CN104569425B - Antigen protein specifically bound with tyrosine phosphatase antibody - Google Patents
Antigen protein specifically bound with tyrosine phosphatase antibody Download PDFInfo
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- G—PHYSICS
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- 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
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- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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- G01N21/76—Chemiluminescence; Bioluminescence
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- G01N2800/00—Detection or diagnosis of diseases
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Abstract
The invention relates to the technical field of antigen-antibody protein, and particularly relates to the technical field of truncated tyrosine phosphatase (IA-2) for detecting diabetes mellitus. One aim of the invention is to screen a truncated antigen protein specifically bound with IA-2 antibody, and the other aim of the invention is to develop antigen protein which is capable of fulfilling a chemiluminiscence closed system and applicable to the chemiluminiscence closed system. According to the antigen protein specifically bound with the IA-2 antibody, the antigen protein is one of 600th-979th, 604th-979th, 604th-969th, 600th-969th and 709th-969th truncated proteins. The invention further provides the truncated protein or IA-2 full-length protein which is coated with nano-magnetic beads, and a kit comprising the protein.
Description
Technical field
The present invention relates to the technical field of Ag-Ab protein, particularly to the cheese of the truncate for detecting diabetes
The technical field of propylhomoserin phosphatase (IA-2).
Background technology
Diabetes be a kind of because of insulin secretion definitely or generation that relative deficiency and the hyperglycemia that causes are principal character
Thanking property disease, is affected by multiple h and E factors.According to ADAs in 1997 and world health group in 1999
Knit (WHO) with regard to diabetes classification and diagnostic criteria, diabetes can be divided into I type, II type, specific type diabetes and glycosuria gravidarum
Disease.
During the immune destruction of type 1 diabetes, can produce in the patient multiple for islet cell autoantigen from
Body antibody.It has been found that multiple insulin autoantibody since 20 century 70s, for example：Insular cellular antibody
(ICA), ICSA (ICSA), insulin antibody (IAA), heat shock protein antibody (HSP), glutamate decarboxylase
65 (GAD65, one of existence form of GAD in mammal) antibody, Protein Tyrosine Phosphatases antibody (IA-2A) etc..Mesh
The front index clinically commonly used in diagnosis type 1 diabetes includes ICA, IAA, GADA, IA-2A.
The current clinically platform used by in-vitro diagnosis type 1 diabetes index is all based on putting exempts from or Enzyme-multiplied immune technique
(ELASA), autoimmunity type 1 diabetes diagnostic reagent needs further improvement in terms of Sensitivity and Specificity.Tradition
Radioimmunity or ELISA method detection time long, at least 6 hours, in addition reaction overnight just can complete test, mainly according to
By sequence of maneuvers such as pure manual sample-addings, efficiency is low, and specificity is not enough.Chemiluminescence system have high sensitivity, high specific excellent
Gesture, the testing result time is fast, efficiency high, and whole full-automatic instrument is automatically brought into operation, and 1 hour can complete to test, and cures to present
The timely diagnoses and treatment of institute plays very big effect.
Application in diagnosing in vitro with the higher chemiluminescence of Sensitivity and Specificity, develops can be suitably used for
The antigen learning luminous closed system becomes to be even more important.
Content of the invention
An object of the present invention is the antigen filtering out with the truncate of tyrosine phosphatase (IA-2) antibody specific bond
Protein.The two of purpose are that exploitation can meet the antigen protein being applied to chemiluminescence closed system, and are applied to chemiluminescence
Closed system.
Compared with the expression of full length protein, the albumen after truncate is easier to express for the expression of the antigen protein of truncate,
Expression is higher；When for detecting diabetes,, compared with full length protein, the albumen sensitivity after truncate is more preferable for it；Non- spy
The opposite sex is lower.
The invention provides a kind of and diabetes specific antibodies specific bond antigen protein, described antigen protein choosing
SEQ ID NO freely:The truncated protein matter of 600-979,604-979,604-969, the 600-969 and 709-969 position in 17
One of.
In a specific embodiment, described antigen protein is selected from such as SEQ ID NO:604-969 in 17,
One of truncated protein matter of 600-969 and 709-969 position.
In a specific embodiment, described antigen protein is selected from such as SEQ ID NO:709-969 position in 17
Truncated protein matter.
In a specific embodiment, the protein that described antigen protein obtains for eukaryotic expression system.
In a specific embodiment, the protein that described antigen protein obtains for insect cell expression system.Elder brother
Worm cell expression system is the more eukaryotic expression system of application, and it can express various exogenous genes in insect cell, bag
Include funguses, plant, antibacterial, the gene of virus.Baculoviruss are the double-stranded DNA viruses with insect cell as natural host for the class,
There is the species specificity of height, do not infect vertebratess, harmless to people, animal.What research was more at present is autographa california
Core polyhedral body (AcNPV), its host is Spodopterafrugiperda.And the host cell of SF9 cell, exactly baculovirus expression system it
One.SF9 cell belongs to half adherent type cell line, can carry out suspension culture it is also possible to carry out adhere-wall culture, be applied to albumen
Expression with prepare in purification, have the advantages that many：1) compared with other eukaryotic expression systems, foreign gene expression levels are high,
Particularly intracellular protein.In most cases, the recombiant protein of expression is solvable, folds and is just truly having post translational modification (as glycosylation
Deng), there is biologic activity, can be easily separated purification；2) utilize insect cell suspension growth, easy amplification culture, be conducive to advising greatly
Mould expresses recombiant protein；3) be easy to expressing heterologous multimeric protein, can by multiple recombinant viruses simultaneously infected insect cell or
Realized with a kind of virus infected insect cell containing multiple expression cassettes；4) insect baculovirus do not infect vertebratess, tool
There is safety.
Present invention also offers a kind of antigen protein, described antigen protein is protein or the IA-2 of above-mentioned truncated-type
Full length protein, and described protein be the coated protein of nanometer magnetic bead.
Invention further provides a kind of test kit for detecting diabetes, described test kit includes a kind of antigen protein
Matter, described antigen protein is selected from such as SEQ ID NO:600-979,604-979,604-969,600-969 in 17 and
One of truncated protein matter of 709-969 position.
In a kind of specific embodiment, the described antigen protein in described test kit is selected from such as SEQ ID NO:In 17
One of the truncated protein matter of 604-969,600-969 and 709-969 position.
In a specific embodiment, described antigen protein is selected from such as SEQ ID NO:709-969 position in 17
Truncated protein matter.It is pointed out that during detection antibody, selected antigen protein not full length protein
It is optimum, because while full length protein contains more antigenic determinants, so easier can identify that it is corresponding
Antibody, but because full length protein comprises more antigenic determinant, also bring along the result that increases of non-specificity；This
Outward, the fragment length of protein is longer, and the difficulty of its expression a series of operation such as difficulty and follow-up purification also can be corresponding
Higher.Accordingly, it is preferred that antigen protein should be first-selection meet susceptiveness and specific under the premise of, the fragment of protein
More short, more it is advantageously applied to reality.For example, in the detecting system of chemoluminescence method, the IA-2 for detecting diabetes resists
The protein of the optimum of body is as SEQ ID NO:The truncated protein matter of the 709-969 position in 17.
In a specific embodiment, the described antigen protein in described test kit is table in eukaryotic expression system
The protein reaching and obtaining.
In a specific embodiment, the described antigen protein in described test kit is in insect cell expression system
Middle expression and obtain protein.
In a specific embodiment, the described antigen protein in described test kit is the coated albumen of nanometer magnetic bead
Matter.
In a specific embodiment, also include the protein A of chemiluminescent labeling in described test kit.Sent out using chemistry
Light method can improve sensitivity and the specificity of product.Wherein, protein A is a kind of vegetarian protein A matter,
Can specifically be combined with the Fc area of people and mammalian antibody (mainly IgG).
In a specific embodiment, in described test kit, also include also including BSA buffer in described test kit.Institute
State the buffer that BSA buffer is high concentration, its concentration can be 3%～7%.
Present invention also offers a kind of gene order of the antigen protein with diabetes specific antibodies specific bond, described
Gene order is to obtain such as SEQ ID NO by expression:600-979,604-979,604-969,600-969 in 17 and
One of described gene order of truncated protein matter of 709-969 position.
In a specific embodiment, described gene order is to obtain such as SEQ ID NO by expression:In 17
One of described gene order of truncated protein matter of 604-969,600-969 and 709-969 position.
In a specific embodiment, described gene order is to obtain such as SEQ ID NO by expression:In 17
The gene order of the described truncated protein matter of 709-969 position.
In a specific embodiment, described gene order is expressed in eukaryotic expression system.
In a specific embodiment, described gene order is expressed in insect cell expression system.
The present invention goes out suitable IA-2 truncated protein matter antigen using eukaryotic expression system expression screening, and is applied to
Chemiluminescence closed system, fills up blank in the market.
Specific embodiment
With reference to embodiment, the present invention is described below.
Embodiment 1
The expression of the gene of the hIA-2 of hIA-2 and truncate
(1) main agents and equipment
PCR kit (comprising Taq enzyme, buffer, dNTP, T4 ligase) and DNA marker are purchased from full formula gold；Albumen
It is biological that Marker is purchased from the green skies；The restricted enzyme such as BamH I, Xho I, EcoR I, Nco I, Hind III are purchased from Takara
(Dalian is precious biological)；Calf intestine alkaline phosphatase (CIAP) test kit (comprises component：Calf intestine alkaline phosphatase, alkaline phosphatase
Enzyme buffer liquid) it is purchased from Takara；Glue reclaim test kit and plasmid extraction kit are purchased from OMEGA company；Kanamycin and ammonia benzyl
Purchased from BBI；IPTG is purchased from BBI；PCR primer and recombiant plasmid sequencing are completed by Hua Da gene or Invitrogen；Remaining reagent
There is provided by Shenzhen New Industries Biomedical Engineering Co., Ltd..
ABI Veriti PCR instrument is purchased from ABI, and horizontal cataphoresis apparatus RDY-SP1Z is purchased from the upper seamount richness limited public affairs of scientific instrument
Department, is purchased from Shanghai than bright Instrument Ltd. than bright constant-temperature table COS-2102C, superclean bench BSC-1000II A2 is purchased from
Shanghai Boxun Industrial Co., Ltd., centrifuge is purchased from Sigma, and inverted microscope is purchased from Olympus, and vertical electrophoresis apparatus are purchased from
BioRad, transferring film instrument is purchased from BioRad, and gel imaging instrument 805 is purchased from upper seamount richness, and remaining equipment is biological by Shenzhen's NPD projects
Engineering in medicine limited company provides.
(2) cell strain, bacterial strain and plasmid
Human IA-2 (hIA-2) is purchased from OriGene；PFastBac1 carrier, SF9 cell, corresponding culture medium and its place
Main DH10Bac is purchased from life technology；Escherichia coli (Escherichia coli) competent cell Trans5 α is purchased from
Beijing full formula gold.
(3) design of primer and synthesis
Using Primer primer5.0, hIA-2 section is expanded according to the cDNA sequence design of hIA-2 (NM_002846.3)
Total length and its specific primer of section, using EcoR I and Sal I as restriction enzyme site, forward primer increases initial design of primers
Codon, reverse primer increases termination codon, and introduces EcoR I and Sal I enzyme action position respectively at the two ends of its sequence respectively
Point, primer sequence such as SEQ ID NO:1-SEQ ID NO:15.Details are shown in Table 1.
Table 1
(4) gene of the hIA-2 of hIA-2 and truncate is expressed in insect cell expression system
With the cDNA plasmid of the people of purchase as template, using corresponding upstream and downstream primer, PCR amplifies purpose band, uses
Glue reclaim test kit reclaims the fragment of genes of interest, then uses EcoR I and Sal I double digestion pFastBac1 carrier and pure respectively
The purpose fragment changed.Enzyme action reclaims hIA-2 fragment and pFastBac1 linear carrier with glue reclaim test kit direct purification after terminating
Digestion products.It is directly added into 1 μ l CIAP in the linear carrier after enzyme action, 37 DEG C of reactions 1 hour (h) carry out dephosphorization process
(choosing is done).With T4 ligase enzyme action purpose fragment after purification and linear carrier fragment, (ratio of both molal quantitys is 1:2～
1:10), linked system is 10 μ l, and 16 DEG C connect overnight.
Linked system is converted escherichia coli (E.coli) Trans5 α.Converted product is coated (the 100 μ g/ of benzyl containing ammonia
Ml in LB solid medium), 37 DEG C of incubated overnight.The random multiple single bacterium colony of picking on the LB flat board containing restructuring rod granule,
Carry out bacterium solution PCR and double digestion sequence verification positive.The positive rod granule carrier obtaining is converted to DH10BacTMIn, from
In filter out positive colony.Then positive restructuring rod granule is transfected to insect cell SF9, incubated cell 72h at 27 DEG C, directly
To observe virus infection sign harvest baculoviruss.
Collect the cell through culture, extract albumen, then adopt affinitive layer purification recombiant protein, then handed over using ion
Change post and be further purified albumen so as to purity >=95%.
Embodiment 2
Measure 14 antigens of above-mentioned insect cell expression sensitivity and specificity to hIA-2 antibody
Chemiluminescence immunoassay sandwich assay principle：Test serum sample, buffer and antigen coat magnetic microsphere add reaction
React in cup, at 37 DEG C, in test serum sample, antibody and the antigen being coated on magnetic microsphere occur immunoreation, magnetic field
In the case of nanometer magnetic bead can be magnetized rapidly, this complex through over cleaning, obtains the complex of antigen and test antibodies, other
Composition is cleaned out, and adds labelled protein A-NHS-ABEI afterwards, and protein A can be tied rapid and test antibodies C-terminal specificity
Close, form " sandwich sandwich " immune complex.When detected antibody content in serum sample is more, the phase that instrument detects
Higher to light intensity RLU, that is, it is proportionate.
(1) being coated of antigen
Measure the concentration of A2, B2, C2, D2, E2, F2, G2, H2, I2, J2, K2, L2, M2 and N2 protein.
From Merck company, the rate of charge of antigen coat nanometer magnetic bead and antigen is 1mg for nanometer magnetic bead buying:10 μ g, room temperature
Temperature bath 2 hours, is placed in 250 turns/min on vortex mixer, after under magnetic field cleaning remove supernatant, obtain magnetic bead-COOH-NHS- antigen.
Wherein, dilute the nanometer magnetic bead of envelope antigen with phosphate buffer, dilution ratio is 1:20 obtain working concentration.
(2) preparation of luminous marker different luminol ABEI
Using Protein A/Protein labelling shiner ABEI, i.e. protein A-NHS-ABEI.
Dilute the luminous marker protein A-NHS-ABEI that labelling completes with phosphate buffer, ratio is 1:1000.
(3) preparation of buffer
Buffer is phosphate buffer, 5%BSA, pH6.8.
(4) test apparatuses
The automatic chemiluminescence immunoassay of Shenzhen New Industries Biomedical Engineering Co., Ltd.'s development ＆ production
Instrument Maglumi 2000Plus, sequence number：2000200068.
(5) sample
Choose regular sample this 20, and 20, the type i diabetes sample of clinical definite.RSR company with Britain
IA-2ELISA test kit be matched group.Measurement result is shown in Table 2 and table 3.
Table 2
Table 3
With the result of the Elisa test kit of RSR company as foundation, in Elisa testing result, the antibody concentration of normal person
Term of reference is<7IU/ml is that is to say, that when antibody concentration is less than 7IU/ml, then judge to be practically free of anti-tyrosine phosphatase
Enzyme antibody, as negative；When antibody concentration is more than or equal to 7IU/ml, then judge to contain anti-tyrosine phosphatase in sample to be tested
Enzyme antibody, as positive.The result of table 2 and table 3 shows, on chemiluminescence platform, the available antigenic domains of IA-2 be A2 or
Separate obvious of the relative light intensity RLU of B2, normal sample and exceptional sample；Preferably antigenic domains are C2 or D2, profit
The RLU of the normal sample being measured with it is respectively less than 20000, and the RLU of weak sun and positive sample is all higher than 45000；More preferably
Antigenic domains are N2, and the RLU of the normal sample being measured using it is respectively less than 10000, and the RLU of positive sample is all higher than 50000,
Non-specific lower, specific reaction becomes apparent from.Other fragments all have missing inspection or normal person's sample virtual height phenomenon.
Embodiment 3
Prokaryotic expression is used for type i diabetes detection with the protein of eukaryotic expression and compares
In the truncated-type protein N 2 of expression in escherichia coli hIA-2, the DNA section of above-mentioned hIA-2 truncate is connected to
On pGEX4T-1 expression vector, escherichia coli expression strain is BL21 (DE3).Expression product through GST label after purification, then is used
After Thrombin excision GST label, obtain the destination protein of no label through affinity chromatograph.
The N2 measuring prokaryotic expression is to the sensitivity of hIA-2 antibody and specificity.
The symbol of the prokaryotic expression section of hIA-2 truncated-type is PN2, and it is corresponding with the N2 of eukaryotic expression.
Detection platform and scheme details, with embodiment 1, the results are shown in Table 4.
Table 4
Compared with the result of N2, the truncated-type hIA-2 proteantigen of prokaryotic expression is used for examining the result of the PN2 in table 4
The non-specific binding surveying normal person's sample is substantially high than eukaryotic expression antigen-non-specific combines, and weakly positive sample and moderate are positive
Property sample signal is also substantially low, and strong positive sample is also more much lower than eucaryon antigen valence.As can be seen here, eukaryotic cell expression is excellent
In procaryotic cell expression.
Embodiment 4
The detection of type i diabetes
Detection platform is with embodiment 1.Truncated-type hIA-2 is N2.
10 points of methods determine standard curve：The original concentration of standard substance (IA-2 antibody, Beijing Bo Aosen company) is 5600IU/
Ml, preserves in -20 DEG C；Press 1 with sandwich assay diluent:20 dilutions.The standard substance of dilution are measured its corresponding values of chemiluminescence,
Obtain the functional relation of standard concentration and RLU according to the relation of standard concentration and its corresponding values of chemiluminescence, that is, mark
Directrix curve.
Calculate the concentration of 228 normal person's samples, and the concentration of the sample of 108 type i diabetes using standard curve
(being shown in Table 5), and detect whether the actual concentrations of World Health Organization (WHO) (WHO) standard substance are consistent with corresponding demarcation concentration.
Wherein, the normal concentration of WHO standard product is 100IU/ml.
With the IA-2Ab ELISA kit of RSR company of Britain for comparison, this kit antigen is eukaryotic cell expression
IA-2 total length antigen.
Result
Table 5 detects the result of 108 type i diabetes
By measuring 228 normal person's testing results, according to statistical analysis, draw the normal person's that judges this test kit
Threshold values is<30IU/ml, and the positive is >=30IU/ml.Meanwhile, the actual concentrations of WHO standard product demarcation concentration corresponding thereto
In the range of error allowing.
Through data analysiss understand (being shown in Table 5), in the detection of the Elisa test kit using RSR company, the 21st, 78,79 and
No. 93 samples are shown as negative；But the N2 albumen of this programme is shown as positive using Chemiluminescence immunoassay testing result, thus
Show the 21st, 78,79 and No. 93 samples actual be type i diabetes patient, this programme improves the sensitive of inspection in other words
Degree and specificity, this also means that this programme can reduce detection leakage phenomenon, it can thus be appreciated that this programme testing result more meets clinic.
By testing result on chemiluminescence immunoassay platform for the hIA-2 antigen of the optimization expression in this programme：
Its positive rate=84/108*100%=77.78%.
And it is used for the IA-2 positive rate=80/108*100%=74.07% in the RSR company contrasting.
Can be drawn by above-mentioned data to draw a conclusion：
Application on the chemiluminescence platform for-N2 of the optimized expression of hIA-2 improves the Detection results of test kit, also
It is to say that its positive rate is better than contrast agent box.Therefore, application on chemiluminescence platform for the N2 improves the sensitive of detection
Degree and specificity, it is to avoid the missing inspection of ELISA or false-positive phenomenon occurs.
Claims (10)
1. a kind of antigen protein with tyrosine phosphatase enzyme antibody specific bond is it is characterised in that described antigen protein selects
SEQ ID NO freely:The truncated protein matter of 600-979,604-979,604-969, the 600-969 and 709-969 position in 17
One of.
2. antigen protein according to claim 1 is it is characterised in that described antigen protein is selected from such as SEQ ID NO:
One of truncated protein matter of 604-969,600-969 and 709-969 position in 17.
3. antigen protein according to claim 2 is it is characterised in that described antigen protein is selected from such as SEQ ID NO:
The truncated protein matter of the 709-969 position in 17.
4. the antigen protein according to claim 1-3 any one is it is characterised in that described antigen protein is true
The protein expressed in nuclear expression system and obtain.
5. antigen protein according to claim 4 is it is characterised in that described antigen protein is in insect cell expression
The protein expressed in system and obtain.
6. a kind of antigen protein is it is characterised in that described antigen protein is according to claim 1-5 any one
Antigen protein, and described protein is the coated protein of nanometer magnetic bead.
7. a kind of test kit for detecting diabetes is it is characterised in that described test kit is included as described in claim 1-6
Antigen protein.
8. test kit according to claim 7 is it is characterised in that also include the egg of chemiluminescent labeling in described test kit
White A.
9. the test kit according to claim 7 or 8 is it is characterised in that also include BSA buffer in described test kit.
10. a kind of gene order of the antigen protein with diabetes specific antibodies specific bond is it is characterised in that described gene
Sequence is one of gene order of antigen protein by expression acquisition as described in claim 1-5 any one.
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| CN107044977A (en) * | 2016-06-30 | 2017-08-15 | 深圳市亚辉龙生物科技股份有限公司 | A kind of tyrosine phosphatase antibody chemical luminescence immunity detection reagent and preparation method thereof |
| CN108414766A (en) * | 2018-01-29 | 2018-08-17 | 上海良润生物医药科技有限公司 | Kit for quantitatively detecting diabetes autoantibody and its application |
| CN110261597A (en) * | 2019-06-06 | 2019-09-20 | 三诺生物传感股份有限公司 | A kind of IA-2 autoantibody detection strip |
| CN114924071A (en) * | 2022-03-29 | 2022-08-19 | 北京世纪沃德生物科技有限公司 | Anti-tyrosine phosphatase antibody determination kit |
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| CN1302899A (en) * | 1999-10-29 | 2001-07-11 | 国家人类基因组南方研究中心 | Human protein tyrosine phosphatase and its coding sequence |
| GB0128583D0 (en) * | 2001-11-28 | 2002-01-23 | Rsr Ltd | Detection of autoantibodies indicative of diabetes |
| CN202502099U (en) * | 2012-03-21 | 2012-10-24 | 山东东兴生物科技股份有限公司 | Joint detection reagent strip using colloidal gold method for diabetes autoantibody |
| CN102967704B (en) * | 2012-11-26 | 2014-05-14 | 深圳市伯劳特生物制品有限公司 | Kit for combined detection of 6 diabetic antibodies |
| CN203672887U (en) * | 2014-01-17 | 2014-06-25 | 中国人民解放军第三军医大学第一附属医院 | Joint detection kit for immunofiltration of diabetes autoantibodies |
| CN103969234B (en) * | 2014-04-17 | 2017-02-15 | 山东东兴汇智生物科技有限公司 | Luciferase- poly-antigen fusion protein and protein A agarose-fusion protein-antibody complex |
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