CN110408639A - Pre-dyed albumen marker and preparation method thereof, application can be exposed - Google Patents
Pre-dyed albumen marker and preparation method thereof, application can be exposed Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/13—Labelling of peptides
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
- G01N33/561—Immunoelectrophoresis
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/583—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with non-fluorescent dye label
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
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Abstract
The invention discloses one kind can expose pre-dyed albumen marker and preparation method thereof, application, includes the following steps: S1, designs corresponding genetic fragment according to each protein band molecular size range, and determines corresponding expression vector；S2, the preparation expression vector, and the genetic fragment is obtained by PCR amplification；Genetic fragment is connected on corresponding expression vector, and is transferred in host cell and expressed, purified；S3, the luminescent protein of different molecular weight size is mixed with dyestuff respectively, to obtain the pre-dyed albumen of different molecular weight；S4, the pre-dyed albumen of different molecular weight size is mixed by preset ratio, obtains that pre-dyed albumen marker can be exposed.Preparation method is simple, easy to operate, and the pre-dyed albumen marker that exposes prepared is visible pre-dyed albumen, and is the luminescent protein that can be shone, and be apparent on X film.
Description
Technical field
The present invention relates to molecular biology and genetic engineering field, can expose pre-dyed albumen marker more particularly, to one kind
And preparation method thereof, application.
Background technique
Protein Marker, often referred to as albumen Marker, the standard quilt as a kind of protein molecular weight size
It is widely used in protein electrophorese.In immunoblotting (Western-blot) experiment, only Marker is accurate, experiment
As a result just convincing.In addition to this, whether successfully etc. whether albumen Marker can also prompt electrophoresis normal, transfer information.
So selecting correct albumen Marker is one of the necessary condition of western-blot Success in Experiment.
Existing albumen Marker research and development experienced common Marker, pre-dyed Marker and exposure tri- development ranks of Marker
Section.Wherein, general proteins Marker is exactly due to not being attached to dye molecule either mark molecule, shown molecular size range
The size of albumen script can accurately judge albumen size in early stage.But because can't see common Marker completely in electrophoresis process,
Finally dyeing together with target protein can just see, can not play indication reference to experimentation.
Pre-dyed Marker is by each albumen (or polypeptide) in general proteins marker by chemical modification, with a kind of indigo plant
Color or the dyestuff covalent coupling of other colors form.The appearance of pre-dyed albumen marker is brought greatly just to Western Blot
Benefit, researcher can determine purpose band according to the size of film blue (or other colors) albumen marker after transferring film
Position Approximate, to carry out the processing such as shearing to film.But pre-dyed albumen Marker can not expose on film, exposure signal
It needs to be judged according to the pre-dyed albumen Marker on film, in addition pre-dyed albumen Marker is after chemical modification in SDS-PAGE
Mobility changes in electrophoresis process, causes molecular weight information no longer accurate, so that the judgement of Western Blot result occurs
Relatively large deviation, so unsuitable accurate positioning albumen.
Exposure Marker can specifically bind primary antibody or secondary antibody, so that the final meeting of Marker band and detection target egg
It is white to appear in together in the picture of colour developing, avoid deviation occur when photo and film are compared.Such as application No. is
The preparation method of pre-dyed luminescent protein marker a kind of is disclosed in 201710707539.4 application for a patent for invention, pass through by
The luminescent protein marker and pre-dyed albumen marker mixed up obtains pre-dyed luminescent protein marker in different ratio mixing, but
The ratio of luminescent protein marker and pre-dyed albumen marker are indefinite in this kind of method, need to adjust according to the actual situation, so that system
Standby process becomes cumbersome.
Summary of the invention
In view of the foregoing drawbacks, the present invention provides one kind can expose pre-dyed albumen marker and preparation method thereof, application,
Preparation method is simple, easy to operate, and the pre-dyed albumen marker that exposes prepared is visible pre-dyed albumen, and being can
With luminous luminescent protein, and it is apparent on X film.
The technical solution that the present invention is proposed with regard to above-mentioned technical problem is as follows:
On the one hand, a kind of preparation method that can expose pre-dyed albumen marker is provided comprising following steps:
S1, corresponding nucleic acid fragment is designed according to protein band molecular size range each in pre-dyed luminescent protein marker, and
Determine corresponding expression vector；
S2, the preparation expression vector, and the genetic fragment is obtained by PCR amplification；By nucleic acid fragment be connected to
Corresponding expression vector on, and be transferred in host cell and expressed, purified, to obtain different molecular weight corresponding with band
The luminescent protein of size；
S3, the luminescent protein of different molecular weight size is mixed with dyestuff respectively, to obtain the pre-dyed egg of different molecular weight
It is white；
S4, the pre-dyed albumen of different molecular weight size is mixed by preset ratio, obtains that pre-dyed albumen marker can be exposed.
Preferably, in the step S1, the amino acid sequence of the protein band includes SEQ ID NO.1~SEQ ID
One or several in NO.6.
Preferably, in the step S1, the expression vector includes pET21a and/or pET28a-his-MBP.
Preferably, the nucleic acid sequence of the pET28a-his-MBP carrier includes sequence shown in SEQ ID NO.7.
Preferably, the host cell includes E.coli.
Preferably, the step S3 includes:
S31, the luminescent protein correspondence of different molecular weight size is placed in carbonate buffer solution, and the carbonate buffer
Liquid concentration be 0.1M, pH 9.5-10.5, with obtain it is several include corresponding molecular size range luminescent protein carbonate buffer
Liquid system；
S32, dyestuff is dissolved in the SDS solution of mass fraction 10%, it is molten for the dyestuff of 1-2% to obtain mass fraction
Liquid；
S33, by it is each include corresponding molecular size range luminescent protein carbonate buffer liquid system it is molten with dyestuff respectively
Liquid is mixed, and 25-35min is incubated under the conditions of 55-65 DEG C；And count by volume, each carbonate buffer liquid system: dyestuff is molten
Liquid=1:(0.5-1.5)；
S34, the addition lysine equal with luminescent protein gross mass act on 10min under the conditions of 55-65 DEG C, remove more
Remaining dyestuff adjusts luminescent protein concentration to about 0.15-0.25mg/ml, to obtain the pre-dyed albumen of different molecular weight.
Preferably, the dyestuff includes Remazol.
Preferably, the molecular weight for exposing pre-dyed albumen marker is 35-200kDa.
On the other hand, it additionally provides and a kind of exposes pre-dyed albumen marker according to what above-mentioned preparation method prepared.
On the other hand, it additionally provides and a kind of above-mentioned exposes pre-dyed albumen marker answering in Western-Blot experiment
With.
Preparation method of the invention is simple, easy to operate, and it is not necessary that luminescent protein is prepared separately, what is prepared exposes pre-dyed
Albumen marker is without being added additional antibody, such as anti-biotin antibodies-HRP or streptavidin-HRP, in combination with source
In the IgG or anti-rabbit of the species such as people, rat, mouse, rabbit and the secondary antibody of mouse, can not only indicate in electrophoresis process and after transferring film
The approximate location of albumen, and can on X film indicator protein position, increase pre-dyed marker swimming lane without additional.
Specific embodiment
For a clearer understanding of the technical characteristics, objects and effects of the present invention, detailed now in conjunction with following embodiments
Describe bright a specific embodiment of the invention in detail.
Embodiment one:
The preparation method for exposing pre-dyed albumen marker in the present embodiment includes the following steps:
S1, corresponding nucleic acid fragment sequence is designed according to protein band molecular size range each in pre-dyed luminescent protein marker
Column, and determine corresponding expression vector, the results are shown in Table 1；Wherein, the amino acid sequence of the protein band includes
One or several in SEQ ID NO.1~SEQ ID NO.6；The expression vector includes pET21a and/or pET28a-his-
MBP；
1 pre-dyed luminescent protein marker band of table composition
| Container name | Albumen composition | Protein sequence | Molecular weight of albumen (kDa) |
| pET21a | rFc1+mFc-1 | SEQ ID NO.1 | 35 |
| pET21a | pA1+pG-1 | SEQ ID NO.2 | 43 |
| pET21a | pA1+rFc1 | SEQ ID NO.3 | 60 |
| pET21a | pA1+rFc1+mFc1 | SEQ ID NO.4 | 90 |
| pET28a-his-MBP | pA2+rFc1 | SEQ ID NO.5 | 120 |
| pET28a-his-MBP | MBP1+MBP2+pA3+rFc2 | SEQ ID NO.6 | 200 |
Note: pA:proteinA；pG:proteinG；RFc: rabbit source heavy chain constant region gene；MFc: small source of mouse antibody
Weight chain constant area gene；
S2, the preparation expression vector, and the nucleic acid fragment is obtained by PCR amplification；By nucleic acid fragment be connected to
Corresponding expression vector on, and be transferred in host cell and expressed, purified, to obtain different molecular weight corresponding with band
The luminescent protein of size；
Specifically, the step S2 includes:
S21, according in pMal-C2T (GenBank:JF795283.1) MBP nucleic acid sequence design his+MBP+ it is polyclonal
Sequence (sequence shown in SEQ ID NO.7) is synthesized by Sangon Biotech (Shanghai) Co., Ltd. and is built into described
PET28a-his-MBP carrier)；
S22, synthesis staphylococcus aureus immunity Lysozyme binding protein A (proteinA) and streptococcus immune globulin
The complete genome sequence of white G binding protein G (proteinG)；Purchase plasmid pFUSEss-CHIg-rG*03 (InvivoGen,
Catalog#pfusess-rchg) make with pFUSEss-CHIg-mG2b (InvivoGen, Catalog#pfusess-mchg2b)
For weight chain constant area gene (mFc) pcr template of rabbit source heavy chain constant region gene (rFc) and small source of mouse antibody；With from structure
The pET28a-his-MBP carrier built is MBP sequence pcr template；
S23, corresponding nucleic acid fragment is obtained with PCR amplification primer amplification shown in table 2, then passes through agarose gel electrophoresis
Separate and recover specific target fragment；Each target fragment is subjected to digestion and connection according to the protein combination in table 1；Recycling connection
It is transferred to host cell (including bacillus coli DH 5 alpha) after connecting after product with expression vector enzyme corresponding in table 1, obtains different molecular
Measure the expression plasmid of the luminescent protein of size；
2 design of primers table of table
S24, correct expression plasmid conversion e. coli bl21 (DE3) will be sequenced, and will carry out the inducing expression of albumen, specifically
Experimental implementation can refer to " molecular cloning handbook "；
And S25, the albumen by carrier for the plasmid expression of pET21a are purified with nickel ion affinity chromatograph；Carrier is
The albumen of the plasmid expression of pET28a-his-MBP maltose affinitive layer purification, the behaviour of specific various affinity chromatography column purifications
Make method and process is referred to the execution of protein purification laboratory manual, to obtain the hair of different molecular weight size corresponding with band
Photoprotein；
S3, the luminescent protein of different molecular weight size is mixed with dyestuff respectively, to obtain the pre-dyed egg of different molecular weight
It is white；
Specifically, the step S3 includes:
S31, the luminescent protein correspondence of different molecular weight size is placed in carbonate buffer solution, and the carbonate buffer
Liquid concentration is 0.1M, and it includes corresponding molecular size range luminescent protein to obtain several that pH, which is 9.5-10.5 (preferably 10.0),
Carbonate buffer liquid system；That is, the luminescent protein for being X1 comprising molecular weight in A kind carbonate buffer liquid system, B kind carbonate
The luminescent protein ... for being X2 comprising molecular weight in buffer solution system, and so on；
S32, dyestuff is dissolved in SDS (i.e. lauryl sodium sulfate) solution of mass fraction 10%, to obtain quality
Score is the dye solution of 1-2% (preferably 1.5%)；
S33, by it is each include corresponding molecular size range luminescent protein carbonate buffer liquid system it is molten with dyestuff respectively
Liquid is mixed, and is incubated for 25-35min (preferably 30min) under the conditions of 55-65 DEG C (preferably 60 DEG C), to realize luminescent protein and dye
The covalent even chain of material；And count by volume, each carbonate buffer liquid system: dye solution=1:(0.5-1.5) (preferably 1:
1)；In the present embodiment, the dyestuff includes Remazol (Lei Masu)；
S34, the addition lysine equal with luminescent protein gross mass act under the conditions of 55-65 DEG C (preferably 60 DEG C)
10min crosses G25 column and removes extra dyestuff, adjustment luminescent protein concentration to about 0.15-0.25mg/ml (preferably 0.2mg/
Ml), to obtain the pre-dyed albumen of different molecular weight；
S4, the luminous intensity for detecting each albumen with Western-Bolt respectively, i.e., to the binding ability of antibody；According to single
Band luminous intensity adjustment by the pre-dyed albumen of different molecular weight size in preset weight ratio (in the present embodiment, different molecular
The weight for measuring the pre-dyed albumen of size is identical) mixing, then detected by Western-Bolt mixed as a result, and according to reality
Situation adjusts mixed proportion, to finally obtain the uniform mixture of band.By be incubated for different genera source different antibodies come
The stability for the marker band that shines is detected, to obtain to expose pre-dyed albumen marker.
The molecular weight for exposing pre-dyed albumen marker prepared in the present embodiment is 35-200kDa, by can
It proteinA, proteinG of binding antibody IgG and can be formed by the antibody Fc district amalgamation and expression of two anti-bindings, it then will fusion
Albumen and reactive dye Lei Masu (remazol) covalent coupling, to realize that same albumen is luminescent protein, and are pre-dyed eggs
White purpose, wherein it is not necessary that additional antibody, such as anti-biotin antibodies-HRP or streptavidin-HRP is added, in combination with next
Derived from the IgG or anti-rabbit of the species such as people, rat, mouse, rabbit and the secondary antibody of mouse, can not only refer in electrophoresis process and after transferring film
Show the approximate location of albumen, and can on X film indicator protein position, increase pre-dyed marker swimming lane without additional.
Embodiment two:
Present embodiments provide that a kind of preparation method according to embodiment one prepares exposes pre-dyed albumen
marker。
Embodiment three:
Pre-dyed albumen marker can be exposed by present embodiments providing described in a kind of embodiment two tests in Western-Blot
In application.
In conclusion preparation method of the invention is simple, easy to operate, it is not necessary that luminescent protein is prepared separately, prepare
Pre-dyed albumen marker can be exposed to be not necessarily to that additional antibody, such as anti-biotin antibodies-HRP or streptavidin-HRP is added,
In combination with from the IgG of species or the secondary antibody of anti-rabbit and mouse such as people, rat, mouse, rabbit, can not only in electrophoresis process and
The approximate location of indicator protein after transferring film, and can on X film indicator protein position, increase pre-dyed marker without additional
Swimming lane.
The foregoing is merely presently preferred embodiments of the present invention, is not intended to limit the invention, it is all in spirit of the invention and
Within principle, any modification, equivalent replacement, improvement and so on be should all be included in the protection scope of the present invention.
Claims (10)
1. the preparation method that one kind can expose pre-dyed albumen marker, which comprises the steps of:
S1, corresponding nucleic acid fragment is designed according to protein band molecular size range each in pre-dyed luminescent protein marker, and determine
Corresponding expression vector；
S2, the preparation expression vector, and the genetic fragment is obtained by PCR amplification；It is right therewith that nucleic acid fragment is connected to
It on the expression vector answered, and is transferred in host cell and expressed, purified, to obtain different molecular weight size corresponding with band
Luminescent protein；
S3, the luminescent protein of different molecular weight size is mixed with dyestuff respectively, to obtain the pre-dyed albumen of different molecular weight；
S4, the pre-dyed albumen of different molecular weight size is mixed by preset ratio, obtains that pre-dyed albumen marker can be exposed.
2. preparation method according to claim 1, which is characterized in that in the step S1, the amino of the protein band
Acid sequence includes one or several in SEQ ID NO.1~SEQ ID NO.6.
3. preparation method according to claim 1, which is characterized in that in the step S1, the expression vector includes
PET21a and/or pET28a-his-MBP.
4. preparation method according to claim 3, which is characterized in that the nucleic acid sequence of the pET28a-his-MBP carrier
Including sequence shown in SEQ ID NO.7.
5. preparation method according to claim 1, which is characterized in that the host cell includes E.coli.
6. preparation method according to claim 1, which is characterized in that the step S3 includes:
S31, the luminescent protein correspondence of different molecular weight size is placed in carbonate buffer solution, and the carbonate buffer solution is dense
Degree be 0.1M, pH 9.5-10.5, with obtain it is several include corresponding molecular size range luminescent protein carbonate buffer liquid
System；
S32, dyestuff is dissolved in the SDS solution of mass fraction 10%, to obtain mass fraction as the dye solution of 1-2%；
S33, by it is each include corresponding molecular size range luminescent protein carbonate buffer liquid system respectively with dye solution into
Row mixes, and is incubated for 25-35min under the conditions of 55-65 DEG C；And it counts by volume, each carbonate buffer liquid system: dye solution=
1:(0.5-1.5)；
S34, the addition lysine equal with luminescent protein gross mass, act on 10min, it is extra to remove under the conditions of 55-65 DEG C
Dyestuff adjusts luminescent protein concentration to about 0.15-0.25mg/ml, to obtain the pre-dyed albumen of different molecular weight.
7. preparation method according to claim 1, which is characterized in that the dyestuff includes Remazol.
8. preparation method according to claim 1, which is characterized in that the molecular weight for exposing pre-dyed albumen marker
For 35-200kDa.
9. what a kind of any one of -8 preparation methods according to claim 1 prepared exposes pre-dyed albumen marker.
10. application of the pre-dyed albumen marker in Western-Blot experiment can be exposed described in a kind of claim 9.
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