CN105541977A - Riemerella anatipestifer OmpH intercepted recombinant protein, and preparation method and application thereof - Google Patents
Riemerella anatipestifer OmpH intercepted recombinant protein, and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a riemerella anatipestifer OmpH intercepted recombinant protein, and a preparation method and application thereof, and belongs to the field of biological engineering. The amino acid sequence of the riemerella anatipestifer OmpH intercepted recombinant protein is shown as SEQ ID NO:1; then, a gene engineering technology is used; on the clone basis, the connection with a prokaryotic expression vector pET-32a (+) is performed; a gene engineering product induced and expressed by a colon bacillus BL21(DE3) expression system is successfully converted; the expression form of the protein is OmpHM/His fusion protein; the molecular weight of the expressed fusion protein is 28kDa; the product is designed into fusion expression, and the effect of being convenient to purify is mainly considered; meanwhile, the expression protein maintains the natural activity of the protein. The product can be applied to the detection of detection antigen of a riemerella anatipestifer antibody by an indirect ELISA (enzyme-linked immuno sorbent assay) method, and can also be used as a gene engineering sigmasubunit vaccine for preventing the riemerella anatipestifer.
Description
Technical field
The invention belongs to bioengineering field, particularly a kind of riemerella anatipestifer OmpH intercept recombinant protein and preparation method and application.
Background technology
Riemerella anatipestifer (Riemerellaanatipestifer, RA) be a kind of Gram-negative bacteria, be attributed to Flavobacterium section (Flavobacteriaceae), it is the pathogenic agent of Riemerella anatipestifer disease, the contagious infection disease of a kind of chronic or acute sepsis shape of duck, goose, turkey and multiple bird can be caused, in worldwide distribution, M & M is high, has become current serious harm and has supported one of Infectious Diseases of duck industry.The main clinical symptom main manifestations of this disease is that spirit is depressed, paralysis ground, necking down, yellow-green colour of the having loose bowels symptom such as just.Characteristics of lesion causes the cellulosic airsacculitis of host, meningitis, pericarditis, serohepatitis and caseous salpingitis, and with septicemia in various degree, there is huge threat to the development of aquaculture.
That has set up at present has agar diffusion experiment, agglutination test, ELISA etc. for the method detecting riemerella anatipestifer serum antibody, and these detection methods or sensitivity, specificity are not high, or come with some shortcomings due to the difference of use antigenic component; And the control of riemerella anatipestifer mainly microbiotic carry out treating or with Bacteria vaccine corresponding serotype infected and prevent.OmpH belongs to porin, has no the correlative study report of riemerella anatipestifer OmpH investigation and application at present.
Summary of the invention
For overcoming the shortcoming of prior art existence with not enough, an object of the present invention is to provide a kind of riemerella anatipestifer OmpH intercept recombinant protein, and this albumen can as the trapping agent etc. of riemerella anatipestifer antibody.
Two of object of the present invention is the preparation method providing above-mentioned riemerella anatipestifer OmpH intercept recombinant protein, and the method can be used for the scale operation of OmpH intercept recombinant protein.
Three of object of the present invention is the application providing the above-mentioned riemerella anatipestifer OmpH intercept recombinant protein prepared.
Object of the present invention is achieved through the following technical solutions: a kind of riemerella anatipestifer OmpH intercept recombinant protein, its aminoacid sequence is as shown in SEQIDNO:1.
The DNA sequence dna of above-mentioned riemerella anatipestifer OmpH intercept recombinant protein is as shown in SEQIDNO:2.
The preparation method of above-mentioned riemerella anatipestifer OmpH intercept recombinant protein, specifically comprises the following steps:
A, with RA-CH-1 genomic dna for template, the upstream and downstream primer PCR amplification of nucleotide sequence respectively as shown in SEQIDNO.3 and SEQIDNO.4 is adopted to obtain riemerella anatipestifer OmpH intercept gene fragment (OmpHM), connection with pMD19-T (simple), obtains pMD19-OmpHM;
B, use restriction enzyme BamHI and XhoI double digestion pMD19-OmpHM plasmid and prokaryotic expression carrier pET32a (+) respectively, connect, obtain recombined pronucleus expression plasmid pET32a-OmpHM;
C, by step B extract recombinant plasmid pET32a-OmpHM be transformed into expressive host bacterium abduction delivering, obtain riemerella anatipestifer intercept recombinant protein crude product.
Further, by step C gained riemerella anatipestifer OmpH intercept recombinant protein crude product after nickel sepharose affinitive layer purification, riemerella anatipestifer OmpH intercept recombinant protein is obtained;
Further, the Host Strains in step C is bacterial strain BL21 (DE3), and at IPTG concentration=1.2mmol/L, inducing temperature is carry out abduction delivering 9 hours under 25 DEG C of conditions, obtains riemerella anatipestifer OmpH intercept recombinant protein.
Beneficial effect of the present invention is: riemerella anatipestifer OmpH intercept recombinant protein is the Main Antigenic Region (81aa-151aa) choosing riemerella anatipestifer OmpH gene coded protein, and called after OmpHM; Then utilize genetic engineering technique, carry out on the basis of cloning at it, it be connected with prokaryotic expression carrier pET-32a (+), successful conversion e. coli bl21 (DE3) expression system carries out the gene engineering product of inducing, expressing.The expression-form of this albumen is OmpHM/His soluble fusion protein, and the fusion protein molecule amount of expression is 28kDa, is that amalgamation and expression is mainly considered to be convenient to purifying by product design; Expressing protein is solubility expression form simultaneously, avoids the process such as sex change, renaturation of albumen in the preparation, purge process of albumen, is beneficial to the natural radioactivity keeping albumen.Product after prokaryotic expression shows to have the immune response activity similar to native protein after Westernblot identifies.
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 detectable antigens of the indirect ELISA method detecting riemerella anatipestifer antibody.
2. can be used as the genetic engineering subunit vaccine of Mo Shi bacillus in prevention pest of duck.
Product advantage applies of the present invention exists:
1. because this detectable antigens is riemerella anatipestifer OmpH intercept recombinant protein, and non-fully bacterium, apply when this detectable antigens detects dangerous without loose bacterium with this.
2. solve riemerella anatipestifer difficulty to cultivate using this albumen as antigen, the problem easily polluted.
3. as ELISA Detection of antigen riemerella anatipestifer antibody, high specificity, no cross reaction.
4. stable performance, production cost are low, and be applicable to factorial praluction, market application foreground is wide.
5. as subunit vaccine, there is good protectiveness.
More beneficial effects, will embody in an embodiment.
Accompanying drawing explanation
In order to make object of the present invention, technical scheme and beneficial effect clearly, the invention provides following accompanying drawing:
Fig. 1 is the agarose gel electrophoresis figure of the pcr amplification of OmpH intercept gene.The wherein 1 specific DNA band obtained through agarose gel electrophoresis for OmpH intercept gene PCR amplified production; M is DNAMarker.
Fig. 2 is the agarose gel electrophoresis figure of recombinant plasmid pMD19-OmpHM.Wherein, M is DNAMarker; 1 is recombinant plasmid pMD19-OmpHM BamH I and Xho I double digestion, two specific bands that digestion products obtains through agarose gel electrophoresis.
Fig. 3 is the agarose gel electrophoresis figure that the enzyme of pET32a-OmpHM cuts qualification.Wherein, M is DNAMarker; 1 is recombinant expression plasmid pET32a-OmpHM Xho I single endonuclease digestion, the specific band that digestion products obtains through agarose gel electrophoresis; 2 is recombinant expression plasmid pET32a-OmpHM BamH I and Xho I double digestion, two specific bands that digestion products obtains through agarose gel electrophoresis; 3 is recombinant expression plasmid pET32a-OmpHM BamH I single endonuclease digestion.
Fig. 4 is at 37 DEG C, the SDS-PAGE electrophorogram of the OmpH intercept recombinant protein that abduction delivering spends the night during IPTG=0.4mmol/L.Wherein M is protein Marker; 1 is through SDS-PAGE electrophoresis acquired results after empty carrier pET-32a (+) expression bacterium IPTG induction; 2 induce for recombinant expression plasmid pET32a-OmpHM expresses bacterium IPTG, the bacterium liquid obtained carries out ultrasonic disruption, centrifugal after, gained supernatant carries out the result of SDS-PAGE electrophoresis; 3 induce for recombinant expression plasmid pET32a-OmpHM expresses bacterium IPTG, the bacterium liquid obtained carries out ultrasonic disruption, centrifugal after, gained precipitation carries out the result of SDS-PAGE electrophoresis.
Host Strains E.coliBL21 (DE3) abduction delivering of recombinant plasmid pET32a-OmpHM is contained under different IP TG concentration after 4 hours, the SDS-PAGE electrophorogram of riemerella anatipestifer OmpHM intercept recombinant protein when Fig. 5 is 37 DEG C.Wherein, M is protein Marker; 1 for the final concentration of IPTG be 0mmol/L; 2 for the final concentration of IPTG be 0.4mmol/L; 3 for the final concentration of IPTG be 0.8mmol/L; 4 for the final concentration of IPTG be 1.2mmol/L.
Fig. 6 contains recombinant plasmid pET32a-OmpHM Host Strains E.coliBL21 (DE3) when being IPTG=0.4mmol/L under differing temps expresses the riemerella anatipestifer OmpHM intercept recombinant protein abduction delivering SDS-PAGE electrophorogram of 4 hours.Wherein, M is protein Marker; 1 for inducing temperature be 25 DEG C; 2 for inducing temperature be 30 DEG C; 3 for inducing temperature be 37 DEG C.
The Host Strains E.coliBL21 (DE3) that Fig. 7 is 37 DEG C containing recombinant plasmid pET32a-OmpHM under different induction time when being 0.4mmol/L with IPTG concentration expresses the SDS-PAGE electrophorogram of riemerella anatipestifer OmpHM intercept recombinant protein.Wherein M is protein Marker; 1 is induction 0h; 2 is induction 5h; 3 is induction 7h; 4 is induction 9h.
Fig. 8 is nickel sepharose (Ni
2+-NAT) riemerella anatipestifer OmpH intercept recombinant protein SDS-PAGE electrophorogram after affinitive layer purification.Wherein M is protein Marker; 1 is the OmpH intercept recombinant protein after nickel sepharose affinitive layer purification.
Fig. 9 is that riemerella anatipestifer OmpH intercept recombinant protein Western-blot schemes.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.In 1:2,1:4,1:8 and Positive control wells, have fine sand shape agglutinating particle respectively, 1:16 does not have agglutinating particle in hole.
Figure 11 is riemerella anatipestifer micro-agglutination experiment picture.Equal visible agglutination particle in 1:8,1:16,1:32 and Positive control wells; Without agglutinating particle in 1:64 hole, only there is part bacterial sediment in bottom, identical with negative control hole.
Figure 12 is the pathological tissues organ after the death of negative control group duckling artificial challenge riemerella anatipestifer.All can see one deck fibrinous exudate on heart, liver, surface, abdominal cavity, form one deck pod membrane.
Figure 13 be survive after the duckling artificial challenge riemerella anatipestifer of Mo Shi bacillus OmpH intercept recombinant protein in immune pest of duck analyse situation.Visible abdominal cavity, liver and heart surface are all normal, and have no cellulosic and ooze out and necrosis region, each organ is normal.
Figure 14 is the PCR qualification that RA attacks that malicious duck is separated the 16sRNA of RA again.M is DNAMarker; 1,2,3, the 4 bacterium colony PCR being respectively isolated strains; 5 is negative control.
Embodiment
Hereinafter with reference to accompanying drawing, the preferred embodiments of the present invention are described in detail.The experimental technique of unreceipted actual conditions in preferred embodiment, usual conveniently condition, the such as Molecular Cloning: A Laboratory guide (third edition, J. the work such as Pehanorm Brooker, Huang Peitang etc. translate, Science Press, 2002) described in condition, or to carry out according to the condition that manufacturer advises.
Partial amino-acid is abridged as follows accordingly:
Glutamine glnQ; Glycine glyG; Serine serS; L-Ala alaA; Threonine thrT; α-amino-isovaleric acid valV; Isoleucine ileI; Leucine leuL; Tyrosine tyrY; Phenylalanine pheF; Histidine hisH; Proline(Pro) proP; L-asparagine asnN; Methionine(Met) metM; L-glutamic acid gluE; Tryptophane trpW; Methionin lysK; Halfcystine cysC; Arginine argR; Aspartic acid AspD.
The preparation of embodiment 1 riemerella anatipestifer OmpH intercept recombinant protein
1 major experimental material
Plasmid T-VectorpMD19 (simple),
maxDNAPolymerase, all purchased from the precious biotechnology company limited in Dalian; Prokaryotic expression plasmid pET32a (+), Novagen Products; Cloning host bacterium E.coliDH5 α, expressive host bacterium E.coliBL21 (DE3) and RA-CH-1 bacterial strain, provided by Sichuan Agricultural University's poultry diease research centre.
2 experimental techniques
The clone of 2.1RA – CH-1OmpH intercept gene
2.1.1 design of primers
Choose Main Antigenic Region wherein (81aa-151aa called after OmpHM) according to the existing RA-CH-1OmpH gene order of GenBank, utilize PrimerPremier5.0 software design pair of primers.Upstream primer as shown in SEQIDNO:3: 5 '-CGC
gGATCCgAGGCTCAGAGAACTGCT-3 ' (dashed part is BamH I site); Downstream primer as shown in SEQIDNO:4: 5 '-CCG
cTCGAGtCCTTTATAGATTAACCCC-3 ' (dashed part is Xho I site).After primer synthesis, with appropriate sterilizing deionized water dissolving, make its final concentration be 20mmol/L ,-20 DEG C save backup.
2.1.2RA the extraction of genomic dna
The cultivation of a, riemerella anatipestifer RA-CH-1 strain: get the bacterial classification that freeze-drying is preserved, with dilution in 2mL pancreas peptone soybean broth substratum (TSB), is then inoculated in blood agar, CO
2the lower 37 DEG C of constant temperature of condition leave standstill 18 ~ 24h, and (bacterium colony is rounded, microprotrusion, neat in edge, butteriness, colony diameter 2mm; Basis of microscopic observation thalline is short-thick type).Observe and microscopy through bacterium colony, the single bacterium colony of picking is cultivated in pancreas peptone soybean broth substratum TSB (containing 1% calf serum).
The enlarged culturing of b, riemerella anatipestifer RA-CH-1 strain: be inoculated in pancreas peptone soybean broth substratum (TSB) by the Riemerellosis Anatipestifer bacterium liquid of overnight incubation by 1:100 (containing 1% calf serum), 37 DEG C of water-bath shaking culture cause bacterial growth to logarithmic phase.
The extraction of c, riemerella anatipestifer RA-CH-1 strain DNA, concrete steps are as follows: (1) gets the bacterium liquid 1ml growing into logarithmic phase, and the centrifugal 2min of 12000rpm, abandons supernatant.(2) 567 μ lTEBuffer, 30 μ l10%SDS and 3 μ l Proteinase Ks are added, 37 DEG C of water-bath 1h, dissolution precipitation.(3) add the phenol/centrifugal 5min of chloroform DNA, 12000rpm of equivalent, get supernatant.Repeat this step once.(4) add equal amounts of chloroform/centrifugal 5min of isoamyl alcohol extracting DNA, 12000rpm, get supernatant.(5) dehydrated alcohol adding two volumes is washed, the sodium-acetate of the 3mol/L of 10% volume, and the centrifugal 15min of ice bath 30min, 12000rpm, abandons supernatant.(6) precipitate twice by the washing with alcohol of 70%, abandon supernatant.(7) seasoning precipitation DNA under room temperature, add appropriate TE (pH8.0) dissolution precipitation DNA ,-20 DEG C save backup.
2.1.3PCR increase riemerella anatipestifer OmpH intercept gene
The reaction system of PCR reaction amplification riemerella anatipestifer OmpH intercept gene is as shown in table 1:
Table 1OmpH intercept gene PCR reaction system
Mix gently, the laggard performing PCR of 4000r/min brief centrifugation.
Reaction parameter: 95 DEG C of 5min, then enters 94 DEG C of 40s, 58 DEG C of 40s, 72 DEG C of 60s, totally 35 circulations, and last 72 DEG C extend 10min, save backup in 4 DEG C.Get 5 μ lPCR products electrophoresis on 1% sepharose, observe the length of amplified fragments.
2.1.4 the recovery of riemerella anatipestifer OmpH intercept gene PCR product
Reclaim test kit specification sheets by OMEGA company DNA to carry out, the DNA after recovery is stored in-20 DEG C and saves backup.
2.1.5T clone
The riemerella anatipestifer OmpH intercept gene of purifying and the connection of pMD19-T (simple)
Ligation system is as shown in table 2:
The reaction system of the connection of table 2OmpH intercept gene and pMD19-T (simple)
Mentioned reagent is added in PCR reaction tubes, carefully mix, after brief centrifugation, spend the night in 16 DEG C of connections.
2.1.6DH5 the preparation of α competent cell
Adopt Calcium Chloride Method to prepare fresh DH5 α strain competent escherichia coli cell, be summarized as follows: on (1) aseptic picking flat board, the DH5 alpha monoclonal colony inoculation of fresh culture is in 5mLLB nutrient solution, spends the night in 37 DEG C of shaking culture; (2) getting the above-mentioned nutrient solution of 1mL is inoculated in 100mLLB nutrient solution, and 37 DEG C of 200r/min shaking culture 2.5 ~ 3h, about making OD600=0.4 ~ 0.5; (3) bacterial cultures is poured into after sterilizing in ice-cold centrifuge tube, ice bath 10min; (4) in 4 DEG C of centrifugal 10min of 4000r/min, supernatant is abandoned; (5) CaCl of the 0.1mol/L of 10mL ice precooling is added
2, gentleness has hanged bacterial precipitation, ice bath 30min; (6) in 4 DEG C of centrifugal 10min of 4000r/min, abandon supernatant, add the CaCl of the 0.1mol/L of 4mL ice precooling
2resuspended precipitation again, adds the sterile glycerol CaCl that final concentration is 15%
2, be packed as 100 μ l/ after mixing and manage, preserve in-4 DEG C of refrigerators and spend the night to improve transformation efficiency.
2.1.7 transformed competence colibacillus cell
10 μ l linked systems are all taken out and is added in 100 μ lDH5 α competent cells, ice bath 30min; Be placed in 42 DEG C of temperature bath 90s again, ice bath 2min more afterwards; Then add 890 μ lLB substratum immediately, 37 DEG C of water-bath shaking culture 45min, get 200 μ l and be applied on the substratum containing Amp, put overnight incubation in 37 DEG C of incubators.The single white colony of picking next day is inoculated in the LB liquid nutrient medium containing Amp, carries out plasmid extraction after 37 DEG C of water-bath shaking culture 18h.
2.1.8T the extracting of cloned plasmids
Operate by OMEGA company plasmid extraction test kit specification sheets, reclaim product storage and save backup in-20 DEG C.
2.1.9T the PCR of cloned plasmids and enzyme cut qualification
By the recombinant plasmid called after pMD19-OmpHM of previous step extracting, double digestedly to digest with Xho I single endonuclease digestion with BamH I/Xho I respectively, BamH I single endonuclease digestion digests, 1.0% gel electrophoresis observations.Do pcr amplification goal gene, it is as shown in table 3 that the enzyme of T cloned plasmids cuts system simultaneously:
The enzyme of table 3T cloned plasmids cuts system
2.1.10 riemerella anatipestifer OmpH intercept gene sequencing
Enzyme is cut and identifies that correct plasmid is sent the biological company limited of Shanghai English fine horse and checked order.
The optimization of the structure of 2.2 prokaryotic expression plasmid pET32a-OmpHM, abduction delivering and expression condition
2.2.1 the Construction and identification of prokaryotic expression plasmid pET32a-OmpHM
The enzyme of a, object fragment is cut and is connected: restriction enzyme BamH I and Xho I is double digestion pMD19-OmpHM plasmid and prokaryotic expression carrier pET32a (+) respectively, and it is as shown in table 4 that enzyme cuts system:
Table 4 object fragment and carrier enzyme cut system
37 DEG C of water-bath 4h, after reclaiming object fragment respectively, spend the night according to the 16 DEG C of connections of the linked system shown in table 5 by the DNA glue recovery test kit operation instruction of OMEGA.
The linked system of table 5 object fragment and prokaryotic expression carrier
The conversion of b recombinant plasmid: adopt Calcium Chloride Method to prepare DH5 α competent cell.Afterwards, get in the centrifuge tube that connecting fluid 10 μ l is added to containing 100 μ l competence DH5 α, ice bath 30min after mixing; Be placed in 42 DEG C of water-bath 90s, then rapid ice bath 2min; Add not containing the LB liquid nutrient medium 890 μ l of Amp, 37 DEG C of joltings (150r/min) cultivate 1 ~ 1.5h; Get the LB that 200 μ l cultures coat containing Amp dull and stereotyped, 37 DEG C of overnight incubation, the single colony inoculation of picking next day contains in the LB liquid nutrient medium of Amp in 5mL, cultivate 12 ~ 16h, set up empty carrier conversion group (empty carrier 10 μ l+ competence DH5 α 100 μ l), carrier free control group (sterilizing ultrapure water 10 μ l+ competence DH5 α 100 μ l) simultaneously for 37 DEG C.
The PCR qualification of c bacterium liquid:
With recombinant plasmid bacterium liquid for template, the upstream primer shown in SEQIDNO:3: 5 '-CGC
gGATCCgAGGCTCAGAGAACTGCT-3 ' (dashed part is BamH I site); Downstream primer as shown in SEQIDNO:4: 5 '-CCG
cTCGAGtCCTTTATAGATTAACCCC-3 ' (dashed part is Xho I site).Carry out PCR reaction, its method and amplification condition the same, get PCR primer electrophoresis detection on 1% sepharose.
The enzyme of d recombinant plasmid cuts qualification: clone's strain inoculation of above-mentioned preservation contained in 5mL in the LB liquid nutrient medium of Amp, 37 DEG C of water-bath jolting overnight incubation, next day extracts recombinant plasmid according to a conventional method, then identify this recombinant plasmid with Xho I single endonuclease digestion, BamH I and Xho I double digestion, it is as shown in table 6 that its enzyme cuts system:
Table 6 subclone enzyme cuts each component concentration in system
Cut and PCR qualification through enzyme, obtain recombined pronucleus expression plasmid pET32a-OmpHM.
2.2.2 the abduction delivering of recombinant expression plasmid pET32a-OmpHM
The extraction of a recombinant plasmid pET32a-OmpHM: picking identified containing positive recombinant plasmid pET32a-OmpHM the streak inoculation of DH5 α bacterial classification in containing Amp LB agar plate on, 37 DEG C of overnight incubation, get single colony inoculation next day in the LB liquid nutrient medium containing Amp, thermal agitation is cultivated 10 ~ 16 hours, collected by centrifugation bacterium liquid, illustrates the extraction and purification carrying out recombinant plasmid by OMEGA plasmid DNA Mini Kit.
B recombinant plasmid pET32a-OmpHM transforms and expresses bacterium: adopt Calcium Chloride Method to prepare E.coliBL21 (DE3) competent cell, and be transformed in expressive host bacterium E.coliBL21 (DE3) by the recombinant plasmid pET32a-OmpHM of said extracted.
The abduction delivering of c recombinant plasmid pET32a-OmpHM: from above-mentioned LB solid medium, picking positive colony bacterium, inoculation LB liquid nutrient medium, 37 DEG C of overnight incubation, get the ratio access of bacterium liquid in 1:50 next day containing in the LB liquid nutrient medium of Amp, when thermal agitation is cultured to OD600=0.4, add IPTG inducing culture respectively, collect 1mL and cultivate bacterium liquid, 4 DEG C of centrifugal 2min of 13000r/min, abandon supernatant, 40 μ l ultrapure waters and 10 μ l10 × SDS sample-loading buffers are added in precipitation, 100 DEG C of heating in water bath sex change 10min, carry out 12%SDS-PAGE gel electrophoresis, observe expression of results.
The soluble analysis of d recombinant plasmid pET32a-OmpHM expression product: each 200ml of bacterium liquid of the bacterium liquid that when IPTG concentration is 0.4mmol/L at 37 DEG C, abduction delivering spends the night and empty carrier, press step process respectively: 4 DEG C, the centrifugal 5min of 10000r/min, bacterial sediment 20mL20mmolTris-HCl (pH8.0) suspends; Put-20 DEG C spend the night after, ultrasonic wave (ice bath) interval broken thalline (600w, 30sec/ time, 10 times), 4 DEG C, 1. the centrifugal 10min of 10000r/min, get supernatant for subsequent use; Precipitation 10mL urea solution (10mmol/LPBS+2mol/L urea) suspends, 4 DEG C, after the centrifugal 10min of 10000r/min, precipitation suspends by 10mL washing lotion again, after repeated washing three times, with appropriate urea soln (10mmol/LPBS+8mol/L urea) dissolution precipitation 2., cryopreservation is for subsequent use.Get respectively appropriate supernatant 1. with urea soln dissolve precipitation 2., add 40 μ l ultrapure waters and 10 μ l10 × SDS sample-loading buffers wherein, 100 DEG C of heating in water bath sex change 10min, carry out 12%SDS-PAGE gel electrophoresis, after gel coomassie brilliant blue staining, observations.
2.2.3 the optimization of recombinant plasmid pET32a-OmpHM expression condition
The concentration optimization of a inductor IPTG: get the expressive host bacterium BL21 (DE3) containing recombinant plasmid pET32a-OmpHM, is inoculated in the LB liquid nutrient medium containing Amp, 37 DEG C of jolting overnight incubation.Turn by 1:50 next day in the LB liquid nutrient medium be inoculated in containing Amp, when 37 DEG C of cultivations are cultured to OD600 value about 0.4, get wherein 4 test tubes, add respectively IPTG to final concentration be 0mmol/L, 0.4mmol/L, 0.8mmol/L, 1.2mmol/L37 DEG C induction 4h after, as stated above sample is processed, 12%SDS-PAGE electrophoresis, observations.
B temperature condition is optimized: get the expressive host bacterium BL21 (DE3) containing recombinant plasmid pET32a-OmpHM, be inoculated in the 5mLLB liquid nutrient medium containing Amp, 37 DEG C of jolting overnight incubation.Turn by 1:50 next day in the LB liquid nutrient medium be inoculated in containing Amp, when 37 DEG C of cultivations are cultured to OD600 value about 0.4, get wherein 3 test tubes, adding IPTG respectively to final concentration is 0.4mmol/L, be placed in 25 DEG C, 30 DEG C, 37 DEG C induction 4h respectively, by above-mentioned ordinary method, sample is processed, 12%SDS-PAGE electrophoresis, observations.
C induction time is optimized: get the expressive host bacterium BL21 (DE3) containing recombinant plasmid pET32a-OmpHM, be inoculated in the LB liquid nutrient medium containing Amp, 37 DEG C of jolting overnight incubation.Turn by 1:50 next day in the LB liquid nutrient medium be inoculated in containing Amp, when continuing to be cultured to OD600 value about 0.4, adding IPTG to final concentration is 0.4mmol/L, 37 DEG C induce 0 respectively, 5,7,9h, draw 1mL nutrient solution, as stated above sample is processed, 12%SDS-PAGE electrophoresis, observations.
A large amount of preparations of 2.3 riemerella anatipestifer OmpH intercept recombinant proteins, purifying
2.3.1 a large amount of preparations (supernatant process) of riemerella anatipestifer OmpH intercept recombinant protein
By the 500mL bacterium liquid of abduction delivering in 4 DEG C of centrifugal 10min of 10000r/min, bacterial sediment 40mL20mmolTris-HCl (pH8.0) is suspended; Ultrasonic wave (ice bath) the broken thalline of interval (200w, 30sec/ time, 5 ~ 10 times), 4 DEG C of centrifugal 10min of 10000r/min.Get supernatant and be solubility riemerella anatipestifer OmpH intercept recombinant protein, 4 DEG C save backup.
2.3.2 the purifying of riemerella anatipestifer OmpH intercept recombinant protein
Nickel sepharose (Ni
2+-NAT) affinitive layer purification riemerella anatipestifer OmpH intercept recombinant protein: the affinity interaction special to nickel ion according to the 6-His label of fusion rotein, the fusion rotein with label is enable to be incorporated on nickel sepharose, change the condition of elutriant by its wash-out, and reach the object of purifying.Concrete operation step is:
(1) fill post: nickel sepharose dress post, column volume is about 40mL;
(2) balance: with level pad about 5 bed volume balance chromatography columns, flow velocity is 1mL/min;
(3) loading: the soluble upper protein sample of 0.45 μm of membrane filtration is about 50mL, adds in chromatography column, flow velocity is 0.4mL/min;
(4) wash: wash 2-5 bed volume again with level pad, flow velocity is 1mL/min;
(5) wash-out: respectively with containing 20,50, the elution buffer of 100mmol imidazoles carries out gradient elution, flow velocity is 1mL/min, collects the elution peak of each gradient, by molecular size range and the purity of SDS-PAGE detection fusion albumen;
(6) clean: with ultrapure washing 5 column volumes, then wash 3 column volumes with the ethanol of 25%, flow velocity is 1mL/min, reclaim nickel sepharose post, preserve in 4 DEG C, obtain the riemerella anatipestifer OmpH intercept recombinant protein of purifying.
3 experimental results
The amplification of 3.1 riemerella anatipestifer OmpH intercept genes, T-cloning and identification result
3.1.1 the pcr amplification result of riemerella anatipestifer OmpH intercept gene
With RA-CH-1 genomic dna for template carries out pcr amplification to OmpH intercept gene, its product, 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 primer reclaims after purifying through glue, and be connected with pMD19-T (simple) carrier and transformed competence colibacillus cell DH5 α, the T clone designation obtained is pMD19-OmpHM.PCR is carried out to pMD19-OmpHM, enzyme cuts that (Fig. 2: recombinant plasmid pMD19-OmpHM BamH I and Xho I double digestion obtain two fragments, the molecular size range of small segment is about 234bp) and order-checking qualification, it is completely the same with the OmpH intercept gene DNA sequence of known riemerella anatipestifer RA-CH-1 that result shows that T clones the OmpH intercept gene DNA sequence (SEQIDNO:2 shown in) obtained, and aminoacid sequence of its coding is as shown in SEQIDNO.1.
The Construction and identification of 3.2 prokaryotic expression plasmid pET32a-OmpHM, abduction delivering and optimum result thereof
3.2.1 the structure of recombinant expression plasmid pET32a-OmpHM and enzyme cut qualification
Object fragment is reclaimed with after BamH I and Xho I double digestion T cloned plasmids, carry out being connected with the pET-32a expression vector cut through same enzyme and transform DH5 α, obtain recombinant expression plasmid pET32a-OmpHM, two bar segment are obtained after BamH I and Xho I double digestion, a bar segment is obtained after Xho I, BamH I single endonuclease digestion, and size conforms to theoretical value, show recombined pronucleus expression plasmid construction success (Fig. 3).
3.2.2 the abduction delivering of recombinant plasmid pET32a-OmpHM
Under 37 DEG C of conditions, it is 0.4mmol/L that IPTG adds concentration, and abduction delivering spends the night, and empty carrier transforms bacterial strain at about 20kDa place's appearance one specific protein band, is the size of carrier label albumen own; The recombination fusion protein that recombinant expression plasmid pET32a-OmpHM expresses, at about 28kDa place, conforms to theoretical value; In addition, expressing protein soluble analysis result shows it and is mainly present in supernatant, illustrates that recombinant expression protein exists (Fig. 4) with solvable form in thalline.
3.2.3 the optimization of recombinant plasmid pET32a-OmpHM expression condition
The optimization of aIPTG concentration: under 37 DEG C of conditions, adding IPTG makes its final concentration be respectively 0mmol/L, 0.4mmol/L, 0.8mmol/L, 1.2mmol/L inducing culture 4h, result shows: with increasing of IPTG concentration, protein induced amount increases, and is that 1.2mmol/L is a little more than other concentration (Fig. 5) in IPTG concentration.Therefore, select the IPTG concentration of 1.2mmol/L as abduction delivering concentration.
The optimization of b inducing temperature condition: IPTG final concentration is 0.4mmol/L, respectively at 25 DEG C, 30 DEG C, 37 DEG C induction 4h, result shows, when temperature is at 37 DEG C, inducible protein amount is less, and when when 25 DEG C and 30 DEG C expression amount higher, and expression amount when 25 DEG C is a little more than 30 DEG C, therefore selective temperature 25 DEG C is as best inducing temperature (Fig. 6).
The optimization of c induction time: be 0.4mmol/L in IPTG concentration, under 37 DEG C of conditions, induce 0h, 5h, 7h, 9h respectively, result shows that the expressing quantity that 3h induces is little, the expression of recombinant proteins amount of 5h-9h is higher, and the expressing quantity of induction 9h is a little more than 5h, 7h (Fig. 7), therefore select 9h as best induction time.
The purifying of embodiment 2 riemerella anatipestifer OmpH intercept recombinant protein
By the expression product containing riemerella anatipestifer OmpH intercept recombinant protein obtained for embodiment 1 after collecting a series of process such as thalline, ultrasonic disruption, collection supernatant, nickel sepharose affinitive layer purification restructuring OmpH intercept recombinant protein.UV graphic representation after protein sample crosses column purification demonstrates 3 peaks, and peak 1 is for penetrating peak, and peak 2 is 50mmol imidazoles elution peak, and peak 3 is 100mmol imidazoles elution peak.Collect different concns imidazoles elution peak simultaneously, carry out SDS-PAGE electrophoresis, inspection purity and concentration, result shows: only have in 100mmol imidazoles elution peak containing a large amount of highly purified OmpH intercept recombinant protein (Fig. 8).By the OmpH intercept recombinant protein of purifying after super filter tube is concentrated, measure the final concentration of albumen with nucleic acid-protein instrument, for subsequent use after packing.
The Western-blot (protein immunoblot) of embodiment 3 riemerella anatipestifer OmpH intercept recombinant protein
One, experimental technique
What embodiment 1 obtained contains riemerella anatipestifer OmpH intercept recombinant protein as the probe detecting riemerella anatipestifer serum antibody.
1SDS-PAGE gel is prepared
The preparation of 1.112% separation gel
Deionized water 1215 μ l, 1.5MTris-HCl (PH=8.8) 950 μ l, 10%SDS37.5 μ l, 10%AP37.5 μ l, TEMED1.5 μ l, 30% acrylamide 1.5ml.
The preparation of 1.25% concentrated glue
Deionized water 700 μ l, 1.0MTris-HCl (pH=6.8) 125 μ l, 10%SDS10.0 μ l, 10%AP10.0ul, TEMED1.0 μ l, 30% acrylamide 165 μ l.
The process of 2 protein samples
Appropriate SDS-PAGE albumen sample-loading buffer (0.5MTris-HCl (PH=6.81.2ml), glycerine 1ml, deionized water 4.8ml, 10%SDS2.0ml, 0.1%BPB0.5ml) is added in OmpH intercept recombinant protein sample.100 DEG C are boiled 10000r/min10min after 10min.
3 electrophoresis
After glue cool to room temperature, albumen sample is directly upper in SDS-PAGE hole, 100v electrophoresis 90min.
4 transferring films
The erection sequence turning sandwich according to electricity is installed, 70v90min.
5 close
After transferring film, immediately protein film is placed in preprepared washings, rinsing 2min.
Hatching of 6 primary antibodies
Primary antibodie is that riemerella anatipestifer serum dilutes by 1:200, and then 4 DEG C of overnight incubation wash 3 times with PBS.
7 two anti-hatching
The two anti-dilutions of 1:3000 to specifications of HRP mark, hatch 1h for 4 DEG C, PBS washs 3 times.
8 substrate colour developings
Add enzyme substrates DAB nitrite ion, use deionized water termination reaction, film dries naturally, is stored in dark place.
Two, experiment conclusion
By Western-blot protein immunoblot experiment, riemerella anatipestifer OmpH intercept recombinant protein can produce good reactionogenicity (as Fig. 9) with the positive serum of riemerella anatipestifer RA-CH-1 strain, can be used as the probe or trapping agent that detect riemerella anatipestifer serum antibody.
Embodiment 4 riemerella anatipestifer OmpH intercept recombinant protein is as the indirect ELISA reagent kit of riemerella anatipestifer antibody capture agent
Riemerella anatipestifer OmpH intercept recombinant protein is described mainly through indirect ELISA reagent kit as the application of riemerella anatipestifer antibody capture agent.
Solid phase carrier: solid phase carrier as sorbent material and container, does not participate in chemical reaction in ELISA mensuration process.The material can making solid phase carrier in ELISA is a lot, and as polystyrene, it must meet the performance with stronger adsorbed proteins, and antibody or proteantigen adsorb after on it and still retain original immunologic competence, can be made into various forms.The shape of ELISA carrier mainly contains three kinds: microtiter plate, globule and small test tube.The most conventional with microtiter plate, the product being exclusively used in EILSA is called elisa plate, and the microtiter plate of standard is 96 cellular types of 8 × 12 in the world.For ease of doing the detection of a small amount of sample, have and make 8 hole bars or 12 hole bars, after putting into mounting, size is identical with standard ELISA plate.Simultaneously the feature of elisa plate to carry out the detection of a large amount of sample, and on special colorimeter, can read result rapidly.Existing multiple self-reacting device detects for the ELISA of microtitration template now, comprises the steps such as application of sample, washing, insulation, colorimetric, very favourable to the stdn of operation.What adopt in the present invention is 96 hole enzyme plates of Corning company.
Antibody capture agent: due to the culture environment of riemerella anatipestifer and nutritional requirement high, be unfavorable for cultivate and easily by other living contaminants, so the antibody capture agent adopted in the present invention is the riemerella anatipestifer OmpH intercept recombinant protein intercept recombinant protein after embodiment 2 purifying.
ELIAS secondary antibody: the ELIAS secondary antibody adopted in the present invention is goat-anti duck IgG-HRP (the goat-anti duck IgG of horseradish peroxidase mark, purchased from American KPL company), also the anti-duck IgG-HRP of other non-duck animals can be adopted, except marking with horseradish peroxidase, also other enzyme labellings can be adopted, as phosphoesterase.
Substrate: the ELIAS secondary antibody adopted due to the present invention is goat-anti duck IgG-HRP, therefore chromogenic substrate is tetramethyl benzidine (TMB), purchased from Tian Gen bio tech ltd.Also can selecting other chromogenic substrate, when adopting other enzyme labellings, binding substances coloured with it can be adopted as substrate.
Confining liquid: close be continue bag by after to wrap the process of quilt again with the irrelevant protein solution of high density.Antigen or antibody bag by time concentration used lower, after absorbing, surface of solid phase carriers still has the space be not occupied, and closing is exactly allow a large amount of incoherent these spaces of protein filling, thus repels adsorbing again of in ELISA step thereafter interfering substance.The formality closed is similar with bag.The most frequently used encapsulant is bovine serum albumin, and also useful calf serum or gelatin are as encapsulant, and skim-milk is also a kind of good encapsulant in addition.
Stop buffer: that the present invention adopts is 2mol/LH
2sO
4.
One experimental technique
1 indirect ELISA step
1. the riemerella anatipestifer OmpH intercept recombinant protein coating buffer of purifying being diluted to final concentration is 4 μ g/mL, adds successively in enzyme plate, l00 μ l/ hole, and 4 DEG C of bags are spent the night;
2. wash three times, abandon liquid, in every hole, add 300 μ l confining liquids, 37 DEG C of closed 30min;
3. wash three times, abandon liquid, will also loading after each serum-dilution to be checked, l00 μ l/ hole, hatches 30min for 37 DEG C;
4. wash three times, abandon liquid, add the goat-anti duck IgG-HRP ELIAS secondary antibody of dilution in every hole, l00 μ l/ hole, hatches 30min for 37 DEG C;
5. wash three times, abandon liquid, add TMB100 μ l in every hole, lucifuge colour developing 20min, then adds 2mol/LH
2sO
4stop buffer 50 μ l/ hole, pats mixing, surveys OD450nm value by microplate reader.
2 antigen the bests wrap the determination by concentration and the suitableeest extension rate of serum
By riemerella anatipestifer OmpH intercept recombinant protein with coating buffer doubling dilution and coated elisa plate, l00 μ l/ hole; RA positive serum and negative serum are used antibody diluent volume doubling dilution respectively, then adds in enzyme plate, l00 μ l/ hole.Test with square formation method, OmpH intercept recombinant protein bag when selecting P/N (positive serum OD450nm value/negative serum OD450nm value) maximum by concentration and serum diluting multiple as the antigen coated concentration of the best and the suitableeest serum diluting multiple.
The optimization of 3 antigen coated times
The riemerella anatipestifer OmpH intercept recombinant protein preparing purifying by embodiment 2 is as antigen and carried out bag quilt by the best bag by concentration, respectively 4 DEG C of bags spent the night, 37 DEG C hatch 1h after put 4 DEG C of bags spent the night and 37 DEG C hatch 2h after put 4 DEG C of bags and spent the night, each sample duplicate detection 3 times, then carry out indirect ELISA test, select the optimum antigen coated time.
The optimization of 4 different confining liquids
The riemerella anatipestifer OmpH intercept recombinant protein preparing purifying by embodiment 2 is as antigen and carried out bag quilt by the best bag by condition, close with the skim-milk of 1%BSA, 5%BSA, 10%BSA and 5% respectively, each sample duplicate detection 3 times, then carry out indirect ELISA test, select optimum confining liquid.
The optimization of 5 primary antibodies and two anti-binding times
The riemerella anatipestifer OmpH intercept recombinant protein of purifying is prepared as antigen and by the best bag by condition bag quilt by embodiment 2, close with best sealing condition, then indirect ELISA test is carried out, primary antibodie and two anti-ly reacts 120min, 90min, 60min and 30min respectively, each sample duplicate detection 3 times, selects the optimum reaction times.
6 enzyme labelled antibody best effort concentration determinations
According to fixed antigen the best bag by concentration and the suitableeest extension rate of serum, by goat-anti duck IgG-HRP (the goat-anti duck IgG of horseradish peroxidase mark, purchased from American KPL company) make different volumes multiple (1:50,1:100,1:200,1:400,1:800,1:1600,1:3200,1:6400) and dilute and carry out indirect ELISA test, each extent of dilution repeats 4 times, obtain average p value and N value, thus determine the best effort concentration of goat-anti duck IgG-HRP ELIAS secondary antibody.
The optimization of 7TMB developing time
The riemerella anatipestifer OmpH intercept recombinant protein of purifying is prepared as antigen and by the best bag by condition bag quilt by embodiment 2, close with best sealing condition, then indirect ELISA test is carried out, the substrate developing time of TMB is respectively 10min, 20min and 30min, each sample duplicate detection 3 times, selects optimum developing time.
The determination of 8 yin and yang attribute threshold value criterion
Get the negative duck serum of 20 parts of RA, detect according to the indirect ELISA method of above-mentioned foundation.Using the mean value (X) of the OD450nm value of 20 parts of negative samples and 3 times of standard variance (SD) sums upper limit as negative sample value, be judged to be the positive during threshold value (X+3SD) of the i.e. OD450nm value > negative sample OD450nm value of measuring samples, otherwise be then negative.
9 specific tests
Virulent duck enteritis virus (DHV) positive serum, avian influenza virus (AIV) positive serum, Salmonella anatis (Salmonellabacillus is detected respectively with the indirect ELISA method set up, SB) positive serum, E. coli isolated from ducks (E.coli) positive serum, duck are swollen head syndrome virus positive serum, riemerella anatipestifer RA-ATCC11845 strain and RA-CH-2 strain positive serum, every part of serum sample duplicate detection 3 times.
10 stability tests
Prepare the riemerella anatipestifer OmpH intercept recombinant protein bag of purifying by constar enzyme plate by embodiment 2, detect 5 parts of serum to be checked according to the indirect ELISA method set up, every increment product repeat 5 holes, judge batch interior repeatability.The constar enzyme plate preparing 5 different batches of the riemerella anatipestifer OmpH intercept recombinant protein bag quilt of purifying again by embodiment 2 detects 5 parts of serum to be checked, and judgement repeats between criticizing.
11 sensitivity tests
RA positive serum is carried out volume doubling dilution, then detect with the indirect ELISA method set up, carry out the indirect cell ELISA of the RA-CH-1 after agar diffusion test, slide agglutination test, microagglutination test and fragmentation with same serum sample simultaneously, all detection experiment repetitive operations 3 times, average, compare its susceptibility.
Two experiment conclusion
1 antigen (antibody capture agent) wraps the determination by concentration and serum dilution
By square formation burette test, the detected result of riemerella anatipestifer RA-CH-1 positive serum and negative serum is respectively as shown in table 7 and table 8, when the bag of antibody capture agent is 4ug/mL by concentration, when serum-dilution volume multiple is 1:200, positive serum OD450nm value is 0.384, corresponding negative serum OD450nm value is that 0.017, P/N value maximum (22.588) is as shown in table 9.Therefore, antibody capture agent the best bag is 4ug/mL by concentration, and antibody is the most fit, and long-pending extension rate is 1:200.
The positive serum detected result of the different antigen coated concentration of table 7 and serum dilution
The negative serum detected result of the different antigen coated concentration of table 8 and serum dilution
The P/N value of the different antigen coated concentration of table 9 and serum dilution
The optimization of 2 antigen coated conditions
The riemerella anatipestifer OmpH intercept recombinant protein preparing purifying by embodiment 2 is as antigen and carried out bag quilt by the best bag by concentration, spent the night respectively at 4 DEG C of bags, 37 DEG C hatch 1h after put 4 DEG C of bags spent the night and 37 DEG C hatch 2h after put 4 DEG C of bags and spent the night, then indirect ELISA test is carried out, each pattern detection 3 times, result is as shown in table 10.4 DEG C of bags are the highest by the positive value detected after spending the night, and the numerical value measured is more stable, therefore select 4 DEG C of bags by the antigen coated condition of the best of spending the night as present method.
The optimization of the antigen coated condition of table 10
The optimization of 3 different confining liquids
The riemerella anatipestifer OmpH intercept recombinant protein preparing purifying by embodiment 2 is as antigen and carried out bag quilt by the best bag by condition, close with the skim-milk of 1%BSA, 5%BSA, 10%BSA and 5% respectively, then indirect ELISA test is carried out, each sample duplicate detection 3 times, select optimum confining liquid, result is as shown in table 11.4 kinds of dissimilar results drawn with confining liquid that is concentration are more or less the same, and in line with the principle of saving, therefore select the confining liquid of this 1%BSA as the best confining liquid of present method.
The optimization of table 11 confining liquid
The optimization of 4 primary antibodies and two anti-binding times
The riemerella anatipestifer OmpH intercept recombinant protein preparing purifying by embodiment 2 is as antigen and carried out bag quilt by the best bag by condition, close with best sealing condition, then indirect ELISA test is carried out, primary antibodie and two anti-ly reacts 120min, 90min, 60min and 30min respectively, each sample duplicate detection 3 times, select the optimum reaction times, result is as shown in table 12 and table 13.OD450nm value all raises along with primary antibodie and two anti-increasing of reaction times, therefore primary antibodie and two action times resisted all select 120min.
Table 12 primary antibodie bag is by the optimization of time
The anti-bag of table 13 two is by the optimization of time
The determination of 5 enzyme labelled antibody optimal concentrations
The results are shown in Table 14, when enzyme mark goat-anti duck IgG antibody makes 1:400 volume dilution, P/N value is 24.114 to the maximum, therefore determines that best enzyme labelled antibody volume dilution multiple is 1:400.
The determination of table 14 enzyme labelled antibody best effort concentration.
The optimization of 6TMB developing time
The riemerella anatipestifer OmpH intercept recombinant protein of purifying is prepared as antigen and by the best bag by condition bag quilt by embodiment 2, close with best sealing condition, then indirect ELISA test is carried out, the substrate developing time of TMB is respectively 10min, 20min and 30min, each sample duplicate detection 3 times, selects optimum developing time.Result is as shown in Table 15, and it is optimum that TMB reacts 10min effect at 37 DEG C, therefore the optimum substrate developing time of present method is 10min.
The optimization of table 15TMB substrate developing time
The determination of 7 yin and yang attribute threshold value criterion
Detect 20 parts of RA negative serum samples by each ELISA condition of above-mentioned optimization, each sample duplicate detection 3 times, acquired results of averaging is shown in table 16.X value is 0.21965, SD value is 0.043646, determines that yin and yang attribute stagnation point is X+3SD=0.21965+3 × 0.043646=0.35, therefore, is judged to be the positive, is then judged to be feminine gender when being less than or equal to when the OD450nm value of testing sample is greater than 0.35.
Table 16RA negative serum detected result
8 specific tests
By the indirect ELISA method set up, the 7 kinds of cause of disease positive serums comprising riemerella anatipestifer are measured respectively, result is shown in table 17, except riemerella anatipestifer RA-ATCC11845 strain and RA-CH-2 strain positive serum, the OD450nm value of all the other 5 kinds of positive serums is all less than 0.35, show except these two kinds of positive serums, this antigen not with other 5 kinds of positive serum generation cross reactions above-mentioned, illustrate that the ELISA method using riemerella anatipestifer OmpH intercept recombinant protein as Detection of antigen RA antibody set up has good specificity thus, and this recombinant protein can as RA-CH-1, the general antigen of RA-ATCC11845 and RA-CH-2 different serotypes RA antibody test.
Table 17 specificity experiments result
9 stability tests
Prepare the riemerella anatipestifer OmpH intercept recombinant protein bag of purifying by constar enzyme plate by embodiment 2, detect 5 parts of serum to be checked according to the indirect ELISA method set up, every part of serum sample repeats 5 holes; The constar enzyme plate preparing 5 different batches of the riemerella anatipestifer OmpH intercept recombinant protein bag quilt of purifying again by embodiment 2 detects 5 parts of serum to be checked, by the OD of gained every part serum
450nmvalue calculates average OD
450nmvalue and standard variance (SD), then batch interior and interassay coefficient of variation (CV) calculating every part of serum, result is as shown in table 18 and table 19.In batch, revision test 5 groups of serum variation coefficient mean values are 3.37%, and all lower than 10%, between batch, the variation coefficient mean value of revision test 5 groups of serum is 3.34%, and all more than 10%, illustrate that the indirect ELISA method based on riemerella anatipestifer OmpH intercept recombinant protein that this research is set up has good stability thus.
Revision test in table 18 batch
Revision test between table 19 batch
10 sensitivity tests
With carry out respectively with a serum sample agar diffusion test, slide agglutination test, microagglutination test and the full bacterium of broken RA-CH-1 ELISA detect and compare with present method.Can not there is precipitation line in agar diffusion test, therefore cannot judge antibody titer.Tiring as 1:8 (as Figure 10) of slide agglutination experiment.Tiring as 1:32 (as Figure 11) of micro-agglutination experiment.Indirect cell ELISA after fragmentation is tired as 1:6400.Utilize this experimental technique, result is shown in table 20, along with RA-CH-1 positive serum dilution increase OD450nm value also obviously declines thereupon, when serum diluting multiple is 1:10400, OD450nm value is still greater than 0.35, and when extension rate is 1:20800, Virus monitory result is worth lower than feminine gender, therefore, be 1:10400 with the riemerella anatipestifer OmpH intercept recombinant protein of purifying of this research preparation as the minimum positive serum extension rate detected of antigen.
Table 20 sensitivity experiments result
The preparation of embodiment 5 riemerella anatipestifer OmpH intercept gene subunit vaccine
1 materials and methods
1.1 experimental animal groupings
By 24 1 age in days Cherry Village Duckss (its RA-CH-1 antibody agglutination is negative) random packet, experimental group 8, negative control group 8, commercialized vaccine positive controls 8.
1.2 immunity and attack poison
The riemerella anatipestifer OmpH intercept recombinant protein purification product obtained by embodiment 2 mixes with freund's adjuvant equal-volume, riemerella anatipestifer OmpH intercept recombination subunit vaccine is obtained after emulsification, the immune programme for children of experimental group is shown in table 21, commercialized vaccine positive controls presses the immunity of vaccine specification sheets, negative control group injecting normal saline.
All animal 17 ages in days (exempting from latter 10 days in OmpH intercept recombination subunit vaccine two) the full bacterium of intramuscular injection riemerella anatipestifer, injection volume is 2.5 × 10
9cFU/ml, often only injects 1ml, observes lesion tissue and the death condition of animal.
Table 21 experimental group immune programme for children
1.3 bacteria distribution qualifications
Liver and the cerebral tissue of getting dead animal rule TSA in 37 DEG C of CO
2overnight incubation under condition, to the bacterium colony grown
Carry out PCR qualification.PCR primers designed is as follows:
Upstream primer: 5'-CGAAAGTGATAAGTTAGCCACCT-3'(SEQIDNO:5);
Downstream primer: 5'-GCAGCACCTTGAAAATTGTCC-3'(SEQIDNO:6);
2 results
Negative group duck is all dead, and mortality ratio is 100%, and experimental group survives 4, and protection ratio is 50%, and commercialized vaccine positive controls protection ratio is 50%.Then analyse all not dead animals, the animal of positive commercialization control group and experimental group does not all find pathology, display normal (as Figure 13).The duck of death group is analysed to the pathology (as Figure 12) can seen in various degree.All death group ducks are carried out to bacteria distribution and the PCR qualification of brain, result (as Figure 14) shows, dead duck all dies from the infection of riemerella anatipestifer.
Above-described embodiment is the present invention's preferably embodiment; but embodiments of the present invention are not restricted to the described embodiments; change, the modification done under other any does not deviate from spirit of the present invention and principle, substitute, combine, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.
Claims (7)
1. a riemerella anatipestifer OmpH intercept recombinant protein, is characterized in that its aminoacid sequence is as shown in SEQIDNO:1.
2. riemerella anatipestifer OmpH intercept recombinant protein according to claim 1, is characterized in that its DNA sequence dna is as shown in SEQIDNO:2.
3. the preparation method of riemerella anatipestifer OmpH intercept recombinant protein according to claim 1, is characterized in that specifically comprising the following steps:
A, with RA genomic dna for template, the upstream and downstream primer PCR amplification of nucleotide sequence respectively as shown in SEQIDNO.3 and SEQIDNO.4 is adopted to obtain riemerella anatipestifer OmpH intercept gene fragment, be connected with pMD19-T (simple), obtain pMD19-OmpHM;
B, use restriction enzyme BamHI and XhoI double digestion pMD19-OmpHM plasmid and prokaryotic expression carrier pET32a (+) respectively, connect, obtain recombined pronucleus expression plasmid pET32a-OmpHM;
C, by step B extract recombinant plasmid pET32a-OmpHM be transformed into expressive host bacterium abduction delivering, obtain riemerella anatipestifer intercept recombinant protein crude product.
4. the preparation method of riemerella anatipestifer OmpH intercept recombinant protein according to claim 3, it is characterized in that: by step C gained riemerella anatipestifer OmpH intercept recombinant protein crude product after nickel sepharose affinitive layer purification, with 100mM imidazoles wash-out, obtain riemerella anatipestifer OmpH intercept recombinant protein.
5. the preparation method of riemerella anatipestifer OmpH intercept recombinant protein according to claim 3, it is characterized in that: the Host Strains in step C is bacterial strain BL21 (DE3), at IPTG concentration=1.2mmol/L, inducing temperature is carry out abduction delivering 9 hours under 25 DEG C of conditions, obtains riemerella anatipestifer OmpH intercept recombinant protein.
6. riemerella anatipestifer OmpH intercept recombinant protein according to claim 1 is applied to the detectable antigens as the indirect ELISA method detecting riemerella anatipestifer antibody.
7. riemerella anatipestifer OmpH intercept recombinant protein according to claim 1 is applied to the genetic engineering subunit vaccine as Mo Shi bacillus in prevention pest of duck.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610070599.5A CN105541977B (en) | 2015-06-16 | 2016-01-29 | Riemerella anatipestifer OmpH intercept recombinant protein and preparation method and application |
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