CN105866421A - Bovine theileria annulata indirect ELISA antibody detection kit and preparation method thereof - Google Patents

Bovine theileria annulata indirect ELISA antibody detection kit and preparation method thereof Download PDF

Info

Publication number
CN105866421A
CN105866421A CN201510974076.9A CN201510974076A CN105866421A CN 105866421 A CN105866421 A CN 105866421A CN 201510974076 A CN201510974076 A CN 201510974076A CN 105866421 A CN105866421 A CN 105866421A
Authority
CN
China
Prior art keywords
glu
serum
pro
asp
leu
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN201510974076.9A
Other languages
Chinese (zh)
Inventor
刘光远
罗金
李凯
田占成
殷宏
罗建勋
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Lanzhou Veterinary Research Institute of CAAS
Original Assignee
Lanzhou Veterinary Research Institute of CAAS
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Lanzhou Veterinary Research Institute of CAAS filed Critical Lanzhou Veterinary Research Institute of CAAS
Priority to CN201510974076.9A priority Critical patent/CN105866421A/en
Publication of CN105866421A publication Critical patent/CN105866421A/en
Pending legal-status Critical Current

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56905—Protozoa
    • G—PHYSICS
    • 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

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Cell Biology (AREA)
  • Analytical Chemistry (AREA)
  • Biotechnology (AREA)
  • Pathology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Virology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The invention relates to an indirect ELISA kit for diagnosis of a bovine theileria annulata serum antibody; the invention discloses an enzyme labeled plate coated by a theileria annulata recombinant antigen and used in the kit and a preparation method of the recombinant antigen. The recombinant antigen only is subjected to a reaction with the theileria annulata antibody, does not generate a cross reaction with an antibody of same-genus theileria orientalis infecting cattle, and has relatively good reactionogenicity and extremely high specificity. In addition, the kit has good reproducibility, and has relatively high coincidence rate with contrast detection of nested PCR. The kit is relatively simple to operate, and can be widely used in detection of bovine theileria annulata in production.

Description

Cattle annular loop detector indirect ELISA antibody assay kit and preparation method thereof
Technical field
This relates to a kind of test kit for detecting parazoon, and the preparation of this test kit and non-diseases diagnose Using method.
Background technology
Tropical theileriosis, is by Taylor worm section (Theileria), the annular loop detector of Theileria (Theileria) The blood borne protozoon that (Theileria annualata) parasitizes the macrophage of animal, lymphocyte and erythrocyte and cause Disease, can cause animal high heat, anemia, jaundice, hemorrhage, dyspnea, become thin and the symptom such as body surface swollen lymph node.This disease also known as Torrid zone theileriosis (Tropical theileriosis) or Mediterranean bank heat (Mediterranean Coast Fever), make For the disease of a kind of Ticks transmissibility, the Ticks mainly by Hyalomma is propagated.This disease Epidemic Scope is extensive, respectively from the Mediterranean in Europe The report that littoral and Africa has this disease to fall ill by Middle East to India and China.And have been found that in China and include newly All there is the generation of tropical theileriosis 13 provinces and regions such as boundary, the Inner Mongol.The main disseminator in China's major part region is residual edge glass Eye Ticks, and the communication media of Southern Xinjiang is mainly hyalomma anatolicum anatolicum.World Organization for Animal Health (OIE.Office International DesEpizooties. 2012) it is classified as B class epidemic disease, China is classified as two class epidemic diseases.Primary disease Season, popularity was very strong, how to pass through based on acute, and M & M is the highest, purebred cattle, Crossbred Cattle or other places cattle pair Infecting reaction sensitivity, mortality rate is higher, can reach 90%, and average case fatality rate, between 16%～60%, seriously restricts China's cowboying The development of industry (Wang Ming. veterinary parasitology [M]. Beijing: Chinese agriculture publishing house, 2008:366 369 14；Neitz W O. Theileriosis,gonderioses and cytauxzoono- ses: a review[M]. Government Printer, South Africa, 1965).At present, the means of diagnosis annular loop detector mainly have following several: (1) is conventional Detection: blood smear microscope inspection, lymph node puncture, animal inoculation method, In vitro culture and clinical diagnosis etc..But blood smear microscope Easily there is missing inspection, flase drop in inspection technology；And animal inoculation rule is costly, the used time is longer, it is impossible to carry out the inspection of batch samples Survey.See Nijhof A M, Pillay V, Steyl J, et al. Molecular characterization of Theileria species asso- ciated with mortality in four species of African antelopes[J]. Journal of Clinical Microbiology, 2005, 43 (12): 5907- 5911Nijhof A M, Pillay V, Steyl J, et al. Mole- cular characterization of Theileria species associated with mortality in four species of African antelopes[J]. Journal of Clinical Microbiology, 2005, 43 (12): 5907-5911; Barnett S F, Theileriasis D W, Ristic M. Infectious Blood Diseases of Man and Animals, [J]. 1968;Steyl J C A, Prozesky L, Stoltsz W H, et al. Theileriosis (Cytaux- zoonosis) in Roan antelope (Hippotragus equinus): Field exposure to infection and identification of potential vectors[J]. Onderstepoort Journal of Veterinary Research, 2012, 79(1): 01-08; Mbizeni S, Potgieter F T, Troskie C, et al. Field and laboratory studies on Corridor disease (Theileria parva infection) in cattle population at the livestock / game interface of uPhongoloMkuze area, South Africa[J]. Ticks and tick-borne diseases, 2013, 4 (3): 227-23.(2) molecular biological testing: gene probe techique, reverse linear marking hybridization technique, real-time quantitative PCR detection technique and nest-type PRC detection technique etc..But PCR detection process is cumbersome, the detection result used time is the most more, and some are special Different PCR instrument device is costly, it is impossible to universal.With reference to Mason PJ, Shiels BR, ait A, et al.Sequence and expression of a gene fromTheileria annulatacoding for a 72-kilodalton heat-shock protein[J]. Mol Biochem Parasitol, 1989, 37 (1): 27-35;Altay K, Aydin M F, Dumanli N, et al. Molecular detection of Theileria and Babesia infections in cattle[J]. Veterinary parasitology, 2008, 158(4): 295-301; Ros- García A, Nicolás A, García-Pérez A L, et al. Develop- ment and evaluation of a real-time PCR assay for the quantitative detection of Theileria annulata in cattle[J]. Parasit Vectors, 2012, 5: 171.(3) Serologic detection: complement fixation test (CFT), indirectly Hemagglutination test (IHA), indirect fluorescent antibody test (IFAT) and elisa (ELISA) etc..Examine relative to routine Surveying and diagnosis of molecular biology, Serologic detection has higher sensitivity and stronger specificity, and detection process is easy quickly, It is more objective that result judges.See Malhotra D V. Comparative ef-ficacy of countercurrent immunoelectrophoresis and Complement fixation tests for the detection of Theileria annulata antibodies in experi- mentally infected bovine calves; Katende J, Morzaria S, Toye P, et al. An enzyme- linked immunosorbent assay for detection of Theileria parva antibodies in cattle using a recombinant polymorphic immunodominant molecule[J]. Parasitology research, 1998, 84(5): 408-416;Renneker S, Kullmann B, Gerber S,et al. Development of a competitive ELISA for detectio of Theileria annulata infection[J].Transboundary and emerging diseases, 2008, 55(5-6):249-256。
Summary of the invention
The present invention provides a kind of that overcome prior art deficiency, and the detection for specific detection cattle annular loop detector tries Agent box.
The detection kit of the detection cattle annular loop detector of the present invention is a kind of cattle annular loop detector indirect ELISA antibody inspection Test agent box, includes the ELISA Plate being coated with GST-TaSP recombiant protein, the core of GST-TaSP recombiant protein in test kit Nucleotide sequence is SEQ ID No.3.
For convenience of using, also have in the cattle annular loop detector indirect ELISA antibody assay kit of the present invention: serum-dilution Liquid, HRP enzyme labelling two are anti-, standard positive control serum, standard negative control serum, substrate solution, stop buffer and PBST concentrate Liquid.
ELISA Plate preparation method in the cattle annular loop detector indirect ELISA antibody assay kit of the present invention is: will be with Infect annular loop detector blood extract genomic DNA as positive template, with primer to SEQ ID No.1 and SEQ ID The product of No.2 amplification is connected to pGEX-4T-1 carrier, then pGEX-4T-1 carrier proceeds to induction table in BL21 escherichia coli Reach, for coated elisa plate after expression product is purified, then carry out vacuum sealing after adding enzyme stabilizers wherein.
The annular loop detector recombinant expression plasmid BL21(DE3 of the present invention)/pGEX-4T-1-TaSP is October 26 in 2015 Day submits Wuhan University's China typical culture collection center preservation of Wuhan, China to, and deposit number is CCTCC NO: M2015645, its preservation Classification And Nomenclature is: e. coli bl21 (DE3)/pGEX-4T-1-TaSP
Escherichia coli BL21(DE3)/pGEX-4T-1-TaSP.
Amplimer used by the present invention is as follows:
In above-mentioned primer: 1～3 base of SEQ ID No.1 is the enzyme action positions protecting base, 4～9 bases to be BamH I Point, 1～3 base of primer SEQ ID No.2 are the restriction enzyme sites protecting base, 4～9 bases to be Xho I.
It is good that the detection kit of the present invention has specificity, the feature that sensitivity is high.Illustrate with TaSP recombiant protein to be anti- Former indirect ELISA detection method can be used for sample detection, carries out inspection and the Epidemiological study of annular loop detector infection.
Accompanying drawing explanation
The mensuration of Fig. 1 ELISA antigen optimum dilution degree, often organizes in column marker in figure: left 1 is TaSP feminine gender OD value, left 2 is TaSP positive OD value, and left 3 is GST feminine gender OD value, and left 4 is GST positive OD value.
The mensuration of Fig. 2 ELISA serum optimum diluting multiple, often organizes in column marker in figure: left 1 is TaSP feminine gender OD value, Left 2 is TaSP positive OD value, and left 3 is GST feminine gender OD value, and left 4 is GST positive OD value.
The mensuration of the optimal diluted concentration of Fig. 3 ELISA enzyme conjugates, often organizes in column marker in figure: left 1 is negative for TaSP OD value, left 2 is TaSP positive OD value.
The selection of the optimal confining liquid of Fig. 4 ELISA.
The determination of Fig. 5 ELISA threshold value, wherein: 0 is negative serum testing result；1 is positive serum testing result.
The test kit of Fig. 6 present invention is to different Virus monitory cross reaction experimental results.
Detailed description of the invention
(1) prepared by antigen
(1) method extracting genome from blood:
A) add 900 l BRC Cell Lysis Solution to be centrifuged in the blood 1.5ml containing 300 l infection annular loop detector Guan Zhong, reverse 10 times, is at room temperature placed into liquid clear.
B) 3000g is centrifuged 3 minutes, abandons supernatant, retains following visible white precipitate and about 10～20 μ l liquid, Vortex 20 seconds.
C) pipe often adds 300 l Cell Lysis Solution, piping and druming mixing.
D) 100 l Protein Preciptation Solution are added.
E) spiral 20 seconds, to brown precipitation granule occur.13000g is centrifuged 3 minutes.Supernatant is moved to new labelling Centrifuge tube in, add 300 l isopropanols, reverse 50 times.
F) 13000g is centrifuged 5 minutes.Abandon supernatant, clean napkin is placed, remaining liquid is blotted.
G) 300 l 70% ethanol are added.13000g is centrifuged 3 minutes, carefully removes ethanol, blots residue in absorbent paper Liquid.
H) it is vacuum dried at Speedcac.
I) 100 μ l DNA Hydration Solution are added.
J) 4 DEG C of overnight or 60 DEG C of incubations 1 hour.
K)-20 DEG C save backup.
(2) acquisition of antigen gene
In the PCR pipe of 200 L, add measuring samples in the most following ratio, set up standard positive, negative genes group DNA simultaneously
10×PCR Buffer (Mg 2+ plus) (TaKaRa) 12.50µL
dNTP Mixture (TaKaRa) 12.50µL
PCR Forward primer(10µmol) 1.00µL
PCR Reveres primer(10µmol) 1.00µL
Premix Taq(Ex TaqVersion 2.0) 0.25µL
DNA sample 2.00 L
Ultra-pure water 20.75 L of sterilizing
Cumulative volume 50.00 L
Whole operating process operates the most under aseptic conditions.PCR reaction condition: 94 DEG C of denaturations 4min；94℃ 1min, 53 DEG C of 1min, 72 DEG C of 1min, 40 circulations；72 DEG C extend 7min.Product 10g/L agarose gel electrophoresis.Its product Overnight it is connected with carrier pGEM-T Easy 4 DEG C.After converting JM109 competent cell, identify.Positive colony send company Order-checking.
(3) structure of recombinant expression carrier and qualification
The purified product obtained by expression primer amplification and pGEX-4T-1 vector plasmid use EcoR I+Xho I double digestion respectively, Reclaim purpose fragment.Enzyme action system is: purified product and each 10 μ L of pGEX-4T-1,10 × H buffer 3 μ L, 1.0mL/L BSA 3 μ L, 1.0 g/L Triton X-100 3 μ L, EcoR I 2 μ L, Xho I 2 μ L, aquesterilisa is mended to 30 μ L.Take carrier segments 10 μ L with Purpose fragment 10 μ L mixes, and adds 10 × connection buffer 1 μ L, T4 DNA ligase 0.5 μ L (350IU/mL), uses sterilizing Water is mended to 25 μ L, connects overnight in 16 DEG C after mixing, proceeds to BL21 competent cell, and picking list bacterium colony extracts plasmid after increasing bacterium, Carry out PCR, enzyme action and order-checking to identify, the correct named BL21(DE3 of recombinant expression plasmid will be identified)/pGEX-4T-1- TaSP, has carried out preservation by it in China typical culture collection center simultaneously, and deposit number is CCTCC NO:M2015645, Its preservation Classification And Nomenclature is: e. coli bl21 (DE3)/pGEX-4T-1-TaSP Escherichia coli BL21(DE3)/ pGEX-4T-1-TaSP。
(2) antigen purification
Then use BugBuster GSTBindpGEX-4T-1-TaSP recombiant protein purification, antigen purification process: (1) from Bacterium solution after the heart press lysate and bacterium solution 1:10 volume ratio mixing, be placed in 4 DEG C cracking 5h, the bacterium solution after cracking in 4 DEG C, 8000rpm is centrifuged 10min, takes supernatant.
(2) balance pillar
A. fully upset shakes up GST-Bind resin, causes in uniform suspension；
B. draw 2ml resin in chromatographic column, wait resin sedimentation；
C. it is down on post bed, along time following, add 1 × GST Bind/ of 5 times of volumes when store buffer liquid (20% ethanol) liquid level Wash Buffer (43mM Na2HPO4, 14.7mM KH2PO4, 1.37M NaCl, 27mM Kcl, Ph7.3) and in post, Clean resin；
D. treating that resin natural subsidence, 1 × GST Bind/Wash Buffer are down to below liquid level, so far prepared by chromatographic column, 4 DEG C save backup.
(3) post absorption is crossed
A. add in the pillar after the supernatant after restructuring propagation bacterium cracking extremely processes；
B. combine 1h under the conditions of 4 DEG C, make resin be in suspended state every the piping and druming of 10min pipettor；
C. wash post with 10 times of volume 1 × GST Bind/Wash Buffer, collect and be stored on ice through component.
With 3 times of volume 1 × GST Elution Buffer elution chromatography posts, collect eluent, be albumen after purification.
(3) coated elisa plate process
(1) by antigen, the most aforementioned albumen after purification, dilute in proportion with carbonate solution (0.1 mol/L, PH9.6), mixing After, the 96 every holes of orifice plate add amount of antigen and are about 3 μ g.After 37 DEG C hatch 1h, 4 DEG C overnight.
(2) abandon and be coated liquid, wash plate 3 times with PBST solution, dry.Add the PBST(phosphate containing 2 ‰ Tween-20 to delay Rush liquid) 2% gelatin that dilutes, 100 μ L/ holes.Close 1h for 37 DEG C.
(3) abandon confining liquid, wash plate 3 times with PBST solution, dry.Adding enzyme mark stabilizer, room temperature places 1h.
(4) abandon enzyme mark stabilizer, ELISA Plate natural air drying.
(5) the ELISA Plate evacuation after air-drying processes, 4 DEG C of preservations.
(4) standard female serum and the preparation of standard positive serum
Laboratory newly purchased from the cattle in ringless-type Taylor's insect infection area, selects three, is detected by blood smear and PCR method detection It is feminine gender, gathers and separate the serum of these three experiment cattle respectively, as standard female serum after mixing.Standard positive serum By two health experiment cattle of laboratory artificial challenge's annular loop detector, smear for microscopic examination combine PCR every day after infecting a week Detection, after being all found to be the infection positive, gathers every 5d and prepares the serum of experiment cattle, selects the serum that antibody titer is higher to mix The serum prepared after even, is
Standard positive serum.
(5) separation of serum to be checked
(1), after the whole blood gathered being stood 1h in 37 DEG C, place 4h for 4 DEG C.
(2) 3500rpm is centrifuged 15min under the conditions of 4 DEG C.
(3) serum separates out in faint yellow clear liquid, can short time preservation under the conditions of 4 DEG C after separating；Under the conditions of-20 DEG C Can preserve for a long time.
(6) detection process
(1) adding one and resist serum to be checked, every piece of ELISA Plate sets holes as one group (repetition), is provided with one group of blank altogether Group (an increase serum diluent), one group of standard female serum control group (serum is known standard female serum), one group of standard Positive serum controls group (serum is known standard positive serum), other serum to be checked a group every part.It is dilute that serum adds serum Releasing liquid and carry out 50 times of dilutions, every hole is put in 37 DEG C after adding 100 μ L serum and hatches 1h.
(2) add HRP ELIAS secondary antibody, abandon serum, wash plate 3 times with PBST solution, dry.(100 times dense for HRP ELIAS secondary antibody Contracting) in serum dilution, carry out 10000 times of dilutions, every hole is put in 37 DEG C after adding 100 μ L and hatches 1h.
(3) add substrate, abandon HRP ELIAS secondary antibody, wash with PBST solution, dry.Every hole adds 100 μ L substrates, and (TMB is molten Liquid) after be put in 37 DEG C of lucifuges and hatch 20min.
(4) terminating reaction, every hole adds 100 μ L stop buffers (0.2M sulfuric acid solution) and terminates reaction.
(5) survey OD value, after shaking gently uniformly, measure OD450nm value.
(7) thresholding criteria
Under optimum reaction condition, field acquisition is infected for the negative isolated feminine gender of Sanguis Bovis seu Bubali through PCR detection annular loop detector Serum, the positive serum that annular loop detector infects, carry out ELISA test simultaneously, testing result is through MedCalc software analysis, really Fixed positive threshold value (AbR value 29.2593).
(8) cross reaction experiment
In order to verify the specificity of the present invention, choose annular loop detector standard female serum, Babesia positive serum, Se Shi Taylor's worm positive serum, Theileria sinensis positive serum, Theileria luwenshuni positive serum, big Babesia positive serum, You Shi Taylor's worm positive serum, as the specificity of the comparison checking present invention, shows that specificity of the present invention is good (shown in Fig. 6), Ke Yizuo Method for Pathogen test.
(9) field sample judges
(1) positive findings:
It is fond of blood serum sample mean OD value >=(standard positive serum mean OD value-standard female serum mean OD value) × AbR Value+standard female serum mean OD value.
(2) negative findings:
Be fond of blood serum sample mean OD value < (standard positive serum mean OD value-standard female serum mean OD value) × AbR value+ Standard female serum mean OD value.
(6) detection example
ELISA detection kit involved in the present invention comprise to be coated detection annular loop detector TaSP antigen ELISA flat board, 5% defatted milk powder solution, annular loop detector positive control serum, rabbit anti-cattle ELIAS secondary antibody, substrate nitrite ion and stop buffer (2M Concentrated sulphuric acid).
Specific implementation process of the present invention:
1. close: in the plate well be coated antigen, add 5% defatted milk powder solution 300 μ L, 37 DEG C of incubation 2h, discard hole Interior solution, with PBST wash 3 times (washing methods: fill it up with PBST in ELISA hole, discards after the static 5min of room temperature, repeatedly for three times), Pat dry.Close and the most thoroughly can cause false positive.
2. serum-dilution and sample-adding: serum to be checked carries out 50 times of dilutions, the serum diluted by 100 μ L adds above-mentioned It is coated in the hole of antigen, has put 37 DEG C and hatch 1h, washed 3 times with PBST, pat dry.
3. add enzyme labelled antibody: in each reacting hole, add fresh rabbit anti-cattle ELIAS secondary antibody 100 μ L, hatch 1h for 37 DEG C, use PBST washs 3 times, pats dry.
4. add substrate nitrite ion: each reacting hole adds 100 μ LTMB, 37 DEG C of lucifuge colour developing 10min.
Note: the TMB of commercialization is the mixture of TMB and hydrogen peroxide, long-term illumination, high temperature all can for the most expired one month Affect the color developing effect of reagent.
5. terminate reaction: in each reacting hole, add 50 μ L 2M concentrated sulphuric acids.
6. result judges: can be in white background, and directly detect by an unaided eye result.In reacting hole, color is the deepest, positive journey Spend the strongest.Negative reaction is colourless or color is the most shallow.OD value can also be measured, on ELISA detector, at 450nm, with Survey each hole OD value after blank control wells zeroing, if measured value is higher than threshold values (AbR value 29.2593), be i.e. judged to the positive.
The method set up is entered by the present invention 95 parts of Ox blood serum samples and 45 parts of Gansu Ox blood serum sample from Xinjiang Row assessment.Wherein Sample Positive recall rate in Xinjiang is 23.11%, and Gansu positive rate is 8.89%.And by Chao Shi PCR couple Result above carries out the checking of coincidence rate, and display, its coincidence rate is 97.9%.
From above example, it is good that the detection kit of the present invention has specificity, the feature that sensitivity is high.Illustrate with TaSP recombiant protein is that the indirect ELISA detection method of antigen can be used for sample detection, carries out the inspection of annular loop detector infection And Epidemiological study.
<110>Lanzhou Veterinary Inst., Chinese Acedemy of Agaricultural Sciences
<120>cattle annular loop detector indirect ELISA antibody assay kit and preparation method thereof
<160> 3
<210> 1
<211> 1092
<212> DNA
<213>annular loop detector (Thaileria annultat) Tasp gene
<400>
atgtccccta tactaggtta ttggaaaatt aagggccttg tgcaacccac tcgacttctt 60
ttggaatatc ttgaagaaaa atatgaagag catttgtatg agcgcgatga aggtgataaa 120
tggcgaaaca aaaagtttga attgggtttg gagtttccca atcttcctta ttatattgat 180
ggtgatgtta aattaacaca gtctatggcc atcatacgtt atatagctga caagcacaac 240
atgttgggtg gttgtccaaa agagcgtgca gagatttcaa tgcttgaagg agcggttttg 300
gatattagat acggtgtttc gagaattgca tatagtaaag actttgaaac tctcaaagtt 360
gattttctta gcaagctacc tgaaatgctg aaaatgttcg aagatcgttt atgtcataaa 420
acatatttaa atggtgatca tgtaacccat cctgacttca tgttgtatga cgctcttgat 480
gttgttttat acatggaccc aatgtgcctg gatgcgttcc caaaattagt ttgttttaaa 540
aaacgtattg aagctatccc acaaattgat aagtacttga aatccagcaa gtatatagca 600
tggcctttgc agggctggca agccacgttt ggtggtggcg accatcctcc aaaatcggat 660
ctggttccgc gtggatccga tcgacaacgg aatcctatcg attttgatcc caatgatgat 720
caacagcctt tggaccctaa tcaactcata gatcaagaag aaccttctga acaacctact 780
caacaagaac caatagaacc acaacaacct acagaactag aaactacaga acccgaagag 840
ttggaaccag aaactgttac agtagaagtt ccagaacccg ttacatcaga agaacctaaa 900
gaatcggatc aaactgaaga acaaaaacac gaagaacctg aagcatttcc agctcctgaa 960
ccagttgatg aacccgcagt tcatgctact gaatctactc ctactaaggc aagttccagt 1020
ggtgatggag cagctgtttg tcatggaaaa catcatgatg atgactctga cggggctcga 1080
gcggccgcat cg 1092
<210> 2
<211> 364
<212> PRT
<213>annular loop detector (Thaileria annultat) Tasp gene
<400>
Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile Lys Gly Leu Val Gln
1 5 10 15
Pro Thr Arg Leu Leu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu Glu
16 20 25 30
His Leu Tyr Glu Arg Asp Glu Gly Asp Lys Trp Arg Asn Lys Lys
31 35 40 45
Phe Glu Leu Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Ile Asp
46 50 55 60
Gly Asp Val Lys Leu Thr Gln Ser Met Ala Ile Ile Arg Tyr Ile
61 65 70 75
Ala Asp Lys His Asn Met Leu Gly Gly Cys Pro Lys Glu Arg Ala
76 80 85 90
Glu Ile Ser Met Leu Glu Gly Ala Val Leu Asp Ile Arg Tyr Gly
91 95 100 105
Val Ser Arg Ile Ala Tyr Ser Lys Asp Phe Glu Thr Leu Lys Val
106 110 115 120
Asp Phe Leu Ser Lys Leu Pro Glu Met Leu Lys Met Phe Glu Asp
121 125 130 135
Arg Leu Cys His Lys Thr Tyr Leu Asn Gly Asp His Val Thr His
136 140 145 150
Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp Val Val Leu Tyr Met
151 155 160 165
Asp Pro Met Cys Leu Asp Ala Phe Pro Lys Leu Val Cys Phe Lys
166 170 175 180
Lys Arg Ile Glu Ala Ile Pro Gln Ile Asp Lys Tyr Leu Lys Ser
181 185 190 195
Ser Lys Tyr Ile Ala Trp Pro Leu Gln Gly Trp Gln Ala Thr Phe
196 200 205 210
Gly Gly Gly Asp His Pro Pro Lys Ser Asp Leu Val Pro Arg Gly
211 215 220 225
Ser Asp Arg Gln Arg Asn Pro Ile Asp Phe Asp Pro Asn Asp Asp
226 230 235 240
Gln Gln Pro Leu Asp Pro Asn Gln Leu Ile Asp Gln Glu Glu Pro
241 245 250 255
Ser Glu Gln Pro Thr Gln Gln Glu Pro Ile Glu Pro Gln Gln Pro
256 260 265 270
Thr Glu Leu Glu Thr Thr Glu Pro Glu Glu Leu Glu Pro Glu Thr
271 275 280 285
Val Thr Val Glu Val Pro Glu Pro Val Thr Ser Glu Glu Pro Lys
286 290 295 300
Glu Ser Asp Gln Thr Glu Glu Gln Lys His Glu Glu Pro Glu Ala
301 305 310 315
Phe Pro Ala Pro Glu Pro Val Asp Glu Pro Ala Val His Ala Thr
316 320 325 330
Glu Ser Thr Pro Thr Lys Ala Ser Ser Ser Gly Asp Gly Ala Ala
331 335 340 345
Val Cys His Gly Lys His His Asp Asp Asp Ser Asp Gly Ala Arg
346 350 355 360
Ala Ala Ala Ser
361 364
<210> 3
<211> 1092
<212> DNA
<213>annular loop detector (Thaileria annultat) Tasp gene
<400>
atg tcc cct ata cta ggt tat tgg aaa att aag ggc ctt gtg caa 45
Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile Lys Gly Leu Val Gln
1 5 10 15
ccc act cga ctt ctt ttg gaa tat ctt gaa gaa aaa tat gaa gag 90
Pro Thr Arg Leu Leu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu Glu
16 20 25 30
cat ttg tat gag cgc gat gaa ggt gat aaa tgg cga aac aaa aag 135
His Leu Tyr Glu Arg Asp Glu Gly Asp Lys Trp Arg Asn Lys Lys
31 35 40 45
ttt gaa ttg ggt ttg gag ttt ccc aat ctt cct tat tat att gat 180
Phe Glu Leu Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Ile Asp
46 50 55 60
ggt gat gtt aaa tta aca cag tct atg gcc atc ata cgt tat ata 225
Gly Asp Val Lys Leu Thr Gln Ser Met Ala Ile Ile Arg Tyr Ile
61 65 70 75
gct gac aag cac aac atg ttg ggt ggt tgt cca aaa gag cgt gca 270
Ala Asp Lys His Asn Met Leu Gly Gly Cys Pro Lys Glu Arg Ala
76 80 85 90
gag att tca atg ctt gaa gga gcg gtt ttg gat att aga tac ggt 315
Glu Ile Ser Met Leu Glu Gly Ala Val Leu Asp Ile Arg Tyr Gly
91 95 100 105
gtt tcg aga att gca tat agt aaa gac ttt gaa act ctc aaa gtt 360
Val Ser Arg Ile Ala Tyr Ser Lys Asp Phe Glu Thr Leu Lys Val
106 110 115 120
gat ttt ctt agc aag cta cct gaa atg ctg aaa atg ttc gaa gat 405
Asp Phe Leu Ser Lys Leu Pro Glu Met Leu Lys Met Phe Glu Asp
121 125 130 135
cgt tta tgt cat aaa aca tat tta aat ggt gat cat gta acc cat 450
Arg Leu Cys His Lys Thr Tyr Leu Asn Gly Asp His Val Thr His
136 140 145 150
cct gac ttc atg ttg tat gac gct ctt gat gtt gtt tta tac atg 495
Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp Val Val Leu Tyr Met
151 155 160 165
gac cca atg tgc ctg gat gcg ttc cca aaa tta gtt tgt ttt aaa 540
Asp Pro Met Cys Leu Asp Ala Phe Pro Lys Leu Val Cys Phe Lys
166 170 175 180
aaa cgt att gaa gct atc cca caa att gat aag tac ttg aaa tcc 585
Lys Arg Ile Glu Ala Ile Pro Gln Ile Asp Lys Tyr Leu Lys Ser
181 185 190 195
agc aag tat ata gca tgg cct ttg cag ggc tgg caa gcc acg ttt 630
Ser Lys Tyr Ile Ala Trp Pro Leu Gln Gly Trp Gln Ala Thr Phe
196 200 205 210
ggt ggt ggc gac cat cct cca aaa tcg gat ctg gtt ccg cgt gga 675
Gly Gly Gly Asp His Pro Pro Lys Ser Asp Leu Val Pro Arg Gly
211 215 220 225
tcc gat cga caa cgg aat cct atc gat ttt gat ccc aat gat gat 720
Ser Asp Arg Gln Arg Asn Pro Ile Asp Phe Asp Pro Asn Asp Asp
226 230 235 240
caa cag cct ttg gac cct aat caa ctc ata gat caa gaa gaa cct 765
Gln Gln Pro Leu Asp Pro Asn Gln Leu Ile Asp Gln Glu Glu Pro
241 245 250 255
tct gaa caa cct act caa caa gaa cca ata gaa cca caa caa cct 810
Ser Glu Gln Pro Thr Gln Gln Glu Pro Ile Glu Pro Gln Gln Pro
256 260 265 270
aca gaa cta gaa act aca gaa ccc gaa gag ttg gaa cca gaa act 855
Thr Glu Leu Glu Thr Thr Glu Pro Glu Glu Leu Glu Pro Glu Thr
271 275 280 285
gtt aca gta gaa gtt cca gaa ccc gtt aca tca gaa gaa cct aaa 900
Val Thr Val Glu Val Pro Glu Pro Val Thr Ser Glu Glu Pro Lys
286 290 295 300
gaa tcg gat caa act gaa gaa caa aaa cac gaa gaa cct gaa gca 945
Glu Ser Asp Gln Thr Glu Glu Gln Lys His Glu Glu Pro Glu Ala
301 305 310 315
ttt cca gct cct gaa cca gtt gat gaa ccc gca gtt cat gct act 990
Phe Pro Ala Pro Glu Pro Val Asp Glu Pro Ala Val His Ala Thr
316 320 325 330
gaa tct act cct act aag gca agt tcc agt ggt gat gga gca gct 1035
Glu Ser Thr Pro Thr Lys Ala Ser Ser Ser Gly Asp Gly Ala Ala
331 335 340 345
gtt tgt cat gga aaa cat cat gat gat gac tct gac ggg gct cga 1080
Val Cys His Gly Lys His His Asp Asp Asp Ser Asp Gly Ala Arg
346 350 355 360
gcg gcc gca tcg 1092
Ala Ala Ala Ser
361 364

Claims (4)

1. a cattle annular loop detector indirect ELISA antibody assay kit, it is characterised in that include in test kit and be coated with The ELISA Plate of TaSP recombiant protein.
Cattle annular loop detector indirect ELISA antibody assay kit the most according to claim 1, it is characterised in that test kit In also have: serum dilution, HRP enzyme labelling two are anti-, standard positive control serum, standard negative control serum, substrate solution, end Only liquid and PBST concentrated solution.
3. the ELISA Plate preparation side in the cattle annular loop detector indirect ELISA antibody assay kit described in claim 1 or 2 Method, it is characterised in that by using infect annular loop detector blood extract genomic DNA as positive template, with primer to SEQ ID The product of No.1 and SEQ ID No.2 amplification is connected to pGEX-4T-1 carrier, then pGEX-4T-1 carrier is proceeded to BL21 large intestine Abduction delivering in bacillus, for coated elisa plate after expression product is purified, then carries out vacuum after adding enzyme stabilizers wherein Close.
4. annular loop detector recombinant expression plasmid BL21(DE3)/pGEX-4T-1-TaSP, it is protected at Chinese Typical Representative culture The deposit number of center, Tibetan preservation is CCTCC NO:M2015645, and its preservation Classification And Nomenclature is: e. coli bl21 (DE3)/ PGEX-4T-1-TaSP Escherichia coli BL21(DE3)/pGEX-4T-1-TaSP.
CN201510974076.9A 2015-12-23 2015-12-23 Bovine theileria annulata indirect ELISA antibody detection kit and preparation method thereof Pending CN105866421A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201510974076.9A CN105866421A (en) 2015-12-23 2015-12-23 Bovine theileria annulata indirect ELISA antibody detection kit and preparation method thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201510974076.9A CN105866421A (en) 2015-12-23 2015-12-23 Bovine theileria annulata indirect ELISA antibody detection kit and preparation method thereof

Publications (1)

Publication Number Publication Date
CN105866421A true CN105866421A (en) 2016-08-17

Family

ID=56624403

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201510974076.9A Pending CN105866421A (en) 2015-12-23 2015-12-23 Bovine theileria annulata indirect ELISA antibody detection kit and preparation method thereof

Country Status (1)

Country Link
CN (1) CN105866421A (en)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108627649A (en) * 2017-08-09 2018-10-09 新疆农业大学 Indirect ELISA testing kit preparation method based on horse Taylor's worm merozoite surface protein-1
CN112630446A (en) * 2020-12-16 2021-04-09 中国人民解放军海军军医大学 Bioactive molecule combined target identification method based on double-head photoaffinity probe
CN113563450A (en) * 2021-07-29 2021-10-29 中国农业科学院兰州兽医研究所 Cyclic Theileria taenii TA04380 truncated protein and application thereof

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DIA ELDIN A.BAKHEIT,ET AL.: "Validation of the indirect Ta SP enzyme-linked immunosorbent assay for diagnosis of Theileria annulata infection in cattle", 《PARASITOL RES.》 *
M.A.BAKHEIT, ET AL.: "Application of the recombinant Theileria annulata surface protein in an indirect ELISA for the diagnosis of tropical theileriosis", 《PARASITOL RES.》 *
郑进峰: "环形泰勒虫Tasp基因的克隆、原核表达及PCR诊断方法的建立", 《中国优秀硕士学位论文全文数据库（电子期刊）农业科技辑》 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108627649A (en) * 2017-08-09 2018-10-09 新疆农业大学 Indirect ELISA testing kit preparation method based on horse Taylor's worm merozoite surface protein-1
CN112630446A (en) * 2020-12-16 2021-04-09 中国人民解放军海军军医大学 Bioactive molecule combined target identification method based on double-head photoaffinity probe
CN113563450A (en) * 2021-07-29 2021-10-29 中国农业科学院兰州兽医研究所 Cyclic Theileria taenii TA04380 truncated protein and application thereof

Similar Documents

Publication Publication Date Title
CN113009154B (en) Novel one-step method coronavirus neutralizing antibody magnetic microsphere detection kit and application thereof
CN105527442B (en) A kind of hog cholera antibody detecting system and preparation method thereof
CN106432440B (en) PPA-ELISA (polymerase chain reaction-enzyme linked immunosorbent assay) detection kit for Brucella melitensis antibody and preparation method of PPA-ELISA detection kit
CN103308684A (en) ELISA (enzyme-linked immuno sorbent assay) detection kit of porcine pseudorabies virus IgM antibody
CN105866421A (en) Bovine theileria annulata indirect ELISA antibody detection kit and preparation method thereof
Rimstad et al. Detection of antibodies to caprine arthritis-encephalitis virus using recombinant gag proteins
CN203838160U (en) ELISA detection kit for gB and gE antibodies of porcine pseudorabies virus
CN108715857A (en) A kind of bovine coronavirus VPN genes, the recombinant protein of coding and application
CN207623349U (en) A kind of porcine pseudorabies virus antibody assay kit
CN105061602B (en) For detecting fusion protein, the preparation method and application of anti-pig enterotoxigenic escherichia coil antibody
CN109307772A (en) A kind of Pseudorabies virus gE and gB IgG antibody double fluorescent microballoon immunological detection method
Wood et al. Evaluation of a commercial monoclonal antibody-based enzyme immunoassay for detection of adenovirus types 40 and 41 in stool specimens
CN109239341A (en) A kind of indirect ELISA reagent kit of ox Mannheimia haemolytica antibody test and its application
CN104155443A (en) Indirect ELISA (enzyme-linked immunosorbent assay) detection kit based on toxoplasma gondii matrix protein 1
LIU et al. A serodiagnostic ELISA using recombinant antigen of swine transmissible gastroenteritis virus nucleoprotein
CN110376388A (en) A kind of haemophilus parasuis antibody detection method and its kit
CN109799342A (en) A kind of O-shaped antibodies against foot-and-mouth disease virus competitive ELISA detection kit
CN109655610A (en) A kind of indirect ELISA testing kit of pseudorabies virus
CN109187952A (en) One boar atypia pestivirus ELISA antibody assay kit
Ferreira et al. Expression and analysis of the diagnostic value of an Echinococcus granulosus antigen gene clone
CN109884314B (en) Indirect ELISA antibody detection method for porcine epidemic diarrhea virus S2 protein and kit thereof
Deng et al. Immuno-PCR for detection of Giardia lamblia cysts in water
CN109142757A (en) A kind of brucella indirect ELISA testing kit
CN112213493B (en) Peste des petits ruminants detection kit capable of being used for distinguishing vaccine immunity from natural infection
CN111273005A (en) Enzyme linked immunosorbent assay kit and method for detecting toxoplasma gondii IgG antibody

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
WD01 Invention patent application deemed withdrawn after publication
WD01 Invention patent application deemed withdrawn after publication

Application publication date: 20160817