CN104974248B - Riemerellosis Anatipestifer OmpA/MotB goes signal peptide recombinant protein, its antibody and preparation method and application - Google Patents

Abstract

The invention discloses a kind of Riemerellosis Anatipestifer OmpA/MotB to go signal peptide recombinant protein, its antibody and preparation method and application.The recombinant protein has immune response activity similar with natural OmpA/MotB albumen, it can be in conjunction with RA antibody specificity, and cross reaction is not generated with Salmonella anatis positive serum, E. coli isolated from ducks positive serum, duck swollen head septicaemia virus positive serum, avian influenza virus (H5) positive serum, duck plague virus positive serum, duck hepatitis virus positive serum etc., it can be used as the detection antigen application of the detection full bacteria antibody of RA, it is dangerous without scattered poison when being detected using it and due to the non-full bacterium of the recombinant protein；In addition, the preparation method of the recombinant protein is simple, it is suitable for industrialization large-scale production, can be realized the standardization of product, compare conducive to the result between different experiments room.

Description

Riemerellosis Anatipestifer OmpA/MotB goes to signal peptide recombinant protein, its antibody and preparation side Method and application

Technical field

The invention belongs to gene engineering technology fields, and in particular to a kind of Riemerellosis Anatipestifer OmpA/MotB removes signal peptide Recombinant protein, its antibody and preparation method and application.

Background technique

Riemerellosis Anatipestifer Disease caused by Riemerellosis Anatipestifer (Riemerella anatipestifer, RA) (Riemerella anatipestifer infection) is a kind of contagiousness of a duck, turkey and a variety of other birds Disease, also referred to as new duck disease ((New duck syndrome), duck septicemia (Duck septicaemia), pest of duck syndrome (Anatipestfer syndrome), pest of duck septicemia (Anatipestifer septicaemia), Pasteurella anatipestipestifer disease (Pasteurella anatipestifer infection) and duck infectious serositis (Infectious serositis). It is the common bacterial infectious disease of one kind of duck, mainly encroaches on 2-8 week old ducklings, with fibrinous pericarditis, perihepatitis, Air bag inflammation is main pathological characters.

The cause of disease for causing Riemerellosis Anatipestifer Disease is Riemerellosis Anatipestifer (Riemerella anatipestifer, RA). This bacterium is Gram-negative dialister bacterium, and no brood cell, thallus cannot move without pili or flagellum, there is pod membrane, and Wright's staining is in the two poles of the earth Dense dye.Pure culture bacterium smear, methylene blue, it is seen that thallus is mostly rod-shaped, often individually exists, a small number of at way double-line, accidental individual Thallus is in long Filamentous arrangement.The serotype of RA is more and complicated, and serotype is more than 21, and lacks effective hand between each serotype Immunoprotection is pitched, this brings certain difficulty to the vaccine control of RA.Relatively effective method is for local Major Epidemic serum Type chooses corresponding bacterial strain and develops vaccine, to reach more effective preventive effect.

Bacterial outer membrane proteins (OMP) are that Gram-negative bacteria is distinctive, be present in it is among outer membrane and related with outer membrane The general name of all albumen.Outer membrane surrounds entire bacterium in the outside of bacterial cytoplasm film (inner membrance) and peptidoglycan layer, is gram-negative Specific structure in property bacterium cell wall constituent.Outer membrane protein is made of 6 families: including OmpA albumen, OmpX albumen, hydrolysis Phospholipase A, common porin (OmpF, PhoE), substrate specificity porin (LamB, ScrY) and metal-dependant iron Transport protein FhuA and FepA albumen shares more than 20, but most functions are unclear.Outer membrane protein A (Outer Membrane Protein A, OmpA) be Gram-negative bacteria major outer membrane protein, OmpA is related with the biofilm formation of bacterium, can maintain Outer membrane it is complete, lack the bacterium of OmpA, cell membrane is unstable.OmpA is a kind of transmembrane protein, and some researches show that the albumen removes Have the function of maintaining outside outer membrane structure, important function is in bacterium infection early stage and cell-matrix interaction, field planting In host cell, to reach suction-operated.It is reported that OmpA is a kind of virulence factor of RA, there are pathogenic effects, therefore, There may be important value in the diagnosis of Riemerellosis Anatipestifer Disease and vaccine development.In addition, OmpA gene nucleic acid sequence height It is conservative, there is good immunogenicity, the humoral immunity and cellular immunity of body can be excited, and genetic engineering technology can be passed through Obtain great expression.For heterologous serotype, OmpA can play the role of immunological cross protection, be a kind of potential common protection Property antigen, it is putative RA high protective antigens.Furthermore OmpA facilitates the immune defense of RA escape body.

Thrombospondin refers to thrombospondin, also known as thrombospondin.Its gene family is divided into two A subfamily, subfamily A and subfamily B.The protein of subgroup A includes TSP-1 and TSP-2, is assembled into tripolymer, includes 1,2,3 Type repeats；The protein of subgroup B refers to TSP-3, TSP-4 and COMP (TSP-5), they are characterized in including unique N-terminal area Domain, lacks procollagen homology region and 1 type repeats, and containing there are four the repetition of 2 types of version, is assembled into five polymer compounds.3 type weights The effect of multiple (Thrombospondin type 3repeats) is cell adhesion (cell attachment), propagates (spreading) and protease inhibition (protease inhibition).Thrombospondin type 3repeat: OmpA/MotB may relate to pathogen with the interaction between host cell, related with pathogen absorption host cell, be Other members of OmpA family.MotB is a kind of flagellar motor albumen of bacterium, related with the movement of bacterium.But it writes from memory in pest of duck Salmonella does not have flagellum, can speculate that MotB and OmpA may have homology, there are identity functions.There is document to point out, OmpA phase The conserved sequence for closing albumen and MotB PROTEIN C end has common function: Ke Nengyu in Gram-negative bacteria and positive bacteria Peptide glycan interacts in relation to (The C-terminal sequence conservation between OmpA-related outer membrane proteins and MotB suggests a common function in both gram- positive and gram-negative bacteria,possibly in the interaction of these domains with peptidoglycan,Molecular microbiology,1994,12(2):333)。

Due to both at home and abroad about Riemerellosis Anatipestifer fibrin ferment sensitivity type III repetitive proteins (Thrombospondin type 3repeats): the research of OmpA/MotB gene and its albumen has not been reported, therefore, to Thrombospondin type The research of 3repeats:OmpA/MotB gene and its albumen helps to open up new thinking for the prevention and control of RA.

Summary of the invention

In view of this, the purpose of the present invention is to provide a kind of Riemerellosis Anatipestifer OmpA/MotB, and signal peptide to be gone to recombinate egg It is white, there is immune response activity similar with natural OmpA/MotB albumen, it can be in conjunction with RA antibody specificity；The present invention The antibody and preparation method and application of above-mentioned recombinant protein are additionally provided simultaneously.

The present invention reaches the purpose by following technical proposals:

1, Riemerellosis Anatipestifer OmpA/MotB removes signal peptide recombinant protein, and amino acid sequence is as shown in SEQ ID NO.2.

2, Riemerellosis Anatipestifer OmpA/MotB removes the encoding gene of signal peptide recombinant protein.

Preferably, nucleotide sequence is as shown in SEQ ID NO.1.

3, the recombinant expression carrier containing above-mentioned encoding gene.

It preferably, is to remove nucleotide sequence OmpA/MotB as shown in SEQ ID NO.1 signal peptide recombinant protein to encode Gene cloning enters in prokaryotic expression plasmid pGEX-4T-1 and obtains.

4, the engineering bacteria of above-mentioned recombinant expression carrier.

It preferably, is recombinant expression carrier to be transferred in e. coli bl21 (DE3) and obtained；The recombinant expression carrier To go signal peptide recombinant protein encoding gene to be cloned into protokaryon nucleotide sequence OmpA/MotB as shown in SEQ ID NO.1 It is obtained in expression plasmid pGEX-4T-1.

5, above-mentioned engineering bacteria prepares the method that Riemerellosis Anatipestifer OmpA/MotB removes signal peptide recombinant protein, including as follows Step:

Engineering bacteria is taken to be inoculated in the LB culture medium containing Amp, with 150-200r/min, 37 DEG C of culture 12h or to OD_600nmFor 0.6, it is then added in the LB culture solution containing Amp for the ratio of 1:100 by volume and continues culture to OD for 37 DEG C_600nmUp to 0.6 Afterwards, 12h is induced under the conditions of 0.4mmol/L IPTG, 30 DEG C；Collect bacterium solution, centrifugation, by the bacterial sediment of acquisition with 1/10 bacterium The Tris-HCl of 20mmol/L, pH 8.0 of liquid product suspends, and ice-water bath ultrasound cracks thallus, and then 12000r/min is centrifuged 10min, the 8mol/L urea liquid dissolution of 1/50 original bacteria liquid volume of precipitating, 8000r/min are centrifuged 10min, take lysate, add Enter 5 × SDS loading buffer to boil, it is the pre- cold dyeing of 0.25%KCl with mass fraction that SDS-PAGE, which separates recombinant protein, Glue is cut after color, blob of viscose of dialysing, until gained protein liquid is concentrated after blob of viscose is transparent to obtain the final product.

6, it is the Anti-TNF-α that antigen-immunized animal obtains that Riemerellosis Anatipestifer OmpA/MotB, which removes signal peptide recombinant protein, Body.

7, Riemerellosis Anatipestifer OmpA/MotB goes signal peptide recombinant protein preparing the sub- list of Riemerellosis Anatipestifer genetic engineering Application in position vaccine or antibody test reagent.

The beneficial effects of the present invention are: present invention selection RAOmpA/MotB gene goes to signal peptide protein domain, utilizes Prokaryotic expression system, which has successfully been made, removes signal peptide OmpA/MotB recombinant protein.The recombinant protein has and natural OmpA/MotB The similar immune response activity of albumen, can in conjunction with RA antibody specificity, and with Salmonella anatis positive serum, duck large intestine bar Bacterium positive serum, duck swell head septicaemia virus positive serum, avian influenza virus (H5) positive serum, duck plague virus positive serum, Duck hepatitis virus positive serum etc. does not generate cross reaction, can be used as detection the full bacteria antibody of RA detection antigen application, and due to The recombinant protein non-full bacterium is dangerous without scattered poison when being detected using it；In addition, the preparation method of the recombinant protein is simple, Suitable for industrialization large-scale production, the standardization of product can be realized, compare conducive to the result between different experiments room.

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 OmpA/MotB gene removes signal peptide PCR product electrophoresis.M swimming lane: DL5000Marker, 1 swimming lane: OmpA/ MotB gene removes signal peptide pcr amplification product.

Fig. 2 OmpA/MotB goes signal peptide construction of recombinant plasmid and its PCR and digestion to identify.A figure is pMD19-T/ in figure OmpA/MotB goes signal peptide plasmid PCR to identify, M swimming lane: DL2000Marker, 1 swimming lane: PCR product；B figure is pMD19- in figure T/OmpA/MotB goes signal peptide plasmid enzyme restriction to identify, M swimming lane: DL15000Marker, 1 swimming lane: BamH I/Xho I double digestion Band；C figure is that pGEX-4T-1/OmpA/MotB goes the PCR of signal peptide plasmid to identify in figure, M swimming lane: DL2000Marker, 1 swimming Road: PCR product；D figure is that pGEX-4T-1/OmpA/MotB goes the digestion of signal peptide plasmid to identify in figure, M swimming lane: DL15000Marker；1 swimming lane: BamH I/Xho I double digestion band.

Fig. 3 OmpA/MotB goes the optimization of the expression analysis and expression condition of signal peptide albumen.M swimming lane is low molecular weight egg White matter standard items.A figure is the analysis of recombinant protein expression-form in figure, and swimming lane 1 is that pGEX-4T-1/OmpA/MotB removes signal peptide Plasmid supernatant expression product, 2-3 swimming lane are respectively inclusion body expression product and pGEX-4T-1 empty plasmid expression product；B in figure Figure is the optimization of inducing expression time, and 1 swimming lane is pGEX-4T-1 empty plasmid expression product, and 2-7 swimming lane is followed successively by recombinant expression egg BL21 (DE3) host strain expression product of white inclusion body induction 2h, 4h, 6h, 8h, 10h, 12h；C figure is inducing expression temperature in figure The optimization of degree, 1-3 swimming lane is respectively 25 DEG C, 30 DEG C, 37 DEG C BL21 (DE3) host strain expression product, and 4 swimming lanes are pGEX-4T- 1 empty plasmid expression product；D figure is IPTG concentration optimization in figure, and 1-4 swimming lane is respectively 0mmol/L, 0.4mmol/L, 0.8mmol/ L, the expression product of the IPTG concentration induction of 1.2mmol/L.

The Western-blot qualification result of Fig. 4 expression albumen.M swimming lane is pre- dsred protein Marker, and 1 swimming lane is OmpA/MotB removes signal peptide western blot.

The OmpA/MotB of Fig. 5 purifying goes signal peptide recombinant protein SDS-PAGE electrophoresis result.Wherein, M swimming lane is low molecule Protein standards are measured, 1 swimming lane is that unpurified OmpA/MotB removes signal peptide recombinant protein, and 2 swimming lanes are to cut glue purification OmpA/MotB removes signal peptide recombinant protein.

Fig. 6 rabbit-anti OmpA/MotB albumen hyper-immune serum fine jade expands bioactivity as a result, the OmpA/ of purifying is added in medium pore Q MotB removes signal peptide recombinant protein, and holes around is separately added into the diluted rabbit-anti OmpA/M otB of 1:2,1:4,1:8,1:16 and 1:32 Remove signal peptide recombinant protein hyper-immune serum and blank control PBS.

Specific embodiment

A preferred embodiment of the present invention will be described in detail below.The experiment side of actual conditions is not specified in embodiment Method, usually according to conventional conditions or according to the manufacturer's recommendations.

Main material: serum 1 type Riemerellosis Anatipestifer bacterial strain (RA-CH-1 bacterial strain), Ecoli DH5 α, Ecoli BL21 (DE3) strain, PGEX-4T-1 carrier are saved by Sichuan Agricultural University's poultry disease prevention and control research center and are provided.1 age in days Growth of Cherry Valley meat Duck 40 is only purchased from Sichuan Agricultural University's poultry trial field.Adult buck 4, is purchased from local warren.

TSA and TSB is purchased from Britain OXOID company；Trypone, Yeast Extract are purchased from Oxoid company； DL2000Marker、DL15000Marker、Prime STAR PCR Mix、PMD19-T cloning kit、ligation Mix connection kit, BamHI and XhoI restriction enzyme, bacillus coli DH 5 alpha competent cell are purchased from precious bioengineering (Dalian) Co., Ltd；Agarose, DAB colour reagent box are purchased from Shanghai Ai Yan Biotechnology Co., Ltd；Plasmid extraction examination Agent box, plastic recovery kit are purchased from OMEGA company；Soluble one pack system tmb substrate solution is purchased from the limited public affairs of Tiangeng biochemical technology Department；Tris alkali, glycine, SDS, acrylamide, methene acrylamide, ammonium persulfate, TEMED, R250, BSA are purchased from Shanghai life Work biotechnology Co., Ltd；The goat-anti duck IgG of HRP label is purchased from KPL company, the U.S.；Low molecular weight protein Marker, pre-dyed albumen Marker, pvdf membrane；IPTG is purchased from Promega company；Freund's complete adjuvant, incomplete Freund's adjuvant It is purchased from Sigma biotechnology company；0.22 μm and 0.45 μm of filter membrane, super filter tube, bag filter are purchased from Millipore company； ELISA enzyme mark strip is purchased from healthy and free from worry (Corning) Co., Ltd.

Embodiment 1 goes the preparation of signal peptide OmpA/MotB recombinant protein

1, OmpA/MotB gene (removing signal peptide) design of primers

According to the OmpA/MotB gene order of the Gene Bank RA-CH-1 bioinformatic analysis announced and supposition (Genbank ID number: CP003787.1) information, the long 1467bp of OmpA/MotB genetic fragment use SignalP 4.1Server Signal peptide prediction is carried out, OmpA/MotB gene 1-63 base is signal peptide sequence.It is soft using Primer Premier5.0 Part is as follows to the conservative region design primer of removal signal peptide, by the primer amplification, obtains OmpA/MotB gene and removes signal peptide The length of fragment gene primer region is 1563bp, in addition the size of PCR product is after restriction enzyme site and protectiveness base 1581bp, OmpA/MotB go the nucleotide sequence of signal peptide expression product to see that SEQ ID NO.1, amino acid sequence are shown in SEQ ID NO.2。

PCR cloning primer:

Upstream primer P1:5'-cgcggatccC agactactag caatccttgg ttaatag-3', (SEQ ID NO.3)；

Downstream primer P2:5'-ccgctcgagT atcccaacga gccatcttag ag-3', (SEQ ID NO.4)；

It wherein, is respectively BamH I and Xho I restriction endonuclease sites at underscore, primer is had by Shanghai Invitrogen The synthesis of limit company.

2, RA full-length genome extracts and OmpA/MotB goes signal peptide section PCR amplification

RA-CH-1 bacterial strain complete genome DNA is extracted referring to Tiangeng bacterial genomes DNA kit specification step.Foundation TaKaRaMax DNA Polymerase specification carries out the processing of primer and template.

It is as follows to expand OmpA/MotB gene (removing signal peptide) PCR reaction system: ddH₂9 μ L, 2x primerstar of O Max12.5 μ L, P1 (10pmol) 1 μ L, P2 (10pmol) 1 μ L, template DNA (100ng/ μ L) 1.5 μ L, 25 μ L of total volume.PCR is anti- Answer condition: 95 DEG C of initial denaturation 5min；94 DEG C of denaturation 40s, 58 DEG C of annealing 40s, 72 DEG C of extension 50s, 30 circulations；72℃10min. Take 5 μ L products to carry out electrophoresis with 1% Ago-Gel, gel imaging system detects amplification, as shown in Figure 1, obtain with it is pre- The OmpA/MotB gene of phase 1581bp size position consistency removes signal fragments of peptide target DNA fragment.It recycles and tries according to OMEGA glue Agent box specification carries out glue recycling.

3, the building of recombinant expression plasmid

Target DNA fragment end after recovery purifying is added into A, is convenient for subsequent connection work.System are as follows: 2xPCR mix5 μ L, 5 μ L of target DNA, 10 μ L of total volume, 72 DEG C of reaction 30min of PCR instrument.T clones system are as follows: 5 μ L of Ligation mix, Purpose OmpA/MotB removes 4 μ L, pMD19-T carrier of signal fragments of peptide, 1 μ L, 10 μ L of total volume.16 DEG C of connections are stayed overnight, sterile lower behaviour Make, in order to avoid influence changing effect.It takes 10 μ L connection products in 100 μ L DH5 α competent cells, mixes gently, ice bath 30min； And 42 DEG C of water-bath thermal shock 90s are placed on, then fast transfer continues ice bath 2min into ice bath；LB liquid medium 890 is added μ L, in 37 DEG C of shaken cultivation 1h；750 μ supernatants are abandoned after low-speed centrifugal, and 250 μ L precipitating culture is taken to be applied to containing ampicillin (Amp+), on the LB agar plate of X-gal, IPTG, 37 DEG C of culture 16h.The single white colony of random picking, is inoculated into 5mlLB In fluid nutrient medium (Amp+), 37 DEG C of shaken cultivations are stayed overnight.

Plasmid is extracted referring to OMEGA company small amount plasmid extraction kit specification, PCR is then carried out and digestion is identified, Digestion identification system is as follows: 1 μ L of 10xK buffer, 6 μ L, BamH I (15U/ μ L) of plasmid, 0.5 μ L, Xho I (15U/ μ L) 0.5 μ L, 2 μ L of ultrapure water, 10 μ L of total volume.Reaction condition is 37 DEG C, water-bath 4h.After completion of the reaction plus 2 10 × Loading of μ L Buffer is to terminate reaction.1% agarose gel electrophoresis detects digestion as a result, as shown in Fig. 2, OmpA/MotB gene is write to After number peptide fragment is connected to pMD19-T carrier, obtained through PCR (A schemes in Fig. 2) and BamHI, XhoI double digestion (B schemes in Fig. 2) It is expected that band.The correct plasmid of Preliminary Identification is finally named as pMD19-T/OmpA/MotB and removes signal peptide, and is sent The sequencing of Invitrogen company.

Sequencing is identified that correct T cloned plasmids pMD19-T/OmpA/MotB removes signal peptide, pGEX-4T-1 carrier difference BamH I/Xho I double digestion, is separately recovered target gene fragment and carrier large fragment, is carried out according to kit specification connection, Linked system is as follows: 4.5 3 μ L, Ligation mix of μ L, pGEX-4T-1 of target gene recovered liquid 7.5 μ L, 15 μ L of total volume. 16 DEG C of connections overnight.Connection product converts DH5 α competent cell, with Amp resistance LB solid and positive gram of fluid nutrient medium screening It is grand, through PCR (C schemes in Fig. 2) and BamHI/XhoI double digestion (D schemes in Fig. 2) identification, respectively obtain expected band.Matter will be recombinated Grain is sent to Invitrogen for sequencing, and will identify that correct plasmid is named as pGEX-4T-1/OmpA/MotB and removes signal peptide.It will Contain correct plasmid DH5 α bacterium conservation.

4, the building and inducing expression of engineering bacteria are recombinantly expressed

Correct pGEX-4T-1/OmpA/MotB, which will be sequenced, goes signal peptide former nuclear expression plasmid to convert to Ecoli.BL21 (DE3) it expresses in bacterium, picking single bacterium is fallen in the test tube of the LB liquid of Amp containing 5mL, 150r/min, 37 DEG C of overnight incubations.Next day Culture 1:100 is inoculated in Amp LB liquid medium, expands culture about 3h, bacterium solution OD_600nmWith 0.4mmol/L when up to 0.6 37 DEG C of induction 4h of IPTG final concentration, bacterium solution 8000r/min is centrifuged, and collects bacterial sediment.1/10 bacterium solution volume of bacterial sediment 20mmol/L Tris-HCl (pH 8.0) suspend, ice-water bath ultrasound crack thallus, then 12000r/min be centrifuged 10min, take Supernatant is added 5xSDS loading buffer and boils sample preparation, referred to as supernatant；Precipitate the 8M urea of 1/50 original bacteria liquid volume Solution dissolution, 8000r/min are centrifuged 10min, take lysate, add 5xSDS loading buffer to boil sample preparation, be known as forgiving Body.The bacterium of plasmid expression containing empty carrier is arranged simultaneously to compare, direct sample preparation after ultrasound, regardless of supernatant and inclusion body.By made sample It carries out SDS-PAGE electrophoresis, as a result purpose band occurs at the place about 76kD or so (A schemes in Fig. 3).

Picking goes BL21 (DE3) single colonie of signal peptide plasmid to be inoculated into the training of LB liquid containing pGEX-4T-1/OmpA/MotB It supports base (Amp+), 37 DEG C of activation are overnight.Next day is seeded to fresh 100ml LB liquid medium (Amp+) by 1:100, and 37 DEG C Shaken cultivation 3h, OD_600nmWhen up to 0.6 or so, IPTG induction is added to shake bacterium.Do IPTG concentration, IPTG induction time, expression temperature etc. The optimization of condition, to obtain the optimum condition of the expression of destination protein.

(1) inducible protein expresses IPTG concentration optimization

Go the expressive host bacterium BL21 (DE3) of signal peptide with 1:100 points pGEX-4T-1/OmpA/MotB containing recombinant plasmid It is not inoculated in the LB containing Amp, 37 DEG C of violent shaken cultivations to OD_600nm=0.6 or so, addition IPTG to final concentration 0mmol/L, 4~6h of 0.4mmol/L, 0.8mmol/L, 1.2mmol/L Fiber differentiation or so, centrifugation obtain thallus, ultrasound cracking, and centrifugation obtains Obtain sample.After sample treatment, SDS-PAGE electrophoresis, as a result as shown in D figure in Fig. 3.

(2) optimization of inducing temperature

Referring to the above method, to bacterium solution culture to OD_600nmAfter=0.6 or so, addition IPTG to final concentration of optium concentration, Respectively in 25 DEG C, 30 DEG C and 37 DEG C 4~6h of Fiber differentiation, it is centrifuged sample preparation, sample is handled, SDS-PAGE electrophoresis, as a result As shown in C figure in Fig. 3.

(3) optimization of induction time

Referring to the above method, bacterium solution culture to be expressed to OD_600nmWhen=0.6 or so, using best IPTG concentration and most suitable Temperature, induction time are set as 2h, 4h, 6h, 8h, 10h, 12h, will carry out SDS-PAGE analysis after expression product sample preparation processing, As a result as shown in B figure in Fig. 3.

From the figure 3, it may be seen that OmpA/MotB goes the optimum condition of the expression of signal peptide albumen to lure for 0.4mmol/L IPTG, 30 DEG C Lead 12h.

5, the Western-blot identification of albumen is expressed

The expression product of the optimal expression bacterium filtered out is subjected to western blot according to a conventional method, process is as follows: will express Sample carries out SDS-PAGE, and then the gel after electrophoresis is transferred on pvdf membrane, and 80V transfers 90min, will after transfer Pvdf membrane takes out, and with 1%BSA in 37 DEG C of incubation 1h, is then incubated for 37 DEG C of shakes of the diluted rabbit-anti RA-CH-1IgG of 1:100 It takes out after 1h, with PBS washing 3 times, each 2min, is then shaken with the goat anti-rabbit igg of the diluted HRP label of 1:3000 in 37 DEG C It is incubated for 1h, develops the color after being washed with PBS according to DAB colour reagent box specification, is rushed when purpose band is high-visible with distilled water Wash color development stopping.Pvdf membrane drying is kept in dark place.Qualification result is shown in Fig. 4, and as shown in Figure 4, the OmpA/MotB of recombinant expression is gone Signal peptide albumen can be specifically bound with RA-CH-1 polyclonal antibody.

6, the great expression and purifying of recombinant protein

Picking goes signal peptide BL21 (DE3) single bacterium to fall within 10ml Amp LB culture medium containing pGEX-4T-1/OmpA/MotB In, 37 DEG C of 200r/min, 5-6h, until OD_600nmIt is 0.6；Above-mentioned 10ml bacterium solution is added to the taper of the LB of Amp containing 1L culture solution In bottle, 37 DEG C, 200r/min continues to cultivate 2-3h.OD_600nmUp to after 0.6, in best IPTG induced concentration, most suitable inducing temperature and It is induced under the conditions of best induction time.Thallus system is collected according to the method when the building of recombinant expression engineering bacteria and inducing expression Sample obtains solubilization of inclusion bodies liquid.SDS-PAGE separates recombinant protein, cuts glue after 0.25%KCl pre-cooling dyeing.By blob of viscose in level The dialysis of gel electrophoresis slot, after blob of viscose is transparent, the protein liquid of bag filter is concentrated with super filter tube, -20 DEG C save backup.It will be resulting Purifying OmpA/MotB goes signal peptide albumen to carry out SDS-PAGE electrophoresis to detect purity of protein, as a result sees Fig. 5.It obtains as shown in Figure 5 The OmpA/MotB for obtaining high-purity removes signal peptide albumen.

Embodiment 2 goes the foundation of signal peptide albumen indirect ELISA method based on OmpA/MotB

1, the reaction condition optimization of signal peptide protein ELISA method is removed based on OmpA/MotB

Optimal conditions are shown in Table 1.

1 indirect ELISA condition optimizing of table

It follows these steps to optimize with each reaction condition of table 1:

(1) coating buffer diluted protein is used, the every hole of microwell plate adds 100 μ L, and 4 DEG C overnight；

(2) next day gets rid of abandoning liquid, and PBST 200 μ L, 37 DEG C of closing 1h containing 1%BSA is added in every hole；

(3) PBST is washed 4 times, and each 5min is patted dry, and serum 100 μ L, 37 DEG C of incubation 1h to be checked is added；

(4) PBST is washed 4 times, is patted dry, and goat-anti duck IgG 100 the μ L, 37 DEG C of incubation 1h of working concentration HRP label is added；

(5) board-washing is same as above, and is patted dry, and 100 μ L of TMB is added in every hole, is protected from light colour developing 15min, terminate liquid (2mol/L is added H₂SO₄) 100 μ L, mixing is patted, surveys OD under microplate reader_450/630nmValue.

OmpA/MotB goes signal peptide proteantigen peridium concentration and serum dilution to select to be shown in Table 2.

2 OmpA/MotB of table goes signal peptide proteantigen peridium concentration and serum dilution to select

As shown in Table 2, OmpA/MotB go signal peptide albumen best peridium concentration be 3.61 μ g/mL (dilution 1: 200), best serum dilution is 1:320.

OmpA/MotB goes signal peptide protease labeling antibody working concentration to optimize: 3 repetitions are arranged in each concentration, are averaged Value is incubated in 37 DEG C, and yin and yang attribute serum is arranged, and P/N value the maximum is best secondary antibody dilution, then OmpA/MotB removes signal peptide The best secondary antibody dilution of albumen is 1:800 (table 3).

3 OmpA/MotB of table goes signal peptide protease labeling antibody working concentration to optimize

Best developing time optimization: each developing time is positive, negative serum is respectively provided with 5 repetitions, reads its average value, Colour developing is protected from light in 37 DEG C.Selecting the number of minutes corresponding to P/N value the maximum is best developing time, as shown in table 4, OmpA/MotB Signal peptide albumen is gone most preferably to develop the color for 10min.

4 OmpA/MotB of table goes the best developing time selection of signal peptide albumen

The selection of best coating condition: each coating condition sets 5 repetitions.P/N value the maximum is best coating condition, by Table 5 is it is found that it is 4 DEG C overnight (12 hours) that OmpA/MotB, which goes signal peptide albumen to be most preferably coated with condition,.

5 OmpA/MotB of table goes signal peptide albumen to be coated with condition optimizing

Best confining liquid, off-period: every kind of confining liquid sets 5 repetitions, closes in 37 DEG C, selects P/N value the maximum As best confining liquid and off-period, it is shown in Table 6 and table 7 in detail, it is 5% that OmpA/MotB, which removes the best confining liquid of signal peptide albumen, Skimmed milk power, best off-period 1h.

6 OmpA/MotB of table goes the best confining liquid selection of signal peptide albumen

7 OmpA/MotB of table goes signal peptide protein blocking selection of time

Best serum, enzyme labelled antibody incubation time: 8 repetitions are arranged in each time point, are incubated at 37 DEG C, select P/N Being worth the maximum and negative value junior is best serum, the best incubation time of enzyme labelled antibody, is shown in Table 8 and 9 in detail, as shown in Table 8, It is 2h that OmpA/MotB, which removes the best serum incubation time of signal peptide albumen, and as shown in Table 9, best secondary antibody incubation time is 0.5h.

8 OmpA/MotB of table goes signal peptide albumen serum incubation time to optimize

9 OmpA/MotB of table goes signal peptide albumen secondary antibody incubation time to optimize

2, positive threshold value determines

It goes signal peptide albumen optimum dilution degree to be coated with enzyme mark strip with OmpA/MotB, is reacted with optimum reaction condition, examined Survey 40 parts of negative serum OD_450/630nmValue calculates positive threshold value=mean value+3 × variance (positive threshold value=X+3 × SD).As a result see Table 14 takes mean value X+3 × SD as positive threshold value, obtain OmpA/MotB go signal peptide protein positive threshold value be 0.082+3 × 0.023=0.151.

14 OmpA/MotB of table removes 40 parts of negative serum OD of signal peptide albumen_450/630nmValue

3, ELISA method specific detection

(1) specific cross is tested: going signal peptide albumen as antigen using OmpA/MotB, with the ELISA method point of foundation It Jian Ce not E. coli isolated from ducks, Salmonella anatis, the swollen head septicaemia virus of duck, avian influenza virus (H5), duck plague virus and duck hepatitis Virus-positive serum, the setting Riemerellosis Anatipestifer positive, negative serum control.3 repetitions of experimental setup are averaged, and are as a result seen Table 10.

10 OmpA/MotB of table goes signal peptide protein ELISA method specific detection

As shown in Table 10, only the OD value of Riemerellosis Anatipestifer positive serum be greater than positive threshold value 0.151 and with negative blood The ratio of clear OD value is greater than 2.1, the positive serum OD value of remaining six kinds of cause of disease to be checked be respectively less than positive threshold value 0.151 and with yin Property serum ratio be respectively less than 2.1, show that the ELISA method established specificity is good.

(2) specific inhibition is tested: with the volume ratio (albumen: serum) of 10:1, Riemerellosis Anatipestifer serum 1 type is positive Property, negative serum go signal peptide albumen to mix with the OmpA/MotB of 1mg/mL to be placed on 37 DEG C of neutralization 1h, be then diluted to best Serum dilution blocks the OD of front and back serum with the ELISA method detection of foundation_450/630nmValue calculates blocking rate.It is positive, negative 8 repetitions are respectively set before serum blocks and after blocking, is averaged, the results are shown in Table 11.

11 OmpA/MotB of table removes signal peptide albumen blocking experiment

As shown in Table 11, it is 70.04% to RA-CH-1 positive serum blocking-up rate that OmpA/MotB, which removes signal peptide albumen, negative Serum blocking rate is 7.87%, shows that OmpA/MotB goes signal peptide albumen that can identify simultaneously with Riemerellosis Anatipestifer serum specificity It is neutralized.

4, ELISA method repeatability detects

With a batch and different batches purifying antigen coated elisa plate, positive blood known to 6 parts is detected with the ELISA method of foundation Clear OD_450/630nm6 repetitions are arranged in value, every part of serum, detect in its plate, the coefficient of variation between plate.Calculate coefficient of variation CV=mark Quasi- difference/average value (SD/M × 100%).It the results are shown in Table 12 and 13.

12 OmpA/MotB of table goes the coefficient of variation in signal peptide albumen plate

13 OmpA/MotB of table goes the coefficient of variation between signal peptide albumen plate

The result shows that OmpA/MotB goes in signal peptide albumen plate the coefficient of variation in 0.81%-4.77%, respectively less than 5% (table 12), the coefficient of variation is in 1.15%-5.39% between plate, respectively less than 10% (table 13), shows that the recombination OmpA/MotB established writes to The ELISA detection method repeatability of number peptide Protein Detection RA antibody is good.

5, ELISA method sensitivity Detection

Serum 1 type Riemerellosis Anatipestifer positive serum known to taking 6 parts, respectively since 1:100, with 2 times of doubling dilutions, With the ELISA method of foundation, positive greatest dilution can be detected for its sensitivity.Foundation is obtained from 15 data of table OmpA/MotB goes signal peptide albumen indirect ELISA method sensitivity are as follows: can still detect when positive serum is diluted to 1:6400.

15 OmpA/MotB of table removes signal peptide albumen indirect ELISA method sensitivity technique

3 rabbit-anti OmpA/MotB of embodiment goes the preparation and application of signal peptide recombinant protein hyper-immune serum

1, the preparation of immunogene

Signal peptide recombinant protein and Freund's complete adjuvant (FCA) or Freund are gone not with OmpA/M otB prepared by embodiment 1 Freund's complete adjuvant (FICA) mixes in equal volume, is placed on ice bath, until forming Water-In-Oil agent through ultrasonic wave effect emulsification.Check emulsification Effect standard, which will emulsify antigen drop, to be occurred complete in ice water and continues indiffusible round oil droplet and kept for 1 minute.

2, animal is immune

Rabbit is immunized by 16 immune programme of table, head exempts from the previous day and draws blood to rabbit, and separation serum is as yin Property serum control.

16 rabbit-anti OmpA/MotB of table goes signal peptide recombinant protein hyper-immune serum to prepare immune programme

3, the preparation and titration of hyper-immune serum

Four exempt from 10 days latter, arteria carotis bloodletting, collect blood in 37 DEG C of placement lh, 4 DEG C are precipitated serum, 5000r/min overnight It is centrifuged 10min and removes red blood cell, collect serum, packing, -20 DEG C of preservations or freeze-drying save.Serum is made, examination is spread with agarose Antibody titer is determined in test.As a result OmpA/MotB goes signal peptide recombinant protein rabbit anti-serum that can also observe under the dilution of 1:16 To white ribbon (Fig. 6).

4 OmpA/MotB of embodiment goes signal peptide recombinant protein to study the immune protective effect of duckling

1, test grouping and immune programme

1 age in days health Cherry Village Duckss 40 are only divided into 4 groups, after respectively OmpA/MotB removes signal peptide protein immunization Attack poison group Y1；RA-CH-1 inactivated vaccine is first immunized attacks poison group Y2 afterwards；RA attacks malicious control group C1, Normal group C2.Specific grouping And immune programme such as table 17.

17 animal packet of table and immune programme

2, duckling attacks malicious protection

Head exempts from rear 14d, attacks poison (10 with RA-CH-1⁸CFU/ is only) difference leg muscle injection Y1, Y2 and C1 each group duck, The physiological saline processing of C2 Normal group sterilizing.It observes and records and attacks condition of morbidity death in poison latter week.

The criterion of morbidity: meeting one of following condition, is judged to fall ill:

(1) there is lassitude in test duck, action is walked haltingly, eye nose has secretion outflow, arranges green loose stools；

(2) the duck infectious serositis clinical symptoms such as thin excrement of yellow green are even dead；

(3) the visible constrictive pericarditis of duck or liver week film inflammation or scrositis lesion or painstaking effort, liver, spleen separation of bacterial when dissect It is positive.

Attack poison after a week, not dead duck, which is cutd open, to be killed, and internal organs lesions situation is recorded, as long as there is fibrinous pericarditis, liver Zhou Yan, air bag it is scorching or in which one kind or riemerella anatipestifer separation it is positive, that is, be recorded as " lesion duck ".

Specific separation and identification method: morbidity dead duckling and not dead duck in dissection one week takes brain, liver, heart group It knits and carries out bacterium separation.It dips above-mentioned tissue inoculation with the oese that alcolhol burner burns red rear cooling to line on TSA plate, 37 DEG C After cultivating 18h, observation bacterium colony size, color and form.And picking single bacterium colony, expand culture, carries out plate agglutination test: Clean slide center is added dropwise aqua sterilisa 1 and drips, and the separation bacterium for dipping centrifuge washing with oese is a little, mixes with aqua sterilisa, drop Undiluted positive serum 1 is added to drip.Glass slide is vibrated or spreadable with oese repeatedly, observes result.Occur after several seconds clear The cotton-shaped agglutination block person of clear milky is determined as positive reaction.Reactor must be repeated 3 times aforesaid operations to reduce false sun Property.

16 ages in days inject RA-CH-1 10 to four groups of duck leg muscles⁸CFU/, it has been observed that: attack poison 48 hours Afterwards, it attacks malicious control group (C1) and begins with the phenomena of mortality.Malicious control group is attacked second day after attacking poison, occurs clinical condition one by one Shape, shows as that spirit is depressed, lie prostrate, circumference of eyes secretion increases, and dietary amount is reduced, and part duck draws green loose stools.With when Between growth, most of waddling gaits are unstable, and neck skew is trembled, the death mostly within 3-5 days.C1 group only has extremely individual ducks Son there are no any symptom.To the dead duck dissect of C1 group, dead early duck can not see apparent dissect lesion, evening death time The duck of (attacking after poison 4-7 days) is also shown " three is scorching " phenomenon through dissect.Then by the tissue of dead duck and organ (brain, the heart, Liver) respectively streak inoculation can be separated to homologous bacterium in cultivating for 24 hours on TSA culture medium.Poison is attacked after 7 days, C1 group is not dead 2 ducks living are slaughtered, and wherein 1 duck Glisson's capsule and pericardium thicken for dissect discovery, carry out RA-CH-1 to its brain, the heart, liver and divide From identification, RA-CH-1 bacterial strain is as a result all obtained；Before other 1 duck is slaughtered, mental status is good, there are no obvious clinical condition Shape, dissect are not shown any abnormalities yet, and are not also separated to Riemerellosis Anatipestifer from respective organization organ.Head attacked after exempting from malicious group (Y1, Y2) the time ratio that death occurs in duck attacks the slow of malicious control group (C1), and the death rate is obviously lower than attacking malicious control group (C1).Attack poison After 1 week, by it is each be first immunized to attack poison afterwards and organize duck living all slaughter, part duck is pericardium and liver envelope through dissect discovery major lesions It thickens, there is cellulosic exudation, can also be separated to homologous bacterium.Remaining most of duck can not be separated to duck without obvious lesion Silent Salmonella in epidemic disease.

Learn that the immune protective rate of Y1, Y2 group is respectively 80% and 90% from protest test result (table 18).

18 incidence of table and immune protective rate

From the above results, OmpA/MotB is right after going signal peptide albumen and RA-CH- commodity inactivated vaccine that duckling is immunized The immune protective rate of RA-CH-1 attack is respectively 80% and 90%, rather than immune duck 100% is fallen ill, i.e., OmpA/MotB removes signal Peptide albumen can be used as the genetic engineering subunit vaccine of prevention riemerella anatipestifer.

Finally, it is stated that preferred embodiment above is only used to illustrate the technical scheme of the present invention and not to limit it, although logical It crosses above preferred embodiment the present invention is described in detail, however, those skilled in the art should understand that, can be Various changes are made to it in form and in details, without departing from claims of the present invention limited range.

Claims (2)

1. Riemerellosis Anatipestifer OmpA/MotB goes signal peptide recombinant protein preparing Riemerellosis Anatipestifer gene engineered subunit epidemic disease Application in seedling or antibody test reagent；It is characterized by: preparing Riemerellosis Anatipestifer OmpA/MotB removes signal peptide recombinant protein Method include the following steps:

Engineering bacteria is taken to be inoculated in the LB culture medium containing Amp, with 150-200r/min, 37 DEG C of culture 12h or to OD_600nmIt is 0.6, Then it is added in the LB culture solution containing Amp for the ratio of 1:100 by volume and continues culture to OD for 37 DEG C_600nmUp to after 0.6, in 0.4mmol/L IPTG, 12h is induced under the conditions of 30 DEG C；Collect bacterium solution, centrifugation, by 1/10 bacteria liquid of the bacterial sediment of acquisition The Tris-HCl of 20 long-pending mmol/L, pH 8.0 suspends, and ice-water bath ultrasound cracks thallus, and then 12000r/min is centrifuged 10min, the 8mol/L urea liquid dissolution of 1/50 original bacteria liquid volume of precipitating, 8000r/min are centrifuged 10min, take lysate, add Enter 5 × SDS loading buffer to boil, SDS-PAGE separates recombinant protein, is 0.25%KCl pre-cooling dyeing with mass fraction After cut glue, blob of viscose of dialysing, until gained protein liquid is concentrated after blob of viscose is transparent to obtain the final product；It is that recombinant expression carrier is transferred to large intestine bar Bacterium BL21(DE3) in and obtain；The recombinant expression carrier is by nucleotide sequence OmpA/ as shown in SEQ ID NO.1 MotB goes signal peptide recombinant protein encoding gene to be cloned into prokaryotic expression plasmid pGEX-4T-1 and obtain.

2. using the polyclonal antibody that Riemerellosis Anatipestifer OmpA/MotB goes signal peptide recombinant protein to obtain as antigen-immunized animal, Include the following steps: it is characterized by: preparing the method that Riemerellosis Anatipestifer OmpA/MotB removes signal peptide recombinant protein

Engineering bacteria is taken to be inoculated in the LB culture medium containing Amp, with 150-200r/min, 37 DEG C of culture 12h or to OD_600nmIt is 0.6, Then it is added in the LB culture solution containing Amp for the ratio of 1:100 by volume and continues culture to OD for 37 DEG C_600nmUp to after 0.6, in 0.4mmol/L IPTG, 12h is induced under the conditions of 30 DEG C；Collect bacterium solution, centrifugation, by 1/10 bacteria liquid of the bacterial sediment of acquisition The Tris-HCl of 20 long-pending mmol/L, pH 8.0 suspends, and ice-water bath ultrasound cracks thallus, and then 12000r/min is centrifuged 10min, the 8mol/L urea liquid dissolution of 1/50 original bacteria liquid volume of precipitating, 8000r/min are centrifuged 10min, take lysate, add Enter 5 × SDS loading buffer to boil, SDS-PAGE separates recombinant protein, is 0.25%KCl pre-cooling dyeing with mass fraction After cut glue, blob of viscose of dialysing, until gained protein liquid is concentrated after blob of viscose is transparent to obtain the final product；It is that recombinant expression carrier is transferred to large intestine bar Bacterium BL21(DE3) in and obtain；The recombinant expression carrier is by nucleotide sequence OmpA/ as shown in SEQ ID NO.1 MotB goes signal peptide recombinant protein encoding gene to be cloned into prokaryotic expression plasmid pGEX-4T-1 and obtain.