CN109406787A - A kind of novel protein-DNA the compound detecting carcinomebryonic antigen and its synthetic method and application - Google Patents
A kind of novel protein-DNA the compound detecting carcinomebryonic antigen and its synthetic method and application Download PDFInfo
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- CN109406787A CN109406787A CN201811198066.0A CN201811198066A CN109406787A CN 109406787 A CN109406787 A CN 109406787A CN 201811198066 A CN201811198066 A CN 201811198066A CN 109406787 A CN109406787 A CN 109406787A
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Abstract
The invention belongs to technical field of biological, and in particular to a kind of novel protein-DNA compound for detecting carcinomebryonic antigen and its synthetic method and application.Novel protein-DNA the compound includes carcinomebryonic antigen secondary antibody, glucose oxidase and the DNA structure of horseradish peroxidase-labeled.The present invention is based on rolling-circle replication technologies to construct a kind of novel protein-DNA composite construction for encapsulating a large amount of ELIAS secondary antibodies and glucose oxidase, as enzyme cascade signal amplifying probe.By using the fixed Cea Monoclonal Antibodies molecule of 96 orifice plates, after carcinomebryonic antigen to be measured is added, sandwich structure is formed.The glycoxidative generation H of grape is catalyzed by using the glucose oxidase of encapsulation2O2, horseradish peroxidase is then catalyzed H2O2The reaction for aoxidizing ABTS, realizes highly sensitive, high specific the colorimetric determination of carcinomebryonic antigen, is of great significance to the early diagnosis and clinical research of cancer.
Description
Technical field
The invention belongs to technical field of biological, and in particular to a kind of novel protein-DNA for detecting carcinomebryonic antigen is multiple
Close object and its synthetic method and application.
Background technique
DNA is other than having the function of storing and conveying hereditary information, since it is with sequence designability, synthesis letter
List is easy to the advantages that functionalization, good biocompatibility, therefore can accurately control DNA nanostructure in DNA assembling process
Molecular structure and dynamic function, to construct two and three dimensions DNA nanostructure.Currently, the method for building DNA structure is main
There are single-stranded tiling assembling, hybridization tiling assembling, DNA paper folding etc., is widely used to bio-sensing, bio-imaging, biological medicine
Equal fields.However the DNA self-assembling technique based on Watson-Crick base pair complementarity principle, in the process of building DNA structure
In generally existing sequence design and the problems such as synthesis is complicated, steric hindrance is larger, molecule efficiency of loading is low.
Rolling-circle replication is a kind of constant temperature nucleic acid amplification technology of enzymatic.The technological borrowing phage DNA molecule rolling ring
Copy mode can amplify specific nucleic acid sequence with index.Different from conventional DNA nanoassemble technology, rolling-circle replication technology
Independent of Watson-Crick base pair complementarity principle, DNA nanostructure can be constructed by crystallization.The technology
The somewhat complex design and synthesis for avoiding DNA sequence dna, in fields such as the building of DNA nanostructure, biomolecule detection, medicals diagnosis on disease
It is widely used.
" DNA sensor detection protein and metal ion based on enzyme amplification " (Xie Xuxu is based on enzyme amplification
DNA sensor detection albumen and metal ion [D] Qingdao University of Science and Technology, 2015.), the research is mainly anti-using hybridization chain type
It answers, the technologies such as the circulation amplification method of rolling-circle replication and enzymic catalytic reaction, detection signal is amplified, scan-type electrochemical is passed through
The methods of microscope (SECM), fluorescence detection are realized to human thrombin albumen, platelet derived growth factor BB (PDGF-BB) egg
Bletilla trace mercury in water solution ion (Hg2+) highly sensitive, highly selective detection.The building of its fluorescence aptamer sensor, it is first
First one section of aptamer of PDGF-BB is fixed on magnetic bead, another section of aptamer carries out rolling circle amplification, in conjunction with PDGF-BB after be made
Sandwich structure, the long-chain after rolling circle amplification are hybridized with primed DNA again, and then the DNA for causing biotin labeling carries out HCR
Reaction, and the horseradish peroxidase of a large amount of Avidin label is introduced, fluorescence signal is amplified by the catalytic action of enzyme, is realized
Detection to PDGF-BB, detection PDGF minimum concentration are 1.0 × 10-11g/mL.Although this method have preferable sensitivity and
Selectivity, but its process is complicated, needing progress, nucleic acid amplification, production cost are higher twice.
Therefore it provides detection sensitivity is high, protein detection technology easy to operate is still to be needed at present.
Summary of the invention
For background above technology, the present invention provide it is a kind of detect carcinomebryonic antigen novel protein-DNA compound and its
Synthetic method and application.
The invention adopts the following technical scheme:
First aspect of the present invention, provides a kind of novel protein-DNA compound, and the novel protein-DNA is compound
Object includes the carcinomebryonic antigen secondary antibody (HRP-Ab2), glucose oxidase (GOD) and DNA structure of horseradish peroxidase-labeled.
The second aspect of the present invention provides the synthetic method of above-described novel protein-DNA compound, the conjunction
At method the following steps are included:
S1. padlock probe and primer strand are subjected to annulation, synthesize rolling circle amplification annular template；The padlock probe
Nucleotide sequence is as shown in SEQ ID NO:1, the terminal modified phosphate group of padlock probe 5'；The nucleotide sequence of the primer strand is such as
Shown in SEQ ID NO:2；
S2. by carcinomebryonic antigen secondary antibody, the grape of the step S1 annular template and horseradish peroxidase-labeled being prepared
After oxidase solution mixing, triggering rolling circle amplification reaction obtains HRP-Ab2/GOD@DNA compound.
Third aspect of the present invention provides above-described novel protein-DNA compound in preparation and detects carcinomebryonic antigen
Application in reagent or kit.
The 4th aspect of the present invention, provides a kind of kit for detecting carcinomebryonic antigen, the reagent includes following component: cancer
Embryonal antigen monoclonal antibody, padlock probe, primer strand, T4DNA ligase, 1 × T4DNA ligase buffer solution, exonuclease
I, 1 × excision enzyme buffer solution, phi29DNA polymerase solution, 1 × phi29DNA polymerase buffer, dNTPs solution, horseradish mistake
The carcinomebryonic antigen two corresponding anti-solution of oxide enzyme label, glucose oxidase solution, carcinomebryonic antigen standard items, acetate buffer solution, Portugal
Grape sugar juice and ABTS solution；
The nucleotide sequence of the padlock probe is as shown in SEQ ID NO:1, the terminal modified phosphate group of padlock probe 5'；Institute
The nucleotide sequence of primer strand is stated as shown in SEQ ID NO:2.
The 5th aspect of the present invention, provides the application method of kit described above, comprising the following steps:
(1) coated 96 orifice plate of Cea Monoclonal Antibodies is prepared；
(2) padlock probe and primer strand are subjected to annulation, synthesize rolling circle amplification annular template；The padlock probe
Nucleotide sequence is as shown in SEQ ID NO:1, the terminal modified phosphate group of padlock probe 5'；The nucleotide sequence of the primer strand is such as
Shown in SEQ ID NO:2；
(3) carcinomebryonic antigen secondary antibody, the grape for the annular template and horseradish peroxidase-labeled that step (2) is prepared
After oxidase solution mixing, triggering rolling circle amplification reaction obtains HRP-Ab2/GOD@DNA compound；
(4) the carcinomebryonic antigen standard items of various concentration the carcinomebryonic antigen monoclonal that step (1) is prepared is added to resist
In coated 96 orifice plate of body and mix after, be added HRP-Ab2/GOD@DNA compound, acetate buffer solution, glucose solution and
ABTS solution reaction measures absorbance, standard curve is prepared；
(5) sample to be tested is added in coated 96 orifice plate of Cea Monoclonal Antibodies that step (1) is prepared
And after mixing, HRP-Ab2/GOD@DNA compound, acetate buffer solution, glucose solution and ABTS solution reaction is added, measurement is inhaled
Luminosity, according to step (4) standard curve calculate sample to be tested concentration to get.
Compared with prior art, present invention has the advantage that
It is constructed the present invention is based on rolling-circle replication technology and a kind of encapsulates the novel of a large amount of ELIAS secondary antibodies and glucose oxidase
Protein-DNA composite construction, as enzyme cascade signal amplifying probe.By using the fixed carcinomebryonic antigen Dan Ke of 96 orifice plates
Grand antibody (Ab1) after carcinomebryonic antigen to be measured is added, forms Ab1/CEA/HRP-Ab2/GOD@DNA sandwich structure.By using
The glucose oxidase of encapsulation is catalyzed the glycoxidative generation H of grape2O2, horseradish peroxidase is then catalyzed H2O2Aoxidize ABTS's
Reaction, realizes highly sensitive, high specific the colorimetric determination of carcinomebryonic antigen, early diagnosis and clinical research tool to cancer
It is significant.
Detailed description of the invention
The Figure of description for constituting a part of the invention is used to provide further understanding of the present invention, and of the invention shows
Examples and descriptions thereof are used to explain the present invention for meaning property, does not constitute improper limitations of the present invention.
Fig. 1 principle of the invention figure.
DNA molecular native polyacrylamide gel electrophoresis figure after the reaction of Fig. 2 rolling circle amplification: swimming lane 1: primer strand；Swimming lane
2: padlock probe；Swimming lane 3: primer strand and padlock probe anneal product；T4DNA is added in 4: Xiang Yongdao 3 product of swimming lane to connect
Connect the end 5' and the end 3' of enzyme connection lock type probe；Exonuclease I is added in 5: Xiang Yongdao 4 product of swimming lane and cuts non-hybridized lock
Formula probe and primer strand；Swimming lane 6: the DNA structure after rolling-circle replication；Swimming lane 7:HRP-Ab2/GOD@DNA compound.
The scanning electron microscope of DNA structure (A) and HRP-Ab2/GOD@DNA compound (B) after Fig. 3 rolling-circle replication
Figure.
Fig. 4 CEA canonical plotting.
Fig. 5 kit specificity verification figure of the present invention.
Specific embodiment
It is noted that described further below be all exemplary, it is intended to provide further instruction to the present invention.Unless another
It indicates, all technical and scientific terms used herein has usual with general technical staff of the technical field of the invention
The identical meanings of understanding.
It should be noted that term used herein above is merely to describe specific embodiment, and be not intended to restricted root
According to exemplary embodiments of the present invention.As used herein, unless the context clearly indicates otherwise, otherwise singular
Also it is intended to include plural form, additionally, it should be understood that, when in the present specification using term "comprising" and/or " packet
Include " when, indicate existing characteristics, step, operation and/or their combination.
As background technique is introduced, detection sensitivity height is provided, protein detection technology easy to operate is still current institute
It needs.
In view of this, it is multiple to provide a kind of novel protein-DNA in the present invention one or some typical embodiments
Object is closed, the novel protein-DNA compound includes that carcinomebryonic antigen secondary antibody, the grape of horseradish peroxidase-labeled are glycoxidative
Enzyme and DNA structure.
Further, the carcinomebryonic antigen secondary antibody and glucose oxidase of horseradish peroxidase-labeled are encapsulated into DNA structure
In, form HRP-Ab2/GOD@DNA compound.
Further, the DNA structure is by rolling-circle replication self assembly, synthetic method are as follows: by padlock probe and primer
Chain carries out annulation, synthesizes rolling circle amplification annular template, and then triggers rolling circle amplification reaction, and it is single-stranded to form long DNA；It is described
Long DNA is single-stranded since local concentration is excessively high, is precipitated to form micron-sized porous DNA knot by anisotropic crystallization mode
Structure.
Further, the nucleotide sequence of the padlock probe is as shown in SEQ ID NO:1, the terminal modified phosphorus of padlock probe 5'
Acid groups；The nucleotide sequence of the primer strand is as shown in SEQ ID NO:2.
Padlock probe used in the present invention, primer sequence high specificity, high sensitivity.
In the present invention one or some typical embodiments, the conjunction of the novel protein-DNA compound is provided
At method, the synthetic method the following steps are included:
S1. padlock probe and primer strand are subjected to annulation, synthesize rolling circle amplification annular template；The padlock probe
Nucleotide sequence is as shown in SEQ ID NO:1, the terminal modified phosphate group of padlock probe 5'；The nucleotide sequence of the primer strand is such as
Shown in SEQ ID NO:2；
S2. by carcinomebryonic antigen secondary antibody, the grape of the step S1 annular template and horseradish peroxidase-labeled being prepared
After oxidase solution mixing, triggering rolling circle amplification reaction obtains HRP-Ab2/GOD@DNA compound.
Further, annular template concentrations used in step S2 are 1.5~1.8 μM.
In the present invention one or some typical embodiments, above-described novel protein-DNA compound is provided
Application in preparation detection carcinomebryonic antigen reagent or kit.
In the present invention one or some typical embodiments, a kind of kit for detecting carcinomebryonic antigen is provided, it is described
Reagent includes following component: Cea Monoclonal Antibodies, padlock probe, primer strand, T4 ligase, 1 × T4DNA ligase are slow
Fliud flushing, exonuclease I and 1 × excision enzyme buffer solution, phi29DNA polymerase solution, 1 × phi29DNA polymerase buffer,
DNTPs solution, the carcinomebryonic antigen two corresponding anti-solution of horseradish peroxidase-labeled, glucose oxidase solution, carcinomebryonic antigen standard
Product, acetate buffer solution, glucose solution and ABTS solution；
The nucleotide sequence of the padlock probe is as shown in SEQ ID NO:1, the terminal modified phosphate group of padlock probe 5'；Institute
The nucleotide sequence of primer strand is stated as shown in SEQ ID NO:2.
In the present invention one or some typical embodiments, the application method of kit described above is provided, including
Following steps:
(1) coated 96 orifice plate of Cea Monoclonal Antibodies is prepared；
(2) padlock probe and primer strand are subjected to annulation, synthesize rolling circle amplification annular template；The padlock probe
Nucleotide sequence is as shown in SEQ ID NO:1, the terminal modified phosphate group of padlock probe 5'；The nucleotide sequence of the primer strand is such as
Shown in SEQ ID NO:2；
(3) carcinomebryonic antigen secondary antibody, the grape for the annular template and horseradish peroxidase-labeled that step (2) is prepared
After oxidase solution mixing, triggering rolling circle amplification reaction obtains HRP-Ab2/GOD@DNA compound；
(4) the carcinomebryonic antigen standard items of various concentration the carcinomebryonic antigen monoclonal that step (1) is prepared is added to resist
In coated 96 orifice plate of body and mix after, be added HRP-Ab2/GOD@DNA compound, acetate buffer solution, glucose solution and
ABTS solution reaction measures absorbance, standard curve is prepared；
(5) sample to be tested is added in coated 96 orifice plate of Cea Monoclonal Antibodies that step (1) is prepared
And after mixing, HRP-Ab2/GOD@DNA compound, acetate buffer solution, glucose solution and ABTS solution reaction is added, measurement is inhaled
Luminosity, according to step (4) standard curve calculate sample to be tested concentration to get.
Further, HRP-Ab2/GOD@DNA complex concentration is 2.4~2.6nM in step (4) and step (5).
Novel protein-DNA compound of the present invention is used to detect the principle of carcinomebryonic antigen: as shown in Figure 1, multiple by rolling ring
Technology self assembly processed obtains the carcinomebryonic antigen secondary antibody of encapsulation horseradish peroxidase-labeled and the novel protein of glucose oxidase
Matter-DNA composite construction.In the present invention, padlock probe is the single stranded DNA that a length is 75 bases, and the end 5' of probe is repaired
Phosphate group is adornd, it is complementary with primer strand that both ends pass through 13 bases respectively.Padlock probe is annealed simultaneously with primer strand,
Under the action of T4DNA ligase, the phosphate group at the end padlock probe 5' and the hydroxyl at the end 3' form phosphodiester bond, to make to lock
The closing of formula probe notch, forms the circular template of rolling-circle replication.Exonuclease I is added by non-hybridized padlock probe and primer
Chain hydrolysis.After phi29DNA polymerase and dNTPs is added, using the probe of cyclization as template, it is single-stranded to form long DNA.Due to poly-
The DNA chain local concentration for closing object shape is excessively high, is precipitated to form micron-sized porous DNA structure by anisotropic crystallization mode.
The carcinomebryonic antigen secondary antibody and glucose oxidase of horseradish peroxidase-labeled is added simultaneously during rolling-circle replication reaction,
Form the DNA structure (HRP-Ab2/ of the carcinomebryonic antigen secondary antibody and glucose oxidase that encapsulate a large amount of horseradish peroxidase-labeleds
GOD@DNA), to realize that enzyme cascade signal amplifies.
In addition, Cea Monoclonal Antibodies are fixed in the orifice plate with the confining liquid containing BSA.Have carcinomebryonic antigen and
In the presence of HRP-Ab2/GOD@DNA compound, Ab1/CEA/ is formed by Ag-Ab specific recognition in the orifice plate
HRP-Ab2/GOD@DNA sandwich structure.Unbonded HRP-Ab2/GOD@DNA compound is washed off, to reduce detection background value.
Then a certain amount of glucose and ABTS, the glucose oxidase catalytic oxidation of glucose in sandwich structure are added into orifice plate
Generate H2O2, then horseradish peroxidase enzyme catalytic oxidation ABTS generates the ABTS of blue·+。ABTS·+Extinction at 420nm
Degree is directly proportional to the concentration of carcinomebryonic antigen, with absorbance of the microplate reader measurement reaction product at 420nm, i.e. realization carcinomebryonic antigen
Colorimetric determination.
In order to enable those skilled in the art can clearly understand technical solution of the present invention, below with reference to tool
The embodiment of the body technical solution that the present invention will be described in detail.
A kind of novel protein-DNA the compound of embodiment 1
A kind of novel protein-DNA compound, the novel protein-DNA compound include horseradish peroxidase mark
Carcinomebryonic antigen secondary antibody, glucose oxidase and the DNA structure of note.The carcinomebryonic antigen secondary antibody and grape of horseradish peroxidase-labeled
Carbohydrate oxidase is encapsulated into DNA structure, forms HRP-Ab2/GOD@DNA compound.
The DNA structure is by rolling-circle replication self assembly, synthetic method are as follows: padlock probe and primer strand are carried out cyclization
Reaction synthesizes rolling circle amplification annular template, and then triggers rolling circle amplification reaction, and it is single-stranded to form long DNA；The DNA of the length is mono-
Chain is precipitated to form micron-sized porous DNA structure by anisotropic crystallization mode since local concentration is excessively high.
The nucleotide sequence of the padlock probe is as shown in SEQ ID NO:1, the terminal modified phosphate group of padlock probe 5'；Institute
The nucleotide sequence of primer strand is stated as shown in SEQ ID NO:2.
1 probe of table and primer strand nucleotide sequence
The synthetic method of the novel protein-DNA compound, comprising the following steps:
S1. padlock probe and primer strand are subjected to annulation, synthesize rolling circle amplification annular template: to 1 × T4DNA connection
Enzyme buffer liquid (40mM Tris-HCl, 10mM MgCl2, 10mM DTT, 0.5mM ATP, pH 7.8) and 10 end μ L5' phosphorus of middle addition
The padlock probe (100 μM) and 20 μ L primer strands (100 μM) of acid groups modification, are heated to 95 DEG C of 5min, then cold with 0.1 DEG C/s
But to 25 DEG C, and continue to stablize 3h at 25 DEG C.10 μ L T4DNA ligases are added into the DNA solution after hybridization, at 16 DEG C
After standing overnight, heating 10min at 65 DEG C inactivates T4DNA ligase.5 μ L exonuclease I (10U/ μ L) are added, 1
× excision enzyme buffer solution (67mM glycine-KOH, 6.7mM MgCl2, 1mM DTT, pH 9.5) in react 1.5h at 37 DEG C, with
Non-hybridized padlock probe and primer strand are hydrolyzed, circular template is obtained.15min is heated at 80 DEG C, inactivates exonuclease I.
The nucleotide sequence of the padlock probe is as shown in SEQ ID NO:1, the terminal modified phosphate group of padlock probe 5'；Institute
The nucleotide sequence of primer strand is stated as shown in SEQ ID NO:2；
S2. by carcinomebryonic antigen secondary antibody, the grape of the step S1 annular template and horseradish peroxidase-labeled being prepared
After oxidase solution mixing, triggering rolling circle amplification reaction obtains HRP-Ab2/GOD@DNA compound, specifically: to 1 ×
Phi29DNA polymerase buffer (33mM Tris-acetate, 10mM Mg (CH3COO)2, 66mM CH3COOK, 1%
Tween20,1mM DTT, pH7.9) in be separately added into circular template solution (1.8 μM), 5 μ L that 2 μ L step S1 are prepared
Phi29DNA polymerase solution (10U/ μ L), 10 μ L dNTPs solution (10mM), 10 μ L horseradish peroxidase-labeleds cancer embryo
Antigen two corresponding anti-solution (5mg/mL), 10 μ L glucoses aoxidize enzyme solutions (5mg/mL), are incubated for 20h at 30 DEG C.Then at 65 DEG C
Heating 10min inactivates phi29DNA polymerase.Obtained HRP-Ab2/GOD@DNA compound ultrapure water centrifuge washing is three times
(i.e. being centrifuged 10min under 8000rpm), saves backup in 4 DEG C.
A kind of kit and its application method for detecting carcinomebryonic antigen of embodiment 2
The kit includes following component: Cea Monoclonal Antibodies, padlock probe, primer strand, T4DNA connection
Enzyme, 1 × T4DNA ligase buffer solution, exonuclease I, 1 × excision enzyme buffer solution, phi29DNA polymerase solution, 1 ×
Phi29DNA polymerase buffer, dNTPs solution, the carcinomebryonic antigen two corresponding anti-solution of horseradish peroxidase-labeled, grape glycosyloxy
Change enzyme solutions, carcinomebryonic antigen standard items, acetate buffer solution, glucose solution and ABTS solution；
The nucleotide sequence of the padlock probe is as shown in SEQ ID NO:1, the terminal modified phosphate group of padlock probe 5'；Institute
The nucleotide sequence of primer strand is stated as shown in SEQ ID NO:2.
1 × T4DNA ligase buffer solution: 40mM Tris-HCl, 10mM MgCl2, 10mM DTT, 0.5mM ATP, pH
7.8。
1 × excision enzyme buffer solution: 67mM glycine-KOH, 6.7mM MgCl2, 1mM DTT, pH 9.5.
1 × phi29DNA polymerase buffer: 33mM Tris-acetate, 10mM Mg (CH3COO)2, 66mM
CH3COOK, 1%Tween 20,1mM DTT, pH7.9.
The application method of the kit, comprising the following steps:
(1) it prepares coated 96 orifice plate of Cea Monoclonal Antibodies: Cea Monoclonal Antibodies carbonate is delayed
Fliud flushing (50mM, pH 9.6) is diluted to 5 μ g/mL, the Cea Monoclonal Antibodies after 100 μ L dilution is added into every hole, and
It is incubated overnight in 4 DEG C.Liquid in hole is poured out, 200 μ L PBS buffer solution (10mM, pH 7.4) are added into every hole, is stood
Then liquid in orifice plate is poured out and dries orifice plate by 5min.200 μ L confining liquids (1%BSA) are added into every hole, in 37 DEG C
Lower incubation 2h.Confining liquid is poured out into drying, 200 μ L, 4% sucrose solution is added into every hole, 30min is incubated at 37 DEG C, so
Liquid in orifice plate is poured out afterwards and dries orifice plate.
(2) padlock probe and primer strand are subjected to annulation, synthesize rolling circle amplification annular template, specifically: to 1 ×
T4DNA ligase buffer solution (40mM Tris-HCl, 10mM MgCl2, 10mM DTT, 0.5mM ATP, pH 7.8) in be added 10
μ L5' end phosphate group modification padlock probe (100 μM) and 20 μ L primer strands (100 μM), be heated to 95 DEG C of 5min, then with
0.1 DEG C/s is cooled to 25 DEG C, and continues to stablize 3h at 25 DEG C.10 μ L T4DNA connections are added into the DNA solution after hybridization
Enzyme, after standing overnight at 16 DEG C, heating 10min at 65 DEG C inactivates T4DNA ligase.5 μ L exonuclease I are added
(10U/ μ L), in 1 × excision enzyme buffer solution (67mM glycine-KOH, 6.7mM MgCl2, 1mM DTT, pH 9.5) at 37 DEG C
It reacts 1.5h and obtains circular template to hydrolyze non-hybridized padlock probe and primer strand.15min is heated at 80 DEG C, is made outside nucleic acid
Enzyme cutting I inactivation.
The nucleotide sequence of the padlock probe is as shown in SEQ ID NO:1, the terminal modified phosphate group of padlock probe 5'；Institute
The nucleotide sequence of primer strand is stated as shown in SEQ ID NO:2；
(3) carcinomebryonic antigen secondary antibody, the grape for the annular template and horseradish peroxidase-labeled that step (2) is prepared
After oxidase solution mixing, triggering rolling circle amplification reaction obtains HRP-Ab2/GOD@DNA compound；Specifically: to 1 ×
Phi29DNA polymerase buffer (33mM Tris-acetate, 10mM Mg (CH3COO)2, 66mM CH3COOK, 1%Tween
20,1mM DTT, pH7.9) in be separately added into circular template solution (1.8 μM), 5 μ L that 2 μ L steps (2) are prepared
Phi29DNA polymerase solution (10U/ μ L), 10 μ L dNTPs solution (10mM), 10 μ L horseradish peroxidase-labeleds cancer embryo
Antigen two corresponding anti-solution (5mg/mL), 10 μ L glucoses aoxidize enzyme solutions (5mg/mL), are incubated for 20h at 30 DEG C.Then at 65 DEG C
Heating 10min inactivates phi29DNA polymerase.Obtained HRP-Ab2/GOD@DNA compound ultrapure water centrifuge washing is three times
(i.e. being centrifuged 10min under 8000rpm), saves backup in 4 DEG C.
(4) the carcinomebryonic antigen standard items of various concentration the carcinomebryonic antigen monoclonal that step (1) is prepared is added to resist
In coated 96 orifice plate of body and mix after, be added HRP-Ab2/GOD@DNA compound, acetate buffer solution, glucose solution and
ABTS solution reaction measures absorbance, standard curve is prepared；Specifically: to Cea Monoclonal Antibodies coated 96
It is added the carcinomebryonic antigen standard solution that 100 μ L concentration are respectively 0,1,5,10,50,100,200,400ng/mL in orifice plate, 37 DEG C
It is washed 3 times after lower incubation 1h with PBST buffer (pH 7.4), 50 μ LHRP-Ab2/GOD@DNA solutions is then respectively added
(2.6nM) is washed 3 times after being incubated for 1h at 37 DEG C with PBST buffer.Be added into every hole 85 μ L acetate buffer solutions (100mM,
PH4.5), 5 μ L glucose solution (5mM) and 10 μ L ABTS solution (5mM), react 30min after measure 420nm at absorbance.
By the absorbance value fitting a straight line of the product that generates to various concentration carcinomebryonic antigen at 420nm, the inspection of carcinomebryonic antigen is obtained
Survey range and detection limit.
As shown in figure 4, when carcinomebryonic antigen concentration is in 1~400ng/mL range, the concentration and reaction product of carcinomebryonic antigen
Absorbance at 420nm has good linear relationship, linear equation are as follows: A=7.24 × 10-3C+0.221, R2=0.999.
By 11 parallel determination background values, the detection that carcinomebryonic antigen is calculated is limited to 63.8pg/mL.
(5) sample to be tested is added in coated 96 orifice plate of Cea Monoclonal Antibodies that step (1) is prepared
And after mixing, HRP-Ab2/GOD@DNA compound, acetate buffer solution, glucose solution and ABTS solution reaction is added, measurement is inhaled
Luminosity, according to step (4) standard curve calculate sample to be tested concentration to get.
Test example
(1) native polyacrylamide gel electrophoresis characterizes
Pass through native polyacrylamide gel electrophoresis validating DNA circular template, the conjunction of HRP-Ab2/GOD@DNA compound
At.As shown in Fig. 2, compared with swimming lane 2 and 3, the biggish band of molecular weight of swimming lane more than 4, it was demonstrated that primer strand and locking-type visit
Hybridization reaction occurs after annealing for needle.Behind the end 5' and the end 3' of T4DNA ligase connection lock type probe, padlock probe cyclization
(swimming lane 5).The display of swimming lane 6 is after exonuclease I cutting, circular template is being cut, it was demonstrated that circular template
It is formed.The band of swimming lane 7 and swimming lane 8 shows the DNA structure that macromolecule is formd after rolling-circle replication.
(2) scanning electron microscope characterizes
As shown in figure 3, the size of the DNA structure and HRP-Ab2/GOD@DNA compound prepared by rolling-circle replication technology
Similar, diameter shows carcinomebryonic antigen secondary antibody and the encapsulation of glucose oxidase of horseradish peroxidase-labeled at 3 μm or so
There is no the progress for hindering rolling-circle replication reaction.In addition, DNA structure and HRP-Ab2/GOD@DNA compound all have classification
Layer structure, core DNA density with higher；With the progress that rolling-circle replication reacts, DNA structure and HRP-Ab2/
The DNA density of GOD@DNA compound surrounding reduces, and has more obvious fold and porous structure, thus for encapsulation horseradish peroxide
The carcinomebryonic antigen secondary antibody and glucose oxidase of compound enzyme label provide a large amount of reaction site, make a large amount of horseradish peroxidases
The carcinomebryonic antigen secondary antibody and glucose oxidase of enzyme label are encapsulated into DNA structure, form HRP-Ab2/GOD@DNA compound.
(3) specificity analysis
In order to verify the specificity of the experimental method, taking concentration respectively is alpha-fetoprotein (AFP), the ferritin of 200ng/mL
(SF), carbohydrate antigen 125 (CA125), prostate specific antigen (PSA) and CEA are that determinand is detected, as a result such as Fig. 5 institute
Show.The absorbance of detection carcinomebryonic antigen is significantly higher than the extinction that other interference of detection albumin A FP, SF, CA125 and PSA are generated
Degree, show Cea Monoclonal Antibodies only with carcinomebryonic antigen and HRP-Ab2/GOD@DNA specific binding and shape in the orifice plate
At Ab1/CEA/HRP-Ab2/GOD@DNA sandwich structure.Since Cea Monoclonal Antibodies can not identify other determinands,
The HRP-Ab2/GOD DNA being thus added in orifice plate is washed off, therefore is only capable of detecting background signal, and it is good to show that this method has
Good selectivity.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention,
It should be equivalent substitute mode, be included within the scope of the present invention.
Claims (10)
1. a kind of novel protein-DNA compound, which is characterized in that the novel protein-DNA compound includes horseradish mistake
Carcinomebryonic antigen secondary antibody, glucose oxidase and the DNA structure of oxide enzyme label.
2. novel protein-DNA compound according to claim 1, which is characterized in that horseradish peroxidase-labeled
Carcinomebryonic antigen secondary antibody and glucose oxidase are encapsulated into DNA structure, form HRP-Ab2/GOD@DNA compound.
3. novel protein-DNA compound according to claim 1 or 2, which is characterized in that the DNA structure is by rolling ring
Replicate self assembly, synthetic method are as follows: padlock probe and primer strand are subjected to annulation, synthesize rolling circle amplification circular die
Plate, and then rolling circle amplification reaction is triggered, it is single-stranded to form long DNA；The DNA of the length is single-stranded since local concentration is excessively high, passes through
Anisotropic crystallization mode is precipitated to form micron-sized porous DNA structure.
4. novel protein-DNA compound according to claim 3, which is characterized in that the nucleotide of the padlock probe
Sequence is as shown in SEQ ID NO:1, the terminal modified phosphate group of padlock probe 5'；The nucleotide sequence of the primer strand such as SEQ ID
Shown in NO:2.
5. the synthetic method of any novel protein-DNA compound of Claims 1 to 4, which is characterized in that including with
Lower step:
S1. padlock probe and primer strand are subjected to annulation, synthesize rolling circle amplification annular template；The nucleosides of the padlock probe
Acid sequence is as shown in SEQ ID NO:1, the terminal modified phosphate group of padlock probe 5'；The nucleotide sequence of the primer strand such as SEQ
Shown in ID NO:2；
S2. by the carcinomebryonic antigen secondary antibody of the step S1 annular template and horseradish peroxidase-labeled being prepared, grape glycosyloxy
After changing enzyme solutions mixing, triggering rolling circle amplification reaction obtains HRP-Ab2/GOD@DNA compound.
6. synthetic method according to claim 5, which is characterized in that annular template concentrations used in step S2 are 1.5~1.8
μM。
7. any novel protein-DNA compound of claim 1~6 is in preparation detection carcinomebryonic antigen reagent or reagent
Application in box.
8. a kind of kit for detecting carcinomebryonic antigen, which is characterized in that including following component: Cea Monoclonal Antibodies, lock
Formula probe, primer strand, T4DNA ligase, 1 × T4DNA ligase buffer solution, exonuclease I, 1 × excision enzyme buffer solution,
Phi29DNA polymerase solution, 1 × phi29DNA polymerase buffer, dNTPs solution, horseradish peroxidase-labeled cancer embryo
Antigen two corresponding anti-solution, glucose oxidase solution, carcinomebryonic antigen standard items, acetate buffer solution, glucose solution and ABTS solution；
The nucleotide sequence of the padlock probe is as shown in SEQ ID NO:1, the terminal modified phosphate group of padlock probe 5'；It is described to draw
The nucleotide sequence of object chain is as shown in SEQ ID NO:2.
9. the application method of kit described in claim 8, which comprises the following steps:
(1) coated 96 orifice plate of Cea Monoclonal Antibodies is prepared；
(2) padlock probe and primer strand are subjected to annulation, synthesize rolling circle amplification annular template；The nucleosides of the padlock probe
Acid sequence is as shown in SEQ ID NO:1, the terminal modified phosphate group of padlock probe 5'；The nucleotide sequence of the primer strand such as SEQ
Shown in ID NO:2；
(3) the carcinomebryonic antigen secondary antibody, grape glycosyloxy for the annular template and horseradish peroxidase-labeled that step (2) is prepared
After changing enzyme solutions mixing, triggering rolling circle amplification reaction obtains HRP-Ab2/GOD@DNA compound；
(4) the carcinomebryonic antigen standard items of various concentration are added to the Cea Monoclonal Antibodies packet that step (1) is prepared
In 96 orifice plates of quilt and after mixing, it is molten that HRP-Ab2/GOD@DNA compound, acetate buffer solution, glucose solution and ABTS is added
Liquid reaction, measures absorbance, standard curve is prepared；
(5) sample to be tested is added in coated 96 orifice plate of Cea Monoclonal Antibodies that step (1) is prepared and is mixed
After even, HRP-Ab2/GOD@DNA compound, acetate buffer solution, glucose solution and ABTS solution reaction is added, measures extinction
Degree, according to step (4) standard curve calculate sample to be tested concentration to get.
10. the application method of kit according to claim 8, which is characterized in that HRP- in step (4) and step (5)
Ab2/GOD@DNA complex concentration is 2.4~2.6nM.
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