CN101556287B

CN101556287B - Novel protein molecular weight standard and preparation method thereof

Info

Publication number: CN101556287B
Application number: CN 200910095982
Authority: CN
Inventors: 余铭恩; 冯俊涛; 吴琼杉; 李晓照; 敖翔; 余卫
Original assignee: HANGZHOU SONGHUA BIOLOGICAL TECHNOLOGY Co Ltd
Current assignee: HANGZHOU SONGHUA BIOLOGICAL TECHNOLOGY Co Ltd
Priority date: 2009-02-26
Filing date: 2009-02-26
Publication date: 2013-05-15
Anticipated expiration: 2029-02-26
Also published as: CN101556287A

Abstract

The invention belongs to the technical field of biological engineering, and discloses a novel protein molecular weight standard and a preparation method thereof. The invention adopts the concrete method that recombinant protein which is obtained by the tandem expression of different numbers of SPA (Staphylococcal protein A) is used as the protein molecular weight standard. The invention also discloses a method for applying the novel protein molecular weight standard so as to cause the novel protein molecular weight standard to be developed on a film by utilizing the capability of being combined with the Fc section of various mammal IgG of SPA. The invention also discloses a method for optimizing an SPA nucleotide sequence by genetic engineering, and an optimized nucleotide sequence. The invention further discloses a method for conducting tandem expression and purification on various SPAs by utilizing BsaI restriction sites.

Description

A kind of novel protein molecular weight standard and preparation method thereof

Technical field

The invention belongs to technical field of bioengineering, be specifically related to a kind of novel protein molecular weight standard of realizing that in protein immunoblot experiment different molecular weight albumen shows simultaneously, this protein molecular weight standard is comprised of the recombinant protein of the different number SPA of a series of series connection.

Background technology

Detected by Western blot (Western Blot) has in immunology extremely widely to be used, it adopts polyacrylamide gel electrophoresis protein isolate quality sample, transfer on solid phase carrier (for example cellulose nitrate film), solid phase carrier is with non-covalent bond form adsorbed proteins, and can keep protein type and the biologic activity thereof of electrophoretic separation constant.As antigen, with corresponding antibody incubation combination, then hatch combination with the second antibody of enzyme labeling with the protein on solid phase carrier, through substrate colour developing or produce fluorescence and egative film exposes to detect the expression of the specific proteins of electrophoretic separation.

Protein molecular weight standard normally by a series of known molecular amounts but the protein that varies in size form, it is mainly used in the albumen polyacrylamide gel electrophoresis, thus the position of the judgement destination protein of the comparison by molecular weight on gel.In the Western blot experiment, due to the protein molecular weight standard of routine can't with antibody and the reaction of corresponding second antibody of destination protein, thereby can't develop the color, develop on egative film thereby especially can't produce fluorescence, cause correctly to judge by the auxiliary of protein molecular weight standard the tram of destination protein on egative film.

Staphylococcal protein A (Staphylococal Protein A, SPA) is a kind of distinctive protein in pathogenic aureus cell surface, has the ability of being combined with the Fc of multiple mammal IgG section, and its gene order is open.Therefore, utilize the peculiar property of SPA albumen, adopt technique for gene engineering to optimize its nucleotide sequence, make SPA expressing in series and the purifying of different numbers, develop and to realize the novel protein molecular weight standard that different molecular weight albumen shows simultaneously in protein immunoblot experiment, for being widely used of detected by Western blot provides a kind of reagent.

Summary of the invention

The purpose of this invention is to provide and a kind ofly can realize different molecular weight albumen simultaneously in conjunction with multiple mammal IgG antibody in protein immunoblot experiment, and the novel protein molecular weight standard that can show on egative film.This protein molecular weight standard is comprised of the recombinant protein of the different number SPA of a series of series connection.

For realizing purpose of the present invention, intend by the following technical solutions: (1) adopts the Escherichia coli preference codon that natural SPA gene nucleotide series is optimized, and is beneficial to the destination protein Expression in Escherichia coli.(2) carry out design of primers, synthetic take the SPA sequence of optimizing as template, obtain the purpose fragment by splicing primer and pcr amplification.(3) the purpose fragment is inserted expression vector PET one 28a (+) in different serial number purpose modes, and in e. coli bl21 (DE3) abduction delivering.(4) by nickel agarose affinity chromatography purifying, obtain the recombinant protein of the different number SPA of a series of series connection, i.e. protein molecular weight standard.(5) confirm that by protein immunoblot experiment this protein molecular weight standard can be combined with multiple mammal IgG antibody.

compared with prior art, the present invention utilizes staphylococcal protein A (Staphylococal Protein A, SPA) has the ability of being combined with the Fc of multiple mammal IgG section, adopt technique for gene engineering to optimize its nucleotide sequence, make SPA expressing in series and the purifying of different numbers, develop the novel protein molecular weight standard that to realize in protein immunoblot experiment that different molecular weight albumen shows simultaneously on egative film, overcome the shortcoming that traditional protein molecular weight standard can't show on film, for being widely used of detected by Western blot provides a kind of reagent, make the result of detected by Western blot have more confidence level and cogency.

Description of drawings

Fig. 1 is plasmid construction process schematic diagram.

Fig. 2 is mouse IgG antibody and the result of rabbit source property IgG antibody after exograph exposure, development, photographic fixing.

Embodiment

Below in conjunction with specific embodiment and accompanying drawing, the present invention is described in further detail, described experimental example is intended to specifically illustrate the present invention with way of example.The value of the concentration of reagent, reagent, temperature and other variable and carrier and host's selection just illustrates application of the present invention, and is not construed as limiting the invention.Below explanation is not used in and limits the scope of the invention.

1. the nucleotide sequence of Optimized Coding Based SPA albumen

In order to improve SPA protein expression amount, under the constant prerequisite of SPA Argine Monohydrochloride sequence, adopt will the encode nucleotide sequence optimization of SPA albumen of Escherichia coli preference codon, the nucleotide sequence after optimization is SEQ ID NO:1 in sequence table.

2.SPA the structure of series multiple plasmid

Classifying template as with the SPA nucleotides sequence after optimizing, to carry out design of primers synthetic, splice and pcr amplification acquisition purpose fragment by primer annealing.The PCR reaction conditions is 94 ℃ of sex change 5 minutes, 55 ℃ of annealing 20 seconds, 72 ℃ were extended 30 seconds, 35 circulations altogether, at last again 72 ℃ extended 10 minutes.Capable 1% agarose gel electrophoresis of PCR product is cut glue and is reclaimed the purpose fragment.Primer sequence is as follows:

F1 of SPA：

5’-GGATCCGTGGATAACAAATTTAACAAAGAACAGCAGAACGCCTTTTATGAAATTCTGCATCT-3’

R1 of SPA：

5’-CAGGCTCTGAATAAAGGCGTTGCGCTGTTCTTCGTTCAGGTTCGGCAGATGCAGAATTTCATA-3’

F2 of SPA：

5’-CGCCTTTATTCAGAGCCTGAAAGATGATCCGAGCCAGAGCGCCAACCTGCTGGCCGAAGCCAA-3’

R2 of SPA：

5’-GAATTCTTAGGTCTCGGATCACTTCGGGGCCTGGGCATCGTTCAGCTTCTTGGCTTCGGCCAGCAG-3’

This purpose fragment is connected the pMD19-T carrier, obtain recombinant plasmid pMD19-T-SPA*1.Because the purpose fragment tip designs of inserting has the BsaI restriction enzyme site, the recognition sequence of this restriction endonuclease is different with the shearing sequence, thus with pMD19-T-SPA*1 for the plasmid that sets out, can realize the insertion again of purpose fragment, obtain recombinant plasmid pMD19-T-SPA*2.In like manner analogize, can obtain successively the restructuring pMD19-T plasmid (the plasmid construction process is referring to Fig. 1 in detail) of SPA series multiple.

3. the structure of recombinant expression carrier

The restructuring pMD-19T plasmid of the SPA series multiple in implementation step 2 is processed with BamHI, EcoRI double digestion, and glue reclaims kit and reclaims the purpose fragment.BamHI, EcoRI double digestion are processed prokaryotic expression plasmid PET-28a (+), and glue reclaims kit and reclaims carrier.With the T4DNA ligase purpose fragment is connected with PET-28a+) connect, after enzyme is cut evaluation, obtain the prokaryotic expression plasmid of a plurality of expression SPA series multiple.

4. the Expression and purification of protein molecular weight standard

The prokaryotic expression plasmid that obtains a plurality of expression SPA series multiple in implementation step 3 is changed in e. coli bl21 (DE3), obtain the bacterial classification of a series of marking protein molecular weight standards.These bacterial classifications are inoculated in respectively in the LB fluid nutrient medium that contains that penicillin of card, 250rpm/min, it is 1.0mmol/L that 37 ℃ of cultivations added derivant isopropylthio-β-D-galactoside to final concentration after 12 hours, continue to cultivate after 4 hours, 5000rpm/min, centrifugal 20 minutes, the enrichment thalline.Thalline is resuspended with 10mMPBS (PH7.4) damping fluid, ice bath, and ultrasonic disruption 5-10 minute, the high speed refrigerated centrifuge was also collected supernatant.After the supernatant of collecting mixes with the nickel agarose respectively, first use low concentration imidazoles solution (50mM) washing foreign protein, use again high concentration imidazoles solution (300mM) wash-out and collect destination protein, standby in-20 ℃ after dialysis, obtain the recombinant protein of the different number SPA of a series of series connection, be protein molecular weight standard, the molecular weight of each albumen is respectively 15kD, 21kD, 30kD, 40kD, 60kD, 95kD.

5.Western Blot

The capable 12%SDS-PAGE of protein molecular weight standard after purifying, electrotransfer is to pvdf membrane, primary antibodie is respectively mouse IgG antibody and the rabbit source property IgG antibody of random choose, two anti-sheep anti-mouse antibody and goat anti-rabbit antibodies for corresponding HRP mark, substrate are Western Blot miaow promise fluorogenic substrate in Shandong commonly used.Exograph exposure, development, photographic fixing (result is referring to Fig. 2).

The related solution formula:

1. electrophoretic buffer:

Gly1 8.8g, Tris 3.03g, SDS 1g adds distilled water and is settled to 1000ml.

2. transferring film damping fluid:

Gly 2.9g, Tris 5.8g, SDS 0.37g, methyl alcohol 200ml adds distilled water and is settled to 1000ml.

3. cleansing solution:

Tris 3g, NaCl 8g, KCl 0.2g, Tween-201ml is settled to 1000ml (pH7.8).

4. fluorogenic substrate:

Pierce company product (article No.: Prod#34075).

Sequence table

＜101〉Hangzhou Songhua Biological Technology Co., Ltd.

＜120〉a kind of novel protein molecular weight standard and preparation method thereof

<160>2

<210>1

<211>174

<212>DNA

<400>1

GTGGATAACA AATTTAACAA AGAACAGCAG AACGCCTTTT ATGAAATTCT 50

GCATCTGCCG AACCTGAACG AAGAACAGCG CAACGCCTTT ATTCAGAGCC 100

TGAAAGATGA TCCGAGCCAG AGCGCCAACC TGCTGGCCGA AGCCAAGAAG 150

CTGAACGATG CCCAGGCCCC GAAG 174

<210>2

<211>58

<212>PRT

<400>2

Val Asp Asn Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu

1 5 10 15

Ile Leu His Leu Pro Asn Leu Asn Glu Glu Gln Arg Asn Ala Phe

20 25 30

Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Ala Asn Leu Leu

35 40 45

Ala Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys

50 55 End

Claims

1. the preparation method of a novel protein molecular weight standard is characterized in that comprising the following steps:

(1) adopt the Escherichia coli preference codon that its nucleotide sequence is optimized natural grape coccus protein A gene, the nucleotide sequence after optimization is SEQ ID NO:1 in sequence table;

(2) the staphylococcal protein A gene order after to optimize is as template design primer and chemosynthesis, and splice also by primer, the method for pcr amplification obtains the purpose fragment;

(3) the purpose fragment is inserted expression vector PET-28a (+) in different serial number purpose modes, and in e. coli bl21 (DE3) abduction delivering;

(4) by nickel agarose affinity chromatography purifying, obtain the recombinant protein of the different number staphylococcal protein A of a series of series connection, i.e. protein molecular weight standard;

(5) confirm that by protein immunoblot experiment this protein molecular weight standard can be combined with multiple mammal IgG antibody.

2. the preparation method of a kind of novel protein molecular weight standard according to claim 1, it is characterized in that all the connect staphylococcal protein A of different numbers of each albumen in protein molecular weight standard, the amino acid sequence of staphylococcal protein A is SEQ ID NO:2 in sequence table.

3. the preparation method of a kind of novel protein molecular weight standard according to claim 1, after it is characterized in that step (3) comprises that also the purpose fragment that step (2) is obtained is inserted expression vector PET-28a (+) in different serial number purpose modes, transform e. coli bl21 (DE3), obtain the bacterial classification of a series of marking protein molecular weight standards.

4. the preparation method of a kind of novel protein molecular weight standard according to claim 3, it is characterized in that step (3) comprises that also the bacterial classification with a series of marking protein molecular weight standards claimed in claim 3 is inoculated in respectively in the LB fluid nutrient medium that contains that penicillin of card, 250rpm/min, it is 1.0mmol/L that 37 ℃ of cultivations added derivant isopropylthio-β-D-galactoside to final concentration after 12 hours, continue to cultivate after 4 hours, 5000rpm/min, centrifugal 20 minutes, the enrichment thalline; Thalline 10mM, the PBS damping fluid of PH7.4 is resuspended, ice bath, ultrasonic disruption 5-10 minute, the high speed refrigerated centrifuge was also collected supernatant.

5. the preparation method of a kind of novel protein molecular weight standard according to claim 4, it is characterized in that step (4) is with the mixed nickel agarose of the described supernatant that obtains of claim 4, first use the low concentration imidazoles solution washing foreign protein of 50mM, again with the high concentration imidazoles eluant solution of 300mM and collect destination protein, obtain the recombinant protein of the different number staphylococcal protein A of a series of series connection, i.e. protein molecular weight standard.

6. the preparation method of a kind of novel protein molecular weight standard according to claim 1, is characterized in that in step (5) protein immunoblot experiment, protein molecular weight standard used is the protein molecular weight standard that step (4) obtains; In immunoblot experiment, primary antibodie is respectively multiple mammal IgG antibody, and two anti-are corresponding HRP mark or the IgG antibody of other mark, and catalytic substrate is the fluorogenic substrates such as luminol, exposure and develop, photographic fixing.

7. the preparation method of a kind of novel protein molecular weight standard according to claim 1, is characterized in that described protein molecular weight standard can be used for the combination of mammal IgG antibody, detection, separation, purifying.