CN107190102A - A kind of digoxigenin-probe and its application for humanized's MANF genetic tests - Google Patents
A kind of digoxigenin-probe and its application for humanized's MANF genetic tests Download PDFInfo
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- CN107190102A CN107190102A CN201610301670.6A CN201610301670A CN107190102A CN 107190102 A CN107190102 A CN 107190102A CN 201610301670 A CN201610301670 A CN 201610301670A CN 107190102 A CN107190102 A CN 107190102A
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Abstract
The invention discloses a kind of digoxigenin-probe for humanized's MANF genetic tests and its application, described digoxigenin-probe sequence such as SEQ NO：Shown in 1.The invention further relates to application of the digoxigenin-probe of described humanized's MANF genetic tests in recombinant virus titre detection method.The invention further relates to a kind of Dot hybridization detection method, this method includes the digoxigenin-probe described in claim 1.The initiative digoxigenin-probe for devising humanized's MANF genetic tests of the invention, the country still belongs to the first time, and the probe specificity is strong.The digoxigenin-probe that the present invention is designed, can be used for the recombinant virus titre detection of correlation, and detection high specificity, sensitivity are high, and great convenience is provided for related experimental study.
Description
Technical field
The invention belongs to gene technology field, it is related to a kind of probe, more particularly to it is a kind of for humanized MANF
The digoxigenin-probe of genetic test and its application.
Background technology
Neurotrophic factor plays an important role to Neuronal Survival, growth, differentiation, thus is used for neural guarantor
The research that shield, regeneration and function are repaired.Middle cerebro-cardiac apoplexy derived neurotrophic factor (Mesencephalic
Astropcyte-derived Neurotrophic Factor, MANF) it is Secondary Culture in vitro in 2003
It is separated to first in the culture medium of Midbrain In The Rat I type astroglias, molecular weight is 20kDa, belongs to guarantor
The MANF protein families of keeping property.The albumen is a kind of secreted protein, is mainly expressed in neuron, and
Expressed in spongiocyte less.Research shows that a variety of er stress can be raised in cerebral ischemia and nerve cell
MANF expression, experiment in vitro confirms that MANF can promote the propagation of nerve cell, and can resist a variety of thorns
Nerve cell apoptosis caused by swashing, experiment in vivo displays that MANF albumen to dopamine caused by 6-OHDA
The damage of serotonergic neuron has preventive and therapeutic action.Because MANF albumen has protection to neure damage
And therapeutic action, many researchs are applied to treatment nerve degenerative diseases, to prevent the development of the course of disease from accelerating
The recovery of disease.Parkinson's (Parkinson Disease, PD) are a kind of typical nerve degenerative diseases,
Its main pathological change is that the denaturation of substantia nigra of midbrain dopamine (dopamine, DA) serotonergic neuron is dead, mesh
Preceding existing many researchers use the research that MANF albumen is treated to PD animal models.
It is blood-brain barrier that MANF, which is used for one of major obstacle of clinical treatment Parkinson's,.Research has shown that, divides
The lipophilic small molecule that protonatomic mass is less than 500Da can just pass through blood-brain barrier, and MANF is used as macromolecular
Albumen, it is impossible to pass through blood-brain barrier, stereotaxis intracerebral injection can cause again operation associated injury can not repeat into
OK, limit it and be applied to clinical possibility.Adeno-associated virus (Adeno-Associated Virus, AAV)
It is one of member of Parvoviridae (Parvoviridae) family.It is used as a kind of Gene delivery system, AAV
Viral vector has security good, and immunogenicity is low, can infect somatoblast and Unseparated Cell, can mediate base
In the advantages of expression steady in a long-term of cause, such as model of one research Parkinson's technological invention mechanism, by rat
Model intracerebral black substance position stereotactic injection can express the restructuring AAV virus researches of the alpha-synapse nucleoprotein eggs
White dopaminergic neuron damage mechanisms, research finds sustainable expression alpha-synapse nucleoprotein in rat brain, and
And rat shows the behavioural characteristic of dopaminergic neuron function damage, illustrate that restructuring AAV viruses can be effective
Functional active alpha-synapse nucleoprotein is mediated in the expression of intracerebral.Therefore AAV viruses are in the nervous system disease
Internal and external research in, receive more and more attention and pay attention to.
Restructuring AAV viruses or other recombinant viral vectors often relate to virus titer really in actual applications
Determine problem, recombinant virus titre is determined according to the content of target gene in packaging recombinant virus, can effectively keep away
Exempt from the interference of gutless protein component, therefore can to the titer determination of the recombinant virus containing MANF genes
Determined by the quantitative detection of MANF genes.Detection method currently for MANF gene quantifications is main
It is fluorescence quantitative PCR method, this method needs specific instrument, and requirement of experiment is high.Dot hybridization is that gene is determined
A kind of reliable method of detection is measured, can be realized by the design of specific probe, it is less demanding to experimental facilities.
This method principle is：Hybridization probe with homology and target gene under certain condition (suitable temperature and
Ionic strength etc.) it can hybridize to form double-strand by base complementrity principle, by detecting hybridization signal to probe development.
Such a method has higher detection specificity, not yet has at present for the miscellaneous of humanized's MANF genetic tests
Hand over probe.
The content of the invention
The present invention is solution above mentioned problem of the prior art, it is proposed that a kind of to be efficiently used for humanized MANF
The specific digoxigenin-probe and its method of genetic test.
In order to solve above-mentioned technical problem, the technical measures that the present invention is taken are：
The first aspect of the invention is related to a kind of digoxigenin-probe for humanized's MANF genetic tests,
Characterized in that, the described digoxigenin-probe sequence such as SEQ NO for humanized's MANF genetic tests：
Shown in 1.
The second aspect of the invention is related to the above-mentioned digoxigenin-probe for humanized's MANF genetic tests
Application in virus titer detection method.
Third aspect of the present invention is related to a kind of Dot hybridization detection method, and the Dot hybridization detection method includes upper
Described digoxigenin-probe is stated, the Dot hybridization detection method is DNA Dot hybridization detection methods, detection side
Method comprises the following steps：
Step 1：Point is added in the surface of nitrocellulose filter respectively after standard items and sample DNA denaturation；
Step 2：Obtained nitrocellulose filter will be handled in step 1 it is put into hybridization buffer and carries out prehybridization；
Step 3：Visited being added to be formed in hybridization buffer after the digoxigenin-probe denaturation described in claim 1
Probe hybridization solution is added on pin hybridization solution, the nitrocellulose filter obtained toward step 2 processing, then gentle vibration
It is incubated 24 hours；
Step 4：The nitrocellulose filter that step 3 processing is obtained is washed with cleaning solution；
Step 5：The nitrocellulose filter that step 4 processing is obtained carries out immune detection.
Preferably, above-mentioned nitrocellulose filter is positively charged nylon membrane.
Preferably, in above-mentioned step 1 standard items and sample DNA Denaturing are：At 95 DEG C, become
Property 5 minutes.
Preferably, the hybridization buffer in above-mentioned step 2 is DIG Easy Hyb buffer solutions.
Preferably, the chemical colour reaction thing of immune detection is CSPD in above-mentioned step 4.
The fourth aspect of the invention is related to above-mentioned Dot hybridization detection method in virus titer detection method
Using.
The present invention uses above-mentioned technical proposal, compared with prior art, have the following technical effect that：
1) high specificity
The initiative digoxigenin-probe for devising humanized's MANF genetic tests of the invention, it is domestic still to belong to first
Secondary, the probe specificity is strong.
2) detection sensitivity is high
Compared with conventional method, the present invention combines probe and dot hybridization, both can ensure that the special of detection
Property, it can also significantly improve detection sensitivity.
3) it is easy to operate simple
The present invention is easy to operate simple, provides a kind of relatively reliable for the measure of the related recombinant virus titres of MANF
Detection method, be that Related Experimental Study is provided convenience.
Brief description of the drawings
Fig. 1 is the titer determination result of rAAV-MANF viruses.
Embodiment
The present invention is described in more detail below by specific embodiment, for a better understanding of this hair
It is bright, but following embodiments are not intended to limit the scope of the invention.
Embodiment 1
The present embodiment is related to a kind of method that virus titer is determined with dot hybridization, comprises the following steps：
First, rAAV8-MANF viral DNAs are extracted
Viral DNA is extracted using commercial kit, and determines concentration, quality OD260/280 is extracted in control
For 1.8.The present embodiment Tiangeng viral DNA extracts kit, concrete operations by specification is carried out.
2nd, dot hybridization
The present embodiment Roche DIG High Prime DNA markers and hybridization check kit II.Concrete operations
It is as follows：
1.DNA is fixed
(1) preparation of standard items：By humanized's MANF recombinant plasmids standard items of linearisation successively 2 times of ladders
Degree dilution；
(2) sample pretreatment：Standard items and sample DNA are placed in into 95 DEG C to be denatured 5 minutes, then place ice
Upper cooling；
(3) point sample：Each DNA sample 1ul is taken, the mat surface in Positively charged Nylon membrane is put respectively；
(4) it is UV-crosslinked：Nylon membrane point sample up, measures 70000 micro- joule/cm2It is UV-crosslinked.
2. hybridization
(1) prehybridization：The DIG Easy Hyb buffer solutions (about 5ml) of appropriate volume are preheating to hybridization temperature
(41℃).Film by DNA point samples and after fixing is put into hybridization buffer, the gentle vibration 30 in hybrid pipe
Minute, carry out prehybridization.
(2) preparation of hybridization solution：Taking the DNA probe of appropriate digoxigenin labeled, (concentration is about in hybridization solution
For 25ng/ml), probe is put into boiling water bath and is denatured, is immediately placed on ice or is cooled down in ice-water bath after 5 minutes.
The digoxin labelled probe of denaturation is added in the DIG Easy Hyb buffer solutions (5ml, 41 DEG C) of preheating,
Fully mix but to avoid producing bubble.
(3) hybridize：Prehybridization solution is poured out, probe hybridization solution is added on film.Gentle oscillation incubation 24 hours
(41℃)。
3. film is washed
With 2 enough × SSC, 0.1%SDS film is washed in 15~25 DEG C of continuous oscillations twice, 5 minutes every time.
At 65 DEG C, with 0.5 enough × SSC, 0.1%SDS (being preheating to wash temperature) continuous oscillation washes film twice,
15 minutes every time.
4. immune detection
(1) film is simply washed：After hybridization and stringent washes step, film is simply rinsed with lavation buffer solution
1-5 minutes.
(2) closing and antibody incubation：Film is incubated 45 minutes for 41 DEG C in 10ml lock solution working solutions.So
Afterwards in 10ml antibody-solutions working solutions 41 DEG C be incubated 45 minutes.
(3) film is washed：Film is washed with 20ml lavation buffer solutions twice, 15 minutes every time.
(4) fluorescence reaction：Film is balanced 2-5 minutes in 20ml detection buffer solutions.Then by DNA point samples
Film surface upward, be placed in folded clip (or hybridization bag), to film on add 1ml CSPD ready-to-use,
Covering capping immediately makes CSPD be evenly distributed in film surface, and prevents the generation of bubble.25 DEG C of incubation 60min.
Wet film is placed in 37 DEG C again and is incubated 10min, is reacted with enhanced chemiluminescence.
(5) image exposure：Under the conditions of 15~25 DEG C, exposed 30 minutes with X-ray, developing fixing,
Obtain image.
Solution formula used refers to hybridization kit specification in this example.
3rd, image result is analyzed
Sample spot is compared with standard items development intensity, virus titer can be drawn.Testing result shows virus
Titre is about 108Copy/μ l, as a result as shown in Figure 1.
The digoxigenin-probe that the present invention is designed can be drawn from embodiment, the virus titer detection of correlation can be used for,
Detect that high specificity, sensitivity are high, great convenience is provided for Related Experimental Study.
The specific embodiment of the present invention is described in detail above, but it is intended only as example, and the present invention is simultaneously
It is not restricted to particular embodiments described above.To those skilled in the art, it is any to present invention progress
Equivalent modifications and substitute also all among scope of the invention.Therefore, the spirit and model of the present invention are not being departed from
Enclose lower made impartial conversion and change, all should be contained within the scope of the invention.
Claims (10)
1. a kind of digoxigenin-probe for humanized's MANF genetic tests, it is characterised in that described is used for people
The digoxigenin-probe sequence of source property MANF genetic tests such as SEQ NO：Shown in 1.
2. the digoxigenin-probe according to claim 1 for humanized's MANF genetic tests containing
Application in the recombinant virus titre detection method of MANF genes.
3. a kind of Dot hybridization detection method, it is characterised in that including the digoxigenin-probe described in claim 1.
4. a kind of Dot hybridization detection method according to claim 3, it is characterised in that described dot hybridization
Detection method is DNA Dot hybridization detection methods.
5. a kind of Dot hybridization detection method according to claim 3, it is characterised in that described dot hybridization
Detection method comprises the following steps：
Step 1：Point is added in the surface of nitrocellulose filter respectively after standard items and sample DNA denaturation；
Step 2：Obtained nitrocellulose filter will be handled in step 1 it is put into hybridization buffer and carries out prehybridization；
Step 3：Formation probe in hybridization buffer will be added to after the denaturation of digoxigenin-probe described in claim 1 miscellaneous
Liquid is handed over, probe hybridization solution is added on the nitrocellulose filter obtained toward step 2 processing, then gentle oscillation incubation
24 hours；
Step 4：The nitrocellulose filter that step 3 processing is obtained is washed with cleaning solution；
Step 5：The nitrocellulose filter that step 4 processing is obtained carries out immune detection.
6. a kind of Dot hybridization detection method according to claim 5, it is characterised in that described cellulose nitrate
Plain film is positively charged nylon membrane.
7. a kind of Dot hybridization detection method according to claim 5, it is characterised in that in described step 1
Standard items and sample DNA Denaturing be：At 95 DEG C, it is denatured 5 minutes.
8. a kind of Dot hybridization detection method according to claim 5, it is characterised in that in described step 2
Hybridization buffer be DIG Easy Hyb buffer solutions.
9. a kind of Dot hybridization detection method according to claim 5, it is characterised in that in described step 4
The chemical colour reaction thing of immune detection is CSPD.
10. a kind of Dot hybridization detection method according to any one in claim 3-9 is containing MANF bases
Application in the recombinant virus titre detection method of cause.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201610301670.6A CN107190102A (en) | 2016-05-09 | 2016-05-09 | A kind of digoxigenin-probe and its application for humanized's MANF genetic tests |
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| CN201610301670.6A CN107190102A (en) | 2016-05-09 | 2016-05-09 | A kind of digoxigenin-probe and its application for humanized's MANF genetic tests |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009133247A1 (en) * | 2008-04-30 | 2009-11-05 | Licentia Oy | Neurotrophic factor manf and uses thereof |
| WO2012162594A1 (en) * | 2011-05-25 | 2012-11-29 | Emory University | Methods of determining vaccination efficacy, devices, and compositions related thereto |
| WO2015100257A1 (en) * | 2013-12-23 | 2015-07-02 | The General Hospital Corporation | Methods and assays for determining reduced brca1 pathway function in a cancer cell |
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- 2016-05-09 CN CN201610301670.6A patent/CN107190102A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009133247A1 (en) * | 2008-04-30 | 2009-11-05 | Licentia Oy | Neurotrophic factor manf and uses thereof |
| WO2012162594A1 (en) * | 2011-05-25 | 2012-11-29 | Emory University | Methods of determining vaccination efficacy, devices, and compositions related thereto |
| WO2015100257A1 (en) * | 2013-12-23 | 2015-07-02 | The General Hospital Corporation | Methods and assays for determining reduced brca1 pathway function in a cancer cell |
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Application publication date: 20170922 |