CN104166000A - A method of indentifying brucella natural infection or immunifaction for livestock - Google Patents

Abstract

A method of indentifying brucella natural infection or immunifaction for livestock is provided. According to the method, Omp31 protein or recombinant Omp31 protein containing a His label is adopted as interspecific difference identification protein, and Bp26 protein or recombinant Bp26 protein containing a His label is adopted as positive control protein. For livestock positive to a brucella infection serum SAT, the Bp26 protein or the recombinant Bp26 protein containing the His label reacts with serums infected by different strains of brucella, while the Omp31 protein or the recombinant Omp31 protein containing the His label only reacts with serums infected by brucella except the bovine brucella, and therefore the livestock which is positive to the SAT is naturally infected or is subjected to immunifaction. Through Western Blot detection, the two kinds of protein can identify the bovine brucella immune serums and non-bovine brucella immune serums, and can identify natural infection and immunifaction of bovine. The Omp31 protein and the Bp26 protein can be used for indentifying nonspecific agglutination reactions of other microorganisms with a brucella SAT antigen.

Description

For the identification of the method for brucella natural infection or immunity inoculation domestic animal

Technical field

The present invention relates to proteomics, specifically, relate to a kind of method for the identification of brucella natural infection or immunity inoculation domestic animal.

Background technology

Brucella (Brucella is called for short cloth Salmonella) is a kind of facultative intracellular parasitic bacteria of Grain stain feminine gender, is the brucellosis pathogen of (Brucellosis is called for short cloth disease).Cloth disease is a kind of Amphixenosis, in China's law on the prevention and control of infectious diseases, belongs to Category B notifiable disease.In the world, cloth disease is classified as category-B infectious disease, worldwide has popularly, and particularly after 2000, the sick epidemic situation of cloth significantly rises, and brings serious Health cost and economic loss to the people.

At present, the goldstandard of brucella diagnosis is that bacterium separates cultivation, and still, brucella cultivation difficulty is large, and the cycle is long, and separation rate is low.The most frequently used Serology test is rose bengal precipitation test (RBPT), tube agglutination test (SAT), complement fixation test (CFT) (CFT) etc., in addition, by the coated ELISA detection method of brucella lipopolysaccharides (lipopolysaccharide, LPS) also progressively for the diagnosis of cloth disease.But it is due to vaccine immunity or natural infection that above method all cannot be distinguished tube agglutination positive serum.Along with the development of genomics and proteomics, many scientific research personnel are conceived to find the differential protein of brucella vaccine strain and street strain, and carried out large quantity research, but so far, do not find one can be from distinguishing in essence the Perfected process of vaccine strain and street strain.

The prevention and control of human world cloth disease are inseparable with the prevention and control of animal cloth disease, and data shows that the fashion trend of human world cloth disease conforms to the fashion trend of animal cloth disease.In China, ox and sheep are the sick main infections sources of cloth, therefore, control the sick epidemic situation of cloth of ox and sheep, are conducive to control the popular of human world cloth disease, and the economic loss that reduces the country and people is significant.One of major measure of the sick prevention of domestic animal cloth is the immunity inoculation of vaccine.But, because traditional detection method cannot be distinguished natural infection and the immunity inoculation of domestic animal, if it is immune domestic animal that sick cloth ill domestic animal is takeed for, can preserve the infection sources, people and healthy domestic animal are brought to great threat, otherwise, be that the sick epidemic disease of cloth is raiseeed and eliminated and slaughter if immune domestic animal is takeed for, can bring massive losses to country and individual economy.The particularly Niu Zuowei sick common transmittable of a kind of cloth source; and its economic worth is higher; so; in current epidemic situation extremely serious period; more needing a kind of method can distinguish ox is natural infection or immunity inoculation; and take accordingly the treatment measures of science, and farthest avoid unnecessary economic loss, differentiate the health of epidemic-infected animal protection people and other domestic animals simultaneously.

At present, the vaccine that China is widely used in ox is most M5 and S2, be respectively the brucellar attenuated live vaccines strain of sheep kind and pig kind, and the sick ox of natural infection is mainly B. abortus.Along with the development of brucella genome sequencing technology, there is report bp26 gene to be present in all brucella kinds, and quite conservative between planting, and omp31 gene is present in all brucella except ox kind, so, using Bp26 albumen as positive control albumen, and Omp31 albumen identifies albumen as interspecific difference, can effectively other brucella kinds such as B. abortus and sheep kind and pig kind be made a distinction from serological method, and then judge that the ox of the SAT positive is natural infection or immunity inoculation.

Summary of the invention

Mainly different according to the brucella kind of domestic animal immunity inoculation vaccine strain and natural infection of the present invention, by distinguishing brucellar interspecific difference, indirectly make a distinction the domestic animal of vaccine inoculation and natural infection.

In order to realize the object of the invention, the invention provides a kind of method for the identification of brucella natural infection or immunity inoculation domestic animal, it is to identify albumen using Omp31 albumen or containing the restructuring Omp31 albumen of His label as brucella interspecific difference, and using Bp26 albumen or containing the restructuring Bp26 albumen of His label as positive control albumen, the domestic animal of being positive for Infected with Brucella Serum SA T, Bp26 albumen or all react containing the restructuring Bp26 albumen of His label and Infected with Brucella serum not of the same race, and Omp31 albumen or only react with the Infected with Brucella serum of other kind except B. abortus containing the restructuring Omp31 albumen of His label, and then judge that the domestic animal that SAT is positive is brucella natural infection or immunity inoculation.

Wherein, described restructuring Omp31 albumen containing His label and the preparation method containing the restructuring Bp26 albumen of His label are:

1) (GenBank of bp26 gene is numbered AY166768.1 to obtain the gene order of bp26 and omp31 from NCBI, the GenBank of omp31 gene is numbered AF366063.1), design respectively primer, pcr amplification bp26 gene and omp31 gene;

2) respectively to step 1) amplified production carry out enzyme and cut, then bp26 genetic fragment is connected with the pET30a plasmid of cutting through same enzyme, omp31 genetic fragment is connected with the pET32a plasmid of cutting through same enzyme, respectively connection product is transformed into bacillus coli DH 5 alpha competent cell, screening positive clone;

3) by step 2) in the positive colony that screens be inoculated into respectively containing in corresponding antibiotic LB fluid nutrient medium, add derivant IPTG to induce, by centrifugal the bacterium liquid after induction, collect thalline, add the resuspended thalline of PBS liquid, centrifugal after ultrasonication, gained precipitation is dissolved with urea, and centrifugal rear collection supernatant, with the membrane filtration of aperture 0.45 μ m, filtrate is crossed His label protein purification column, obtains the recombinant protein containing His label.

Primer for pcr amplification bp26 gene comprises:

Upstream primer: 5 '-CCG gAATTCaTGAACACTCGTGCTAGCAA-3 ' and

Downstream primer: 5 '-CCG cTCGAGtTACTTGATTTCAAAAACGACAT-3 '.

Primer for pcr amplification omp31 gene comprises:

Upstream primer: 5 '-CCG gAATTCaTGAAATCCGTAATTTTGGC-3 ' and

Downstream primer: 5 '-CCG gAATTCaTGAAATCCGTAATTTTGGC-3 '.

Aforesaid method, utilizes detected by Western blot (Western Blot, abbreviation WB) to detect reacting between the specific antibody of anti-corresponding protein in albumen and Infected with Brucella serum.

The domestic animal relating in the present invention includes but not limited to ox.

The present invention also provides a kind of kit for the identification of brucella natural infection or immunity inoculation domestic animal, and it is with Omp31 albumen or containing the restructuring Omp31 albumen of His label, and Bp26 albumen or containing the restructuring Bp26 albumen of His label as envelope antigen.

The present invention has successfully built two kinds of objects and has identified the recombined pronucleus expression plasmid of albumen Omp31 and Bp26, and successfully expresses and be purified into two kinds of objects mark albumen.WB test findings shows, in the serum of M5 vaccine immunity, 6/7 shows as Bp26 and the equal reacting positive of Omp31, in S2 vaccine immunity serum, 5/6 shows as two kinds of albumino reaction positives, and in the serum of natural infection, 9/10 shows as Bp26 reacting positive, and Omp31 reaction negative.Detect by WB, proved that two kinds of albumen have the ability of distinguishing ox kind and non-B. abortus immune serum, also can distinguish natural infection and the vaccine immunity of ox; Omp31 and Bp26 albumen also can be used for differentiating the non-specific agglutinating reaction of other microorganisms and brucella SAT antigen.

Brief description of the drawings

Fig. 1 is two kinds of brucella gene PCR amplification figure in the embodiment of the present invention; Wherein, A is bp26 gene magnification figure: 1.DNA marker, 2.bp26; B is omp31 gene magnification figure: 1.DNA marker, 2.omp31.

Fig. 2 is that in the embodiment of the present invention, two kinds of recombinant plasmid enzymes are cut qualification figure; Wherein, 1.DNA Marker; 2.pET30a/bp26 recombinant plasmid enzyme is cut result; 3.pET32a/omp31 recombinant plasmid enzyme is cut result.

Fig. 3 is the expression of recombinant plasmid PET30a/bp26 in BL21 (DE3) in the embodiment of the present invention; In A: 1 albumen Marker; After BL21 (DE3) the competent cell induction of 2 unconverted plasmids; 3 do not insert aim sequence PET30a carrier is transformed into after the induction of BL21 (DE3) competent cell; 4 recombinant plasmid PET30a/bp26 are not induction in BL21 (DE3); After 5 recombinant plasmid transformed product inductions; The recombinant plasmid ultrasonication postprecipitation of 6 inductions; Supernatant after the recombinant plasmid ultrasonication of 7 inductions; In B: 1 albumen Marker; After BL21 (DE3) the competent cell induction of 2 unconverted plasmids; 3 do not insert aim sequence PET32a carrier is transformed into after the induction of BL21 (DE3) competent cell; 4 recombinant plasmid PET32a/omp31 are not induction in BL21 (DE3); After 5 recombinant plasmid transformed product inductions; The recombinant plasmid ultrasonication postprecipitation of 6 inductions; Supernatant after the recombinant plasmid ultrasonication of 7 inductions.

Fig. 4 is that in the embodiment of the present invention, different urea concentrations dissolve Bp26 recombinant protein inclusion body design sketch; In A: 1 albumen Marker; 2 not induction contrasts; Supernatant after 3 cracking; Supernatant after 4 2M urea dissolve; Supernatant after 5 4M urea dissolve; Supernatant after 6 6M urea dissolve; Supernatant after 7 8M urea dissolve; 8 dissolve postprecipitation; In B: 1 albumen Marker; 2 not induction contrasts; Supernatant after 3 cracking; Contrast after 4 inductions; Supernatant after 5 2M urea dissolve; Supernatant after 6 4M urea dissolve; Supernatant after 7 6M urea dissolve; Supernatant after 8 8M urea dissolve.

Fig. 5 is two kinds of recombinant proteins SDS-PAGE electrophoresis result after His label post purifying in the embodiment of the present invention; In A: 1 Marker; The recombinant protein of 2 inductions; Bacterium liquid ultrasonication postprecipitation after 3 inductions; Supernatant after the ultrasonication of bacterium liquid after 4 inductions; 5-8 eluent 1.-4.; In B: 1 Marker; 2-9 Omp31 albumen eluent 1.-8..

Fig. 6 is two kinds of protein antigenicity results of Western Blot checking in the embodiment of the present invention; Wherein, A, B, C are followed successively by blank, negative control, sheep kind 16M immune serum; Wherein, blank is to replace primary antibodie to hatch with PBS, and negative control is to hatch as primary antibodie from the healthy cow's serum of Pest-or disease-free area; In A-C, 1-3 represents albumen Marker, Bp26 and Omp31 successively.

Fig. 7 is that in the embodiment of the present invention, Western Blot detects known Serum Antibody result; Wherein, A, B, C are followed successively by the sick cow's serum of vaccine strain M5 immune serum, vaccine strain S2 immune serum, the infection of ox kind bacterium; In A-C, 1-3 represents albumen Marker, Bp26 and Omp31 successively.

Embodiment

Following examples are used for illustrating the present invention, but are not used for limiting the scope of the invention.If do not specialize, embodiment is all according to normal experiment condition, as Sambrook equimolecular cloning experimentation handbook (Sambrook J & Russell DW, Molecular cloning:a laboratory manual, 2001), or according to the condition of manufacturer instructions suggestion.

Embodiment is for the identification of the method for brucella natural infection or immunity inoculation domestic animal

Application state according to the poultry of current China with vaccine, the sick prevention approach of cows cloth is mainly inoculation No. 5 brucella vaccines of sheep (M5) and No. 2 brucella vaccines of pig (S2), and cattle infected is mainly B. abortus.So, different by Omp31 and the Bp26 existence of mark between not of the same race, can study in serological level brucella ox kind bacterium and Fei Niu kind bacterium are differentiated, be due to natural infection or vaccine inoculation thereby indirectly distinguish ox, provide necessary foundation for further it being carried out to antidiastole processing.

1. the clonal expression of brucella albumen bp26 and omp31 gene

The amplification of 1.1 genes of interest

1.1.1 design of primers is obtained the gene order of bp26 and omp31 from NCBI (GenBank of bp26 gene is numbered AY166768.1, the GenBank of omp31 gene is numbered AF366063.1), detect 2 genes from website and whether have signal peptide, it is 0 that 2 genes exist the possibility of signal peptide simultaneously.Then design restriction endonuclease sites, the present embodiment is selected EcoR I and Xho I, uses primer5 Software for Design primer, and the information of 2 expressing genes and primer are in table 1 and table 2.

The basic condition of two kinds of gene orders of table 1

Two kinds of brucella gene cloning and expression design primers of table 2

1.1.2 pcr amplification and product reclaim

(1) PCR20 μ L amplification system: HiFi Mix10 μ L, the each 0.5 μ L of upstream and downstream primer, template (16M) 0.5 μ L, ddH ₂o8.5 μ L.

PCR cycling condition: 95 DEG C of 5min; 95 DEG C of 1min, 62 DEG C of 1min, 72 DEG C of 2min, 30 circulations; 72 DEG C of 5min; PCR product is in 4 DEG C of preservations.

(2) the PCR product of amplification gained carries out 1.0% agarose gel electrophoresis, observations under gel imaging instrument.The PCR product of having identified is cut to glue purification, use gel to reclaim kit and reclaim.Pcr amplification the results are shown in Figure 1.

1.2 genes of interest connect T carrier

1.21 connect

10 μ L linked systems: PMD18-T carrier 1 μ L, PCR product 3 μ L, connect solution I 5 μ L, ddH ₂o1 μ L, spends the night in 16 DEG C of connections.

1.2.2 transform

Getting 10 μ L connection products is transformed in the bacillus coli DH 5 alpha competent cell of 50 μ L.Method for transformation: DH5 α competent cell is put in 10min on ice, treats that it melts, and 10 μ L are connected after products and 50 μ L bacillus coli DH 5 alpha competent cells mix and are positioned over 30min on ice.42 DEG C of heat shock 90s, are put in 90s on ice at once.The LB nutrient culture media that adds 500 μ L, mixes, and 37 DEG C, 220rpm shakes bacterium 45～60min, takes out 200 μ L bacterium liquid and is evenly coated on the flat board that contains resistance, 37 DEG C of overnight incubation.

1.2.3 positive colony qualification

Taking-up incubated overnight flat board, the random several monoclonals of picking, plant and preserve in new resistant panel, and on oese, residual bacterium is added to and in PCR system, does bacterium colony PCR qualification.Pcr amplification system: HiFi Mix10 μ L; The each 0.5 μ L of upstream and downstream primer; ddH ₂o9 μ L.Pcr amplification condition: 95 DEG C of 10min; 95 DEG C of 1min, 62 DEG C of 1min, 72 DEG C of 2min, 30 circulations; 72 DEG C of 5min; Preserve amplified production for 4 DEG C.Amplified production is carried out to 1% agarose gel electrophoresis, and whether observations under ultraviolet, amplified the object band of expecting.

Positive colony is inoculated into fresh containing in the fresh LB fluid nutrient medium of 0.1mg/ml Amp (kana), spends the night and shakes bacterium, extracts plasmid, taking plasmid as template, it is carried out to plasmid PCR qualification, and plasmid PCR system is identical with regular-PCR.Be accredited as positive clone and be labeled as T/bp26 and T/omp31.

Order-checking: by T/bp26 and the sub-plasmid of T/omp31 positive colony, deliver to Tian Yihuiyuan bio tech ltd, Beijing, utilize the order-checking of T carrier universal primer.Sequence after order-checking and original series are compared with blast, see and have or not sudden change, select without the clone of any sudden change and proceed follow-up test.

The structure of 1.3 recombinant plasmids

1.3.1 enzyme cuts back to close object fragment and expression plasmid

By T/bp26 and T/omp31; carry out double digestion with two kinds of restriction enzyme EcoR I and Xho I with expression vector kana resistance and Amp resistance reacts simultaneously; the enzyme system of cutting is 30 μ L systems: restructuring T vector plasmid or the each 20 μ L of expression vector, 10 × damping fluid H3 μ L, ddH ₂o5 μ L, the each 1 μ L of restriction enzyme EcoR I and Xho I.Enzyme tangent condition: 37 DEG C of 4h.Enzyme is cut product and is carried out 1% agarose gel electrophoresis enzyme analysis and cut effect, and under uviol lamp, is cutting object band, reclaims kit carry out nucleic acid recovery according to the ordinary gel of Tian Gen company.Fragment after recovery is carried out 1% agarose gel electrophoresis and is checked recovering effect.Deposit in-20 DEG C for subsequent use.

1.3.2 connect object fragment and expression vector

Genes of interest after cutting according to enzyme and its concentration ratio of expression vector band brightness guestimate, the mol ratio of cutting carrier according to the genes of interest fragment being inserted into and enzyme is 3-6:1, calculates its volume ratio, by the two connection.If the two band brightness is suitable, linked system is (10 μ L): genes of interest 6 μ L, expression vector 2 μ L, T4DNA ligase 1 μ L, T4DNA ligase damping fluid 1 μ L.Condition of contact: 16 DEG C, the connection of spending the night.

1.3.3 transform

Connection product is transformed into DH5 α competent cell: from-80 DEG C of ultra low temperature freezers, take out DH5 α competent cell and be put in 10min on young ice, treat its thawing, in competent cell, add connection product 10 μ L, rotating centrifugal pipe is to mix content gently, in ice bath, leave standstill 30min, centrifuge tube is placed in to 42 DEG C of water-baths and places heat shock 90s, then fast pipe is transferred to 90s in ice bath, this process is not shaken centrifuge tube.Then in centrifuge tube, add in the LB nutrient culture media of 500 μ L, mix and be placed on 37 DEG C of shaking table shaken cultivation 45min, object is to make tolerant gene expression relevant on plasmid, makes thalline recovery; Then centrifuge tube content is mixed, draw the competent cell that transformed of 100 μ L and be added to containing on corresponding antibiotic LB nutrient culture media, cell is evenly coated with and is opened with aseptic inoculation ring.Flat board is placed in to room temperature until liquid is absorbed, is inverted flat board, 37 DEG C of overnight incubation.

The screening and identification of 1.4 positive colony

The random some single bacterium colony kinds of picking in new resistant panel to preserve, on oese, residual bacterium is added to and in PCR system, does bacterium colony PCR qualification, and amplified production is carried out to 1% Ago-Gel and carry out electrophoresis, whether observations under ultraviolet, amplified the object band of expecting.

Be accredited as positive clone through bacterium colony PCR and be inoculated in the fresh LB fluid nutrient medium containing corresponding resistant, 220rpm, 37 DEG C are spent the night and shake bacterium, extract recombinant plasmid with the little extraction reagent kit of plasmid of Tian Gen company, carry out plasmid PCR qualification.Amplified production carries out electrophoresis with 1% Ago-Gel, and whether observations under ultraviolet amplifies the object band of expection.

Bacterium colony PCR is identified to positive clone is carried out plasmid PCR and double digestion is identified.

Plasmid PCR system: HiFi Mix10 μ L, the each 0.5 μ L of upstream and downstream primer, plasmid 1 μ L, ddH ₂o8.5 μ L.PCR cycling condition: 95 DEG C of 5min; 95 DEG C of 1min, 62 DEG C of 1min, 72 DEG C of 2min, 30 circulations; 72 DEG C of 5min;

Enzyme is cut system (10 μ L): recombinant plasmid 5 μ L, 10 × damping fluid H1 μ L, distilled water 3 μ L, the each 0.5 μ L of restriction enzyme EcoR I and Xho I.Enzyme tangent condition: 37 DEG C of 4h.

Whether plasmid PCR product and enzyme are cut product and are carried out 1% agarose gel electrophoresis, under ultraviolet light, observe and cut and obtained corresponding band at desired location enzyme.Qualification result as shown in Figure 2.

Recombinant plasmid transformed is arrived in BL21 (DE3) competent cell, get competent cell and be placed in ice bath, in competent cell, add recombinant plasmid 1 μ L, place on ice after 30min, then in centrifuge tube, add in the LB nutrient culture media of 800 μ L, mix and be placed on 37 DEG C of shaking table shaken cultivation 45min; Draw the competent cell that 100 μ L have transformed and be added to containing on corresponding antibiotic LB culture plate, cell is evenly coated with and is opened, 37 DEG C of overnight incubation with aseptic inoculation ring.Select single colony inoculation and contain in the LB fluid nutrient medium of corresponding resistant in 5mL, 37 DEG C, 200rpm shaking table spends the night, with the little extraction reagent kit extracting of plasmid plasmid, and plasmid PCR qualification.The recombinant plasmid that success builds is labeled as PET30a/bp26 and PET32a/omp31.

The induction of 1.5 recombinant plasmids and expression

Picking positive colony is inoculated in the LB fluid nutrient medium containing corresponding resistant, spends the night and shakes bacterium.Add 5ml to contain in the fresh LB fluid nutrient medium of corresponding resistant in the ratio of 1:100 the bacterium liquid spending the night, 37 DEG C, 220rpm shakes and is about at 0.6～1.0 o'clock to bacterium liquid OD value and adds derivant IPTG, and making its final concentration is 1.0mM.37 DEG C, 220rmp induces 4h.By the centrifugal 5min of bacterium liquid 12000rpm after induction, abandon supernatant, with PBS washing 2 times, precipitation suspends with 30 μ L PBS, adds 10 μ L4 × albumen sample-loading buffers to mix, and boils 8min, and SDS-PAGE electrophoresis is identified, observes expression of results.Choose the IPTG concentration of inducible protein amount maximum, after again bacterium liquid being induced, wash bacterium 2 times with the PBS of 0.01M, 200 μ L PBS suspendible thalline, ice-bath ultrasonic break process, ultrasonic 2s, intermittently 3s, altogether super 2min; 4 DEG C, centrifugal 5 minutes of 12000rpm, cleer and peaceful precipitation in separation, precipitation adds PBS to suspend, and gets 30 μ L precipitations or supernatant and mixes with 10 μ L4 × albumen sample-loading buffers, boils rear race SDS-PAGE electrophoresis, observations.SDS-PAGE the results are shown in Figure 3.

SDS-PAGE electrophoresis method:

First, preparation SDS-PAGE gel, resolving gel concentration 15%, concentrated gum concentration 5%, prepares SDS-PAGE electrophoresis; By the protein sample of handling well and protein standard substance--the albumen marker dying in advance adds in well in certain sequence, determines application of sample amount by protein content wherein; Voltage 80V, 30min, after bromophenol blue enters separation gel, changes 120V and continues electrophoresis 2h left and right; In the time that arriving apart from 1cm left and right, gel bottom, bromophenol blue stops.Take out gel, the Coomassie brilliant blue dye liquor that the is placed in 5 times of volumes 2h that dyes, then with the destainer 4h that decolours, visual inspection or with the imaging of gel imaging instrument.

Recombinant plasmid abduction delivering result: the size of two kinds of gene bp26 and omp31 expressing protein should be respectively 27.6kDa and 26.5kDa.The tag size of pET30a is 5kDa, and the label of pET32 is the fusion of a 18kDa, therefore Bp26 protein band should be positioned at 32kDa left and right in theory, and Omp31 protein band should be positioned at 44kDa left and right.Result demonstration, pET30a/bp26 recombinant clone and pET32a/omp31 recombinant clone all have albumen to be induced out at destination locations, after ultrasonic degradation, find that two kinds of recombinant proteins are all mainly present in precipitation with the form of inclusion body.

The purifying of 1.6 albumen

1.6.1 the optimization of the best urea concentration of solubilization of inclusion bodies

The microbionation that contains positive colony is entered to 10ml containing in the fresh LB fluid nutrient medium of corresponding resistant, carry out abduction delivering according to above method, after having induced, collect as stated above thalline, washing ultrasonication, centrifugal, collect respectively upper cleer and peaceful precipitation, precipitation is suspended in 2M urea liquid and is dissolved, place 30 minutes, 10000rpm is centrifugal, cleer and peaceful precipitation in collection, the precipitation of gained continues to dissolve with 4M urea, then successively by the precipitation 6M of gained last time, 8M urea liquid dissolves, collect supernatant and the last centrifugal precipitation of each centrifugal gained, carry out SDS-PAGE electrophoresis, determine the urea concentration the strongest to the dissolution of inclusion body, SDS-PAGE the results are shown in Figure 4.

By after the bacterium liquid ultrasonication after induction, precipitation is dissolved acquired results as shown in Figure 4 with the urea of 2M, 4M, 6M and 8M successively, under the dissolving of the urea of Cmax 8M, still there is Partial Protein stripping, the urea soluble protein that 8M is described is the most thorough, therefore selects 8M as the suitableeest urea concentration that dissolves inclusion body.

1.6.2 protein purification

The microbionation that contains positive colony is entered 600ml containing inducing in a large number in the LB fluid nutrient medium of corresponding resistant, and the centrifugal 30min of 8000rpm collects thalline, and PBS washes three times.Thalline is suspended in 50mlPBS to ice-bath ultrasonic fragmentation, ultrasonic 5s, 10 seconds, interval, 60W, ultrasonic 20min.The centrifugal 20min of 10000rpm.Gained precipitation is dissolved after 30min with 8M urea, and the centrifugal 20min of 10000rpm collects supernatant, and for subsequent use with the membrane filtration of 0.45 μ m.

Syringe hand push purifying: His label protein purification column (5ml) is crossed post with pure water and the binding buffer liquid of 5-10 column volume successively, cleans and balance pillar; Protein injection device hand push after filtering is crossed to post, and flow control is within 0.5-5ml/min scope; After completion of the sample, use at least binding buffer liquid of 5-10 column volume to cross post, washing away can not be with the foreign protein of pillar combination; Then use the elution buffer wash-out destination protein of 5-10 column volume, while collecting eluent, be in charge of collection, to ensure the concentration of albumen; After wash-out completes, crossing post with 10 volume elution buffers in addition, thoroughly wash away residual albumen, then cross post with the pure water of 10-15 column volume, finally cross post with 5 volume 20% ethanol, preserve pillar for subsequent use.

After BP26 albumen is crossed post purifying, eluent divides four pipes to collect, wherein 1., 2. tubulin content is more, concentration is suitable, 3., 4. tubulin is little, almost can ignore, collect liquid so discard this two pipe, merge 1., the 2. collection liquid of pipe, give over to follow-up test for subsequent use.After Omp31 protein purification, eluent divides 8 pipes to collect, and is designated as 1.-8. pipe, wherein 2., 3. tubulin content is more, merges for subsequent usely, discards other several pipes and collects liquid.

Will be in charge of each tubulin of collection run SDS-PAGE electrophoresis, contrast purification effect, and detect each tubulin concentration with Bradford method protein quantification kit, chooses the optimum albumen of concentration and carries out follow-up test, SDS-PAGE the results are shown in Figure 5.

2Western Blot analyzes

The antigenicity checking of 2.1 two kinds of destination proteins

The albumen obtaining after clonal expression and purifying carries out SDS-PAGE electrophoresis, after electrophoresis completes, ready NC film is soaked to 5min in transferring film liquid, with " sandwich method ", membrane-transferring device is installed, from top to bottom according to the order stationary installation of sponge, filter paper, SDS-PAGE glue, NC film, filter paper, sponge, note can not there be bubble between each layer.NC film is towards positive pole, and constant current 200mA wets and turns 2h.

NC film after transferring film, washs a little with PBS, then proceeds to confining liquid (PBS containing 0.5%Tween is made into 5% skimmed milk power), 4 DEG C of sealings of spending the night.

The primary antibodie sheep kind bacterium 16M immunity sheep serum dilution that NC film after sealing is placed in to 1:100 dilution, 37 DEG C, 50rpm slowly shake is hatched 2h, after having hatched, outwells primary antibodie dilution, and PBS washes film three times, each 10min.Add the two anti-dilutions (mouse-anti sheep/goat/ox IgG of HRP mark) of 1:30000 dilution, 37 DEG C, 50rpm slowly shake is hatched 2h, after having hatched, outwells two anti-dilutions, and PBS washes film three times, each 10min.After washing film, add nitrite ion DAB, slowly shake colour developing 1-2min, the reaction of adding distil water color development stopping.The WB of various immune serums the results are shown in Figure 6 and Fig. 7.

2.2 Western Blot methods detect serum sample

Serum sample is originated in Table 3-table 5.

Table 3 M5 immune serum

Table 4 S2 immune serum

Table 5 natural infection cow's serum

The testing result of blood serum sample: from the testing result of each blood serum sample, detect altogether 9 parts of M5 vaccine immunity serum, 10 parts of S2 vaccine immunity serum, the cow's serum of 10 parts of natural infections, in the serum of M5 immunity, have 7 parts of SAT test positive, wherein 6 parts of Bp26 and Omp31 are all positive, and 1 part shows as that the two is all negative, the western blot test of SAT negative serum, two kinds of albumen all show as feminine gender; In S2 immune serum, there is 6 parts of SAT test positive, 5 parts of Bp26 and Omp31 are wherein all positive, and in 4 parts of SAT negative serums, Bp26 and Omp31 are all negative.In 10 parts of cow's serums of natural infection, have 9 parts to occur expected results, Bp26 is positive and Omp31 is negative, and 1 part of Bp26 and Omp31 all show feminine gender.

The various brucella positive serum of table 6 Western Blot result

Be difficult to for natural infection ill domestic animal and vaccine immunity animal the problem of distinguishing, the analysis that research is before carried out differential protein from the wild toxic bacterial strain 16M of brucella melitensis and vaccine strains M5 is started with, result of study is found the phenomenon that expression rise has occurred or lowered in the mutation process of partial function albumen from street strain to vaccine strain, grow out of nothing or from having to unconverted albumen but fail to find to show, therefore, also just fail and find the differential protein with discriminating street strain and vaccine strain ability.

In the present invention, based on existing achievement in research, by consulting pertinent literature, on to the basis of brucella protein 2-D electrophoresis and mass spectrophotometry, grasp the characteristic of various albumen, in conjunction with the brucellar rule of cattle infected and strain characteristics, and state's intradermal vaccine behaviour in service, dividing the serum of the brucella positive from the angular area of application is that ox kind bacterium is also that non-ox kind bacterium infects the interspecific difference forming, and is that natural infection or vaccine immunity distinguish indirectly with this by ox.

Two kinds of expression vector pET30a and pET32a that the present invention is used all belong to pET series, and object fragment is inserted after this series expression vector, is subject to phage t7 to transcribe by force and translate (can select) signal controlling; The t7 rna polymerase being provided by host cell is provided induces.T7 rna polymerase mechanism is tool selectivity very effectively also: while fully induction, nearly all cell resource is all for expressing destination protein; After abduction delivering only several hours, destination protein can account for the more than 50% of total protein of cell conventionally.And under non-inductive condition, can make genes of interest not transcribe in silence state completely.PET30a is by T7 promoter, T7 terminator, and card is received resistant gene, lac controlling gene, His label gene and S label gene and each restriction enzyme site composition.

The restriction enzyme site that the present invention chooses is two kinds of EcoR I and Xho I, and therefore expression product should be the fusion with His label; PET32a is made up of T7 promoter, T7 terminator, ammonia benzyl resistant gene, lac controlling gene, His label (histidine-tagged) gene, Trx label (thioredoxin label) gene and S label gene and each restriction enzyme site, expression product between two restriction enzyme sites, inserts object fragment, so should be the fusion with above three kinds of labels.His label is protein purification label, and the albumen particularly existing with inclusion body form, after albumen can being dissolved under the condition of sex change completely, carries out affinity purification; Trx label can increase the dissolubility of expressing protein, and thioredoxin can drive the formation of disulfide bond, promotes the correct folding of albumen, increases its solubility.

In the present invention, initial by amplification omp31 and bp26 genes of interest, prokaryotic expression recombinant plasmid pET30a/bp26, pET30a/omp31 are built, in full accord with order-checking proof Insert Fragment and object fragment through double digestion, after IPTG induction e. coli bl21 (DE3) competent cell, successful expression has gone out Bp26 fusion, but pET30a/omp31 fails to induce albumen.Further the conditional parameters such as derivant concentration, inducing temperature, induction time are explored, successful expression goes out Omp31 fusion.

In two kinds of destination proteins of the present invention, Bp26 identifies albumen as positive control, and its antibody should be present in the serum of all brucella positives.From test findings, can find out, in 23 parts of tube agglutination test positive serums, there are 21 parts of serum all to produce antigen-antibody reaction with Bp26 albumen, coincidence rate is 91%, accuracy rate 90% this result that detects the sick antigen of cloth as antigen with the bp26 of bibliographical information conforms to, and has reached the object of expection.

Omp31 is as the identification mark albumen of interspecific difference, and ox kind bacterium infects the antibody that can not produce this albumen, but not all has its antibody in ox kind bacterium infection serum, has also verified in the present invention this theory.In the immune serum of 13 parts of non-ox kind bacterium, there are 11 parts all to produce immune band with Omp31 albumen, and in the cow's serum of 10 parts of natural infections, all do not have, with Omp31, specific antigen-antibody reaction occurs, prove that it has the ability of differentiating that ox kind bacterium and Fei Niu kind bacterium infect serum.

There is the animal blood serum of partial immunity to fail to produce the antibody of cloth disease, and SAT test is also negative, analyze reason, may be due to the immunization ways of each animal, individual difference and blood sampling time difference, after vaccine inoculation, can both there is antibody in not all animal, there is result of study to show, after S2 oral immunity milk cow 15d, can detect Brucella antibody, 30d peaks, after 45～90d, antibody titer and property rate present slow decreasing trend, and in different research, due to immunization method difference, the antibody titer difference that tube agglutination test detects is larger, from 5%～60% not etc.Therefore, the antibody horizontal that the induction of oral vaccination S2 strain brucella vaccine forms is lower, disappears slower.In addition, these domestic animals that do not produce antibody may be more because although it is inoculated according to normal dose, brucella vaccine inoculation is unsuccessful, does not produce corresponding Brucella antibody.In the present invention, immunization method has had no way of investigating, but two kinds of albumen of this part serum of SAT feminine gender are all negative, so do not affect conclusion.

Remove outside SAT negative serum do not consider, 6/7 M5 vaccine immunity positive serum, all there is expected results in 5/6 S2 immunity positive serum, all there is positive reaction band in Bp26 and Omp31; And in natural infection ox positive serum, 9/10 serum is the Bp26 positive and Omp31 negative reaction.So, obtaining by the western blot verification experimental verification of serum, Bp26 and Omp31 can more effectively distinguish the infection of ox kind bacterium and non-B. abortus.

In addition, in natural infection serum, the SAT test findings of No. 5 samples is positive, and being Bp26 and Omp31, Western Blot result all there is negative reaction, analyze its reason, this serum may not be the serum of Infected with Brucella, but the cross agglutination that the antigen of the antibody occurring after other infected by microbes and SAT occurs, thereby show as the SAT positive.Because the serum of the present invention's natural infection used is directly to pick up from infection animal, positive with its of current Positive judgement standards SAT test judgement, be not separated to pathogen.And SAT antigen used is somatic antigen,, likely there is cross agglutination with the serum of a lot of infected by microbes in its comparison of ingredients complexity.The present invention's antigen used is the peculiar two kinds of albumen of brucella, therefore, if can not there is not special antigen-antibody reaction with these two kinds of albumen in the antibody that other infected by microbes produce.As can be seen here, by the further detection test of Bp26 and Omp31, can get rid of the cross agglutination phenomenon of other microorganism and brucella SAT antigen, this point also needs further evidence.

In the present invention, adopt WB method to detect reacting of albumen and Serum Antibody, mainly consider that it is highly sensitive, be applicable to the detection of qualitative results.

Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, all belong to the scope of protection of present invention.

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Claims (7)

1. the method for the identification of brucella natural infection or immunity inoculation domestic animal, it is characterized in that, it is to identify albumen using Omp31 albumen or containing the restructuring Omp31 albumen of His label as brucella interspecific difference, and using Bp26 albumen or containing the restructuring Bp26 albumen of His label as positive control albumen, the domestic animal of being positive for Infected with Brucella Serum SA T, Bp26 albumen or all react containing the restructuring Bp26 albumen of His label and Infected with Brucella serum not of the same race, and Omp31 albumen or only react with the Infected with Brucella serum of other kind except B. abortus containing the restructuring Omp31 albumen of His label, and then judge that the domestic animal that SAT is positive is brucella natural infection or immunity inoculation.

2. method according to claim 1, is characterized in that, utilizes detected by Western blot to detect reacting between the specific antibody of anti-corresponding protein in albumen and Infected with Brucella serum.

3. method according to claim 1, is characterized in that, described restructuring Omp31 albumen containing His label and the preparation method containing the restructuring Bp26 albumen of His label are:

1) obtain the gene order of bp26 and omp31 from NCBI, design respectively primer, pcr amplification bp26 gene and omp31 gene;

2) respectively to step 1) amplified production carry out enzyme and cut, then bp26 genetic fragment is connected with the pET30a plasmid of cutting through same enzyme, omp31 genetic fragment is connected with the pET32a plasmid of cutting through same enzyme, respectively connection product is transformed into bacillus coli DH 5 alpha competent cell, screening positive clone;

3) by step 2) in the positive colony that screens be inoculated into respectively containing in corresponding antibiotic LB fluid nutrient medium, add derivant IPTG to induce, by centrifugal the bacterium liquid after induction, collect thalline, add the resuspended thalline of PBS liquid, centrifugal after ultrasonication, gained precipitation is dissolved with urea, and centrifugal rear collection supernatant, with the membrane filtration of aperture 0.45 μ m, filtrate is crossed His label protein purification column, obtains the recombinant protein containing His label.

4. method according to claim 3, is characterized in that, comprises for the primer of pcr amplification bp26 gene:

Upstream primer: 5 '-CCG gAATTCaTGAACACTCGTGCTAGCAA-3 ' and

Downstream primer: 5 '-CCG cTCGAGtTACTTGATTTCAAAAACGACAT-3 '.

5. method according to claim 3, is characterized in that, comprises for the primer of pcr amplification omp31 gene:

Upstream primer: 5 '-CCG gAATTCaTGAAATCCGTAATTTTGGC-3 ' and

Downstream primer: 5 '-CCG gAATTCaTGAAATCCGTAATTTTGGC-3 '.

6. according to the method described in claim 1-5 any one, it is characterized in that, described domestic animal includes but not limited to ox.

7. for the identification of the kit of brucella natural infection or immunity inoculation domestic animal, it is characterized in that, with Omp31 albumen or containing the restructuring Omp31 albumen of His label, and Bp26 albumen or containing the restructuring Bp26 albumen of His label as envelope antigen.