CN104862332A

CN104862332A - Preparation method of protein exposure marker

Info

Publication number: CN104862332A
Application number: CN201510307032.0A
Authority: CN
Inventors: 华权高; 沈鹤霄; 马峰
Original assignee: CUSABIO BIOTECH Co Ltd
Current assignee: CUSABIO BIOTECH Co Ltd
Priority date: 2015-06-05
Filing date: 2015-06-05
Publication date: 2015-08-26

Abstract

The invention discloses a preparation method of a protein exposure marker. The preparation method comprises the steps of selecting expression vectors with different fusion tags according to the molecular weights of required protein exposure markers, carrying out PCR amplification to obtain protein G genes, carrying out enzyme digestion and enzyme link on the protein G genes and the expression vectors with the different fusion tags to obtain expression vectors of different-sized protein G recombinant proteins, converting host cells, carrying out induced expression to obtain the different-sized protein G recombinant proteins, and finally carrying out purification to obtain the protein exposure markers of different molecular weights. By virtue of the preparation method, the prepared protein exposure marker is good in batch repeatability, and the experimental accuracy is guaranteed.

Description

A kind of albumen exposure Marker preparation method

Technical field

The present invention relates to molecular biology and genetically engineered field, especially relate to the preparation method of a kind of albumen exposure Marker.

Background technology

Protein Marker, is often referred to as albumen Marker.In immunoblotting (Western blot) test, though molecular weight of albumen Marker is unremarkable, significant, being positive control, is the standard of size, is necessary.Only have Marker accurately errorless, experimental result is just convincing.In addition, whether albumen Marker can also point out electrophoresis normal, the transfer whether information such as successfully.So select correct albumen Marker to be one of prerequisite of western blot Success in Experiment.

Existing albumen Marker experienced by common Marker, pre-dyed Marker and exposure Marker tri-developmental stage.

Wherein, general proteins Marker, or be referred to as the Protein Marker that is unstained, also premix (pre mixed) molecular weight of albumen Marker is, being the simplest, is also the most a kind of, owing to not attaching dye molecule or tagged molecule, shown size is just in time albumen size originally, is accurately judge albumen size essential product in early days.Common Marker majority all selects the Marker of premix, facilitates the comparison of the albumen of different size.At present, common Marker is handy not as pre-dyed Marker, because can't see completely in electrophoresis process, will wait until that last dyeing could be shown in true appearance together with target protein, cannot play indication with reference to effect to experimentation.Belong to " knowing aftersensation afterwards " type completely, certainly should than " unconsciously " do not do contrast will.

Pre-dyed molecular weight of albumen (prestained Marker), is also pre-dyed Marker, is by Marker albumen, by being coupled with dyestuff covalency, in electrophoresis process or transferring film time can a kind of molecular weight of albumen Marker of arriving of direct visual perception.Pre-dyed Marker brings a lot of convenience, can help us when electrophoresis, after electrophoresis, and monitors electrophoresis situation after transferring film and estimate mobility.Because pre-dyed albumen Marker and dyestuff covalency are coupled, under different buffer conditions, during electrophoresis may there is some change in migrate attribute, may cause some deviations, so be not too applicable to accurate positioning protein.

Common Marker and pre-dyed Marker also has a shortcoming to be exactly develop the color in the film or scanned photograph that can not expose at final Western, final judgement Western result, also need photo and film to compare, be easy to like this cause experimental result to occur deviation or mistake.Exposure Marker (Exposure Marker) is exactly that final Marker band meeting and detection target protein can together appear in the picture of colour developing, avoids occurring deviation when photo and film being compared.Therefore, research and develop high-quality exposure Marker and will provide certain booster action for the progress of scientific experiment.

Summary of the invention

The object of this invention is to provide the preparation method of a kind of albumen exposure Marker.

For achieving the above object, the technical solution used in the present invention is:

The preparation method of a kind of albumen exposure maker, it is characterized in that: select the expression vector with different fusion tag according to desirable proteins exposure marker molecular weight, pcr amplification obtains protein G gene, protein G is cut by enzyme the expression vector that enzyme gets the protein G recombinant protein of different size continuously from the described expression vector with different fusion tag, then transformed host cell, abduction delivering obtains the protein G recombinant protein of different size, obtains the albumen exposure marker of different molecular weight finally by purifying.

Further, described albumen exposure maker molecular weight is between 10-120KD.

Further, the described expression vector with different fusion tag comprises: pET28a, pET32a, pGEX 6P-1 and pMAL-c2x.

Further, described host cell is intestinal bacteria, is preferably e. coli bl21 (DE3).

Further, after obtaining the albumen exposure marker of different molecular weight, detect molecular weight of albumen and purity by SDS-PAGE, and albumen is quantitatively stored.

The application of the albumen exposure maker prepared according to above-mentioned preparation method, needs stripe size needed for albumen exposure Marker, selects the corresponding large small protein after purifying to carry out mixing, packing, namely can be used as the tracing protein of exposure Marker.

The invention provides the preparation method of a kind of albumen exposure Marker, by building the different size fusion tag expression vectors of restructuring containing proteinG, respectively after abduction delivering, purifying, select the protein G recombinant protein of different size as required, then different exposure Marker is prepared after carrying out separation and purification, mixing, between batch, repeatability can ensure completely, ensure that the accuracy of experiment.

Embodiment

The preparation method of the protein fragments of different molecular weight in albumen exposure Marker of the present invention, comprise: select the carrier containing the different fusion tag of band, as prokaryotic expression carriers such as pET28a, pET32a, pGEX 6P-1, pMAL-c2x according to the different sizes of molecular weight of albumen in required exposure Marker; Primer sequence is depending on the design of plasmid and insertion sequence, and cut to lengthen is at about 20bp, and restriction enzyme site is determined according to different plasmids and protein G gene fragment order; By plasmid routine transformation E.Coli BL21DE3 (novagen) respectively built, the protein G recombinant protein of the different size of abduction delivering respectively, then the recombinant protein of escherichia coli expression is carried out purifying by different affinity chromatography methods respectively, can obtain molecular weight be between 10-120KD albumen exposure marker, SDS-PAGE detects molecular weight of albumen and purity, and carries out corresponding condition storage quantitatively, for subsequent use to albumen.

The present invention prepares the protein G fusion rotein of various different size, is mixed in accordance with the appropriate ratio by the protein G fusion rotein of these different sizes, exposure Marker.In WB experiment, exposure Marker and sample carry out SDS-PAGE electrophoresis on same glue, then through transferring film, add antibody incubation, and development, namely can be used as the contrast of experiment.

Preparation method of the present invention can by following specific embodiment detailed description in addition.

Embodiment 1

One, molecular size range is the preparation method of the albumen exposure marker of 10.03kD, 24.19kD, 33.1kD, 49.57kD.

1, synthesize the IgG calmodulin binding domain CaM of streptococcus immune globulin G, the aminoacid sequence in this region is as described in sequence table 5.The nucleotide sequence of the IgG calmodulin binding domain CaM of streptococcus immune globulin G is as described in sequence table 6.This fragment is the IgG binding domains of a molecular weight, all can be in conjunction with the IgG of various common Species origin.And eliminating the interval of other serum proteins combination, its specificity is stronger.

2, following primer is designed, and by the nucleotide sequence in primer amplification sequence table 6.Primer sequence is as follows respectively:

Primer sequence is as follows:

Upstream: CGC gGATCcACCACCTACAA (BamH I)

Downstream: GC gTCGACtTAGGTAACCATT (Sal I)

First by the above-mentioned primer amplification object fragment of method that pcr gene increases, then specific object fragment is reclaimed by SDS-PAGE glue, detect target sequence, then the object fragment increased is reclaimed by separation and purification, obtain object fragment solution, then through and the enzyme of carrier cut and connect process with enzyme and can obtain specific expression vector, for the expression process of recombinant protein.

Expression vector, the restriction enzyme site selected, the characteristic of albumen obtained and the information such as the protein sequence of correspondence and size that the albumen exposure Marker of concrete various different molecular weights is suitable for are as shown in table 1 below.

The albumen exposure Marker of table 1 different molecular weight

By the expression vector transformation of E. coli built, concrete experimental implementation with reference to " molecular cloning handbook ", can carry out the expression of colibacillary cultivation and each specific protein.

Two, molecular size range is the preparation method of the albumen marker of 76.26kD.

Redesign primer with the pGEX 6P-1-protein G obtained for template, increase above-mentioned gained GST-protein G gene, then with the GST-protein G gene insertion pMAL-c2x carrier that Eco R I/sal I will increase for restriction enzyme site.

Wherein primer sequence is as follows:

Upstream: CG gAATTCaTGAGCGATAA (EcoR I)

Downstream: GC gTCGACtTAGGTAACCATT (Sal I)

The recombinant protein characteristic of gained is MBP-GST-protein G, and molecular weight of albumen size is 76.26kD, and the aminoacid sequence of concrete expressed recombinant protein is as shown in sequence table 7.

By the expression vector transformation of E. coli built, concrete experimental implementation with reference to " molecular cloning handbook ", can carry out the expression of colibacillary cultivation and specific protein.

Three, molecular size range is the preparation method of the albumen marker of 111.91kD.

With intestinal bacteria K13 genome for template amplification archaeal dna polymerase 1 gene order (gene order number: 85676195) with NdeI/BamH I for restriction enzyme site inserts in the pet 28a-proteinG plasmid of 1 step gained.

Wherein primer sequence is as follows:

Upstream primer: CGC cATATGgTTCAGATCC

Downstream primer: CGC gGATCcGTGCGCCTGA

Gained recombinant protein characteristic is 6*His-polA-protein G, and the size of molecular weight of albumen is 111.91kD, and the aminoacid sequence of concrete expressed recombinant protein is as shown in sequence table 8.

Four, the separation and purification of the albumen of various different molecular weight

Protein specificity is 6*His-protein G, Trx-6*His-protein G, 6*His-polA-proteinG, above three kinds of albumen nickel ion metal chelate chromatography purifying;

Protein specificity is MBP-protein G, MBP-GST-protein G, and above two kinds of albumen are with maltose affinitive layer purification;

Protein specificity is GST-protein G, with gsh affinitive layer purification.

Working method and the flow process of concrete various affinity chromatography column purification can perform with reference to laboratory manual.

Five, the acquisition of albumen exposure marker

The albumen of the different molecular weight after the purifying obtain step 4, according to waiting mole mixing, namely can be used for WB experiment, as using by albumen exposure marker.

It should be noted last that, above embodiment is only in order to illustrate technical scheme of the present invention and unrestricted, although with reference to example to invention has been detailed description, those of ordinary skill in the art is to be understood that, can modify to technical scheme of the present invention or equivalent replacement, and not departing from the spirit and scope of technical solution of the present invention, it all should be encompassed in the middle of right of the present invention.

SEQUENCE LISTING

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Thr

Claims

1. the preparation method of an albumen exposure maker, it is characterized in that: select the expression vector with different fusion tag according to desirable proteins exposure marker molecular weight, pcr amplification obtains protein G gene, protein G is cut by enzyme the expression vector that enzyme gets the protein G recombinant protein of different size continuously from the described expression vector with different fusion tag, then transformed host cell, abduction delivering obtains the protein G recombinant protein of different size, obtains the albumen exposure marker of different molecular weight finally by purifying.

2. preparation method as claimed in claim 1, is characterized in that: described albumen exposure maker molecular weight is between 10-120KD.

3. preparation method as claimed in claim 1, is characterized in that: the described expression vector with different fusion tag comprises: pET28a, pET32a, pGEX 6P-1 and pMAL-c2x.

4. preparation method as claimed in claim 1, is characterized in that: described host cell is intestinal bacteria.

5. preparation method as claimed in claim 1, is characterized in that: after obtaining the albumen exposure marker of different molecular weight, detect molecular weight of albumen and purity by SDS-PAGE.

6. the albumen prepared according to the preparation method of claim 1 exposes the application of maker, it is characterized in that: stripe size needed for albumen exposure Marker, select the corresponding large small protein after purifying to carry out mixing, packing, namely can be used as the tracing protein of exposure Marker.