CN102558316B

CN102558316B - High-stability Recomb Protein A having antibody binding capacity and preparation thereof

Info

Publication number: CN102558316B
Application number: CN201010614100.5A
Authority: CN
Inventors: 李素霞; 赵致; 冯矗
Original assignee: SHANGHAI YAXIN BIOTECH CO Ltd
Current assignee: SHANGHAI YAXIN BIOTECH CO Ltd
Priority date: 2010-12-30
Filing date: 2010-12-30
Publication date: 2015-07-01
Anticipated expiration: 2030-12-30
Also published as: CN102558316A

Abstract

The invention relates to high-stability Recomb Protein A having an antibody binding capacity and preparation thereof. The invention further provides affinity chromatography columns which contain the high-stability Recomb Protein A having an antibody binding capacity. Compared with wide-type Protein A, the high-stability Recomb Protein A having an antibody binding capacity is greatly improved on stability and has a higher antibody binding capacity.

Description

The recombinant protein A of the tool antibody binding capacity of high stability and production thereof

Technical field

The invention belongs to biological technical field; More specifically, the present invention relates to recombinant protein A and the production thereof of the tool antibody binding capacity of high stability.

Background technology

Albumin A (Protein A) derives from streptococcus aureus (Staphylococcus aureus), is a kind of membranin on its surface.Albumin A has the ability be combined with antibodies specific, protein A affinity chromatography post has become the affinity column of the widely used antibody purification of biological technical field, can from ascites, in serum and cells and supernatant or cell extract the multiple Mammals different subtype of abstraction and purification antibody or comprise the genetically engineered recombinant protein of antibody Fc fragment.Albumin A can be produced by genetically engineered recombinant technology.

But the albumin A that existing market is sold has unstable defect, thus affect its use.This area also needs to be optimized albumin A and production technology thereof, to improve its stability and antigen binding capacity, and then improves its using value.

Summary of the invention

The object of the present invention is to provide a kind of recombinant protein A and production method thereof of tool antibody binding capacity of new high stability.

In a first aspect of the present invention, provide a kind of recombinant protein A of tool antibody binding capacity, it is:

The albumen of the aminoacid sequence shown in (a) SEQ ID NO:2; Or

B () is passed through by the aminoacid sequence shown in (a) and is replaced, lacks or add one or several amino acid (as 1-10, preferably 1-5, more preferably 1-3) and there is the albumen derivative by (a) of the identical function of albumen that (a) limit.

In another preference, described disappearance or interpolation occur in the carboxyl terminal of albumen, aminoterminal or the two have concurrently.

In another aspect of this invention, provide a kind of polynucleotide, it is selected from lower group:

The polynucleotide of the recombinant protein A of the tool antibody binding capacity described in (i) coding; Or

(ii) polynucleotide of the polynucleotide complementation and in (i).

In another preference, the albumen of aminoacid sequence shown in this polynucleotide encoding SEQ ID NO:2.

In another preference, the nucleotide sequence of these polynucleotide is as shown in SEQ ID NO:1.

In another aspect of this invention, provide a kind of carrier, it contains described polynucleotide.

In another aspect of this invention, provide a kind of genetically engineered host cell, it contains described carrier, or is integrated with described polynucleotide in genome.

In another aspect of this invention, a kind of method of producing the recombinant protein A of described tool antibody binding capacity is provided, comprises step:

(1) host cell described in cultivation, obtains culture; With

(2) from culture, be separated the recombinant protein A of described tool antibody binding capacity.

In another preference, at temperature 20 ± 10 DEG C, cultivate described host cell.

In another preference, at temperature 20 ± 7 DEG C, cultivate described host cell; More preferably at temperature 20 ± 5 DEG C, cultivate described host cell; More preferably at temperature 15 ± 3 DEG C, cultivate described host cell.

In another preference, described host cell is Bacillus coli cells, cultivates in described host cell process and adopts IPTG or lactose-induced expression.

In another preference, adopt the IPTG abduction delivering of 0.05-2.0mM concentration.

In another aspect of this invention, the purposes of the recombinant protein A of described tool antibody binding capacity is provided, for binding antibody; Or for the preparation of antibody affinity column; Or for the purifying of antibody.

In another aspect of this invention, provide a kind of affinity column of antibody purification, it comprises affinity media, and is attached to the recombinant protein A of described tool antibody binding capacity of described affinity media.

In another preference, described affinity media is selected from (but being not limited to): sepharose, dextrane gel, Mierocrystalline cellulose, macroporous adsorbent resin etc.

Other side of the present invention, due to disclosure herein, is apparent to those skilled in the art.

Accompanying drawing explanation

Fig. 1, left figure are staphylococcus aureus gene group extracting result; Right figure is the electroresis appraisal result of total length SPA (FL) and truncate SPA (32-327) PCR primer.In figure, SPA-32 refers to truncate SPA (32-327).

Fig. 2, bacterium colony PCR qualification result.

Fig. 3, this recombinant expression vector is transformed into competent e. coli bl21 (DE3), and carries out abduction delivering.

Wherein, Lane1: before induction; After Lane2,3,4,5:0.5mM IPTG induces.

Fig. 4 left figure: SDS-PAGE detects and under different temperature condition, cultivates Recombinant staphylococcus protein A (32-327) BL21 (DE3) cell, the culture supernatant of acquisition and the expression of inclusion bodies albumen.

The right figure of Fig. 4: for SDS-PAGE detects the expression cultivating albumen in restructuring BL21 (DE3) cell under different inductive conditions.Wherein Lane 1: before induction; Lane 2:0.1mM IPTG; Lane 3:0.5mM IPTG; Lane 4:1.0mM IPTG; Lane 5:10mM IPTG.Sup: supernatant; Pel: precipitation.

The growth curve chart of SPA (32-327) under Fig. 5, differing temps.

Fig. 6, course of fermentation curve.

Fig. 7, SDS-PAGE electroresis appraisal DEAE-FF purifying protein.Each swimming lane is respectively: 1:SPA (32-327) supernatant; 2: effluent liquid; 3,4,5: target protein.

Fig. 8, Ni-NTA purifying SPA (32-327).

Repeated pruning before and after Fig. 9, SPA (32-327) freeze-drying.Wherein each swimming lane is respectively: before 1:SPA (32-327) freeze-drying; Before 2:SPA (32-327)-His freeze-drying; After 3:SPA (32-327) freeze-drying; After 4:SPA (32-327)-His freeze-drying.

The SPA (32-327) of Figure 10, different protein concentration or the Dottern experimental result of SPA (32-327)-His.

Embodiment

The present inventor, through extensive and deep research, discloses a kind of recombinant protein A of tool antibody binding capacity of high stability first, and compared with wild-type protein A, this recombinant protein A stability improves greatly, has stronger antibody binding capacity.Complete the present invention on this basis.

As used herein, unless otherwise indicated, described " recombinant protein A of tool antibody binding capacity ", " SPA (32-327) ", " truncate Protein S PA (32-327) ", " truncate albumen " etc. are used interchangeably, all refer to relative to wild-type protein A, its aminoterminal lacks 31 amino acid whose albumen; More preferably, relative to wild-type protein A, its carboxyl terminal also lacks 100-200 amino acid (as 173 amino acid).

If desired represent the albumen of wild-type, unless otherwise indicated, it will be denoted as " wild-type protein A ", " SPA (FL) full-length proteins ".

As used herein, " separation " refers to that material is separated from its primal environment (if natural substance, namely primal environment is natural surroundings).As the polynucleotide under the native state in active somatic cell and albumen do not have separation and purification, but same polynucleotide or albumen as from native state with in other materials existed separately, then for separation and purification." restructuring " refers to albumen, engineering carrier or the cell etc. that obtain (or preparing in a large number) by genetic engineering means.

Albumen of the present invention can be recombinant protein, native protein, synthetic proteins, preferred recombinant protein.Albumen of the present invention can be native purified product, or the product of chemosynthesis, or uses recombinant technology to produce from protokaryon or eucaryon host (such as, bacterium, yeast, higher plant, insect and mammalian cell).The host used according to recombinant production scheme, albumen of the present invention can be glycosylated, can be maybe nonglycosylated.Albumen of the present invention also can comprise or not comprise initial methionine residues.

The present invention also comprises the fragment of the recombinant protein A of described tool antibody binding capacity, derivative and analogue.As used herein, term " fragment ", " derivative " and " analogue " refer to the albumen substantially keeping biological function that the recombinant protein A of tool antibody binding capacity of the present invention is identical or activity.Protein fragments of the present invention, derivative or analogue can be the albumen that (i) has one or more conservative or non-conservative amino acid residue (preferred conservative amino acid) and be substituted, and the amino-acid residue of such replacement can may not be and encoded by genetic code, or (ii) has the albumen of substituted radical in one or more amino-acid residue, or (iii) maturation protein and another compound (such as extend the compound of protein half-life, such as polyoxyethylene glycol) merge the albumen formed, or (iv) additional aminoacid sequence is fused to this protein sequence and the albumen formed (as leader sequence or secretion sequence or be used for the sequence of this albumen of purifying or proprotein sequence, or fusion rotein).The known scope of those skilled in the art is belonged to according to these fragments of definition herein, derivative and analogue.But, in the recombinant protein A of described tool antibody binding capacity and the aminoacid sequence of fragment, derivative and analogue thereof, relative to wild-type protein A, its aminoterminal lacks 31 amino acid whose albumen more preferably, relative to wild-type protein A, its carboxyl terminal also lacks 100-200 amino acid (as 173 amino acid).

In the present invention, term " recombinant protein A of tool antibody binding capacity " refers to the albumen of the SEQ ID NO:2 sequence of the recombinant protein A activity with tool antibody binding capacity of the present invention.This term also comprise have with the recombinant protein A identical function of described tool antibody binding capacity, the variant form of SEQ ID NO:2 sequence.These variant forms comprise (but being not limited to): several (are generally 1-20, more preferably 1-10, also better for 1-8,1-5,1-3 or 1-2) amino acid whose disappearance, insertion and/or replacement, and (be generally within 20 in C-terminal interpolation or disappearance one or several, within being preferably 10, within being more preferably 5) amino acid.Such as, in the art, when replacing with similar nature or similar amino acid, the function of protein can not usually be changed.Again such as, add at C-terminal the function that or several amino acid also can not change protein usually.This term also comprises active fragments and the reactive derivative of the recombinant protein A of tool antibody binding capacity.Best, in the variant form of these SEQ ID NO:2, relative to wild-type protein A, its aminoterminal lacks 31 amino acid whose albumen; More preferably, relative to wild-type protein A, its carboxyl terminal also lacks 100-200 amino acid (as 173 amino acid).

The variant form of albumen comprises: homologous sequence, conservative variant, allelic variant, natural mutation, induced mutants, under high or low stringency condition can with the albumen coded by the DNA of the DNA hybridization of the recombinant protein A of tool antibody binding capacity and the albumen utilizing the antiserum(antisera) of the recombinant protein A of described anti-tool antibody binding capacity to obtain or albumen.Present invention also offers other albumen, as the fusion rotein of the recombinant protein A or its fragment that comprise tool antibody binding capacity.

Invention also provides the recombinant protein A of tool antibody binding capacity or the analogue of albumen.The difference of the recombinant protein A of these analogues and tool antibody binding capacity can be the difference on aminoacid sequence, can be also the difference do not affected on the modified forms of sequence, or have both at the same time.These albumen comprise genetic variant that is natural or induction.Induce variation body can be obtained by various technology, as by radiation or be exposed to mutagenic compound and produce random mutagenesis, also by site-directed mutagenesis or the biological technology of other known moleculars.Analogue also comprises the analogue with the residue (as D-amino acid) being different from natural L-amino acids, and has the analogue of amino acid (as β, gamma-amino acid) that is that non-natural exists or synthesis.Should be understood that albumen of the present invention is not limited to the above-mentioned representational albumen exemplified.

(usually the not changing primary structure) form of modification comprises: the chemically derived form of the albumen that body is interior or external is as acetylize or carboxylated.Modify and also comprise glycosylation.Modified forms also comprises the sequence with phosphorylated amino acid residue (as Tyrosine O-phosphate, phosphoserine, phosphothreonine).Also comprise and modified thus improve its anti-proteolysis performance or optimize the albumen of solubility property.

In the present invention, " the recombinant protein A conservative variation albumen of tool antibody binding capacity " refers to compared with the aminoacid sequence of SEQ IDNO:2, there are 10 at the most, preferably at the most 8, more preferably at the most 5, best at the most 3 (as 1,2 or 3) amino acid replace by the similar or close amino acid of character and form albumen.These conservative variation's albumen preferably carry out amino acid replacement according to table 1 and produce.

Table 1

Initial residue	Representational replacement	Preferred replacement
			Ala(A)	Val；Leu；Ile	Val

Arg(R)	Lys；Gln；Asn	Lys
			Asn(N)	Gln；His；Lys；Arg	Gln
Asp(D)	Glu	Glu
			Cys(C)	Ser	Ser
Gln(Q)	Asn	Asn
			Glu(E)	Asp	Asp
Gly(G)	Pro；Ala	Ala
			His(H)	Asn；Gln；Lys；Arg	Arg
Ile(I)	Leu；Val；Met；Ala；Phe	Leu
			Leu(L)	Ile；Val；Met；Ala；Phe	Ile
Lys(K)	Arg；Gln；Asn	Arg
			Met(M)	Leu；Phe；Ile	Leu
Phe(F)	Leu；Val；Ile；Ala；Tyr	Leu
			Pro(P)	Ala	Ala
Ser(S)	Thr	Thr
			Thr(T)	Ser	Ser
Trp(W)	Tyr；Phe	Tyr
			Tyr(Y)	Trp；Phe；Thr；Ser	Phe
Val(V)	Ile；Leu；Met；Phe；Ala	Leu

Present invention also offers the recombinant protein A of code book invention tool antibody binding capacity or the polynucleotide sequence of its conservative variation's albumen.

Polynucleotide of the present invention can be DNA form or rna form.DNA form comprises the DNA of cDNA, genomic dna or synthetic.DNA can be strand or double-strand.DNA can be coding strand or noncoding strand.The coding region sequence of encoding mature albumen can the varient of or degeneracy identical with the coding region sequence shown in SEQ ID NO:1.As used herein, " varient of degeneracy " refers to that coding has the protein of SEQ ID NO:2 in the present invention, but with the differentiated nucleotide sequence of coding region sequence shown in SEQ ID NO:1.

The polynucleotide of the maturation protein of coding SEQ ID NO:2 comprise: the encoding sequence of an encoding mature albumen; The encoding sequence of maturation protein and various additional coding sequence; The encoding sequence (with optional additional coding sequence) of maturation protein and non-coding sequence.

Term " polynucleotide of proteins encoded " can be the polynucleotide comprising this albumen of coding, also can be the polynucleotide also comprising additional code and/or non-coding sequence.

The invention still further relates to the varient of above-mentioned polynucleotide, its coding has the albumen of identical aminoacid sequence or fragment, the sum analogous to general Dedekind sum of albumen with the present invention.The varient of these polynucleotide can be the allelic variant of natural generation or the varient of non-natural generation.These nucleotide variants comprise and replace varient, Deletion variants and insertion varient.As known in the art, allelic variant is the replacement form of polynucleotide, and it may be the replacement of one or more Nucleotide, disappearance or insertion, but can not from the function of albumen changing in fact its coding.

The invention still further relates to and above-mentioned sequence hybridization and have at least 50% between two sequences, preferably at least 70%, the more preferably polynucleotide of at least 80% (as 85%, 90%, 95%, 99%) homogeny.The present invention be more particularly directed to polynucleotide interfertile with polynucleotide of the present invention under strict conditions.In the present invention, " stringent condition " refers to: (1) compared with the hybridization under low ionic strength and comparatively high temps and wash-out, as 0.2 × SSC, 0.I%SDS, 60 DEG C; Or be added with denaturing agent during (2) hybridization, and as 50% (v/v) methane amide, 0.1% calf serum/0.1%Ficoll, 42 DEG C etc.; Or (3) homogeny only between two sequences, at least more than 90%, is just hybridized when being more preferably more than 95%.Further, the albumen of interfertile polynucleotide encoding has identical biological function and activity with the maturation protein shown in SEQID NO:2.

The invention still further relates to the nucleic acid fragment with above-mentioned sequence hybridization.As used herein, the length of " nucleic acid fragment ", at least containing 15 Nucleotide, is better at least 30 Nucleotide, is more preferably at least 50 Nucleotide, preferably more than at least 100 Nucleotide.The amplification technique (as PCR) that nucleic acid fragment can be used for nucleic acid is to determine and/or to be separated the polynucleotide of recombinant protein A of coding tool antibody binding capacity.

The recombinant protein A Nucleotide full length sequence of tool antibody binding capacity of the present invention or its fragment can obtain by the method for pcr amplification method, recombination method or synthetic usually.For pcr amplification method, can be disclosed according to the present invention about nucleotide sequence, especially open reading frame sequence designs primer, and with commercially available cDNA storehouse or by the cDNA storehouse prepared by ordinary method well known by persons skilled in the art as template, amplification and relevant sequence.When sequence is longer, usually needs to carry out twice or repeatedly pcr amplification, and then the fragment that each time amplifies is stitched together by proper order.

Once obtain relevant sequence, just relevant sequence can be obtained in large quantity with recombination method.This is normally cloned into carrier, then proceeds to cell, is then separated from the host cell after propagation by ordinary method and obtains relevant sequence.

In addition, also relevant sequence can be synthesized, when especially fragment length is shorter by the method for synthetic.Usually, by first synthesizing multiple small segment, and then carry out connect can obtain the very long fragment of sequence.

At present, the DNA sequence dna of code book invention albumen (or its fragment, or derivatives thereof) can be obtained completely by chemosynthesis.Then this DNA sequence dna can be introduced in various existing DNA molecular (or as carrier) as known in the art and cell.In addition, also by chemosynthesis, sudden change is introduced in protein sequence of the present invention.

The present invention also relates to the carrier comprising polynucleotide of the present invention, and with the host cell that the recombinant protein A encoding sequence of carrier of the present invention or tool antibody binding capacity produces through genetically engineered, and the method for albumen of the present invention is produced through recombinant technology.

By recombinant DNA technology (Science, 1984 of routine; 224:1431), polynucleotide sequence of the present invention can be utilized express or the recombinant protein A of tool antibody binding capacity of Restruction.In general following steps are had:

(1). with the polynucleotide (or varient) of the recombinant protein A of coding tool antibody binding capacity of the present invention, or transform or suitable host cell of transduceing with the recombinant expression vector containing these polynucleotide;

(2). the host cell cultivated in suitable substratum;

(3). separation, protein purification from substratum or cell.

In the present invention, the recombinant protein A polynucleotide sequence of tool antibody binding capacity can be inserted in recombinant expression vector.Term " recombinant expression vector " refers to bacterial plasmid well known in the art, phage, yeast plasmid, vegetable cell is viral, mammalian cell is viral or other carriers.In a word, as long as can copy in host and stablize, any plasmid and carrier can be used.A key character of expression vector is usually containing replication orgin, promotor, marker gene and translation controlling elements.

Method well-known to those having ordinary skill in the art can be used for building the recombinant protein A DNA sequences encoding containing tool antibody binding capacity and the suitable expression vector of transcribing/translating control signal.These methods comprise recombinant DNA technology in vi, DNA synthetic technology, In vivo recombination technology etc.Described DNA sequence dna can be effectively connected in the suitable promotor in expression vector, synthesizes to instruct mRNA.Expression vector also comprises ribosome bind site and the transcription terminator of translation initiation.

In addition, expression vector preferably comprises one or more selected marker, to be provided for the phenotypic character selecting the host cell transformed, as Tetrahydrofolate dehydrogenase, neomycin resistance and green fluorescent protein (GFP) that eukaryotic cell is cultivated, or for colibacillary kantlex or amicillin resistance.

Comprise the carrier of above-mentioned suitable DNA sequence dna and suitably promotor or control sequence, may be used for transforming suitable host cell, with can marking protein.

Host cell can be prokaryotic cell prokaryocyte, as bacterial cell; Or the eukaryotic cell such as low, as yeast cell; Or higher eucaryotic cells, as vegetable cell.Representative example has: intestinal bacteria, streptomyces, Agrobacterium; Fungal cell is as yeast; Vegetable cell etc.

When polynucleotide of the present invention are expressed in higher eucaryotic cells, if will make to transcribe to be enhanced when inserting enhancer sequence in the carrier.Enhanser is the cis-acting factors of DNA, and nearly 10 to 300 base pairs, act on promotor transcribing with enhancing gene usually.

Persons skilled in the art all know how to select suitable carrier, promotor, enhanser and host cell.

Can carry out with routine techniques well known to those skilled in the art with recombinant DNA transformed host cell.When host be prokaryotic organism as intestinal bacteria time, the competent cell that can absorb DNA can be gathered in the crops at exponential growth after date, uses CaCl ₂method process, step used is well-known in this area.Another kind method uses MgCl ₂.If needed, transform and also can be undertaken by the method for electroporation.When host is eukaryote, can select following DNA transfection method: calcium phosphate precipitation, conventional mechanical methods is as microinjection, electroporation, liposome packaging etc.

The transformant obtained can be cultivated by ordinary method, expresses the albumen of coded by said gene of the present invention.According to host cell used, substratum used in cultivation can be selected from various conventional medium.Cultivate under the condition being suitable for host cell growth.When after host cell growth to suitable cell density, the promotor selected with the induction of suitable method (as temperature transition or chemical induction), cultivates for some time again by cell.

As another example of the present invention, the recombinant protein A of tool antibody binding capacity is produced by genetic engineering means, such as utilize the recombinant protein A of the tool antibody binding capacity described in the production of any suitable genetic engineering bacterium, the recombinant protein A of the tool antibody binding capacity described in separation.

Recombinant protein in the above methods can be expressed or be secreted into extracellular in cell or on cytolemma.If needed, can utilize its physics, the albumen of being recombinated by various separation method abstraction and purification with other characteristic of chemistry.These methods are well-known to those skilled in the art.The example of these methods includes, but are not limited to: conventional renaturation process, combination by protein precipitant process (salting-out method), centrifugal, the broken bacterium of infiltration, super process, ultracentrifugation, sieve chromatography (gel-filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography (LC) technology and these methods.

As a specific embodiment of the present invention, the recombinant protein A of described tool antibody binding capacity is according to Genbank (GI:88193823) Staphylococcus aureus complete genome sequence design primer, the SPA transferred from staphylococcus aureus gene group (32-327) gene fragment.Described gene fragment, after being connected with expression vector, is expressed in intestinal bacteria, obtains the clone that expression amount is higher.And purifying has been carried out to the SPA after expressing, the albumen after purifying has comparatively powerful antibody binding ability.

As optimal way of the present invention, the recombinant protein A of described tool antibody binding capacity is preferably selected to express at low temperatures, its at low temperatures expression amount be higher than at high temperature as the expression at 37 DEG C.

The recombinant protein A of tool antibody binding capacity of the present invention preferably may be used for the affinity column preparing antibody purification.Therefore, the invention provides a kind of affinity column of antibody purification, it comprises affinity media, and is attached to the recombinant protein A of tool antibody binding capacity of the present invention of described affinity media.

The present invention does not have special to described affinity media but limits, and any affinity media being suitable for attachment protein A is all available, such as but not limited to: agarose (pearl), dextrane gel, Mierocrystalline cellulose, macroporous adsorbent resin etc.

Utilize the method for the affinity column described in the preparation of the recombinant protein A of tool antibody binding capacity of the present invention to have no particular limits, method well known to those skilled in the art and condition can be adopted.

Major advantage of the present invention is:

The recombinant protein A of tool antibody binding capacity of the present invention has good stability, feature that antibody binding capacity is strong.And the poor stability of the albumin A of total length.

The recombinant protein A of tool antibody binding capacity of the present invention has high antibody binding capacity.Can be used for the antibody affinity column preparing albumin A, for the purifying of antibody.In an embodiment of the present invention, the SPA (32-327) of verified 6ng just can with antibodies, and along with the increase of protein content, more with antibodies.

Below in conjunction with specific embodiment, set forth the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usual conveniently condition is as people such as Sambrook, molecular cloning: lab guide (New York:Cold Spring Harbor Laboratory Press, 2001) condition described in, or according to the condition that manufacturer advises.Unless otherwise indicated, otherwise per-cent and number calculate by weight.

Unless otherwise defined, all specialties used in literary composition and scientific words and one skilled in the art the same meaning be familiar with.In addition, any method similar or impartial to described content and material all can be applicable in the present invention.The use that better implementation method described in literary composition and material only present a demonstration.

Embodiment 1, gene order and corresponding protein sequence

Described SPA (32-327) gene order following (SEQ ID NO:1,891bp):

CCTGCTGCAAATGCTGCGCAACACGATGAAGCTCAACAAAATGCTTTTTATCAAGTTTTAAATATGCCTAACTTAAATGCTGATCGACGCAATGGTTTTATCCAAAGCCTTAAAGATGATCCAAGCCAAAGTGCTAACGTTTTAGGTGAAGCTCAAAAACTTAATGACTCTCAAGCTCCAAAAGCTGATGCGCAACAAAATAACTTCAACAAAGATCAACAAAGCGCCTTCTATGAAATTTTGAACATGCCTAACTTAAACGAAGCGCAACGCAATGGTTTCATTCAAAGTCTTAAAGACGATCCAAGCCAAAGCACTAACGTTTTAGGTGAAGCTAAAAAATTAAACGAATCTCAAGCACCGAAAGCTGACAACAATTTCAACAAAGAACAACAAAATGCTTTCTATGAAATCTTGAACATGCCTAACTTGAACGAAGAACAACGCAATGGTTTCATCCAAAGCTTAAAAGATGACCCAAGTCAAAGTGCTAACCTATTGTCAGAAGCTAAAAAGTTAAATGAATCTCAAGCACCGAAAGCGGATAACAAATTCAACAAAGAACAACAAAATGCTTTCTATGAAATCTTACATTTACCTAACTTAAACGAAGAACAACGCAATGGTTTCATCCAAAGCTTAAAAGATGACCCAAGCCAAAGCGCTAACCTTTTAGCAGAAGCTAAAAAGCTAAATGATGCACAAGCACCAAAAGCTGACAACAAATTCAACAAAGAACAACAAAATGCTTTCTATGAAATTTTACATTTACCTAACTTAACTGAAGAACAACGTAACGGCTTCATCCAAAGCCTTAAAGACGATCCTTCAGTGAGCAAAGAAATTTTAGCAGAAGCTAAAAAGCTAAACGATGCTCAAGCACCAAAATAA

The protein sequence following (SEQ ID NO:2,296aa) of described SPA (32-327):

P A A N A A Q H D E A Q Q N A F Y Q V L N M P N L N A D R R N G F I Q S L K D D P S Q S AN V L G E A Q K L N D S Q A P K A D A Q Q N N F N K D Q Q S A F Y E I L N M P N L N E AQ R N G F I Q S L K D D P S Q S T N V L G E A K K L N E S Q A P K A D N N F N K E Q Q N AF Y E I L N M P N L N E E Q R N G F I Q S L K D D P S Q S A N L L S E A K K L N E S Q A P KA D N K F N K E Q Q N A F Y E I L H L P N L N E E Q R N G F I Q S L K D D P S Q S A N L L AE A K K L N D A Q A P K A D N K F N K E Q Q N A F Y E I L H L P N L T E E Q R N G F I Q S LK D D P S V S K E I L A E A K K L N D A Q A P K

SPA (32-327) gene order obtained and corresponding protein sequence (Query 1; SEQ ID NO:2) and as follows to the comparison of the sequence (Sbjct1, SEQ ID NO:3) in corresponding site in Genbank (GI:88193823) Staphylococcus aureus sequence:

Query 1 PAANAAQHDEAQQNAFYQVLNMPNLNADRRNGFIQSLKDDPSQSANVLGEAQKLNDSQAP 60

PAANAAQHDEAQQNAFYQVLNMPNLNAD+RNGFIQSLKDDPSQSANVLGEAQKLNDSQAP

Sbjct 1 PAANAAQHDEAQQNAFYQVLNMPNLNADQRNGFIQSLKDDPSQSANVLGEAQKLNDSQAP 60

Query 61 KADAQQNNFNKDQQSAFYEILNMPNLNEAQRNGFIQSLKDDPSQSTNVLGEAKKLNESQA 120

KADAQQNNFNKDQQSAFYEILNMPNLNEAQRNGFIQSLKDDPSQSTNVLGEAKKLNESQA

Sbjct 61 KADAQQNNFNKDQQSAFYEILNMPNLNEAQRNGFIQSLKDDPSQSTNVLGEAKKLNESQA 120

Query 121 PKADNNFNKEQQNAFYEILNMPNLNEEQRNGFIQSLKDDPSQSANLLSEAKKLNESQAPK 180

PKADNNFNKEQQNAFYEILNMPNLNEEQRNGFIQSLKDDPSQSANLLSEAKKLNESQAPK

Sbjct 121 PKADNNFNKEQQNAFYEILNMPNLNEEQRNGFIQSLKDDPSQSANLLSEAKKLNESQAPK 180

Query 181 ADNKFNKEQQNAFYEILHLPNLNEEQRNGFIQSLKDDPSQSANLLAEAKKLNDAQAPKAD 240

ADNKFNKEQQNAFYEILHLPNLNEEQRNGFIQSLKDDPSQSANLLAEAKKLNDAQAPKAD

Sbjct 181 ADNKFNKEQQNAFYEILHLPNLNEEQRNGFIQSLKDDPSQSANLLAEAKKLNDAQAPKAD 240

Query 241 NKFNKEQQNAFYEILHLPNLTEEQRNGFIQSLKDDPSVSKEILAEAKKLNDAQAPK 296

NKFNKEQQNAFYEILHLPNLTEEQRNGFIQSLKDDPSVSKEILAEAKKLNDAQAPK

Sbjct 241 NKFNKEQQNAFYEILHLPNLTEEQRNGFIQSLKDDPSVSKEILAEAKKLNDAQAPK 296

SPA (FL) full-length gene order following (1503bp, SEQ ID NO:4):

TTGAAAAAGAAAAACATTTATTCAATTCGTAAACTAGGTGTAGGTATTGCATCTGTAACTTTAGGTACATTACTTATATCTGGTGGCGTAACACCTGCTGCAAATGCTGCGCAACACGATGAAGCTCAACAAAATGCTTTTTATCAAGTCTTAAATATGCCTAACTTAAATGCTGATCAACGCAATGGTTTTATCCAAAGCCTTAAAGATGATCCAAGCCAAAGTGCTAACGTTTTAGGTGAAGCTCAAAAACTTAATGACTCTCAAGCTCCAAAAGCTGATGCGCAACAAAATAACTTCAACAAAGATCAACAAAGCGCCTTCTATGAAATTTTGAACATGCCTAACTTAAACGAAGCGCAACGCAATGGTTTCATTCAAAGTCTTAAAGACGATCCAAGCCAAAGCACTAACGTTTTAGGTGAAGCTAATAAATTAAACGAATCTCAAGCACCGAAAGCTGACAACAATTTCAACAAAGAACAACAAAATGCTTTCTATGAAATCTTGAACATGCCTAACTTGAACGAAGAACAACGCAATGGTTTCATCCAAAGCTTAAAAGATGACCCAAGTCAAAGTGCTAACCTATTGTCAGAAGCTAAAAAGTTAAATGAATCTCAAGCACCGAAAGCGGATAACAAATTCAACAAAGAACAACAAAATGCTTTCTATGAAATCTTACATTTACCTAACTTAAACGAAGAACAACGCAATGGTTTCATCCAAAGCTTAAAAGATGACCCAAGCCAAAGCGCTAACCTTTTAGCAGAAGCTAAAAAGCTAAATGATGCACAAGCACCAAAAGCTGACAACAAATTCAACAAAGAACAACAAAATGCTTTCTATGAAATTTTACATTTACCTAACTTAACTGAAGAACAACGTAACGGCTTCATCCAAAGCCTTAAAGACGATCCTTCAGTGAGCAAAGGAATTTTAGCAGAAGCTAAAAAGCTAAACGATGCTCAAGCACCAAAAGAGGAAGACAACAACAAACCTGGCAAAGAAGACAACAACAAGCCTGGTAAAGAAGACAACAACAAGCCTGGCAAAGAAGACGGCAACAAGCCTGGTAAAGAAGACAACAAAAAACCTGGTAAGGAAGACAACAAAAAACCTGGTAAAGAAGACAACAAAAAACCTGGTAAAGAAGATGGCAACAAGCCTGGCAAAGAAGACAACAAAAAACCTGGTAAAGAAGACGGCAACGGAGTACATGTCGTTAAACCTGGTGATACAGTAAATGACATTGCAAAAGCAAACGGCACTACTGCTGACAAAATTGCTGCAGATAACAAATTAGCTGATAAAAACATGATCAAACCTGGTCAAGAACTTGTTGTTGATAAGAAGCAACCAGCAAACCATGCAGATGCTAACAAAGCTCAAGCATTACCAGAAACTGGTGAAGAAAATCCATTCATCGGTACAACTGTATTTGGTGGATTATCATTAGCGTTAGGTGCAGCGTTATTAGCTGGACGTCGTCGCGAACTATAA

SPA (FL) full-length proteins sequence following (500aa, SEQ ID NO:5):

L K K K N I Y S I R K L G V G I A S V T L G T L L I S G G V T P A A N A A Q H D E AQ Q N A F Y Q V L N M P N L N A D Q R N G F I Q S L K D D P S Q S A N V L G E A Q K LN D S Q A P K A D A Q Q N N F N K D Q Q S A F Y E I L N M P N L N E A Q R N G F I Q SL K D D P S Q S T N V L G E A N K L N E S Q A P K A D N N F N K E Q Q N A F Y E I L NM P N L N E E Q R N G F I Q S L K D D P S Q S A N L L S E A K K L N E S Q A P K A D NK F N K E Q Q N A F Y E I L H L P N L N E E Q R N G F I Q S L K D D P S Q S A N L L A EA K K L N D A Q A P K A D N K F N K E Q Q N A F Y E I L H L P N L T E E Q R N G F I Q SL K D D P S V S K G I L A E A K K L N D A Q A P K E E D N N K P G K E D N N K P G K ED N N K P G K E D G N K P G K E D N K K P G K E D N K K P G K E D N K K P G K E D GN K P G K E D N K K P G K E D G N G V H V V K P G D T V N D I A K A N G T T A D K I AA D N K L A D K N M I K P G Q E L V V D K K Q P A N H A D A N K A Q A L P E T G E E NP F I G T T V F G G L S L A L G A A L L A G R R R E L

SPA (FL) full-length proteins sequence (Query 1; SEQ ID NO:5) and as follows with the comparison result of Genbank (GI:88193823) Staphylococcus aureus sequence (Sbjct 1, SEQ ID NO:6):

Query 1 LKKKNIYSIRKLGVGIASVTLGTLLISGGVTPAANAAQHDEAQQNAFYQVLNMPNLNADQ 60

+KKKNIYSIRKLGVGIASVTLGTLLISGGVTPAANAAQHDEAQQNAFYQVLNMPNLNADQ

Sbjct 1 MKKKNIYSIRKLGVGIASVTLGTLLISGGVTPAANAAQHDEAQQNAFYQVLNMPNLNADQ 60

Query 61 RNGFIQSLKDDPSQSANVLGEAQKLNDSQAPKADAQQNNFNKDQQSAFYEILNMPNLNEA 120

RNGFIQSLKDDPSQSANVLGEAQKLNDSQAPKADAQQNNFNKDQQSAFYEILNMPNLNEA

Sbjct 61 RNGFIQSLKDDPSQSANVLGEAQKLNDSQAPKADAQQNNFNKDQQSAFYEILNMPNLNEA 120

Query 121 QRNGFIQSLKDDPSQSTNVLGEANKLNESQAPKADNNFNKEQQNAFYEILNMPNLNEEQR 180

QRNGFIQSLKDDPSQSTNVLGEA KLNESQAPKADNNFNKEQQNAFYEILNMPNLNEEQR

Sbjct 121 QRNGFIQSLKDDPSQSTNVLGEAKKLNESQAPKADNNFNKEQQNAFYEILNMPNLNEEQR 180

Query 181 NGFIQSLKDDPSQSANLLSEAKKLNESQAPKADNKFNKEQQNAFYEILHLPNLNEEQRNG 240

NGFIQSLKDDPSQSANLLSEAKKLNESQAPKADNKFNKEQQNAFYEILHLPNLNEEQRNG

Sbjct 181 NGFIQSLKDDPSQSANLLSEAKKLNESQAPKADNKFNKEQQNAFYEILHLPNLNEEQRNG 240

Query 241 FIQSLKDDPSQSANLLAEAKKLNDAQAPKADNKFNKEQQNAFYEILHLPNLTEEQRNGFI 300

FIQSLKDDPSQSANLLAEAKKLNDAQAPKADNKFNKEQQNAFYEILHLPNLTEEQRNGFI

Sbjct 241 FIQSLKDDPSQSANLLAEAKKLNDAQAPKADNKFNKEQQNAFYEILHLPNLTEEQRNGFI 300

Query 301 QSLKDDPSVSKGILAEAKKLNDAQAPKEEDNNKPG----------------KEDNNKPGK 344

QSLKDDPSVSKILAEAKKLNDAQAPKEEDNNKPG KEDNNKPGK

Sbjct 301 QSLKDDPSVSKEILAEAKKLNDAQAPKEEDNNKPGKEDNNKPGKEDNNKPGKEDNNKPGK 360

Query 345 EDNNKPGKEDGNKPGKEDNKKPGKEDNKKPGKEDNKKPGKEDGNKPGKEDNKKPGKEDGN 404

EDNNKPGKEDGNKPGKEDNKKPGKED KPGKEDNKKPGKEDGNKPGKED KPGKEDGN

Sbjct 361 EDNNKPGKEDGNKPGKEDNKKPGKEDGNKPGKEDNKKPGKEDGNKPGKEDGNKPGKEDGN 420

Query 405 GVHVVKPGDTVNDIAKANGTTADKIAADNKLADKNMIKPGQELVVDKKQPANHADANKAQ 464

GVHVVKPGDTVNDIAKANGTTADKIAADNKLADKNMIKPGQELVVDKKQPANHADANKAQ

Sbjct 421 GVHVVKPGDTVNDIAKANGTTADKIAADNKLADKNMIKPGQELVVDKKQPANHADANKAQ 480

Query 465 ALPETGEENPFIGTTVFGGLSLALGAALLAGRRREL 500

ALPETGEENPFIGTTVFGGLSLALGAALLAGRRREL

Sbjct 481 ALPETGEENPFIGTTVFGGLSLALGAALLAGRRREL 516

The cloning and expression qualification of embodiment 2, SPA (FL) full-length gene and SPA (32-327) gene

1, pcr amplification obtains truncate SPA (32-327) and total length SPA (FL)

Cultivate streptococcus aureus, its genomic dna of ordinary method extracting.

Design the encoding gene of following amplimer for truncate SPA (32-327) that increase:

Forward: CGCCATATGCCTGCTGCAAATGCTGCGC (SEQ ID NO:7)

Reverse: CCGCTCGAGTTATTTTGGTGCTTGAGC (SEQ ID NO:8)

Design the encoding gene of following amplimer for the total length SPA that increases (FL):

Forward: CGCCATATGTTGAAAAAGAAAAACATTTATTC (SEQ ID NO:9)

Reverse: CCGCTCGAGTTATAGTTCGCGACGACG (SEQ ID NO:10)

Adopt above primer respectively, with staphylococcus aureus gene group DNA for template, amplification obtains total length SPA (FL) and truncate SPA (32-327) respectively.The electroresis appraisal of PCR primer the results are shown in Figure 1.

The total length SPA (FL) of above-mentioned acquisition is cut with NdeI/Xho I enzyme with truncate SPA (32-327) PCR primer, digestion products is connected to pGEM-T carrier (purchased from Progema), to be binned in and be transformed into competent e. coli bl21 (DE3) day, picking mono-clonal shakes and spends the night in LB substratum, and bacterium colony PCR qualification result is as Fig. 2.Correct sequence is obtained through sequence verification.

Be inserted in the corresponding site of expression vector pET-24a (purchased from Progema) after utilizing Nde I/Xho I double digestion to cut out DNA fragmentation from pGEM-T carrier, obtain pET-24a/SPA (32-327) and pET-24a/SPA (FL).Above-mentioned recombinant expression vector is transformed into competent e. coli bl21 (DE3), and carries out abduction delivering.As a result, in competent cell, SPA (32-327) has expression, and SPA (FL) does not express successfully, sees Fig. 3.

The expression optimization of embodiment 3, SPA (32-327)

Please provide the concrete grammar cultivating restructuring BL21 (DE3) cell: the mono-clonal bacterium colony on picking transformation plate, be seeded to (containing 100 μ g/mL Amp) in 30mL LB nutrient solution, after being placed in 37 DEG C of incubator overnight cultivation (10 ~ 12h), proceed in secondary bottle with 2% inoculum size, continue to cultivate.Strain density to OD600 0.5 ~ 0.6 time, at 37 DEG C and 12 DEG C, add final concentration 0.5mmol/L IPTG respectively induce 4 hours, 10000rpm collected by centrifugation thalline, abandons supernatant, and thalline ultrasonication is centrifugal, carries out electroresis appraisal respectively to centrifugal rear cleer and peaceful precipitation.

Based on above method, investigate differing temps to the impact of the expression amount of SPA (32-327), and the impact that different IP TG concentration is expressed induction SPA (32-327).

BL21 (DE3) cell having proceeded to pET-24a/SPA (32-327) by aforementioned preparation is expressed at different temperature (15 DEG C, 30 DEG C and 37 DEG C), and observation of cell expresses the situation of SPA (32-327).The IPTG concentration expressing employing is at these tem-peratures 0.5mM.The results are shown in Figure 4 left figure and Fig. 5.Visible, reconstitution cell post-induction growth at 30 DEG C with 37 DEG C is slow, its possible cause be SPA (32-327) at higher temperatures too fast expression have restraining effect to thalli growth, and comparatively under low temperature induction, it is slow that SPA (32-327) expresses speed, restraining effect is weak, therefore thalline is able to continued growth.

BL21 (DE3) cell having proceeded to pET-24a/SPA (32-327) of aforementioned preparation gives different IPTG concentration (0.1mM, 0.5mM, 1.0mM and 10mM) inductions.Observation of cell expresses the situation of SPA (32-327).The temperature expressing employing under above-mentioned induced concentration is 37 DEG C.The results are shown in Figure 4 right figure.Visible, under different IPTG induced concentrations, the difference of protein expression is little.

The high density fermentation of the 10L fermentor tank of embodiment 4, SPA (32-327)

Seed culture medium: Tryptones 1% (w/v), yeast extract 0.5% (w/v), NaCl 1% (w/v), adjusts pH to 7.0 with 5M NaOH;

Fermention medium: peptone 10g/L, yeast extract 10g/L, NaCl 10g/L, NH ₄cl 2g/L, K ₂hPO ₄10.7g/L, KH ₂pO ₄5.2g/L, MgSO ₄1g/L, adjusts pH to 7.0 with 10M NaOH;

First order seed is cultivated: seed liquor accesses in the triangular flask of the LB substratum (50ug/ml Kan) that liquid amount is 30ml/250ml by the inoculum size with 1%, and shaking speed is 180r/min, cultivates 6 ~ 8h for 37 DEG C.

Secondary seed is cultivated: first order seed is accessed liquid amount and is respectively in the triangular flask of LB substratum (50ug/ml Kan) of 30ml/250ml and 200ml/500ml by the inoculum size with 1%, and shaking speed is 180r/min, 37 DEG C of incubated overnight.

Earlier fermentation air flow controls at 5L/min, and mixing speed controls at 150r/min, and in whole process, control DO is about 30%, so strengthen air flow and mixing speed afterwards, to maintain DO level, finally carries air flow and mixing speed to maximum.In whole fermenting process, temperature controls at 37 DEG C.When reducing sugar exhausts, start stream and add formula feed supplement.Add 0.5mM IPTG to induce when thalline is in the logarithmic growth later stage, after induction 5h, put tank, collected by centrifugation thalline.

Course of fermentation curve is shown in Fig. 6.

The thalline weight in wet base that above-mentioned fermentation obtains is about 450g.Albumen is expressed, and expresses with soluble form.

The ion-exchange purification research of embodiment 5, SPA (32-327)

Adopt ion exchange column DEAE Sepharose FF purifying SPA (32-327).

10g restructuring BL21 (DE3) thalline 100ml lysis buffer (50mM Tris.Hcl, pH 8.0) suspend, ultrasonication, after diluting 4 times, 400ml supernatant is splined on 30ml DEAE Sepharose FF post, after steady to baseline by 50mmol/L Tris-HCl pH 8.0 damping fluid balance, continuous gradient wash-out is carried out with the NaCl elutriant of 0 ~ 0.3mmol/L, through SDS-PAGE electrophoresis (Fig. 7), albumen collected after purifying is SPA (32-327).

The affinity chromatography purifying of embodiment 6, SPA (32-327)

Build SPA (FL) and the SPA (32-327) of band His-Tag, prepare to adopt affinity chromatography purifying protein.The method of structure and protein expression is:

Forward: CGCGAATTCCCTGCTGCAAATGCTGCGC (SEQ ID NO:11)

Reverse: CCGCTCGAGTTATTTTGGTGCTTGAGC (SEQ ID NO:8)

Wherein contain EcoR I in forward primer, be building up to the pET-24a that same enzyme is cut, when translating, the N end of albumen just contains the protein sequence of the 6His that one section of plasmid itself is encoded, thus can carry out purifying with Ni affinity column.

His label protein Purification Resin (Ni-NTA Resin) is a kind of purification media for purifying 6 × His label recombinant protein, 6 × His can with Ni ²⁺chelating, thus make His label protein be combined in Ni-NTA purification media, unconjugated albumen is washed down, and the albumen be combined on medium elutes through the gentleness of certain density imidazoles or low PH damping fluid, thus obtains highly purified target protein.

15 DEG C of inductions of spending the night above restructuring BL21 (DE3) the thalline 1L obtained, thalline 40ml lysis buffer (50mM NaH of acquisition ₂pO ₄, 300mM NaCl) and ultrasonication.Ni-NTA agarose gel volume is after 3ml, 40ml supernatant upper prop, balances with the lysis buffer of 5 times of column volumes.Use the 10mM of 4 times of column volumes respectively, 20mM, 40mM, 80mM, 250mM imidazoles wash-out.As shown in Figure 8, SPA (32-327) just can by wash-out under 50mM imidazoles existent condition, and the imidazoles of more than 60mM concentration can obtain the very high SPA of purity (32-327).

The freeze-drying stability of embodiment 7, SPA (32-327)

The freeze drying process adopted is: the SPA (32-327) after purifying, SPA (32-327)-His are divided in 5ml freeze-drying pipe ,-80 DEG C of pre-freezes are spent the night, next day, put into and vacuumize freeze-drying 48h in vacuum freeze drying.

Identify as follows through the method for the stability of the SPA (32-327) of freeze-drying process: take a small amount of dried frozen aquatic products, be made into 1mg/ml with pure water, solvability is fabulous, contrasts and carries out SDS-PAGE electrophoresis detection, have no degraded with sample before freeze-drying.

Result is as Fig. 9.Visible, SPA (32-327) is very stable in freeze-drying process.

Embodiment 8, SPA (32-327)-His and the research of IgG antibody affinity

1.ELISA tests

Get sample (aforementioned expression the SPA of purifying (32-327)-His protein sample) 100 μ l (20ng), the sample concentration 0.2mg/ml of dilution 1000 times.ELISA kit is purchased from Roche company.

(1) point sample: get 3 μ l point samples on NC film by after sample after purifying step by step 10 times of dilutions, hatch 2h in 37 DEG C after sealing with preservative film;

(2) close: NC film is placed in confining liquid 4 DEG C close spend the night;

(3) wash: wash film 3 times with washings PBST, each 10min;

(4) answer with two anti-reflective: by two anti-diluted 4000 times, and NC film is immersed, hatches 2h for 37 DEG C;

(5) wash: wash film 3 times with washings PBST, each 10min;

(6) develop the color: NC film is immersed in DAB nitrite ion, after color development at room temperature 5min, uses deionized water termination reaction.

(7) HRP-ECL luminescence method: by A, B luminescent solution dilution mixture in proportion.Film deionized water rinsing a little, filter paper pastes angle and blots, anti-subsides method is overlying on A, B mixing drop, extremely visible light green fluorescent bands (about 5min) of turning off the light is blotted at filter paper subsides angle afterwards, be placed in preservative film internal fixtion in film magazine, cover film rapidly, close glue box, expose according to finding fluorescence intensity.Take out film and immerse 1-2min in developing solution completely immediately, clear water rinsing is placed in stop bath completely fixing to egative film once afterwards, and clear water washes down and dries, and carries out analyzing and scans.

Result is as table 2.Wherein SPA-32 represents SPA (32-327), and contrast represents the positive control that test kit provides.

Table 2

As seen from table, SPA-32 and sheep anti-mouse igg avidity through expressing also purifying are higher, lower with goat-anti rabbit avidity.

2.Dottern tests

By the SPA (32-327) of different concns or SPA (32-327)-His point sample on NC film, 4 DEG C close spend the night after PBST wash 3 times, each 10 minutes; 10ml PBS (0.1% skim-milk) adds two anti-HRP-sheep anti-mouse igg (0.4mg/ml, purchased from Roche) 2 μ l, hatches 2h; PBSR washs 3 times, and colour developing working fluid 1.5ml covers NC film, hatches 5min; Exposure, development.More with antibodies, color is darker.

As shown in Figure 10, the SPA (32-327) of 6ng just can with antibodies, and along with the increase of protein content, bonding force strengthens.

The all documents mentioned in the present invention are quoted as a reference all in this application, are just quoted separately as a reference as each section of document.In addition should be understood that those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.

Claims

1. a recombinant protein A for tool antibody binding capacity, it is the albumen of the aminoacid sequence shown in SEQ ID NO:2.

2. polynucleotide, it is the polynucleotide of recombinant protein A of tool antibody binding capacity according to claim 1 of encoding.

3. polynucleotide as claimed in claim 2, it is characterized in that, the nucleotide sequence of these polynucleotide is as shown in SEQ ID NO:1.

4. a carrier, is characterized in that, it contains the arbitrary described polynucleotide of Claims 2 or 3.

5. a genetically engineered host cell, is characterized in that, it contains carrier according to claim 4, or is integrated with the arbitrary described polynucleotide of Claims 2 or 3 in genome.

6. produce a method for the recombinant protein A of the tool antibody binding capacity described in claim 1, it is characterized in that, comprise step:

(1) cultivate host cell according to claim 5, obtain culture; With

(2) from culture, be separated the recombinant protein A of tool antibody binding capacity according to claim 1.

7. method as claimed in claim 6, is characterized in that, at temperature 20 ± 10 DEG C, cultivate host cell according to claim 5.

8. method as claimed in claim 6, it is characterized in that, described host cell is Bacillus coli cells, cultivates in described host cell process and adopts IPTG or lactose-induced expression.

9. the purposes of the recombinant protein A of tool antibody binding capacity according to claim 1, for binding antibody; Or for the preparation of antibody affinity column; Or for the purifying of antibody.

10. an affinity column for antibody purification, it comprises affinity media, and is attached to the recombinant protein A of tool antibody binding capacity according to claim 1 of described affinity media.