent is added according to the ratio of 1:1 to be uniformly mixed. Subpackaging with sterile penicillin bottles according to a ratio of 3 mL/bottle, freeze-drying, sealing and packaging. The final product is light yellow loose solid, namely Brucella gene engineering subunit vaccine of 15 parts/bottle (for sheep and goat) or 3 parts/bottle (for cattle), and is preserved at 2-8 deg.C. The vaccine is diluted by 15mL of sterile physiological saline or aluminum hydroxide solution, so that the brucella abortus genetic engineering subunit vaccine containing 1mg/mL of Mutie antigen is used for sheep and goats, and each sheep or goat can stimulate good immune response by intramuscular or subcutaneous injection of 1mL at 0 day and 21 days. The vaccine is diluted by 3mL of sterile normal saline or aluminum hydroxide solution, and is a Brucella vaccine for cattle containing 5mg/mL of Mutie antigen, and each cattle can stimulate good immune response by intramuscular injection or subcutaneous injection of 1mL in 0 day and 21 days.

Experimental example 7 immune response experiment of Brucella gene engineering subunit vaccine on experimental animal

The vaccines prepared in example 6 were diluted with sterile physiological saline and aluminum hydroxide solution, respectively, to prepare experimental groups, and the vaccines were used as control groups to immunize 4-6 month-old lambs and 6 month-old calves intramuscularly or subcutaneously on days 0 and 21, respectively. Peripheral blood and serum of each group of experimental sheep and experimental cattle are collected 14 days after the last immunization. The serum antibody titer is determined to be more than 1:12800 by an ELISA method, and the vaccine can generate good antibody level regardless of the addition of an adjuvant or the absence of the adjuvant. The results of the antibody level detection in the experimental sheep and cattle are shown in fig. 3(V0 is before the first immunization, V1 is 14 days after the first immunization, and V2 is 14 days after the second immunization), and fig. 4(V0 is before the first immunization, V1 is 14 days after the first immunization, and V2 is 14 days after the second immunization).

Experimental example 8 safety experiment of Brucella genetic engineering subunit vaccine

(1) Sterility, mycoplasma assay: the vaccine prepared in example 6 was inoculated into thioglycollate medium, nutrient agar slant medium and modified Martin medium for 14d, respectively, and sterile physiological saline was used as negative control, and the culture temperature was 25 deg.C and 35 deg.C, so that no bacteria would grow. The vaccine is primarily cultured for 21 days and secondarily cultured for 21 days in a semi-fluid culture medium and a broth culture medium at 37 ℃ respectively, and sterile physiological saline is used as a negative control, so that no mycoplasma grows obviously.

(2) Hemolytic test: a guinea pig with a weight of about 350g was selected, and 1mL of fresh guinea pig blood was collected, washed 3 times with PBS, and the blood cell volume was recovered and diluted 10-fold. The vaccine prepared in example 6 was diluted 2 times, 4 times, and 8 times with physiological saline, guinea pig blood cells were added to the diluted adjuvant to be tested, and after 8 hours, hemolyzation was evaluated based on visual observation or detection of the supernatant concentration, and absorbance was detected at 570 nm. As a result, there should be no rupture of the blood cell and no hemolysis.

(3) Acute toxicity test: the vaccine prepared in example 6 was diluted with 1.5mL of sterile physiological saline, and 1.5mL of 12-18 g Balb/C mice were intraperitoneally injected, 10 mice were administered per group, and a physiological saline control group was used, so that all experimental mice survived for 14 days without adverse symptoms such as piloerection, listlessness, and bradykinesia, and the weight of the experimental mice increased. After 14 days, the patient was sacrificed and examined for dissection, and no pathological changes of the organs were observed. The vaccine is safe to mice under 10 times of sheep dosage and 1.5 times of cattle dosage.

(5) Rabbit pyrogen test: fixing 3 rabbits with the weight of 2-3 kg qualified by pre-detection, measuring the body temperature after 30 minutes, measuring for 2 times in total at intervals of 30 minutes, wherein the temperature difference of 2 times is not more than 0.2 ℃, and the average temperature of each rabbit for 2 times is 38.6-39.5 ℃. The vaccine prepared in example 6 was diluted with 1.5mL of sterile physiological saline, warmed to 38 ℃ and injected slowly into each of the ear veins of rabbits at a rate of 0.5 mL/body within 15 minutes after the 2 nd temperature measurement. Body temperature was measured 1 time every 30 minutes after injection and 6 times in succession. As a result, no fever reaction of the rabbits should be caused, and the temperature rise of the rabbit individuals should not exceed 0.2 ℃.

Experimental example 9 stability experiment of Brucella genetic engineering subunit vaccine

The vaccine prepared in example 6 was allowed to stand at 2-8 deg.C, room temperature (20-25 deg.C) and 37 deg.C for 1 week, 2 weeks, 1 month, 3 months, 6 months, 12 months and 15 months, respectively. The appearance, pH value, sterility and safety of the sample are all normal. After the animal is placed at room temperature for 6 months and at 37 ℃ for 1 month, the antibody level is 1: 12800. The animal is immunized at 2-8 deg.C for 15 months, and the antibody level should reach 1: 12800. The validity period of the vaccine of the invention is not less than 12 months.

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, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Sequence listing

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gcggcggatg cgattgtggc gccggaaccg gaagcggtgg aatatgtgcg cgtgtgcgat 60

gcgtatggcg cgggctattt ttatattccg ctgcagtttg cgtatattac cctgggcggc 120

tttaaagtgg gcattattgc gggcgtggtg gcgtatgata gcgtgattga agaagcggtg 180

accgcgaacg tggcgtatga actggtgccg ggctttaccg tgaccccgga agtgagctat 240

accaccagcg cgctggtgcc ggcgattgcg gtgggcaccg gcgtgaacgt gattgcggcg 300

ctgtatgtgg tgaccgatgt gaacgtggaa gtggcgagcg cggtgagcgt ggcgaacgcg 360

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gatgcgattg tggcgcagga accggcgccg attgcgattg cgccgagctt tagctgggcg 480

ggcgcgtata ccgataacgt gctgctgcgc ctggaatatc gctttatgcc gtatattgcg 540

ggcggcgtgg cgtttggcga tcagattgtg tatggcgtgg aacgcgcgcg cgtgggctat 600

gatctgaacc cggtgatgcc gtatctgacc gcgggccgcg cgcgcgtggg ctatgatctg 660

aacccggtga tgccgtatct gaccgcgggc gcggcggatg tggtggtgag cgaaccggtg 720

attctggcga gcattgcggc gatgtttgcg gataacggcg tggtgctggg cgcggaaagc 780

aaagtgaact ttcataccgt gcgcgtgggc ctgaacagcg gcagcctgga tgtgaccgcg 840

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gcgctgagcg gcccgctgag cgtgaaagcg gaaggcggca ttgtggtggg caaaaacgtg 960

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Ala Ala Asp Ala Ile Val Ala Pro Glu Pro Glu Ala Val Glu Tyr Val

1 5 10 15

Arg Val Cys Asp Ala Tyr Gly Ala Gly Tyr Phe Tyr Ile Pro Leu Gln

20 25 30

Phe Ala Tyr Ile Thr Leu Gly Gly Phe Lys Val Gly Ile Ile Ala Gly

35 40 45

Val Val Ala Tyr Asp Ser Val Ile Glu Glu Ala Val Thr Ala Asn Val

50 55 60

Ala Tyr Glu Leu Val Pro Gly Phe Thr Val Thr Pro Glu Val Ser Tyr

65 70 75 80

Thr Thr Ser Ala Leu Val Pro Ala Ile Ala Val Gly Thr Gly Val Asn

85 90 95

Val Ile Ala Ala Leu Tyr Val Val Thr Asp Val Asn Val Glu Val Ala

100 105 110

Ser Ala Val Ser Val Ala Asn Ala Lys Asn Leu Leu Gly Ala Ser Leu

115 120 125

Val Ala Val Ile Thr Ser Thr Ser Ala Tyr Ala Ala Asp Ala Ile Val

130 135 140

Ala Gln Glu Pro Ala Pro Ile Ala Ile Ala Pro Ser Phe Ser Trp Ala

145 150 155 160

Gly Ala Tyr Thr Asp Asn Val Leu Leu Arg Leu Glu Tyr Arg Phe Met

165 170 175

Pro Tyr Ile Ala Gly Gly Val Ala Phe Gly Asp Gln Ile Val Tyr Gly

180 185 190

Val Glu Arg Ala Arg Val Gly Tyr Asp Leu Asn Pro Val Met Pro Tyr

195 200 205

Leu Thr Ala Gly Arg Ala Arg Val Gly Tyr Asp Leu Asn Pro Val Met

210 215 220

Pro Tyr Leu Thr Ala Gly Ala Ala Asp Val Val Val Ser Glu Pro Val

225 230 235 240

Ile Leu Ala Ser Ile Ala Ala Met Phe Ala Asp Asn Gly Val Val Leu

245 250 255

Gly Ala Glu Ser Lys Val Asn Phe His Thr Val Arg Val Gly Leu Asn

260 265 270

Ser Gly Ser Leu Asp Val Thr Ala Gly Gly Phe Val Gly Gly Val Gln

275 280 285

Ala Gly Gly Val Leu Ile Gly Ala Gly Val Glu Gln Ala Leu Ser Gly

290 295 300

Pro Leu Ser Val Lys Ala Glu Gly Gly Ile Val Val Gly Lys Asn Val

305 310 315 320

Ser Val Asn Val Val Phe Ile Gln Pro Ile Tyr Val Tyr Pro Asp

325 330 335

Claims

1. a Brucella outer membrane protein tandem epitope protein, is characterized in that, the aminoacid sequence of described Brucella outer membrane protein tandem epitope protein is composed of sheep species Omp2a protein, sheep species Omp2b albumen, cattle species Omp2b Protein, pig Omp2b protein, bovine Omp25 protein, dog Omp25 protein, pig Omp25 protein, sheep testis Omp25 protein, sheep Omp31 protein, dog Omp31 protein, sheep testis Omp31 protein, pig Omp31 protein, sheep The amino acid sequence composition of testis Omp22 protein, bovine Omp22 protein, sheep Omp28 protein, and bovine Omp28 protein.

2. Brucella outer membrane protein tandem epitope protein according to claim 1, is characterized in that, the aminoacid sequence of described Brucella outer membrane protein tandem epitope protein is composed of 22-51 of sheep species Omp2a protein. 233-245 and 314-336 of sheep Omp2b protein; 4-29 of bovine Omp2b protein; 41-53 of pig Omp2b protein; 4-46 of bovine Omp25 protein , 187-197, 143-154; canine Omp25 protein 87-94; pig Omp25 protein 120-137; sheep testis Omp25 protein 10-27; sheep Omp31 protein 19-27 Positions 4-14 of Omp31 protein of canine species; positions 95-102 and 224-237 of Omp31 protein of sheep testis; positions 72-89 of Omp31 protein of pig species; 157-177 positions of Omp22 protein of sheep testis 74-80 of Omp22 protein; 240-247 of sheep Omp28 protein and 103-111 of bovine Omp28 protein.

3. Brucella outer membrane protein tandem epitope protein according to claim 1, is characterized in that, the aminoacid sequence of described Brucella outer membrane protein tandem epitope protein is as shown in SEQ ID NO.2.

4. A Brucella outer membrane protein tandem epitope protein, characterized in that the nucleotide sequence of the Brucella outer membrane protein tandem epitope protein is shown in SEQ ID NO.1.

5. a recombinant plasmid expressing Brucella outer membrane protein series epitope protein, it is characterized in that, described recombinant plasmid comprises the nucleotide of the described Brucella outer membrane protein series epitope protein of claim 4 sequence.

6. A genetically engineered strain prepared by utilizing the recombinant plasmid of claim 5.

7. the Brucella subunit vaccine that utilizes the genetic engineering strain described in claim 6 to prepare.

8. the preparation method of the described Brucella subunit vaccine of claim 7, is characterized in that, comprises the steps:

(1) fermenting and culturing the genetically engineered strain;

(2) adding vaccine preparation buffer to resuspend, crush and centrifuge to obtain supernatant;

(4) adding a freeze-drying protective agent and freeze-drying to obtain a Brucella subunit vaccine.

9. method according to claim 8 is characterized in that, described freeze-drying protective agent is made up of Tris 6.057g/L, EDTA0.146g/L, NaCl 2.922g/L, sucrose 100g/L, skimmed milk powder 200g/L and water composition.

10. the application of the described brucella subunit vaccine of claim 7 in the preparation of anti-brucella antibody.