CN107043427A - A kind of fusion protein and the ELISA method based on the fusion protein - Google Patents
A kind of fusion protein and the ELISA method based on the fusion protein Download PDFInfo
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- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
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- C12Y302/0101—Oligo-1,6-glucosidase (3.2.1.10), i.e. sucrase
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Abstract
The invention discloses a kind of fusion protein and the ELISA method based on the fusion protein.The amino acid sequence of the fusion protein is as shown in SEQ ID NO.1.The present invention applies to blood glucose meter in ELISA detections, instead of spectrophotometer for the access threshold for simplifying the experimentation of ELISA detections, reduction is monitored.Streptavidin sucrose enzyme fusion proteins are connected on the antibody of biotin labeling by the present invention, then add into test system sucrose.Blood glucose meter by measure sucrase breaks down cane obtain glucose amount, so as to obtain the amount of surveyed antigen or antibody.
Description
Technical field
The invention belongs to immunological technique field, and in particular to a kind of fusion protein and the ELISA based on the fusion protein
Method.
Background technology
ELISA（Enzyme linked immunosorbent assay, enzyme linked immunosorbent assay (ELISA)）As a kind of ripe
Antigen and antibody technology, be commercially available and be widely applied.However, because detection process needs to use ELIASA
Or spectrophotometer, detecting instrument is limited by, site of deployment environment is stricter, also causes the ageing fixing by one of detection
Ring.Therefore, invent a kind of convenient, portable, low while also having highly sensitive ELISA detection method concurrently to site environment requirement
The applicable scope of detection can greatly be expanded and the convenient degree of operation is improved.
The content of the invention
It is contemplated that overcoming the deficiencies in the prior art there is provided a kind of fusion protein and the ELISA based on the fusion protein
Method.
In order to achieve the above object, the technical scheme that provides of the present invention is：
The amino acid sequence of the fusion protein is as shown in SEQ ID NO.1：
MLLQAFLFLLAGFAAKISASMTNETSDRPLVHFTPNKGWMNDPNGLWYDEKDAKWHLYFQYNPNDTVWGTPLF
WGHATSDDLTNWEDQPIAIAPKRNDSGAFSGSMVVDYNNTSGFFNDTIDPRQRCVAIWTYNTPESEEQYISYSLDGG
YTFTEYQKNPVLAANSTQFRDPKVFWYEPSQKWIMTAAKSQDYKIEIYSSDDLKSWKLESAFANEGFLGYQYECPGL
IEVPTEQDPSKSYWVMFISINPGAPAGGSFNQYFVGSFNGTHFEAFDNQSRVVDFGKDYYALQTFFNTDPTYGSALG
IAWASNWEYSAFVPTNPWRSSMSLVRKFSLNTEYQANPETELINLKAEPILNISNAGPWSRFATNTTLTKANSYNVD
LSNSTGTLEFELVYAVNTTQTISKSVFADLSLWFKGLEDPEEYLRMGFEVSASSFFLDRGNSKVKFVKENPYFTNRM
SVNNQPFKSENDLSYYKVYGLLDQNILELYFNDGDVVSTNTYFMTTGNALGSVNMTTGVDNLFYIDKFQVREVKAAA
AAAMDPSKDSKAQVSAAEAGITGTWYNQLGSTFIVTAGADGALTGTYESAVGNAESRYVLTGRYDSAPATDGSGTAL
GWTVAWKNNYRNAHSATTWSGQYVGGAEARINTQWLLTSGTTEANAWKSTLVGHDTFTKVKPSAASIDAAKKAGVNN
GNPLDAVQQ。
The fusion protein can be used for preparing ELISA detection reagents and kit.
ELISA method based on above-mentioned fusion protein comprises the following steps：
（1）Detection sample is combined with corresponding antibodies：Antibody with selectivity is fixed on plastic cement porose disc, works as fix stably
Unnecessary antibody is washed away afterwards；Then add detection sample to be measured, detection sample is fully combined with antibody, it is to be combined firmly after
Wash away unnecessary detection sample；
（2）Add the antibody of biotin labeling：Add and antibody-testing sample-biotin mark is formed after the antibody of biotin labeling
Remember the sandwich complex of antibody；After biotin labelled antibodies and testing sample are firmly combined with, unnecessary biotin labeling is washed away
Antibody；
（3）Described fusion protein is added in plastic cement porose disc, the fusion protein is obtained most after being combined with sandwich complex
Whole compound, it is to be combined firmly after wash away unnecessary fusion protein；Described fusion protein is Streptavidin-invertase fusion
Albumen, Streptavidin has the ability with some protein formation covalent bond, can be with according to this characteristic of Streptavidin
Streptavidin is set to merge to form Streptavidin-sucrose enzyme fusion proteins with invertase；
（4）The detection of testing sample：Into resulting composite add concentration be 50 mM sucrose solution, wherein sucrose solution with
The volume ratio of resulting composite is 1:1, and the amount of glucose is determined, the amount of glucose is detection sample.Because sucrose is in sugarcane
Glucose can be resolved into the presence of carbohydrase, it is possible to determine that what is built is finally combined according to the amount of obtained glucose
The amount of thing, and resulting composite is 1 with detection sample：1 relation, you can determine to detect sample with the amount by glucose
Content.Due to examining the method for glucose very easy, can in time it be detected by equipment such as blood glucose meters, so passing through this method
The index of correlation of detection sample can be detected rapidly.
The invention will be further described below：
（1）The antibody of biotin labeling.
Applicating biotin labelled antibody is the class new bio reaction amplifying technique of the Later development seventies.Biotin is
The coenzyme of widely distributed a kind of carboxylase in animal and plant.Its miaow ring group is the portion combined with Avidin trp residue
Point, the carboxyl on its pentanoic acid side chain is the site combined with antibody or enzyme.Biotin is biology through hydroxysuccinimide activation
Element-N- maloyls Asia ester, its ester group can be combined from different groups without influenceing the latter's activity.It is incorporated on antibody molecule
Biotin can be combined with multiple Avidins, so as to produce multistage enlarge-effect.
Extraction and purifying of the whole process Jing Guo monoclonal antibody, SDS-PAGE electroresis appraisal monoclonal antibody purifications it is pure
Degree, ELISA method determine the steps such as antibody activity, the preparation of the antibody of biotin labeling and determination of activity.
It should be noted that, using High Purity antibody, reaction system should be free of the materials such as-NH2, otherwise in biotin labelled antibodies
These materials can be combined with biotin and influence the crosslinking of biotin and antibody.If the other not enough amplification efficiency of biotin
It is low, do not reach required sensitiveness.If cross at most can blocking antibody antigen binding site.
Experimental method test phase, can directly buy commercialized biotin labelled antibodies.
（2）Streptavidin is merged with invertase.
The design and expression of Streptavidin and sucrose enzyme fusion proteins are the keys of this project.The overall knot of fusion protein
Structure need to not influence the difference function of two domains, the i.e. biotin binding capacity of streptavidin and Hydrolysis of Sucrose By Sucrase
Ability.Streptavidin and saccharase gene are inserted directly into one section of soft Polyalanine long-chain by plan, make what it was connected
Two domains are relatively independent.
Streptavidin be with a kind of affine protein for have similar biological properties, be the secretion of Streptavidin bacterium
The ability of thing, its molecular weight and combination biotin is similar to the Avidin in egg, isoelectric point 6.0, and non-specific binding is remote
It is lower than Avidin.Streptavidin isoelectric point is low, without glycosyl.Combined with biotin high specific.
（3）The combination of antibody biotin and Streptavidin-sucrose enzyme fusion proteins
The biotin set up on the basis of Streptavidin-Streptavidin system（BAS）Have in actual applications significant
Superiority：
I, sensitivity is high.Biotin is easily combined with the large biological molecule such as protein and nucleic acid, the biotin derivative of formation,
Original bioactivity of macromolecular substances is not only maintained, and than moderately high, has polyvalency.In addition, each avidin molecule
There are four biotin binding sites, biotinylated macromolecular derivatives and label can be combined simultaneously with multivalent forms.Therefore,
BAS has multistage amplification, it is greatly enhanced the sensitivity of detection method in application.
II, specificity is high.Combination between Avidin and biotin has high affinity, and it is reacted in highly single-minded
Property.Therefore, BAS multi-level amplification does not increase nonspecific interference while sensitivity is improved.Moreover, BAS is tied
Closing characteristic will not be impacted because of the high dilution of reaction reagent, it is reduced reaction examination to greatest extent in actual applications
The non-specific effect of agent.
III, stability is high.The affinity costant of Avidin combination biotin can be million times of antigen-antibody reaction, the two
The dissociation constant very little of compound is combined to form, in irreversible reaction；And acid, alkali, denaturant, protein resolvase and have
Machine solvent does not influence it to combine.Therefore, in actual applications, the stability of product is high, so as to reduce operating error by BAS,
Improve the accuracy determined.
In summary, Site Detection can not only improve result judgement efficiency, also greatly reduce and preserving with being produced in transport
Raw extra cost.ELISA after the improvement can be used for multiple infectious disease antigen or antibody（Indirect elisa method detects antibody）'s
Site Detection, sensitivity is higher, is with a wide range of applications and prospect.
The present invention, which is exactly based on, replaces the conventional horseradish peroxidase of enzyme labelled antibody or antigen in ELISA method for sugarcane
Carbohydrase, it is deep that the concentration conversion horseradish peroxidase enzyme catalytic of determined antigen or antibody is produced substrate colors by conventional ELISA method
Shallow difference, and the concentration of antigen or antibody is converted into the signal of more sensitive concentration of glucose by this method, and then can be used
Portable, personal blood glucose meter detects its signal.The detection method is more convenient compared to other antigen and antibody methods, to instrument
Device and environmental requirement are relatively low, can apply to Site Detection.Due to the sample transport not easy to maintain that part field is gathered, scene
Detection can not only improve result judgement efficiency, also greatly reduce and preserving the extra cost with being produced in transport.After the improvement
ELISA can be used for multiple infectious disease antigen or antibody（Indirect elisa method detects antibody）Site Detection, sensitivity is higher,
It is with a wide range of applications and prospect.
In a word, the present invention transports blood glucose meter for the access threshold for simplifying the experimentation of ELISA detections, reduction is monitored
Use in ELISA detections, instead of spectrophotometer.Streptavidin-sucrose enzyme fusion proteins are connected to biotin by the present invention
On the antibody of mark, then add into test system sucrose.Blood glucose meter is by measuring the glucose that sucrase breaks down cane is obtained
Amount, so as to obtain the amount of surveyed antigen or antibody.
Embodiment
The sequence composition of fusion protein：Saccharase gene C-terminal connects Streptavidin gene, and one section is connected between the two
6 polyalanine sequences ensure the functional independence in two-end structure domain to increase pliability.In addition, synthetic gene two ends connect respectively
It is connected toBamHI HeEcoRI restriction enzyme site.Synthesized gene is connected into carrier pETDuet after double digestion through T4 DNA ligases.
Connection product converts freshly prepared competent escherichia coli cell BL21 (DE3), with the LB containing 100 μ g/mL penicillin
Solid medium plate screening.Induced expression is carried out to recombinant plasmid using e. coli bl21 (DE3), lactose is chosen and lures
Lead agent（0.3 g/100mL）, 37 DEG C of cultures are induced when to bacterium solution OD600 being about 0.6.Dodecyl sulphate is used after induction
Sodium polyacrylamide gel electrophoresis（SDS-PAGE）Detect the expression of institute's inducible protein.Under the conditions of ice-water bath, to centrifugation
The Bacillus coli cells of collection carry out ultrasonication with 200W power, and protease inhibitors is added into cracked thalline
To prevent protein degradation.By ultrasonication thing, 11000g centrifuges 40min under the conditions of 4 DEG C, takes supernatant.
The purifying of fused protein
Inclusion body in E. coli lysate is with denaturing liquid（6M guanidine hydrochlorides, 500 8.5,10 mM bis- of mM Tris-HCl, pH
Sulphur threitol, 10 mM mercaptoethanols, 1 mM diphenyl phosphates and 2 mM MgCl2）Dissolving.
Fusion protein after dissolving loads in bag filter, in 4 DEG C in elution buffer（100 mM Tris-HCl, pH
8.5,10 mM dithiothreitol (DTT)s, 5 mM reductive glutathiones, 1 mM GSSGs, 1 mM diphenyl phosphates, 2
mM MgCl2, 500 mM L-arginines）Middle stirring dialysis, changes once new dialyzate in every 3 hours, changes altogether 5 times.
Carry out purifying protein using Avidin affinity chromatography.Post is filled with 2- iminobiotins Ago-Gel.Use pH
11.0 0.05mol/L sodium carbonate liquors pretreatment affinity column.By albumen crude extract loading, 0.05mol/L carbonic acid is used
Sodium solution elutes foreign protein.After the UV absorption values OD280 of eluent is down to below 0.05, then with pH 4.0 0.1mol/L vinegar
Sour sodium buffer solution dissociation, collects and includes destination protein eluent, with the fusion protein purified.
Detection method one（Detect the antigen in sample）
200 μ l testing samples or sample solution are added in the elisa plate aperture for being coated with antibody, after 37 DEG C incubate 30 minutes,
With lavation buffer solution（PBS-Tween）Wash plastic ware 3 times.The μ l of antibody 200 of Avidin mark are added into plastic ware again,
After 37 DEG C incubate 30 minutes, washed 3 times with PBS-Tween again.Fusion protein is added, after 10 minutes, PBS-Tween washings 3
It is secondary, add sucrose solution（4 mmol/L）.After 5 minutes, dropping liquid drop is taken to use portable glucose meter reading.The horizontal direct ratio of reading
The determined antigen concentration in sample.
Detection method two（Detect the antibody in sample）
200 μ l testing samples or sample solution are added in the elisa plate aperture for being coated with recombinant antigen, 37 DEG C incubate 30 points
Zhong Hou, with lavation buffer solution（PBS-Tween）Wash plastic ware 3 times.The anti-human igg of Avidin mark is added into plastic ware again
The μ l of secondary antibody 200 at Fc ends, after 37 DEG C incubate 30 minutes, are washed 3 times with PBS-Tween again.Add fusion protein, 10 minutes
Afterwards, PBS-Tween is washed 3 times, adds sucrose solution（4 mmol/L）.After 5 minutes, dropping liquid drop is taken to use portable glucose meter
Reading.Reading level is proportional to determined antigen concentration in sample.
Application example：Detect the anti-HIV-1 antibody in mucous membrane of mouth diffusate
1. wiping gum with buccal swab, mucous membrane of mouth diffusate is obtained.
2. swab is put into immediately in the eppendorf pipes for 500 μ l PBS being equipped with dilute sample, and swab is tight
Tube wall is pasted, is scraped 6-10 times up and down.
3. drawing the sample of the 200 above-mentioned dilutions of μ l, addition is coated with the aperture of 96 orifice plates of HIV-1 p24 antigens, 37
DEG C incubate 30 minutes.Meanwhile, if the positive control and the negative control of non-specific antibody of anti-HIV-1 p24 antibody.
4. 96 orifice plate is washed 3 times with the PBS containing 0.01% Tween-20.Discard after cleaning solution, added into plastic ware
The μ l of secondary antibody 100 of the anti-human igg of Avidin mark（1:20000）, incubated 30 minutes in 37 DEG C.
The μ l of fusion protein 100 of Streptavidin and invertase are added in the aperture of 5.96 orifice plates（0.01 μg/L）, after
It is continuous to be placed in 37 DEG C and incubate 30 minutes.
20 μ l sucrose solutions are added in every hole（0.2 mol/L）, after 5 minutes, with blood glucose meter reading, if reading is more than 5
Mg/dL, then test sample is the positive（Contain anti-HIV-1 p24 antibody）, and reading is proportional to anti-HIV-1 p24 antibody in sample
Concentration.
SEQUENCE LISTING
<110>Central South University
<120>A kind of fusion protein and the ELISA method based on the fusion protein
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 698
<212> PRT
<213>It is artificial synthesized
<400> 1
Met Leu Leu Gln Ala Phe Leu Phe Leu Leu Ala Gly Phe Ala Ala Lys
1 5 10 15
Ile Ser Ala Ser Met Thr Asn Glu Thr Ser Asp Arg Pro Leu Val His
20 25 30
Phe Thr Pro Asn Lys Gly Trp Met Asn Asp Pro Asn Gly Leu Trp Tyr
35 40 45
Asp Glu Lys Asp Ala Lys Trp His Leu Tyr Phe Gln Tyr Asn Pro Asn
50 55 60
Asp Thr Val Trp Gly Thr Pro Leu Phe Trp Gly His Ala Thr Ser Asp
65 70 75 80
Asp Leu Thr Asn Trp Glu Asp Gln Pro Ile Ala Ile Ala Pro Lys Arg
85 90 95
Asn Asp Ser Gly Ala Phe Ser Gly Ser Met Val Val Asp Tyr Asn Asn
100 105 110
Thr Ser Gly Phe Phe Asn Asp Thr Ile Asp Pro Arg Gln Arg Cys Val
115 120 125
Ala Ile Trp Thr Tyr Asn Thr Pro Glu Ser Glu Glu Gln Tyr Ile Ser
130 135 140
Tyr Ser Leu Asp Gly Gly Tyr Thr Phe Thr Glu Tyr Gln Lys Asn Pro
145 150 155 160
Val Leu Ala Ala Asn Ser Thr Gln Phe Arg Asp Pro Lys Val Phe Trp
165 170 175
Tyr Glu Pro Ser Gln Lys Trp Ile Met Thr Ala Ala Lys Ser Gln Asp
180 185 190
Tyr Lys Ile Glu Ile Tyr Ser Ser Asp Asp Leu Lys Ser Trp Lys Leu
195 200 205
Glu Ser Ala Phe Ala Asn Glu Gly Phe Leu Gly Tyr Gln Tyr Glu Cys
210 215 220
Pro Gly Leu Ile Glu Val Pro Thr Glu Gln Asp Pro Ser Lys Ser Tyr
225 230 235 240
Trp Val Met Phe Ile Ser Ile Asn Pro Gly Ala Pro Ala Gly Gly Ser
245 250 255
Phe Asn Gln Tyr Phe Val Gly Ser Phe Asn Gly Thr His Phe Glu Ala
260 265 270
Phe Asp Asn Gln Ser Arg Val Val Asp Phe Gly Lys Asp Tyr Tyr Ala
275 280 285
Leu Gln Thr Phe Phe Asn Thr Asp Pro Thr Tyr Gly Ser Ala Leu Gly
290 295 300
Ile Ala Trp Ala Ser Asn Trp Glu Tyr Ser Ala Phe Val Pro Thr Asn
305 310 315 320
Pro Trp Arg Ser Ser Met Ser Leu Val Arg Lys Phe Ser Leu Asn Thr
325 330 335
Glu Tyr Gln Ala Asn Pro Glu Thr Glu Leu Ile Asn Leu Lys Ala Glu
340 345 350
Pro Ile Leu Asn Ile Ser Asn Ala Gly Pro Trp Ser Arg Phe Ala Thr
355 360 365
Asn Thr Thr Leu Thr Lys Ala Asn Ser Tyr Asn Val Asp Leu Ser Asn
370 375 380
Ser Thr Gly Thr Leu Glu Phe Glu Leu Val Tyr Ala Val Asn Thr Thr
385 390 395 400
Gln Thr Ile Ser Lys Ser Val Phe Ala Asp Leu Ser Leu Trp Phe Lys
405 410 415
Gly Leu Glu Asp Pro Glu Glu Tyr Leu Arg Met Gly Phe Glu Val Ser
420 425 430
Ala Ser Ser Phe Phe Leu Asp Arg Gly Asn Ser Lys Val Lys Phe Val
435 440 445
Lys Glu Asn Pro Tyr Phe Thr Asn Arg Met Ser Val Asn Asn Gln Pro
450 455 460
Phe Lys Ser Glu Asn Asp Leu Ser Tyr Tyr Lys Val Tyr Gly Leu Leu
465 470 475 480
Asp Gln Asn Ile Leu Glu Leu Tyr Phe Asn Asp Gly Asp Val Val Ser
485 490 495
Thr Asn Thr Tyr Phe Met Thr Thr Gly Asn Ala Leu Gly Ser Val Asn
500 505 510
Met Thr Thr Gly Val Asp Asn Leu Phe Tyr Ile Asp Lys Phe Gln Val
515 520 525
Arg Glu Val Lys Ala Ala Ala Ala Ala Ala Met Asp Pro Ser Lys Asp
530 535 540
Ser Lys Ala Gln Val Ser Ala Ala Glu Ala Gly Ile Thr Gly Thr Trp
545 550 555 560
Tyr Asn Gln Leu Gly Ser Thr Phe Ile Val Thr Ala Gly Ala Asp Gly
565 570 575
Ala Leu Thr Gly Thr Tyr Glu Ser Ala Val Gly Asn Ala Glu Ser Arg
580 585 590
Tyr Val Leu Thr Gly Arg Tyr Asp Ser Ala Pro Ala Thr Asp Gly Ser
595 600 605
Gly Thr Ala Leu Gly Trp Thr Val Ala Trp Lys Asn Asn Tyr Arg Asn
610 615 620
Ala His Ser Ala Thr Thr Trp Ser Gly Gln Tyr Val Gly Gly Ala Glu
625 630 635 640
Ala Arg Ile Asn Thr Gln Trp Leu Leu Thr Ser Gly Thr Thr Glu Ala
645 650 655
Asn Ala Trp Lys Ser Thr Leu Val Gly His Asp Thr Phe Thr Lys Val
660 665 670
Lys Pro Ser Ala Ala Ser Ile Asp Ala Ala Lys Lys Ala Gly Val Asn
675 680 685
Asn Gly Asn Pro Leu Asp Ala Val Gln Gln
690 695
Claims (5)
1. a kind of fusion protein, it is characterised in that the amino acid sequence of the fusion protein is as shown in SEQ ID NO.1.
2. the ELISA method based on fusion protein described in claim 1, it is characterised in that methods described comprises the following steps：
(1) detection sample is combined with corresponding antibodies：Antibody with selectivity is fixed on plastic cement porose disc, works as fix stably
Unnecessary antibody is washed away afterwards；Then add detection sample to be measured, detection sample is fully combined with antibody, it is to be combined firmly after
Wash away unnecessary detection sample；
(2) antibody of biotin labeling is added：Add and antibody-testing sample-biotin mark is formed after the antibody of biotin labeling
Remember the sandwich complex of antibody；After biotin labelled antibodies and testing sample are firmly combined with, unnecessary biotin labeling is washed away
Antibody；
(3) fusion protein described in claim 1 is added in plastic cement porose disc, the fusion protein is combined with sandwich complex
After obtain resulting composite, it is to be combined firmly after wash away unnecessary fusion protein；
(4) detection of testing sample：Sucrose solution is added into resulting composite, and determines the amount of glucose, the amount of glucose
As detect sample.
3. the method as described in claim 1, it is characterised in that step（4）In, the body of the sucrose solution and resulting composite
Product is than being 1:1.
4. a kind of ELISA detection kit, it is characterised in that contain fusion egg as claimed in claim 1 in the kit
In vain.
5. application of the fusion protein as claimed in claim 1 in ELISA detection reagents are prepared.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2023024066A1 (en) * | 2021-08-27 | 2023-03-02 | 中国科学院深圳先进技术研究院 | Proximity labeling complex, proximity labeling method, and intermolecular interaction analysis method |
-
2017
- 2017-03-10 CN CN201710139408.0A patent/CN107043427A/en active Pending
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| WO2023024066A1 (en) * | 2021-08-27 | 2023-03-02 | 中国科学院深圳先进技术研究院 | Proximity labeling complex, proximity labeling method, and intermolecular interaction analysis method |
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