CN104560911B - A kind of fusion antigen protein matter - Google Patents
A kind of fusion antigen protein matter Download PDFInfo
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- CN104560911B CN104560911B CN201410802191.3A CN201410802191A CN104560911B CN 104560911 B CN104560911 B CN 104560911B CN 201410802191 A CN201410802191 A CN 201410802191A CN 104560911 B CN104560911 B CN 104560911B
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
- C12N9/0014—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4)
- C12N9/0016—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4) with NAD or NADP as acceptor (1.4.1)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y104/00—Oxidoreductases acting on the CH-NH2 group of donors (1.4)
- C12Y104/01—Oxidoreductases acting on the CH-NH2 group of donors (1.4) with NAD+ or NADP+ as acceptor (1.4.1)
- C12Y104/01002—Glutamate dehydrogenase (1.4.1.2)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y301/00—Hydrolases acting on ester bonds (3.1)
- C12Y301/03—Phosphoric monoester hydrolases (3.1.3)
- C12Y301/03048—Protein-tyrosine-phosphatase (3.1.3.48)
Abstract
The present invention relates to a kind of fused proteins.The fused protein is that the fusion antigen protein matter is the protein that 65 gene of glutamte dehydrogenase or 65 gene of truncated glutamte dehydrogenase are obtained with tyrosine phosphatase enzyme gene or truncated tyrosine phosphatase enzyme gene by amalgamation and expression.In addition, the present invention also provides the kits for being used to detect diabetes including the fusion antigen protein matter.
Description
Technical field
The present invention relates to a kind of fusion antigen protein matter, more particularly to glutamte dehydrogenase 65 or truncated glutamate dehydrogenase
The fused protein technical field of enzyme 65 and tyrosine phosphatase or the amalgamation and expression of truncated tyrosine phosphatase.
Background technology
Diabetes be it is a kind of because insulin secretion definitely or relative deficiency caused by hyperglycemia be main feature generation
Thanking property disease is influenced by a variety of h and E factors.According to American Diabetes Associations in 1997 and world health group in 1999
I types, II types, specific type diabetes and glycosuria gravidarum can be divided on diabetes classification and diagnostic criteria, diabetes by knitting (WHO)
Disease.
During the immune destruction of type 1 diabetes, patient's body can generate it is a variety of for islet cell autoantigen from
Body antibody.Since the 1970s, it has been found that a variety of insulin autoantibodies, such as：Islet cell antibodies
(ICA), islet cell surface antibody (ICSA), insulin antibody (IAA), heat shock protein antibody (HSP), glutamate decarboxylase
65 (GAD65, one of existence form of GAD in mammal) antibody, protein tyrosine phosphatase antibody (IA-2A) etc..Mesh
It is preceding clinically to include ICA, IAA, GADA and IA-2A in the diagnosis common index of type 1 diabetes.
Platform clinically used in in-vitro diagnosis type 1 diabetes index, which is all based on putting, at present exempts from or Enzyme-multiplied immune technique
(ELASA), autoimmune type 1 diabetes diagnostic reagent needs further improvement in terms of sensibility and specificity.Tradition
Radio-immunity or ELISA method detection time it is long, when at least six is small or even reaction overnight could be completed to test, mainly according to
By the pure sequence of maneuvers such as sample-adding by hand, efficiency is low, specificity deficiency.Chemiluminescence system has highly sensitive, high specific excellent
Gesture, the testing result time is fast, efficient, and whole full-automatic instrument is automatically brought into operation, and test can be completed in 1 hour, to curing now
The timely diagnoses and treatment of institute serves very big.
Can be suitably used for is developed in application in being diagnosed in vitro with the higher chemiluminescence of sensibility and specificity
Learning the antigen for the closed system that shines becomes to be even more important.
The content of the invention
An object of the present invention is to filter out and glutamic acid decarboxylase 65 (GAD65) antibody and tyrosine phosphatase (IA-
2) the fusion antigen protein matter of antibody specific bond.The second purpose is that exploitation can meet applied to the anti-of chemiluminescence closed system
Former albumen, and applied to chemiluminescence closed system.
When for detecting diabetes, the sensitivity of fusion antigen protein matter is more preferable, and non-specific lower.
The present invention provides a kind of fusion antigen protein matter, the fused protein is 65 gene of glutamte dehydrogenase or cuts
Short 65 gene of glutamte dehydrogenase passes through amalgamation and expression with tyrosine phosphatase enzyme gene or truncated tyrosine phosphatase enzyme gene
The protein of acquisition.
In a specific embodiment, the fusion antigen protein matter be selected from as A2-B1, A2-A1, B2-A1, B2-B1,
C2-B1、C2-G1、C2-H1、C2-I1、C2-J1、C2-K1、C2-L1、D2-M1、D2-B1、D2-G1、D2-H1、D2-I1、D2-
One kind in J1, D2-K1, D2-L1 and D2-M1；Wherein, symbol A1 is such as SEQ ID NO:36 glutamte dehydrogenase 65 it is complete
Long protein, B1 are such as SEQ ID NO:The 36 truncated protein matter of 46-585, G1 are SEQ ID NO:36 66-
The truncated protein matter of 585, H1 are SEQ ID NO:The 36 truncated protein matter of 90-585, I1 are SEQ ID NO:36
The truncated protein matter of 110-585, J1 are SEQ ID NO:The 36 truncated protein matter of 130-585, K1 are SEQ ID
NO:The 36 truncated protein matter of 150-585, L1 are SEQ ID NO:The 36 truncated protein matter of 160-585, M1 are
SEQ ID NO:The 36 truncated protein matter of 180-585, A2 are SEQ ID NO:38 truncated protein of 600-979
Matter, B2 are SEQ ID NO:The 38 truncated protein matter of 604-979, C2 are SEQ ID NO:604-969 s' of 38
Truncated protein matter, D2 are SEQ ID NO:The 38 truncated protein matter of 600-969, and the amalgamation and expression of the protein
Order is the order of connection of the symbol of the protein.Such as A2-B1, the nitrogen end of the protein is A2, carbon teminal B1.
In a specific embodiment, the fusion antigen protein matter be selected from as C2-B1, C2-G1, C2-H1, C2-I1,
In C2-J1, C2-K1, C2-L1, D2-M1, D2-B1, D2-G1, D2-H1, D2-I1, D2-J1, D2-K1, D2-L1 and D2-M1
It is a kind of.
In a specific embodiment, the one kind of the fusion antigen protein matter in such as C2-H1 or D2-H1.
In a specific embodiment, the fusion antigen protein matter is expressed in eukaryotic expression system and obtained
Protein.
In a specific embodiment, the fusion antigen protein matter obtains to be expressed in insect cell expression system
The protein obtained.
Insect cell expression system is using more eukaryotic expression system, it can express a variety of outer in insect cell
Source gene includes the gene of fungi, plant, bacterium and virus.Baculoviral is a kind of using insect cell as the double of natural host
Chain DNA virus, has the species specificity of height, does not infect vertebrate, harmless to people, animal.It is lucerne to study at present more
Mu three-spotted plusia core polyhedral body (AcNPV), host are Spodopterafrugiperdas.And SF9 cells, it is exactly baculovirus expression system
One of host cell.SF9 cells belong to half adherent type cell line, can carry out suspension culture, can also carry out adhere-wall culture,
Applied to albumen expression with prepare purifying in, there are many advantages of：1) compared with other eukaryotic expression systems, foreign gene table
Up to horizontal height, particularly intracellular protein.In most cases, the recombinant protein of expression is solvable, and folding just truly has posttranslational modification
(such as glycosylating), has biological activity, can be easily separated purifying；2) using insect cell suspension growth, easily amplification culture, has
Beneficial to extensive expression recombinant protein；3) it is easy to expressing heterologous multimeric protein, elder brother can be infected simultaneously by a variety of recombinant viruses
Worm cell is realized with a kind of viral infected insect cell containing multiple expression cassettes；4) insect baculovirus does not infect vertebra
Animal has security.
In a specific embodiment, the fusion antigen protein matter is the coated protein of nanometer magnetic bead.
The present invention also provides a kind of for detecting the kit of diabetes, the kit includes glutamte dehydrogenase
65 genes or 65 gene of truncated glutamte dehydrogenase lead to tyrosine phosphatase enzyme gene or truncated tyrosine phosphatase enzyme gene
Cross the protein of amalgamation and expression acquisition.
In a specific embodiment, the fused protein in the kit be selected from as A2-B1, A2-A1,
B2-A1、B2-B1、C2-B1、C2-G1、C2-H1、C2-I1、C2-J1、C2-K1、C2-L1、D2-M1、D2-B1、D2-G1、D2-
One kind in H1, D2-I1, D2-J1, D2-K1, D2-L1 and D2-M1.
In a specific embodiment, the fused protein in the kit be selected from as C2-B1, C2-G1,
C2-H1、C2-I1、C2-J1、C2-K1、C2-L1、D2-M1、D2-B1、D2-G1、D2-H1、D2-I1、D2-J1、D2-K1、D2-L1
With one kind in D2-M1.
In a specific embodiment, the fusion antigen protein matter in the kit is selected from such as C2-H1 or D2-
One kind in H1.
In a specific embodiment, the albumin A of chemiluminescent labeling is further included in the kit.It is sent out using chemistry
Light method can improve the sensitivity and specificity of product.Wherein, albumin A is a kind of vegetarian protein A matter,
Fc areas that can be specifically with people and mammalian antibody (mainly IgG) are combined.
In a specific embodiment, BSA buffer solutions are further included in the kit.The BSA buffer solutions are highly concentrated
The buffer solution of degree, such as the concentration are 3%~7%.
The present invention also provides a kind of gene order of fusion antigen protein matter, the gene order is to be obtained by expressing
Such as A2-B1, A2-A1, B2-A1, B2-B1, C2-B1, C2-G1, C2-H1, C2-I1, C2-J1, C2-K1, C2-L1, D2-M1, D2-
One in the gene order of the fusion antigen protein matter of B1, D2-G1, D2-H1, D2-I1, D2-J1, D2-K1, D2-L1 and D2-M1
Kind.
In a specific embodiment, the gene order be by express obtain as C2-B1, C2-G1, C2-H1,
C2-I1, C2-J1, C2-K1, C2-L1, D2-M1, D2-B1, D2-G1, D2-H1, D2-I1, D2-J1, D2-K1, D2-L1 and D2-
One kind in the gene order of the fusion antigen protein matter of M1.
In a specific embodiment, the gene order is by expressing the gene sequence obtained such as C2-H1 or D2-H1
One kind in row.
In a specific embodiment, the gene order is expressed in eukaryotic expression system.
In a specific embodiment, the gene order is expressed in insect cell expression system.
The present invention goes out the fusion antigen protein matter piece of suitable GAD65 and IA-2 using eukaryotic expression system expression screening
Section, and applied to chemiluminescence closed system, fill up blank currently on the market.
Specific embodiment
The present invention is described below with reference to embodiment.
Embodiment 1
The fusion and expression of gene
(1) main agents and equipment
PCR kit (including Taq enzyme, buffer, dNTP, T4 ligases) and DNA marker are purchased from full formula gold；Albumen
Marker is purchased from green skies biology；The restriction enzymes such as BamH I, Xho I, EcoR I, Nco I, Hind III are purchased from Takara
(the precious biology in Dalian)；Calf intestine alkaline phosphatase (CIAP) kit (includes component：Calf intestine alkaline phosphatase, alkaline phosphatase
Enzyme buffer liquid) it is purchased from TaKaRa；Plastic recovery kit and plasmid extraction kit are purchased from OMEGA companies；Kanamycins and ammonia benzyl
Purchased from BBI；IPTG is purchased from BBI；PCR primer and recombinant plasmid sequencing are completed by Hua Da gene or Invitrogen；Remaining reagent
It is provided by Shenzhen New Industries Biomedical Engineering Co., Ltd..
ABI Veriti PCR instruments are purchased from ABI, and horizontal cataphoresis apparatus RDY-SP1Z is purchased from the upper limited public affairs of seamount richness scientific instrument
Department, than bright constant-temperature table COS-2102C purchased from Shanghai than bright Instrument Ltd., superclean bench BSC-1000II A2 are purchased from
Shanghai Boxun Industrial Co., Ltd., centrifuge are purchased from Sigma, and inverted microscope is purchased from Olympus, and vertical electrophoresis apparatus is purchased from
BioRad, transferring film instrument are purchased from BioRad, and gel imager 805 is rich purchased from upper seamount, remaining equipment is provided by our company.
(2) cell line, bacterial strain and plasmid
Human GAD65 (hGAD65) and Human IA-2 (hIA-2) are purchased from OriGene；PFastBac1 carriers, SF9 are thin
Born of the same parents, corresponding culture medium and its host DH10Bac are purchased from life technology；Competent cell Trans5 α are complete purchased from Beijing
Formula gold.
(3) design and synthesis of primer
To ensure to obtain the optimal combination of the two Combined expression, first the segment of hGAD65 and hIA-2 single expressions is distinguished
It is screened：
Using Primer primer5.0 according to cDNA sequence (the SEQ ID NO of hGAD65 (NM_000818.2):35) set
The specific primer of meter amplification hGAD65 sections overall length and its section, design of primers is using BamH I and EcoR I as digestion position
Point, forward primer increase initiation codon, and reverse primer increases terminator codon, and is introduced respectively at the both ends of its sequence respectively
BamH I and EcoR I restriction enzyme sites, primer sequence such as SEQ ID NO:1-SEQ ID NO:19.Details are shown in Table 1.
Table 1
Using Primer primer5.0 according to cDNA sequence (the SEQ ID NO of hIA-2 (NM_002846.3):37) set
Meter amplification hIA-2 sections overall length and its section specific primer, design of primers using EcoR I and Sal I as restriction enzyme site,
Forward primer increases initiation codon, and reverse primer increases terminator codon, and is introduced respectively at the both ends of its sequence respectively
EcoR I and Sal I restriction enzyme sites, primer sequence such as SEQ ID NO:20-SEQ ID NO:34.Details are shown in Table 2.
Table 2
(4) antigen protein is expressed in insect cell expression system
Using the people cDNA plasmids of purchase as template, using corresponding upstream and downstream primer, PCR amplification goes out purpose band, uses glue
QIAquick Gel Extraction Kit recycle target gene segment, then respectively double digestion pFastBac1 carriers and purifying target fragment.Digestion
After with plastic recovery kit direct purification recycling hGAD65 segments and pFastBac1 linear carriers digestion products.To enzyme
1 μ l CIAP, 37 DEG C of reaction 1h is directly added into linear carrier after cutting and carries out dephosphorization processing (choosing is done).With T4 ligase digestions
(ratio of the molal quantity of the two is 1 to target fragment and linear carrier segment after purification:2~1:10), linked system is 10 μ l,
16 DEG C of connections are overnight.
By linked system conversion Escherichia coli (E.coli) Trans5 α.Converted product is coated on (the 100 μ g/ of benzyl containing ammonia
Ml in LB solid mediums), 37 DEG C are incubated overnight.The random multiple single bacterium colonies of picking on the LB tablets containing restructuring rod granule,
Carry out bacterium solution PCR and double digestion and sequence verification positive.By the positive rod granule carrier of acquisition convert toDH10Bac TMIn, from
In filter out positive colony.Then positive restructuring rod granule is transfected into insect cell SF9, incubated cell 72h at 27 DEG C, directly
To observe virus infection sign harvest baculoviral.
The cell by culture is collected, extracts albumen, then using affinitive layer purification recombinant protein, then is handed over using ion
It changes column and albumen is further purified, make its purity >=95%.
Combination of two is carried out on the basis of the GAD65 sections of above-mentioned screening and the experimental result of IA-2 sections, it is specific as follows：
First group：A1-A2, A1-B2, A1-C2, A1-D2, A1-N2；B1-A2, B1-B2, B1-C2, B1-D2, B1-N2；
G1-A2, G1-B2, G1-C2, G1-D2, G1-N2；H1-A2, H1-B2, H1-C2, H1-D2, H1-N2；I1-A2, I1-B2, I1-
C2, I1-D2, I1-N2；J1-A2, J1-B2, J1-C2, J1-D2, J1-N2；K1-A2, K1-B2, K1-C2, K1-D2, K1-N2；L-
1A2, L1-B2, L1-C2, L1-D2, L1-N2；M1-A2, M1-B2, M1-C2, M1-D2, M1-N2；N1-A2, N1-B2, N1-C2,
N1-D2, N1-N2；O1-A2, O1-B2, O1-C2, O1-D2, O1N2；
Second group：A2-A1, B2-A1, C2-A1, D2-A1, N2-A1；A2-B1, B2-B1, C2-B1, D2-B1, N2-B1；
A2-G1, B2-G1, C2-G1, D2-G1, N2-G1；A2-H1, B2-H1, C2-H1, D2-H1, N2-H1；A2-I1, B2-I1, C2-
I1, D2-I1, N2-I1；A2-J1, B2-J1, C2-J1, D2-J1, N2-J1；A2-K1, B2-K1, C2-K1, D2-K1, N2-K1；
A2-L1, B2-L1, C2-L1, D2-L1, N2-L1；A2-M1, B2-M1, C2-M1, D2-M1, N2-M1；A2-N1, B2-N1, C2-
N1, D2-N1, N2-N1；A2-O1, B2-O1, C2-O1, D2-O1, N2-O1.
Wherein, the design of primer sequence used and the design of individually amplification GAD65 or IA-2 are essentially identical, difference
It is in for expanding the sense primer of 5 ' end DNA fragmentations containing minimum codon, primer is without terminator codon downstream；With
Initiation codon can be free of in the upstream of 3 ' end DNA fragmentation of amplification, but primer contains terminator codon (such as combination A2- downstream
O1, the sense primer of amplification A2 segments contains minimum codon, but primer is free of terminator codon downstream；Expand O1 segments
Sense primer can be free of initiation codon, but primer contains terminator codon downstream).Two DNA fragmentations can pass through overlapping
The mode of PCR or the corresponding position of first rear clone and identical carrier obtain the DNA fragmentation that can carry out amalgamation and expression.Next behaviour
Make the step of step is with above-mentioned protein expression.
Embodiment 2
The antigen protein for measuring above-mentioned insect cell expression is verified the sensitivity and specificity of its corresponding antibody
Chemiluminescence immunoassay sandwich method principle：Test serum sample, buffer solution and antigen coat magnetic microsphere add in reaction
It is reacted in cup, at 37 DEG C, immune response, magnetic field occur for antibody and the antigen being coated on magnetic microsphere in test serum sample
In the case of nanometer magnetic bead can be magnetized rapidly, which obtains the compound of antigen and test antibodies through over cleaning, other
Ingredient is cleaned out, and adds labelled protein A-NHS-ABEI afterwards, and albumin A can rapid and test antibodies C-terminal specificity knot
It closes, forms " sandwich sandwich " immune complex.When in serum sample be detected antibody content it is more, the phase that instrument detects
It is higher to luminous intensity RLU, that is, it is proportionate.
(1) coating of antigen
The concentration of the protein of above-mentioned expression is measured respectively.
From Merck companies, the rate of charge of antigen coat nanometer magnetic bead and antigen is 1mg for nanometer magnetic bead buying：10 μ g, room temperature
When warm bath 2 is small, be placed in 250 turns/min on vortex mixer, after under magnetic field cleaning remove supernatant, obtain magnetic bead-COOH-NHS- antigens.
Wherein, with the nanometer magnetic bead of phosphate buffer dilution envelope antigen, dilution ratio 1:20 obtain working concentration.
(2) preparation of luminous marker different luminol ABEI
Shiner ABEI, i.e. albumin A-NHS-ABEI are marked using Protein A/Protein.
Luminous marker albumin A-the NHS-ABEI of mark completion, ratio 1 are diluted with phosphate buffer:1000.
(3) preparation of buffer solution
Buffer solution is phosphate buffer, 5% BSA, pH6.8.
(4) test apparatus
The automatic chemiluminescence immunoassay of Shenzhen New Industries Biomedical Engineering Co., Ltd.'s development ＆ production
Instrument Maglumi 2000Plus, sequence number：2000200068.
(5) sample
Choose 20, the type-1 diabetes mellitus sample of regular sample sheet 20 and clinical definite.With the RSR companies of Britain
GAD65, IA-2 ELISA kit for control group, the measurement result of the antigen protein of non-fusion expression is shown in Table 3-6.20
Selection the 1st, 2,3,4 and 5 amounts to 5 normal samples and I -1, I- in example normal sample and 20 type-1 diabetes mellitus samples
3rd, I-5, I-8 and No. I-12 amount to 5 type-1 diabetes mellitus positive samples.
Table 3
Table 4
Using RSR Elisa results as foundation, in Elisa testing results, the antibody concentration term of reference of normal person is<
6IU/ml, that is to say, that when antibody concentration is less than 6IU/ml, judgement is practically free of glutamic acid decarboxylase antibody, for feminine gender；When
When antibody concentration is greater than or equal to 6IU/ml, then judge to contain glutamic acid decarboxylase antibody in sample to be tested, for the positive.Passing through will
The hGAD65 antigens and overall length hGAD65 of above-mentioned 17 truncated-types measure the numerical value of normal sample and type-1 diabetes mellitus sample with
RSR Elisa results are compared, it is known that the region for the hGAD65 that sensitivity reduces successively for H1, B1, G1, A1, K1, M1, L1, J1,
I1、O1、N1.Wherein, available antigenic domains are O1 or N1, and the relative light intensity RLU of normal sample and exceptional sample is separated
It is obvious；Preferred antigenic domains are K1, M1, L1, J1, I1 or A1, and the RLU of normal sample is respectively less than 25000, weakly positive and
The RLU of positive sample is all higher than 30000；More preferably antigenic domains are H1, B1 or G1, and the RLU of normal sample is respectively less than 10000,
The RLU of positive sample is all higher than 32000, and non-specific lower, specific reaction becomes apparent from.Other segments have missing inspection or normal
Proper manners this virtual height phenomenon.
Table 5
Table 6
Using RSR Elisa results as foundation, in Elisa testing results, the antibody concentration term of reference of normal person is<
7IU/ml, that is to say, that when antibody concentration is less than 7IU/ml, judgement is practically free of tyrosine phosphatase enzyme antibody, for feminine gender；When
When antibody concentration is greater than or equal to 7IU/ml, then judge containing tyrosine phosphatase enzyme antibody in sample to be tested, for the positive.Above-mentioned knot
Fruit shows that on chemiluminescence platform the available antigenic domains of IA-2 are A2 or B2, are detected using the antigen of the two expression
Separated obvious of the relative light intensity RLU of its corresponding antibody, normal sample and exceptional sample；Preferably antigenic domains are
C2 or D2 is respectively less than 20000 using the RLU of the normal sample of its measure, and the RLU of weakly positive and positive sample is all higher than
45000；Preferred antigenic domains are N2, and 10000 are respectively less than using the RLU of the normal sample of its measure, and positive sample
RLU is all higher than 50000, and non-specific lower, specific reaction becomes apparent from.Other segments have missing inspection or normal person's sample virtual height
Phenomenon.
Table 7
Using RSR Elisa results as foundation, when the experimental result for screening GAD65 sections and IA-2 sections carries out group two-by-two
During conjunction, shown by the testing result on chemiluminescence platform：The non-specific binding of normal person is higher in first group of data, most
Height is close to 100000, and the RLU of two weakly positive samples of I-8, I-9 is not high enough, only 80000-90000 and normal person's sample
It can not distinguish；Normal person's non-specific binding of second group of data is apparent lower than first group, two weakly positive samples of I-8, I-9
RLU and normal person distinguish apparent；Second group of Data Detection effect is generally better than first group, i.e., when GAD65 antigen section and
When the antigen section of IA-2 carries out amalgamation and expression, IA-2 antigenic regions section is located at the N-terminal of protein and GAD65 antigenic regions section is located at egg
During the C-terminal of white matter, fused protein detection antibody detection result better than GAD65 antigenic regions section be located at protein N-terminal and
IA-2 antigenic regions section is located at the fused protein during C-terminal of protein.Meanwhile experimental result filters out GAD65 and IA-2 fusions
The antigenic domains of expression can be A2-B1, A2-A1, B2-A1 or B2-B1；It is preferred that amalgamation and expression antigenic domains are C2-B1, C2-
G1, C2-I1, C2-J1, C2-K1, C2-L1, D2-M1, D2-B1, D2-G1, D2-I1, D2-J1, D2-K1, D2-L1 or D2-M1；
Particularly preferred amalgamation and expression antigenic domains are C2-H1 or D2-H1.
Embodiment 3
Prokaryotic expression is compared with the protein of eukaryotic expression is for type-1 diabetes mellitus detection
In expression in escherichia coli hIA-2 and its truncated-type, the optimization section that above-mentioned hIA-2 is screened is connected respectively to
On pGEX4T-1 expression vectors, Bacillus coli expression strain is BL21 (DE3).Expression product after purification, then is used through GST labels
After Thrombin excision GST labels, the destination protein without label is obtained through affinity chromatography.
Measure sensitivity and specificity of the hIA-2 of prokaryotic expression to hIA-2 antibody.
The prokaryotic expression section of hGAD65 and its truncated-type, number consecutively PA1, PB1, PG1 and PH1, with eukaryotic expression
A1, B1, G1 and H1 are corresponded.
The sign bit PN2 of the prokaryotic expression section of the truncated-type of hIA-2-, it is corresponding with the N2 of eukaryotic expression.
Detection platform and scheme details the results are shown in Table 8 with embodiment 1.
Table 8
Using the result of the Elisa kits of RSR companies as foundation.The result and A1 of PA1, PB1, PG1 and PH1 in table 8,
B1, G1 are compared with the result of H1, and prokaryotic expression GAD65 antigens detection normal person and glutamic acid decarboxylase antibody positive sample are distinguished
Effect is poor, and non-specific binding is higher, and positive reaction is weaker；The PN2 proteantigens of prokaryotic expression are normal for detecting
Proper manners sheet, non-specific binding of the non-specific binding substantially than the N2 proteantigens of eukaryotic expression is high, weakly positive sample
It is also apparent relatively low with moderate positive sample signal.It can be seen that eukaryotic cell expression is better than procaryotic cell expression.
Embodiment 4
The detection of type-1 diabetes mellitus
Detection platform is the same as embodiment 1.Amalgamation and expression albumen is C2-H1.
10 methods determine standard curve：The original concentration of standard items (GAD65 antibody, Beijing Bo Aosen companies) is
5600IU/ml, in -20 DEG C of preservations；The original concentration of standard items (IA-2 antibody, Beijing Bo Aosen companies) is 5600IU/ml,
In -20 DEG C of preservations；1 is pressed with sandwich method dilution:20 dilutions.The standard items isoconcentration of GAD65 antibody and IA-2 antibody is mixed,
And be diluted, its corresponding values of chemiluminescence is measured, is obtained according to the relation of standard concentration values of chemiluminescence corresponding with its
To standard concentration and the functional relation of RLU, i.e. standard curve.
The concentration of 228 normal person's samples and the concentration of the sample of 108 type-1 diabetes mellitus are calculated using standard curve
Using the GAD65Ab ELISA kits of RSR companies of Britain and IA-2Ab ELISA kits as control, the kit
Antigen is the GAD65 overall lengths antigen of eukaryotic cell expression and IA-2 overall length antigens.
As a result
Table 9 detects the result of 108 type-1 diabetes mellitus
By measuring 228 normal person's testing results, according to statistical analysis, draw the normal person's that judges this kit
The threshold values of the antibody of GAD65 and IA-2 fusion proteins is<28IU/ml, and it is positive for >=28IU/ml.
Through data analysis understand (being shown in Table 9), in the detection of Elisa kits for using RSR companies, the 20th, 23,43,
58th, 62,69,79,105 and No. 107 samples are shown as negative；But the C2-H1 albumen of this programme is examined using Chemiluminescence immunoassay
The results show is surveyed as the positive, is suffered from so as to show that the sample of the 20th, 23,43,58,62,69,79,105 and 107 is actual for type-1 diabetes mellitus
Person, this programme improves the sensitivity and specificity of inspection in other words, this also means that this programme can reduce detection leakage phenomenon, by
This understands that this programme testing result more meets clinic.
Pass through testing result of the C2-H1 antigens on chemiluminescence immunoassay platform in this programme：Its positive detection
Rate=88/108*100%=81.5%.
And GAD65 positive rates=83/108*100%=76.9% in the RSR companies compared is used for, IA-2 sun
Property recall rate=80/108*100%=74.07%.
It can draw to draw a conclusion by above-mentioned data：
Applications of the C2-H1 of optimized expression on chemiluminescence platform improves the detection result of kit, that is,
Say that its positive rate is better than contrast agent box.Therefore, applications of the C2-H1 on chemiluminescence platform improves the sensitive of detection
Degree and specificity, avoid a section that individual GAD65 and IA-2 antigens section is expressing protein and cannot examine completely
Measure the autoantibody problem in patients serum.
Claims (10)
1. a kind of fusion antigen protein matter, which is characterized in that the fusion antigen protein matter for 65 gene of glutamte dehydrogenase or
Truncated 65 gene of glutamte dehydrogenase is with tyrosine phosphatase enzyme gene or truncated tyrosine phosphatase enzyme gene by merging table
Up to the protein of acquisition；
Tyrosine phosphatase section is located at the N-terminal of fusion antigen protein matter, glutamte dehydrogenase in the fusion antigen protein matter
65 sections are located at the C-terminal of fusion antigen protein matter；
The fusion antigen protein matter be selected from as A2-B1, A2-A1, B2-A1, B2-B1, C2-B1, C2-G1, C2-H1, C2-I1,
In C2-J1, C2-K1, C2-L1, D2-M1, D2-B1, D2-G1, D2-H1, D2-I1, D2-J1, D2-K1, D2-L1 and D2-M1
It is a kind of；Wherein, symbol A1 is such as SEQ ID NO:The full length protein of 36 glutamte dehydrogenase 65, B1 are such as SEQ ID NO:
The 36 truncated protein matter of 46-585, G1 are SEQ ID NO:The 36 truncated protein matter of 66-585, H1 SEQ
ID NO:The 36 truncated protein matter of 90-585, I1 are SEQ ID NO:The 36 truncated protein matter of 110-585, J1
For SEQ ID NO:The 36 truncated protein matter of 130-585, K1 are SEQ ID NO:The 36 truncation egg of 150-585
White matter, L1 are SEQ ID NO:The 36 truncated protein matter of 160-585, M1 are SEQ ID NO:180-585 of 36
Truncated protein matter, A2 be SEQ ID NO:The 38 truncated protein matter of 600-979, B2 are SEQ ID NO:The of 38
The truncated protein matter of 604-979, C2 are SEQ ID NO:The 38 truncated protein matter of 604-969, D2 are SEQ ID
NO:The 38 truncated protein matter of 600-969, and the symbol that the order of the amalgamation and expression of the protein is the protein
The order of connection.
2. fusion antigen protein matter according to claim 1, which is characterized in that the fusion antigen protein matter is selected from such as
C2-B1、C2-G1、C2-H1、C2-I1、C2-J1、C2-K1、C2-L1、D2-M1、D2-B1、D2-G1、D2-H1、D2-I1、D2-
One kind in J1, D2-K1, D2-L1 and D2-M1.
3. fusion antigen protein matter according to claim 2, which is characterized in that the fusion antigen protein matter is selected from such as
One kind in C2-H1 and D2-H1.
4. fusion antigen protein matter according to claim 1, which is characterized in that the fusion antigen protein matter is in eucaryon
The protein expressed and obtained in expression system.
5. fusion antigen protein matter according to claim 4, which is characterized in that the fusion antigen protein matter is thin in insect
The protein expressed and obtained in cellular expression system.
6. according to the fusion antigen protein matter described in claim 1-5 any one, which is characterized in that the fusion antigen protein
Matter is the coated protein of nanometer magnetic bead.
7. a kind of kit for being used to detect diabetes, which is characterized in that the kit includes being appointed according to claim 1-6
Fusion antigen protein matter described in meaning one.
8. kit according to claim 7, which is characterized in that the egg of chemiluminescent labeling is further included in the kit
White A.
9. the kit according to claim 7 or 8, which is characterized in that BSA buffer solutions are further included in the kit.
A kind of 10. gene order of antigen protein with diabetes specific antibodies specific bond, which is characterized in that the gene
Sequence is that the gene order of the fusion antigen protein matter as described in claim 1-5 any one is obtained by expressing.
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