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
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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
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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.
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