CN102558315A - Staphylococcal protein A, preparation method thereof and use thereof - Google Patents
Staphylococcal protein A, preparation method thereof and use thereof Download PDFInfo
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- CN102558315A CN102558315A CN2010105814173A CN201010581417A CN102558315A CN 102558315 A CN102558315 A CN 102558315A CN 2010105814173 A CN2010105814173 A CN 2010105814173A CN 201010581417 A CN201010581417 A CN 201010581417A CN 102558315 A CN102558315 A CN 102558315A
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Abstract
The invention discloses a staphylococcal protein A, a nucleotide sequence coding the staphylococcal protein A, and an affine chromatography media comprising the staphylococcal protein A and a chromatography media carrier. The amino acid sequence of the staphylococcal protein A provided in the invention is represented by SEQ ID NO:1, 5, 7 or 11. The affine chromatography media prepared by using the staphylococcal protein A provided in the invention has the advantages of strong protein combination specificity, high affinity and substantial improvement of the purification efficiency, and the dynamic adsorption load of the affine chromatography media is obviously higher than dynamic adsorption loads of Protein A Sepharose 4Fast Flow sold in the market.
Description
Technical field
The invention belongs to biological technical field, specifically, relate to a kind of SP.
Background technology
(Staphylococal Protein A is a kind of from the isolating protein of aureus cell wall SPA) to SP.1940, Vevwey found in some streptococcus aureus, to contain a kind of material, in double diffusion test, can form deposition with normal human serum.Nineteen fifty-nine, Jensen has also found similar phenomenon, with its called after A antigen; Nineteen sixty Grov is called for short the SPA albumin A with its called after SP, and Lofkvist in 1963 etc. have separated A antigen, and prove that it is a kind of protein, and have any different with sugar.
SP is the important medium of antibody purification.Along with China's biotechnology industry, with the monoclonal antibody drug fast development of the bio-pharmaceuticals industry of representative particularly, the market requirement of SP will increase sharply.
But when use contains the affinity chromatography technology of SP; There are problems such as avidity is low, purification efficiency is low, therefore when using this affinity chromatography technology, need a kind of improved SP; Realize higher avidity, improve the effect of purifying.
Summary of the invention
One of the object of the invention is, a kind of SP is provided, to overcome the deficiency of prior art.
Second purpose of the present invention is, the gene order of this SP of coding is provided.
The 3rd purpose of the present invention is, the carrier that contains this SP gene is provided.
The 4th purpose of the present invention is, provides and contains this SP expression carrier transformed host cell.
The 6th purpose of the present invention is, the purposes of this SP is provided.
The 7th purpose of the present invention is, a kind of affinity chromatography medium of being made up of this SP and chromatography media carrier is provided.
The aminoacid sequence of SP provided by the invention is like SEQ ID NO:1, shown in 5,7 or 11.
The nucleotide sequence of coding provided by the invention SP as claimed in claim 1 is like SEQ ID NO:2, shown in 6,8 or 12.
The expression regulation sequence that recombinant expression vector provided by the invention contains nucleotide sequence provided by the invention and links to each other with this series of operations property.
Host cell provided by the invention is transformed by expression vector provided by the invention, and preferred, host cell is intestinal bacteria, and is preferred, and intestinal bacteria are BL21 (DE3).
The purposes of SP provided by the invention is for being used for antibody test, separation, purifying.
Affinity chromatography medium provided by the invention is made up of SP provided by the invention and chromatography media carrier, and preferred chromatography media carrier is sepharose, VISOSE, Mierocrystalline cellulose, silica gel or hydroxyl high molecular polymer.
SP expression of gene product SP of the present invention can be used for becoming the separation and purification that affinity chromatography medium is used for antibody with various chromatography media carrier like couplings such as: sepharose, VISOSE, Mierocrystalline cellulose, hydroxyl high molecular polymer, silica gel.
Use the affinity chromatography medium of SP preparation provided by the invention to have the protein binding high specificity; Avidity is high; The advantage that purification efficiency improves is greatly compared with commercially available ProteinA Sepharose 4Fast Flow, and its dynamic adsorption carrying capacity obviously improves.
Embodiment
Further specify the present invention below in conjunction with embodiment, following embodiment should not be construed as limitation of the present invention.Embodiment does not comprise the detailed description to traditional method; Be used for the method for carrier construction and plasmid like those, the gene of proteins encoded is inserted into the method for such carrier and plasmid or plasmid is introduced the method for host cell and classical cytogamy is screened with monoclonal antibody and the method for purifying etc.Such method is well-known for the personnel that have ordinary skill in this area, and in many publications, all describes to some extent, comprises Sambrook; J., Fritsch, E.F.and Maniais; T. (1989) MolecularCloning:A Laboratory Manual, 2
NdEdition, Cold spring Harbor Laboratory Press.
In following embodiment of the present invention, use:
Buffer A is the PBS solution of pH7.4;
Buffer B is the 20mM citrate buffer solution of pH4.0, and its compound method is following: Hydrocerol A 2.1g adds water 950ml, transfers to pH4.0 with 5M NaOH, adds water to 1000ml;
Damping fluid C is the 0.5M acetate buffer solution of pH3.0, and its compound method is following: the 0.5M acetum transfers to pH3.0 with solid NaOH.
In following embodiment of the present invention, the human IgG1 of use presses disclosed method preparation among the Chinese invention patent 01132225.X.
Embodiment 1, synthetic SP gene monomer
Method through chemosynthesis; The sequence of the synthetic repeated fragment of SP gene of design, its sequence comprises that length is 174bp, front end adds the NcoI restriction enzyme site that links to each other with expression vector; 6 His (are used for affinity chromatography; Combine with the nickel post, reduce purification step), EK restriction enzyme site (be used for His and DDDDK are cut, form correct Protein A) and AccI restriction enzyme site (being used to be connected into a plurality of repeated fragments).In addition, also comprise terminator codon TAA, and the clone is total to 9bp with BamH I restriction enzyme joint.
Through the method for chemosynthesis, the sequence of the synthetic repeated fragment of SP gene of design, its sequence comprises that length is 174bp, two ends are the AccI restriction enzyme site, are used to make up the sequence that contains a plurality of repeated fragments.
Wherein, the repeated fragment sequence is shown in the 41-214 position Nucleotide of SEQ ID NO:2.
Embodiment 2, make up the pET32a carrier contain a SP gene monomer
With restricted type restriction endonuclease NcoI and BamH I above-mentioned SP gene monomer is downcut; Be connected with the carrier pET32a that cuts through NcoI and BamH I enzyme; Transformed into escherichia coli; Screening has the transformant of amicillin resistance, extracts through plasmid, and enzyme is cut and identified that back proof SP gene monomer is cloned among the pET32a.
Embodiment 3, contain the structure of the pET32a-P carrier of SP gene
Above-mentioned pET32a carrier and the SP gene monomer that contains a SP gene monomer cut with the AccI enzyme; Connect after reclaiming respective segments, transformed into escherichia coli, screening has the transformant of amicillin resistance; Extract through plasmid; Enzyme is cut the pET32a-P carrier that screening obtains to contain 3 monomeric SPs of protein A gene, and through detecting, its gene order is shown in SEQ ID No:2.
Embodiment 4, express the structure of the coli strain of SP
With the CaC12 method pET32a-P is transformed BL21 (DE3), screen transformant containing on the LB flat board of penbritin, detect the sub-BL21/pET32a of recombinant conversion that with restriction analysis acquisition contains pET32a through plasmid.
Embodiment 5, produce SP
5.1, culture of strains fermentation
Picking colibacillus engineering BL21/pET32a; Be inoculated in the LB substratum, inoculum size 1～2%V/V is in 30 ℃ of overnight cultures; Next day under aseptic condition with above-mentioned cultured seed substratum by 1: 10-1: 5 are inoculated on the fermention medium; 30 ℃ ferment to O.D600 and reach 0.4～0.8, are warming up to 42 ℃ and induce, centrifugal receipts bacterium after 3～5 hours.
The cell that takes a morsel adds 2 * sample-loading buffer, carries out the SDS-PAGE detected through gel electrophoresis, and the result shows that recombinant protein A induced soluble-expression in BL21/pET32a.
5.2, the purifying of the SP of expressing
The thalline of collecting in the step 5.1 is suspended with phosphate buffered saline buffer (0.2M, pH7.0-8.0 contain 0.1MNaCl), ultrasonication, 4 ℃ are centrifugal, collect supernatant, get crude extract.
Crude extract is used the SDS-PAGE electrophoresis detection; The result shows that most of SP is slightly carried; The amount that remains in thalline is atomic: crude extract is carried out Sephacryl 5200 molecular sieve purification; Collect the SP (called after Protein A 1) that characteristic peak (second elution peak) is purifying, its purity can reach more than 90%, and its aminoacid sequence is shown in SEQ ID No:1.
Adopt and the same or analogous method of embodiment 1-5; Respectively preparation, purifying 5 kinds of different SPs (called after ProteinA2-6 respectively); Nucleotide sequence respectively like SEQ ID No:4, shown in 6,8,10 and 12, corresponding amino acid sequence respectively like SEQ ID No:3, shown in 5,7,9 and 11.
Embodiment 6, ELISA detects SP and antibody binding activity
Adopt the ELISA method to detect SP and antibody binding activity, detailed process is following:
1) encapsulate: every hole encapsulates SP sample 100ul, 37 ℃, 1h in 96 orifice plates;
2) sealing: every hole is with 1% the BSA sealing of 100ul, 37 ℃, 1h;
3) adding one resists: every hole adds about 100ug human IgG antibody, 37 ℃, 1h;
4) adding two resists: every hole adds about 100ul, 1: 1000 horseradish peroxidase-labeled antibody, 37 ℃, 1h;
5) colour developing.
Detect demonstration through ELISA; Protein A 1,3,4,6 combines active strong with the human IgG antibody; Every hole encapsulates the 2.5-4.5ng SP can obtain tangible detection signal; Therefore can be used for detection of antibodies, and the effect of ProteinA 2 and ProteinA 5 is relatively poor relatively, needs 8.2 just can obtain detection signal with the 9.6ng SP respectively.
Embodiment 7, SP sepharose 5S CP preparation
Use the Protein A 1-6 of embodiment 5 preparations to prepare SP sepharose 5S CP respectively, detailed process is following:
1, react in water medium with epoxy chloropropane, sodium hydroxide and agarose (5% cross-linked agarose gel), reaction is 2-3 hour under 30-60 ℃ temperature, has reacted back the cleaning to neutrality with zero(ppm) water and has drained;
2, above-mentioned product was reacted 15-20 hour under 5-25 ℃ of temperature with ProteinA 1-6 respectively, reacted the back cleaning and drained again, promptly obtain SP sepharose 5S CP, respectively corresponding Protein A 1-6 called after 5S CP-1-5S CP-6.
Through detecting, the SP sepharose 5S CP1-6 characteristic of preparation is as shown in table 1:
The characteristic of the 5S CP of table 1, preparation
| 5S?CP-1 | 5S?CP-2 | 5S?CP-3 | |
| Aglucon density (SP/ml) | ≈6mg | ≈5mg | ≈6mg |
| Absorption carrying capacity (mouse IgG2a/ml) | 3～35mg | 3～26mg | 4～33mg |
| The particle size of affinity media (μ m) | 30-180 | 30-280 | 30-160 |
| Peak Flow Rate (cm/h) | 270 | 240 | 310 |
| The pH scope | 2-11 | 2-11 | 2-11 |
| Storage temperature | 4-8℃ | 4-8℃ | 4-8℃ |
| Preserve liquid | 20% ethanol | 20% ethanol | 20% ethanol |
| 5S?CP-4 | 5S?CP-5 | 5S?CP-6 | |
| Aglucon density (SP/ml) | ≈6mg | ≈6mg | ≈6mg |
| Absorption carrying capacity (mouse IgG2a/ml) | 3～36mg | 3～22mg | 3～37mg |
| The particle size of affinity media (μ m) | 30-180 | 40-250 | 40-150 |
| Peak Flow Rate (cm/h) | 275 | 285 | 295 |
| The pH scope | 2-11 | 2-11 | 2-11 |
| Storage temperature | 4-8℃ | 4-8℃ | 4-8℃ |
| Preserve liquid | 20% ethanol | 20% ethanol | 20% ethanol |
If treat that the antibody of separation and purification and the adsorptive power between affinity chromatography medium are more weak, then can suitably improve the pH value and the salt concn of adsorption-buffering liquid, can obtain separating effect preferably.
Embodiment 8, SP sepharose 5S CP separation and purification IgG2a from mouse ascites
Use the 5S CP1-5S CP-6 separation and purification IgG2a from mouse ascites that obtains among the embodiment 7 respectively, detailed process is following:
1, SP sepharose 5S CP dress post, 1.6 * 20cm, column volume are 10ml;
2, with 5-10 bed volume of buffer A balance, flow velocity is 1ml/min;
3, the 2ml mouse ascites is diluted to 20ml with buffer A, 0.45 μ m membrane filtration, last appearance, flow velocity is 1ml/min;
4, wash 5-10 bed volume again with buffer A, flow velocity is 1ml/min;
5, use the buffer B wash-out, flow velocity is 1ml/min, collects elution peak;
6, wash 10 column volumes with pure water stream, wash 10 column volumes with 20% ethanol stream again, flow velocity is 2ml/min, and pillar places 4-8 ℃ of environment to preserve;
7, IgG2a and the reference substance with separation and purification carries out the SDS-PAGE electrophoretic analysis simultaneously.
Should wash 1 time in order to damping fluid C stream after the every use several times of pillar, so that will adsorb the more albumen removal in jail.
The SDS-PAGE electrophoresis result shows, the 5SCP affinity chromatography medium that uses Protein A 1,3,4,6 preparations of the present invention can obtain purity greater than 95% IgG2a once going on foot, and wherein the effect of 5S CP-6 is best.
Embodiment 9, the SP polydextran gel preparation
Use the Protein A 1-6 of embodiment 5 preparations to prepare SP polydextran gel 1-6 respectively, detailed process is following:
1, react in water medium with epoxy chloropropane, sodium hydroxide and polydextran gel, reaction is 2-3 hour under 30-60 ℃ temperature, has reacted back the cleaning to neutrality with zero(ppm) water and has drained;
2, above-mentioned product and SP of the present invention were reacted 15-20 hour under 5-25 ℃ of temperature; Having reacted the back cleaning drains again; Promptly obtain the SP polydextran gel, respectively corresponding Protein A 1-6 called after SP polydextran gel 1-6.
Through detecting, the SP polydextran gel characteristic of preparation is as shown in table 2:
Table 2, SP polydextran gel 1-6 characteristic
Annotate: the corresponding SP polydextran gel 1-6 of 1-6 difference in the table.
If treat that the antibody of separation and purification and the adsorptive power between affinity chromatography medium are more weak, then can suitably improve the pH value and the salt concn of adsorption-buffering liquid, can obtain separating effect preferably.
Embodiment 10, SP polydextran gel separation and purification IgG2a from mouse ascites
Use the SP polydextran gel 1-6 separation and purification IgG2a from mouse ascites that obtains among the embodiment 9 respectively, detailed process is following:
1, SP polydextran gel dress post, 1.6 * 20cm, column volume are 10ml;
2, with 5-10 bed volume of buffer A balance, flow velocity is 1ml/min;
3, the 2ml mouse ascites is diluted to 20ml with buffer A, 0.45 μ m membrane filtration, last appearance, flow velocity is 1ml/min;
4, wash 5-10 bed volume again with buffer A, flow velocity is 1ml/min;
5, use the buffer B wash-out, flow velocity is 1ml/min, collects elution peak;
6, wash 10 column volumes with pure water stream, wash 10 column volumes with 20% ethanol stream again, flow velocity is 2ml/min, and pillar places 4-8 ℃ of environment to preserve;
7, IgG2a and the reference substance with separation and purification carries out the SDS-PAGE electrophoretic analysis simultaneously;
Should wash 1 time in order to damping fluid C stream after the every use several times of pillar, so that will adsorb the more albumen removal in jail.
The SDS-PAGE electrophoresis result shows; The SP polydextran gel affinity chromatography medium that uses Protein A 1,3,4,6 preparations can obtain purity greater than 95% IgG2a once going on foot, and wherein the effect of the SP polydextran gel affinity chromatography medium for preparing of Protein A 4 is best.
Embodiment 11, SP sepharose, polydextran gel and commercially available ProteinASepharose 4 Fast Flow to the mensuration of human IgG1's dynamic adsorption carrying capacity relatively
Detect SP sepharose that previous embodiment prepares, polydextran gel and commercially available ProteinA Sepharose 4 Fast Flow to human IgG1's dynamic adsorption carrying capacity, concrete steps are following:
1, with 5-10 bed volume of buffer A balance, flow velocity is 1ml/min;
2, the concentration known human IgG1 goes up appearance, and flow velocity is 1ml/min, when 10% penetrates, stops;
3, per sample concentration, go up the dynamic adsorption carrying capacity that appearance volume and column volume calculate 10% each gel when penetrating.
The result shows; The SP sepharose of use Protein A 1,3,4,6 preparations and SP polydextran gel all are higher than commercially available ProteinA Sepharose 4Fast Flow (about 18mg/ml) to human IgG1's dynamic adsorption carrying capacity (about 25-30mg/ml); And use the SP sepharose of Protein A 2,5 preparations similar with commercially available ProteinA Sepharose 4Fast Flow to human IgG1's dynamic adsorption carrying capacity (about 18mg/ml), but the SP polydextran gel that use Protein A 2,5 prepares to human IgG1's dynamic adsorption carrying capacity (about 15mg/ml) a little less than commercially available ProteinA Sepharose 4 Fast Flow.
Claims (10)
1. a SP is characterized in that, the aminoacid sequence of said SP is like SEQ ID NO:1, shown in 5,7 or 11.
2. the encode nucleotide sequence of SP as claimed in claim 1 is characterized in that said nucleotide sequence is like SEQ ID NO:2, shown in 6,8 or 12.
3. a recombinant expression vector is characterized in that, the expression regulation sequence that said expression vector contains the described nucleotide sequence of claim 2 and links to each other with this series of operations property.
4. expression vector as claimed in claim 3 is characterized in that said expression vector is pET32a-P.
5. a host cell is characterized in that, said host cell transforms have the right requirement 3 or 4 described expression vectors.
6. host cell as claimed in claim 5 is characterized in that the host cell of being addressed is intestinal bacteria.
7. host cell as claimed in claim 6 is characterized in that, the intestinal bacteria of being addressed are BL21 (DE3).
8. SP as claimed in claim 1 is used for the application of antibody test, separation, purifying.
9. an affinity chromatography medium is characterized in that, said affinity chromatography medium is made up of described SP of claim 1 and chromatography media carrier.
10. affinity chromatography medium as claimed in claim 10 is characterized by, and said chromatography media carrier is sepharose, VISOSE, Mierocrystalline cellulose, silica gel or hydroxyl high molecular polymer.
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Application publication date: 20120711 |