CN108359683A - A kind of Wdwardsiella tarda outer membrane protein OmpA with immanoprotection action - Google Patents
A kind of Wdwardsiella tarda outer membrane protein OmpA with immanoprotection action Download PDFInfo
- Publication number
- CN108359683A CN108359683A CN201810132575.7A CN201810132575A CN108359683A CN 108359683 A CN108359683 A CN 108359683A CN 201810132575 A CN201810132575 A CN 201810132575A CN 108359683 A CN108359683 A CN 108359683A
- Authority
- CN
- China
- Prior art keywords
- ompa
- outer membrane
- recombinant
- wdwardsiella tarda
- recombinant protein
- 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
-
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Zoology (AREA)
- General Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Microbiology (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Biomedical Technology (AREA)
- Physics & Mathematics (AREA)
- Immunology (AREA)
- Plant Pathology (AREA)
- Mycology (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Gastroenterology & Hepatology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Peptides Or Proteins (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
Applications of the Wdwardsiella tarda outer membrane protein OmpA in subunit vaccine with immanoprotection action that the invention discloses a kind of, is prepared the recombinant protein using prokaryotic expression method, specifically includes:It is arranged as template with Wdwardsiella tarda ET CL genome sequences, PCR amplification ompA genes;PMD18 T ompA carriers are built, sequencing is carried out to ompA genes;Build recombinant expression carrier pET 32a ompA;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 outer membrane protein OmpA of large-scale purification using prokaryotic expression method; and the immunogenicity of the recombinant protein and the protection for animal pattern further are assessed using zoopery, show that the recombinant protein can be used in developing Wdwardsiella tarda subunit vaccine and nucleic acid vaccine target spot.
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 bacterial outer membrane protein OmpA.
Background technology
Wdwardsiella tarda (Edwardsiella tarda, E.tarda) is that cause of disease very common in aquaculture is thin
One of bacterium can cause a variety of seawater and freshwater fish infection morbidity, cause high mortality, system infections and septicemia, the shoal of fish
Overall immunity declines, and seriously affects aquaculture development.Currently, E.tarda has become China flounder, flounder, sea eel class most
One of important pathogen causes heavy losses to aquaculture.In addition to this, E.tarda can cause the multiple types of people to infect,
Septicemia, threat to life can be seriously caused, therefore there is important public hygienics meaning to the research of this bacterium.
Vaccine is widely used good to the control effect of epidemic disease on livestock and poultry cultivation, and reference is provided for aquatic products control and prevention of disease.
For E.tarda, subunit vaccine and live vector vaccine research based on protective antigens screening study are as research
Hot spot.Studies have shown that outer membrane protein (outermembrane proteins, Omps) has excellent immunogenicity, can lure
It leads body and generates very strong immune response, animal, which is immunized, using outer membrane protein crude extract shows extraordinary protecting effect.
The present invention is expressed using the external memebrane protein main component OmpA of prokaryotic expression system (outer-membrane proteinA)
And purifying, protection of the recombinant protein for animal pattern is assessed by zoopery, shows that the recombinant protein can be used in
Develop Wdwardsiella tarda subunit vaccine and nucleic acid vaccine target spot.
Invention content
The purpose of the present invention is to provide a kind of Wdwardsiella tarda outer membrane protein OmpA with immanoprotection action, tool
Body obtains Wdwardsiella tarda outer membrane protein OmpA 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 outer membrane protein OmpA in subunit vaccine with immanoprotection action.
Further, the preparation method of the Wdwardsiella tarda outer membrane protein OmpA 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 ompA genes;
The reaction system of PCR amplification is 20 μ L, wherein 2 × Taq PCR Mix 10 μ L, primer P1 (20 μM) and P2 (20 μM) each 1 μ L,
Template 1 μ L, ddH27 μ L of O, the response procedures of PCR amplification are 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’-ATGAAAAAAACAGCGATCGCA-3’;SEQ ID NO:1;
P2:5’-TTAAGCCTGCGGCTGAGAAACTTC-3’;SEQ ID NO:2;
(2) pMD18-T-ompA carriers are built, sequencing is carried out to ompA genes;
(3) the correct bacterial strain of sequencing result expands culture, extracts plasmid, using pMD18-T-ompA as template, P3 (20 μM)
(20 μM) are primer with P4, and PCR amplification ompA genes obtain purifying target gene;The reaction system of PCR amplification is 20 μ L,
In 2 × TaqPCR Mix each 1 μ L of 10 μ L, primer P3 and P4, template 1 μ L, ddH2The response procedures of 7 μ L of O, PCR amplification are 94
DEG C pre-degeneration 5min, 94 DEG C of 30s, 53 DEG C of 30s, 72 DEG C of 60s, 30 cycles, 72 DEG C of 10min;Wherein primer P3 and P4 are:
(4)P3:5’-GGAATTCGCTCCGAAAGACGACACCTGG-3 ', EcoR I;SEQ ID NO:3;
(5)P4:5’-ACGCGTCGACAGCCTGCGGCTGAGAAACTTC-3 ', Sal I;SEQ ID NO:4;
Wherein underscore part is restriction enzyme site;
(6) double enzymes are carried out respectively to purifying target gene and pET-32a carriers using restriction enzyme EcoR I and Sal I
It cuts, structure recombinant expression carrier pET-32a-ompA;
(7) 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;
(8) recombinant expression carrier pET-32a-ompA and empty carrier pET-32a are converted into BL21 engineering bacterias respectively, carries out weight
The prokaryotic expression of histone;
(9) SDS-PAGE is utilized to analyze recombinant protein expression;
(10) it is secondary antibody by primary antibody, HRP-IgG of His-Mab, Western blot verifications is carried out to recombinant protein.
The beneficial effects of the invention are as follows:The present invention is obtained using prokaryotic expression method outside the Wdwardsiella tarda of large-scale purification
The recombinant protein of memebrane protein OmpA, 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.
Description of the drawings:
Fig. 1 is different strains ompA Genetic homology of carbapenem-resistant in the embodiment of the present invention 1;
Fig. 2 is the PCR amplification of ompA genes in the embodiment of the present invention 2;
M.DL2000plus Marker;1.ompA gene magnifications;
Fig. 3 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.pET-32a-ompA/BL21 cellular lysate objects;2.pET-32a/BL21 zero loads compare;
Fig. 4 is that Western blot analyze expression of the His-OmpA in Escherichia coli in the embodiment of the present invention 4;
1.pET-32a-ompA/BL21 cellular lysate objects;2.pET-32a/BL21 zero loads compare;
Fig. 5 is the soluble analysis of recombinant protein in the embodiment of the present invention 4;
M. albumen Marker;1.pET-32a-ompA/BL21 cellular lysate objects;
2.pET-32a-ompA/BL21 cellular lysate object supernatants;3.pET-32a-ompA/BL21 cellular lysate objects precipitate;
Fig. 6 is the SDS-PAGE of the destination protein purified in the embodiment of the present invention 4;
M. albumen Marker;1.pET-32a-ompA/BL21 cellular lysate objects precipitate;2. purification of recombinant proteins;
Fig. 7 is that Westernblot analyzes identification of the positive serum to recombinant protein in the embodiment of the present invention 4;
1. recombinant protein;2. negative control (His- σ C reovirus capsid proteins);
Fig. 8 is recombinant protein in the embodiment of the present invention 5 to the immune protective effect of mouse;
Fig. 9 is the antibody level variation of immune serum in the embodiment of the present invention 5.
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 ompA gene sequencings of embodiment
According to E.tarda reference strain ET-1 genome sequences (CP001135.1) the ompA genes logged on GenBank
Design primer, primer P1 and P2 are:
P1:5’-ATGAAAAAAACAGCGATCGCA-3’;SEQ ID NO:1;
P2:5’-TTAAGCCTGCGGCTGAGAAACTTC-3’;SEQ ID NO:2;
ET-CL genomes are extracted using bacterial genomes extracts kit, and using it as template PCR amplifications ompA genes;
Wherein the reaction system of PCR amplification is 20 μ L, wherein 2 × Taq PCR Mix 10 μ L, primer P1 (20 μM) and P2 (20 μM) each 1
μ L, template 1 μ L, ddH2O 7μL;Pcr amplification reaction program: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;Target gene is recycled, pMD18-T carriers, 10 μ L systems are connected:Target gene 4.5 μ L, pMD18-
0.5 μ L, 2 × Connect buffer of T Vector, 5 μ L, 16 DEG C of connections are overnight;It is thin that connection product is transformed into DH5 α competence
Born of the same parents;Picking single bacterium colony carries out PCR detections, carries out sequencing by Sheng Gong biotech firms, is referred to reference to E.tarda in GenBank
Sequencing result is compared in bacterial strain and other enterobacteriaceae lactobacteriaceae ompA gene orders, and the results are shown in Figure 1, Edward
OmpA genes are highly conserved between Pseudomonas member, and higher with Aeromonas homology.
2 Wdwardsiella tarda ompA gene magnifications of embodiment
Using the OmpA albumen basic structure characteristics of UniProt web analytics E.tarda, designed according to sequencing result special
Property amplimer P3 and P4, remove OmpA signal peptide moieties, OmpA protein signal peptides influence protein expression.
The correct PMD18-T-ompA bacterial strains of 1 sequencing result of embodiment are expanded into culture, extract plasmid, and using it as mould
(20 μM) of plate, P3 (20 μM) and P4 are primer, and PCR amplification ompA genes, acquisition size is the target fragment of 990bp (such as Fig. 2 institutes
Show), purify target gene;Wherein the reaction system of PCR amplification be 20 μ L, wherein 2 × Taq PCR Mix 10 μ L, primer P3 and
Each 1 μ L of P4, template 1 μ L, ddH27 μ L of O, the response procedures of PCR amplification are 94 DEG C of pre-degenerations 5min, 94 DEG C of 30s, 53 DEG C of 30s,
72 DEG C of 60s, 30 cycles, 72 DEG C of 10min;Wherein primer P3 and P4 are:
P3:5’-GGAATTCGCTCCGAAAGACGACACCTGG-3 ', EcoR I;SEQ ID NO:3;
P4:5’-ACGCGTCGACAGCCTGCGGCTGAGAAACTTC-3 ', Sal I;SEQ ID NO:4;
Wherein, underscore part is restriction enzyme site.
The structure of embodiment 3pET-32a-ompA recombinant expression carriers
Double digestion, structure recombination table are carried out to purifying target gene and pET-32a carriers respectively using EcoR I and Sal I
Up to plasmid pET-32a-ompA, DH5 α competent cells, ice bath 30min, 42 DEG C of heating bath 90s, ice bath 5min are converted;1mL is added
LB liquid medium, 37 DEG C of rejuvenation 1h are coated on the tablet containing the final concentration of 100 μ g/mL of ammonia benzyl, are inverted in 37 DEG C and cultivate
Night;Picking monoclonal carries out PCR and double digestion verification, double digestion system totally 30 μ L:Recombinant plasmid/pET-32a 25 μ L, EcoR
Each 1 μ L, 10 × Green Buffer of I/Sal, I restriction enzymes 3 μ L, 37 DEG C of 1h, positive colony extract plasmid and carry out sequence
It measures, it is ensured that without mutation and frameshit.
The prokaryotic expression of 4 recombinant protein of embodiment
Recombinant vector pET-32a-ompA 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, 37 DEG C of shake cultures to OD600It is 0.6-0.8 to be worth, addition IPTG to final concentration of 0.5mmol/L, 37 DEG C of shake culture 4h~
6h, thalline were collected by centrifugation, and PBS buffer solution is washed 3 times;Albumen sample-loading buffer is added, 10min is boiled after mixing well, centrifuges,
It is cellular lysate object to collect supernatant.
It is identified using SDS-PAGE, after loading, voltage is adjusted to 80V, after sample is by separation gel and concentration glue separator bar,
Voltage is adjusted to 120V, until electrophoresis terminates, film coomassie brilliant blue staining 1min is taken out, with the multiple boiling decoloring of distilled water;Knot
At 58kD as shown in figure 3, there is destination protein band in fruit.
Further confirm that destination protein is expressed with Western blot methods.After SDS-PAG electrophoresis, film is taken out, is utilized
Transferring film instrument goes to albumen on NC films (80V 1h);After transferring film, NC films are taken out, PBST is washed 3 times, 5min/ times;5% is de-
Fat milk powder 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 as one
It is anti-, it is incubated at room temperature 1h;PBST is washed 3 times, 5min/ times;5% skimmed milk power presses 1:5000 dilution HRP-IgG are as secondary antibody, room temperature
It is incubated 30min;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 4,
Further verification shows recombinant protein OmpA successful expressions.
Thalline were collected by centrifugation, and albumen sample-loading buffer is added, and is placed in ultrasound 20min, ultrasonic 3s in ice chest, suspends 3s;It splits
It is centrifuged after solution, collects supernatant precipitation;SDS-PAGE analyses are carried out, the results are shown in Figure 5, and recombinant protein is primarily present in precipitation
In, it is expressed with inclusion bodies.
Inclusion body is purified using urea extraction method, the sediment after ultrasound is cracked is dissolved in by every gram (weight in wet base)
In 1mL water, 4 DEG C of high speed centrifugation 15min;Precipitation is resuspended and contains the 0.1mol/LTris-Cl (pH8.5) of 1M urea with 1mL, 4
DEG C high speed centrifugation 15min;Supernatant is taken to obtain more pure destination protein, the results are shown in Figure 6, and it is a concentration of to measure albumen
0.56mg/mL。
With E.tarda infecting mouses positive serum (1:100) it is primary antibody, HRP-IgG (1:5000) it is secondary antibody, utilizes
Identification of the Western blot verification positive serums to recombinant protein, the results are shown in Figure 7, E.tarda infecting mouse positive bloods
OmpA albumen can be recombinated with specific recognition clearly.
5 recombinant protein immunoprotection experiment of embodiment
40 healthy Kunming mouses are randomly divided into two groups, every group 20 at random.Head exempts from 30 μ g recombinant proteins of intramuscular injection
OmpA (Freund's complete adjuvant emulsification), two exempt from (incomplete Freund's adjuvant emulsification) after being spaced 2 weeks, are not exclusively helped with Freund after 30d
Agent emulsified protein booster immunization;Control group injects isometric PBS (corresponding Freund's adjuvant emulsification).The 48d pairs two groups of mouse peritoneal
It is inoculated with 5LD50(2.0×107Cfu) Wdwardsiella tarda ET-CL bacterium solutions observe 4d after infection, count The dead quantity.As a result it shows
Show, control group mice is all dead, 9 death of immune group mouse, remaining mouse undergo of short duration spirit is depressed, do not eat after it is gradual
It gets well.The results are shown in Figure 8, shows there is certain protection, protective rate 55% after mouse is immunized in recombinant protein OmpA.
The different time points before and after mouse immune take mouse blood with sinus under socket of the eye and detach serum, with ultrasonication
Wdwardsiella tarda whole bacterial protein detects the potency of antibody in mice serum using indirect ELISA method as envelope antigen, with
Negative serum OD450Average value adds 3 times of standard deviations as yin and yang attribute criterion, and it is 0.160 to calculate critical value, when blood to be checked
Clear OD values are determined as the positive when being more than critical value;It is determined as feminine gender when serum OD values to be checked are less than critical value.4 times are acquired
Serum carries out indirect ELISA detection, and the results are shown in Figure 9, and it is special to show that the immune mouse of recombinant protein can generate higher level
Property antibody, reaches highest level 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 outer membrane protein OmpA with immanoprotection action
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
<212> DNA
<213> Artificial
<400> 1
atgaaaaaaa cagcgatcgc a 21
<210> 2
<211> 24
<212> DNA
<213> Artificial
<400> 2
ttaagcctgc ggctgagaaa cttc 24
<210> 3
<211> 28
<212> DNA
<213> Artificial
<400> 3
ggaattcgct ccgaaagacg acacctgg 28
<210> 4
<211> 31
<212> DNA
<213> Artificial
<400> 4
acgcgtcgac agcctgcggc tgagaaactt c 31
Claims (6)
1. applications of the Wdwardsiella tarda outer membrane protein OmpA with immanoprotection action in subunit vaccine.
2. the preparation side of the Wdwardsiella tarda outer membrane protein OmpA 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 ompA genes;Wherein
Primer P1 and P2 are:
P1:5’-ATGAAAAAAACAGCGATCGCA-3’SEQ ID NO:1;
P2:5’-TTAAGCCTGCGGCTGAGAAACTTC-3’SEQ ID NO:2;
(2) pMD18-T-ompA carriers are built, sequencing is carried out to ompA genes;
(3) the correct bacterial strain of sequencing result expands culture, extracts plasmid, and using pMD18-T-ompA as template, P3 and P4 are primer,
PCR amplification ompA genes obtain purifying target gene;Wherein primer P3 and P4 are:
P3:5’-GGAATTCGCTCCGAAAGACGACACCTGG-3 ', EcoR I;SEQ ID NO:3;
P4:5’-ACGCGTCGACAGCCTGCGGCTGAGAAACTTC-3 ', Sal I;SEQ ID NO:4;
(4) double digestion is carried out respectively to purifying target gene and pET-32a carriers using restriction enzyme EcoR I and Sal I,
Build recombinant expression carrier pET-32a-ompA;
(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-ompA 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 outer membrane protein OmpA according to claim 2 with immanoprotection action
Method, which is characterized in that the reaction system of step (1) described PCR amplification is 20 μ L, wherein 2 × Taq PCR Mix 10 μ L, 20
μM P11 μ L, 20 μM of P21 μ L, template 1 μ L, ddH27 μ L of O, 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 outer membrane protein OmpA according to claim 2 with immanoprotection action
Method, which is characterized in that the reaction system of step (3) described PCR amplification is 20 μ L, wherein 2 × Taq PCR Mix 10 μ L, 20
μM P31 μ L, 20 μM of P41 μ L, template 1 μ L, ddH27 μ L of O, the response procedures of PCR amplification are 94 DEG C of pre-degeneration 5min, 94 DEG C
30s, 53 DEG C of 30s, 72 DEG C of 60s, 30 cycles, 72 DEG C of 10min.
5. the prokaryotic expression of the Wdwardsiella tarda outer membrane protein OmpA according to claim 2 with immanoprotection action
Method, which is characterized in that step (6) the conversion BL21 engineering bacterias are as follows:
5 μ L recombinant vectors pET-32a-ompA and 5 μ L empty carriers pET-32a are added in 100 μ L BL21 engineering bacterias respectively;
Ice bath 30min, 42 DEG C of heating bath 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 LB tablets of the final concentration of 100 μ g/mL of ammonia benzyl are inverted, 37 DEG C of overnight incubations.
6. the prokaryotic expression of the Wdwardsiella tarda outer membrane protein OmpA 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 |
---|---|---|---|
CN201810132575.7A CN108359683B (en) | 2018-02-09 | 2018-02-09 | Edwardsiella tarda outer membrane protein OmpA with immune protection effect |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201810132575.7A CN108359683B (en) | 2018-02-09 | 2018-02-09 | Edwardsiella tarda outer membrane protein OmpA with immune protection effect |
Publications (2)
Publication Number | Publication Date |
---|---|
CN108359683A true CN108359683A (en) | 2018-08-03 |
CN108359683B CN108359683B (en) | 2021-07-06 |
Family
ID=63005583
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201810132575.7A Active CN108359683B (en) | 2018-02-09 | 2018-02-09 | Edwardsiella tarda outer membrane protein OmpA with immune protection effect |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN108359683B (en) |
Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103830746A (en) * | 2014-03-18 | 2014-06-04 | 贵州大学 | Riemerella anatipestifer deoxyribonucleic acid (DNA) vaccine based on OmpA (octamethyl pyrophosphoramide) gene and preparation method of vaccine |
CN103930131A (en) * | 2011-05-13 | 2014-07-16 | 美国加州大学洛杉矶海滨分校医学中心的洛杉矶生物医学研究所 | Compositions and methods for immunization against drug resistant acinetobacter baumannii |
CN104961811A (en) * | 2015-06-29 | 2015-10-07 | 郑宗林 | Aeromonas hydrophila outer membrane protein gene prokaryotic expression protein and application thereof |
CN104962572A (en) * | 2015-06-29 | 2015-10-07 | 郑宗林 | Preparation method for aeromonas hydrophila outer membrane protein gene prokaryotic expression protein |
CN105497885A (en) * | 2014-09-25 | 2016-04-20 | 普莱柯生物工程股份有限公司 | Subunit vaccine, and preparation method and application thereof |
CN105566461A (en) * | 2015-12-25 | 2016-05-11 | 中山大学 | Bacterial outer membrane protein ompAs-19 after DNA shuffling and application thereof as an immunomodulator |
CN105968212A (en) * | 2016-05-24 | 2016-09-28 | 集美大学 | Eel Vibrio vulnificus/Edwardsiella tarda duplex recombinant protein and preparation method thereof |
-
2018
- 2018-02-09 CN CN201810132575.7A patent/CN108359683B/en active Active
Patent Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103930131A (en) * | 2011-05-13 | 2014-07-16 | 美国加州大学洛杉矶海滨分校医学中心的洛杉矶生物医学研究所 | Compositions and methods for immunization against drug resistant acinetobacter baumannii |
CN103830746A (en) * | 2014-03-18 | 2014-06-04 | 贵州大学 | Riemerella anatipestifer deoxyribonucleic acid (DNA) vaccine based on OmpA (octamethyl pyrophosphoramide) gene and preparation method of vaccine |
CN105497885A (en) * | 2014-09-25 | 2016-04-20 | 普莱柯生物工程股份有限公司 | Subunit vaccine, and preparation method and application thereof |
CN104961811A (en) * | 2015-06-29 | 2015-10-07 | 郑宗林 | Aeromonas hydrophila outer membrane protein gene prokaryotic expression protein and application thereof |
CN104962572A (en) * | 2015-06-29 | 2015-10-07 | 郑宗林 | Preparation method for aeromonas hydrophila outer membrane protein gene prokaryotic expression protein |
CN105566461A (en) * | 2015-12-25 | 2016-05-11 | 中山大学 | Bacterial outer membrane protein ompAs-19 after DNA shuffling and application thereof as an immunomodulator |
CN105968212A (en) * | 2016-05-24 | 2016-09-28 | 集美大学 | Eel Vibrio vulnificus/Edwardsiella tarda duplex recombinant protein and preparation method thereof |
Non-Patent Citations (4)
Title |
---|
BISWAJIT MAITI ET AL: "Recombinant outer membrane protein A (OmpA) of Edwardsiella tarda, a potential vaccine candidate for fish, common carp", 《MICROBIOLOGICAL RESEARCH》 * |
CHAO WANG ET AL: "Identification of plasma-responsive outer membrane proteins and their vaccine potential in Edwardsiella tarda using proteomic approach", 《JOURNAL OF PROTEOMICS》 * |
CHAO WANG ET AL: "TolC plays a crucial role in immune protection conferred by Edwardsiella tarda whole-cell vaccines", 《SCIENTIFIC REPORTS》 * |
SAURABH DUBEY ET AL: "Edwardsiella tarda OmpA Encapsulated in Chitosan Nanoparticles Shows Superior Protection over Inactivated Whole Cell Vaccine in Orally Vaccinated Fringed-Lipped Peninsula Carp (Labeo fimbriatus)", 《VACCINES》 * |
Also Published As
Publication number | Publication date |
---|---|
CN108359683B (en) | 2021-07-06 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Fu et al. | Protective immunity against iridovirus disease in mandarin fish, induced by recombinant major capsid protein of infectious spleen and kidney necrosis virus | |
CN103172749B (en) | Preparation of African swine fever protein engineering vaccine | |
CN106220716B (en) | Infectious coryza of chicken subunit vaccine and preparation method thereof | |
Gong et al. | Immune efficacy of DNA vaccines based on oprL and oprF genes of Pseudomonas aeruginosa in chickens | |
Feng et al. | Identification and characterization of a novel conserved 46 kD maltoporin of Aeromonas hydrophila as a versatile vaccine candidate in European eel (Anguilla anguilla) | |
CN104693310B (en) | A kind of chimeric protein, virus-like particle and its application | |
CN101451145B (en) | Tuberculosis gene vaccine based on T cell epitope as well as preparation method and use thereof | |
CN101016541A (en) | Method of producing brucella vaccine antigen protein | |
CN101838658B (en) | O type foot-and-mouth disease virus variant as well as coding gene and application thereof | |
CN103275228A (en) | K99-987P-F41 recombinant protein and application thereof | |
CN105039233B (en) | A kind of B. abortus molecular marker vaccine strain and its application | |
CN107936123B (en) | Swine transmissible gastroenteritis virus fusion protein and preparation method and application thereof | |
Hashish et al. | A multiepitope fusion antigen elicits neutralizing antibodies against enterotoxigenic Escherichia coli and homologous bovine viral diarrhea virus in vitro | |
CN109568572A (en) | A kind of preparation method and applications of Aeromonas Multivalent DNA Vaccine | |
Gong et al. | Evaluation of immunogenicity and protective efficacy of recombinant ptfA of avian Pasteurella multocida | |
CN108330142A (en) | A kind of Mermaid luminous bacillus hemolysin Hly with immanoprotection actionchAlbumen | |
CN108530522A (en) | A kind of OmpK multi-epitopes polypeptide, construction method and its application of recombination | |
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 | |
CN108359682B (en) | Edwardsiella tarda outer membrane protein PagC with immune protection effect | |
CN103243105B (en) | A kind of Trichina recombinant protein and application | |
CN103387604B (en) | CD8+T cell epitope polypeptide of S1 protein of chicken IBV (Infectious Bronchitis Virus) S1 protein | |
CN108359683A (en) | A kind of Wdwardsiella tarda outer membrane protein OmpA with immanoprotection action | |
TWI614026B (en) | Recombinant avibacterium paragallinarum flfa fimbrium subunit vaccine for infectious coryza of chickens, preparation method and use thereof | |
CN109517044B (en) | Porcine epidemic diarrhea virus genetic engineering antigen and antibody | |
TW201236693A (en) | Method of inducing antibody in birds by the OmpA of Riemerella anatipestifer and the Riemerella anatipestifer vaccine |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
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 |