CN109568572A - A kind of preparation method and applications of Aeromonas Multivalent DNA Vaccine - Google Patents
A kind of preparation method and applications of Aeromonas Multivalent DNA Vaccine Download PDFInfo
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Abstract
The invention discloses a kind of preparation method and applications of Aeromonas Multivalent DNA Vaccine, belong to disease control of aquatic animal technical field.Technical solution of the present invention main points are as follows: designed for expanding the specific primer of the main HpaA gene of Aeromonas hydrophila, primer sequence mah-F:5 ' CGCGGATCCATGAAAAAGACAATTCTGGC 3'；Mah-R:5 ' CCCAAGCTTTTAGAAGTTGTATTGCAGGG 3 ', underscore are restriction enzyme site；Bacteria Culture simultaneously extracts Aeromonas hydrophila DNA；The main HpaA gene segment of Aeromonas hydrophila, using pcDNA3.1 plasmid as carrier, construction recombination plasmid and sequence verification are expanded, mah-pcDNA3.1 recombinant plasmid i.e. Aeromonas Multivalent DNA Vaccine is named as.DNA vaccination of the invention is contained by cultured fishes immunity inoculationmahThe recombinant vaccine of gene generates main adhesin antibodies in fish body and regulates and controls the expression of anti-infectious immunity related gene, to effectively prevent the infection of a variety of Aeromonas.
Description
Technical field
The invention belongs to disease control of aquatic animal technical fields, and in particular to a kind of Aeromonas Multivalent DNA Vaccine
Preparation method and applications.
Background technique
Aeromonas is the cause of disease disease that a major class is widely present in various freshwater aquiculture water bodys, it can cause a variety of aquatic
Animal morbidity, to lead to aquatic livestock haemorrhage, causes serious economic loss to culture fishery, is China's cultured freshwater fish
The main pathogen of class Outbreak-infective disease.In addition, Aeromonas not only infects freshwater fish or what people-poultry-fish suffered from altogether causes a disease
Microorganism can cause animal local infection or systemic sepsis, or even cause the acute gastroenteritis, septicemia, wound sense of people
Dye, tympanitis, peritonitis etc..As people are continuously increased Aquatic products consumption amount, the status as pathogenic microorganisms is
It is determined, and has become the important object of current food public health concern, by aquatic products educational circles, animal doctor educational circles and medicine
The extensive attention on boundary.Therefore, aquatic animal disease caused by effective prevention and control Aeromonas pacifies aquaculture and aquatic product quality
There is extremely important meaning entirely.
Aeromonas hydrophila is one of most common pathogen in Aeromonas, course of infection include adherency, intrusion,
Breeding etc..It causes a disease in view of Aeromonas hydrophila to heaviness economic loss caused by freshwater aquaculture industry, people have been devoted to seek
Look for a kind of safe and effective prevention method.Fish vaccine has safe, hygienic, efficient spy as a kind of novel prophylactic measures
Property, the increasingly approval and attention by countries in the world becomes and gradually replaces traditional antibiotic diseases prevention mode.Traditional vaccine
It is mainly attenuated live seedling and inactivated vaccine, these vaccines is produced and used extensively, but there is also many disadvantages.Nucleic acid vaccine
As third generation of vaccine technology, refers to and candidate antigen gene is cloned into carrier, rather than in engineering strain or cell,
Express antigen protein by being transferred in host cell, so induce host itself specific immune protection function, reach to
The effect of imperial pathogenic bacterial infection.A large number of studies show that DNA vaccination can induce the intracorporal antigen specific immune of fish and cell to exempt from
Epidemic disease, in addition, the expression of antigen protein can also induction of lymphocyte differentiate cytotoxic T-Lymphocyte (CTLs), cytotoxic T-Lymphocyte
Pathogenic cell can be killed or pathogenic bacterial infection is inhibited by non-cytolytic mode.As newest vaccine technologies, DNA vaccination was both
Safety has the ability for inducing comprehensive immune response again, and has and be easy to construct, and preparation is simple, and stability is high, can generate
Efficient immanoprotection action and can use of large-scale production the advantages of, receive significant attention.
Aeromonas hydrophila serotype is numerous, and biochemical phenotype and antigenicity are complicated, selects protective antigens particularly important.Mesh
It is preceding to be concentrated mainly on Bacterial outer membrane proteins, such as OmpA, OmpW, porin (porin) and Omp38 egg about nucleic acid vaccine research
It is white.Adhesin plays important adhesive attraction and Protection of antigen during Aeromonas hydrophila infects host, not only can
The panimmunity mechanism including humoral immunity of body is excited, immunological cross can also be generated with different serotypes pathogen
Reaction, is the common protective antigens of Aeromonas hydrophila.Some researches show that clonal expression Aeromonas hydrophila adhesins to merge
Albumen has good antigenicity and immunogenicity, and adhesin can be significantly reduced bacterium to the adhesive attraction of host, realize resistance
Disconnected invasion of the pathogenetic bacteria to host break through the numerous obstacle of Aeromonas hydrophila serotype, effectively prevent Aeromonas hydrophila
Infection.However, there is presently no the reports of adhesin nucleic acid vaccine.Therefore, the present invention provides a kind of Aeromonas mah
The construction method of DNA vaccination, and demonstrate application of the DNA vaccination in fish disease prevention and cure.
Summary of the invention
The technical problem to be solved by the present invention is to provide a kind of preparation method of novel Aeromonas Multivalent DNA Vaccine and its
Using the Multivalent DNA Vaccine is generated in fish body main by the recombinant vaccine of cultured fishes immunity inoculation gene containing mah
Adhesin antibodies and the expression for regulating and controlling anti-infectious immunity related gene, to effectively prevent the infection of a variety of Aeromonas.
The present invention adopts the following technical scheme that solve above-mentioned technical problem, a kind of system of Aeromonas Multivalent DNA Vaccine
Preparation Method, it is characterised in that detailed process are as follows:
Step S1: the specific primer designed for expanding the main HpaA gene of Aeromonas hydrophila, primer sequence are
Mah-F:5 ' CGCGGATCCATGAAAAAGACAATTCTGGC 3'；Mah-R:5 '
CCCAAGCTTTTAGAAGTTGTATTGCAGGG 3 ', underscore are restriction enzyme site；
Step S2: Bacteria Culture simultaneously extracts Aeromonas hydrophila DNA；
Step S3: the amplification main HpaA gene segment of Aeromonas hydrophila, using pcDNA3.1 plasmid as carrier, building weight
Group plasmid and sequence verification, are named as mah-pcDNA3.1 recombinant plasmid i.e. Aeromonas Multivalent DNA Vaccine.
Further preferably, the preparation method of the Aeromonas Multivalent DNA Vaccine, it is characterised in that specific steps are as follows:
Step S1: the extraction of Aeromonas hydrophila genomic DNA
Aeromonas hydrophila is obtained by this laboratory from separation in bacterial enteritis disease fish is suffered from, and bacterium is in brain heart infusion broth
Culture, with conventional DNA extraction kit purification of bacterial genomic DNA；
Step S2: the specific primer design of the main HpaA gene of Aeromonas hydrophila
Designed for expanding the specific primer of the main HpaA gene mah of Aeromonas hydrophila, primer sequence mah-
F:5 ' CGCGGATCCATGAAAAAGACAATTCTGGC 3'；Mah-R:5 ' CCCAAGCTTTTAGAAGTTGTATTGCAGGG
3 ', underscore is restriction enzyme site；
Step S3: the PCR amplification of Aeromonas hydrophila genomic DNA
Take 1 μ L Aeromonas hydrophila genomic DNA for template, each 2.5 μ L of 1 μ L, dNTP of target gene upstream and downstream primer,
PCR Buffer 2.5 μ L, ExTaq 0.25 μ L, ddH2O complement to 25 μ L, PCR reaction conditions: 95 DEG C of 5min；94 DEG C of 30s, 55
DEG C 30s, 72 DEG C of 1min, 30 circulations；72 DEG C of extension 10min, with the Ago-Gel testing goal clip size of 1wt%, glue
Recycle mah target gene；
The building of step S4:mah-pcDNA3.1 recombinant plasmid
By after purification mah target gene and carrier pcDNA3.1 plasmid use restriction enzyme BamH I and Hind respectively
III carries out double digestion, digestion system are as follows: each 0.5 μ L, mah target gene of BamH I and Hind III and carrier pcDNA3.1 matter
Grain is respectively 5 μ L, 10 × buffer 2 μ L, ddH2O supplies 25 μ L, 37 DEG C, digestion 3h in metal bath, is recycled try with glue again
Agent box respectively recycles digestion products, linked system are as follows: 5 μ L of double digestion target gene fragment after purification, double enzymes after purification
Cut 1 μ L, T4 DNA Ligase of carrier, 0.5 μ L, 10 × T4 DNA buffer 1 μ L, ddH2O supplies 10 μ L, in metal bath
16 DEG C of connections overnight, 10 μ L products of overnight connection are added in 20 μ L competent escherichia coli cell DH5 α, on ice ice
30min, 42 DEG C of thermal shock 90s in water-bath are bathed, immediately ice bath 5min, 200 μ L LB liquid mediums are added, bacterium solution is coated onto
In plate with ampicillin, 37 DEG C are incubated overnight, until with macroscopic single colonie；
Step S5: the PCR detection of recombinant plasmid and sequence verification
5 single bacteriums of picking are fallen in the LB culture medium of 600 μ L, and 200r/min is expanded culture, using 1 μ L bacterium solution as mould
Plate carries out PCR detection with the universal primer on pcDNA3.1, PCR system: 2 × PCR mix, 5 μ L, the universal primer on carrier
Each 1 μ L of F/R, 1 μ L of bacterium solution, enzyme 0.25 μ L, ddH2O supplies 10 μ L, PCR conditions: 95 DEG C of 5min, 95 DEG C of 30s, 50 DEG C of 30s, and 72 DEG C
80s, 30 circulations, 72 DEG C of 10min, 1wt% agarose gel electrophoresis detects PCR product, and send the bacterium solution being proved to be successful to survey
Sequence, wherein the nucleic acid sequence of Aeromonas hydrophila mah gene is as shown in SEQ ID NO.15；
Step S6: the extraction of plasmid
Correct recombinant plasmid bacterium will be sequenced to expand culture, spend endotoxin plasmid extraction kit extraction plasmid and obtain
To mah-pcDNA3.1 recombinant plasmid, that is, Aeromonas Multivalent DNA Vaccine.
Application of the Aeromonas Multivalent DNA Vaccine of the present invention as preventing and curing of fish disease vaccine.
Further preferably, the Aeromonas Multivalent DNA Vaccine, that is, mah-pcDNA3.1 recombinant plasmid passes through dorsal fin muscle
The Yellow River carp is immunized in the mode of injection, for improving the survival rate of fish body after infected by Aeromonas hydrophila.
Further preferably, the Aeromonas Multivalent DNA Vaccine, that is, mah-pcDNA3.1 recombinant plasmid is for regulating and controlling fish body
The immune response of IgM gene, TNF α gene, c- type lysozyme, g- type lysozyme and IL-1 β gene in liver organization, into
And host is improved to the anti-infection ability of extraneous pathogen.
Further preferably, the Aeromonas Multivalent DNA Vaccine, that is, mah-pcDNA3.1 recombinant plasmid is for prevention and gas
Monad mah gene has the infection of the aeromonas salmonicida, Aeromonas veronii or/and Aeromonas sobria of homology.
Further preferably, the Aeromonas Multivalent DNA Vaccine, that is, mah-pcDNA3.1 recombinant plasmid uses intramuscular injection
Mode carry out fish immunity, or using oral or be inoculated with carry out fish immunity by the way of impregnating.
Compared with the prior art, the present invention has the following advantages:
1, the present invention develops a kind of novel DNA vaccination, compared to traditional inactivated vaccine or live bacterial vaccines, the DNA epidemic disease
Seedling has better development trend, and immune protective effect is more safe and efficient.
2, the main adhesin of Aeromonas hydrophila has good antigenicity and immunogenicity, invades host and Jie in thallus
It leads in host processes and plays a significant role, compare present Outer membrane protein antigen, main adhesin vaccine breaks through Aeromonas serum
The numerous obstacle of type effectively prevent the infection of a variety of Aeromonas pathogens.
3, the mah-pcDNA3.1 DNA vaccination that the present invention selects is while playing high immunogenicity, in immunological regulation side
Face also plays unique effect, and what the present invention constructed is a kind of recombinant plasmid, is immunized in a manner of injection in embodiment
Inoculation, as the extension of profession of the invention, which can also be fused in bacillus subtilis or saccharomycete and prepare carrier
Vaccine, to realize efficient immunity inoculation by being added in feed with oral way, therefore with before more extensive application
Scape.
Detailed description of the invention
Fig. 1 is the PCR amplification of Aeromonas hydrophila mah gene；
Fig. 2 is the building schematic diagram of mah-pcDNA3.1 recombinant plasmid；
Fig. 3 is Mah protein expression situation in musculature after vaccine immunity injection fish body；
Fig. 4 is IgM gene, TNF α gene, c- type lysozyme, g- type in liver organization after DNA vaccination inoculation fish body
The relative expression quantity of lysozyme and IL-1 β gene；
Fig. 5 is the systematic evolution tree of Aeromonas hydrophila HpaA gene (mah)；
Fig. 6 is DNA vaccination to Aeromonas veronii, the cross-protection of aeromonas salmonicida and Aeromonas sobria.
Specific embodiment
Above content of the invention is described in further details by the following examples, but this should not be interpreted as to this
The range for inventing above-mentioned theme is only limitted to embodiment below, and all technologies realized based on above content of the present invention belong to this hair
Bright range.
Embodiment 1
The construction method of Aeromonas hydrophila gene mah-pcDNA3.1 recombinant plasmid
Step S1: the extraction of Aeromonas hydrophila genomic DNA
Aeromonas hydrophila is obtained by this laboratory from separation in bacterial enteritis disease fish is suffered from, and bacterium is in brain heart infusion broth
Culture, with conventional DNA extraction kit purification of bacterial genomic DNA；
Step S2: the specific primer design of the main HpaA gene of Aeromonas hydrophila
Designed for expanding the specific primer of the main HpaA gene mah of Aeromonas hydrophila, primer sequence mah-
F:5 ' CGCGGATCCATGAAAAAGACAATTCTGGC 3'；Mah-R:5 ' CCCAAGCTTTTAGAAGTTGTATTGCAGGG
3 ', underscore is restriction enzyme site；
Step S3: the PCR amplification of Aeromonas hydrophila genomic DNA
Take 1 μ L Aeromonas hydrophila genomic DNA for template, each 2.5 μ L of 1 μ L, dNTP of target gene upstream and downstream primer,
PCR Buffer 2.5 μ L, ExTaq 0.25 μ L, ddH2O complement to 25 μ L, PCR reaction conditions: 95 DEG C of 5min；94 DEG C of 30s, 55
DEG C 30s, 72 DEG C of 1min, 30 circulations；72 DEG C of extension 10min, with the Ago-Gel testing goal clip size of 1wt%, glue
Recycle mah target gene, the result is shown in Figure 1；
The building of step S4:mah-pcDNA3.1 recombinant plasmid
The construction step of mah-pcDNA3.1 recombinant plasmid is shown in Fig. 2, by mah target gene and carrier after purification
PcDNA3.1 plasmid carries out double digestion, digestion system with restriction enzyme BamH I and Hind III respectively are as follows: BamH I and
Each 0.5 μ L, mah target gene of Hind III and carrier pcDNA3.1 plasmid are respectively 5 μ L, 10 × buffer 2 μ L, ddH2O is mended
25 μ L of foot again respectively recycle digestion products with plastic recovery kit, linked system 37 DEG C, digestion 3h in metal bath are as follows:
5 μ L of double digestion target gene fragment after purification, 1 μ L, T4 DNA Ligase of double digestion carrier 0.5 μ L after purification, 10 ×
T4 DNA buffer 1 μ L, ddH2O supplies 10 μ L, and 16 DEG C of connections overnight, 10 μ L products of overnight connection are added in metal bath
Enter into 20 μ L competent escherichia coli cell DH5 α, on ice ice bath 30min, 42 DEG C of thermal shock 90s in water-bath, immediately ice
5min is bathed, 200 μ L LB liquid mediums are added, bacterium solution are coated onto plate with ampicillin, 37 DEG C are incubated overnight, directly
To with macroscopic single colonie；
Step S5: the PCR detection of recombinant plasmid and sequence verification
5 single bacteriums of picking are fallen in the LB culture medium of 600 μ L, and 200r/min is expanded culture, using 1 μ L bacterium solution as mould
Plate carries out PCR detection with the universal primer on pcDNA3.1, PCR system: 2 × PCR mix, 5 μ L, the universal primer on carrier
Each 1 μ L of F/R, 1 μ L of bacterium solution, enzyme 0.25 μ L, ddH2O supplies 10 μ L, PCR conditions: 95 DEG C of 5min, 95 DEG C of 30s, 50 DEG C of 30s, and 72 DEG C
80s, 30 circulations, 72 DEG C of 10min, 1wt% agarose gel electrophoresis detects PCR product, and send the bacterium solution being proved to be successful to survey
Sequence, wherein the nucleic acid sequence of Aeromonas hydrophila mah gene is as shown in SEQ ID NO.15；
Step S6: the extraction of plasmid
Correct recombinant plasmid bacterium will be sequenced to expand culture, spend endotoxin plasmid extraction kit extraction plasmid and obtain
To mah-pcDNA3.1 recombinant plasmid, that is, Aeromonas Multivalent DNA Vaccine, while empty carrier pcDNA3.1 is extracted as control matter
Grain.
Embodiment 2
Evaluation of the immunity inoculation mah-pcDNA3.1 DNA vaccination to carp immune protective effect
The Yellow River carp is immunized in DNA vaccination: healthy Yellow River carp (50 ± 10g) being randomly divided into six groups, every group of 30 tails.Every tail fish is pressed
According to the dosage of 10 μ g (ultrapure water for being dissolved in 50 μ L), immunity inoculation mah-pcDNA3.1 is recombinated by way of dorsal fin intramuscular injection
Plasmid, pcDNA3.1 zero load is as a control group.Fish after vaccinating, which continues to be placed in 25 DEG C of constant temperature water tanks, to be raised 28 days.
Fish body Mah Protein Detection: the 1st after injection, the musculature of a tail fish is respectively taken within 7,14,21,28 days to extract protein.
20mg musculature is added RIPA lysate and is cracked, and the total protein after then taking 100 μ g to crack carries out SDS-PAGE electrophoresis,
In electrotransformation to pvdf membrane, Western blot detection is carried out with the primary antibody of Mah albumen and commercialization secondary antibody.The results show that the
The expression of destination protein, the immune mah-pcDNA3.1 DNA epidemic disease of this explanation can be detected in 14 days, 21 days and 28 days samples
Seedling can express Mah albumen (as shown in Figure 3) in fish body.
The Efficacy evaluation of DNA vaccination: Aeromonas hydrophila cultivates 16h on plate, and picking single bacterium falls on new LB
In culture medium, 180r/min is incubated overnight, with physiological saline tune OD600=0.6 (about 5 × 108CFU/mL).After 28 days immune, often
Endnote penetrates 0.1mL 5x107The Aeromonas hydrophila bacterium solution of CFU/mL carries out attacking poison, and the fish after injection is placed in 25 DEG C of water tanks, even
Continuous observation 7d, records the morbidity and death condition of fish daily, finally counts cumulative mortality.Immune protective rate=[1-(immune group
The death rate/control group the death rate)] × 100%.
As described in Table 1, immune guarantor of the mah-pcDNA3.1 DNA vaccination that prepared by embodiment 1 to infected by Aeromonas hydrophila
Shield rate reaches 75%.It can be seen that the DNA vaccination has good control efficiency, the Yellow River carp can be effectively protected to thermophilic aqueous vapor
Monad infects, and has good application value.
1 DNA vaccination of table analyzes the immune protective rate of the Yellow River carp
Embodiment 3
The expression of mah-pcDNA3.1 DNA vaccination adjusting fish body immunogene
RNA is extracted and reverse transcription: after mah-pcDNA3.1 DNA vaccination immune 28 days, every group of liver organization for taking 3 tail fishes
It is extracted for RNA.Fish body total serum IgE is extracted according to Trizol method, DNase I is added, 37 DEG C of digestion 1h are to remove genomic DNA dirt
Dye, then reverse transcription is carried out with random primer, fluorogenic quantitative detection is carried out by template of reverse transcription product.
QRT-PCR detect fish body in gene involved in immunity expression: fluorescence quantitative RT-RCR method detection IgM, TNF α,
The expression of c- type lysozyme, g- type lysozyme and IL-1 β totally 5 genes, using β-actin gene as internal reference.Specificity is drawn
Object is respectively as follows: IgM-F:5 ' GCGCGTGAGGAAAAGTGATT 3 ', IgM-R:5 ' GAAAACCGCTGGGCTAAACA 3 '；TNF
α-F:5 ' GAAGTCCGGCTCGTCAAAGT 3 ', TNF α-R:5 ' GATGGCTGCCTTGGAAGTGA 3 '；C- type lysozyme-F:
5 ' GGCACTCCAGGTGGAAAGAA 3 ', c- type lysozyme-R:5 ' AGTAACTTCCCCAGGTATCCCA 3 '；G- type bacteriolyze
Enzyme-F:5 ' GAATGGGTGGGAACCCGTAG 3 ', g- type lysozyme-R:5 ' CATTGGGCTCTGGCAACAAC 3 '；IL-1β-
F:5 ' AGCTGTCTTCGCATCCTCAC 3 ', IL-1 β-R:5 ' CGAGCAGCTCCTCATCACAA 3 '；β-actin-F:5 '
GAGTGATGGTTGGCATGGGA 3 ', β-actin-R:5 ' CCCAGTTGGTCACAATACCGT 3 '.
The results show that IgM, TNF α, c- type lysozyme, g- type lysozyme and IL-1 β gene expression quantity dramatically increase (P <
0.05 or P < 0.01), wherein 2 times or so (P < 0.01) IgM and TNF α expression up-regulation, this illustrates to cause after vaccine injection host anti-
The increase of body content, the enhancing of immune response.Most notably g- type lysozyme gene, expression increase about for expression variation
4.5 times, the gene is directly related with the antibacterial defense function of host, and illustrating DNA vaccination, immune host may to be greatly improved external
The anti-infection ability of boundary pathogen.
Embodiment 4
Cross-protection of the mah-pcDNA3.1 DNA vaccination to other Aeromonas
The analysis of mah sequence: the Aeromonas hydrophila mah gene order obtained according to sequencing, with online software BLAST
(http://blast.ncbi.nlm.nih.gov/Blast.cgi) searches for its homologous amino acid sequence, and soft with MEGA 6.0
Part adjacent method constructs systematic evolution tree, and the bacterium being related in chadogram has Aeromonas hydrophila (Aeromonas
Hydrophila), aeromonas salmonicida (Aeromonas salmonicida), Aeromonas veronii (Aeromonas
Veronii), Aeromonas caviae (Aeromonas caviae) and Aeromonas sobria (Aeromonas sobria).As a result
It was found that Aeromonas hydrophila adhesin is highly conserved in Aeromonas, with the main adhesin of Aeromonas hydrophila WC10-1 bacterial strain
Have highest homology, mainly adhered to BSK-10 bacterial strain be known as 98% homology.Its amino acid sequence and Aeromonas
Other bacterium ahaI, ompTS, ompF, omp38, ompK40 gene also have the homology of 89%-99%, as shown in Figure 5.Leather
The immunogenicity of Lan Shi negative bacteria outer membrane albumen and as having carried out a large amount of research, mah in the present embodiment in the exploitation of vaccine
Gene is as a kind of important adhesin molecules, with a variety of outer membrane protein very high homologies of Aeromonas bacterium.Therefore, we recognize
For mah gene vaccine development prospect with higher.
Immanoprotection action of the DNA vaccination to other Aeromonas: the Yellow River carp is immunized after 28 days in DNA vaccination, cultivates respectively
Aeromonas veronii, aeromonas salmonicida and Aeromonas sobria, with 2 × LD50The bacterium solution of dosage carries out attacking poison, the fish after injection
It is placed in 25 DEG C of water tanks, 7d, the morbidity and death condition of every 12h record fish, the survival rate of last statistical test fish is observed continuously.
Fig. 6 result is it can be found that the mah-pcDNA3.1 DNA vaccination of the preparation of embodiment 1 kills salmon to Aeromonas veronii
Aeromonas and Aeromonas sobria protective rate with higher, especially to the immune protective rate highest of Aeromonas veronii.
Therefore, the DNA vaccination that prepared by embodiment 1 can not only protect infection of the fish body to Aeromonas hydrophila, also to Aeromonas veronii,
The infection of aeromonas salmonicida and Aeromonas sobria has good cross-protection, is a kind of efficient multivalent dna epidemic disease
Seedling has good application value.
Embodiment above describes basic principles and main features of the invention and advantage, the technical staff of the industry should
Understand, the present invention is not limited to the above embodiments, and the above embodiments and description only describe originals of the invention
Reason, under the range for not departing from the principle of the invention, various changes and improvements may be made to the invention, these changes and improvements are each fallen within
In the scope of protection of the invention.
Sequence table
Sequence table
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<210> 9
<211> 20
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 9
agctgtcttc gcatcctcac 20
<210> 10
<211> 20
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 10
cgagcagctc ctcatcacaa 20
<210> 11
<211> 20
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 11
gagtgatggt tggcatggga 20
<210> 12
<211> 21
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 12
cccagttggt cacaataccg t 21
<210> 13
<211> 29
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 13
cgcggatcca tgaaaaagac aattctggc 29
<210> 14
<211> 29
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 14
cccaagcttt tagaagttgt attgcaggg 29
<210> 15
<211> 1122
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 15
atgaaaaaga caattctggc tattgctatc ccggctctgt tcgcatctgc cgctaacgct 60
gcagtggttt atgacaaaga cggtaccact tttgacgtat acggccgtgt tcaggctaac 120
tactatggtg accacaacaa atctgtagct gctactgatg gatcttgggg cttcagtgaa 180
actggtactc cggaatatac tcctggtacc gcggctcatt actctgatgt cgatggtgag 240
ctggttggtt cttcccgtct gggttggtcc ggcaagattg ccctgaacaa cacctggtcc 300
ggtatcgcca agaccgagtg gcaagtttct gctgaaaact ctgccaacaa gtttgactcc 360
cgtcacatct acgttggttt cgacggcaca cagtacggca agatcatctt cggtcagact 420
gacactgcgt tctacgacgt gctggaaccg accgatatct tcaacgaatg gggtgatgta 480
ggtaacttct atgacggtcg tcaagaaggt caggtcatct actccaacac ctacggcggc 540
ttcaaaggca aactgtccta tcaaaccaat gatgacaaag ccgttaaagt taccgacgtt 600
ggtcaaggta tcaaagaaaa agcggtgtac ggcaaggatg ttaagcgtaa ctacggttat 660
gccgctgctg ccggttatga cttcgacttc ggtctgggtc tgaacgcagg ttacgcctac 720
tccgatctgg aaaataccgc aaccaacaac actggtgaca agaaagagtg ggcaccgggt 780
gcacactacg ccatcaacgg tttctacttc gccggtgtct atacccaagc agatctgagc 840
tatgacacca ccaccggtgg tgacaaggac aagggccgtg gctacgagct ggctgcttcc 900
tacaacgttg atgcttggac tttcctggcg ggttacaact tcactgaagg taaagttgct 960
tccaacaaag ctggtgctga gtaccaagac atcgttgacg aaaccctgct gggcgtacag 1020
tacgctttca cttccaagct gaaagcctac accgagtaca aaatccaggg tatcgacaag 1080
atggacgacg agtggaccgt tgccctgcaa tacaacttct aa 1122
Claims (7)
1. a kind of preparation method of Aeromonas Multivalent DNA Vaccine, it is characterised in that detailed process are as follows:
Step S1: the specific primer designed for expanding the main HpaA gene of Aeromonas hydrophila, primer sequence mah-
F:5 ' CGCGGATCCATGAAAAAGACAATTCTGGC 3'；Mah-R:5 ' CCCAAGCTTTTAGAAGTTGTATTGCAGGG
3 ', underscore is restriction enzyme site；
Step S2: Bacteria Culture simultaneously extracts Aeromonas hydrophila DNA；
Step S3: the amplification main HpaA gene segment of Aeromonas hydrophila, using pcDNA3.1 plasmid as carrier, building recombination matter
Grain and sequence verification, are named as mah-pcDNA3.1 recombinant plasmid i.e. Aeromonas Multivalent DNA Vaccine.
2. the preparation method of Aeromonas Multivalent DNA Vaccine according to claim 1, it is characterised in that specific steps are as follows:
Step S1: the extraction of Aeromonas hydrophila genomic DNA
Aeromonas hydrophila is obtained by this laboratory from separation in bacterial enteritis disease fish is suffered from, and bacterium is trained in brain heart infusion broth
It supports, with conventional DNA extraction kit purification of bacterial genomic DNA；
Step S2: the specific primer design of the main HpaA gene of Aeromonas hydrophila
Designed for expanding the main HpaA gene of Aeromonas hydrophilamahSpecific primer, primer sequence mah-F:5 '
CGCGGATCCATGAAAAAGACAATTCTGGC 3'；Mah-R:5 ' CCCAAGCTTTTAGAAGTTGTATTGCAGGG 3 ', under
It is marked as restriction enzyme site；
Step S3: the PCR amplification of Aeromonas hydrophila genomic DNA
Taking 1 μ L Aeromonas hydrophila genomic DNA is template, target gene upstream and downstream primer each 1 μ L, dNTP 2.5 μ L, PCR
Buffer 2.5 μ L, ExTaq 0.25 μ L, ddH2O complement to 25 μ L, PCR reaction conditions: 95 DEG C of 5min；94 DEG C of 30s, 55 DEG C
30s, 72 DEG C of 1min, 30 circulations；72 DEG C of extension 10min, with the Ago-Gel testing goal clip size of 1wt%, glue is returned
It receivesmahTarget gene；
The building of step S4:mah-pcDNA3.1 recombinant plasmid
It will after purificationmahTarget gene and carrier pcDNA3.1 plasmid use restriction enzyme BamH I and Hind III respectively
Carry out double digestion, digestion system are as follows: each 0.5 μ L of BamH I and Hind III,mahTarget gene and carrier pcDNA3.1 plasmid point
It Wei not 5 μ L, 10 × buffer 2 μ L, ddH2O supplies 25 μ L, and 37 DEG C in metal bath, digestion 3h uses plastic recovery kit again
Digestion products are recycled respectively, linked system are as follows: 5 μ L of double digestion target gene fragment after purification, double digestion after purification carry
1 μ L, T4 DNA Ligase of body, 0.5 μ L, 10 × T4 DNA buffer 1 μ L, ddH2O supplies 10 μ L, 16 DEG C in metal bath
Connection overnight, 10 μ L products of overnight connection is added in 20 μ L competent escherichia coli cell DH5 α, on ice ice bath
30min, 42 DEG C of thermal shock 90s in water-bath, ice bath 5min, is added 200 μ L LB liquid mediums, bacterium solution is coated onto and is contained immediately
In the plate of ampicillin, 37 DEG C are incubated overnight, until with macroscopic single colonie；
Step S5: the PCR detection of recombinant plasmid and sequence verification
5 single bacteriums of picking are fallen in the LB culture medium of 600 μ L, and 200r/min is expanded culture, and using 1 μ L bacterium solution as template, are used
Universal primer on pcDNA3.1 carries out PCR detection, PCR system: 2 × PCR mix, 5 μ L, the universal primer F/R each 1 on carrier
μ L, 1 μ L of bacterium solution, enzyme 0.25 μ L, ddH2O supplies 10 μ L, PCR conditions: 95 DEG C of 5min, 95 DEG C of 30s, 50 DEG C of 30s, and 72 DEG C
80s, 30 circulations, 72 DEG C of 10min, 1wt% agarose gel electrophoresis detects PCR product, and send the bacterium solution being proved to be successful to survey
Sequence, wherein Aeromonas hydrophilamahThe nucleic acid sequence of gene is as shown in SEQ ID NO.15；
Step S6: the extraction of plasmid
Correct recombinant plasmid bacterium will be sequenced to expand culture, spend endotoxin plasmid extraction kit extraction plasmid and obtain
Mah-pcDNA3.1 recombinant plasmid, that is, Aeromonas Multivalent DNA Vaccine.
3. application of the Aeromonas Multivalent DNA Vaccine as preventing and curing of fish disease vaccine made from the method according to claim 11.
4. application according to claim 3, it is characterised in that: the Aeromonas Multivalent DNA Vaccine, that is, mah-
The Yellow River carp is immunized in pcDNA3.1 recombinant plasmid by way of dorsal fin intramuscular injection, for improving fish after infected by Aeromonas hydrophila
The survival rate of body.
5. application according to claim 3, it is characterised in that: the Aeromonas Multivalent DNA Vaccine, that is, mah-
PcDNA3.1 recombinant plasmid is for regulating and controlling IgM gene, TNF α gene, c- type lysozyme, g- type lysozyme in fish liver tissue
With the immune response of IL-1 β gene, and then host is improved to the anti-infection ability of extraneous pathogen.
6. application according to claim 3, it is characterised in that: the Aeromonas Multivalent DNA Vaccine, that is, mah-
PcDNA3.1 recombinant plasmid is for prevention and AeromonasmahGene has aeromonas salmonicida, the Vickers gas unit cell of homology
The infection of bacterium or/and Aeromonas sobria.
7. application according to claim 3, it is characterised in that: the Aeromonas Multivalent DNA Vaccine, that is, mah-
PcDNA3.1 recombinant plasmid carries out fish immunity by the way of intramuscular injection, or is inoculated by the way of oral or immersion
Row fish immunity.
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| CN114015711A (en) * | 2021-10-25 | 2022-02-08 | 中国农业科学院饲料研究所 | Recombinant protein for inhibiting Aeromonas veronii infection and preparation method and application thereof |
| CN114164159A (en) * | 2021-06-21 | 2022-03-11 | 湖南师范大学 | Bivalent vaccine for preventing and treating fish salmon gas killing and Edwardsiella tarda infection and preparation method and application thereof |
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