CN108330143A - A kind of Wdwardsiella tarda flagellin FliC with immanoprotection action - Google Patents
A kind of Wdwardsiella tarda flagellin FliC with immanoprotection action Download PDFInfo
- Publication number
- CN108330143A CN108330143A CN201810134891.8A CN201810134891A CN108330143A CN 108330143 A CN108330143 A CN 108330143A CN 201810134891 A CN201810134891 A CN 201810134891A CN 108330143 A CN108330143 A CN 108330143A
- Authority
- CN
- China
- Prior art keywords
- flic
- recombinant protein
- flagellin
- wdwardsiella tarda
- recombinant
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/02—Bacterial antigens
- A61K39/025—Enterobacteriales, e.g. Enterobacter
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
- A61K2039/552—Veterinary vaccine
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55511—Organic adjuvants
- A61K2039/55544—Bacterial toxins
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Medicinal Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Epidemiology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Molecular Biology (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Biomedical Technology (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Immunology (AREA)
- Mycology (AREA)
- Pharmacology & Pharmacy (AREA)
- Plant Pathology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Physics & Mathematics (AREA)
- Gastroenterology & Hepatology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Peptides Or Proteins (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
Applications of the Wdwardsiella tarda flagellin FliC in subunit vaccine with immanoprotection action that the invention discloses a kind of, is prepared the recombinant protein using prokaryotic expression method, specifically includes：PCR amplification fliC genes；PMD18 T fliC carriers are built, sequencing is carried out to fliC genes；The correct bacterial strain of sequencing result expands culture, extracts plasmid, obtains purifying target gene；Build recombinant expression carrier pET 32a fliC；Recombinant expression carrier is converted into DH5 α competent cells, obtains positive colony, extracts plasmid, sequencing；Carry out the prokaryotic expression of recombinant protein；Recombinant protein expression is analyzed using SDS PAGE；Western blot verifications are carried out to recombinant protein.The present invention obtains the recombinant protein of the Wdwardsiella tarda flagellin FliC of large-scale purification by prokaryotic expression method; and the immunogenicity of the recombinant protein and the protection for animal pattern further are assessed using zoopery, show that Wdwardsiella tarda flagellin FliC can be used in developing subunit vaccine.
Description
Technical field
The present invention relates to technical field of bioengineering, specifically, being related to a kind of slow love with immanoprotection action
Moral China bacterium flagellin FliC.
Background technology
Wdwardsiella tarda (Edwardsiella tarda, E.tarda) endangers seriously in aquaculture, infection of marine fishes
After the bacterium death rate it is high, easily repeatedly, hypoimmunity, conventional antibiotic and chemicals uncertain therapeutic efficacy cut, be badly in need of exploitation it is novel,
Efficiently, safe control method.Vaccine provides many references using the immense success in Production of Livestock and Poultry to aquaculture, a variety of
Aquatic products vaccine shows good prospect by development and application, regrettably, there is no commercialized vaccine for the slow Edward of prevention and control at present
Bacterium infects.In addition to this, Wdwardsiella tarda can cause the multiple types of people to infect, and messenger is infected by food source approach in recent years
Report be on the increase, disclose this bacterium public hygienics value.
Flagellin is one of major structural protein of bacterial flagellum, in bacteria motility, adheres to and infects in host processes
It plays a significant role, is the important virulence factor of bacterium.The study found that bacterial flagellin can activate host immune response,
Play the role of vaccine adjuvant, further study show that, flagellin can induce inflammatory reaction to occur simultaneously by TLR5 accesses
Promote maturing dendritic cell.The albumen is either mixed with antigen or amalgamation and expression, shows good immune effect.
In addition to this, flagellin itself has stronger immunogenicity, has certain immune protective efficiency.FliC is to constitute mastigoneme
Primary protein component, be the focus albumen of current flagellum research, biological function obtains to be parsed to a certain extent.The present invention is logical
The recombinant protein that prokaryotic expression method obtains the Wdwardsiella tarda flagellin FliC of large-scale purification is crossed, and further using dynamic
The immunogenicity of the object experimental evaluation recombinant protein and the protection for animal pattern, show Wdwardsiella tarda flagellin
FliC can be used in developing subunit vaccine.
Invention content
The purpose of the present invention is to provide a kind of Wdwardsiella tarda flagellin FliC with immanoprotection action, tool
Body obtains Wdwardsiella tarda flagellin FliC recombinant proteins by prokaryotic expression method, to further develop slow love
Moral China bacterium subunit vaccine.
To achieve the above object, the present invention adopts the following technical scheme that：
Applications of the Wdwardsiella tarda flagellin FliC in subunit vaccine with immanoprotection action.
Further, the preparation method of the Wdwardsiella tarda flagellin FliC with immanoprotection action, tool
Steps are as follows for body：
(1) it is arranged as template with Wdwardsiella tarda ET-CL genome sequences, P1 and P2 are primer, PCR amplification fliC genes；
The reaction system of PCR amplification totally 20 μ L, wherein 2 × Taq PCR Mix, 10 μ L, template 1 μ L, primer P1 (20 μM) and P2 (20 μ
M) each 1 μ L, ddH2O 7μL；The response procedures of PCR amplification be 94 DEG C of pre-degenerations 5min, 94 DEG C of 30s, 54 DEG C of 30s, 72 DEG C of 60s,
25 cycles, 72 DEG C of 10min；Wherein primer P1 and P2 are：
P1：5’-ATGGCACAAGTAATTAATACC-3’；SEQ ID NO：1；
P2：5’-ACGCAGCAGAGACAGGACG-3’；SEQ ID NO：2；
(2) pMD18-T-fliC carriers are built, sequencing is carried out to fliC genes；
(3) the correct bacterial strain of sequencing result expands culture, extracts plasmid, using pMD18-T-fliC as template, P3 and P4 are
Primer, PCR amplification fliC genes obtain purifying target gene；The reaction system of PCR amplification totally 20 μ L, wherein 2 × TaqPCR
Mix 10 μ L, primer P3 (20 μM) and P4 (20 μM) each 1 μ L, template 1 μ L, ddH2O 7μL；The response procedures of PCR amplification are 94
DEG C pre-degeneration 5min, 94 DEG C of 30s, 52 DEG C of 30s, 72 DEG C of 60s, 30 cycles, 72 DEG C of 10min；Wherein primer P3 and P4 are：
P3：5’-CGGGATCCATGGCACAAGTAATTAATACC-3 ', BamH I；SEQ ID NO：3；
P4：5’-GCGTCGACACGCAGCAGAGACAGGACG-3 ', Sal I；SEQ ID NO：4；
Wherein, underscore part is restriction enzyme site；
(4) double enzymes are carried out respectively to purifying target gene and pET-32a carriers using restriction enzyme BamH I and Sal I
It cuts, structure recombinant expression carrier pET-32a-fliC；
(5) recombinant expression carrier is converted into DH5 α competent cells, picking monoclonal carries out PCR and double digestion verification, obtains
Positive colony is obtained, plasmid, sequencing are extracted；
(6) recombinant expression carrier pET-32a-fliC and empty carrier pET-32a are converted into BL21 engineering bacterias respectively, carries out weight
The prokaryotic expression of histone, is as follows：Respectively by 5 μ L recombinant vectors pET-32a-fliC and 5 μ L empty carriers pET-32a
It is added in 100 μ L BL21 engineering bacterias；Ice bath 30min, 42 DEG C of thermal shock 90s, ice bath 5min；1mL LB liquid mediums are added,
After 37 DEG C of rejuvenation 1h, 5 μ L is respectively taken to be coated on the solid LB tablets of the final concentration of 100 μ g/mL of ammonia benzyl, is inverted, 37 DEG C of overnight incubations；
(7) SDS-PAGE is utilized to analyze recombinant protein expression；
(8) it is secondary antibody by primary antibody, HRP-IgG of His-Mab, Westernblot verifications is carried out to recombinant protein.
The beneficial effects of the invention are as follows：The present invention obtains the Wdwardsiella tarda whip of large-scale purification using prokaryotic expression method
The recombinant protein of hairless protein FliC, and zoopery is further utilized to assess the immunogenicity of the recombinant protein and moved for model
The protection of object, to further develop Wdwardsiella tarda subunit vaccine and nucleic acid vaccine target spot, meanwhile, slow Edward
Bacterium FliC albumen also has adjuvanting properties.
Description of the drawings：
Fig. 1 is the PCR amplification of fliC genes in the embodiment of the present invention 2；
M.DL2000Marker；1.fliC gene magnifications；
Fig. 2 is that SDS-PAGE analyzes expression of the recombinant protein in Escherichia coli in the embodiment of the present invention 4；
M. albumen Marker；1,2.pET-32a-fliC/BL21 cellular lysate object；3.pET-32a/BL21 zero loads compare；
Fig. 3 is that Westernblot identifies expression of the recombinant protein in Escherichia coli in the embodiment of the present invention 4；
1. albumen Marker；2.pET-32a-fliC/BL21 cellular lysate objects；
Fig. 4 is the soluble analysis of recombinant protein in the embodiment of the present invention 5；
M. albumen Marker；1.pET-32a-fliC/BL21 cellular lysate objects；2. lysate supernatant；3. lysate precipitates；
Fig. 5 is the SDS-PAGE of the destination protein purified in the embodiment of the present invention 5；
M. albumen Marker；1. non-purification of recombinant proteins；2. purification of recombinant proteins；
Fig. 6 is that Westernblot analyzes identification of the positive serum to His-FliC albumen in the embodiment of the present invention 6；
1. recombinant protein；2. negative control (His- σ C reovirus capsid proteins)；
Fig. 7 is His-FliC albumen in the embodiment of the present invention 6 to the immune protective effect of mouse；
Fig. 8 is that BSA antibody titers monitor under different adjuvant immunities in the embodiment of the present invention 7；
Fig. 9 is anti-FliC antibody titers monitoring in the embodiment of the present invention 7 after the immune mouse of His-FliC.
Specific implementation mode
The present invention is described in further detail below in conjunction with attached drawing, the given examples are served only to explain the present invention, not
For limiting the scope of the present invention.
1 Wdwardsiella tarda fliC gene sequencings of embodiment
According to E.tarda reference strain ET-1 genome sequences (CP001135.1) the fliC genes logged on GenBank
Design primer, primer P1 and P2 are：
P1：5’-ATGGCACAAGTAATTAATACC-3’；SEQ ID NO：1；
P2：5’-ACGCAGCAGAGACAGGACG-3’；SEQ ID NO：2；
ET-CL genomes are extracted using OMEGA genome extracts kits, and PCR amplification is carried out using it as template；PCR
The reaction system of amplification is 2 × TaqPCR Mix 10 μ L, primer P1 (20 μM) and P2 (20 μM) each 1 μ L, template 1 μ L, ddH2O
7 μ L, totally 20 μ L；The response procedures of PCR amplification are 94 DEG C of pre-degeneration 5min, and 94 DEG C of 30s, 54 DEG C of 30s, 72 DEG C of 60s, 25 are followed
Ring, 72 DEG C of 10min；FliC genetic fragments are recycled, PMD18-T carriers are connected, linked system is as follows：4.5 μ L of target gene,
0.5 5 μ L of μ L, 2 × Connectbuffer of pMD18-T Vector, totally 10 μ L, 16 DEG C of connections are overnight.Connection product is transformed into
DH5 α competent cells；Picking single bacterium colony carries out PCR detections, and positive bacterium colony is sent by Beijing life work sequencing.
With reference to E.tarda reference strains (E.tardaET-1, E.tarda ETW41, E.tarda FL6- in GenBank
And other enterobacteriaceaes other member (S.marcescens str.SmUNAM836, Klebsiella aerogenes 60)
Str.AR_0009, S.typhimurium str.LT2, E.coli str.K-12, S.enterica str.SGB23,
Kosakonia cowaniistr.Esp_Z) bacterial strain fliC gene orders, fliC gene conservatives are analyzed, as a result, it has been found that, like moral
FliC genes are highly conserved between magnificent Pseudomonas member.
2 Wdwardsiella tarda fliC gene magnifications of embodiment
The correct PMD18-T-fliC bacterial strains of 1 sequencing result of embodiment are expanded into culture, extract plasmid, and using it as mould
Plate, P3 and P4 are primer, PCR amplification fliC genes, reaction system totally 20 μ L, wherein 2 × Taq PCR Mix 10 μ L, primer P3
(20 μM) and P4 (20 μM) each 1 μ L, template 1 μ L, ddH2O 7μL；Response procedures are 94 DEG C of pre-degeneration 5min, 94 DEG C of 30s, 52 DEG C
30s, 72 DEG C of 60s, 30 cycles, 72 DEG C of 10min；The target fragment (as shown in Figure 1) that size is 1248bp is obtained, purpose is purified
Target gene is connect structure recombinant expression carrier by gene with prokaryotic expression carrier pET-32a；Wherein primer P3 and P4 are：
P3：5’-CGGGATCCATGGCACAAGTAATTAATACC-3 ', BamH I；SEQ ID NO：3；
P4：5’-GCGTCGACACGCAGCAGAGACAGGACG-3 ', Sal I；SEQ ID NO：4；
Wherein, underscore part is restriction enzyme site.
The structure of embodiment 3pET-32a-fliC recombinant expression carriers
Double digestion, digestion system are carried out to purifying target gene and pET-32a carriers respectively using BamH I and Sal I：Mesh
I each 3 μ L of 1 μ L, 10 × Greenbuffer of gene/pET-32a 25 μ L, BamH I and Sal.According to 3 after recycling:1 ratio
22 DEG C of connection 30min, whole connection products are transferred in 100 μ LDH5 α competent cells, ice bath 30min, 42 DEG C of heating bath 90s,
Ice bath 5min, is added 1mL LB liquid mediums, and 37 DEG C of rejuvenation 1h are coated on the solid containing the final concentration of 100 μ g/mL of ammonia benzyl
LB tablets are inverted in 37 DEG C of overnight incubations.Next day, picking monoclonal carry out PCR verifications with P3, P4 primer.Positive colony extracts
Plasmid carries out double digestion verification and sequencing, and no mutation and frameshit obtain recombinant expression carrier pET-32a-fliC.
The prokaryotic expression of 4 recombinant protein of embodiment
Recombinant vector pET-32a-fliC and empty carrier are transformed into respectively in BL21 engineering bacterias, ice bath 30min, 42 DEG C of heat
Bathe 90s, ice bath 5min；1mL LB liquid mediums are added, 37 DEG C of rejuvenation 1h are coated on containing the final concentration of 100 μ g/mL of ammonia benzyl
Tablet, be inverted in 37 DEG C of overnight incubations.Picking single bacterium colony is inoculated in the LB liquid medium containing 100 μ g/L ampicillins
In, IPTG to final concentration of 0.5mmol/L, induced expression 5h is added in 37 DEG C of shake cultures to logarithmic phase.It is heavy that thalline were collected by centrifugation
It forms sediment, PBS buffer solution is washed 3 times；Albumen sample-loading buffer is added, 10min is boiled after mixing well, centrifuges, collecting supernatant is
It for protein sample, is analyzed using SDS-PAGE, after loading, voltage is adjusted to 80V, waits for that sample passes through separation gel and concentration glue point
After line, voltage is adjusted to 120V, until electrophoresis terminates, film coomassie brilliant blue staining 1min is taken out, is repeatedly boiled with distilled water
Decoloration；The results are shown in Figure 2, and induced expression goes out destination protein at 60kD；Using His-MAb as primary antibody, HRP-IgG is secondary antibody,
Respectively 1 is pressed with 5% skimmed milk power:1000 and 1:5000 dilutions, destination protein table is further confirmed that with Westernblot methods
It reaches, is as follows：After SDS-PAG electrophoresis, film is taken out, is gone to albumen on NC films using transferring film instrument, 80V transferring films 1h；
After transferring film, NC films are taken out, PBST is washed 3 times, 5min/ times；5% skimmed milk power room temperature closes 1h, and PBST is washed 3 times,
5min/ times；1 is pressed with 5% skimmed milk power:1000 dilution His-Mab are incubated at room temperature 1h as primary antibody；PBST is washed 3 times, 5min/
It is secondary；5% skimmed milk power presses 1:5000 dilution HRP-IgG are incubated at room temperature 30min as secondary antibody；PBST is washed 3 times, 5min/ times；
Developer solution is added, is protected from light 5min, finally shine development；The results are shown in Figure 3, occurs destination protein band at 60kD, it was demonstrated that
His-FliC recombinant proteins are expressed successfully.
The soluble analysis of 5 destination protein of embodiment and purifying
Express express target protein bacterial strain is enlarged culture according to above-mentioned condition, thalline were collected by centrifugation；Utilize ultrasonication
Instrument is crushed thalline, centrifuges precipitation, supernatant, and bacterial lysate, supernatant and precipitation are prepared protein sample respectively, carried out
SDS-PAGE, the results are shown in Figure 4, occurs protein band at 60kD.
Induced expression supernatant is taken, Ni is utilized2+Affinity chromatography method is purified, and the results are shown in Figure 5, is purified at 60kD
Go out His-FliC, purifying protein concentration is measured using BCA albumen concentration detection kits, it is a concentration of as a result to measure albumen
0.36mg/mL。
Embodiment 6 recombinates FliC protein immunization Protections
E.tarda infecting mouse positive serums are prepared, with positive serum (1：500) it is primary antibody, HRP-IgG (1:5000) it is
Secondary antibody, the identification using Western blot verification positive serums to recombinant protein, the results are shown in Figure 6, and E.tarda infection is small
Mouse positive serum can recombinate His-FliC albumen with specific recognition.
40 healthy Kunming mouses are randomly divided into two groups, control group and immune group, every group 20.With the His-FliC of purifying
For albumen as immunogene, immune group mouse is immune for the first time, 30 μ g recombination FliC albumen (Freund's complete adjuvant breasts of intramuscular injection
Change), interval is after 2 weeks, second immune (incomplete Freund's adjuvant emulsification), is added with incomplete Freund's adjuvant emulsified protein after 30d
It is strong immune；Control group mice is immunized injects isometric PBS (adding corresponding adjuvant) three times.The 48d pairs two groups of mouse peritoneal connects
Kind Wdwardsiella tarda ET-CL bacterium solutions, dosage 5LD50(2.0×107Cfu), 4d is observed after infection, counts The dead quantity.Knot
Fruit shows, control group mice is all dead, immune group dead mouse 6, remaining mouse undergo of short duration spirit is depressed, do not eat after
It gradually recovers one's health.The results are shown in Figure 7, shows there is certain protection, protection after mouse is immunized in His-FliC recombinant proteins
Rate is 70%.
7 Wdwardsiella tarda FliC albumen of embodiment is assessed as adjuvant effect
40 healthy Kunming mouses are randomly divided into 4 groups, every group 10.Using the bovine serum albumin(BSA) BSA of purchase as assessment
Albumen, setting BSA immune groups, BSA+FliC protein immunizations group and BSA+ Freund's adjuvant immune groups, while setting up PBS group conducts
Control.Immunization route is to be subcutaneously injected, immune to carry out at twice, and interval 10d carries out second of immune, albumen after being immunized for the first time
Immunizing dose is 100 μ g/.BSA albumen oil emu immune group first time is immunized plus complete Freund's adjuvant, immune for the second time to add
Incomplete Freund's adjuvant is immunized, and blank control group is substituted with PBS.
The tail point blood sampling in the 0th, 10,20 and 30 day after immune of each immune group, and serum is detached, with the recombination FliC eggs of purifying
It is used as envelope antigen in vain, the potency of antibody in mice serum is detected using indirect ELISA method, with negative serum OD450Average value
Add 3 times of standard deviations as yin and yang attribute criterion, it is 0.176 to calculate critical value, when serum OD values to be checked are more than critical value
It is determined as the positive；It is determined as feminine gender when serum OD values to be checked are less than critical value.
The serum that different adjuvant+BSA are immunized to 4 acquisitions of mouse is diluted by 1: 100, with 500ng/ml BSA albumen
Coated elisa plate carries out indirect ELISA detection by secondary antibody of sheep anti-mouse igg-HRP, and the results are shown in Figure 8.The result shows that：Merely
BSA is immunized mouse and is nearly free from specific antibody, and Freund's adjuvant or FliC mixed immunities can stimulate body generation higher
Specific antibody, it was demonstrated that the adjuvanting properties of Wdwardsiella tarda FliC albumen.
The serum that FliC is immunized to 4 acquisitions of mouse is diluted by 1: 100, and enzyme mark is coated with 500ng/ml recombinant proteins
Plate carries out indirect ELISA detection by secondary antibody of sheep anti-mouse igg-HRP, and the results are shown in Figure 9.The result shows that：Recombinant protein is immune
Mouse can generate higher level specific antibody, and highest level is reached after booster immunization.
The foregoing is merely presently preferred embodiments of the present invention, is not intended to limit the invention, it is all the present invention spirit and
Within principle, any modification, equivalent replacement, improvement and so on should all be included in the protection scope of the present invention.
Sequence table
<110>Hebei Science ＆ Technology Normal College
<120>A kind of Wdwardsiella tarda flagellin FliC with immanoprotection action
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
<212> DNA
<213> Artificial
<400> 1
atggcacaag taattaatac c 21
<210> 2
<211> 19
<212> DNA
<213> Artificial
<400> 2
acgcagcaga gacaggacg 19
<210> 3
<211> 29
<212> DNA
<213> Artificial
<400> 3
cgggatccat ggcacaagta attaatacc 29
<210> 4
<211> 27
<212> DNA
<213> Artificial
<400> 4
gcgtcgacac gcagcagaga caggacg 27
Claims (6)
1. applications of the Wdwardsiella tarda flagellin FliC with immanoprotection action in subunit vaccine.
2. the preparation side of the Wdwardsiella tarda flagellin FliC according to claim 1 with immanoprotection action
Method, which is characterized in that be as follows：
(1) it is arranged as template with Wdwardsiella tarda ET-CL genome sequences, P1 and P2 are primer, PCR amplification fliC genes；Wherein
Primer P1 and P2 are：
P1：5’-ATGGCACAAGTAATTAATACC-3’SEQ ID NO：1；
P2：5’-ACGCAGCAGAGACAGGACG-3’SEQ ID NO：2；
(2) pMD18-T-fliC carriers are built, sequencing is carried out to fliC genes；
(3) the correct bacterial strain of sequencing result expands culture, extracts plasmid, and using pMD18-T-fliC as template, P3 and P4 are primer,
PCR amplification fliC genes obtain purifying target gene；Wherein primer P3 and P4 are：
P3：5’-CGGGATCCATGGCACAAGTAATTAATACC-3 ', BamH I；SEQ ID NO：3；
P4：5’-GCGTCGACACGCAGCAGAGACAGGACG-3 ', Sal I；SEQ ID NO：4；
(4) double enzymes are carried out respectively to purifying target gene and pET-32a carriers using restriction enzyme BamH I and Sal I
It cuts, structure recombinant expression carrier pET-32a-fliC；
(5) recombinant expression carrier is converted into DH5 α competent cells, picking monoclonal carries out PCR and double digestion verification, obtains sun
Property clone, extract plasmid, sequencing；
(6) recombinant expression carrier pET-32a-fliC and empty carrier pET-32a are converted into BL21 engineering bacterias respectively, carries out recombination egg
White prokaryotic expression；
(7) SDS-PAGE is utilized to analyze recombinant protein expression；
(8) Westernblot verifications are carried out to recombinant protein.
3. the prokaryotic expression of the Wdwardsiella tarda flagellin FliC according to claim 2 with immanoprotection action
Method, which is characterized in that the reaction system of step (1) described PCR amplification totally 20 μ L, wherein 2 × Taq PCR Mix 10 μ L, 20
μM P1 1 μ L, 20 μM of 1 μ L of P2, template 1 μ L, ddH2O 7μL；The response procedures of PCR amplification are 94 DEG C of pre-degeneration 5min, 94
DEG C 30s, 54 DEG C of 30s, 72 DEG C of 60s, 25 cycles, 72 DEG C of 10min.
4. the prokaryotic expression of the Wdwardsiella tarda flagellin FliC according to claim 2 with immanoprotection action
Method, which is characterized in that the reaction system of step (3) described PCR amplification totally 20 μ L, wherein 2 × Taq PCR Mix 10 μ L, 20
μM P3 1 μ L, 20 μM of 1 μ L of P4, template 1 μ L, ddH2O 7μL；The response procedures of PCR amplification are 94 DEG C of pre-degeneration 5min, 94
DEG C 30s, 52 DEG C of 30s, 72 DEG C of 60s, 30 cycles, 72 DEG C of 10min.
5. the prokaryotic expression of the Wdwardsiella tarda flagellin FliC according to claim 2 with immanoprotection action
Method, which is characterized in that step (6) the conversion BL21 engineering bacterias are as follows：Respectively by 5 μ L recombinant vectors pET-
32a-fliC and 5 μ L empty carriers pET-32a are added in 100 μ L BL21 engineering bacterias；Ice bath 30min, 42 DEG C of thermal shock 90s, ice bath
5min；1mL LB liquid mediums are added, after 37 DEG C of rejuvenation 1h, 5 μ L are respectively taken to be coated on the solid of the final concentration of 100 μ g/mL of ammonia benzyl
LB tablets are inverted, 37 DEG C of overnight incubations.
6. the prokaryotic expression of the Wdwardsiella tarda flagellin FliC according to claim 2 with immanoprotection action
Method, which is characterized in that step (8) the Western blot verifications are secondary antibody by primary antibody, HRP-IgG of His-Mab.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810134891.8A CN108330143B (en) | 2018-02-09 | 2018-02-09 | Edwardsiella tarda flagellin FliC with immune protection effect |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810134891.8A CN108330143B (en) | 2018-02-09 | 2018-02-09 | Edwardsiella tarda flagellin FliC with immune protection effect |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108330143A true CN108330143A (en) | 2018-07-27 |
| CN108330143B CN108330143B (en) | 2021-07-09 |
Family
ID=62927494
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810134891.8A Active CN108330143B (en) | 2018-02-09 | 2018-02-09 | Edwardsiella tarda flagellin FliC with immune protection effect |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108330143B (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105920593A (en) * | 2016-06-08 | 2016-09-07 | 山东省海洋生物研究院 | Edwardsiella tarda subunit oral microencapsule vaccine for aquatic product |
| CN106075418A (en) * | 2016-06-08 | 2016-11-09 | 类延乐 | A kind of fliC albumen and GAPDH albumen microcapsule vaccine |
-
2018
- 2018-02-09 CN CN201810134891.8A patent/CN108330143B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105920593A (en) * | 2016-06-08 | 2016-09-07 | 山东省海洋生物研究院 | Edwardsiella tarda subunit oral microencapsule vaccine for aquatic product |
| CN106075418A (en) * | 2016-06-08 | 2016-11-09 | 类延乐 | A kind of fliC albumen and GAPDH albumen microcapsule vaccine |
Non-Patent Citations (6)
| Title |
|---|
| JUN OKUDA ET AL: "Base changes in the fliC gene of Edwardsiella tarda: possible effects on flagellation and motility", 《DISEASES OF AQUATIC ORGANISMS》 * |
| MENGZHANG ET AL: "Edwardsiella tarda flagellar protein FlgD：A protective immunogen against edwardsiellosis", 《VACCINE》 * |
| XU-DONG JIAO ET AL: "Construction and evaluation of DNA vaccines encoding Edwardsiella tarda antigens", 《VACCINE》 * |
| YANG HE ET AL: "FliC, a Flagellin Protein, Is Essential for the Growth and Virulence of Fish Pathogen Edwardsiella tarda", 《PLOS ONE》 * |
| 焦绪栋: "迟缓爱德华氏菌基因工程疫苗的构建及免疫效果的研究", 《中国博士学位论文全文数据库 医药卫生科技辑》 * |
| 贺扬: "迟缓爱德华氏菌（Edwardsiella tarda）的多样性分析及其鞭毛蛋白的功能研究", 《中国博士学位论文全文数据库 农业科技辑》 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108330143B (en) | 2021-07-09 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103172749B (en) | Preparation of African swine fever protein engineering vaccine | |
| CN106220716B (en) | Infectious coryza of chicken subunit vaccine and preparation method thereof | |
| CN110760006A (en) | African swine fever immune system targeted genetic engineering vaccine | |
| CN104593397B (en) | A kind of enterotoxigenic escherichia coil polyvalent antigen gene order of optimization and its application in preventing post-weaning diarrhea | |
| CN102276730B (en) | Preparation method for staphylococcus aureus Iron-regulated surface determinant B immunodominant fragment (IsdBid)-target of RNAIII activating protein (TRAP) fusion protein and application thereof | |
| CN104693310B (en) | A kind of chimeric protein, virus-like particle and its application | |
| CN107653260A (en) | A kind of preparation method and application of Recombinant Lactococcus lactis | |
| CN113354743A (en) | Multi-epitope antigen and vaccine for piglet diarrhea as well as preparation method and application of multi-epitope antigen and vaccine | |
| CN101016541A (en) | Method of producing brucella vaccine antigen protein | |
| CN102816246B (en) | Human cytomegalo virus immunogen fusion protein as well as preparation method and usage thereof | |
| CN104628865B (en) | A kind of pseudo- mad dog epitope polypeptide recombinant vaccine | |
| CN103275228A (en) | K99-987P-F41 recombinant protein and application thereof | |
| CN107936123B (en) | Swine transmissible gastroenteritis virus fusion protein and preparation method and application thereof | |
| CN109568572A (en) | A kind of preparation method and applications of Aeromonas Multivalent DNA Vaccine | |
| CN104894045A (en) | Recombinant lactobacillus for coexpression of foot and mouth disease virus VP1 gene and immunoadjuvant cattle IL-6 gene, and preparation method and application of recombinant lactobacillus | |
| CN106117365B (en) | Anti-streptococcus suis is sick and with the active fusion protein of autoimmunity and its preparation and application | |
| CN104250304B (en) | The vaccine combination of a kind of fusion protein and its coding and application | |
| CN105602981B (en) | Preparation method and application of porcine epidemic diarrhea virus genetic engineering subunit oral combined vaccine | |
| CN108330143A (en) | A kind of Wdwardsiella tarda flagellin FliC with immanoprotection action | |
| CN107827986B (en) | Pig O/Mya98 and O/PanAsia type foot-and-mouth disease gene engineering inactivated vaccine | |
| CN107625960B (en) | Universal swine colibacillosis vaccine and preparation method thereof | |
| CN112843225A (en) | RA OmpA gene-based Riemerella anatipestifer DNA vaccine and preparation method and identification method thereof | |
| CN108359683B (en) | Edwardsiella tarda outer membrane protein OmpA with immune protection effect | |
| CN104497148B (en) | A kind of preparation for recombinating ubiquitination blue-ear disease vaccine | |
| CN116574160B (en) | Streptococcus suis antigen protein and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |