CN103981139B - One strain brucella low virulent strain and vaccine thereof - Google Patents
One strain brucella low virulent strain and vaccine thereof Download PDFInfo
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Abstract
The present invention relates to strain brucella low virulent strain and a vaccine thereof.The domestication that this vaccine strain is combined with A factor serum by antibiotic and triage techniques, filter out a strain rough type B. abortus low virulent strain RA343.The security of this rough type low virulent strain significantly improves, but still remains the immune effect good to brucellosis.Utilize this low virulent strain to be prepared into brucella disease vaccine, change brucella vaccine immune animal and street strain's infection animal are difficult to the present situation distinguished, and effectively improve the security of existing vaccine.
Description
Technical field the present invention relates to strain brucella low virulent strain and a vaccine thereof.This vaccine is ox, sheep and the pig prevention living vaccine to brucellosis, belongs to veterinary biologics field.
Technical background
Brucellosis (cloth sick) is by brucella or what claim brucella (Brucella) to cause is the Amphixenosis of feature with miscarriage and heating, seriously threatens the life and health of people and many animals.This disease not only has serious harm to the breeding of animal and production performance, the more important thing is, after people infects brucella, is often difficult to cure, thus causes serious public health problem.Therefore in the country that brucella is popular, eliminate cloth disease is one of most important target in public health program always.The main method eliminating this disease in current worldwide slaughters to combine with immunity, and to cloth disease, relatively serious China occurs, because the cost slaughtered is high, vaccine prevention becomes this sick major control means.
According to the difference of brucella phenotype, brucella can be divided into smooth type (S) and rough type (R) two class, the cloth disease live-vaccine used in China is at present smooth type bacterial strain, the subject matter that its immunity brings to distinguish the animal of vaccine immunity and natural infection after using, which greatly limits using and promoting of brucella vaccine, bring certain difficulty also to effective control of cloth disease and removing.
Although brucella exists two kinds of different phenotypes, no cross reaction in aggegation antibody-like level, has good intersecting protective, and the brucella in source not of the same race exists cross-protection ability each other equally.If with the animal of Rough Anti-Brucella vaccine immunity, its serum only with rough generation agglutination reaction, and not react with smooth type serum, production of vaccine bacterial strain and street strain can be distinguished with this.For these reasons, the research and development of people to Rough Anti-Brucella vaccine strain were never interrupted.Up to the present, successfully develop and there is good immunogenic Rough Anti-Brucella vaccine strain but rarely have report.
The earliest prepared by used Rough Anti-Brucella vaccine 45/20 bacterial strain, but this bacterial strain extremely unstable, often there will be the variation from R type to S type, cause virulence to return by force, thus this vaccine does not re-use substantially.The nineties, American scientist was by the mutagenic obtained Rough Anti-Brucella RB51 bacterial strain of Rifampin, and this bacterial strain has good immunizing power, widely use in multiple countries of the U.S. and Latin America.But its security is still disputable.
Existing research confirms that the O chain composition in brucella cell wall lipopolysaccharides (LPS) determines the phenotype of its smooth type or rough type, and some composition disappearance of O chain, can cause bacterial strain to become rough type by smooth type.Have now found that gene that is multiple and brucella smooth type phenotypic correlation, comprise gmd, per, pgm, wbkA, lpx, wa, wbkC, wz and wbkC.But the restructuring brucella low virulent strain and the vaccine thereof that up to the present, there is no transgenosis acquisition both at home and abroad go through and apply.
Summary of the invention
Between 2007-2012 years, this laboratory is separated to B. abortus 41 strain, brucella melitensis 74 strain, Br. cants 9 strain successively in the different animal body such as province and regional ox, sheep, dog and deer of China.In research process, we have tentatively found strain B. abortus (Brucellaabortus) strain isolated, its virulence is between strong poison and vaccine virus, and patent applicant has carried out comprehensive biochemical assays to it, and determines its virulence with mouse and cavy respectively.Found that its virulence is between strong poison and weak poison, is determined as mesogenic brucella, called after Abortus343.The induction method adopting low-level chlorinated mycin (1 μ g/ml) to combine with smooth type B. abortus antiserum(antisera) (A factor serum) of the present invention, screening obtains the Rough Anti-Brucella called after RA343 of a strain inheritance stability.RA343 strain security is high, good immune effect, and after the vaccine immunity animal of preparing with this bacterium, its serum can be distinguished with natural infection mutually with the agglutination test of routine.
Technical scheme of the present invention
1. strain B. abortus (Brucellaabortus) less-virulent strain RA343, it is characterized in that the screening method adopting paraxin to combine with A factor serum, screening obtains the rough type B. abortus that the sick clinical diagnosis of cloth is not disturbed in a strain, after the vaccine immunity animal of preparing with this bacterium, its serum can be distinguished with natural infection mutually with the agglutination test of routine, B. abortus (Brucellaabortus) RA343 bacterial strain delivered the China Microbiological preservation council of Institute of Microorganism, Academia Sinica common micro-organisms preservation center, No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City on 05 20th, 2014, preserving number is: CGMCCNo.8886.
2. a production method for Brucella live vaccine, is characterized in that with the substratum of pancreas peptone soybean broth as brucella CGMCCNo.8886 strain; By 1% ~ 2% access brucella CGMCCNo.8886 strain kind daughter bacteria liquid cultivating base unit weight after medium sterilization; 37 DEG C; fermentation culture 28 ~ 38h according to a conventional method; add the lyophilized vaccine that veterinary biologics is conventional; through vacuum freezedrying after in abundant mixing, packing vaccine bottle, namely become rough type RA343 strain B. abortus disease vaccine.
The present invention is realized by following steps:
1. screen mesogenic brucella strain isolated from 41 strain B. abortus (Brucellaabortus) field strain isolateds, obtained the Candidate Strain of 10 strain form stables by limiting dilution assay and bacterium colony violet staining.Refer again to " regulations " (The Ministry of Agriculture of the People's Republic of China, MOA compile. People's Republic of China (PRC) regulations .2000 version, Chemical Industry Press, 2001) brucella disease live-vaccine kind poison toxicity test method in, adopt cavy spleen bacteria containing amount to measure the virulence of 10 strain candidate strain, screen the Candidate Strain of the moderate brucella (A343 strain) of virulence as induction.
2. pair candidate's strain isolated is induced and is tamed and first gone down to posterity containing on the TSA substratum of low-level chlorinated mycin (1 μ g/ml) by A343, passed for 35 generations altogether, consequently the positive rate of bacterium colony violet staining by A343 (F0) lower than 1% to A343 (F40) about 6%.Then smooth type B. abortus antiserum(antisera) (A factor serum) is used to tame further, to repeatedly not go down to posterity with the bacterium of A factor serum generation aggegation, when the rough colony ratio of this bacterium reaches about 90%, use double A factor serum (B. abortus specific serum, the preparation of this laboratory and preservation) of tiring instead and continue induction.The Rough Anti-Brucella of the inheritance stability obtained the most at last, called after RA343 strain.
3. couple Rough Anti-Brucella RA343 carries out the calibrating of detailed bacterial classification and comprises form and biochemical characteristic, serology and PCR atopic, and the virulence to cavy.
4. prepare vaccine and prepare Rough Anti-Brucella disease live-vaccine by the classical way of cloth disease vaccine, for preventing animal brucellosis.
The detailed description of invention
1. the screening of mesogenic brucella strain isolated
(1) according to B. abortus 41 strain that this laboratory was separated from field from 2007-2012 years with stability preliminary screening by form, single bacterium colony is cultivated by limiting dilution assay, choosing colony size is even, violet staining bacterium colony positive rate, lower than candidate brucella 10 strain of 1%, surveys poison for cavy.
(2) by again screening with reference to brucella vaccine kind poison toxicity test method in " veterinary biological product code " to mensuration cavy virulence, cavy spleen bacteria containing amount is adopted to measure the virulence of 10 strain candidate strain, by 10 strain brucella respectively on TSA flat board after multiplication culture, collect thalline, by stroke-physiological saline solution, each bacterial concentration is adjusted to 1 × 10
9cFU/ml.Get 350 ~ 400g Female guinea pigs 50, be divided into 10 groups, 5/group, corresponding 10 strain candidate bacterium.By candidate strain respectively with 1ml/ each test group cavy of inguinal region subcutaneous injection, within 14th, observe organ disease situation afterwards, get spleen, mixing is weighed simultaneously, makes emulsion, and inoculation TSA is dull and stereotyped, calculates every 1 gram of spleen bacteria containing amount according to bacterial growth quantity.Result only has 1 strain (A343) strain isolated virulence minimum, is 1.2 × 10
6cFU/g, all the other 9 strains are all 6 × 10
6more than CFU/g.Choose A343 in the present invention as the original strain of inducing in the present invention, be labeled as A343 (F0).
The induction of 2.A343 strain and domestication
(1) the A343 strain filtered out is being gone down to posterity containing on the TSA substratum of paraxin (1 μ g/ml) by the induction of paraxin, when going down to posterity toward the next generation, adopting limiting dilution assay to carry out, and carrying out violet staining to the bacterium colony on flat board simultaneously.Choose the most obvious bacterium colony of violet staining coloring degree to continue to go down to posterity, if there is not the bacterium colony of the violet staining positive, then random picking.Passed for 35 generations altogether in this approach, consequently the positive rate of bacterium colony violet staining by A343 (F0) lower than 1% to A343 (F40) about 6%.
(2) the single A343 of domestication picking (F40) colony inoculation of smooth type B. abortus antiserum(antisera) (A factor serum) cultivates 48h in TSB liquid nutrient medium, regulates bacterial concentration about 20 ~ 3,000,000,000 CFU/ml (now OD600=0.1) by stroke-physiological saline solution.Get 0.5ml bacterium liquid and add 0.5mlA factor serum (sterile filtration, containing 2 aggegation unit/ml), fully after mixing, 37 DEG C of stationary incubation 2h, then transfer to 4 DEG C of refrigerators and leave standstill 12h.Gently suct layer UA bacterium liquid 20 μ l, transfer is inoculated in fresh TSB substratum, continues to cultivate, so repeatedly induces screening 30 generation.In every 5 generations, obtain single bacterium colony by limiting dilution assay, and carry out bacterium colony violet staining, and the painted single bacterium colony of picking Viola crystallina continues to go down to posterity.To the 30th generation, i.e. A343 (F35+30), the bacterium colony ratio about 90% that A343 violet staining can be painted.A factor serum is tired and is adjusted to 4 aggegation unit/ml, continue induction as stated above.Carry out violet staining to the 6th generation (always going down to posterity was the 71st generation) bacterium colony, now all bacterium colonies are all-round painted.The A factor serum of 4 aggegation unit/ml is still used to continue induction 6 generation (always going down to posterity was the 77th generation) on this basis, by Rough Anti-Brucella called after B. abortus (Brucellaabortus) the RA343 strain of the inheritance stability of acquisition.
3. Rough Anti-Brucella RA343 strain bacterial classification calibrating
(1) form and biochemical characteristic RA343 strain bacterium colony edge neat, mellow and full, dew drop-wise, skew ray irradiate, backlight observes micro-band blue-opalescent.Dyeing form is coccobacillus, is singlely dispersed in, and does not form gemma and pod membrane.Thalline size is between 0.3 ~ 0.6 μm.Gramstaining is negative.The growth of this bacterium does not rely on CO
2, can grow on the substratum of sulfur-bearing a beautiful gem and basic fuchsin, H
2s tests strong positive.
(2) specificity
1) serological specificity makes antigen with the culture of RA343 strain bacterium, should not occur aggegation, should occur aggegation with rough type serum (prepared by this laboratory) with smooth type brucella positive serum (middle prison institute, 201101 batches).
2) PCR specificity following 4 primers of synthesis (sequence 1, sequence 2, sequence 3 and sequence 4), be made into primer mixing storage liquid, each primer concentration is 25 μMs.
Feri：5’-GCGCCGCGAAGAACTTATCAA-3’
Reri:5’-CGCCATGTTAGCGGCGGTGA-3’
F
abortus:5’-GACGAACGGAATTTTTCCAATCCC-3’
R
IS711:5’-TGCCGATCACTTAAGGGCCTTCAT-3’
Template: the genomic dna extracting RA343 strain bacterium with the cultivation bacterium colony of RA343 strain bacterium or test kit.
PCR reaction system: in the reaction system of 50 μ L, containing 5 μ L10 × Buffer, 8 μ L2.5mMdNTPs, mix primer storage liquid 2 μ L, Taq enzyme 2U, template DNA 1 μ L (or picking colony is a little).
PCR response procedures: after 95 DEG C of 5min, carries out 28 circulations, last 72 DEG C of 10min by 94 DEG C of 1min, 60 DEG C of 1.5min, 72 DEG C of 1.5min.
, should there are 2 specific PCR bands in result: amplified production 1.5% sepharose carries out electroresis appraisal, size is respectively 178bp and 494bp (see Fig. 1).
(3) under the RA343 strain bacterium culture cultivating 48-72h on solid medium is washed by virulence stroke-physiological saline solution, be diluted to every milliliter of suspension containing viable bacteria 1,000,000,000 CFU, the cavy (Hartley strain) 5 of inguinal region subcutaneous injection body weight 350 ~ 400g, 1ml/ only.Cutd open after 14 ~ 15 days and kill, get spleen, mixing is weighed, and makes emulsion, and inoculation TSA is dull and stereotyped, according to the colony number of its growth, calculates the bacteria containing amount of every gram of spleen, and result spleen is 8.9 × 10 containing bacterium
3cFU, is no more than 200,000 CFU (the judgement mark of virulent strain).
(4) rough type characteristic thermoagglutination test, trypaflavine agglutination test, bacterium colony violet staining test-results meet the feature of Rough Anti-Brucella, and namely thermoagglutination test is positive, and trypaflavine agglutination test is positive, can by violet staining.
(5) RA343 viable bacteria normal saline dilution becomes 1ml to contain viable bacteria 1,000,000,000 CFU by safety verification, the small white mouse of subcutaneous injection body weight 18 ~ 20g 5, and every 0.1ml observes 6, all strong alive.
(6) RA343 is diluted to every milliliter of suspension containing 5,000,000,000 CFU bacterium by immunogenicity physiological saline, the cavy of subcutaneous injection body weight 350 ~ 400g 10, every 0.1ml.After 30 days, (CVCC788 strain is with the strong malicious B. abortus 2308 strain bacterium of 3 minimal infecting doses, by Chinese veterinary microorganism culture presevation administrative center's preservation and supply) about 30 CFU inoculations attack poison, attack malicious cuing open for latter 30 days to kill, get spleen to smash to pieces and make brucella separation and Culture, the cavy of more than 80% is not all separated to bacterium.
(7) genetic stability RA343 uploaded for 30 generations at TSA substratum, in every 5 generations, test to its form and biochemical characteristic, cultural characters, rough type characteristic, virulence, PCR specificity, security and immunogenicity, and result does not all change (table 1).
Table 1 brucella RA343 strain genetic stability measurement result
| Survey malicious generation | Form and biochemical characteristic | Cultural characters | Rough type characteristic | PCR specificity | Spleen bacteria containing amount | Protection ratio |
| Primary | G-, coccobacillus, H 2S+ | TAB well-grown | Rough type | Ox kind | 8.9×10 3CFU | 80％ |
| 5 | G-, coccobacillus, H 2S+ | TAB well-grown | Rough type | Ox kind | 7.7×10 3CFU | 80％ |
| 10 | G-, coccobacillus, H 2S+ | TAB well-grown | Rough type | Ox kind | 7.9×10 3CFU | 80％ |
| 15 | G-, coccobacillus, H 2S+ | TAB well-grown | Rough type | Ox kind | 6.1×10 3CFU | 80％ |
| 20 | G-, coccobacillus, H 2S+ | TAB well-grown | Rough type | Ox kind | 8.4×10 3CFU | 90％ |
| 25 | G-, coccobacillus, H 2S+ | TAB well-grown | Rough type | Ox kind | 7.2×10 3CFU | 80％ |
| 30 | G-, coccobacillus, H 2S+ | TAB well-grown | Rough type | Ox kind | 6.3×10 3CFU | 90％ |
4. vaccine preparation and quality standard
(1) produce the ordinary method of brucella disease live-vaccine as production bacterial strain by brucella S2 strain; be inoculated in appropriate media; results culture; add suitable lyophilized vaccine; through vacuum freezedrying make living vaccine (The Ministry of Agriculture of the People's Republic of China, MOA. People's Republic of China's regulations in 2000 version. Chemical Industry Press; 2001, the present invention is called for short " code ").For preventing animal brucellosis.Production of vaccine substratum of the present invention is all can be used as with pancreas peptone soybean broth (TSB, U.S. company BD) or for brucella S2 bacterial strain production of vaccine substratum (as Martin's soup, Yi Shi meat soup, liver soup etc.).By 1% ~ 2% access RA343 kind daughter bacteria liquid cultivating base unit weight after medium sterilization, cultivate 48 ~ 72h for 37 DEG C, add the sucrose gelatine stabiliser that veterinary biologics is conventional, abundant mixing, be sub-packed in after in vaccine bottle and carry out vacuum freezedrying immediately, namely RA343 living vaccine is become, called after brucella disease live-vaccine (RA343 strain).Vaccine after vacuum freezedrying carries out inspection after construction immediately by quality standard.
(2) the related check method that relates to of quality standard this standard by People's Republic of China's veterinary drug allusion quotation (the Chinese veterinary pharmacopoeia council. People's Republic of China (PRC) veterinary drug allusion quotation 201 〇 version (three). Chinese agriculture press, 2011, the present invention claims " Chinese veterinary pharmacopoeia ") method carry out.
1) vaccine should be loose group soon, can (in 1min) dissolve rapidly after adding diluent;
2) vaccine should purely without other bacteriums;
3) vaccine is diluted to every 1ml containing 1 × 10
9cFU viable bacteria, the Kunming white mouse of inguinal region subcutaneous vaccination 18 ~ 20g 5, each 0.25ml/ only, observes 6, all should be good for and live;
4) live bacterial count is carried out to vaccine, the head number appraised and decided should be not less than.Pig: 20,000,000,000/head part, sheep: 20,000,000,000/head part, ox: 80,000,000,000/head part.
The Microbial resources information that the present invention relates to
The Microbial resources related in the present invention are B. abortus RA34 strain, this strain bacterium is that this laboratory in 2009 is separated to the induction of strain B. abortus (Brucellaabortus) isolate A 343 strain and domestication and obtains a strain rough type less-virulent strain in diary farm, Shandong miscarriage ox body, this strain delivered the China Microbiological preservation council of Institute of Microorganism, Academia Sinica common micro-organisms preservation center, No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City all on 05 20th, 2014, preserving number is: CGMCCNo.8886; B. abortus 2308 strain bacterium (is CVCC788 strain, by Chinese veterinary microorganism culture presevation administrative center's preservation and supply, see China Veterinery Drug Inspection Office, veterinary microorganism culture presevation administrative center of China writes. Chinese animal doctor's bacterial classification catalogue second edition, Chinese agriculture Science Press, 2002, p24.)
Accompanying drawing explanation
1:Mareker2000 in Fig. 1 RA343 strain PCR Species estimation result figure; 2: Escherichia coli O 157 contrasts; 3.RA343 bacterium PCR result.
Advantage of the present invention
The present invention relates to strain brucella low virulent strain and a vaccine thereof.This brucella low virulent strain is the induction method combined by antibiotic and mono-specific antiserum, the B. abortus of the strain mesogenic by clinical separation is induced into the Rough Anti-Brucella RA343 strain of inheritance stability.RA343 not only remains the immune effect good to brucellosis, and effectively improves its security.With this bacterial strain production vaccine, change brucella vaccine immune animal and street strain's infection animal are difficult to the present situation distinguished.
Embodiment
The present embodiment, for further illustrating technical scheme of the present invention, is not construed as limiting the present invention.
Embodiment 1
Pancreas peptone soybean broth (TSB, U.S. company BD) or other appropriate media as the substratum of Rough Anti-Brucella vaccine strain (RA343), appropriate defoamers is added by cultivation base unit weight, by 1% ~ 2% access RA343 kind daughter bacteria liquid cultivating base unit weight after sterilizing, air flow is strengthened gradually at 36 ~ 37 DEG C, fermentation culture 36h according to a conventional method, can add the glucose solution of 50% in culturing process as required, each add-on is 1% ~ 2% of substratum total amount.
Cultivation completes, and sampling tryptose soya agar does pure inspection by the method that " Chinese veterinary pharmacopoeia " specifies, is pure.Sampling TSA substratum carries out live bacterial count simultaneously, for reference time concentrated.
By the bacterium liquid be purely up to the standards, add Xylo-Mucine by total amount 0.1% ~ 0.2%, make bacterial sediment, suck supernatant liquor, put 2 ~ 8 DEG C and keep in, to be packed.
Get the method that concentrated bacterium liquid specifies by " Chinese veterinary pharmacopoeia " purely to check, for purely.
Get the method TSA that concentrated bacterium liquid specifies by " Chinese veterinary pharmacopoeia " and carry out live bacterial count, as reference when joining seedling.
Bacterium liquid through being up to the standards; the conventional pH7.0 sucrose gelatine stabiliser (see " code ") of veterinary biologics is added by its bacterium number; its final content is made to be 5% ~ 10% sucrose and 1% ~ 1.5% gelatin; add the thiocarbamide that final content is 1% ~ 3%, also can use other suitable lyophilized vaccine.Abundant mixing is afterwards by the dosage quantitative separating of 80,000,000,000 ~ 1,000 hundred million/ml.Namely Rough Anti-Brucella disease live-vaccine (RA343 strain) is become after the vacuum-drying of vaccine bottle quick freeze after packing.
Embodiment 2
Inspection after construction, vaccine is wherein prepared by technical scheme provided by the invention.
The related check method that the present invention relates to is undertaken by the method for " Chinese veterinary pharmacopoeia "
(1) physical behavior vaccine should be loose group soon, all can dissolve rapidly in 1min after adding diluent;
(2) pure inspection vaccine is all pure in other bacteriums;
(3) vaccine is diluted to every 1ml containing 1 × 10 by safety verification
9cFU viable bacteria, the Kunming white mouse of inguinal region subcutaneous vaccination 18 ~ 20g 5, each 0.25ml/ only, observes 6, all strong (the results are shown in Table 2) alive;
Table 2 brucella disease live-vaccine (RA343 strain) safety verification result
-: healthy survival, injection site is without exception; +: dead or morbidity.
(4) live bacterial count TSA substratum carries out live bacterial count and carries out live bacterial count to vaccine, and every part viable count is all not less than the head number appraised and decided.
(5) RA343 strain living vaccine is diluted to every milliliter of suspension containing 5,000,000,000 CFU bacterium by immunogenicity physiological saline, the cavy of subcutaneous injection body weight 350 ~ 400g 10, every 0.1ml.After 30 days, attack poison with the inoculation of strong malicious B. abortus 2308 bacterial strain (about 30 CFU) of 3 minimal infecting doses, attack poison and within latter 30 days, cut open and kill, get spleen and smash to pieces and make brucella separation and Culture, the cavy of more than 80%, all without strong malicious appearance, the results are shown in Table 3.
Table 3 brucella disease live-vaccine (RA343 strain) immunogenicity assay
Note :+: be separated to bacterium, be judged to and do not protect;-: be not separated to bacterium, be judged to protection.*: mouse number.
Claims (2)
1. a strain B. abortus less-virulent strain RA343, it is characterized in that the screening method adopting paraxin to combine with A factor serum, screening obtains the rough type B. abortus that the sick clinical diagnosis of cloth is not disturbed in a strain, after the vaccine immunity animal of preparing with this bacterium, its serum can be distinguished with natural infection mutually with the agglutination test of routine, B. abortus (Brucellaabortus) RA343 bacterial strain delivered the China Microbiological preservation council of Institute of Microorganism, Academia Sinica common micro-organisms preservation center, No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City on 05 20th, 2014, preserving number is: CGMCCNo.8886.
2. a production method for Brucella live vaccine, is characterized in that with the substratum of pancreas peptone soybean broth as brucella CGMCCNo.8886 strain; By 1% ~ 2% access brucella CGMCCNo.8886 strain kind daughter bacteria liquid cultivating base unit weight after medium sterilization; 37 DEG C; fermentation culture 28 ~ 38h according to a conventional method; add the lyophilized vaccine that veterinary biologics is conventional; through vacuum freezedrying after in abundant mixing, packing vaccine bottle, namely become rough type RA343 strain B. abortus disease vaccine.
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| CN104862242B (en) * | 2015-03-20 | 2018-03-16 | 中国食品药品检定研究院 | A kind of Brucella vaccine |
| CN104845916B (en) * | 2015-05-28 | 2017-12-12 | 中国兽医药品监察所 | One plant of rough type brucella melitensis low virulent strain and its vaccine |
| CN105039233B (en) * | 2015-08-25 | 2018-10-23 | 内蒙古华希生物科技有限公司 | A kind of B. abortus molecular marker vaccine strain and its application |
| PE20201285A1 (en) * | 2017-11-24 | 2020-11-24 | Consejo Superior Investigacion | MODIFIED BRUCELLA VACCINAL STRAIN FOR THE TREATMENT OF BRUCELLOSIS |
| CN110013547B (en) * | 2019-04-10 | 2023-02-03 | 中国兽医药品监察所 | Rough brucella of recombinant peste des petits ruminants virus H gene and vaccine production method thereof |
| CN109999194B (en) * | 2019-04-10 | 2023-01-31 | 中国兽医药品监察所 | Rough brucella of recombinant A-type foot-and-mouth disease virus VP1 gene and vaccine production method thereof |
| CN109939225B (en) * | 2019-04-10 | 2022-04-26 | 中国兽医药品监察所 | Rough brucella abortus of recombinant chlamydia psittaci outer membrane protein MOMP gene and vaccine production method thereof |
| CN110016457B (en) * | 2019-04-10 | 2021-03-26 | 中国兽医药品监察所 | Rough brucella abortus for recombining echinococcus granulosus Eg95gene and vaccine production method thereof |
| CN110004100A (en) * | 2019-04-10 | 2019-07-12 | 中国兽医药品监察所 | The Rough Anti-Brucella and its immunogenic production process of the one O-shaped VP 1 Gene of Foot-and-Mouth Disease virus of plant weight group |
| CN116218707B (en) * | 2022-12-14 | 2023-10-31 | 北京科牧丰生物制药有限公司 | Rough sheep brucella attenuated strain, vaccine and application thereof |
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