CN106771190B - A kind of sheep anaplasmosis indirect ELISA diagnostic reagent kit and preparation method thereof - Google Patents

A kind of sheep anaplasmosis indirect ELISA diagnostic reagent kit and preparation method thereof Download PDF

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CN106771190B
CN106771190B CN201611220771.7A CN201611220771A CN106771190B CN 106771190 B CN106771190 B CN 106771190B CN 201611220771 A CN201611220771 A CN 201611220771A CN 106771190 B CN106771190 B CN 106771190B
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serum
sheep
kit
antibody
anaplasmosis
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CN106771190A (en
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刘志杰
殷宏
王振国
杨吉飞
牛庆丽
罗建勋
关贵全
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Lanzhou Veterinary Research Institute of CAAS
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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms

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Abstract

The present invention relates to a kind of detection sheep without slurry（Anaplasmaovis）Serum antibody using the P35 of recombinant expression as the indirect ELISA diagnostic reagent kit of antigen, P35 recombinant antigens and preparation method thereof.The kit only withA.ovisSpecific reaction occurs for positive serum, has good specificity；The kit has good sensibility, can detect the antibody that sheep generates without slurry early infection, and can be from 1：Antibody is detected in 800 diluted positive serums.Kit repeatability is good, operates relative ease, can be applied to the epidemiological survey of sheep anaplasmosis and the detection of immune antiboidy level.

Description

A kind of sheep anaplasmosis indirect ELISA diagnostic reagent kit and preparation method thereof
Technical field
The present invention relates to field of immunological detection, specially a kind of sheep anaplasmosis indirect ELISA diagnostic reagent kit and its Preparation method.
Background technology
No slurry be one kind by tick-borne obligate intracellular bacterial parasite, main parasitic is in the ruminants red blood cell such as cattle and sheep It is interior, lead to anaplasmosis, is once called as anaplasmosis.No slurry is classified as deformation Gammaproteobacteria, Rickettsiales with Human ehrlichia （Rickettsiaies）, no slurry section（Anaplasmatacea）, no slurry category（Anaplasma）, wherein sheep is without slurry（A. ovis）It is important forming member, the anaplasmosis of small ruminant can be caused.The disease clinical symptoms are fever, anaemia, Huang It is subcutaneous ulcer, weak and gradual become thin.Blood picture detected representation be erythrocyte number, packed cell volume and content of hemoglobin it is notable under Drop, whens severe infections, can lead to death.The disease is widely current all over the world, also happens occasionally in many areas in China, sternly The development for hindering animal husbandry again causes huge economic loss to animal husbandry.Therefore, quick, sensitive, accurate behaviour again is established It is very necessary to make convenient detection method.
Domestic and foreign scholars have carried out a series of research work to detection method of the sheep without slurry, and microscopic examination of perpheral blood smear method is mended Body combines experiment（CF）, card agglutination test（CAT）, indirect fluorescent antibody test（IFAT）, PCR method etc., microscopic examination of perpheral blood smear Method is to apply most methods in practice.However this method needs stronger professional knowledge to come and the blood such as Taylor worm, Babesia Liquid parasitic infection is distinguished, and this method is unfavorable for infection early stage and infects the extremely low situation of duration Infestation rat.Though Right complement fixation test (CFT)（CF）, card agglutination test（CAT）, indirect fluorescent antibody test（IFAT）Deng the report for also having application, But complement fixation test (CFT) is complicated for operation, is not easy to promote；Card agglutination test is easy to operate, but sensibility is poor, these serology Method rarely has the report largely used in practice, and it is still necessary to further evaluate its detection performance for these methods.Though PCR method is one Kind compares the molecular diagnosis method of rapid sensitive, but it needs agarose gel electrophoresis analysis result, required reagent such as bromination second Ingot, operation slightly just generates murder by poisoning to user and ambient enviroment accidentally, and PCR instrument and gel imaging system are expensive, it is difficult to It is promoted in grass-roots unit.So far, the world is still without the serology kit for commercialization of the sheep without slurry infection detection.
Invention content
The present invention provides one kind and can overcome the shortage of prior art, only withA. ovisAntibody reacts, without with other ticks Cross reaction occurs for the antibody for passing cause of disease or Related Bacteria, for detectingA. ovisThe indirect ELISA reagent kit of serum antibody, It provides and is prepared used in the method and kit of this detection kit simultaneouslyA. ovisP35 recombinant antigens and this heavy The preparation method of group antigen.
The present invention sheep include without slurry indirect ELISA antibody assay kit；Envelope antigen, vacuum-packed enzyme Target（2 pieces）；Standard positive serum 1（100μl）；Standard female serum 1（100μl）；The rabbit-anti goat-anti of 50 times of HRP labels Body 1（100μl）；1 bottle of serum dilution（20ml）1 bottle of enzyme labelled antibody dilution（20ml）；1 bottle of TMB developing solutions（20ml）；Eventually Only 1 bottle of liquid（20ml）；50 times of 1 bottle of concentrated cleaning solutions（20ml）.4 DEG C of preservations of kit, storage life are 6 months.Kit can be examined Survey 180 parts of serum.
Antigen of the sheep without dotey's ELISA Plate in slurry indirect ELISA antibody assay kit of the present invention is recombinant protein, For Bacillus coli expression albumen, the gene of expression is located at the 73bp-969 bp of P35 genes, and through sequencing, sequence is SEQ ID No.1, length are 897 bp, include the epitope of P35 specificity.
The preparation method of the recombinant antigen is：With forward primer SEQ ID No.2, reverse primer SEQ ID No.3, withA. ovisFull-length genome is the gene that template amplification goes out P35, and length is the segment of 897 bp, by aforementioned amplified fragments and pGEM-T Easy Vector connections, transformed competence colibacillus DH5a Escherichia coli, screening positive clone bacterial strain send sequencing, identify correctly recombination Plasmid is named as pGEM-P35.Correct clone strain extraction recombinant plasmid will be sequenced, then by plasmid pET30a and pGEM-P35 Restriction endonuclease is used respectivelyEcoR I andHindTarget DNA fragment and pET30a carrier segments is separately recovered in III double digestion, connection, Transformed competence colibacillus BL21 (DE3) Escherichia coli, screening positive clone bacterial strain carry out induction table with the IPTG of 1 mMol/L of final concentration It reaches, recombinant protein is obtained after expression product is purified, the P35 recombinant proteins after expression are 40KDa.
The present invention kit include；Envelope antigen, vacuum-packed ELISA Plate（2 pieces）；Standard positive serum 1 （100μl）；Standard female serum 1（100μl）；The rabbit-anti goat-anti body 1 of 50 times of HRP labels（100μl）；Serum dilution 1 Bottle（20ml）1 bottle of enzyme labelled antibody dilution（20ml）；1 bottle of TMB developing solutions（20ml）；1 bottle of terminate liquid（20ml）;50 times of concentrations are washed Wash 1 bottle of liquid（20ml）.4 DEG C of preservations of kit, storage life are 6 months.Kit can detect 180 parts of serum.
The present invention has found through overtesting, part specific fragment of the sheep without slurry P35 genes, i.e. the 73-969 of P35 genes Bp, length be 897 bp segment, only react without slurry positive serum with sheep, and to the positive serum of other related cause of diseases No cross reaction.Experiments have shown that, serum is detected for this segment, result has preferable special by further Property.
The present invention superiority be：
A. it selectsA. ovisThe specific fragment of gene carries out clonal expression, selected genetic fragment and belong to its The homology of its bacterium gene homologous segment is low, and contains the specific antigen epitope in P35 genes.Therefore, this pure The recombinant protein of change as envelope antigen, only withA. ovisPositive serum reacts, and ensure that specificity；And the antigen Epitope is linear epitope, and recombinant protein has good reactivity, ensure that the sensibility of reaction.
B. the carrier expressed used in target gene fragment is pET-30a, and obtained recombinant protein carries histidine tag.Though Right recombinant protein exists with inclusion bodies, but can be purified using AKTA systems with NI HisTrap FF after dissolving, purity On ensure that specificity with antibody response.
C. kit application indirect ELISA technology of the present invention, opening operation, with currently used for detection sheep without slurry The round pcr of body is compared with other detection serum, and technology requirement is more relaxed, has great operability.
Description of the drawings
Fig. 1：A. ovisThe PCR amplification of P35 genes.
Fig. 2：The SDS-PAGE of P35 recombinant proteins is analyzed, wherein M：Protein Marker；1：Full bacterium；2：Precipitation；3：On Clearly；4 purified products.
Fig. 3：The Western-blotting of P35 recombinant proteins is analyzed, wherein M:Protein molecular standard；1：Anti- His labels Antibody；2：Sheep is without slurry positive serum；3 negative serums；4-11：Mycoplasma ovine pneumoniae, mycoplasma capri goat pneumonia subspecies, Sheep Babesia, T.uilenbergi, Theileria luwenshuni, anaplasma bovis, Niu Babeisi, annular loop detector.
Specific implementation mode
Experimental method used in following embodiments is conventional method unless otherwise specified.
Material, reagent used in following embodiments etc., are commercially available unless otherwise specified.
It is prepared by embodiment one, recombinant antigen.
Pair of primers, forward primer are designed according to selected genetic fragment（SEQ ID No.2）Sequence is 5 '- CCGGAATTCAGGGTACTGGTAATGGGC-3 ', reverse primer（SEQ ID No.3）Sequence is 5 '- CCCAAGCTTCTAAATAGCAAGACTT TGCGTATTAG-3'.Underscore in primer is restriction enzyme site, before restriction enzyme site 3, face base is protectiveness base, and digestion is that point is in sense primerEcoRI, restriction enzyme site is in downstreamHind III.With complete Blood genome extracts kit infects extraction whole blood genome in blood without slurry from sheep and is passed through with above-mentioned primer as template PCR method expands target fragment.It is 897 bp to expand obtained target fragment length, includes P35 epitopes.It will The target fragment expanded recycles target DNA into row agarose gel electrophoresis, purifying, by target DNA fragment and pGEM-T Easy Vector connections, transformed competence colibacillus DH5a Escherichia coli, screening positive clone bacterial strain send sequencing, will be sequenced correct gram Grand bacterial strain extracts recombinant plasmid, then plasmid pET30a and pGEM-P35 are used restriction endonuclease respectivelyEcoR I andHindIII double digestion, Target DNA fragment and pET30a carrier segments is separately recovered, in connection, transformed competence colibacillus BL21 (DE3) Escherichia coli, screening sun Property clone strain, induced expression is carried out with the IPTG of 1 mMol/L of final concentration.
Bacterium solution centrifugation after induction is washed precipitation with the PBS of PH7.4, is resuspended, then ultrasonication thalline, then from The destination protein of the heart, expression is that inclusion body is located in precipitation.Precipitation is dissolved with 8 M urea, is then crossed NI HisTrap FF and is adopted Purified with AKTA systems, recombinant protein is obtained after purified.Operation carries out to specifications.
Embodiment two, antigen coat ELISA Plate.
Purified recombinant protein is as antigen coat ELISA Plate.Through gridiron pattern method titrate test, it is ensured that antigen it is best A concentration of 2.5 μ g/ml, 100 holes μ l/;Serum diluting multiple is 1：100；It is 1 that HRP, which marks rabbit-anti goat-anti body extension rate,： 20000, it is first diluted to 50 times of preservations, working concentration does 400 times of dilutions again.
The coating program of antigen is：- 20 DEG C of antigens frozen are melted, are diluted to the carbonate buffer solution of PH9.6 dense Degree is 2.5 μ g/ml, and ELISA Plate is added, and 100 holes μ l/ are transferred to 4 DEG C overnight.Next day gets rid of antigen liquid, after washing with 1% it is bright Glue is closed, 100 holes μ l/, 37 DEG C of incubation 1h, then is washed.Then protein stabiliser is added, 100 holes μ l/ are incubated at room temperature 30min, Drying, blows 1h in super-clean bench.It is finally putting into packaging bag, is evacuated.
Standard positive serum in kit of the present invention is usedA. ovisBacterium blood vains inject 1 years old sheep 10ml, warp It is prepared when result is the positive after blood film dyeing microscopic examination, PCR method detection.Negative serum is menses smear staining microscopy, PCR Result is negative healthy sheep serum after method detection.50 times of concentrate HRP in box of the present invention mark rabbit-anti goat-anti body It buys in Sigma companies；Serum dilution, enzyme labelled antibody dilution, TMB developing solutions, terminate liquid, 50 times of concentrated cleaning solutions are purchased From Lanzhou Veterinary Inst., Chinese Acedemy of Agaricultural Sciences's biological products diagnostic center.
Embodiment three, assembling kit.
The detection that assembly cost is invented after prepared by various reagentsA. ovisThe indirect ELISA reagent kit of serum antibody（Below Abbreviation kit）.Kit includes；Envelope antigen, vacuum-packed ELISA Plate（2 pieces）；Standard positive serum 1（100 μl）；Standard female serum 1（100μl）；The rabbit-anti goat-anti body 1 of 50 times of HRP labels（100μl）；1 bottle of serum dilution （20ml）1 bottle of enzyme labelled antibody dilution（20ml）；1 bottle of TMB developing solutions（20ml）；1 bottle of terminate liquid（20ml）;50 times of thickening and washings 1 bottle of liquid（20ml）.4 DEG C of preservations of kit, storage life are 6 months.Kit can detect 180 parts of serum.
Example IV, the application method of kit show row.
1. kit is taken out from 4 DEG C of refrigerators, balance to room temperature.50 times of cleaning solutions are diluted to 1 times with double distilled water Working concentration.
2. taking out ELISA Plate, determine that the ELIAS strip needed, remaining lath are put into packaging bag by the quantity of serum to be checked In, 4 DEG C of preservations.The ELISA Plate of opening should use in one week.
3. increase serum dilution:Serum dilution, the holes 5-6 of first row are added in ELISA Plate hole（Blank control wells）For 100 holes μ l/, remaining is 99 holes μ l/.
4. increase serum：Standard positive serum is added in the holes 1-2 of first row, and standard female serum, 5-6 is added in the holes 3-4 Hole is blank control.Serum to be checked is added in remaining each hole, and every part of serum is added 1 hole, does not do repetition.Each serum addition is 1 μ The holes l/（The final dilution of serum is 1：100）, blank control wells not increase serum.Gently shake mixing.
5. incubating：ELISA Plate is sealed with sealing plate film, is put into 37 DEG C of incubators and is incubated 1h.
6. washing：Careful removal sealing plate film, is washed 3 times with board-washing machine washing, is patted dry.
7. and two anti-bindings：Rabbit-anti sheep ELIAS secondary antibody HRP antibody is done into 400 times of dilutions with enzyme labelled antibody dilution, is added In enzyme mark hole, 100 holes μ l/ are added.
8. incubating：Operation is the same as 5.
9. washing：Operation is the same as 6.
10. colour developing：TMB developing solutions are added, 100 holes μ l/ are added, 37 DEG C are protected from light colour developing 8min.
11. terminating：100 holes μ l/ of terminate liquid are added.
12. measuring：Measure the absorbance in each hole under 450nm wavelength with microplate reader immediately（OD450Value）.
13. result judgement：（OD samples-OD is negative）÷（The OD positives-OD is negative）* 100,
The yin and yang attribute threshold value of judgement sample：If being less than 6.0, for feminine gender；Potency is more than 6.0 for the positive.
Embodiment five, the sensibility of kit, specificity, repeatability measure.
By laboratory infectionA. ovis3 sheep positive serum, respectively according to 1：20,1：40,1:80,1：160, 1:320 totally 5 dilutions detect respectively, be 1 by antibody dilution:20 with 1:320 every part of dilution Virus monitory is The positive illustrates that kit has higher sensibility.
With kit detection sheep T.uilenbergi, Theileria luwenshuni, Babesia, mycoplasma ovine pneumoniae, mycoplasma mountain The positive serum of sheep subspecies is feminine gender.Illustrate that cross reaction is not present between positive serum and other serum.Kit has Good specificity.
Take 20 partsA. ovisPositive serum detected 3 times under different time the same terms with same batch kit, judge As a result identical, illustrate kit repeatability preferably.
Embodiment six, kit detect sample.
Have detected the serum of 3735 parts of acquisitions with the method, including artificial challenge's animal blood serum, field animal blood serum and strong Health animal blood serum.As a result 100% infection animal serum is the positive, and 35.25% field sample is the positive, and healthy animal serum is It is negative.Illustrate that the ELISA kit of the present invention is functional, can be used for the investigation of field sample.
Above example shows the P35 recombinant proteins synthesized for the first time in the present invention, for detecting sheep anaplasmosis meaning weight Greatly.Use P35 recombinant proteins for the sheep anaplasmosis indirect ELISA antibody assay kit of envelope antigen, high sensitivity, specifically Property it is good, it is easy to use, filled up domestic and international sheep without slurry infection detection commercialization serology kit blank.
SEQUENCE LISTING
<110>Lanzhou Veterinary Inst., Chinese Acedemy of Agaricultural Sciences
<120>A kind of sheep anaplasmosis indirect ELISA diagnostic reagent kit and preparation method thereof
<160> 3
<210> 1
<211> 897
<212> DNA
<213>Recombinant protein P35
<400> 1
ttgttgatgt cggggggttc ccctacaggc cttggttctg ttgttgccgc tcttaaagcc 60
gctgttgtgc ttggggaagc ggctaaagct ctacttgcta ctgtggatcg tgcagaactc 120
gagcaggagc tgagggccat agatgccaag tggcgttctc aagtttcgga ggaagctaaa 180
ctggcactag gtaaggaact taaagccata gatgccaagt ggcgttctca agtttcggag 240
gaagctaaac tggcactagg taaggaactt aaagccatag atgccaagtg gcgttctcaa 300
gtttcggagg aagctaaact ggcactaggt aaggaactta aagccataga tgccaagtgg 360
cgtcctgaag ttctcaataa ggaactgagg gccatagatg ccaagtggcg ttctcaagtt 420
tcggaggaag ctaaactggc actaggtaag gaacttaaag ccatagatgc caagtggcgt 480
cctgaagttc tcaataagga actgagggcc atagatgcca agtggcgtcc tcaagttctc 540
aatgaggagc tgaggaccat agatgccaag tggcgtcctc aagtttcgga ggaagctaaa 600
ctggcactag gtaaggaact gagggccata gatgccaagt ggcgtcctca agtttcgggg 660
gaagctaaac tggcactagg tgaggaactg agggccatag atgccaagtg gaagtctaga 720
attgcggagg aagctcaaca ggaacttgcc cgcgttgcgg agaggtttct cgctgcagca 780
cagaagtgcg gacagcttgg cgttataggc aaacaggctg tacccagcac tgatatgata 840
agtgttgttg cacaatctgt ttctgctact aatactaata cgcaaagtct tgctatt 897
<210> 2
<211> 27
<212> DNA
<213>Artificial sense primer
<400> 2
ccggaattca gggtactggt aatgggc 27
<210> 3
<211> 35
<212> DNA
<213>Artificial downstream primer
<400> 3
cccaagcttc taaatagcaa gactttgcgt attag 35

Claims (1)

1. a kind of sheep anaplasmosis indirect ELISA diagnostic reagent kit, include by the ELISA Plate of antigen coat, standard positive serum, Standard female serum, HRP label rabbit-anti goat-anti body, serum dilution, enzyme labelled antibody dilution, TMB developing solutions, terminate liquid and Concentrated cleaning solution, it is characterised in that the antigen is recombinant protein P35, and nucleotides sequence is classified as SEQ No. 1.
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