CN101092605A - Mutant strain of Brucella bacterin with weak poison, constructing method, and application - Google Patents
Mutant strain of Brucella bacterin with weak poison, constructing method, and application Download PDFInfo
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Abstract
This invention discloses attenuated Brucella mutant, its construction method and its application. The attenuated Brucella mutant is constructed by deleting bp26, wboA, omp31, and P39 or pgm gene from attenuated Brucella. It is proven by experiments that attenuated Brucella mutant has good immune protection effects and the same virulence as attenuated Brucella. Because certain antigen is delected in the attenuated Brucella mutant, vaccine immunity and natural infection can be differentiated according to the immunobiology of the deleted gene. The attenuated Brucella mutant can be used to manufacture preventative Brucella vaccine and marked attenuated vaccine, and prevent epidemic diseases caused by Brucella.
Description
Technical field
The present invention relates to vaccine mutant strain and construction process thereof and application, particularly relate to a kind of brucella attenuated vaccine mutant strain and construction process thereof and its application in preparation brucella preventative vaccine.
Background technology
Brucellosis (cloth disease) is a kind of popular extensive, the very harmful zoonosis that is caused by brucella, be the Category B notifiable disease in 35 kinds of transmissible diseases putting down in writing in " People's Republic of China's law on the prevention and control of infectious diseases ", and be listed in first of two class transmissible diseases of putting down in writing in " domestic animals and fowls epidemic prevention regulations detailed rules for the implementation ".Up to now, this disease has fed through to five continents, the world, and there is the existence of people and animals' cloth disease and popular existing more than 160 countries and regions in more than 200 countries.The cloth disease is popular in the world at present the widest, and one of zoonosis that harm is maximum seriously affects people ' s health and animal husbandry development.The people suffers from this disease, and stadium is several months, several years, even the more than ten years, severe patient even disability.Domestic animal suffers from cloth and occurs miscarriage, stillborn foetus, infertile, mazoitis and testitis etc. after being ill, and the annual financial loss that causes because of domestic animal cloth disease is up to multi-million dollar.The cloth disease is wide in the popular scope of China, harm is serious, has carried out the control of comprehensive system after new China sets up, thereby has controlled the generation of cloth disease and popular effectively.Yet since the nineties, the domestic and international epidemic situation of this disease is obvious ascendant trend, is listed in the transmissible disease of wreaking havoc once again, causes extensive concern both domestic and external from eighties of last century.Be control animal cloth disease, China successfully develops brucella sheep kind M
5Attenuated vaccine and pig kind S
2Attenuated vaccine, and carried out large-area applying has played enormous function to the anti-system of animal cloth diseases such as ox, sheep, pig, deer, and economic benefit and social benefit are very remarkable.In addition, the people is adopted professional population and compromised high risk population in the 104M attenuated live vaccine immunity epidemic-stricken area of external introduction, also controlled brucellar infection effectively.But, the process that is used for preventing and treating the cloth disease at cloth Salmonella vaccine, also exist the problem that natural infection and vaccine immunity are difficult to differentiate, extremely similar because people and animals use the various sero-reactions that produced after the live vaccine with the various serological reactions that natural infection is produced.Differential diagnosis is not only concerning the preventing and controlling of cloth disease, and is also significant to epidemiology survey simultaneously, and this also is one of difficult problem of the sick prevention and control field of cloth.
The subject matter of cloth disease is between brucella attenuated live vaccines control people, animal: the antibody that antibody that the inoculation back produces and natural infection produce can't differentiate that this brings difficulty for the diagnosis of cloth disease.At present, the brucella attenuated live vaccines of widespread use comprises international S19, Rev-1 and 104M, and the brucella sheep kind M of China's development
5Attenuated vaccine and pig kind S
2All there is this problem (WHO.The Development of New/ImprovedBrucellosis Vaccines:Report of WHO Meeting, 1997.) in attenuated vaccine.Recently, the U.S. filters out a kind of rough type ox kind brucella mutant strain living vaccine RB51 of attenuation from virulent strain 2308, this mutant strain does not produce corresponding antibody owing to lack O-antigen, therefore do not disturb conventional serodiagnosis, obtained government permission in the U.S. and formally used (WHO.The Development of New/Improved Brucellosis Vaccines:Report of WHOMeeting, 1997.).But, nearest bibliographical information, RB51 also produces low-level O-antigen, mean that making vaccine with RB51 still has possibility (the Cloeckaert A that disturbs conventional serology and ELISA diagnosis, Zygmunt M S, Guilloteau L A.Brucella abortus vaccine strain RB51 produces low levels ofM-like O-antigen.Vaccine.2002,20:1820-1822.).
Bp26 (GenBank number: AF242532), wboA (relevant) (GenBank number: AF107768) with the polymerization of O-antigen, omp31 (GenBank number: U39453, the function of this gene is: the albumen that forms the epicyte duct), P39 (GenBank number: AF360361, the movement system that participates in conjugated protein dependence) and pgm (GenBank number: AF232056 the function of this gene is:, phosphoglucomutase, Phosphoglucomutase, with the O-antigen group pass is housed) be the gene that derives from pathogenic agent, it is nonessential that these genes are that bacterium is duplicated, simultaneously their immunodominant antigens of encoding again.
Summary of the invention
It is good to the purpose of this invention is to provide immanoprotection action, and can infect the brucella attenuated vaccine mutant strain of differentiating with not mutated strain.
For solving the problems of the technologies described above, the present invention takes following technical scheme: brucella attenuated vaccine mutant strain, be with the gene knockout of coding immunodominant antigen albumen bp26, wboA, omp31, P39 or pgm in the brucella attenuated vaccine strain, obtain brucella attenuated vaccine mutant strain.
Described brucella attenuated vaccine strain can be brucella sheep kind M
5Strain, human 104M strain or pig kind S2 strain (above-mentioned brucella attenuated vaccine strain all can available from Chinese pharmaceutical biological product institute).
Will be wherein strain immune effect brucella sheep kind M preferably
5The bp26 genetically deficient mutant strain called after M5-26 of vaccine strain, this cell strain has been preserved in the Chinese typical culture collection center (CCTCC) that is positioned at Chinese Luojiashan, Wuchang, Wuhan City, Hubei Province Wuhan University on 03 30th, 2007, deposit number is CCTCC M207030.
Second purpose of the present invention provides a kind of method that makes up above-mentioned brucella attenuated vaccine mutant strain.
The construction process of brucella attenuated vaccine mutant strain provided by the present invention can may further comprise the steps:
1) design primer: respectively according to the nucleotide sequence of the upstream and downstream known region of target gene bp26, wboA, omp31, P39 or pgm in the brucella whole genome sequence, each designs two pairs of special PCR primers, the homology nucleotide fragments that two length in amplifying target genes upstream and downstream are 500bp to 1000bp; Connect into the homology nucleotide fragments that contains damaged gene for orientation, 5 ' end of segmental reverse primer in described amplification upstream and the segmental forward primer in amplification downstream adds a kind of identical restriction enzyme enzyme recognition site and protectiveness base thereof, 5 ' end of segmental forward primer in described amplification upstream and the segmental reverse primer in amplification downstream adds another kind of identical restriction enzyme enzyme recognition site and protectiveness base thereof, in addition, for preventing to change the single open reading frame of downstream gene, 5 ' terminal interval base number at disappearance goal gene respective regions of the forward primer of the reverse primer of amplification upstream homology nucleotide fragments and amplification downstream homology nucleotide fragments is 3 multiple;
2) acquisition of goal gene defective type homology nucleotide fragments: with brucellar genomic dna is template, under the guiding of two pairs of primers that step 1) designs, carry out pcr amplification respectively, obtain two homology nucleotide fragments, again these two upstream and downstream homology nucleotide fragments orientations are coupled together, obtain goal gene defective type homology nucleotide fragments;
3) with step 2) the goal gene defective type homology nucleotide fragments that obtains connects in the suicide vector, obtains containing the reorganization suicide vector of goal gene defective type homology nucleotide fragments;
4) the reorganization suicide vector that contains goal gene defective type homology nucleotide fragments that step 3) is made up transforms brucella, and screening positive clone obtains lacking the brucella attenuated vaccine mutant strain of goal gene.
In the construction process of above-mentioned brucella attenuated vaccine mutant strain, can be sequence 1 and sequence 2 in the sequence table according to the PCR primer of the nucleotide sequence design of the upstream known region of bp26 in the step 1), can be sequence 3 and sequence 4 in the sequence table according to the PCR primer of the nucleotide sequence design of the downstream known region of bp26; The restriction enzyme enzyme recognition site that 5 ' end of the segmental forward primer of amplification upstream segmental reverse primer and amplification downstream adds can be selected arbitrarily, and as Bgl II recognition site etc., its protectiveness base can be sequence 5 in the sequence table.The restriction enzyme enzyme recognition site that 5 ' end of the segmental reverse primer of segmental forward primer in described amplification upstream and amplification downstream adds can be definite according to carrier, as can be the BamHI recognition site, and its protectiveness base can be sequence 6 in the sequence table.
The carrier that sets out that is used to make up the reorganization suicide vector that contains goal gene defective type homology nucleotide fragments in the step 3) can be suicide vectors such as pKNG101 or pCVD442, be preferably pKNG101 (Kaniga K, DelorI, Cornelis GR.A wide-host-range suicide vector for improving reverse geneticsin gram negative bacteria:inactivation of the blaA gene of Yersiniaenterocolitica.Gene.1991,109 (1): 137-41).
The brucella starting strain that is used to transform the reorganization suicide vector that contains goal gene defective type homology nucleotide fragments in the step 4) can be brucella sheep kind M
5Strain, human 104M strain or pig kind S2 strain.
The present invention is directed to existing brucella attenuated vaccine and have problems such as being difficult to differential diagnosis; the mutant strain that has prepared them by the mode of gene knockout; the experimentation on animals result who with the mouse is model shows; the immanoprotection action of mutant strain is good; and can infect with not mutated strain and differentiate; the cloth Salmonella live vaccine mutant strain that makes up has lacked some gene of pathogenic agent; these genes are that bacterium is duplicated their immunodominant antigens of encoding again of nonessential while; and kept the immunizing power of maternal bacterial strain and virulence does not increase; in addition; because the mutant strain that the present invention makes up has lacked certain antigenic protein gene, therefore can also set up the differential diagnosis method of distinguishing vaccine immunity and natural infection according to the immunobiology characteristic of missing gene.The weak malicious mutant strain of brucella of the present invention is expected to develop into the attenuated vaccine of tape label, and popular, the propagation of control cloth disease and the sound development of national economy are had important and practical meanings, has a extensive future.
Below in conjunction with specific embodiment the present invention is described in further details.
Description of drawings
Fig. 1 is the schema of brucella attenuated vaccine mutant strain building process
Fig. 2 is the F1 and the segmental agarose gel electrophoresis detected result of F2 of pcr amplification
Fig. 3 is F1 is connected product with F2 an agarose gel electrophoresis detected result
Embodiment
Method therefor is ordinary method if no special instructions among the following embodiment, and concrete steps can be referring to " Molecular Cloning:A Laboratory Manual " (Sambrook, J., Russell, David W., Molecular Cloning:A Laboratory Manual, 3
RdEdition, 2001, NY, Cold SpringHarbor).It is synthetic that the primer is given birth to the worker by Shanghai.
One, brucella sheep kind M
5The structure of vaccine strain bp26 genetically deficient mutant strain
Select the condition of disappearance target gene foundation: the first, do not influence duplicating of brucella vaccine mutant after the genetically deficient, and virulence do not increase, and immunizing power does not reduce; The second, the missing gene immunodominant antigen protein of should encoding.Select wboA, bp26, omp31, P39 or pgm as the disappearance target gene that is used to make up brucella attenuated vaccine mutant strain according to top condition.Bp26 is an example with disappearance, makes up brucella sheep kind M referring to Fig. 1 with method of the present invention
5The bp26 deletion mutantion strain of vaccine strain, detailed process may further comprise the steps:
1, PCR design of primers
Whole genome sequence according to brucella sheep kind bacterium 16M strain; at two couples of special PCR primer P1A/P1B and P2A/P2B of bp26 gene upstream and downstream design; segmental reverse primer P1B adds identical restricted Nucleotide restriction endonuclease Bgl II recognition site and protectiveness base (GAAGATCT thereof with 5 of the segmental forward primer P2A in downstream '-end in the upstream; sequence 5 in the sequence table); in the upstream 5 of segmental forward primer P1A and the segmental reverse primer P2B in downstream '-end; add BamHI restriction enzyme site and protectiveness base (CGGGATCC thereof respectively; sequence 6 in the sequence table), primer sequence is as follows:
P1A:5 '-CGGGATCCGGTTTGTAACGATTTTAAATTA-3 ' (sequence 1 in the sequence table)
P1B:5 '-GAAGATCTTATCGACCCCATACTGCGGGAA-3 ' (sequence 2 in the sequence table);
P2A:5 '-GAAGATCTTTTTAGAGGATGTCCTTTTC-3 ' (sequence 3 in the sequence table)
P2B:5 '-CGGGATCCAAGACCACTGCCGCATACCG-3 ' (sequence 4 in the sequence table).
2, the acquisition of goal gene defective type homology nucleotide fragments
1) pcr amplification
Genomic dna with brucella sheep kind bacterium 16M strain (available from Chinese pharmaceutical biological product institute) is a template, under the guiding of two pairs of primers that step 1 designs, carry out pcr amplification respectively, 50 μ l reaction systems are: 10 * PCR damping fluid, 5 μ l, upstream primer (5uM) 2 μ l, downstream primer (5uM) 2 μ l, dNTPs (10 μ M) 0.5 μ l, rTaq (5u/ul) 0.4 μ l, water 38. μ l, brucella genomic dna (20ng/ μ l) 2 μ l.The pcr amplification condition is: 95 ℃ of 3min of elder generation; 94 ℃ of 30sec then, 58 ℃ of 40sec, 72 ℃ of 1min, totally 30 circulations, last 72 ℃ of 10min.After reaction finishes, pcr amplification product is carried out 1.0% agarose gel electrophoresis to be detected, (swimming lane 1 is the F1 fragment to detected result as shown in Figure 2, swimming lane 2 is the F2 fragment, swimming lane 3 is DNA Marker DL2000), obtained two homology nucleotide fragments through amplification, promptly length is about the F1 of 700bp and the F2 that length is about 500bp, conforms to expected results.
2) purified pcr product
PCR product purification/sepharose DNA recovery test kit of producing with Nuo Dejin biotech firm carries out purifying to the pcr amplification product of step 1), method is: get PCR product to be purified 200 μ l, with isopyknic abundant mixing of liquid that combines, of short durationly transfer in the adsorption column after centrifugal, the centrifugal 1min of 12000rpm behind the room temperature placement 1min, in post, add 600 μ l rinsing liquid AW after abandoning waste liquid, the centrifugal 1min of 12000rpm, add 500 μ l rinsing liquid AW again, behind the centrifugal 1min of 12000rpm adsorption column placed a new centrifuge tube, uncap and place 1min, thoroughly dry rinsing liquid remaining in the sorbing material, add 50 μ l sterilized water wash-outs, place 2min for 37 ℃, the centrifugal 1min of last 12000rpm collects purified product.
3) enzyme is cut the PCR product
With restriction enzyme Bgl II to step 2) two purified dna fragmentations carry out enzyme respectively and cut, the 20 μ l enzyme systems of cutting are: dna fragmentation (1000ng) 4 μ l, Bgl II restriction endonuclease (TAKARA company) 1 μ l, 10 * enzyme cutting buffering liquid, 2 μ l, water 13 μ l.The enzyme tangent condition is: 37 ℃ act on 4 hours.After reaction finished, the fragment after cutting with GeneacidPCR product test kit purifying enzyme was used the water elution (repeating to wash 2 times) of 20 μ l at last.
4) enzyme is cut the connection of product
Under the effect of T4DNA ligase enzyme, two endonuclease bamhi F1 are connected spend the night (12-24 hour) with F2 under 4 ℃, linked system is: fragment F1 enzyme is cut product 200ng, and fragment F2 enzyme is cut product 200ng, T
4Dna ligase 1 μ l, 10 * ligase enzyme damping fluid, 1 μ l mends and adds water to 10 μ l.After ligation finishes, carry out 1% agarose gel electrophoresis to connecting product, (swimming lane 1 is for connecting product as shown in Figure 3 for the result, swimming lane 2 is DNA MarkerDL2000), reclaim test kit with PCR product purification/sepharose DNA and cut glue recovery F1-F2 target DNA fragment, reclaim and purification process is: weigh and write down the weight of little centrifuge tube, from sepharose, cut F1-F2 purpose fragment under the ultraviolet lamp and put into the little centrifuge tube that weighs up, weigh, calculate blob of viscose weight, every 10mg blob of viscose adds 10 μ l in conjunction with liquid BS, the vortex vibration evenly, 50-65 ℃ of water-bath effect 10min, during shake frequently, blob of viscose is dissolved fully.Instantaneous centrifugal, all be transferred to solution in the adsorption column, room temperature is placed 1min, and the centrifugal 1min of 12000rpm abandons waste liquid, adsorption column is put back in the collection tube, add 600 μ l rinsing liquid AW again, the centrifugal 1min of 12000rpm abandons waste liquid, adsorption column is put back in the collection tube, add 500 μ l rinsing liquid AW, the centrifugal 1min of 12000rpm abandons waste liquid, adsorption column is put back in the collection tube, the centrifugal 2min of 12000rpm goes to adsorption column in the new centrifuge tube, uncaps and places 1min, thoroughly dry rinsing liquid remaining in the sorbing material, add 50 μ l sterilized waters to adsorption column, room temperature is placed 2min, the last centrifugal 1min of 12000rpm, obtain F1-F2 target DNA purification of samples, with this as goal gene defective type homology nucleotide fragments.
3, make up the suicide vector that contains goal gene defective type homology nucleotide fragments
1) enzyme is cut F1-F2 fragment and suicide plasmid
Digest the F1-F2 fragment of about 500ng and the suicide plasmid pKNG101 of about 1000ng respectively with BamHI (50U), 20 μ l endonuclease reaction systems and reaction conditions are: F1-F2 or pKNG101 (500ng or 1000ng) 4 μ l, BamHI restriction endonuclease 10 * damping fluid 2 μ l, water 13 μ l, behind effect 12h under 30 ℃, use Geneacid PCR product test kit purifying digestion product respectively, method is: 100 μ l reaction product are placed an Eppendorf tube, the DF damping fluid that adds 5 times of volumes, the vibration mixing, be added in the centrifugal post of the DF that collection tube is housed, the centrifugal 30sec of 13000rpm installs to the DF post on the collection tube after abandoning waste liquid again, Xiang Zhuzhong adds 600 μ l Wash damping fluids, the centrifugal 30sec of 13000rpm installs to the DF post on the collection tube after abandoning waste liquid again, the dry centrifugal post of the centrifugal 3min of 13000rpm, dried centrifugal post is installed on the new centrifuge tube, add 15-50 μ l aseptic deionized water, room temperature is placed 3min, and the centrifugal 2min of last 13000rpm collects the DNA sample of purifying.
2) the F1-F2 fragment is connected with plasmid
Under the effect of T4DNA ligase enzyme, with in the step 1) through enzyme cut and purifying after F1-F2 fragment and pKNG101 enzyme cut product and be connected at 4 ℃ and spend the night, fragment F1-F2 is inserted the BamHI site of suicide plasmid, obtain containing the segmental recombinant vectors of F1-F2, called after pKNG101/F1-F2, the ligation system is: F1-F2 fragment enzyme is cut product 200ng, and the pKNG101 enzyme is cut product 100ng, T
4Dna ligase 1 μ l, 10 * damping fluid, 1 μ l mends and adds water to 10 μ l.
3) preparation intestinal bacteria SPY372 λ pir competence
Inoculation recipient bacterium intestinal bacteria SPY372 λ pir (Millert VL, Mekalanos JJ.A Novel SuicideVector and Its Use in Construction of Insertion Mutations:Osmoregulation ofOuter Membrane Proteins and Virulence Determinants in Vibrio cholerae RequirestoxR.Journal of Bacteriology.170 (6): 2575-2583) in 3mL LB broth culture (tryptone 10g, yeast extract 5g, NaCl10g, the 950mL deionized water) in, shaking bacterium at 37 ℃ of following 150rpm (controls the OD value and is 0.4-0.6 to muddy, be that the bacterium amount can not be too many, can not be very little), the centrifugal 5min collection of 4500rpm bacterium is with the 0.1M CaCl of 1mL
2Wash bacterium twice (with kapillary bacterium is hanged gently, 4 ℃ of centrifugal 5min of following 5000rpm abandon supernatant), add the 0.1M CaCl of 1mL
2Resuspended thalline, 4 ℃ are spent the night, standby in 24 hours.The competence bacteria transformation efficiency of preparation of spending the night is the highest, but the shelf-time can not oversize (being no more than 48h), otherwise the competence bacterium is easily aging.Get the competence bacteria of 100 μ l preparation, insert the LB meat soup (Ap that 3mL contains 40 μ g/mL penbritins
R) in, incubated overnight does not have thalli growth, determines not pollute.
4) transformed into escherichia coli SPY372 λ pir
Get the intestinal bacteria SPY372 λ pir competence bacterium of 100-200 μ l step 3) preparation, join in the centrifuge tube of 1.5mL, add the connection product that 10 μ l step 3) obtain again, behind the ice bath 30min, in 42 ℃ of (water-bath) thermal shockings 90 seconds, ice bath 3min, the LB meat soup that adds 400 μ l, the centrifugal mouth of pipe is tight with sealing film envelope, shakes 2 hours under 37 ℃, gets 150 μ l and is applied to the LB resistant panel (Ap that contains 40 μ g/mL penbritins
R), be inverted in 37 ℃ of incubators, cultivated 12-16 hour, picking 10-20 clone, cultivated 12 hours in increase bacterium on the LB agar plate under 37 ℃, the PCR reaction that guides with special primer P1A and P2B filters out positive colony, can amplify the segmental positive clone of 1200bpDNA.
4, transform brucella
1) extracts the suicide plasmid that contains goal gene defective type homology nucleotide fragments
Extract with GENEAID plasmid trace the positive colony of the intestinal bacteria SPY372 λ pir that test kit filters out from step 3 and extract suicide plasmid, will identify that correct intestinal bacteria SPY372 λ pir positive colony is inoculated into the LB meat soup (Ap of 6mL
R) in the substratum, cultivate 6-8 hour (OD value 0.6-0.8) down at 37 ℃, the centrifugal 1min collection of 12000rpm bacterium abandons supernatant, adds 200 μ lPD1 (RNase A added), abundant mixing; Add 200 μ lPD2 again, spin upside down 4-6 time, this moment, solution became clear; Add 300 μ l PD3, spin upside down 4-6 back and produce a large amount of white flockss, the centrifugal 10min of 12000rpm; Supernatant carefully is transferred in the centrifugal post, avoids having the bacterial sediment particle, the centrifugal 1min of 12000rpm outwells centrifugal liquid in pipe; Add 400 μ lW1 solution in centrifugal post, the centrifugal 1min of 12000rpm outwells centrifugal liquid in pipe; Add 600 μ lWash Buffer solution in centrifugal post, the centrifugal 1min of 12000rpm outwells centrifugal liquid in pipe; Centrifugal again 1min dries washing lotion in the post; Add 20 μ l aseptic deionized waters, the centrifugal 1min of 12000rpm collects plasmid.Get 2 μ l plasmids, add 2 μ l load sample damping fluids, moisturizing to 10 μ l carries out 1% agarose gel electrophoresis and detects, and the result is the bright fine and close band of about 8400bp as seen.
2) brucella melitensis M
5Strain electricity transformed competence colibacillus cell preparation
With brucella melitensis M
5Strain (available from Chinese pharmaceutical biological product institute) is inoculated in the 5mL LB liquid nutrient medium, 37 ℃ of low-speed oscillations cultivate 6 hours to the OD value be 0.6-0.8, to shock by electricity the cup (1mm) and an aseptic deionized water precooling on ice bath in advance, collect thalline at 4 ℃ of centrifugal 5min of following 4000rpm, with isopyknic precooling aseptic deionized water (5mL) washed twice, centrifugal collection bacterium, use isopyknic precooling aseptic deionized water (5mL) to suspend evenly again, be distributed into 50 μ l systems, put on the ice bath standby.
3) electricity transforms
Get the recombinant plasmid (about 100ng) of 1 μ l pKNG101/F1-F2, join step 2) in the 50 μ l competence bacteriums (precooling 10min) of prepared fresh, slight mixing, put and continue to place 15min on ice, join (adherent adding avoids bubble to produce) in the electric shock cup of precooling, under the shock parameters of 25 μ F, 200 Ω, 1.8Kv, shock by electricity.Add the LB liquid nutrient medium suspension bacterium of 500 μ l after the electric shock immediately, the cell that will shock by electricity forwards in the centrifuge tube, and 37 ℃ of low-speed oscillations were cultivated 2 hours, got 100 μ l and were applied on the LB agar plate (claiming LB-sucrose flat board again) that contains 5% sucrose 37 ℃ of incubated overnight.
6) mutant strain screening and evaluation
Each picking 5-6 clone from each LB-sucrose flat board, spread upon LB-sucrose and increase the bacterium cultivation after 12 hours for dull and stereotyped last 37 ℃, respectively get about 2mg bacterium and place the tubule that contains 50 μ l sterile distilled waters, mixing, after boiling 5 minutes, the centrifugal 10min of 10000rpm, with the supernatant liquor is template, carry out pcr amplification with primer P1A/P2B, select the cloning of small fragment that only amplifies 1200bp F1-F2 length, on LB-sucrose flat board, dilute cultivation again, respectively get 5-6 clone and repeat PCR and identify, will be continuous be positive colony as the goal gene defective mutant through the PCR evaluation 3 times.Mutant strain is carried out sequencing, show to have obtained the correct brucella M5 vaccine strain bp26 genetically deficient mutant strain of sequence.
Two, brucella M
5The virulence of vaccine strain bp26 genetically deficient mutant strain and immanoprotection action are measured
1, the toxicity test of mutant strain
Get TSA (tryptose soy agar) substratum (Tryptones 17.0g, soya peptone 3.0g, glucose 2.5g, NaCl 5.0g, K
2HPO
42.5g, agar 20.0g, distilled water 1000mL) go up the brucella M of growth
5The mutant strain bacterium colony of the bp26 genetically deficient that vaccine strain and step 1 obtain makes 1 * 10 with PBS respectively
5The CFU bacteria suspension is divided into three groups with all female BALB/c mouse of 75 6-8, more at random respectively at every injected in mice 0.2mL of intraperitoneal brucella M
5Vaccine strain and bp26 transgenation strain bacterium liquid (M
5Mutant strain), make blank at every injected in mice 0.2mL physiological saline of another group.Respectively the metainfective the 8th, 15,28,42 and 60 days, from every group, get 5 mouse and put to death, aseptic condition takes out spleen down, weighs, adds then the PBS homogenate of 5mL, get 0.1mL behind 10 times of serial dilutions and be seeded on the TSA plate culture medium, cultivate bacterium numeration (CFU) after 5 days for 30 ℃, calculate the seedling number of living in the full spleen, be converted into logarithmic value at last, the result as shown in Table 1 and Table 2.Table 1 has shown brucella M
5Vaccine strain and M
5Bacterium numeration situation in the vaccine mutant strain spleen, table 2 has shown the inflammatory response situation behind the two infecting mouse.Behind the two infecting mouse in the spleen viable bacteria numeration and spleen weight as can be seen, brucella M
5Vaccine strain and M of the present invention
5The virulence of vaccine mutant strain does not have significant difference, and promptly the virulence of mutant strain does not increase.
Table 1 brucella M
5Vaccine strain and M
5Bacterium numeration result in the vaccine mutant strain spleen
| Group | Log 10The CFU/ spleen | ||||
| 8 days | 15 days | 28 days | 42 days | 60 days | |
| M5 vaccine strain M5 mutant strain blank | 8.21 7.84 0.00 | 8.28 7.67 0.00 | 5.76 5.37 0.00 | 4.63 4.24 0.00 | 2.98 2.18 0.00 |
Annotate: every group of measurement result is the mean value of 5 mouse.
Table 2 brucella M
5Vaccine strain and M
5The spleen re-computation result of vaccine mutant strain
| Group | Spleen heavy (mg) | ||||
| 8 days | 15 days | 28 days | 42 days | 60 days | |
| M 5Vaccine strain M 5The mutant strain blank | 375.12 364.45 345.23 | 578.12 532.67 327.14 | 523.25 576.38 297.47 | 417.78 394.29 302.76 | 334.45 332.37 327.85 |
Annotate: every group of measurement result is the mean value of 5 mouse.
2, measure brucella sheep kind M
5The immanoprotection action of vaccine strain bp26 genetically deficient mutant strain
Get the brucella S2308 that grows on TSA (the tryptose soy agar) substratum (available from Chinese pharmaceutical biological product institute) bacterium colony, make 5 * 10 with PBS
4The CFU bacteria suspension is got brucella sheep kind M
5Vaccine strain or bp26 genetically deficient mutant strain immune mouse are after 10 weeks, and 10 mouse are distinguished 5 * 10 of peritonaeum inside fire attack poison 0.2mL in each group
4CFU brucella strain S2308, establish the blank group simultaneously, attack poison after 15 days, put to death mouse, take out spleen, PBS homogenate with 5mL, behind the PBS serial dilution, get 0.1mL inoculation TSA flat board (add 0.1% red tinea sugar alcohol in the flat board and guarantee to have only S2308 to grow), cultivate bacterium numeration (CFU) after 5 days for 30 ℃, calculate viable count in the full spleen, the results are shown in Table 3.As can be seen from Table 3, respectively by M
5Vaccine strain and M
5Vaccine mutant strain mice immunized group is compared with the blank group, and the viable bacteria numeration obviously reduces in the spleen, shows that vaccine strain and vaccine mutant strain all have good immunoprotective effect.The immune effect that will obtain with aforesaid method is brucella sheep kind M preferably
5Vaccine strain bp26 genetically deficient mutant strain called after M5-26, this cell strain has been preserved in the Chinese typical culture collection center (CCTCC) that is positioned at Chinese Luojiashan, Wuchang, Wuhan City, Hubei Province Wuhan University on 03 30th, 2007, deposit number is CCTCC M207030.
Table 3 brucella M
5Vaccine strain and M
5Vaccine mutant strain immanoprotection action measurement result
| Group | Intermediate value (CFU/ spleen) | Scope (CFU/ spleen) |
| M 5Vaccine strain M 5Mutant strain blank group | 2.6×10 4 1.3×10 4 1.6×10 7 | 2.6×10 3-3.7×10 57.4×10 3-7.5×10 54.8×10 6-2.7×10 8 |
Annotate: every group of result who measures 10 mouse.
Sequence table
<160>6
<210>1
<211>29
<212>PRT
＜213〉artificial sequence
<220>
<223>
<400>1
cgggatccggt ttgtaacgat tttaaatta 29
<210>2
<211>29
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>2
gaagatcttat cgaccccata ctgcgggaa 29
<210>3
<211>27
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>3
gaagatctttt tagaggatgt ccttttc 27
<210>4
<211>27
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>4
cgggatccaag accactgccg cataccg 27
<210>5
<211>7
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>5
gaagatct 7
<210>6
<211>7
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>6
cgggatcc 7
Claims (10)
1, brucella attenuated vaccine mutant strain is the brucella attenuated vaccine mutant strain that obtains behind brucella attenuated vaccine strain disappearance bp26, wboA, omp31, P39 or the pgm gene.
2, brucella attenuated vaccine mutant strain according to claim 1 is characterized in that: described brucella attenuated vaccine strain is sheep kind M
5Strain, human 104M strain or pig kind S
2Strain.
3, brucella attenuated vaccine mutant strain according to claim 1 is characterized in that: be the brucella attenuated vaccine mutant strain M5-26 of CCTCC M207030 for biological preserving number.
4, a kind of method that makes up the described brucella attenuated vaccine of claim 1 mutant strain may further comprise the steps:
1) respectively according to the nucleotide sequence of the upstream and downstream known region of target gene bp26, wboA, omp31, P39 or pgm in the brucella whole genome sequence, each designs two pairs of special PCR primers, the homology nucleotide fragments that two length in amplifying target genes upstream and downstream are 500bp to 1000bp; Connect into the homology nucleotide fragments that contains damaged gene for orientation, 5 ' end of segmental reverse primer in described amplification upstream and the segmental forward primer in amplification downstream adds a kind of identical restriction enzyme enzyme recognition site and protectiveness base thereof, 5 ' end of segmental forward primer in described amplification upstream and the segmental reverse primer in amplification downstream adds another kind of identical restriction enzyme enzyme recognition site and protectiveness base thereof, in addition, for preventing to change the single open reading frame of downstream gene, 5 ' terminal interval base number at disappearance goal gene respective regions of the forward primer of the reverse primer of amplification upstream homology nucleotide fragments and amplification downstream homology nucleotide fragments is 3 multiple;
2) be template with brucellar genomic dna, under the guiding of two pairs of primers that step 1) designs, carry out pcr amplification respectively, obtain two homology nucleotide fragments, again these two upstream and downstream homology nucleotide fragments orientations are coupled together, obtain goal gene defective type homology nucleotide fragments;
3) with step 2) the goal gene defective type homology nucleotide fragments that obtains connects in the suicide vector, obtains containing the reorganization suicide vector of goal gene defective type homology nucleotide fragments;
4) the reorganization suicide vector that contains goal gene defective type homology nucleotide fragments that step 3) is made up transforms brucella, and screening positive clone obtains lacking the brucella attenuated vaccine mutant strain of goal gene.
5, construction process according to claim 4; it is characterized in that: the restriction enzyme enzyme recognition site that 5 ' end of the segmental forward primer of segmental reverse primer in amplification upstream and amplification downstream adds in the described step 1) is the BglII recognition site; its protectiveness base is the sequence 5 in the sequence table; the restriction enzyme enzyme recognition site that 5 ' end of the segmental reverse primer of segmental forward primer in amplification upstream and amplification downstream adds is a BamH I recognition site, and its protectiveness base is the sequence 6 in the sequence table.
6, construction process according to claim 5, it is characterized in that: being sequence 1 and sequence 2 in the sequence table according to the PCR primer of the nucleotide sequence design of the upstream known region of bp26 in the described step 1), is sequence 3 and sequence 4 in the sequence table according to the PCR primer of the nucleotide sequence design of the downstream known region of bp26.
7, according to claim 4 or 5 or 6 described construction processs, it is characterized in that: the carrier that sets out that is used to make up the reorganization suicide vector that contains goal gene defective type homology nucleotide fragments in the described step 3) is pKNG101 or pCVD442.
8, according to claim 4 or 5 or 6 described construction processs, it is characterized in that: the brucella starting strain that is used to transform the reorganization suicide vector that contains goal gene defective type homology nucleotide fragments in the described step 4) is brucella sheep kind M
5Strain, human 104M strain or pig kind S
2Strain.
9, claim 1 or the 2 or 3 described brucella attenuated vaccine mutant strains application in preparation brucella preventative vaccine.
10, claim 1 or the 2 or 3 described brucella attenuated vaccine mutant strains application in preparation discriminating brucella vaccine immunity and natural infection reagent.
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| CN102258775A (en) * | 2011-07-20 | 2011-11-30 | 河南亚卫动物药业有限公司 | Recombinant vaccinia virus vaccine for treating brucella abortus disease and preparation method thereof |
| CN102327606A (en) * | 2011-09-06 | 2012-01-25 | 中国兽医药品监察所 | Brucella live vaccine and production method thereof |
| CN104845916A (en) * | 2015-05-28 | 2015-08-19 | 中国兽医药品监察所 | Rough type Brucella melitensis attenuated strain and vaccine thereof |
| CN107267430A (en) * | 2016-04-07 | 2017-10-20 | 中国人民解放军军事医学科学院生物工程研究所 | Brucella 104M vaccine strains knock out recombinant bacterium and the application of Omp25 genes |
| CN108037277A (en) * | 2017-12-07 | 2018-05-15 | 中国兽医药品监察所 | It is a kind of to distinguish Brucella abortus vaccine immunity and the method for natural infection state |
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| CN112852698A (en) * | 2021-01-30 | 2021-05-28 | 军事科学院军事医学研究院军事兽医研究所 | Construction method and application of asd gene deletion strain of Brucella A19 strain |
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| CN102258775A (en) * | 2011-07-20 | 2011-11-30 | 河南亚卫动物药业有限公司 | Recombinant vaccinia virus vaccine for treating brucella abortus disease and preparation method thereof |
| CN102327606A (en) * | 2011-09-06 | 2012-01-25 | 中国兽医药品监察所 | Brucella live vaccine and production method thereof |
| CN102327606B (en) * | 2011-09-06 | 2013-05-01 | 中国兽医药品监察所 | Brucella live vaccine and production method thereof |
| 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 |
| CN104845916A (en) * | 2015-05-28 | 2015-08-19 | 中国兽医药品监察所 | Rough type Brucella melitensis attenuated strain and vaccine thereof |
| CN107267430B (en) * | 2016-04-07 | 2020-04-10 | 中国人民解放军军事医学科学院生物工程研究所 | Recombinant bacterium of Brucella 104M vaccine strain with Omp25 gene knocked out and application |
| CN107267430A (en) * | 2016-04-07 | 2017-10-20 | 中国人民解放军军事医学科学院生物工程研究所 | Brucella 104M vaccine strains knock out recombinant bacterium and the application of Omp25 genes |
| CN108037277A (en) * | 2017-12-07 | 2018-05-15 | 中国兽医药品监察所 | It is a kind of to distinguish Brucella abortus vaccine immunity and the method for natural infection state |
| CN108392498A (en) * | 2018-05-24 | 2018-08-14 | 中国人民解放军军事科学院军事医学研究院 | Mouse model is immunized in the bacterium attenuated live vaccine liquid aersol lung delivering of cloth Shandong |
| CN110484484A (en) * | 2019-08-29 | 2019-11-22 | 西北农林科技大学 | One plant of brucella S2 vaccine strain ArsR gene deletion strains, its construction method and application |
| CN112852698A (en) * | 2021-01-30 | 2021-05-28 | 军事科学院军事医学研究院军事兽医研究所 | Construction method and application of asd gene deletion strain of Brucella A19 strain |
| CN112852698B (en) * | 2021-01-30 | 2022-11-29 | 军事科学院军事医学研究院军事兽医研究所 | Construction method and application of Brucella A19 strain asd gene deletion strain |
| CN116218707A (en) * | 2022-12-14 | 2023-06-06 | 北京科牧丰生物制药有限公司 | Rough sheep brucella attenuated strain, vaccine and application thereof |
| 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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Open date: 20071226 |