CN105541977B - Riemerella anatipestifer OmpH intercept recombinant protein and preparation method and application - Google Patents
Riemerella anatipestifer OmpH intercept recombinant protein and preparation method and application Download PDFInfo
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- CN105541977B CN105541977B CN201610070599.5A CN201610070599A CN105541977B CN 105541977 B CN105541977 B CN 105541977B CN 201610070599 A CN201610070599 A CN 201610070599A CN 105541977 B CN105541977 B CN 105541977B
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- 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
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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Abstract
The invention discloses a kind of riemerella anatipestifer OmpH intercept recombinant protein and preparation method and applications, belong to bioengineering field.The amino acid sequence of riemerella anatipestifer OmpH intercept recombinant protein of the invention is as shown in SEQ ID NO:1；Then it is connect with prokaryotic expression carrier pET-32a (+) on the basis of it is cloned using technique for gene engineering, the gene engineering product that successful conversion e. coli bl21 (DE3) expression system is induced, expressed；The expression-form of the albumen is OmpHM/His fusion protein, and the fusion protein molecule amount of expression is 28kDa, is that amalgamation and expression mainly considers convenient for purifying by product design；The natural activity that albumen keeps albumen is expressed simultaneously.Product can be applied to the detection antigen of the indirect ELISA method of detection riemerella anatipestifer antibody, also can be used as the genetic engineering subunit vaccine of prevention riemerella anatipestifer.
Description
Technical field
The invention belongs to bioengineering field, in particular to a kind of riemerella anatipestifer OmpH intercept recombinant protein and system
Preparation Method and application.
Background technique
Riemerella anatipestifer (Riemerella anatipestifer, RA) is a kind of Gram-negative bacteria, is attributed to Huang
Bacteriaceae (Flavobacteriaceae) is the pathogen of Riemerella anatipestifer disease, can cause duck, goose, turkey and a variety of
Chronic or acute sepsis shape the contagious infection disease of one kind of birds, is in worldwide distribution, morbidity and mortality are high,
As current one of the Infectious Diseases for seriously endangering duck culturing industry.The main clinical symptoms of the disease are mainly shown as that spirit is depressed,
Paralysed ground, draws the symptoms such as yellow green loose stools at necking down.Lesion characteristics are to cause cellulosic air bag inflammation, the meningitis, pericardium of host
Scorching, perihepatitis and caseous salpingitis, and with different degrees of septicemia, to the development of aquaculture, there is huge
Threat.
The current established method for detecting riemerella anatipestifer serum antibody has AGP test experiment, agglutination examination
It tests, ELISA etc., these detection methods or sensitivity, specificity be not high, or exists due to using the difference of antigenic component
Some shortcomings；And the prevention and treatment of riemerella anatipestifer is mainly that antibiotic carries out treatment or with Bacteria vaccine come to corresponding serotype
Infection is prevented.OmpH belongs to porin, has no the correlative study report of riemerella anatipestifer OmpH research and application at present
Road.
Summary of the invention
To overcome shortcoming and deficiency of the existing technology, one of the objects of the present invention is to provide Mo Shi in a kind of pest of duck
Bacillus OmpH intercept recombinant protein, the albumen can capturing agents etc. as riemerella anatipestifer antibody.
The second object of the present invention is to provide the preparation method of above-mentioned riemerella anatipestifer OmpH intercept recombinant protein,
This method can be used for the large-scale production of OmpH intercept recombinant protein.
The third object of the present invention is to provide the above-mentioned riemerella anatipestifer OmpH intercept recombinant protein prepared
Application.
The purpose of the invention is achieved by the following technical solution: a kind of riemerella anatipestifer OmpH intercept recombinant protein,
Its amino acid sequence is as shown in SEQ ID NO:1.
The DNA sequence dna of above-mentioned riemerella anatipestifer OmpH intercept recombinant protein is as shown in SEQ ID NO:2.
The preparation method of above-mentioned riemerella anatipestifer OmpH intercept recombinant protein, specifically includes the following steps:
A, using RA-CH-1 genomic DNA as template, using nucleotide sequence respectively such as SEQ ID NO.3 and SEQ ID
Upstream and downstream primer PCR shown in NO.4 expands to obtain riemerella anatipestifer OmpH intercept genetic fragment (OmpHM), with
The connection of pMD19-T (simple), obtains pMD19-OmpHM；
B, double digestion pMD19-OmpHM plasmid and prokaryotic expression carrier are distinguished with restriction enzyme BamH I and Xho I
PET32a (+), is attached, and obtains recombined pronucleus expression plasmid pET32a-OmpHM；
C, the recombinant plasmid pET32a-OmpHM that step B is extracted is transformed into expressive host bacterium inducing expression, obtained in pest of duck
Mo Shi bacillus intercept recombinant protein crude product.
Further, by riemerella anatipestifer OmpH intercept recombinant protein crude product obtained by step C through nickel Ago-Gel parent
After chromatographic purifying, riemerella anatipestifer OmpH intercept recombinant protein is obtained；
Further, the host strain in step C is bacterial strain BL21 (DE3), in IPTG concentration=1.2mmol/L, inducing temperature
It is carried out inducing expression 9 hours under the conditions of being 25 DEG C, obtains riemerella anatipestifer OmpH intercept recombinant protein.
The beneficial effects of the present invention are: riemerella anatipestifer OmpH intercept recombinant protein is to choose Mo Shi bar in pest of duck
The Main Antigenic Region (81aa-151aa) of bacterium OmpH gene coded protein, and it is named as OmpHM；Then genetic engineering skill is utilized
It connect, successful conversion e. coli bl21 by art on the basis of it is cloned with prokaryotic expression carrier pET-32a (+)
(DE3) gene engineering product that expression system is induced, expressed.The expression-form of the albumen is that OmpHM/His solubility is melted
Hop protein, the fusion protein molecule amount of expression are 28kDa, are that amalgamation and expression mainly considers convenient for purifying by product design；Together
When expression albumen be solubility expression form, the denaturation of albumen is avoided in the preparation of albumen, purification process, renaturation waited
Journey, conducive to the natural activity for keeping albumen.Product after prokaryotic expression is after Western blot is identified
Show that there is immune response activity similar with native protein.
The application of the above-mentioned riemerella anatipestifer OmpH intercept recombinant protein prepared is mainly reflected in:
1. this product can be used as the detection antigen of the indirect ELISA method of detection riemerella anatipestifer antibody.
2. can be used as the genetic engineering subunit vaccine of prevention riemerella anatipestifer.
Product advantage of the invention is embodied in:
1. not full bacterium should with this application since the detection antigen is riemerella anatipestifer OmpH intercept recombinant protein
Detection antigen is dangerous without scattered bacterium when being detected.
2. solving riemerella anatipestifer difficulty culture, problem easy to pollute using the albumen as antigen.
3. detecting riemerella anatipestifer antibody, high specificity, no cross reaction as ELISA antigen.
4. performance is stable, production cost is low, it is suitble to the factorial production, market application prospect is wide.
5. there is good protectiveness as subunit vaccine.
More beneficial effects will embody in embodiment.
Detailed description of the invention
In order to keep the purpose of the present invention, technical scheme and beneficial effects clearer, the present invention provides following attached drawing:
Fig. 1 is the agarose gel electrophoresis figure of the PCR amplification of OmpH intercept gene.Wherein 1 expands for OmpH intercept gene PCR
Increase production object through agarose gel electrophoresis specific DNA band obtained；M is DNA Marker.
Fig. 2 is the agarose gel electrophoresis figure of recombinant plasmid pMD19-OmpHM.Wherein, M is DNA Marker；1 is recombination
Plasmid pMD19-OmpHM I double digestion of BamH I and Xho, digestion products are special through agarose gel electrophoresis two obtained
Property band.
The agarose gel electrophoresis figure that the digestion that Fig. 3 is pET32a-OmpHM is identified.Wherein, M is DNA Marker；1 is
Recombinant expression plasmid pET32a-OmpHM I single endonuclease digestion of Xho, digestion products are through an agarose gel electrophoresis spy obtained
Anisotropic band；2 be recombinant expression plasmid pET32a-OmpHM I double digestion of BamH I and Xho, and digestion products are solidifying through agarose
Gel electrophoresis two specific bands obtained；3 be recombinant expression plasmid pET32a-OmpHM I single endonuclease digestion of BamH.
Fig. 4 is the SDS- of the overnight OmpH intercept recombinant protein of inducing expression when IPTG=0.4mmol/L at 37 DEG C
PAGE electrophoretogram.Wherein M is protein Marker；1 expresses after bacterium is induced with IPTG for empty carrier pET-32a (+) through SDS-
PAGE electrophoresis acquired results；2 express bacterium for recombinant expression plasmid pET32a-OmpHM is induced with IPTG, and obtained bacterium solution is surpassed
After sonication, centrifugation, gained supernatant carries out the result of SDS-PAGE electrophoresis；3 be recombinant expression plasmid pET32a-OmpHM table
It is induced up to bacterium with IPTG, after obtained bacterium solution carries out ultrasonic disruption, centrifugation, gained precipitating carries out the knot of SDS-PAGE electrophoresis
Fruit.
Host strain E.coli BL21 containing recombinant plasmid pET32a-OmpHM under difference IPTG concentration when Fig. 5 is 37 DEG C
(DE3) after inducing expression 4 hours, the SDS-PAGE electrophoresis of riemerella anatipestifer OmpHM intercept recombinant protein.Wherein, M is
Protein Marker；1 is the final concentration of 0mmol/L of IPTG；2 be the final concentration of 0.4mmol/L of IPTG；3 be the end of IPTG
Concentration is 0.8mmol/L；4 be the final concentration of 1.2mmol/L of IPTG.
Fig. 6 contains the host strain of recombinant plasmid pET32a-OmpHM when being IPTG=0.4mmol/L under different temperatures
E.coli BL21 (DE3) expresses 4 hours SDS-PAGE of riemerella anatipestifer OmpHM intercept recombinant protein inducing expression electricity
Swimming figure.Wherein, M is protein Marker；1 be inducing temperature is 25 DEG C；2 be inducing temperature be 30 DEG C；3 be inducing temperature be 37
℃。
Fig. 7, which is 37 DEG C, contains recombinant plasmid pET32a- under different induction times when with IPTG concentration being 0.4mmol/L
The SDS-PAGE electricity of host strain E.coli BL21 (DE3) the expression riemerella anatipestifer OmpHM intercept recombinant protein of OmpHM
Swimming figure.Wherein M is protein Marker；1 is induction 0h；2 be induction 5h；3 be induction 7h；4 be induction 9h.
Fig. 8 is nickel Ago-Gel (Ni2+- NAT) riemerella anatipestifer OmpH intercept recombination after affinitive layer purification
Protein SDS-PAGE electrophoretogram.Wherein M is protein Marker；1 is the OmpH after nickel Ago-Gel affinitive layer purification
Intercept recombinant protein.
Fig. 9 is riemerella anatipestifer OmpH intercept recombinant protein Western-blot figure.Wherein M is pre- dsred protein
Marker；1 is the immunoblotting band of OmpH intercept recombinant protein.
Figure 10 is riemerella anatipestifer slide agglutination test result figure.Divide in 1:2,1:4,1:8 and Positive control wells
There is not fine sand shape agglutinating particle, without agglutinating particle in the hole 1:16.
Figure 11 is that riemerella anatipestifer micro-agglutination tests picture.In 1:8,1:16,1:32 and Positive control wells
Visible agglutination particle；Without agglutinating particle in the hole 1:64, only part bacterial sediment is identical as negative control hole in bottom.
Figure 12 is the pathological tissues organ after negative control group duckling artificial challenge riemerella anatipestifer is dead.In the heart
Dirty, liver, abdominal cavity surface can see one layer of fibrinous exudate, form one layer of pod membrane.
Figure 13 is duckling artificial challenge's riemerella anatipestifer of immune riemerella anatipestifer bacillus OmpH intercept recombinant protein
What is survived afterwards analyses situation.It can be seen that abdominal cavity, liver and heart surface are normal, cellulosic exudation and necrosis are had no
Stove, each organ are normal.
Figure 14 is the PCR identification that RA attacks that malicious duck separates the 16sRNA of RA again.M is DNA Marker；1,2,3,4 difference
For the bacterium colony PCR of isolated strains；5 be negative control.
Specific embodiment
Hereinafter reference will be made to the drawings, and the preferred embodiment of the present invention is described in detail.Tool is not specified in preferred embodiment
The experimental method of concrete conditions in the establishment of a specific crime, usually according to normal condition, such as Molecular Cloning:A Laboratory guide (third edition, J. Pehanorm Brooker etc.
Write, Huang Peitang etc. translates, Science Press, 2002) described in condition, or carry out according to the normal condition proposed by manufacturer.
Partial amino-acid is abridged as follows accordingly:
Glutamine gln Q；Glycine gly G；Serine ser S；Alanine ala A；Threonine thr T；Valine val
V；Isoleucine ile I；Leucine leu L；Tyrosine tyr Y；Phenylalanine phe F；Histidine his H；Proline pro
P；Asparagine asn N；Methionine met M；Glutamic acid glu E；Tryptophan trp W；Lysine lys K；Cysteine cys
C；Arginine arg R；Aspartic acid Asp D.
The preparation of 1 riemerella anatipestifer OmpH intercept recombinant protein of embodiment
1 main experimental materials
Plasmid T-Vector pMD19 (simple),Max DNA Polymerase, is purchased from Dalian
Precious bioengineering Co., Ltd；Prokaryotic expression plasmid pET32a (+), Novagen Products；Cloning host bacterium E.coli DH5
α, expressive host bacterium E.coli BL21 (DE3) and RA-CH-1 bacterial strain are provided by Sichuan Agricultural University's poultry diease research center.
2 experimental methods
The clone of 2.1RA-CH-1OmpH intercept gene
2.1.1 design of primers
Choosing Main Antigenic Region therein according to the existing RA-CH-1OmpH gene order of GenBank, (81aa-151aa is simultaneously
It is named as OmpHM), utilize Primer Premier5.0 software design pair of primers.Draw the upstream as shown in SEQ ID NO:3
Object: 5 '-CGCGGATCCGAGGCTCAGAGAACTGCT-3 ' (dashed part is I site BamH)；As shown in SEQ ID NO:4
Downstream primer: 5 '-CCGCTCGAGTCCTTTATAGATTAACCCC-3 ' (dashed part is I site Xho).After primer synthesis,
With the dissolution of appropriate sterile deionized water, make its final concentration of 20mmol/L, -20 DEG C save backup.
2.1.2RA the extraction of genomic DNA
A, riemerella anatipestifer RA-CH-1 plants of culture: the strain for taking freeze-drying to save, with 2mL Triptic soya meat
Dilution, is then inoculated in blood plate, CO in soup culture medium (TSB)2Under the conditions of 37 DEG C of constant temperature stand 18~for 24 hours (bacterium colony is rounded,
Microprotrusion, neat in edge, butyrous, colony diameter 2mm；Microscopically observation thallus is in short-thick type).By bacterium colony observation and mirror
Inspection, the culture in pancreas peptone soybean broth culture medium TSB (containing 1% calf serum) of picking single bacterium colony.
B, the Riemerellosis Anatipestifer bacterium solution of overnight incubation riemerella anatipestifer RA-CH-1 plants of expansion culture: is pressed 1:
100 (containing 1% calf serum) were inoculated into pancreas peptone soybean broth culture medium (TSB), and 37 DEG C of water-bath shaken cultivations cause bacterium
Grow into logarithmic growth phase.
C, the extraction of riemerella anatipestifer RA-CH-1 plants of DNA, the specific steps are as follows: (1) take the bacterium for growing into logarithmic phase
Liquid 1ml, 12000rpm are centrifuged 2min, abandon supernatant.(2) 567 μ l TE Buffer, 30 μ l10%SDS and 3 μ l Proteinase Ks are added,
37 DEG C of water-bath 1h, dissolution precipitating.(3) phenol/chloroform DNA, 12000rpm centrifugation 5min of equivalent is added, takes supernatant.It repeats
The step is primary.(4) equal amounts of chloroform/isoamyl alcohol extracting DNA, 12000rpm centrifugation 5min is added, takes supernatant.(5) twice of body is added
Long-pending dehydrated alcohol is washed, the sodium acetate of the 3mol/L of 10% volume, and ice bath 30min, 12000rpm are centrifuged 15min, abandons supernatant.
(6) it is precipitated twice with 70% ethanol washing, abandons supernatant.(7) precipitating DNA is spontaneously dried at room temperature, and appropriate TE (pH8.0) is added
Dissolution precipitating DNA, -20 DEG C save backup.
2.1.3PCR riemerella anatipestifer OmpH intercept gene is expanded
The reaction system of PCR reaction amplification riemerella anatipestifer OmpH intercept gene is as shown in table 1:
1 OmpH intercept gene PCR reaction system of table
It mixes gently, PCR is carried out after 4000r/min brief centrifugation.
Response parameter: 95 DEG C of 5min, subsequently into 94 DEG C of 40s, 58 DEG C of 40s, 72 DEG C of 60s, totally 35 are recycled, and last 72 DEG C
Extend 10min, is saved backup in 4 DEG C.5 μ l PCR products electrophoresis on 1% Ago-Gel is taken, the length of amplified fragments is observed
Degree.
2.1.4 the recycling of riemerella anatipestifer OmpH intercept gene PCR product
It is carried out by OMEGA company DNA QIAquick Gel Extraction Kit specification, DNA after the recovery is stored in -20 DEG C and saves backup.
2.1.5T clone
The connection of the riemerella anatipestifer OmpH intercept gene and pMD19-T (simple) of purifying
Coupled reaction system is as shown in table 2:
The reaction system that 2 OmpH intercept gene of table is connect with pMD19-T (simple)
Mentioned reagent is added in PCR reaction tube, it is careful to mix, after brief centrifugation, overnight in 16 DEG C of connections.
2.1.6DH5 the preparation of α competent cell
Prepare α plants of competent escherichia coli cells of fresh DH5 using Calcium Chloride Method, be summarized as follows: (1) sterile picking is flat
The DH5 alpha monoclonal colony inoculation of fresh cultured is stayed overnight in 5mL LB culture solution in 37 DEG C of shaken cultivations on plate；(2) 1mL is taken
Above-mentioned culture solution is inoculated in 100mL LB culture solution, 37 DEG C of 200r/min 2.5~3h of shaken cultivation, make OD600=0.4~
0.5 or so；(3) bacterial cultures is poured into after sterilizing in ice-cold centrifuge tube, ice bath 10min；(4) in 4 DEG C of 4000r/
Min is centrifuged 10min, abandons supernatant；(5) add the CaCl of the 0.1mol/L of 10mL ice pre-cooling2, mildly hanged bacterial precipitation, ice bath
30min；(6) it is centrifuged 10min in 4 DEG C of 4000r/min, abandons supernatant, the CaCl for the 0.1mol/L for adding 4mL ice to be pre-chilled2It is resuspended again
Final concentration of 15% sterile glycerol CaCl is added in precipitating2, it is packed as 100 μ l/ pipe after mixing, is saved overnight in -4 DEG C of refrigerators
To improve transformation efficiency.
2.1.7 transformed competence colibacillus cell
10 μ l linked systems are all taken out and are added in 100 μ l DH5 α competent cells, ice bath 30min；It is placed in 42 DEG C again
Warm bath 90s, later ice bath 2min again；890 μ l LB culture mediums are added immediately after, 37 DEG C of water-bath shaken cultivation 45min take 200
μ l is applied on the culture medium containing Amp, sets overnight incubation in 37 DEG C of incubators.The single white colony of next day picking is inoculated in containing Amp
LB liquid medium in, carry out plasmid extraction after 37 DEG C of water-bath shaken cultivation 18h.
2.1.8T the extracting of cloned plasmids
It is operated by OMEGA company plasmid extraction kit specification, recovery product is stored in -20 DEG C and saves backup.
2.1.9T the PCR of cloned plasmids and digestion identification
The recombinant plasmid that previous step extracts is named as pMD19-OmpHM, it is double digested with I/Xho of BamH I respectively
It is digested with the digestion of I single endonuclease digestion of Xho, I single endonuclease digestion of BamH, result is observed in 1.0% gel electrophoresis.PCR amplification purpose base is done simultaneously
The digestion system of cause, T cloned plasmids is as shown in table 3:
The digestion system of 3 T cloned plasmids of table
2.1.10 riemerella anatipestifer OmpH intercept gene sequencing
Digestion is identified that correct plasmid is sent Shanghai English fine horse biology Co., Ltd and is sequenced.
The building of 2.2 prokaryotic expression plasmid pET32a-OmpHM, the optimization of inducing expression and expression condition
2.2.1 the building and identification of prokaryotic expression plasmid pET32a-OmpHM
A, the digestion and connection of target fragment: restriction enzyme BamH I and Xho I distinguishes double digestion pMD19-OmpHM matter
Grain and prokaryotic expression carrier pET32a (+), digestion system are as shown in table 4:
4 target fragment of table and carrier digestion system
37 DEG C of water-bath 4h, after target fragment is separately recovered by the DNA plastic recovery kit operation instruction of OMEGA, according to table 5
Shown in 16 DEG C of linked system connection overnight.
The linked system of table 5 target fragment and prokaryotic expression carrier
The conversion of b recombinant plasmid: DH5 α competent cell is prepared using Calcium Chloride Method.Later, connection 10 μ l of liquid is taken to be added to
In centrifuge tube containing 100 μ l competence DH5 α, ice bath 30min after mixing；42 DEG C of water-bath 90s are placed in, then rapid ice bath 2min；
The 890 μ l of LB liquid medium without Amp is added, 37 DEG C of shakings (150r/min) cultivate 1~1.5h；200 μ l cultures are taken to apply
It is distributed in the LB plate containing Amp, 37 DEG C of overnight incubations, next day picking single bacterium colony is inoculated in LB liquid medium of the 5mL containing Amp
In, 37 DEG C of 12~16h of culture, while setting up empty carrier conversion group (10 μ l+ competence DH5 α of empty carrier, 100 μ l), carrier-free pair
According to group (sterilizing 10 μ l+ competence DH5 α of ultrapure water, 100 μ l).
The PCR of c bacterium solution is identified:
Using recombinant plasmid bacterium solution as template, upstream primer shown in SEQ ID NO:3: 5 '-
CGCGGATCCGAGGCTCAGAGAACTGCT-3 ' (dashed part is I site BamH)；Draw in the downstream as shown in SEQ ID NO:4
Object: 5 '-CCGCTCGAGTCCTTTATAGATTAACCCC-3 ' (dashed part is I site Xho).Carry out PCR reaction, method
It is same as above with amplification condition, takes PCR product in electrophoresis detection on 1% Ago-Gel.
The digestion of d recombinant plasmid is identified: clone's strain of above-mentioned preservation is inoculated in LB liquid medium of the 5mL containing Amp
In, 37 DEG C of water-baths shake overnight incubation, and next day extracts recombinant plasmid according to a conventional method, then use I single endonuclease digestion of Xho, I and of BamH
I double digestion of Xho identifies the recombinant plasmid, and digestion system is as shown in table 6:
Table 6 is subcloned each component content in digestion system
It is identified by digestion and PCR, obtains recombined pronucleus expression plasmid pET32a-OmpHM.
2.2.2 the inducing expression of recombinant expression plasmid pET32a-OmpHM
The extraction of a recombinant plasmid pET32a-OmpHM: picking has identified the DH5 of the pET32a-OmpHM containing positive recombinant plasmid
For the streak inoculation of α strain on the LB agar plate containing Amp, 37 DEG C of overnight incubations, next day takes single bacterium colony to be inoculated in the LB containing Amp
In fluid nutrient medium, bacterium solution is collected by centrifugation in violent shaken cultivation 10~16 hours, by OMEGA Plasmid DNA Mini Kit
Illustrate the extraction and purification for carrying out recombinant plasmid.
B recombinant plasmid pET32a-OmpHM conversion expression bacterium: using Calcium Chloride Method preparation E.coli BL21 (DE3) impression
State cell, and the recombinant plasmid pET32a-OmpHM of said extracted is transformed into expressive host bacterium E.coli BL21 (DE3).
The inducing expression of c recombinant plasmid pET32a-OmpHM: from above-mentioned LB solid medium, picking positive colony bacterium,
It is inoculated with LB liquid medium, 37 DEG C of overnight incubations, next day takes bacterium solution to access the LB liquid medium containing Amp in the ratio of 1:50
In, when violent shaken cultivation is to OD600=0.4, it is separately added into IPTG Fiber differentiation, 1mL is collected and cultivates bacterium solution, 4 DEG C of 13000r/
Min is centrifuged 2min, abandons supernatant, 40 μ l ultrapure waters and 10 μ l 10 × SDS sample-loading buffers, 100 DEG C of heating water baths are added in precipitating
It is denaturalized 10min, carries out 12%SDS-PAGE gel electrophoresis, observes expression of results.
The soluble analysis of d recombinant plasmid pET32a-OmpHM expression product: IPTG concentration is 0.4mmol/L at 37 DEG C
When the overnight bacterium solution of inducing expression and empty carrier each 200ml of bacterium solution, press step process respectively: 4 DEG C, 10000r/min centrifugation
5min, bacterial sediment are suspended with 20mL 20mmol Tris-HCl (pH8.0)；After setting -20 DEG C overnight, ultrasonic wave (ice bath) interval
Broken thallus (600w, 30sec/ times, 10 times), 4 DEG C, 10000r/min is centrifuged 10min, takes supernatant spare 1.；Precipitating uses 10mL
Urea solution (10mmol/L PBS+2mol/L urea) suspends, and 4 DEG C, after 10000r/min is centrifuged 10min, precipitating uses 10mL again
Washing lotion suspends, 2. with appropriate urea liquid (10mmol/L PBS+8mol/L urea) dissolution precipitating, low after washing repeatedly three times
Temperature saves backup.2., 40 μ l ultrapure waters and 10 μ are added in the precipitating for taking suitable supernatant 1. to dissolve with urea liquid respectively thereto
L 10 × SDS sample-loading buffer, 100 DEG C of heating water baths are denaturalized 10min, carry out 12%SDS-PAGE gel electrophoresis, gel is used
After coomassie brilliant blue staining, result is observed.
2.2.3 the optimization of recombinant plasmid pET32a-OmpHM expression condition
The concentration optimization of a inducer IPTG: taking the expressive host bacterium BL21 (DE3) of the pET32a-OmpHM containing recombinant plasmid,
It is inoculated in the LB liquid medium containing Amp, 37 DEG C of shaking overnight incubations.Next day is turned to be inoculated in the LB liquid containing Amp by 1:50
In culture medium, when 37 DEG C of cultures culture to OD600 value about 0.4, wherein 4 test tubes are taken, are separately added into IPTG to final concentration of
After 37 DEG C of induction 4h of 0mmol/L, 0.4mmol/L, 0.8mmol/L, 1.2mmol/L, sample is handled according to the above method,
12%SDS-PAGE electrophoresis observes result.
B temperature condition optimizing: the expressive host bacterium BL21 (DE3) of the pET32a-OmpHM containing recombinant plasmid is taken, is inoculated in and contains
In the 5mL LB liquid medium of Amp, 37 DEG C of shaking overnight incubations.Next day is turned to be inoculated in the LB Liquid Culture containing Amp by 1:50
In base, when 37 DEG C of cultures culture to OD600 value about 0.4, wherein 3 test tubes are taken, are separately added into IPTG to final concentration of
0.4mmol/L is respectively placed in 25 DEG C, 30 DEG C, 37 DEG C of induction 4h, handles by above-mentioned conventional method sample, 12%SDS-
PAGE electrophoresis observes result.
The optimization of c induction time: the expressive host bacterium BL21 (DE3) of the pET32a-OmpHM containing recombinant plasmid is taken, is inoculated in and contains
In the LB liquid medium of Amp, 37 DEG C of shaking overnight incubations.Next day is turned to be inoculated in the LB liquid medium containing Amp by 1:50,
When continuing culture to OD600 value about 0.4, IPTG to final concentration of 0.4mmol/L is added, 37 DEG C induce 0,5,7,9h respectively, inhale
1mL culture solution is taken, sample is handled according to the above method, 12%SDS-PAGE electrophoresis observes result.
A large amount of preparations, the purifying of 2.3 riemerella anatipestifer OmpH intercept recombinant proteins
2.3.1 a large amount of preparations (supernatant processing) of riemerella anatipestifer OmpH intercept recombinant protein
The 500mL bacterium solution of inducing expression is centrifuged 10min in 4 DEG C of 10000r/min, bacterial sediment is used
40mL20mmolTris-HCl (pH8.0) suspends；The broken thallus of ultrasonic wave (ice bath) interval (200w, 30sec/ times, 5~10
It is secondary), 4 DEG C of 10000r/min are centrifuged 10min.Taking supernatant is soluble riemerella anatipestifer OmpH intercept recombinant protein, and 4 DEG C
It saves backup.
2.3.2 the purifying of riemerella anatipestifer OmpH intercept recombinant protein
Nickel Ago-Gel (Ni2+- NAT) affinitive layer purification riemerella anatipestifer OmpH intercept recombinant protein: according to
The 6-His label of the fusion protein affinity interaction special to nickel ion enables the fusion protein with label to be incorporated into nickel fine jade
On sepharose, the condition for changing eluent is eluted, and achievees the purpose that purifying.Concrete operation step are as follows:
(1) fill column: nickel Ago-Gel fills column, and bed volume is about 40mL；
(2) it balances: balancing chromatographic column, flow velocity 1mL/min with about 5 bed volumes of equilibration buffer；
(3) loading: the soluble upper protein sample about 50mL of 0.45 μm of membrane filtration is added in chromatographic column, flow velocity
For 0.4mL/min；
(4) it washs: washing 2-5 bed volume, flow velocity 1mL/min again with equilibration buffer；
(5) elute: respectively with contain 20,50, the elution buffer of 100mmol imidazoles carry out gradient elution, flow velocity 1mL/
Min collects the eluting peak of each gradient, with the molecular size range and purity of SDS-PAGE detection fusion albumen；
(6) it cleans: washing 3 bed volumes, flow velocity 1mL/ with 5 bed volumes of ultrapure washing, then with 25% ethyl alcohol
Min recycles nickel agarose Gel column, saves in 4 DEG C, obtain the riemerella anatipestifer OmpH intercept recombinant protein of purifying.
3 experimental results
Amplification, T- clone and the qualification result of 3.1 riemerella anatipestifer OmpH intercept genes
3.1.1 the PCR amplification result of riemerella anatipestifer OmpH intercept gene
PCR amplification is carried out to OmpH intercept gene using RA-CH-1 genomic DNA as template, product is through 1.0% agarose
Gel electrophoresis obtains the specific DNA band (Fig. 1) of a treaty 234bp.
3.1.2 riemerella anatipestifer OmpH intercept gene T clone identification result
PCR product after purification by gel, simultaneously transformed competence colibacillus cell DH5 α is connect with pMD19-T (simple) carrier,
Obtained T clone designation is pMD19-OmpHM.PCR, digestion (Fig. 2: recombinant plasmid pMD19- are carried out to pMD19-OmpHM
OmpHM obtains two segments with BamH I and I double digestion of Xho, and the molecular size range of small fragment is about 234bp) and sequencing identification,
The result shows that T clones OmpH intercept gene DNA sequence (shown in SEQ ID NO:2) obtained with Mo Shi bar in known pest of duck
The OmpH intercept gene DNA sequence of bacterium RA-CH-1 is completely the same, and the amino acid sequence of coding is as shown in SEQ ID NO.1.
The building of 3.2 prokaryotic expression plasmid pET32a-OmpHM and identification, inducing expression and its optimum results
3.2.1 building Yu the digestion identification of recombinant expression plasmid pET32a-OmpHM
With target fragment is recycled after I double digestion T cloned plasmids of BamH I and Xho, with the pET-32a table through identical digestion
DH5 α is attached and converted up to carrier, obtains recombinant expression plasmid pET32a-OmpHM, after I double digestion of BamH I and Xho
Two bar segments are obtained, obtain a bar segment after Xho I, I single endonuclease digestion of BamH, and size is consistent with theoretical value, show to recombinate protokaryon
Expression plasmid constructs successfully (Fig. 3).
3.2.2 the inducing expression of recombinant plasmid pET32a-OmpHM
Under the conditions of 37 DEG C, it is 0.4mmol/L that concentration, which is added, in IPTG, and inducing expression is overnight, and empty carrier converts bacterial strain about
Occur a specific protein band at 20kDa, is label protein itself size on carrier；Recombinant expression plasmid pET32a-OmpHM
The recombination fusion protein of expression is consistent at about 28kDa with theoretical value；In addition, expression albumen soluble analysis is as the result is shown
It is primarily present in supernatant, illustrates that recombinant expression protein has (Fig. 4) in the form of soluble in thallus.
3.2.3 the optimization of recombinant plasmid pET32a-OmpHM expression condition
The optimization of a IPTG concentration: under the conditions of 37 DEG C, be added IPTG make its final concentration be respectively 0mmol/L,
0.4mmol/L, 0.8mmol/L, 1.2mmol/L Fiber differentiation 4h, as the result is shown: with increasing for IPTG concentration, protein induced amount
Increase, and is that 1.2mmol/L is slightly above other concentration (Fig. 5) in IPTG concentration.Therefore, the IPTG concentration of 1.2mmol/L is selected
As inducing expression concentration.
The optimization of b inducing temperature condition: the final concentration of 0.4mmol/L of IPTG, respectively at 25 DEG C, 30 DEG C, 37 DEG C of induction 4h,
The results show that inducible protein amount is less when temperature is at 37 DEG C, and when in 25 DEG C and 30 DEG C expression quantity it is higher, and at 25 DEG C
Expression quantity be slightly above 30 DEG C, therefore select 25 DEG C of temperature as best inducing temperature (Fig. 6).
The optimization of c induction time: being 0.4mmol/L in IPTG concentration, under the conditions of 37 DEG C, induces 0h, 5h, 7h, 9h respectively,
The result shows that the expressing quantity of 3h induction is seldom, the recombinant protein expression quantity of 5h-9h is higher, and induces the expressing quantity of 9h
Slightly above 5h, 7h (Fig. 7), therefore select 9h as best induction time.
The purifying of 2 riemerella anatipestifer OmpH intercept recombinant protein of embodiment
By the expression product made from embodiment 1 containing riemerella anatipestifer OmpH intercept recombinant protein by collecting bacterium
After a series of processing such as body, ultrasonic disruption, collection supernatant, nickel Ago-Gel affinitive layer purification recombinates the recombination of OmpH intercept
Albumen.The UV curve graph that protein sample is crossed after column purification shows that 3 peaks, peak 1 are to penetrate peak, and peak 2 is that 50mmol imidazoles elutes
Peak, peak 3 are 100mmol imidazoles eluting peak.It is collected simultaneously various concentration imidazoles eluting peak, carries out SDS-PAGE electrophoresis, is examined pure
Degree and concentration, as the result is shown: the OmpH intercept recombinant protein (figure only in 100mmol imidazoles eluting peak containing a large amount of high-purities
8).By the OmpH intercept recombinant protein of purifying after super filter tube is concentrated, the final concentration of albumen is measured with nucleic acid-protein instrument, after packing
It is spare.
The Western-blot (protein immunoblot) of 3 riemerella anatipestifer OmpH intercept recombinant protein of embodiment
One, experimental method
Contain riemerella anatipestifer OmpH intercept recombinant protein as Mo Shi bar in detection pest of duck for what embodiment 1 obtained
The probe of bacterium serum antibody.
1SDS-PAGE gel is prepared
The preparation of 1.1 12% separation gels
1215 μ l, 1.5M Tris-HCl (PH=8.8) of deionized water, 950 37.5 μ l, 10%AP 37.5 of μ l, 10%SDS
μ l, TEMED1.5 μ l, 30% acrylamide 1.5ml.
The preparation of 1.2 5% concentration glue
700 μ l, 1.0M Tris-HCl (pH=6.8) of deionized water 125 μ l, 10%SDS 10.0 μ l, 10%AP
10.0ul, TEMED1.0 μ l, 30% acrylamide, 165 μ l.
The processing of 2 protein samples
Suitable SDS-PAGE albumen sample-loading buffer (0.5M Tris- is added in OmpH intercept recombinant protein sample
HCl (PH=6.81.2ml), glycerol 1ml, deionized water 4.8ml, 10%SDS 2.0ml, 0.1%BPB 0.5ml).100 DEG C are boiled
10000r/min 10min after boiling 10min.
3 electrophoresis
After glue is cooled to room temperature, albumen sample is directly above arrived in the hole SDS-PAGE, 100v electrophoresis 90min.
4 transferring films
Turn the erection sequence installation of sandwich, 70v 90min according to electricity.
5 closings
After transferring film, protein film is placed into preprepared cleaning solution immediately, rinses 2min.
The incubation of 6 primary antibodies
Primary antibody is that riemerella anatipestifer serum is diluted by 1:200, then 4 DEG C of overnight incubations are washed 3 times with PBS.
The incubation of 7 secondary antibodies
1:3000 dilutes the secondary antibody of HRP label to specifications, 4 DEG C of incubations 1h, PBS washing 3 times.
The colour developing of 8 substrates
Zymolyte DAB developing solution is added, is terminated and is reacted with deionized water, film naturally dry is stored in dark place.
Two, experiment conclusion
By Western-blot protein immunoblot experiment, riemerella anatipestifer OmpH intercept recombinant protein can be with
The positive serum that RA-CH-1 plants of riemerella anatipestifer generates good reactionogenicity (such as Fig. 9), can be used as and writes from memory in detection pest of duck
The probe or capturing agent of family name's bacillus serum antibody.
4 riemerella anatipestifer OmpH intercept recombinant protein of embodiment is as between riemerella anatipestifer antibody capture agent
Connect ELISA kit
Riemerella anatipestifer OmpH intercept recombinant protein is main as the application of riemerella anatipestifer antibody capture agent
It is illustrated by indirect ELISA reagent kit.
Solid phase carrier: solid phase carrier is used as adsorbent and container in ELISA continuous mode, is not involved in chemical reaction.It can
There are many material for making solid phase carrier in ELISA, such as polystyrene, must satisfy the performance with stronger adsorbed proteins,
Still retain original immunologic competence after antibody or proteantigen absorption thereon, can be made into various forms.The shape of ELISA carrier
There are mainly three types of shapes: microtiter plate, globule and small test tube.The most commonly used with microtiter plate, the product for being exclusively used in EILSA claims
For elisa plate, 96 cellular types that the microtiter plate of standard is 8 × 12 in the world.For the detection convenient for making a small amount of sample, it is made
8 hole items or 12 hole items, after being put into mounting, size is identical as standard ELISA plate.The characteristics of elisa plate is can be simultaneously
The detection of a large amount of samples is carried out, and result can be read rapidly on special colorimeter.There are many self-reacting devices to use now
It detects in the ELISA of microtitration template, including sample-adding, washing, heat preservation, colorimetric, extremely has to the standardization of operation
Benefit.Using 96 hole elisa Plates of Corning company in the present invention.
Antibody capture agent: since the culture environment and nutritional requirement of riemerella anatipestifer are high, it is unfavorable for culture and easily quilt
Other living contaminants, so riemerella anatipestifer OmpH of the antibody capture agent used in the present invention for embodiment 2 after purification
Intercept recombinant protein intercept recombinant protein.
ELIAS secondary antibody: the ELIAS secondary antibody used in the present invention is the goat-anti duck IgG-HRP (goat-anti of horseradish peroxidase label
Duck IgG is purchased from U.S. KPL company), the anti-duck IgG-HRP of other non-duck animals can also be used, in addition to horseradish peroxidase mark
Note can also be marked, such as phosphate using other enzymes.
Substrate: since the ELIAS secondary antibody that the present invention uses is goat-anti duck IgG-HRP, therefore chromogenic substrate is tetramethyl benzidine
(TMB), it is purchased from Tiangeng Biotechnology Co., Ltd.Also other chromogenic substrates can be selected, when using other enzymes label, can be used
With its coloured conjugate as substrate.
Confining liquid: closing is after coating with the irrelevant protein solution of high concentration coated process again.Antigen is anti-
Body concentration used when being coated with is lower, the gap that surface of solid phase carriers Shang Youwei is occupied after absorption, and closing is exactly to allow largely not
Relevant protein fills these gaps, thus repel ELISA thereafter the step of in interfering substance again adsorb.Closed hand
It is continuous similar with coating.Most common sealer is bovine serum albumin(BSA), and also useful calf serum or gelatin be as sealer,
Furthermore skimmed milk power is also a kind of good sealer.
Terminate liquid: the present invention is using 2mol/L H2SO4。
One experimental method
1 indirect ELISA step
1. the riemerella anatipestifer OmpH intercept recombinant protein of purifying is diluted to final concentration of 4 μ g/mL with coating buffer,
It sequentially adds in ELISA Plate, the hole l00 μ l/, 4 DEG C of coatings are overnight；
2. washing three times, liquid is abandoned, 300 μ l confining liquids, 37 DEG C of closing 30min are added in every hole；
3. washing three times, abandon liquid, will simultaneously loading after each serum to be checked dilution, the hole l00 μ l/, 37 DEG C of incubation 30min；
4. washing three times, liquid is abandoned, is added diluted goat-anti duck IgG-HRP ELIAS secondary antibody in every hole, the hole l00 μ l/, 37 DEG C
It is incubated for 30min；
5. washing three times, liquid is abandoned, TMB100 μ l is added in every hole, colour developing 20min is protected from light, 2mol/L is then added
H2SO450 hole μ l/ of terminate liquid, pats mixing, surveys OD450nm value with microplate reader.
The determination of the best peridium concentration of 2 antigens and the most suitable extension rate of serum
By riemerella anatipestifer OmpH intercept recombinant protein coating buffer doubling dilution and coated elisa plate, l00 μ l/
Hole；RA positive serum and negative serum are used into antibody diluent volume doubling dilution respectively, are then added in ELISA Plate, l00 μ l/
Hole.It is tested with square matrix method, OmpH when selecting P/N (positive serum OD450nm value/negative serum OD450nm value) maximum
Intercept recombinant protein peridium concentration and serum diluting multiple are as best antigen coat concentration and most suitable serum diluting multiple.
The optimization of 3 antigen coat times
Use the riemerella anatipestifer OmpH intercept recombinant protein of the preparation purifying of embodiment 2 as antigen and by best coating
Concentration is coated with, and 4 DEG C of coatings put 4 DEG C of coatings overnight, after 37 DEG C of incubation 1h and put 4 DEG C of coatings overnight and after 37 DEG C of incubation 2h respectively
Overnight, each sample repeats detection 3 times, then carries out indirect ELISA test, selects the optimal antigen coat time.
The optimization of 4 different confining liquids
Use the riemerella anatipestifer OmpH intercept recombinant protein of the preparation purifying of embodiment 2 as antigen and by best coating
Condition is coated with, and is closed respectively with the skimmed milk power of 1%BSA, 5%BSA, 10%BSA and 5%, and each sample repeats
Then detection 3 times carries out indirect ELISA test, selects optimal confining liquid.
The optimization of 5 primary antibodies and secondary antibody binding time
Use the riemerella anatipestifer OmpH intercept recombinant protein of the preparation purifying of embodiment 2 as antigen and by best coating
Condition coating, is closed with best sealing condition, then carries out indirect ELISA test, primary antibody and secondary antibody react respectively
120min, 90min, 60min and 30min, each sample repeat detection 3 times, select the optimal reaction time.
6 enzyme labelled antibody best effort concentration mensurations
According to the best peridium concentration of fixed antigen and the most suitable extension rate of serum, by goat-anti duck IgG-HRP (horseradish mistake
The goat-anti duck IgG of oxidase label, be purchased from U.S. KPL company) make different volumes multiple (1:50,1:100,1:200,1:400,
1:800,1:1600,1:3200,1:6400) it dilutes and carries out indirect ELISA test, each dilution is repeated 4 times, and is found out average
P value and N value, so that it is determined that the best effort concentration of goat-anti duck IgG-HRP ELIAS secondary antibody.
The optimization of 7TMB developing time
Use the riemerella anatipestifer OmpH intercept recombinant protein of the preparation purifying of embodiment 2 as antigen and by best coating
Condition coating, is closed with best sealing condition, then carries out indirect ELISA test, and the substrate developing time of TMB is respectively
10min, 20min and 30min, each sample repeat detection 3 times, select optimal developing time.
The determination of 8 yin and yang attribute critical value criterion
It takes 20 parts of RA feminine gender duck bloods clear, is detected according to the indirect ELISA method of above-mentioned foundation.By 20 parts of negative samples
OD450nm value average value (X) and the sum of 3 times of standard variances (SD) upper limit as negative sample value, i.e. measuring samples
It is determined as the positive when critical value (X+3SD) of OD450nm value > negative sample OD450nm value, it is on the contrary then be feminine gender.
9 specific tests
Virulent duck enteritis virus (DHV) positive serum, avian flu are detected respectively with established indirect ELISA method
Malicious (AIV) positive serum, Salmonella anatis (Salmonella bacillus, SB) positive serum, E. coli isolated from ducks (E.coli)
Positive serum, duck swollen 11845 plants and RA-CH-2 plants head syndrome virus positive serum, riemerella anatipestifer RA-ATCC positive bloods
Clearly, every part of serum sample repeats detection 3 times.
10 stability tests
It is coated with constar ELISA Plate with the riemerella anatipestifer OmpH intercept recombinant protein of the preparation purifying of embodiment 2, is pressed
5 parts of serum to be checked are detected according to the indirect ELISA method of foundation, every part of sample repeats 5 holes, determines batch interior repeatability.Again with implementation
The constar ELISA Plate inspection of coated 5 different batches of riemerella anatipestifer OmpH intercept recombinant protein of the preparation purifying of example 2
5 parts of serum to be checked are surveyed, determine to repeat between criticizing.
11 sensitivity tests
RA positive serum is subjected to volume doubling dilution, is then detected with the indirect ELISA method having built up, together
When with same serum sample carry out agar gel diffusion test, slide agglutination test, microagglutination test and broken RA-CH-1
Indirect cell ELISA, all detections test repetitive operation 3 times, are averaged, compare its sensibility.
Two experiment conclusions
The determination of 1 antigen (antibody capture agent) peridium concentration and serum dilution
By square matrix burette test, the testing result point of riemerella anatipestifer RA-CH-1 positive serum and negative serum
Not as shown in table 7 and table 8, when the peridium concentration of antibody capture agent is 4ug/mL, and serum dilution volume multiple is 1:200, sun
Property serum OD450nm value be 0.384, corresponding negative serum OD450nm value is 0.017, P/N value maximum (22.588) such as table 9
It is shown.Therefore, the best peridium concentration of antibody capture agent is 4ug/mL, and antibody most aptamer product extension rate is 1:200.
The positive serum testing result of table 7 different antigen coat concentration and serum dilution
The negative serum testing result of table 8 different antigen coat concentration and serum dilution
The P/N value of table 9 different antigen coat concentration and serum dilution
The optimization of 2 antigen coat conditions
Use the riemerella anatipestifer OmpH intercept recombinant protein of the preparation purifying of embodiment 2 as antigen and by best coating
Concentration is coated with, and is put 4 DEG C of coatings overnight, after 37 DEG C of incubation 1h respectively at 4 DEG C of coatings and is put 4 DEG C of packets overnight and after 37 DEG C of incubation 2h
It is stayed overnight, then carries out indirect ELISA test, each pattern detection 3 times, the results are shown in Table 10.4 DEG C of coatings detect after overnight
Positive value highest out, and the numerical value measured is more stable, therefore select 4 DEG C of coatings overnight as the best antigen coat of this method
Condition.
The optimization of 10 antigen coat condition of table
The optimization of 3 different confining liquids
Use the riemerella anatipestifer OmpH intercept recombinant protein of the preparation purifying of embodiment 2 as antigen and by best coating
Condition is coated with, and is closed respectively with the skimmed milk power of 1%BSA, 5%BSA, 10%BSA and 5%, is then carried out indirect
ELISA test, each sample repeat detection 3 times, select optimal confining liquid, as a result as shown in table 11.4 kinds of different types and dense
The result that the confining liquid of degree is obtained is not much different, and in line with the principle of saving, therefore selects the confining liquid of the 1%BSA as we
The best confining liquid of method.
The optimization of 11 confining liquid of table
The optimization of 4 primary antibodies and secondary antibody binding time
Use the riemerella anatipestifer OmpH intercept recombinant protein of the preparation purifying of embodiment 2 as antigen and by best coating
Condition is coated with, and is closed with best sealing condition, then carries out indirect ELISA test, primary antibody and secondary antibody react respectively
120min, 90min, 60min and 30min, each sample repeat detection 3 times, select the optimal reaction time, as a result such as 12 He of table
Shown in table 13.OD450nm value increasing and increase with primary antibody and secondary antibody reaction time, therefore the action time of primary antibody and secondary antibody
Select 120min.
The optimization of 12 primary antibody of table coating time
The optimization of 13 secondary antibody of table coating time
The determination of 5 enzyme labelled antibody optimum concentrations
It the results are shown in Table 14, when enzyme mark goat-anti duck IgG antibody makees 1:400 volume dilution, P/N value is up to 24.114, because
This determines that best enzyme labelled antibody volume dilution multiple is 1:400.
The determination of 14 enzyme labelled antibody best effort concentration of table.
The optimization of 6TMB developing time
Use the riemerella anatipestifer OmpH intercept recombinant protein of the preparation purifying of embodiment 2 as antigen and by best coating
Condition coating, is closed with best sealing condition, then carries out indirect ELISA test, and the substrate developing time of TMB is respectively
10min, 20min and 30min, each sample repeat detection 3 times, select optimal developing time.As a result as shown in Table 15, TMB
It is optimal that 10min effect is reacted at 37 DEG C, therefore the optimal substrate developing time of this method is 10min.
The optimization of 15 tmb substrate developing time of table
The determination of 7 yin and yang attribute critical value criterion
20 parts of RA negative serum samples are detected with each ELISA condition of above-mentioned optimization, each sample repeats detection 3
It is secondary, it is as shown in table 16 to be averaged acquired results.X value is that 0.21965, SD value is 0.043646, determines that yin and yang attribute critical point is X
Therefore+3SD=0.21965+3 × 0.043646=0.35 is determined as sun when the OD450nm value of sample to be tested is greater than 0.35
Property, less than or equal to when be then determined as feminine gender.
16 RA negative serum testing result of table
8 specific tests
7 kinds of cause of disease positive serums including riemerella anatipestifer are distinguished by established indirect ELISA method
Be measured, as a result as shown in table 17, except riemerella anatipestifer RA-ATCC11845 plant with RA-CH-2 plants of positive serums with
Outside, the OD450nm value of remaining 5 kinds of positive serum is respectively less than 0.35, shows in addition to both positive serums, the antigen not with it is upper
State other 5 kinds of positive serums and cross reactions occur, thus illustrate to establish with riemerella anatipestifer OmpH intercept recombinant protein
ELISA method as antigen detection RA antibody has preferable specificity, and the recombinant protein can be used as RA-CH-1, RA-
The general antigen of ATCC 11845 and RA-CH-2 different serotypes RA antibody test.
17 specificity experiments result of table
9 stability tests
It is coated with constar ELISA Plate with the riemerella anatipestifer OmpH intercept recombinant protein of the preparation purifying of embodiment 2, is pressed
5 parts of serum to be checked are detected according to the indirect ELISA method of foundation, every part of blood serum sample repeats 5 holes；Again with the preparation purifying of embodiment 2
Coated 5 different batches of riemerella anatipestifer OmpH intercept recombinant protein constar ELISA Plate detect 5 parts of blood to be checked
Clearly, pass through the OD of every part of serum of gained450nmValue calculates average OD450nmValue and standard variance (SD), then calculate every part of serum
Batch in and interassay coefficient of variation (CV), as a result as shown in table 18 and table 19.It is average that 5 groups of serum coefficient of variation of test are repeated in batch
Value is 3.37%, and is below 10%, and the coefficient of variation average value for repeating to test 5 groups of serum between batch is 3.34%, and is not surpassed
10% is crossed, thus illustrates the indirect ELISA method based on riemerella anatipestifer OmpH intercept recombinant protein that this research is established
With preferable stability.
18 batches, table interior repetitions are tested
It repeats to test between 19 batches, table
10 sensitivity tests
Agar gel diffusion test, slide agglutination test, microagglutination test are carried out respectively with same a serum sample and are crushed
The ELISA of the full bacterium of RA-CH-1 is detected and is compared with this method.There can be no precipitation lines for agar diffusion test, therefore can not determine to resist
Body potency.The potency of class agglutination test is 1:8 (such as Figure 10).The potency of micro-agglutination experiment is 1:32 (such as Figure 11).It is broken
Indirect cell ELISA potency afterwards is 1:6400.Using this experimental method, as a result as shown in table 20, as RA-CH-1 positive blood is thin
The increase OD450nm value for degree of releasing also significantly depending on decline, when serum diluting multiple be 1:10400 when, OD450nm value is still greater than
0.35, when extension rate is 1:20800, serum testing result is lower than negative value, therefore, with the duck of purifying prepared by this research
Mo Shi bacillus OmpH intercept recombinant protein is 1:10400 as the minimum detectable positive serum extension rate of antigen in epidemic disease.
20 sensitivity experiments result of table
The preparation of 5 riemerella anatipestifer OmpH intercept gene subunit vaccine of embodiment
1 materials and methods
The grouping of 1.1 experimental animals
24 1 age in days Cherry Village Duckss (its RA-CH-1 antibody agglutination is feminine gender) are grouped at random, experimental group 8,
Negative control group 8, commercialized vaccine positive controls 8.
1.2 are immunized and attack poison
Riemerella anatipestifer OmpH intercept recombinant protein purification product and Freund's adjuvant made from embodiment 2 are isometric
Mixing obtains riemerella anatipestifer OmpH intercept recombination subunit vaccine after emulsifying, and the immune programme of experimental group is such as
Shown in table 21, commercialized vaccine positive controls are immune by vaccine specification, negative control group injecting normal saline.
In all 17 age in days of animal (10 days after OmpH intercept recombination subunit vaccine two is exempted from) intramuscular injection pest of ducks
The full bacterium of Mo Shi bacillus, injection volume are 2.5 × 109CFU/ml, every injection 1ml, observes the lesion tissue and death condition of animal.
21 experimental group immune programme of table
The separation identification of 1.3 bacteriums
The liver and brain tissue scribing line TSA for taking dead animal are in 37 DEG C of CO2Under the conditions of overnight incubation, to the bacterium colony grown
Carry out PCR identification.PCR identifies that primer is as follows:
Upstream primer: 5'-CGAAAGTGATAAGTTAGCCACCT-3'(SEQ ID NO:5)；
Downstream primer: 5'-GCAGCACCTTGAAAATTGTCC-3'(SEQ ID NO:6)；
2 results
Feminine gender group duck is all dead, and the death rate 100%, experimental group is survived 4, protective rate 50%, and epidemic disease is commercialized
Seedling positive controls protective rate is 50%.Then all not dead animals are analysed, positive commercialization control group and reality
The animal for testing group does not find lesion, shows normal (such as Figure 13).The duck of death group analyse it can be seen that different degrees of
Lesion (such as Figure 12).All dead group ducks are carried out with the bacterium separation and PCR identification of brain, as a result (such as Figure 14) shows
Dead duck dies of the infection of riemerella anatipestifer.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention,
It should be equivalent substitute mode, be included within the scope of the present invention.
Claims (6)
1. riemerella anatipestifer OmpH intercept recombinant protein is applied to as the indirect of detection riemerella anatipestifer antibody
The detection antigen of ELISA method, it is characterised in that: the amino acid sequence of the riemerella anatipestifer OmpH intercept recombinant protein
As shown in SEQ ID NO:1.
2. application described in claim 1, it is characterised in that: the DNA sequence dna of riemerella anatipestifer OmpH intercept recombinant protein
As shown in SEQ ID NO:2.
3. application described in claim 1, it is characterised in that: the preparation of the riemerella anatipestifer OmpH intercept recombinant protein
Method specifically includes the following steps:
A, with riemerella anatipestifer (Riemerella anatipestifer) genomic DNA be template, using nucleotides sequence
The upstream and downstream primer PCR as shown in SEQ ID NO.3 and SEQ ID NO.4 expands to obtain riemerella anatipestifer column respectively
OmpH intercept genetic fragment connect with T-Vector pMD19 (simple), obtains pMD19-OmpHM；
B, double digestion pMD19-OmpHM plasmid and prokaryotic expression carrier are distinguished with restriction enzyme BamH I and Xho I
PET32a (+), is attached, and obtains recombined pronucleus expression plasmid pET32a-OmpHM；
C, the recombinant plasmid pET32a-OmpHM that step B is extracted is transformed into expressive host bacterium inducing expression, obtains Mo Shi in pest of duck
Bacillus intercept recombinant protein crude product.
4. application according to claim 3, it is characterised in that: by riemerella anatipestifer OmpH intercept weight obtained by step C
Histone crude product is eluted after nickel Ago-Gel affinitive layer purification with 100mM imidazoles, is obtained riemerella anatipestifer OmpH and is cut
Section recombinant protein.
5. application according to claim 3, it is characterised in that: the host strain in step C is bacterial strain BL21 (DE3),
IPTG concentration=1.2mmol/L, inducing temperature carry out inducing expression 9 hours under the conditions of being 25 DEG C, obtain riemerella anatipestifer OmpH
Intercept recombinant protein.
6. riemerella anatipestifer OmpH intercept recombinant protein is applied to sub- as the genetic engineering of prevention riemerella anatipestifer
Subunit vaccine, it is characterised in that: the amino acid sequence such as SEQ ID of the riemerella anatipestifer OmpH intercept recombinant protein
Shown in NO:1.
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| CN106434701B (en) * | 2016-10-17 | 2019-08-06 | 四川农业大学 | The rpsL mutated gene of riemerella anatipestifer and its application |
| CN112402598A (en) * | 2019-08-20 | 2021-02-26 | 管庆丰 | General subunit vaccine for riemerella anatipestifer infection |
| CN112062819A (en) * | 2020-09-10 | 2020-12-11 | 海南大学 | Riemerella anatipestifer BamHI D recombinant protein and preparation method and application thereof |
| CN112180087B (en) * | 2020-10-23 | 2022-06-14 | 温氏食品集团股份有限公司 | ELISA method for detecting riemerella anatipestifer antibody, kit and application thereof |
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