CN1982439A - Reagent kit for inspecting bovine antibody and its use - Google Patents

Abstract

Reagent kit for inspecting bovine tuberculosis and its production are disclosed. The reagent kit consists of kit, 96-hole reactive board with v-shaped bottom, bovine tuberculosis mycobacterium infectious standard positive blood serum, health bovine negative blood serum, serum diluent and 1% allergic sheep red blood cell suspension. The allergic sheep red blood cell suspension is obtained by bovine mycobacterium specific secretory protein MPB70 expressed by colon bacillus. It has excellent specificity and sensitivity. It can be used to inspect bovine tuberculosis antibody.

Description

A kind of test kit and application that is applicable to that bovine tuberculosis antibody detects

Technical field

The present invention relates to the animal bacteria field that learns a skill.Be specifically related to a kind of test kit and application thereof that is applicable to that bovine tuberculosis antibody detects.

Background technology

Bovine tuberculosis is a kind of chronic expendable zoonosis that is caused by Mycobacterium bovis, and (OIE) is decided to be category-B zoonosis by the International Animal Health tissue.The part statistics of calendar year 2001 and 2002 shows that the indivedual provinces and cities of China bovine tuberculosis positive rate is up to more than 10%.The anti-system of bovine tuberculosis mainly is to adopt the Niu Tichun tuberculin to carry out the transformation reactions quarantine, and the positive ox that detects is isolated or slaughters, and stops pathophoresis from the source.But because Niu Tichun tuberculin complicated component, its specificity is influenced by other non-virulent mycobacteriums and false positive occurs, and susceptibility is also lower simultaneously, and its diagnostic result confidence level is not high, and the implementation result of " quarantining-slaughter " policy is had a greatly reduced quality.The research tendency of diagnostic reagent is to use the antigen protein of definite ingredients to replace PPD to carry out diagnosing bovine tuberculosis (Pollock JM etc., Tuberculosis, 2001,81 (1/2): 65-69) in the world at present.MPB70 is a Mycobacterium bovis specific secretion protein, because of its relative mobility is 0.7 (the Infect Immun such as Harboe M that gains the name, 1986,52 (1): 293-302), secretory volume accounts for virulence Mycobacterium bovis culture medium protein total amount 10%, is body fluid and cellular immunization target antigen (Harboe M etc., Am Rev Resp Dis important in the Mycobacterium bovis course of infection, 1984,129:444-452) also be one of the main component of PPD.MPB70 is obtained by people such as Nagai purifying from the substratum of BCG Tokyo 172 strains at first, and recording molecular weight by SDS-PAGE is 18kD (Infect Immun such as Nagai S, 1981,31 (3): 1152-1160.).Its gene order 1989 is determined, infer that its protein sequence has to contain 30 amino acid whose signal peptides, mature protein has 163 amino acid (Terasaka K etc., Complete nucleotide sequence of immunogenic protein MPB70 fromMycobacterium bovis BCG, FEMS Microbiol Lett, 1989,49 (2-3): 273-276 has thermostability, and iso-electric point is 4.8.People such as Hewinson use MPB70 self initiator codon to carry out the heterogenous expression of MPB70 for the first time in intestinal bacteria, the result has observed the MPB70 protein of 26kD and two kinds of molecular weight of 22kD, 22kD albumen is present in the cell pericentral siphon in a large number, therefore infer that 26kD is that signal peptide is not sheared product (Hewinson RG, Deng J Gen Microbiol, 1993,139 (6): 1253-1259).All do not find existing or trace expression (the Miura K etc. only of MPB70 in other inferior strains outside the inferior strain of BCG Tokyo and Moreau, Infect Immun, 1983,39 (2): 540-545), also trace expression (Wiker HG etc. only in mycobacterium tuberculosis, J.Immunol, 1996,43:374-380).To discovering of isolated 49 strain environment mycobacteriums in the positive ox of PPD intracutaneous transformation reactions, comprise that none contains MPB70 protein (Hughes MS etc. in the various mycobacteriums of avain tuberculosis, paratuberculosis, Veterinary Microbiology, 2005,108:101-112).The difference of MPB70 expression amount between each mycobacterium strain mainly is because a point mutation of the Sigma factor (SigK gene) initiator codon causes (Charlet D etc., Molecular Microbiology, 2005,56 (5): 1302-1313).

In view of the characteristic of mycobacterium bovis BCG specific secretion albumen mpb70, utilize this albumen to develop quick, accurate, safe, cheap diagnostic kit, will provide effective tool for the anti-system and the elimination of bovine tuberculosis.

Summary of the invention

The object of the invention is to overcome the deficiencies in the prior art, prepares a kind of test kit and application thereof that is applicable to that bovine tuberculosis antibody detects.The present invention is by setting up the indirect hemagglutination method, prepare a specific specificity good, highly sensitive, detect the test kit of the antibody of bovine tuberculosis quickly and accurately, for the anti-system of China's bovine tuberculosis provides a kind of novel agent box and novel method.

The present invention is achieved through the following technical solutions:

The present invention is by setting up the indirect hemagglutination method, prepared a kind of test kit that is applicable to that bovine tuberculosis antibody detects, this test kit comprises box body, be located at 96 hole Sptting plates at the bottom of the intravital V-arrangement of box, bovine tuberculosis mycobacterium infectious standard positive serum, healthy ox negative serum, serum dilution and 1% sensitization sheep red blood cell (SRBC) suspension.Wherein bovine tuberculosis mycobacterium infectious standard positive serum (diagnostic antigen) is to utilize the proteic recombination bacillus coli PHVO14Escherichia of Mycobacterium bovis MPB70 coliPHVO14, be deposited in CCTCC, deposit number: CCTCC-M205135 obtains.

1% sensitization sheep red blood cell (SRBC) suspension wherein is that it prepares by following method with the red corpuscle of Mycobacterium bovis specific antigen protein MPB70 sensitization: with adding isopyknic antigen protein MPB70 in the red cell suspension, through water-bath, stir; The centrifugal supernatant of abandoning is with PBS washing 2 times, again with the PBS washing that contains 1% rabbit anteserum once, be made into 1% sensitization sheep red blood cell (SRBC) suspension, add 0.01% Thiomersalate, put 2-8 ℃ of preservation after the packing, standby, promptly obtain 1% sensitization sheep red blood cell (SRBC) suspension of the present invention.

(1) comprise the clone of mycobacterium bovis BCG specific secretion albumen mpb70 gene and the preparation method who expresses in recombination bacillus coli, it comprises the following steps:

(1) with mycobacterium bovis BCG type strain (Mycobacterium bovis AF2122/97, the gene order of this bacterial strain has been logined Genebank, accession number is: NC002945) bacterial cultures adds a small amount of distilled water and boils 10min cracking thalline, get supernatant liquor after centrifugal as pcr template, obtain the cDNA sequence of mycobacterium bovis BCG mpb70 gene fragment by the PCR method amplification; The directed cDNA sequence of inserting mycobacterium bovis BCG mpb70 gene fragment in the EcoRI of prokaryotic expression carrier pET-28a and HindIII multiple clone site makes up and obtains prokaryotic expression carrier pET-28a-mpb70;

(2) the said prokaryotic expression carrier pET-28a-mpb70 of step (1) is converted into the e. coli bl21 competent cell,, induces this bacterial strain, obtain specifically expressing mycobacterium bovis BCG mpb70 albumen with the single reorganization bacterium of kalamycin resistance screening;

(3) the expressed MPB70 albumen of purification step (2) obtains required diagnostic antigen.

(2) concrete steps of mycobacterium bovis BCG specific secretion albumen mpb70 expression and purification:

Enzyme is cut the recombinant plasmid transformed intestinal bacteria BL correct with Sequence Identification ₂₁DE3 (available from Stratagene company) strain, the positive single bacterium colony on the kalamycin resistance plate of picking 50 μ g/mL adds in the LB substratum contain kalamycin resistance and is cultured to logarithmic phase (OD at 37 ℃ of following thermal agitations ₆₀₀=0.6-0.8), in substratum, add IPTG (sec.-propyl-β-D thiogalactoside) and carry out abduction delivering, two IPTG concentration gradients of 0.4mM and 0.8mM are set.37 ℃ are continued shaking culture, inducing back the 0th respectively, 1,2, got the 1mL bacterial cultures in 3 hours, the centrifugal collection thalline of 12000r/min, phosphate buffered saline buffer (PBS) rinsing precipitation is also resuspended with 40uL, add 50uL 2 * SDS sample-loading buffer and 10uL dithiothreitol (DTT) (DTT), 100 ℃ are boiled 10min, get a certain amount of SDS-PAGE of carrying out of 20uL electrophoretic analysis (Sa nurse Brooker J, Ritchie EF not, Manny A Disi T edits (Jin Dongyan, Li Mengfeng etc. translate). the molecular cloning experiment guide. and second edition, Beijing, Science Press, 1992 editions), observation analysis exogenous gene expression situation.The inductive thalline is through centrifugal, ultrasonic disruption (Sa nurse Brooker J, Ritchie EF not, Manny A Disi T edits (Jin Dongyan, Li Mengfeng etc. translate). the molecular cloning experiment guide. second edition, Beijing, Science Press, 1992 editions) back extracts inclusion body, and purified, sex change, renaturation handle that to be sub-packed in-20 ℃ of preservations after (referring to embodiment) standby.

(3) application of bovine tuberculosis antibody test kit:

(1) utilize above-mentioned mycobacterium bovis BCG specific secretion albumen mpb70 albumen to set up indirect hemagglutination test;

(2) prepare the antibody assay kit that is used for bovine tuberculosis with the said method of step (1).

More detailed technical scheme is as described below:

(1) the Mycobacterium bovis type strain (Mycobacterium bovis AF2122/97 is so kind as to give by the Guo Mingxing researcher of Hubei Entry-Exit Inspection and Quarantine Bureau, and the gene order of this bacterial strain has been logined Genebank,, accession number is: NC002945).The present invention is a template with Mycobacterium bovis type strain (Mycobacterium bovis AF2122/97) genome, obtain the cDNA of mpb70 gene by pcr amplification, determine that through sequencing analysis the gene order that the cDNA sequence of this mpb70 gene and Genebank announce compares, prove the gene order that obtains as the gene order size unanimity of expection referring to accompanying drawing 2.

(2) structure of prokaryotic expression carrier pET-28a-mpb70: the EcoRI and the HindIII multiple clone site that the cDNA sequence orientation of mycobacterium bovis BCG specific secretion albumen mpb70 are inserted into prokaryotic expression carrier pET-28a (available from invitrogen company).

(3) structure of recombination bacillus coli Escherichia coli BL21/pET-28a-mpb70: the described prokaryotic expression carrier of step (2) is converted into the e. coli bl21 competent cell, obtains positive recombinant bacterial strain through the kalamycin resistance screening.This bacterial strain is deposited in CCTCC, preservation date: on November 25th, 2005, deposit number: CCTCC-M205135.

(4) expression of mycobacterium bovis BCG specific secretion albumen mpb70 protein gene in e. coli bl21 and the purification process of expressing protein: the single bacterium colony of the described recombination bacillus coli Escherichia of above-mentioned steps (3) coli BL21/pET-28a-mpb70 picking to LB substratum is added kantlex to final concentration 50 μ g/mL, and 37 ℃ of shaking tables are cultivated after 12 hours and are put 4 ℃ of refrigerator overnight; With the LB substratum that contains 50 μ g/mL kantlex with this bacterium liquid by after 1: 100 dilution proportion, divide and be filled in the bacteria culture bottle, put 37 ℃ of shaking tables and be cultured to OD600 ≈ 0.6, isopropylthio-with 0.4mmol/L is induced, induce after 3 hours and carry out the SDS-PAGE electrophoretic analysis, the inductive thalline extracts inclusion body behind centrifugal, ultrasonic disruption, it is standby to be sub-packed in-20 ℃ of preservations after the processing such as purified, sex change, renaturation.With reference to " molecular cloning experiment guide " (Sa nurse Brooker J, Ritchie EF not, Manny A Disi T edits (Jin Dongyan, Li Mengfeng etc. translate). second edition, Beijing Science Press, 1992 editions) albumen of purifying is carried out the dot hybridization analysis, confirm that this recombination bacillus coli can express mycobacterium bovis BCG specific secretion albumen mpb70 specifically, expressed proteins exists in the e. coli bl21 with the form of inclusion body, and has immunologic competence.

(5) mycobacterium bovis BCG specific secretion albumen mpb70 protein antibodies detects and the foundation of bovine tuberculosis indirect hemagglutination diagnostic method and the preparation of test kit:

1) preparation of 1% sensitization sheep red blood cell (SRBC) suspension: from step (4), obtain purifying mpb70 expressing protein in the described inclusion body; With Mycobacterium bovis specific antigen protein MPB70 sensitized erythrocyte, it prepares by following method, get the content for preparing and be that to add isopyknic content in 2.5% the red cell suspension be the antigen protein MPB70 of 100 μ g/ml, put 37 ℃ of following water-baths 30 minutes, constantly stir the centrifugal supernatant of abandoning therebetween; With the PBS suspension blood cell of pH7.2, hematocrit centrifuge washing 2 times; With containing 1% normal rabbit serum PBS washing once, be made into 1% sensitization sheep red blood cell (SRBC) suspension with this liquid at last again, add 0.01% Thiomersalate, put 2-8 ℃ of preservation, standby after the packing, be 1% sensitization sheep red blood cell (SRBC) suspension of the present invention.

2) A Shi (is called for short A Shi liquid: referring to chief editors such as He Zhaoyang, " animal immunology experimental technique " Changchun: Jilin science tech publishing house, 2002) preparation of alserver's solution: get glucose 2.05g, Sodium Citrate (Na3C6H507.2H2O) 0.8g, Citric Acid 0.55g, NaCl0.42g is with distilled water constant volume to 100 milliliter, 121 ℃ of sterilizations 15 minutes, put 4 degree refrigerators and preserve.

3) preparation of negative control sera: the healthy ox through intracutaneous transformation reactions, microbial culture and PCR checking is carried out aseptic collection blood, adopt blood leave standstill 1h, 4 ℃ of standing over night, separation of serum in 37 ℃; Sterile filtration is that to add 0.01% sodium azide in serum anticorrosion, adds fashionablely fully to shake up.

4) preparation of positive control serum: in the diary farm after skin test reaction preliminary screening, 10 of the milk cows that the Mycobacterium bovis that discovery is made a definite diagnosis through microbial culture, PCR check infects, and cut open inspection and histopathology by pathology and confirm that wherein 8 is typical mycobacterium tuberculosis var bovis grave illness ox, gather its blood and be prepared into serum, this serum is as the positive serum control sample of this test kit.

Preparation specific as follows:

5) preparation of antigenic dilution: PBS (phosphate buffered saline buffer) 0.15mol/L pH7.2

First liquid is received phosphoric acid hydrogen two and is mixed with the solution that content is 0.15mol/L

Concrete grammar is: get phosphoric acid hydrogen two and receive 21.3g, adding distil water is settled to 1000ml.

It is 0.15mol/L solution that second liquid is mixed with content with potassium primary phosphate

Concrete grammar is: get potassium primary phosphate 20.42g, adding distil water is settled to 1000ml.

As with containing the phosphoric acid salt of crystal water, then to multiply by 0.15 weighing by the gram molecular weight of band water during weighing.Above-mentioned first liquid for preparing and second liquid are according to ordinary method sterilization (121 ℃ were sterilized 15 minutes).

Above-mentioned first liquid, second liquid and sodium chloride are mixed according to following proportioning:

First liquid 87ml

Second liquid 13ml

Sodium chloride 0.85g

6) assembling of test kit: indirect hemagglutination plate, bovine tuberculosis mycobacterium infectious standard positive serum, healthy ox negative serum, serum dilution are assembled into the mycobacterium bovis BCG antibody assay kit.

Beneficial effect of the present invention:

1, test kit biological safety height of the present invention.Involved in the present invention to prokaryotic expression carrier pET-28a be prokaryotic expression carrier commonly used in the molecular biology, do not have bio-hazard, the prokaryotic expression carrier pET-28a-mpb70 of Gou Jianing is also without any bio-hazard on this basis.Recombination bacillus coli BL21/pET-28a-mpb70 is converted in the molecular biology e. coli bl21 competent cell commonly used with prokaryotic expression carrier pET-28a-mpb70 after the kalamycin resistance screening obtains, and does not also have a bio-hazard.The used antigen of the present invention does not relate to ox type mycobacterium viable bacteria with mycobacterium bovis BCG specific secretion albumen mpb70 protein Preparation in the preparation process, do not have therefore that ox type mycobacterium escapes, the potentially dangerous of diffusion.

2, test kit production cost of the present invention is low.Mycobacterium bovis BCG specific secretion albumen mpb70 antibody diagnosis method provided by the present invention and the required antigen of diagnostic kit are mycobacterium bovis BCG specific secretion albumen mpb70 albumen.This albumen can obtain a large amount of expression external by recombination bacillus coli BL21/pET-28a-mpb70, is fit to large-scale production.And the present domestic test kit that does not have special detection ox type mycobacterium serum antibody.

3, test kit high specificity of the present invention, highly sensitive, operation is simple, is fit to very much the clinical extensive detection of mycobacterium bovis.

Description of drawings

Fig. 1: be main technological route of the present invention.

Fig. 2: be vector construction physics figure of the present invention.

Fig. 3: mpb70 gene PCR amplification.

The molecular weight standard 1,2,3 of M:DL 2000: the gene that amplifies

Fig. 4: recombinant plasmid pET28a-mpb70 enzyme is cut qualification result

M1: be the DL15000 molecular weight standard; M2: be the DL2000 molecular weight standard; 1: for the EcoRI enzyme is cut processing;

2: for the HindIII enzyme is cut processing; 3: be EcoRI and the processing of HindIII double digestion.

Fig. 5: be the SDS-PAGE electrophorogram of the mycobacterium bovis BCG specific secretion albumen mpb70 of abduction delivering of the present invention, different induced concentrations and induction time condition MPB70 expression.

Fig. 6: be that the result is cutd open in the positive cattle disease understanding of clinical detection of the present invention.

Among the figure: (A) greater omentum (cud portion): see that there is the part cluster in the dispersivity tubercle; (B) lung outer wall: the calcification kitchen range of two faint yellow walnut sizes, dissectd husky sense; (C) pleura: a large amount of beans sample tubercles are arranged on the rib inwall; (D) every flesh: be coated with a large amount of pearl-like tubercles.

Fig. 7: be the clinical serum detected result that one of them embodiment of the present invention carries out on same block of blood-coagulation-board plate

Embodiment

Embodiment 1

The clone of mycobacterium bovis BCG specific secretion albumen mpb70 albumen cNDA sequence

1, the PCR design of primers is with synthetic

Mpb70 gene order (accession number: 1008916) design upstream and downstream primer according to the Genbank announcement.Upstream primer is introduced EcoRI restriction enzyme site (shown in the underscore in the primer), and downstream primer is introduced HindIII restriction enzyme site (shown in the underscore in the primer).Primer of the present invention is synthetic by Beijing AudioCodes biotech firm.

Mpb70 upstream primer: 5 '-AGTT CAGAATTCATGAAGGTAAAGAACACAATTGC-3 '

Mpb70 downstream primer: 5 '-ACGTCG AAGCTTTTACGCCGGAGGCATTAGC-3 '

2, amplification of MPB70 protein coding gene and processing

With mycobacterium bovis BCG type strain ((Mycobacterium bovis AF2122/97, this bacterial strain is so kind as to give by the Guo Mingxing researcher of Hubei Entry-Exit Inspection and Quarantine Bureau, Genebank, accession number is: NC002945) bacterial cultures adds a small amount of distilled water and boils 10min cracking thalline, gets supernatant liquor as pcr template after centrifugal.The amplified reaction of mpb70 is by 25 circulations, 94 ℃ of pre-sex change 5min, and 94 ℃ of sex change 30s, 60 ℃ of annealing 30s, 72 ℃ are extended 40s, and last 72 ℃ are extended the 7min program fully and carry out.The PCR product uses dna gel to reclaim test kit (available from Shanghai bio-engineering corporation) purifying mpb70 gene fragment fast with 0.8% agarose gel electrophoresis detection reaction result.

Embodiment 2

The structure of mycobacterium bovis BCG specific secretion albumen mpb70 gene prokaryotic carrier

With EcoRI and HindIII double digestion pET28a (+) (available from invitrogen company) and PCR product, reclaim fast with dna gel and glue terminal the connection structure recombinant expression vector pET28a-mpb70 after test kit (available from Shanghai bio-engineering corporation) purifying reclaims.Vector construction as shown in Figure 2.

Embodiment 3

The structure of recombination bacillus coli Escherichia coli BL21/pET-28a-mpb70

Recombinant expression vector pET-28a-mpb70 is converted into e. coli bl21 (coli strain is available from Stratagene company) competent cell, coating LB kantlex plate, selecting a plurality of single bacterium colonies puts into 37 ℃ of LB liquid nutrient mediums and cultivates after 8 hours and use isopropylthio-(IPTG) abduction delivering respectively, carry out SDS-PAGE and dot hybridization analysis (Sa nurse Brooker J then, Ritchie EF not, Manny A Disi T chief editor, the molecular cloning experiment guide, Jin Dongyan, Li Mengfeng etc. translate, second edition, Science Press, Beijing, 1992 editions), therefrom filter out can be in e. coli bl21 the recombination bacillus coli Escherichia coli BL21/pET-28a-mpb70 of abduction delivering mycobacterium bovis BCG specific secretion albumen mpb70.This bacterial strain is deposited in the China typical case and cultivates Wu Baozang center (CCTCC), and preserving number is M205135.

Embodiment 4

Determining of best inductor concentration and best induction time

The single recombination bacillus coli Escherichia of picking coli BL21/pET-28a-mpb70 bacterium colony is to 5mL LB substratum, and Jia Kana penicillin (Kana) to final concentration is 50 μ g/mL, and 37 ℃ of shaking tables are cultivated after 8 hours and put 4 ℃ of refrigerator overnight.With the LB substratum that contains 50 those penicillin of μ g/mL card (Kana) with this bacterium liquid by after 1: 100 dilution proportion, divide and be filled to (5mL/ bottle) in 5 bacteria culture bottles, putting 37 ℃ of shaking tables cultivates, add inductor isopropylthio-(IPTG) to final concentration and be respectively 0.2,0.4,0.6,0.8 and 1.0mmol/L, after continuing to cultivate 3h, SDS-PAGE is carried out in the equivalent sampling.Determine that according to MPB70 protein expression situation the best induced concentration of IPTG is 0.4mmol/L.

The single recombination bacillus coli BL21/pET-28a-mpb70 of picking bacterium colony is to 5mL LB substratum, and adding to final concentration is 50 μ g/mL, and 37 ℃ of shaking tables are cultivated after 8 hours and put 4 ℃ of refrigerator overnight.With the LB substratum that contains 50 μ g/mL kantlex with this bacterium liquid by after 1: 100 dilution proportion, divide and be filled to (5mL/ bottle) in 5 bacteria culture bottles, put 37 ℃ of shaking tables and be cultured to OD600 ≌ 0.60-0.80, induce with best inductor concentration (IPTG concentration is 0.4mmol/L), 1mL per hour takes a sample, coinduction carry out after 5 hours the SDS-PAGE electrophoretic analysis (the molecular cloning experiment guide. Sa nurse Brooker J, Ritchie EF not, Manny A Disi T edits (Jin Dongyan, Li Mengfeng etc. translate). second edition, Beijing Science Press, 1992 editions), determine that according to the mpb70 expression best induction time is 3 hours.

Embodiment 5

A large amount of abduction deliverings and the protein purification of mycobacterium bovis BCG specific secretion albumen mpb70

Press 1% inoculation recombination bacillus coli BL21/pET-28a-mpb70 of LB culture volume, cultivated 12 hours down at 37 ℃, putting 4 ℃ spends the night, next day 2 milliliters of bacterium liquid are inoculated in 200 milliliters of fresh LB substratum that are added with 50 μ g/mL kantlex enlarged culturing and reach 0.60-0.80 to 0D600, add final concentration and be the IPTG abduction delivering 3 hours of 0.4mmol/L.With 8000r/min centrifugal 10 minutes, abandon supernatant, collect thalline.The thalline of collecting is contained the buffer A of 1%Trinton-X100 (by 50mmol/l Tris-cl pH8.0 with 1/10 nutrient solution volume, 0.5mmol/L EDTA pH8.0,50mmol/L NaCL, 0.5mmol/L DTT (dithiothreitol (DTT)), the preparation of 5% glycerine) after the suspension, room temperature left standstill 0.5 hour, used big ultrasonic head, set the pitch time of 10 circulations and 50%, in ice bath ultrasonication to solution limpid till.Solution after the fragmentation is through 10000r/min, centrifugal 12min, and collecting precipitation is abandoned supernatant.Precipitation suspends with the buffer A that 1/10 nutrient solution volume contains 0.2%DOC (Sodium desoxycholate), and put room temperature and left standstill 0.5 hour, 10000r/min, centrifugal 12min, collecting precipitation is abandoned supernatant.Precipitation contains the buffer A suspension of 0.3% dodecyl base amino acid sodium with 1/10 nutrient solution volume, puts room temperature and leaves standstill and dissolved fully to inclusion body in 1.5 hours.Then through 10000r/min, behind the centrifugal 12min, abandon precipitation (containing cell debris), collect supernatant, adding final concentration is 0.2% Macrogol 4000 (PEG4000), and 1mmol/L Sleep-promoting factor B and 2mmol/L reduced glutathion are put 4 ℃ and spent the night, use 10mmol/L TE (pH8.0) dialysis 2-3 days at last, change liquid 3-4 every day.It is standby that solution after the dialysis is distributed into tubule postposition-20 ℃ preservation, as erythrocyte sensitizing substance antigen.

Embodiment 6

The preparation of hydroformylation red corpuscle diagnostic reagent

1, the preparation and the check of quick hydroformylation sheep red blood cell (SRBC) diagnostic reagent

(1) preparation of sheep red blood cell (SRBC): the healthy ram of selection suis 2 type negative antibodies, after fibre is taken off in the aseptic technique blood sampling, add the A Shi liquid (prescription is the same) of equivalent, put 2-8C and stablize 3-5 day, use physiological saline centrifuge washing 5 times.

(2) red corpuscle hydroformylation (fixing): be made into 5% red cell suspension with 0.15mol/L pH7.2 PBS, slowly 2.5% glutaraldehyde solution that adds 1/5 capacity on the discharge magnetic stirrer, about 1 hour, add, continue then to stir 10 minutes, the centrifugal supernatant of abandoning is again with PBS washing 1 time, be made into 10% suspension with PBS at last, add 0.01% Thiomersalate, put 2-8 ℃ of preservation, this red cell suspension usage period is no more than 6 months.

(3) erythrocytic the tanning of hydroformylation: the earlier centrifugal back of hydroformylation red corpuscle is suspended washing once with PBS, become 2.5% suspension with PBS then, the tannic acid (preparing) of 1: 20000 concentration of capacity such as adding with PBS, put 37 ℃ of water-baths 15 minutes, stir during this time for several times, the centrifugal supernatant of abandoning suspends washing once with PBS again, is made into 2.5% suspension with pH6.4 PBS liquid at last and is used for sensitization.

Embodiment 7

Assembling of mycobacterium bovis BCG specific secretion albumen mpb70 antibody diagnosing reagent kit and preparation method

1, the mpb70-IHA test kit comprises:

The sheep red blood cell (SRBC) antigen 1 bottle/box of sensitization

Each 1 bottle of (0.5m1)/box of positive serum and negative serum

1 bottle of (5m1)/box of serum dilution

2 (96 hole)/boxes of V-type blood-coagulation-board

2mpb70-IHA test kit related solution prescription

The preparation of serum dilution (being phosphoric acid buffer):

First liquid is received phosphoric acid hydrogen two and is mixed with the solution that content is 0.15mol/L.

Concrete grammar: get phosphoric acid hydrogen two and receive 21.3g, adding distil water is settled to 1000ml.

Second liquid is mixed with the solution that content is 0.15mol/L with potassium primary phosphate.

Concrete grammar: get potassium primary phosphate 20.42g, adding distil water is settled to 1000ml.

As with containing the phosphoric acid salt of crystal water, then to multiply by 0.15 weighing by the gram molecular weight of band water during weighing.The equal 121 pounds of autoclavings of first liquid and second liquid 15 minutes.

First liquid 87ml

Second liquid 13ml

Sodium-chlor 0.85g

3, best antigen concentration, antigen sensibilization time and result judge the selection of Best Times: the antigen of getting 1 part of 5mL/L hydroformylation tannic acid red corpuscle and 1 part of broken optimum concn of ultrasonic grinding instrument, in 37 ℃ of water-baths, act on 30min, continuous shake gently therebetween, at last with the centrifugal 3min of 3000r/min, abandon supernatant liquor, after containing healthy rabbit anteserum 1%PBS washing, be made into 1% sensitized erythrocyte suspension.Test-results shows that hemagglutinative titer is low when the antigen diluent degree is 1: 128; Raise with antigen concentration, hemagglutinative titer also improves.But the antigen diluent degree surpasses 1: 8 o'clock hemagglutinative titer no longer obviously to be improved, thereby determines that the suitableeest antigen concentration is 1: 8-1: higher between 16 with 1% sensitized erythrocyte hemagglutinative titer of this concentration antigen prepd, and clear picture, through trial effect is also stable repeatedly.Test-results shows under the situation of determining sensitization antigen amount, change the sensitization time, when the sensitization time is 60 minutes, with the positive serum reaction, susceptibility is the highest, and the highest hemagglutinative titer of serum can reach 1: 1024, at this moment, 1% sensitized erythrocyte hemagglutinative titer of preparation is the highest, and clear picture, through trial effect is also stable repeatedly.With "-", "+", " ++ ", " +++", " ++ ++ " erythrocytic aggegation intensity of expression, when positive control and negative control are set up fully, the highly diluted multiple of serum that "+" occur is the hemagglutinative titer of this serum, result of determination is a Best Times when verifying in this test 45 minutes repeatedly, this moment, positive serum went out a tangible agglutination phenomenon, and not aggegation of negative serum.

Embodiment 8

Mpb70-IHA detects step and judging criterion

Operation steps:

(1) take out test kit of the present invention from 4 ℃ of refrigerators, each component of balance is to room temperature.

(2) draw diluent 25 μ L with micropipet, drip in 96 holes " V " type micro-reaction plate hole;

(3) get tested inactivated serum 25 μ L in the 1st hole of micro-reaction plate with micropipet, do doubling dilution to the 11 holes then successively.The 12nd hole is a blank, only adds diluent;

(4) set the row contrast in addition: sensitized erythrocyte adds positive serum, sensitized erythrocyte adds diluent and negative serum;

(5) every hole adds the red cell suspension of 25 μ L antigen sensibilizations;

(6) 1min that gently shakes on microoscillator makes its thorough mixing, and room temperature is placed observations after 45 minutes.

Determining of mpb70-IHA criterion as a result:

Establishment condition as a result: sensitized erythrocyte adds positive serum and 100% aggegation occurs, and sensitized erythrocyte adds diluent and not aggegation of negative serum, shows that the result sets up;

The judgement of agglutination titer: with "-", "+", " ++ ", " +++", " ++ ++ " erythrocytic aggegation intensity of expression, the highly diluted multiple of serum that " ++ " occur is the hemagglutinative titer of this serum.

" ++ ++ " 100% aggegation, around red corpuscle is distributed at the bottom of the hole equably;

" +++" 75% aggegation, around red corpuscle is distributed at the bottom of the hole equably, but there is the big point of needle point that red corpuscle forms at the center at the bottom of the hole;

" ++ ": represent 50% aggegation, uneven red cell distribution is arranged, the point that has red corpuscle to avale at the bottom of the hole on every side at the bottom of the hole;

"+": represent 25% aggegation, uneven red cell distribution is arranged on every side at the bottom of the hole, but most of red corpuscle is deposited at the bottom of the hole.

"-": represent not aggegation, red corpuscle forms a round dot at the bottom of being deposited on the hole fully.

Embodiment 9

Effect of the present invention for example

The mpb70-IHA detection method of indication is with reference to embodiment 8 described operation stepss in this example, its detected result such as table 1 and table 2,

1, specificity test

The hydroformylation red corpuscle reagent mycobacterium tuberculosis var bovis mycobacterial diseases reaction that sensitization is good is strong positive, does not all react with johne's disease bovine serum, the sick bovine serum of cloth, toxoplasmosis bovine serum and foot and mouth disease bovine serum, has reached specificity preferably.The results are shown in Table 1.

Table 1 specificity test (unit: log2)

Kinds of Diseases	Paratuberculosis	The cloth disease	Toxoplasma gondii	Foot and mouth disease	The negative control sample
						Detected result	0	0	0	0	0

2, the susceptibility of test kit:

The comparison of mycobacterium tuberculosis var bovis MPB70 albumen indirect hemagglutination test antibody diagnosing reagent kit and agar diffusion test susceptibility, to use mycobacterium tuberculosis var bovis MPB70 albumen indirect hemagglutination test antibody diagnosing reagent kit and agar diffusion test (AGIP) to detect behind 10 parts of mycobacterium tuberculosis var bovis male bovine serum doubling dilutions respectively, it be 1～4 that the AGIP of serum tires; IHA tires and is 128-512.The susceptibility of IHA is 128-512 times (the seeing Table 2) of AGIP.

10 parts of positive serum IHA of table 2 doubling dilution and the detected result of AGIP

Serum	1	2	3	4	5	6	7	8	9	10
											AGIP (tiring)	1	2	1	1	2	2	1	2	1	2
IHA (tiring)	256	512	512	512	512	512	128	512	512	512
											IHA/AGIP	256	256	512	512	256	256	128	256	512	256

Although content of the present invention is to describe in conjunction with present embodiment, can not think limitation of the scope of the invention, scope of the present invention is limited by appended claims.In addition, those skilled in the art carries out various changes or modification to the present invention in the appended claims restricted portion, and these changes or modified forms drop in protection scope of the present invention equally.

Claims (6)

1, expresses the proteic recombination bacillus coli Escherichia of Mycobacterium bovis MPB70 coli BL21/pET-28a-MPB70, be deposited in CCTCC, deposit number: CCTCC-M205135.

2, the detection kit that comprises claim 1.

3, the described test kit of claim 2 is characterized in that, described test kit is the test kit that is used to detect bovine tuberculosis antibody.

4, claim 2 or the 3 described test kits that detect bovine tuberculosis antibody that are applicable to, it comprises box body, be located at 96 hole Sptting plates at the bottom of the intravital V-arrangement of box, bovine tuberculosis mycobacterium infectious standard positive serum, healthy ox negative serum, serum dilution and 1% sensitization sheep red blood cell (SRBC) suspension.

5, the described test kit of claim 4,1% sensitization sheep red blood cell (SRBC) suspension wherein is the red corpuscle with Mycobacterium bovis specific antigen protein MPB70 sensitization, it prepares by following method: with adding isopyknic antigen protein MPB70 in the red cell suspension, through water-bath, stir; The centrifugal supernatant of abandoning with PBS washing 2 times, with the PBS washing that contains 1% rabbit anteserum once, is made into 1% sensitization sheep red blood cell (SRBC) suspension again, adds 0.01% Thiomersalate, puts 2-8 ℃ of preservation after the packing, and is standby.

6, the application of each described test kit of claim 2-5 in detecting bovine tuberculosis antibody.

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