CN109142757A - A kind of brucella indirect ELISA testing kit - Google Patents

Abstract

The invention provides a Brucella indirect ELISA detection kit, which uses the Brucella wzt recombinant protein as a coating antigen. The kit of the invention can detect Brucella with high sensitivity and high specificity, and can be used for the diagnosis and scientific research of Brucella.

Description

A kind of brucella indirect ELISA testing kit

Technical field

The invention belongs to technique for gene engineerings and diagnostic reagent technical field, and in particular to a kind of brucella is indirect ELISA detection kit.

Background technique

Brucellosis is the zoonosis that one kind as caused by brucella can infect people and domestic animal, master in domestic animal Infected cattle, sheep, pig, dog are wanted, wherein the infection conditions of ox and sheep are the most serious, and animal suffering from disease can cause pregnant animal miscarriage, produce The symptoms such as milk decline, infertility, people's illness will appear the symptoms such as the tired, fever of arthralgia, whole body.Brucellosis is broadcast to Animal husbandry and human health bring very big harm, and the annual new infections number in the whole world is about 500,000 people, except a small number of flourishing It is national outer, with the presence of more than 170 a country's brucellosis.Infecting brucellosis is mainly and infected animal especially illness Animal pregnancy, unprocessed meat products or dairy products contact.

The pathogenic mechanism of brucella is their ability to survive in host's mononuclear macrophage, though hypoxemia, nutrition not Foot, acidic environment and there are remain to survive under conditions of oxide.And brucella is bacterial parasite intracellular, once infection is difficult To pass through antibiotic treatment.Even if infection animal self-healing still has the danger of lasting discharge of bacteria.

In terms of brucellosis diagnosis, ELISA kit have high sensitivity, high specificity, stability is good, operation is simple Single, result is easy to the advantages that determining.

Summary of the invention

The purpose of the present invention is to provide a kind of brucella indirect ELISA testing kits, can be used in brucella The qualitative detection of sick antibody has the advantages that high specificity, sensibility are high, reproducible, easily operated.

The present invention provides a kind of brucella indirect ELISA testing kit, and the kit is with brucella wzt recombination Albumen is envelope antigen.

Preferably, the amino acid sequence of the recombinant protein is SEQ ID No.2 in sequence table.

Preferably, the recombinant protein the preparation method comprises the following steps: by brucella wzt gene fragment clone to pET30a At the multiple cloning sites of (+) expression vector, recombinant plasmid pET-30a-wzt is obtained, recombinant plasmid pET-30a-wzt is transformed into Inducing expression is carried out in e. coli bl21 competent cell, is collected supernatant and is purified, obtains recombinant protein after purification.

Preferably, the kit further includes confining liquid, the confining liquid is Casein.

Preferably, the kit further includes coating buffer, the carbon that the coating buffer is 0.025M pH9.6 Phthalate buffer.

Preferably, the kit further includes serum dilution, the serum dilution is Casein.

Preferably, the kit further includes ELIAS secondary antibody, the ELIAS secondary antibody is horseradish peroxidase-labeled The anti-sheep IgG enzyme labelled antibody of mouse.

The present invention also provides application of the mentioned reagent box in detection brucella, the application not with the diagnosis of disease and For the purpose for the treatment of.

The present invention also provides the indirect ELISA detection method of above-mentioned brucella, the detection method not examining with disease For the purpose of disconnected and treatment, steps are as follows:

(1) coated elisa plate: by pET-30a-wzt recombinant protein described in claim any one of 1-3 after purification It is coated with microwell plate；Peridium concentration is 15 μ g/mL；Coating condition is that 4 DEG C of coatings are stayed overnight；

(2) ELISA Plate is closed；The confining liquid being closed as when 37 DEG C of closing 3h, closing is Casein；

(3) serum to be checked after dilution is added, in 37 DEG C of reaction 1h；It is described to be diluted to serum serum dilution to be checked It is diluted with 1:80；

(4) ELIAS secondary antibody after dilution is added, in 37 DEG C of reaction 30min or 90min；It is described to be diluted to use ELIAS secondary antibody ELIAS secondary antibody dilution is diluted with 1:5000；

(5) it develops the color, terminates, and read OD in microplate reader₄₅₀Nm value.

The detection brucella that kit of the invention can be high sensitive and height is special, it is anti-to can be used in brucellosis The qualitative detection of body has the advantages that high specificity, sensibility are high, reproducible, easily operated.It can also be used to brucella Diagnosis and scientific research.

Detailed description of the invention

Attached drawing is used to provide further understanding of the present invention, and constitutes part of specification, with reality of the invention It applies example to be used to explain the present invention together, not be construed as limiting the invention.In the accompanying drawings:

Fig. 1 is unpurified brucella wzt recombinant protein SDS-PAGE electrophoresis provided in an embodiment of the present invention.

Fig. 2 is the brucella wzt recombinant protein SDS-PAGE electrophoresis of purifying provided in an embodiment of the present invention.

Specific embodiment

Embodiment below facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments Method is unless otherwise specified conventional method.Test material as used in the following examples is unless otherwise specified certainly What routine biochemistry reagent shop was commercially available.

Embodiment 1

1, experiment reagent and instrument

Reagent: serum；Serum dilution, serum dilution are casein (SIGMA company)；The anti-sheep IgG- horseradish peroxide of mouse Compound enzyme conjugates；It is coated with buffer；Cleaning solution, cleaning solution are the phosphate buffer of 0.5 ‰ Tween-20 containing mass fraction； Block buffer is casein (SIGMA company)；Substrate developing solution is that every 500mL adds disodium ethylene diamine tetraacetate 0.2g, lemon Sour 0.95g, glycerol 50mL and TMB powder 0.15g；Terminate liquid is the sulfuric acid solution of 2mol/L.

Instrument: enzyme-linked immunosorbent assay instrument, single track pipettor, 8 Dao Huo, 12 pipettor, sample injector, board-washing machine etc..

Consumptive material: ELISA micro-reaction plate selects the micro ELISA Plate of 96 hole polystyrenes；Serum dilutes plate, suction pipette head Deng.

2, the preparation of brucella wzt recombinant protein indirect ELISA testing kit

The expression and purification of 2.1 brucella wzt recombinant proteins

Lipopolysaccharides is the Major Virulence Factors of brucella, to the integrality of the structure and function of Gram-negative bacteria outer membrane It is most important.Wherein, wzt gene is the chief component of abc transport system, while being the synthesis key gene of LPS again.It grinds Study carefully discovery, the missing of the gene will cause the biosynthesis block of LPS.Use individual gene wzt in LPS as antigen compared to LPS is that antigentic specificity can be higher, avoids cross reaction.

PCR amplification wzt genetic fragment first, then using EcoR I and SalI while digestion PCR product and expression vector PET-30a is attached after glue recycling target fragment and carrier, construction recombination plasmid, then by recombinant plasmid transformed to competence E. coli bl21 (DE3) pLys cell, through IPTG inducing expression, the condition of inducing expression are as follows: 25 DEG C of shaking table 180rpm inductions 5h takes supernatant to carry out SDS-PAGE electrophoretic analysis, and pre- as a result as shown in Figure 1, occurring specific band at about 30kDa Phase is consistent, and electrophoretic analysis result prompt destination protein is mainly expressed with soluble form.Recombinant protein through Ni-NTA after purification, it is pure Degree is up to 90% or more, as shown in Figure 2.The nucleotide sequence and amino acid sequence of recombinant protein are respectively as follows:

The amino acid sequence of recombinant protein are as follows:

MIQPSITLSNVHLHYAASAFKERSVKTLVNALLSMRRSAGANIEDIHALKGISVDIARGERVALIGHNG AGKSTFLKTIAGLYPISSGTLKVTGTVRSLFDIGLGFEPDATGRENILYRGLLLGLTPRFMREIEDEIIEFADLGDF IDYPIKTYSAGMQVRLAFAISTAVDGDILLLDEVIGAGDAAFMTKAKARIMNMVEKAEIMVLASHDLANVRQLCTRA LVFKAGTIAFDGRVEDAISFYNSGMGAIA

Wherein, PCR amplification wzt genetic fragment method particularly includes:

Design primer

Upstream primer:

5'-CCGGAATTCGCCACCATGATCCAGCCATCGATTACCCT-3'；

Downstream primer:

5'-CCGGTCGACTTAATGATGATGATGATGATGTGCTATAGCTCCCATTCCCG-3'；

PCR reaction system: each 2 μ L of 2 μ L of template, upstream and downstream primer, 25 μ L of Taq Mastermix enzyme, dd H₂O 19μL。

Wherein, Taq Mastermix enzyme is purchased from TaKaRa company.

Using brucella genome as the PCR reaction condition of template are as follows: 95 DEG C of initial denaturation 5min, 94 DEG C of denaturation 40s, 55 DEG C Anneal 40s, and 72 DEG C of extension 2min, 72 DEG C extend 10min eventually.

Wherein, the purification process of recombinant protein specifically:

Collect thallus, ultrasound after 4 DEG C, 8000rpm be centrifuged 45min, then use His Trap FF affinity chromatography column purification egg It is white, it is operated according to the step of balance, upper albumen, elution foreign protein, elution destination protein.Elute foreign protein and elution purpose Albumen is using imidazole solution, wherein the concentration for eluting the imidazole solution of foreign protein is 40mM, elutes the imidazoles of destination protein The concentration of solution is 250mM.

Fig. 1 is unpurified brucella wzt recombinant protein SDS-PAGE electrophoresis provided in an embodiment of the present invention；Figure In, M: Protein Marker；1,3: other albumen of expression；2: recombination wzt albumen, arrow meaning are recombinant protein.

Fig. 2 is the brucella wzt recombinant protein SDS-PAGE electrophoresis of purifying provided in an embodiment of the present invention；In figure, M: Protein Marker；1,2: elution albumen, arrow meaning are recombinant protein.

The determination of the best peridium concentration of 2.2 antigens

It is using recombinant protein wzt obtained in step 2.1 as envelope antigen, envelope antigen is dilute with coating buffer multiple proportions It is interpreted into 30 μ g/mL, 15 μ g/mL, 7.5 μ g/mL, 3.75 μ g/mL, 1.875 μ g/mL and 0.9375 μ g/mL, each hole of ELISA Plate adds Enter 100 μ L, i.e., each final antigen total amount in hole is followed successively by 3 holes μ g/, 1.5 holes μ g/, 0.75 hole μ g/, 0.375 hole μ g/, 0.1875 The hole μ g/ and 0.09375 hole μ g/.

With 96 hole elisa Plates are coated with after coating buffer dilution, 4 DEG C of coatings are gone to overnight after setting 37 DEG C of incubation 2h.With Cleaning solution is washed 3 times and is patted dry, and 200 μ L of confining liquid is added in 37 DEG C of closing 2h, washing.By 2 parts of negative serums (being denoted as N1 and N2) After being serially diluted respectively with serum dilution with 1:100 with 4 parts of positive serums (being denoted as P1, P2, P3 and P4), every hole is added 100 μ L, every part of serum are loaded twice under same antigen coat concentration conditions, i.e., multiple holes are loaded；Then 37 DEG C of reaction 1h, washing It pats dry for 3 times, 100 μ L ELIAS secondary antibody (extension rate 1:10000), 37 DEG C of reaction 1h are added in every hole；It washs 3 times and pats dry.TMB is added 100 hole μ L/ of substrate developing solution, 37 DEG C are protected from light 15min, and 2mol/L H then is added with the amount in 50 holes μ L/₂SO₄It terminates anti- It answers, sets enzyme mark detector measurement OD₄₅₀Nm value, measurement result are as shown in table 1 below.

1 antigen coat concentration screening result of table

By the experimental data measured in table 1, Computation immunity goat-anti serum (i.e. positive serum) is with respect to OD₄₅₀Nm value and yin Property control serum is with respect to OD₄₅₀Nm value, i.e. P/N value select relatively large 15 μ g/mL of antigen coat concentration (the 1.5 μ g/ of P/N value Hole) it is best peridium concentration.

The determination of 2.3 antigen coat optimum conditions

Brucella recombinant antigen wzt albumen coating is carried out according to the peridium concentration of 2.2 selections, coating plate is respectively placed in It is coated under 3 conditionals below: (1) 37 DEG C of 2h；(2) 4 DEG C overnight；It is stayed overnight for 4 DEG C after (3) 37 DEG C of 2h.Positive serum is sheep cloth Lu Shi Bacterium positive serum, negative serum is Brucella melitensis negative serum, right respectively by 2.2 method for coating and best peridium concentration It is carried out using the 2 parts of negative serums (being denoted as N1 and N2) and 4 parts of positive serums (being denoted as P1, P2, P3 and P4) of above-mentioned coating condition ELISA test, each sample are repeated 2 times, and measure each hole OD₄₅₀Nm value, measurement result are as shown in table 2 below.

2 antigen coat optimum condition of table

P/N value is calculated, as shown in Table 2, it is maximum that the P/N value measured overnight is closed under the conditions of 4 DEG C, therefore selects 4 DEG C to stay overnight It is closed as antigen coat optimum condition.

The determination of 2.4 serum optimum dilution degrees

4 parts of positive serums (P1, P2, P3 are diluted with dilution 1:25,1:50,1:100,1:200 respectively with serum dilution And P4) and 2 parts of negative serums (N1 and N2) be primary antibody.ELISA test is carried out according to 2.2 method, each sample repeats 2 It is secondary, measure each hole OD₄₅₀Nm value, measurement result are as shown in table 3.

The selection of the best serum dilution of table 3

Calculate P/N value, as shown in Table 3, the serum dilution of reacting hole when P/N value is larger as best operating condition, Determine that serum optimum dilution degree is 1:80 on this basis.

The selection of 2.5 confining liquids and sealing condition

(1) selection of confining liquid: select containing mass fraction be respectively 5% skimmed milk power, 10% skimmed milk power, 5%BSA, The PBS confining liquid of 10%BSA and from SIGMA company buy confining liquid (Casein) closed, according to 2.3 determine most Good sealing condition is closed, and the serum optimum diluting multiple determined by 2.4 is to 2 parts of negative serums (being denoted as N1 and N2) and 5 parts Positive serum (being denoted as P1, P2, P3, P4 and P5) dilution, each sample are repeated 2 times, other ELISA measuring methods are measured with 2.2 Each hole OD₄₅₀Nm value, measurement result are as shown in table 4.

The best confining liquid the selection result of table 4

P/N value is calculated, the results are shown in Table 4, it is known that the P/N value of the closed elisa plate of confining liquid Casein is maximum, determines that it is Best confining liquid.

(2) it the selection of best off-period: with most suitable antigen concentration coated elisa plate, is carried out with selected best confining liquid Closing, off-period are respectively 1h, 2h and 3h, and the serum optimum diluting multiple that then positive serum is determined by 2.4 dilutes, each Sample is repeated 2 times, other ELISA measuring methods are the same as 2.2.Measure each hole OD₄₅₀Nm value, measurement result are as shown in table 5.

The best off-period the selection result of table 5

Compare the OD of each group negative serum and positive serum₄₅₀Nm value calculates P/N value, as shown in Table 5 in 37 DEG C of temperature The P/N value that lower closing 3h is measured is maximum, therefore selects 37 DEG C of 3h for best sealing condition.

The determination of 2.6 blood serum sample optimum reacting times

On the basis of the optimum test condition measured above, the optimum reacting time of blood serum sample is screened, is added After entering serum, respectively in 37 DEG C of reactions 15min, 30min, 60min, 90min, by 2.2 method to 2 parts of negative serum (N1 And N2) and 5 parts of positive serums (P1, P2, P3, P4 and P5) carry out ELISA tests, each sample is repeated 3 times, and measures each hole OD₄₅₀Nm value, measurement result are as shown in table 6.

The screening of 6 blood serum sample optimum reacting time of table

Calculate P/N value, as shown in Table 6, the P/N value highest that seroreaction 60min is measured, it is thus determined that under the conditions of 37 DEG C Seroreaction Best Times are 60min.

2.7 enzymes mark conjugate optimum dilution degree and the determination in reaction time

(1) on the basis of the optimum test condition measured above, to enzyme label optimum dilution degree and the reaction time into Row screening.By enzyme labelled antibody by extension rate be respectively 1:5000,1:10000,1:15000 dilution, then in 37 DEG C respectively temperature 30min is educated, other operating procedures carry out ELISA test, each sample to 2 parts of negative serums, 5 parts of positive serums by 2.2 method It is repeated 3 times, measures each hole OD450nm value, measurement result is as shown in table 7.

7 enzyme of table marks the screening of conjugate optimum dilution degree

P/N value and average P/N value are calculated, table 7 shows enzyme label conjugate in 1:5000 dilution times, and P/N value reaches most Greatly, so 1:5000 is that enzyme marks conjugate optimum dilution degree.

(2) after being diluted according to enzyme conjugates optimum dilution degree screen in 2.7 (1), in 37 DEG C respectively incubation 30min, 60min, 90min carry out 2 parts of negative serums (N1, N2) and 5 parts of positive serums (P1, P2, P3, P4 and P5) by 2.2 method ELISA test, each sample are repeated 2 times, and measure each hole OD₄₅₀Nm value, measurement result are as shown in table 8.

The screening of 8 enzyme of table label conjugate optimum reacting time

Calculate negative average value, positive average value and average P/N value, as shown in Table 8 enzyme mark conjugate effect 30min when with P/N value difference is not little when 90min, to keep convenient experimental operation quick, therefore, when 30min being selected to act on for best enzyme labelled antibody Between.

The optimum reacting time of 2.8 substrates

On the basis of the optimum test condition measured above, enzyme labelled antibody by the aforementioned time effect optimized, washing, 5min, 10min, 15min and 30min, the method that other operating procedures press 2.2 are reacted respectively in 37 DEG C after substrate developing solution is added ELISA test is carried out to 6 parts of negative serums and 6 parts of positive serums, each sample is repeated 2 times, and measures each hole OD₄₅₀Nm value, measurement The results are shown in Table 9.

The optimum reacting time of 9 substrate of table screens

P/N value is calculated, the results are shown in Table 9, P/N value highest when substrate reactions 15min, therefore the selected 15min of selected substrate is Optimum reacting time.

The foundation of 2.9 brucella indirect ELISA testing kits

(1) ELISA Plate of pre-coated brucella wzt recombinant protein of the invention；

(2) it is coated with buffer: the carbonate buffer solution of 0.025M pH9.6: Na2CO3 0.4779g, NaHCO30.4620g, distilled water are settled to 100mL after completely dissolution；

(3) confining liquid: Casein (SIGMA company, article No. are as follows: B6429)；

(4) serum dilution: Casein (SIGMA company, article No. are as follows: B6429)；

(5) negative control: the healthy sheep blood serum for being uninfected by brucellosis；

(6) positive control: the sheep blood serum containing Brucella antibody；

(7) ELIAS secondary antibody: the anti-sheep IgG enzyme labelled antibody of the mouse of horseradish peroxidase-labeled；

(8) ELIAS secondary antibody dilution: Casein (SIGMA company, article No. are as follows: B6429)；

(9) substrate developing solution: every 500mL adds disodium ethylene diamine tetraacetate 0.2g, citric acid 0.95g, glycerol 50mL and TMB Powder 0.15g；

(10) terminate liquid: 2mol/L H₂SO₄Solution；

(11) cleaning solution: the phosphate buffer of 0.5 ‰ Tween-20 containing mass fraction, pH 7.4.

The foundation of the indirect ELISA method of 2.10 detection brucella

Using recombinant protein wzt of the invention as envelope antigen, 96 holes are coated with after being diluted to 15 μ g/mL with coating buffer 100 μ L are added in ELISA Plate, every hole, and 4 DEG C of coatings are overnight.It is washed 3 times with cleaning solution, each 2min is patted dry, and confining liquid is added, often 200 μ L are added in hole, in 37 DEG C of closing 3h.It is then washed 3 times with cleaning solution, each 2min.By serum to be checked (to serum to be checked Type does not require, for example can be sheep, ox, pig, dog or human serum etc.), negative control and positive control use serum respectively After dilution is diluted with volume ratio 1:80,100 μ L are added in every hole, and twice, i.e., multiple holes are loaded every part of serum sample-adding；Then 37 DEG C reaction 1h, washing pat dry for 3 times, every hole be added 100 μ L ELIAS secondary antibodies (use ELIAS secondary antibody diluted, extension rate 1: 5000) 37 DEG C of reaction 30min；It washs 3 times and pats dry.100 hole μ L/ of tmb substrate developing solution is added, 37 DEG C are protected from light 15min, so 2mol/L H is added with the amount in 50 holes μ L/ afterwards₂SO₄Reaction is terminated, enzyme mark detector measurement OD is set₄₅₀Nm value.

The performance test of the brucella indirect ELISA testing kit of the invention of embodiment 2

The determination of 1.1 critical values

Taking 24 parts of negative sheep blood serums by extension rate is that 1:100 dilutes, and indirect ELISA is carried out under the optimum condition of screening Measurement carries out statistical analysis to testing result, obtains OD₄₅₀Average value (X) and standard deviation (SD).It will When, it is judged to the positive；When, it is judged to feminine gender.

10 24 parts of brucella negative serums of table

Result judgement: the condition for testing establishment is that Positive control wells are averaged OD₄₅₀Value >=0.3 nm, negative control hole are average OD₄₅₀Nm value must < 0.3；As sample OD₄₅₀Value >=0.3 nm, is judged to the positive；Sample OD₄₅₀When nm value < 0.3, it is judged to feminine gender.

1.2 susceptibility results

11 sensitivity tests result of table

When P/N value is more than or equal to 2.0, corresponding serum diluting multiple is highest dilution, highest dilution 1: 1600。

1.3 specific

Yersinia enterocoltitica serum, Escherichia coli serum, three parts of brucella positive serums, three parts of brucella are taken respectively Negative serum is detected according to the dilution screened with the indirect ELISA condition optimized, and every part of serum does 2 in parallel A repetition, observes whether each serum and system have cross reaction, and test result is as shown in table 12.

12 specific test result of table

As shown in Table 12, with recombination wzt albumen coated elisa plate of the invention, positive control average value OD₄₅₀Nm value is 0.844, negative control is averaged OD₄₅₀Nm value is 0.266, with Yersinia enterocoltitica serum, Escherichia coli seroreaction, OD₄₅₀nm Value is respectively 0.179 and 0.101, lower than negative control.Illustrate that the kit has good specificity.

1.4 repeated

Repetitive test in batch:

5 parts of positive serums (being denoted as P1~P5) are taken to carry out indirect ELISA measurement, every part of serum under conditions of above-mentioned optimization Sample does 10 repetitions in parallel, calculates the coefficient of variation (coefficient of variance, CV) as follows: CV=[sample This OD₄₅₀It is worth standard deviation (S)/sample OD₄₅₀It is worth average] × 100%, test result is as shown in table 13.

13 batches, table interior repetitive test results

Test result is as shown in table 13, elisa plate is coated with the recombination wzt albumen of the invention of same a batch preparation, to 5 kinds Sample is detected, and every kind of sample repeats 10 holes, and the coefficient of variation mean value that test is repeated in batch is 3.62%~5.35%, is lower than 10%, show that the ELISA detection method has repeatability in good batch.

Repetitive test between batch

The recombination wzt albumen coating of the invention for taking 3 parts of different batches, in the case where selecting optimal conditions to 5 kinds of serum into The measurement of row indirect ELISA.Every kind of sample is repeated 3 times, and is averaged, and is observed its CV value with batch method of interior repetitive test, is tried It is as shown in table 14 to test result.

14 batches of repetitive test results of table

Test result is shown in Table 14, is detected with the albumen of 3 different batches coating plate to 5 kinds of samples, every kind of serum weight Again twice, the OD that 5 kinds of serum are measured in 3 batch elisa plates₄₅₀The nm value coefficient of variation is 2.36%~4.59%, is lower than 10%, Show repeated between the ELISA detection method has good batch.

Finally, it should be noted that the foregoing is only a preferred embodiment of the present invention, it is not intended to restrict the invention, Although the present invention is described in detail referring to the foregoing embodiments, for those skilled in the art, still may be used To modify the technical solutions described in the foregoing embodiments or equivalent replacement of some of the technical features. All within the spirits and principles of the present invention, any modification, equivalent replacement, improvement and so on should be included in of the invention Within protection scope.

Sequence table

<110>Lanzhou Veterinary Inst., Chinese Acedemy of Agaricultural Sciences

<120>a kind of brucella indirect ELISA testing kit

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 759

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 1

atgatccagc catcgattac cctgtcaaat gttcatctgc actacgctgc atcagcgttc 60

aaagaacgct cagtcaaaac tctagtaaac gccttattga gtatgcggcg cagcgcagga 120

gcaaacattg aagacatcca tgccctaaag ggtatttctg tagatatagc gcggggcgaa 180

cgcgttgccc tgataggtca caacggggct ggcaaaagta cgttcttgaa aactatagcc 240

ggtctctacc ctatatcatc tgggacatta aaagtgacgg gtaccgtaag atccctgttc 300

gatattggtc ttgggtttga gcctgatgca actggccgtg agaatattct ttaccgtggg 360

ttgcttctcg gactaacgcc acgtttcatg cgagagatcg aggatgagat catcgagttc 420

gcggatctcg gcgattttat cgattatcca atcaaaactt attctgccgg catgcaagtt 480

cggctcgcct tcgcgatttc gacagcagtc gacggcgaca tactccttct agacgaagtt 540

ataggtgcag gtgatgcggc attcatgact aaggcgaagg cccgcataat gaatatggtc 600

gagaaggctg agataatggt tctagcaagc catgaccttg cgaacgtccg tcagctttgc 660

acacgagcat tggttttcaa agccggcaca attgcatttg atggcagggt agaagacgcg 720

atttccttct ataactcggg aatgggagct atagcatga 759

<210> 2

<211> 252

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 2

Met Ile Gln Pro Ser Ile Thr Leu Ser Asn Val His Leu His Tyr Ala

1 5 10 15

Ala Ser Ala Phe Lys Glu Arg Ser Val Lys Thr Leu Val Asn Ala Leu

20 25 30

Leu Ser Met Arg Arg Ser Ala Gly Ala Asn Ile Glu Asp Ile His Ala

35 40 45

Leu Lys Gly Ile Ser Val Asp Ile Ala Arg Gly Glu Arg Val Ala Leu

50 55 60

Ile Gly His Asn Gly Ala Gly Lys Ser Thr Phe Leu Lys Thr Ile Ala

65 70 75 80

Gly Leu Tyr Pro Ile Ser Ser Gly Thr Leu Lys Val Thr Gly Thr Val

85 90 95

Arg Ser Leu Phe Asp Ile Gly Leu Gly Phe Glu Pro Asp Ala Thr Gly

100 105 110

Arg Glu Asn Ile Leu Tyr Arg Gly Leu Leu Leu Gly Leu Thr Pro Arg

115 120 125

Phe Met Arg Glu Ile Glu Asp Glu Ile Ile Glu Phe Ala Asp Leu Gly

130 135 140

Asp Phe Ile Asp Tyr Pro Ile Lys Thr Tyr Ser Ala Gly Met Gln Val

145 150 155 160

Arg Leu Ala Phe Ala Ile Ser Thr Ala Val Asp Gly Asp Ile Leu Leu

165 170 175

Leu Asp Glu Val Ile Gly Ala Gly Asp Ala Ala Phe Met Thr Lys Ala

180 185 190

Lys Ala Arg Ile Met Asn Met Val Glu Lys Ala Glu Ile Met Val Leu

195 200 205

Ala Ser His Asp Leu Ala Asn Val Arg Gln Leu Cys Thr Arg Ala Leu

210 215 220

Val Phe Lys Ala Gly Thr Ile Ala Phe Asp Gly Arg Val Glu Asp Ala

225 230 235 240

Ile Ser Phe Tyr Asn Ser Gly Met Gly Ala Ile Ala

245 250

Claims (9)

1. a kind of brucella indirect ELISA testing kit, it is characterised in that: the kit is with brucella wzt recombination Albumen is envelope antigen.

2. a kind of brucella indirect ELISA testing kit according to claim 1, it is characterised in that: the recombination The amino acid sequence of albumen is SEQ ID No.2 in sequence table.

3. a kind of brucella indirect ELISA testing kit according to claim 1, it is characterised in that: the recombination Albumen the preparation method comprises the following steps: by the multiple cloning sites of brucella wzt gene fragment clone to pET30a (+) expression vector, Obtain recombinant plasmid pET-30a-wzt, by recombinant plasmid pET-30a-wzt be transformed into e. coli bl21 competent cell into Row inducing expression is collected supernatant and is purified, obtains recombinant protein after purification.

4. a kind of brucella indirect ELISA testing kit according to claim 1, it is characterised in that: the reagent Box further includes confining liquid, and the confining liquid is Casein.

5. a kind of brucella indirect ELISA testing kit according to claim 1, it is characterised in that: the reagent Box further includes coating buffer, the carbonate buffer solution that the coating buffer is 0.025M pH9.6.

6. a kind of brucella indirect ELISA testing kit according to claim 1, it is characterised in that: the reagent Box further includes serum dilution, and the serum dilution is Casein.

7. a kind of brucella indirect ELISA testing kit according to claim 1, it is characterised in that: the reagent Box further includes ELIAS secondary antibody, and the ELIAS secondary antibody is the anti-sheep IgG enzyme labelled antibody of mouse of horseradish peroxidase-labeled.

8. application of the described in any item kits of claim 1-7 in detection brucella, the application is not with disease For the purpose of diagnosing and treating.

9. the indirect ELISA detection method of brucella, the detection method is special not for the purpose of the diagnosing and treating of disease Sign is: steps are as follows:

(1) coated elisa plate: pET-30a-wzt recombinant protein described in claim any one of 1-3 after purification is coated with Microwell plate；Peridium concentration is 15 μ g/mL；Coating condition is that 4 DEG C of coatings are stayed overnight；

(2) ELISA Plate is closed；The confining liquid being closed as when 37 DEG C of closing 3h, closing is Casein；

(3) serum to be checked after dilution is added, in 37 DEG C of reaction 1h；It is described to be diluted to serum serum dilution to be checked with 1: 80 are diluted；

(4) ELIAS secondary antibody after dilution is added, in 37 DEG C of reaction 30min or 90min；It is described to be diluted to ELIAS secondary antibody enzyme mark Secondary antibody diluent is diluted with 1:5000；

(5) it develops the color, terminates, and read OD in microplate reader₄₅₀Nm value.