CN1966526A - Evolved immunoglobulin binding molecule, and its preparation method and uses - Google Patents

Evolved immunoglobulin binding molecule, and its preparation method and uses Download PDF

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CN1966526A
CN1966526A CN 200610118694 CN200610118694A CN1966526A CN 1966526 A CN1966526 A CN 1966526A CN 200610118694 CN200610118694 CN 200610118694 CN 200610118694 A CN200610118694 A CN 200610118694A CN 1966526 A CN1966526 A CN 1966526A
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immunoglobulin
evolved
antibody
binding molecule
immunoglobulin binding
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CN100486993C (en
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潘卫
蒋少华
徐容
贾建安
沈毅珺
杨华
王锦红
陈秋莉
何俊
陈璐
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Second Military Medical University SMMU
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Abstract

The invention relataes to novel evolutionary immunoglobulin binding molecules, as well as their preparation methods and applications. The invention discloses separated evolutionary immunoglobulin binding molecules, which are proteins with amino acid sequences as shown by SEQ ID NO : 1, or conservative variant proteins with immunoglobulin binding activity. The invention also discloses the gene encoding, genetic engineering preparation methods and applications of immunoglobulin binding molecules. The disclosed immunoglobulin molecule broad-spectrum combined with various immuneglobulin shows high immunoglobulin whole molecule binding activity, and can be used in large-scale purification of genetic engineering antibodies, purification of natural antibodies and monoclonal antibodies, enzyme-linked immunosorbent assay, and immuno-chromatography and immunohistochemical methods for immune antibody detection and diagnosis.

Description

A kind of evolved immunoglobulin binding molecule, its preparation method and application
Technical field
The immune globulin binding molecule that the present invention relates to recombinate relates in particular to the Ig binding domains of s. aureus protein and peptostreptococcus magnus surface protein is reset immune globulin binding molecule, its preparation method and the application that obtains.
Background technology
Immune globulin binding molecule (Ig binding proteins, IBP) be a class be expressed in bacterium surface can with host Ig privileged site bonded albumen, regulate the immune response of host to bacterium, be one of important virulence factor of bacterium.Study maximum IBP and mainly contain three kinds: the albumen L (PpL) on staphylococcus aureus protein A (SpA), people suis (C and G group) Protein G (SpG) and part peptostreptococcus magnus surface.
The about 57KDa of SpA molecular weight, constitute by 524 left and right sides amino acid, its born of the same parents' outside part contains 5 long 58-61 amino acid Disabled base and sequence height homologous Ig binding domains (called after E, D, A, B and C respectively), single Ig binding domains is made up of the α spiral of three strands of fold backs, and each single structure territory has independently Ig in conjunction with activity.Comprise two to three sequence height homologous Ig binding domainss (C1, C2, C3) in the SpG molecule, its single structure territory is made up of two pairs of antiparallel β lamellas and α spiral of intermediary.Comprise in the PpL molecule by 4-5 (different according to different strains) by 72-76 amino-acid residue composition sequence height homologous Ig binding domains (B1-B5), though aminoacid sequence and SpG differ greatly, its single structure territory conformation is similar.The peptide chain that crystal structure analysis contains four Ig binding domainss of B1-B4 of PpL has the characteristic of while in conjunction with two kappa light chains of same Ig molecule.SpA and SpG Fc section main and IgG has stronger avidity, SpA can also (not overlap with antigenic determinant in conjunction with the VH district that belongs to the VH3 gene family simultaneously, do not influence with antigenic and combine), therefore, it except have with the stronger avidity of IgG (except the IgG3), can also be in conjunction with the Ig of other class.SpG can also be in conjunction with the CH1 district (CH1 γ) of IgG, and it is only in conjunction with IgG, and has higher avidity.And PpL by with the light chain of Ig, 1,3,4 hypotypes that mainly are the κ light chain interact and binding domain-immunoglobulin, binding site is positioned at the variable region of light chain beyond the antigenic determinant, and the characteristic of the strong avidity of the irrelevant SpA of its binding ability and heavy chain subclass and SpG and IgG-Fc section makes it be widely used in detecting and IgG purification.In addition, SpA also is used as the assisting therapy of some autoimmune disorder, and its mechanism is to remove circulating immune complex and immunomodulatory.On these three kinds each comfortable Ig bind profile of main natural Ig binding molecule all is limited.SpA debond human IgG 3, because the VH3 gene family is in the distribution difference of inhomogeneity Ig, SpA is only in conjunction with about 15%, 13% and 40% human IgG Fab, IgA and IgM.In addition, also very big in the binding characteristic difference between the Mammals of system of the same race not: to the Ig of rat in conjunction with a little less than, very big to IgG difference between different subclass of mouse, and strong to the Ig associativity of rabbit.Similar to SpA, because PpL is only in conjunction with 1,3,4 hypotypes of κ light chain,, the Ig that derives from rabbit, ox in conjunction with very weak, and is had strong associativity to the Ig of mouse therefore only in conjunction with 60% people Ig.
SpA, SpG and PpL have been widely used in the quantitative and qualitative analysis of immuno-chemical reaction.When coupled during to radioactive, enzymatic property an or fluorescence labels, SpA be one well, detect and quantitatively and the reagent of this protein high-affinity bonded antibody, enzyme mark SpA has been widely applied in the antibody test of ELISA method, during the specific antibody of especially many transmissible diseases detects.SpA combines the location that can be used for IgG in the immuno-electron microscope with the particulate of gold grain, and the Electronic Speculum of many special constructions and pathogenic agent is detected becomes possibility, and the SpA colloid gold particle also has been widely used in immunochromatographic method and carries out immunologic antibody test in addition.Be fixed on SpA on the solid-phase media and can be used for the large scale purification of multiple natural animal antibody and mouse monoclonal antibody, in addition, immobilised SpA also is widely used in and collects immunocomplex, antigen or complete cell.Although developed many technology that are used for the IgG purification molecule, preferable methods is still adsorbed and wash-out with the ball of SpA parcel.Utilize the SpA of purifying and the engineering fusion rotein of the IgG binding domains that comprises SpA to adopt class ELISA sandwich technique, can purifying and detection dna fragmentation.The same with SpA, SPG has been widely used in immunoassay and some treatments in purifying antibody.But it has a potential inferior position in collecting immunocomplex and IgG purification reagent be that it can be consumingly in conjunction with bovine serum albumin.Yet the IgG binding site of SPG and bovine serum albumin binding site structurally are distinct.And the SPG engineering version, that lacked the bovine serum albumin binding site also can obtain by commercial sources.In addition, the serum albumin structural domain of SPG is always as the purification tag in the fused protein.PpL is an instrument of great use, compare with SpG with SpA, its monoclonal antibody purification from the medium that adds placenta bovine serum or bovine serum albumin and from transgenic animal aspect such as purifying humanized antibody have more advantage.
Because the antibody combining site difference of three kinds of Ig binding molecules, protein A and protein G mainly combine with the Fc section constant region of IgG and protein L combines with the kappa light chain variable district of Ig, therefore can gather and optimize two kinds of proteic Ig combined function, construct novel I g binding molecule and be used for the Ig detection, improving the susceptibility and the specificity of immunodiagnosis, is the focus that the foreign scholar pays close attention to.1992, the Sweden scholar constructed the fusion rotein Protein LG (hereinafter to be referred as pLG) of protein L and protein G, found that Protein LG has that antibody binding activity can be in conjunction with Fc, Fab section and the Ig light chain of most of Ig widely.Compare with single protein L or G, its antibodies characteristic is more complete, except that human Ig, also can be used for the detection of mouse endogenous antibody.1998, the Sweden scholar combines four structural domains in conjunction with Ig κ light chain of protein L the structural domain fusion of IgG with four of protein A, construct a kind of novel hybrid protein Protein LA (hereinafter to be referred as pLA), bind profile is wide, can not only be in conjunction with people and some mammiferous dissimilar Ig and all subclass of IgG, can also not influence the antigen binding capacity of antibody in conjunction with phage single chain antibody Fv (ScFv) fragment, and be improved with the avidity of antibody, except that being used for immunodetection, but, prove that Protein LA is a kind of multi-functional antibody-binding molecules by affinity chromatography also all kinds of Ig of purifying and Fv (ScFv) fragment.1999, the fusion rotein ProteinLA in protein A antibodies district and protein L (1-3) antibodies district is developed as the commercialization reagent of detection and antibody purification by CLONTECH company, have the dual-use function of protein A and protein L concurrently, Protein LA can solve the drawback of traditional method IgM detection difficult in conjunction with various types of antibody.That ProteinLA has is soluble, HRP-enzyme mark, or in conjunction with the preparation of agarose form, can not only improve the susceptibility of immunodetection, can also single stage method carry out antibody purification, simple and easy to do.Domestic do not have analogous products to occur as yet.
Though pLA and pLG simple concatenation have kept source molecule function separately, this structure may not reach brings into play the accurate conformation requirement of dibit point bonded simultaneously.The fundamental unit that we select the IBP molecule to play a role is that Ig is in conjunction with the basic object of single structure territory as research, to the reorganization splicing at random of single structure domain dna, and be main molecular evolution means screening in order to phage display, in the hope of obtaining having the recombinant molecule of new combined function.Be specially: the single Ig binding domains of clone SpA and PpL, single structure domain dna fragment is spliced at random external, use display technique of bacteriophage again these recombinant molecules are expressed in phage surface, phage display library is made up in the single structure territory that has made up SpA and PpL at random, with people Ig is that bait carries out affine screening to the library, the result has obtained a kind ofly being spaced novel I g binding molecule form (MDPL-MDPA) n (MDPL, the mono-domain of proteinL that forms by the B3 structural domain of PpL and D, A, B, C (the being mainly D) structural domain of SpA; MDPA, mono-domain of protein A), this molecular form does not exist in natural IBP, in the IBP molecule of reorganization, do not appear in the newspapers yet, therefore be a kind of new IBP molecular architecture, because be to obtain by the molecular evolution means, we are referred to as evolved immunoglobulin binding molecule (evolved Ig-binding molecules), evolved immunoglobulin binding molecule LD5 (L-D-L-D-L to this quasi-molecule; L is the B3 structural domain of PpL, D is the D structural domain of SpA) be wherein the most representative advantage clone, this content is in " phage display SpA and PpL Ig are in conjunction with combinatorial library and the affine screening at random of single structure territory ", biophysics and biological chemistry progress, have detailed description in 2005 the 32nd volume the 535th～543 page of the 6th phase, but this piece article is not set forth further to the various functional performances of LD5.
The present invention chooses from multiple (MDPL-MDPA) n sequence and represents sequence LD5 to be cloned into prokaryotic expression carrier pET32a (+) structure prokaryotic expression system, produce and the novel evolved immunoglobulin binding molecule LD5 of purifying, binding characteristic to this albumen and each immunoglobulin (Ig) has carried out detailed mensuration, set up the large scale purification that the affinity column of using this molecule preparation carries out genetic engineering antibody, the method of natural antibody and Purification of Monoclonal Antibodies, set up and used this molecule marker enzyme and carry out the diagnostic method that immunological method such as enzyme linked immunosorbent assay detects antibody, set up and used this molecule and prepare the diagnostic method that the colloid gold label enzyme carries out immunochromatographyassay assay antibody.
Summary of the invention
An object of the present invention is to provide a kind of novel evolved immunoglobulin binding molecule and encoding gene thereof.
Another object of the present invention provides carrier, transformant and the gene engineering preparation method thereof of above-mentioned evolved immunoglobulin binding molecule.
A further object of the present invention provides the application of above-mentioned evolved immunoglobulin binding molecule.
A first aspect of the present invention discloses a kind of evolved immunoglobulin binding molecule of reorganization, and it is albumen or its conservative property variant protein with the aminoacid sequence shown in the SEQ ID NO:1.Preferable, it has the aminoacid sequence shown in the SEQ ID NO:1.
Above-mentioned molecule is that the contriver has made up a phage display SpA and PpL Ig in conjunction with the single structure territory at random behind the combinatorial library, find by affine screening, concrete process is in " phage display SpA and PpL Ig are in conjunction with combinatorial library and the affine screening at random of single structure territory ", biophysics and biological chemistry progress had a detailed description in the 535th～543 page of 2005 the 32nd the 6th phase of volume.Through a series of tests of contriver, find that this molecule has outstanding immunoglobulin (Ig) binding characteristic: can not only be in conjunction with IgG, IgM, IgA, IgG Fab or IgG Fc, can also be in conjunction with scFv.In addition, embodiment has confirmed that also it has the very high activity that combines with the full molecule of human normal immunoglobulin IgG; There is enhanced to combine activity with human normal immunoglobulin IgG Fab segment than SpA and PpL; There is enhanced to combine activity with human normal immunoglobulin IgM than SpA and PpL; There is enhanced to combine activity with human normal immunoglobulin IgA than SpA and PpL.And it and each immunoglobulin (Ig) are the dibit point binding pattern bonded with Ig κ light chain and VH3 heavy chain.
A second aspect of the present invention discloses a kind of isolating polynucleotide, its above-mentioned evolved immunoglobulin binding molecule of encoding.Preferable, it has the nucleotide sequence shown in the SEQ ID:NO 2.
A third aspect of the present invention discloses a kind of expression vector, the expression regulation sequence that it contains above-mentioned polynucleotide and links to each other with this polynucleotide sequence operability.Preferable, above-mentioned carrier is a prokaryotic vector, wherein preferred PET-32a (+).
A fourth aspect of the present invention discloses a kind of host cell, and it is transformed by above-mentioned expression vector.Preferable, above-mentioned host cell is a prokaryotic cell prokaryocyte, wherein preferred intestinal bacteria are better, are e. coli bl21-TRXB.
A fifth aspect of the present invention discloses the preparation method of above-mentioned evolved immunoglobulin binding molecule, and this method comprises: express under the condition of evolved immunoglobulin binding molecule being fit to (1), cultivates above-mentioned host cell; (b) from culture, isolate and have immunoglobulin (Ig) in conjunction with active polypeptide.Among the embodiment, the contriver is cloned into novel evolved immunoglobulin binding molecule among the prokaryotic expression carrier pET32a (+), make up its escherichia coli expression bacterial classification, this bacterial classification is carried out large scale culturing, induce the production evolved immunoglobulin binding molecule, carry out purifying with the Ni affinity column, prepare the novel evolved immunoglobulin binding molecule LD5 of purifying.
A sixth aspect of the present invention, disclose above-mentioned evolved immunoglobulin binding molecule has been used for external binding domain-immunoglobulin, evolved immunoglobulin binding molecule and external binding domain-immunoglobulin bonded mode are dibit point binding pattern, promptly with the binding pattern in immunoglobulin kappa light chain and these two sites of VH3 heavy chain.Preferable, above-mentioned immunoglobulin (Ig) is selected from one or more among IgG, IgM, IgA, IgG Fab or the IgG Fc, and is better, and above-mentioned IgG, IgM, IgA, IgG Fab or IgG Fc derive from the people.Same preferable, above-mentioned immunoglobulin (Ig) is scFv.
Preferably, the above-mentioned external immunology detection that evolved immunoglobulin binding molecule is used for antibody that is applied as, wherein immunology detection preferred enzyme connection immunosorption or immunochromatography or immunohistochemical methods.
Same preferred, above-mentioned being applied as is used for antibody purification with evolved immunoglobulin binding molecule, wherein antibody preferred gene engineered antibody or natural antibody or monoclonal antibody.
The invention has the advantages that:
Evolved immunoglobulin binding molecule disclosed by the invention, can wide spectrum in conjunction with various immunoglobulin (Ig)s, compare with immune globulin binding molecule of the prior art, its combination is wide spectrum more, not only can be in conjunction with IgG, IgM, IgA, IgG Fab or IgG Fc, can also be in conjunction with scFv, in addition, with the bonding force of immunoglobulin (Ig) also than existing immune globulin binding molecule height.On the other hand, the invention discloses the gene engineering preparation method of above-mentioned evolved immunoglobulin binding molecule, make low-cost, scale operation become possibility.Again on the one hand, the invention discloses the external immunology detection and the purifying antibody that above-mentioned evolved immunoglobulin binding molecule are used for antibody, compared with prior art, used evolved immunoglobulin binding molecule of the present invention can improve the sensitivity and the accuracy of detection, the effect of purifying is also better.
Description of drawings
The structure schema of Fig. 1 recombinant expression plasmid pET32a (+)-LD5
The amplification of Fig. 2 LD5 cDNA sequence
m：DL2000 Marker
The 1-8:pCANTAB5S-LD5 pcr amplification product
The PCR product of Fig. 3 LD5 expressed sequence reclaims
m：DL2000 Marker
1-4:pCANTAB5S-LD5 PCR reclaims product
The NcoI+Sal I double digestion of Fig. 4 pET32a (+)-LD5 is identified
M：DL15000Marker
1:LD5/PET32 (a) plasmid/Nco I+Sal I
2:LD5/PET32 (a) plasmid
The expression of Fig. 5 fusion rotein LD5
M：14400-97400 Daltons Marker
1-4:LD5/PET32 (a)/BL21-TRXB mono-clonal 1-4#
The IPTG abduction delivering
5: empty bacterium BL21-TRXB contrast
97400,66200,43000,31000,20100,14400: represent 14400-97400 Daltons Marker electrophoresis colour developing band corresponding molecular weight respectively
The purifying of Fig. 6 fusion rotein LD5
M：14400-97400 Daltons Marker
7：LD5
1-6: other IBP molecules
Fig. 7 LD5 and PpL and SPA are to biotin labeling people polyclone IgG binding ratio
X-coordinate: log (IgG-biotin) (nm), i.e. IgG-biotin concentration logarithmic value
Ordinate zou: OD450nm, i.e. the OD value of wavelength 450nm
Band " ◆-" lines: corresponding PpL
Band " △-" lines: corresponding LD5
Band " *-" lines: corresponding SPA
Fig. 8 LD5 and PpL and SPA are to biotin labeling people polyclone IgM binding ratio
X-coordinate: log (IgM-biotin) (nm), i.e. IgM-biotin concentration logarithmic value
Ordinate zou: OD450nm, i.e. the OD value of wavelength 450nm
Band " ◆-" lines: corresponding PpL
Band " △-" lines: corresponding LD5
Band " *-" lines: corresponding SPA
Fig. 9 LD5 and PpL and SPA are to biotin labeling people polyclone IgA binding ratio
X-coordinate: log (IgA-biotin) (nm), i.e. IgA-biotin concentration logarithmic value
Ordinate zou: OD450nm, i.e. the OD value of wavelength 450nm
Band " ◆-" lines: corresponding PpL
Band " △-" lines: corresponding LD5
Band " *-" lines: corresponding SPA
Figure 10 LD5 and PpL and SPA are to biotin labeling people polyclone IgG Fab binding ratio
X-coordinate: log (IgFab-biotin) (nm), i.e. IgFab-biotin concentration logarithmic value
Ordinate zou: OD450nm, i.e. the OD value of wavelength 450nm
Band " ◆-" lines: corresponding PpL
Band " △-" lines: corresponding LD5
Band " *-" lines: corresponding SPA
Figure 11 LD5 and PpL and SPA are to biotin labeling people polyclone IgG Fc binding ratio
X-coordinate: log (IgG Fc-biotin) (nm), i.e. IgG Fc-biotin concentration logarithmic value
Ordinate zou: OD450nm, i.e. the OD value of wavelength 450nm
Band " ◆-" lines: corresponding PpL
Band " △-" lines: corresponding LD5
Band " *-" lines: corresponding SPA
Figure 12: biotin labeling LD5 combines with scFv KM36, KM38, KM41's
X-coordinate: log (LD5-biotin) (nm), i.e. IgLD5-biotin concentration logarithmic value
Ordinate zou: OD450nm, i.e. the OD value of wavelength 450nm
Band " ◆-" lines: correspondence contains the TG1 bacterium lysate supernatant of KM36
Band "-" lines: correspondence contains the TG1 bacterium lysate supernatant of KM38
Band " △-" lines: correspondence contains the TG1 bacterium lysate supernatant of KM41
Band " *-" lines: corresponding empty TG1 bacterium lysate supernatant
Figure 13 LD5 suppresses in conjunction with the competition of people's polyclone IgGFab
X-coordinate: concentration of inhibitor (nm), i.e. supressor concentration (nm)
Ordinate zou: percent of inhibitin, promptly suppress percentage ratio
Band "-" lines: corresponding PpL
Band " △-" lines: corresponding LD5
Band " *-" lines: corresponding SpA
Band " zero-" lines: corresponding PpL+SpA
Figure 14 LD5 suppresses in conjunction with the competition of people's polyclone IgM
X-coordinate: concentration of inhibitor (nm), i.e. supressor concentration (nm)
Ordinate zou: percentof inhibitin, promptly suppress percentage ratio
Band "-" lines: corresponding PpL
Band " △-" lines: corresponding LD5
Band " *-" lines: corresponding SpA
Band " zero-" lines: corresponding PpL+SpA
Figure 15 LD5 suppresses in conjunction with the competition of people's polyclone IgA
X-coordinate: concentration of inhibitor (nm), i.e. supressor concentration (nm)
Ordinate zou: percent of inhibitin, promptly suppress percentage ratio
Band "-" lines: corresponding PpL
Band " △-" lines: corresponding LD5
Band " *-" lines: corresponding SpA
Band " zero-" lines: corresponding PpL+SpA
Figure 16 purifying gene engineering antibody electrophorogram
Figure 17 purifying natural antibody electrophorogram
Figure 18 monoclonal antibody purification electrophorogram
Figure 19 whose anti-HCV immune chromatograph testing strip
The negative blood examination result of A:HCV Ab
B: blank
The positive blood sample detected result of C, D:HCV Ab
1：chicken anti-Protein A
2：Core Ag+NS4 Ag
3:NS5 Ag, NS are the Nonstructural Protein (non structure protein) of hepatitis C virus
4：NS4 Ag
5：NS3 Ag
6：Core Ag
Embodiment
In the present invention, term " its conservative property variant protein " is meant the variant form with the high affine active evolved immunoglobulin binding molecule of immunoglobulin (Ig), these variant forms comprise (but being not limited to): several (are generally 1-30, preferable 1-10, better 1-5) amino acid whose disappearance, insert and/or replacement, and add one or several at C-terminal and/or N-terminal and (be generally in 20, preferably be in 10, more preferably be in 5) amino acid, for example, in the art, close or when similarly amino acid replaces with performance, can not change albumen quality function usually, as replacing Ala etc. with Val or Leu or Ile, again such as, add one and several amino acid at C-terminal and/or N-terminal and also can not change proteinic function usually.In addition, these variant forms comprise that also maturation protein and another compound are (such as the compound that prolongs the polypeptide transformation period, polyoxyethylene glycol for example) merges formed albumen, additional aminoacid sequence is fused to this peptide sequence and the albumen that forms (as leader sequence or secretion sequence or be used for the sequence or the proteinogen sequence of this polypeptide of purifying, such as GST).In addition, variant form also includes, but is not limited to evolved immunoglobulin binding molecule and above-mentioned albumen through the form after modifying: chemically derived form such as acetylize or carboxylated; Glycosylation, carry out glycosylation modified and albumen that produce in the procedure of processing as those in the synthetic of polypeptide and processing or further, this modification can be carried out glycosylated enzyme (as mammiferous glycosylase or deglycosylating enzyme) and finishes by albumen is exposed to; Sequence with phosphorylated amino acid residue (as Tyrosine O-phosphate, phosphoserine, phosphothreonine).Thereby also comprise the albumen that has been improved its anti-proteolysis performance or optimized solubility property by modifying.These fragments, derivative and analogue belong to this area and are familiar with the known scope of technician.
The full length sequence of the polynucleotide of coding evolved immunoglobulin binding molecule of the present invention or its fragment can obtain with the method for pcr amplification method, recombination method or synthetic usually.For the pcr amplification method, can be disclosed according to the present invention relevant nucleotide sequence, especially open reading frame sequence designs primer, and with disclosed plasmid as template, amplification and must be about sequence.Use the PCR method and be optimized for acquisition gene of the present invention.The primer that is used for PCR can suitably be selected according to sequence information of the present invention disclosed herein, and available ordinary method is synthetic.Available ordinary method is as the polynucleotide passage by gel electrophoresis separation and purifying amplification.
Among the present invention, the polynucleotide sequence of coding evolved immunoglobulin binding molecule can be inserted in the various carriers, described carrier comprises various expression vectors, comprise that bacterial plasmid well known in the art, phage, yeast plasmid, vegetable cell virus, mammalian cell virus are as adenovirus, retrovirus or other carriers, preferred prokaryotic vector, such as pKK223-3, pBR322, pGEX carrier or the like, the preferred PET-32a of the carrier of Shi Yonging (+) in the present invention.In a word, as long as can duplicate in host and stablize, any plasmid and carrier can be used.
Among the present invention, term " expression regulation sequence " is often referred to and participates in the sequence that the control nucleotide sequence is expressed.Expression regulation sequence comprises promotor and the termination signal that links to each other with target nucleotide sequence operability.They also comprise the suitably required sequence of translation of nucleotide sequence usually." operability links to each other " is meant that some part of linear DNA sequence can influence the activity of same other parts of linear DNA sequence.For example, if promotor or enhanser have increased transcribing of encoding sequence, then it is that operability links to each other with encoding sequence.
The host cell that transforms recombinant vectors among the present invention can be a prokaryotic cell prokaryocyte, as bacterial cell; Or eukaryotic cell such as low, as yeast cell; Or higher eucaryotic cells, as mammalian cell.Representative example has: intestinal bacteria, streptomyces; The bacterial cell of Salmonella typhimurium; Fungal cell such as yeast; Vegetable cell; The insect cell of fruit bat S2 or Sf9; CHO, the zooblast of COs.293 cell or Bowes melanoma cells etc.E. coli bl21-TRXB in the preferred prokaryotic cell prokaryocyte of the present invention.
Can carry out with routine techniques well known to those skilled in the art with the recombinant vectors transformed host cell.When the host was prokaryotic organism such as intestinal bacteria, the competent cell that can absorb DNA can be used CaGl in exponential growth after date results 2Method or thermal shock method are handled, and used step is well-known in this area.Another kind method is to use MgCl 2If desired, transforming also the method for available electroporation carries out.When the host is an eukaryote, can select following DNA transfection method for use: coprecipitation of calcium phosphate method, conventional mechanical method such as microinjection, electroporation, liposome packing etc.
The condition of suitable expression evolved immunoglobulin binding molecule of the present invention is with to express used host cell relevant, the transformant that the recombinant vectors transformed host cells obtains can be cultivated with ordinary method, expresses evolved immunoglobulin binding molecule of the present invention.According to used host cell, used substratum can be selected from various conventional substratum in the cultivation.Under the condition that is suitable for the host cell growth, cultivate.After host cell grows into suitable cell density, induce the promotor of selection with suitable method (as temperature transition or chemical induction), cell is cultivated for some time again.The extracellular can be expressed or be secreted into to recombinant protein in the aforesaid method in cell or on cytolemma.If desired, can utilize its physics, the separating by various separation methods with other characteristic and the albumen of purification of Recombinant 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 handles, with protein precipitant handle (salt analysis method), centrifugal, the broken bacterium of infiltration, superly handle, the combination of super centrifugal, sieve chromatography (gel-filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography (LC) technology and these methods.
One, a kind of novel evolved immunoglobulin binding molecule protokaryon preparation
A kind of novel evolved immunoglobulin binding molecule LD5 of the present invention uses the molecular evolution method to screen the new immune globulin binding molecule form that obtains from the molecular combinations library that the B3 structural domain of phage display bacterium SpA A, B, C, D single structure territory and PpL is recombinated at random, details is in " phage display SpA and PpL Ig are in conjunction with combinatorial library and the affine screening at random of single structure territory ", biophysics and biological chemistry progress has detailed description in the 535th～543 page of 2005 the 32nd the 6th phase of volume.The present invention is to contain coding evolved immunoglobulin binding molecule LD5 (L-D-L-D-L; L is the B3 structural domain of PpL, and D is the D structural domain of SpA) cDNA sequence clone pCANTAB5S-LD5 is template, the dna segment of pcr amplification LD5, and introduce NcoI and SalI restriction endonuclease recognition site in amplification segment upstream by the primer synthetic method.The amplification segment reclaims test kit with the PCR product and reclaims back NcoI and SalI endonuclease digestion, reclaiming test kit with glue behind the disconnected row agarose gel electrophoresis of enzyme section reclaims, be cloned among the NcoI and SalI site of prokaryotic expression carrier PET-32a (+), successfully construct prokaryotic expression carrier PET32a (+)-LD5, measure the dna sequence dna of cloned sequence LD5 with the downstream primer T7ter of PET32a (+) carrier, The sequencing results confirm the insertion sequence of PET-32a (+) between NcoI and SalI site and PALn at random the positive colony LD5 sequence of library of molecules phage selection conform to fully, frame is entirely true, initial son and terminator add that it is in full accord to insert upstream and downstream sequence and PET32a (+) carrier sequence.Get recombinant plasmid PET32a (+)-LD5200ng transformed into escherichia coli BL21-TRXB, obtain the prokaryotic expression bacterial classification of LD5.With cultivating back IPTG abduction delivering in this bacterium, to establish the sky bacterium and make negative control, the SDS electrophoresis showed protein band that the contrast bacterium does not have occurs at the about 59000 Daltons places of molecular weight, meets the molecular weight of LD5.To cultivate in a large number behind the actication of culture, IPTG abduction delivering, Ni-NTA column purification obtain purifying LD5 fusion rotein, only occur a protein band behind the SDS electrophoresis showed purifying, meet its corresponding molecular weight.
Two, novel evolved immunoglobulin binding molecule and each antibody-like and antibody fragment combines
The B3 structural domain that a kind of novel evolved immunoglobulin binding molecule LD5 of the present invention is bacterium PpL and the interval tumor-necrosis factor glycoproteins (L-D-L-D-L in SpA D single structure territory; L is the B3 structural domain of PpL, D is the D structural domain of SpA), PpL B3 structural domain wherein has the combined function with the immunoglobulin kappa light chain, SpA D structural domain have with the combined function of immunoglobulin (Ig) VH3 heavy chain and with immunoglobulin IgG Fc section bonded function.Therefore, LD5 can be in conjunction with each immunoglobulin like protein (IgG, IgM, IgA, IgD, IgE) Fab and immunoglobulin IgG Fc in theory.The LD5 fusion rotein that the present invention uses above-mentioned expressed purifying wraps quilt, respectively immunoglobulin IgG, immunoglobulin IgG Fab, immunoglobulin IgG Fc, Immunoglobulin IgM and Immunoglobulin IgA are carried out biotin labeling, detect the situation that combines of LD5 and immunoglobulin IgG, immunoglobulin IgG Fab, immunoglobulin IgG Fc, Immunoglobulin IgM and Immunoglobulin IgA respectively with the ELISA method, and compare with combining of above-mentioned immunoglobulin molecules with PpL and SpA.The result confirms that the binding ratio PpL and the SpA of LD5 and immunoglobulin IgG Fab, Immunoglobulin IgM and Immunoglobulin IgA strengthen to some extent, and is suitable with SpA with combining of immunoglobulin IgG and immunoglobulin IgG Fc.
Three, the confirmation of the dibit point binding pattern of novel evolved immunoglobulin binding molecule κ light chain and VH3 heavy chain
A kind of novel evolved immunoglobulin binding molecule LD5 of the present invention uses the molecular evolution method to screen the new immune globulin binding molecule form that obtains from the molecular combinations library that the B3 structural domain of phage display bacterium SpA A, B, C, D single structure territory and PpL is recombinated at random, details is in " phage display SpA and PpL Ig are in conjunction with combinatorial library and the affine screening at random of single structure territory ", biophysics and biological chemistry progress has detailed description in the 535th～543 page of 2005 the 32nd the 6th phase of volume.Such immune globulin binding molecule form is the advantage clone that library screening obtains, there are 33 to contain this class formation among weak the clone who is measured, as bait combinatorial library is carried out the molecular evolution screening owing to use human normal immunoglobulin binding molecule (comprising IgG, IgM, IgA, IgD and IgE type), therefore, can infer this structure and IgF ab have independent combined function than PpL B3 and immunoglobulin kappa light chain and SpA and immunoglobulin (Ig) VH3 heavy chain stronger combine active, the above-mentioned this point that also proves in conjunction with test-results.Can possess this enhancing promptly is dibit point binding pattern with immune globulin binding molecule κ light chain and VH3 heavy chain in conjunction with active most probable combining form.The dibit point bonded test that confirms above-mentioned and immune globulin binding molecule κ light chain and VH3 heavy chain is for combining competition experiments.
We have used competition and suppress ELISA and test combining of the dibit point that confirms LD5 and immune globulin binding molecule κ light chain and VH3 heavy chain among the present invention.Specifically to LD5 mark vitamin H, use immunoglobulin IgG Fab, Immunoglobulin IgM and Immunoglobulin IgA coated elisa plate respectively, add the biotin labeled LD5 of fixed concentration and the various competition albumen (LD5, PpL, SpA and PpL+SpA) of doubling dilution simultaneously, wash plate behind 37 ℃ of 45min, add avidin-HRP mixture, 37 ℃ of 15min wash TMB colour developing 5-10min behind the plate, 2M sulfuric acid stops, and microplate reader reads OD450nm.Calculate the OD average in each multiple hole, Excel draws competition and suppresses curve.If dibit point bonded synergistic effect, then unite with PpL and SpA LD5 and IgG Fab, IgM, IgA (IgG Fab shortage Fc section, the Fc section of IgM and IgA can not combine with SpA and LD5, got rid of the interference effect of Fc section to the competition inhibition test like this) to be significantly less than the restraining effect of LD5 itself with the bonded restraining effect; If not dibit point bonded synergistic effect, but respectively to the independent combination of variable region VH3 heavy chain and κ light chain, then unite with PpL and SpA to LD5 and IgGFab, IgM, IgA bonded restraining effect should be suitable with the restraining effect of LD5 itself.As seen the result unites with PpL and SpA will be significantly less than the restraining effect of LD5 itself to LD5 and IgG Fab, IgM, IgA and bonded restraining effect, illustrates that LD5 combines with immunoglobulin (Ig) to be and the combining of the dibit point of κ light chain and VH3 heavy chain.
Four, use novel evolved immunoglobulin binding molecule large scale purification genetic engineering antibody, natural antibody and monoclonal antibody
A kind of novel evolved immunoglobulin binding molecule LD5 of the present invention uses the molecular evolution method to screen the new immune globulin binding molecule form that obtains from the molecular combinations library that the B3 structural domain of phage display bacterium SpA A, B, C, D single structure territory and PpL is recombinated at random, in conjunction with having experimental results show that this molecule not only has SpA and these two kinds of proteic binding characteristics of PpL, and other bacterial immune globulin binding molecules (comprising SpA and PpL) the κ light chain that is not had and the dibit point binding characteristic of VH3 heavy chain have been produced.Some bacterial immune globulin binding molecules such as SpA, SpG have been widely used in the purifying of natural antibody, monoclonal antibody and genetic engineering antibody in actual applications, and novel evolved immunoglobulin binding molecule LD5 also is proved to be the purifying that can be applied to natural antibody, monoclonal antibody and genetic engineering antibody in invention.
We are prepared into the affinity purification post with LD5 molecule covalent coupling among the present invention on CNBr-Sepharose 4B particle, utilize this affinity purification post that the various types of antibody in the CHO-K1 engineering cell strain nutrient solution that comes autoblood, mouse ascites and genetic engineering antibody are carried out purifying.Specifically be the LD5 affinity column to be crossed in the serum dilution back that comprises various antibody make antibodies on pillar, with acid the antibody elution on the pillar got off promptly to obtain antibody purified again; The hybridoma cell strain that can produce monoclonal antibody is cultivated back injection mouse peritoneal, preparation ascites, be that the back LD5 of mistake of the ascites dilution affinity column that will comprise monoclonal antibody makes antibodies on pillar, with acid the antibody elution on the pillar got off promptly to obtain antibody purified again; The external large scale fermentation of the CHO-K1 engineering cell strain of genetic engineering antibody is cultivated, the preparation nutrient solution, be that the direct LD5 of mistake of the cell culture fluid affinity column that will comprise monoclonal antibody makes antibodies on pillar, with acid the antibody elution on the pillar got off promptly to obtain antibody purified again.
Five, use novel evolved immunoglobulin binding molecule ELISA method and detect specific antibody
A kind of novel evolved immunoglobulin binding molecule LD5 of the present invention can combine with the various human immunoglobulin molecules comprising antibody such as immunoglobulin IgG Fab, immunoglobulin IgG Fc, Immunoglobulin IgM, Immunoglobulin IgAs, therefore, with enzyme molecules such as horseradish peroxidase or alkaline phosphatase on this molecule marker, can be used as enzyme complex (ELIAS secondary antibody) and carry out ELISA method detection specific antibody.
We are with LD5 molecule marker horseradish peroxidase (HRP) among the present invention, enzyme prozyme in the whose anti-HCV antibody ELISA detection kit of substitute goodsization (the anti-human normal immunoglobulin of the animal of HRP mark) carries out the whose anti-HCV antibody test, and carry out parallel control with commercial whose anti-HCV antibody ELISA detection kit and detect, estimate the detection effect of LD5 molecule marker horseradish peroxidase as enzyme complex.
Six, use novel evolved immunoglobulin binding molecule immunochromatographyassay assay specific antibody
A kind of novel evolved immunoglobulin binding molecule LD5 of the present invention can combine with the various human immunoglobulin molecules, comprising antibody such as immunoglobulin IgG Fab, immunoglobulin IgG F Fc Immunoglobulin IgM, Immunoglobulin IgAs, therefore, this molecule and colloid gold particle are fully adsorbed preparation LD5 Radioactive colloidal gold, and the antibody molecule that can adsorb in the detected sample is used for the immunochromatographyassay assay specific antibody.
We fully adsorb preparation LD5 Radioactive colloidal gold with LD5 Radioactive colloidal gold molecule among the present invention, rule (1mm is wide) with HCV antigen (1.5mg/L) and human IgG (1mg/L) on nitrocellulose filter, respectively as detection line and control line.Antibody in the test sample (serum) at first combines with LD5 molecule on the LD5 Radioactive colloidal gold, and be fixed on the colloid gold particle, this particle motion is used line place of HCV antigen to nitrocellulose filter, as containing whose anti-HCV antibody in the test sample, the colloid gold particle that then adsorbs antibody just combines with HCV antigen and is fixed on line place, form mauve Radioactive colloidal gold lines, test positive, as not containing whose anti-HCV antibody in the test sample, then colloid gold particle just can not be by HCV antigen in conjunction with being fixed on line place, can not form mauve Radioactive colloidal gold lines, detect negative.Use immunochromatographyassay assay whose anti-HCV antibody specific antibody, and carry out parallel control with commercial whose anti-HCV antibody ELISA detection kit and detect, estimate LD5 molecular colloid gold and detect effect.
Further set forth the present invention below in conjunction with embodiment.Should be understood that these embodiment only are used to illustrate the present invention, but not limit the scope of the invention.The reagent of the experimental technique of unreceipted actual conditions and undeclared prescription is according to people such as normal condition such as Sambrook in the following example, molecular cloning: the condition of the condition described in the test handbook (New York:Cold Spring Harbor Laboratory Press, 1989) or manufacturers's suggestion is carried out or is disposed.
The method of 1 one kinds of novel evolved immunoglobulin binding molecule protokaryon preparations of embodiment
1. novel evolved immunoglobulin binding molecule cDNA sequence and clone
Contain coding evolved immunoglobulin binding molecule LD5 (L-D-L-D-L; L is the B3 structural domain of PpL, D is the D structural domain of SpA) the reorganization phagemid carrier pCANTAB5S-LD5 of cDNA sequence clone, be to obtain by the screening of molecular evolution method, the concrete steps of preparation pCANTAB5S-LD5 are in " phage display SpA and PpL Ig are in conjunction with combinatorial library and the affine screening at random of single structure territory ", biophysics and biological chemistry progress, full disclosure in 2005 the 32nd volume the 535th～543 page of the 6th phase, it is 4#/33# cloning vector that the 3rd 27#/44# and the 4th that takes turns screening takes turns screening in the table 4 in the document, its cDNA sequence is shown in SEQ ID No.1, and aminoacid sequence is shown in SEQ ID No.2.
Evolved immunoglobulin binding molecule LD5 cDNA sequence (SEQ ID NO:2):
AAAGAAAAAACACCAGAAGAACCAAAAGAAGAAGTTACTATTAAAGCAAACTTAATCTATGCAGATGGAAAAACACA
AACAGCAGAATTCAAAGGAACATTTGAAGAAGCAACAGCAGAAGCATACAGATATGCTGACTTATTAGCAAAAGAAA
ATGGTAAATATACAGTAGACGTTGCAGATAAAGGTTATACTTTAAATATTAAATTTGCTGGAGAGCTCGCTGATGCG
CAACAAAATAACTTCAACAAAGATCAACAAAGCGCCTTCTATGAAATTTTGAACATGCCTAACTTAAACGAAGCGCA
ACGCAATGGTTTCATTCAAAGTCTTAAAGACGATCCAAGCCAAAGCACTAACGTTTTAGGTGAAGCTAAAAAATTAA
ACGAATCTCAAGCACCGAAAGAGCTCAAAGAAAAAACACCAGAAGAACCAAAAGAAGAAGTTACTATTAAAGCAAAC
TTAATCTATGCAGATGGAAAAACACAAACAGCAGAATTCAAAGGAACATTTGAAGAAGCAACAGCAGAAGCATACAG
ATATGCTGACTTATTAGCAAAAGAAAATGGTAAATATACAGTAGACGTTGCAGATAAAGGTTATACTTTAAATATTA
AATTTGCTGGAGAGCTCGCTGATGCGCAACAAAATAACTTCAACAAAGATCAACAAAGCGCCTTCTATGAAATTTTG
AACATGCCTAACTTAAACGAAGCGCAACGCAATGGTTTCATTCAAAGTCTTAAAGACGATCCAAGCCAAAGCACTAA
CGTTTTAGGTGAAGCTAAAAAATTAAACGAATCTCAAGCACCGAAAGAGCTCAAAGAAAAAACACCAGAAGAACCAA
AAGAAGAAGTTACTATTAAAGCAAACTTAATCTATGCAGATGGAAAAACACAAACAGCAGAATTCAAAGGAACATTT
GAAGAAGCAACAGCAGAAGCATACAGATATGCTGACTTATTAGCAAAAGAAAATGGTAAATATACAGTAGACGTTGC
AGATAAAGGTTATACTTTAAATATTAAATTTGCTGGA
Evolved immunoglobulin binding molecule LD5 aminoacid sequence (SEQ ID NO:1):
KEKTPEEPKEEVTIKANLIYADGKTQTAEFKGTFEEATAEAYRYADLLAKENGKYTVDVADKGYTLNIKFAGELADA
QQNNFNKDQQSAFYEILNMPNLNEAQRNGFIQSLKDDPSQSTNVLGEAKKLNESQAPKELKEKTPEEPKEEVTIKAN
LIYADGKTQTAEFKGTFEEATAEAYRYADLLAKENGKYTVDVADKGYTLNIKFAGELADAQQNNFNKDQQSAFYEIL
NMPNLNEAQRNGFIQSLKDDPSQSTNVLGEAKKLNESQAPKELKEKTPEEPKEEVTIKANLIYADGKTQTAEFKGTF
EEATAEAYRYADLLAKENGKYTVDVADKGYTLNIKFAG
2. the structure flow process of prokaryotic expression carrier pET32a (+)-LD5 is seen Fig. 1
2.1 the synthetic primer that is used for the cDNA sequence of pcr amplification evolved immunoglobulin binding molecule LD5 of primer is: upstream 5SNco-u:5 '-TAT CCATGGCTGCGGCCCAGCCGGCCTCT-3 ', downstream 5SNoG-d:5 '-CCTGCGGCCGCAACTGCCGCCGCC-3 '.Introduced NcoI restriction enzyme site (underscore part) in the upstream primer, the primed DNA sequence is synthetic by Shanghai Bo Ya biotech company (now being Ying Jun biotech company).
2.2 the pcr amplification of LD5 expressed sequence is template with pCANTAB5S-LD5, increases with synthetic upstream and downstream primer, the reaction volume of PCR is 50 μ l, adds plasmid template 5ng, each 1 μ mol of upstream and downstream primer, dNTP 100 μ mol, Mg ++3mmol, Taq enzyme 1U adds ddH 2O to 50 μ l.Reaction conditions: 94 ℃ 30s-60 ℃ 30s-72 ℃ of 45s; 35 circulations, reaction finish preceding 72 ℃ and extend 5min, and product detects with 1.2% agarose gel electrophoresis, and rubber tapping is reclaimed test kit and reclaimed purifying.The LD5 cDNA fragment of pcr amplification is having (G4S) 3 joints (for inherent on the pCANTAB5S carrier) behind the SalI site.As seen from Figure 2, PCT product size meets about 1156bp.Glue cuts (G4S) 3 joints with NcoI and SalI double digestion after reclaiming purifying, obtains LD5 expressed sequence 1103bp, and the result sees Fig. 3 respectively.
2.3 the structure of recombinant expression plasmid and determined dna sequence prokaryotic expression carrier pET32a (+) are available from Novagen company, pET-32a (+) reclaims enzyme and cuts product with NcoI and SalI double digestion, test kit.Above-mentioned PCR purified product is with NcoI and SalI double digestion, gets 400ng after test kit reclaims and cuts product with 400ng PET-32a (+) carrier NcoI with the SalI enzyme and be connected.Select the recon enzyme after the conversion and cut evaluation, obtain recombinant plasmid pET-32a (+)-LD5.Extract pET32a (+)-LD5 plasmid,, meet theory expectation and measure the segmental dna sequence dna of insertion with its downstream primer with NcoI and SalI double digestion (see figure 4).
2.4 LD5 Expression of Fusion Protein
2.4.1 the conversion Calcium Chloride Method of recombinant expression vector pET-32a (+)-LD5 prepares e. coli bl21 competent cell (E.coli BL21trxB (DE3) is available from Novagen company), get pET-32a (+)-LD5 200ng and add competence BL21-TRXB100 μ l, ice-water bath 30min, 42 ℃ of 90s are coated with 37 ℃ of overnight incubation of LB flat board (containing Amp100ng/ml and Kana35 ng/ml) after the 37 ℃ of 150rpm of SOC substratum that add 900 μ l behind the ice-water bath 1-2min cultivate 1h.
2.4.2 the preparation of the abduction delivering of LD5 and SDS-PAGE sample from pET-32a (+)-LD5/BL21 transform dull and stereotyped go up 4 mono-clonal colony inoculations of picking shaking table to the 0.5mlLB (containing Amp100ng/ml) and cultivate 3.5h after, take out 100 μ l and be connected to and add 200 μ l glycerine behind the cultivation 4h in 700 μ lLB (Amp) centrifuge tubes and prepare the glycerine bacterial classification; Surplus bacterium liquid adds IPTG and got 300 μ l bacterium liquid 10000rpm centrifugal 1 minute to final concentration 1mM inducing culture 3.5h, abandons supernatant, adds 2 * SDS sampling liquid, 25 μ l and 0.01PBS25 μ l, and mixing is made the SDS sample.Collect the same ratio of empty bacterium BL21 nutrient solution and make the SDS sample.
2.4.3 the expression of SDS-PAGE electrophoresis detection target protein prepares the 12%SDS polyacrylamide gel, with above-mentioned sample and lower molecular weight standard protein boiling water bath 5 minutes, application of sample, initial press 5V/cm, treat to be increased to 12V/cm after tetrabromophenol sulfonphthalein enters separation gel, reach the separation gel bottom until tetrabromophenol sulfonphthalein.Electrophoresis finishes, and takes out gel and spends the night with coomassie brilliant blue staining, places methyl alcohol-glacial acetic acid solution to decolour again 3-4 hour.The results are shown in Figure 5, four of visible institute picking are cloned in the target protein band are all arranged about 59kd, and empty BL21 there is no this band.
2.5 the great expression of fusion rotein LD5 and purifying
2.5.1 express pET-32a (+)-LD5/BL21 glycerine bacterial classification 500 μ l be connected to 50mlLB (containing Amp) overnight incubation, go among the 500mlLB 3.5h37 ℃ of shaking table again and cultivate the IPTG500 μ l inducing culture 3.5h that adds 1mol/l behind the 3.5h, take out in the packing 250ml centrifuge tube 6000rpm4 ℃ centrifugal 10 minutes, remove supernatant, the PBS that adds the 0.1mol/l of 20ml is resuspended in the 50ml centrifuge tube, 11000rpm4 ℃ centrifugal 20 minutes, remove supernatant, the urea of the 8mol/l of precipitation adding 20ml is resuspended, and 4 ℃ are spent the night.
11000rpm4 ℃ of urea lysate is centrifugal 20 minutes 2.5.2 purifying spends the night, get supernatant and cross Ni-NTA post (metal chelate affinity chromatography medium (Ni-NTA) available from German QIAGEN company), the Ni-NTA post is with 8M urea (pH8.0) balance, urea lysate supernatant upper prop, 8M urea (pH7.0) is washed post, with 8M urea (pH4.5) wash-out, collect elution peak, 0.1M Tris neutralization, dialyse with PBS, SDS-PAGE analyzes the purification effect (see figure 6), and as seen the band of LD5 is arranged at 59kd, does not have obvious foreign protein band.The protein sequencing result also shows the sequence that contains SEQ ID NO:1.
Embodiment 2 novel evolved immunoglobulin binding molecules combine with each antibody-like and antibody fragment
1. with the combining of people's polyclone IgG:
1.1 people's polyclone IgG is available from Sigma company, PpL and SpA purchase Sigma company.
1.21mg/ml people's polyclone IgG dialyses with PBS (pH7.2).Get the long-armed activation vitamin Hs of 50 μ L 3mg/ml (available from PIERCE company) and add 1mL IgG (1mg/mL), add dialysis tubing 4 ℃ of dialysed overnight in PBS behind the 4h that slightly vibrates at room temperature, add equivalent glycerine after having dialysed and in-20 ℃, preserve.
1.3 LD5, PpL and SpA all adjust concentration and wrap by 96 orifice plates with carbonate buffer solution (pH9.6) dilution in 1: 200 to 1mg/ml, every kind of albumen all wraps the multiple hole by 3 rows, PBST washes 4-5 time behind 4 ℃ of 24h, wash plate behind 37 ℃ of 1h of confining liquid (2%BSA 0.05%TWEEN-20) sealing, biotin labeled IgG begins doubling dilution with finite concentration, last Kong Bujia, wash plate behind 37 ℃ of 45min, add avidin-HRP mixture (available from PIERCE company), 37 ℃ of 15min, wash TMB colour developing 5-10min behind the plate, 2M sulfuric acid stops, and microplate reader reads OD450nm; Computation of mean values and standard deviation, Excel draws binding curve.
The visible LD5 of result is suitable with SpA with combining of people's polyclone IgG, apparently higher than the combine (see figure 7) of PpL with people's polyclone IgG.
2. with the combining of people's polyclone IgM:
2.1 people's polyclone IgM is available from Sigma company, biotin labeling is with 1.2.
2.2 with LD5 PpL SpA difference wrapper sheet, combine with biotin labeled IgM, method is with 1.3.The visible LD5 of result combines apparently higher than SpA and PpL (see figure 8) with people's polyclone IgM's.
3. with the combining of people's polyclone IgA:
3.1 people's polyclone IgA is available from Sigma company, biotin labeling is with 1.2.
3.2 the ELISA method is with 1.3.The visible LD5 of result combines apparently higher than SpA and PpL (see figure 9) with people's polyclone IgA's.
4. with the combining of people's polyclone IgG Fab:
4.1 being carried out papoid, IgG cuts acquisition Fab and Fc:
4.1.1 halfcystine veraldon: polyclone human IgG IgM IgA is all available from Sigma company.Papoid (magnificent Bioisystech Co., Ltd) is dissolved into concentration 1mg/ml with PBS, adds EDTA and L-halfcystine all to final concentration 0.01M, removes halfcystine and EDTA with the dialysis of 0.1M sodium-acetate behind 37 ℃ of 30min.
Cut 4.1.2 people's polyclone IgG and activatory papoid carry out enzyme by 30: 1 mass ratio, add iodo-acetamide behind 37 ℃ of 5h to the 0.03M termination reaction.
Separate Fab and Fc 4.1.3 product is crossed the SpA post: wash post with the 20mM sodium phosphate buffer, the enzyme reaction solution upper prop is collected the stream that contains Fab and is worn the peak, and the back is with the Fc of the sodium-acetate elution of bound of 0.1M pH3.0.
4.2 the biotin labeling of IgGFab IgGFc is with 1.2
4.3 carry out combining with LD5, SpA and the PpL of bag quilt with biotin labeled IgGFab, method is with 1.3, the visible LD5 of result combines apparently higher than SpA and PpL (see figure 10) with people's polyclone Ig Fab's.
5. with the combining of people's polyclone IgG Fc: carry out and the combining of various IBP molecules with biotin labeled IgG Fc, method is with 1.3.The visible LD5 of result is suitable with SpA with combining of people's polyclone IgG Fc, PpL and people's polyclone IgG Fc debond (seeing Figure 11).
6. with the combining of genetically engineered scFv:
6.1 expression and the purifying of scFv in intestinal bacteria
6.1.1 the abduction delivering of scFv
The expression vector of three kinds of scFv, KM36 (VH3-V λ) KM38 (VH3-V κ) KM41 (VH1-V κ) provides for the Dutch Sanquil r Jan Voorberg of D. Lab, its detailed preparation method is by document Edward N.van den Brink, EllenA.M.Turenhout, Niels Bovenschen, Bram G.A.D.H.Hei jnen, Koen Mertens, Mar joleinPeters, and Jan Voorberg.Multiple VH genes are used to assemble human antibodiesdirected toward the A3-C1 domains of factor VIII. BLOOD, 2001; 97 (4): the 966-972 full disclosure.Its N of scFv end have 6 histidine-tagged.Transformed E Coli TG1, the picking mono-clonal is transferred in 20ml 2 * YTG (Amp100mg/ml), after spending the night, 37 ℃ of 250rpm are expanded to 500ml 2 * YT (Amp100mg/ml 0.1%Glu), add IPTG to 0.1mM during 37 ℃ of 250rpm to OD600=0.5, centrifuging and taking precipitation bacterium behind 30 ℃ of 220rpm 3h.
6.1.2 induce cracking of bacterium and the purifying of scFv
The 50mMTris-HCl (20% sucrose 1mMEDTA 1mMPMSF) that the precipitation bacterium adds the ice precooling suspends, behind the 20min, 12000rpm is centrifugal, and 10min gets supernatant, and precipitation is got supernatant with centrifugal 12000rpm 10min behind 5mM MgSO4 (1mMPMSF) 20min of ice precooling again.To the cellular lysate supernatant that obtains with 20mM imidazoles (500mMNaCl 50mM sodium phosphate pH7.5), 4 ℃ of dialysed overnight; The Ni-NTA post is with the dialyzate balance, and the sample upper prop is washed behind the post with 100mM imidazoles wash-out with the 20mM imidazoles, collects the scFv that elutriant is purifying.
6.2 three kinds of scFv and biotin labeling LD5 bonded ELISA
With mono-clonal scFv KM36, KM38 and the KM41 wrapper sheet of three kinds of purifying, simultaneously with the TG1 bacterium cracking supernatant wrapper sheet that does not contain the scFv expression vector.Capable of experiment with biotin labeled LD5 to immobilization Ig.The TG1 of KM36 KM38 KM41 and empty TG1 bacterium cracking supernatant wrap by 96 orifice plates with carbonate buffer solution dilution in 1: 200, every kind of scFv all wraps the multiple hole by 3 rows, PBST washes 3-4 time behind 4 ℃ of 24h, wash plate behind 37 ℃ of 1h of confining liquid (2%BSA 0.05%TWEEN-20) sealing, the biotin labeling LD5 of 2mg/ml was with 1: 200 beginning doubling dilution, last Kong Bujia, surplus step is with 1.3.The visible LD5 of result and KM38 (VH3-V κ) combine much larger than with combine (Figure 12) of KM41 (VH1-V κ) and KM36 (VH3-V λ).
The confirmation of the dibit point binding pattern of embodiment 3 novel evolved immunoglobulin binding molecule κ light chains and VH3 heavy chain
The Fc section of SpA and IgG has stronger avidity, SpA can also be in conjunction with the VH district (SassoEH that belongs to the VH3 gene family simultaneously, Silverman GJ, and Mannik M.Human IgA and IgG F (ab ') 2 that bind to staphylococcalSpA belong to the VHIII subgroup.J Immunol.1991; 147:1877-1883).PpL by with the light chain of Ig, 1,3,4 hypotypes that mainly are the κ light chain interact and binding domain-immunoglobulin (Nilson BH, Solomon A, BjorckL, et al.PpL from peptostreptococcus magnus binds to the kappa light chain variabledomain; J Biol Chem.1992; 267 (4): 2234-9).From the combine experiment of LD5 with all kinds of Ig molecules, LD5 has IgGFab IgM IgA than SpA and the higher avidity of PpL, infers that LD5 has the dibit point binding characteristic to VH3 heavy chain and kappa light chain variable district.The competition inhibition test can verify that LD5 whether has the dibit point binding characteristic to VH3 heavy chain and kappa light chain variable district, if dibit point bonded synergistic effect, then unite with PpL and SpA LD5 and IgG Fab, IgM, IgA (IgG Fab shortage Fc section, the Fc section of IgM and IgA can not combine with SpA and LD5, got rid of the interference effect of Fc section to the competition inhibition test like this) the bonded restraining effect will be significantly less than the restraining effect of LD5 itself; If not dibit point bonded synergistic effect, but respectively to the independent combination of variable region VH3 heavy chain and κ light chain, then unite with PpL and SpA to LD5 and IgGFab, IgM, IgA bonded restraining effect should be suitable with the restraining effect of LD5 itself.
1. LD5 suppresses the ELISA experiment in conjunction with the competition of IgG Fab
2mg/ml IgG Fab wraps by 96 orifice plates with carbonate buffer solution (pH 9.6) dilution in 1: 200, be used for four classes competition albumen (LD5 self, PpL+SpA, PpL, SpA) each bag of multiple hole is by three rows, other wraps a row and is used for not adding the competition protein groups, PBST washes 2-3 time behind 4 ℃ of 24h, wash plate behind sealing (2%BSA 0.05%TWEEN-20) 37 ℃ of 1h, add the biotin labeled LD5 of fixed concentration and the various competition albumen of doubling dilution (starting point concentration is 10 times of vitamin H label L D5) simultaneously, another row only adds the vitamin H mark LD5 of same fixed concentration, wash plate behind 37 ℃ of 45min, add avidin-HRP mixture, 37 ℃ of 15min, wash TMB colour developing 5-10min behind the plate, 2M sulfuric acid stops, and microplate reader reads OD450nm.Calculate the OD average in each multiple hole, Excel draws competition and suppresses curve, suppresses the 100/OD (% of unit, OD represent the absorbancy average of unconstrained protein groups, and ODx is illustrated in the OD average in three multiple holes under the various inhibition concentrations) of percentage ratio=(OD-ODx).
The visible PpL+SpA of result, PpL, SpA to biotin labeled LD5 IgG Fab bonded restraining effect much smaller than LD5 itself (Figure 13), illustrate that κ light chain and combining of VH3 heavy chain among LD5 and the IgG Fab are dibit point combinations simultaneously, rather than combine with each site is independent respectively.
2. LD5 suppresses the ELISA experiment in conjunction with the competition of IgM
With the IgM wrapper sheet, method is with 1.The visible PpL+SpA of result, PpL, SpA to biotin labeled LD5 IgG Fab bonded restraining effect much smaller than LD5 itself (Figure 14), illustrate that κ light chain and combining of VH3 heavy chain among LD5 and the IgM are dibit point combinations simultaneously, rather than combine with each site is independent respectively.
3. LD5 suppresses the ELISA experiment in conjunction with the competition of IgA
With the IgA wrapper sheet, method is with 1.The visible PpL+SpA of result, PpL, SpA to biotin labeled LD5 IgG Fab bonded restraining effect much smaller than LD5 itself (Figure 15), illustrate that κ light chain and combining of VH3 heavy chain among LD5 and the IgA are dibit point combinations simultaneously, rather than combine with each site is independent respectively.
Embodiment 4 uses novel evolved immunoglobulin binding molecule
Large scale purification genetic engineering antibody, natural antibody and monoclonal antibody
1. agarose activation
(1) claim 4g CNBr-Sepharose 4B (available from Pharmacia company), use 4mM HCl, pH2.5 solution soaking 15min uses this solution repetitive scrubbing 5 times again.
(2) the activatory agarose is poured into rapidly in the B, taken out with frozen water and wash neutrality, take out with cold 0.1Mol/L pH 9.0 NaHCO3 of 250ml rapidly again and wash.
2. coupling protein
(1) in advance LD5 protein 12 0mg is placed 0.1Mol/L pH 9.0 NaHCO3 liquid dialyse a few hours (general coupling amount be 10 ~ 30mg/g carrier).
(2) the activatory agarose is poured into rapidly in the protein liquid and (in 1.5min, washed coupling from taking out), 4 ℃ are slowly stirred and spend the night, and albumen is combined with activatory agar.
3. dress post
To pack in the chromatography column with the agarose of albumen coupling, tighten the end opening folder, allow its sinking, unclamp the end opening folder after several minutes, solution is flowed out with the speed of 1ml/min approximately.Till washing OD280＜0.02 with 0.2Mol/L pH 9.0 NaHCO3 (containing 0.1Mol/L NaGl) to elutant.With 0.01Mol/L pH 7.4PB liquid or physiological saline washing, after the balance, 0.02%NaN3 is standby in 4 ℃ of preservations in adding
4. large scale purification genetic engineering antibody
(1) the CHO-K1 engineering cell strain producer gene engineered antibody of the genetic engineering antibody of the anti-Her2 of expression of Zhangjiang biological medicine company limited of use Fudan University reorganization.In the structure of engineering cell strain, with goal gene and the empty host cell of glutamine synthetase gene (GS) gene co-transfection, utilize GS gene amplification system, use (sulphur ammonia methionine(Met) (MSX)) as the strain of screening of medicaments screening high expressing cell.Carry out mono-clonalization, the adaptation serum-free suspension culture of high expressing cell strain at last, increase, build the storehouse and be used for GMP production.Bio-reactor adopts New Brunswick Scientific Co., CELLIGEN PLUS type.Working volume is 1.4L; Leavening temperature is 37 ℃; Mixing speed is 150rpm; By adding CO2 and 8%NaHCO 3PH is 7.20 in control; (ring sparger) carries out the gas supply by annular air feeder, and the control ventilation flow rate is at 0.1-0.2vvm; Control dissolved oxygen in 50% air saturation by air, oxygen, nitrogen.Engineering cell strain is seeded to the 2.2L bio-reactor after the suspension amplification cultivation, cultivated 25 days through continous pouring, and the results culture supernatant is carried out purifying and evaluation.
(2) the genetic engineering antibody cell culture fluid directly slowly adds purification column by 100 times of bed volumes, and with 0.01Mol/L pH7.4PBS liquid wash-out, flow velocity is 1ml/min, till elutant 0D280＜0.02.
(3) add 0.1Mol/L pH3.0 glycine-HCl damping fluid, flow velocity 1ml/min collects the composition that desorption is got off, immediately with 1Mol/L NaHCO3 neutralization, in order to avoid protein denaturation.
(4) with the 7Mol/L urea washing of two volumes, with 0.01Mol/L pH 7.4PB liquid or physiological saline washing, after the balance, can continue to use again.Preservation can add 0.02%NaN3 in 4 ℃ of preservations.Prevent freezing and dry and cracked.The albumen of purifying is dialysed to PBS ,-40 ℃ of preservations.The capable SDS PAGE of purifying protein gel electrophoresis analysis, visible purity reaches (Figure 16) more than 90%.
5. large scale purification natural antibody
(1) human serum is pressed bed volume 1/10 application of sample, and it is 20% that serum is diluted to concentration with PBS, slowly adds purification column, and with 0.01Mol/L pH7.4PBS liquid wash-out, flow velocity is 1ml/min, till elutant OD280＜0.02.
(2) add 0.1Mol/L pH3.0 glycine-HCl damping fluid, flow velocity 1ml/min collects the composition that desorption is got off, immediately with 1Mol/L NaHCO3 neutralization, in order to avoid protein denaturation.
(3) with the 7Mol/L urea washing of two volumes, with 0.01Mol/L pH 7.4PB liquid or physiological saline washing, after the balance, can continue to use again.Preservation can add 0.02%NaN3 in 4 ℃ of preservations.Prevent freezing and dry and cracked.The albumen of purifying is dialysed to PBS ,-4O ℃ of preservation.The capable SDS PAGE of purifying protein gel electrophoresis analysis, visible purity reaches (Figure 17) more than 90%.
6. large scale purification monoclonal antibody
(1) will resist recombinant human interferon-'s monoclonal antibody hybridoma cell strain recovery, the detail file in this cell strain source are seen " recombinant human IFN-monoclonal antibody hybridoma cell is built strain research ", the Shanghai Journal of Immunology, 1986 the 6th the 6th phases the 35th～43 of volume, RPMI-1640 nutrient solution (available from GIBCO company) is cultivated.In mouse peritoneal injection 0.5ml pristane (available from SIAMA company), injection back inoculation hybridoma in the 3rd week, every abdominal injection 1ml (contains 1.0 * 10 7Individual cell/ml), plant back 10 days left and right sides temporal lesion volume maximums, can extract ascites this moment by the abdominal cavity, got 1 time every 1 ~ 3 day, and desirable 10 times, the institute's ascites of getting-40 ℃ preservation is standby.
(2) monoclonal antibody ascites is pressed bed volume 1/10 application of sample, after mouse ascites dilutes 4 times with cold PBS liquid, in 1.00 * 10 5Leave heart 30min, go precipitation, supernatant is slowly added purification column, with 0.01Mol/L pH7.4PBS liquid wash-out, flow velocity is 1ml/min, till elutant OD280＜0.02.Add 0.1Mol/L pH3.0 glycine-HCl damping fluid, flow velocity 1ml/min collects the composition that desorption is got off, immediately with 1Mol/L NaHCO3 neutralization, in order to avoid protein denaturation.
(3) with the 7Mol/L urea washing of two volumes, with 0.01Mol/L pH 7.4PB liquid or physiological saline washing, after the balance, can continue to use again.Preservation can add 0.02%NaN3 in 4 ℃ of preservations.Prevent freezing and dry and cracked.The albumen of purifying is dialysed to PBS ,-40 ℃ of preservations.The capable SDS PAGE of purifying protein gel electrophoresis analysis, visible purity reaches (Figure 18) more than 90%.
Embodiment 5 uses novel evolved immunoglobulin binding molecule ELISA method and detects specific antibody
1, LD5 molecule marker horseradish peroxidase (HRP)
Get 10mg HRP (available from SIAMA company) and add 1ml0.1mol/L sodium-acetate or water dissolution, fully mixing adds 0.08mol/L NaIO after about 5 minutes 4After aqueous solution 1.0ml mixes, put refrigerator interior 20 minutes, add 0.4mol/L ethylene glycol solution 0.5ml and stop oxidizing reaction, add 21%NaCL solution 0.3ml after 30 minutes, add the ice-cold dehydrated alcohol of 1.2ml (AR) precipitation hydroformylation enzyme again, the centrifugal supernatant that goes, after the precipitation enzyme washs once with 6ml 80% ice-cold alcohol solution dipping again, the centrifugal ethanol that inclines (as far as possible removing ethanol), the precipitation 0.05mol/L sodium carbonate buffer 2ml dissolving of PH9.6, add 2ml LD5 (about inner protein amount 20mg) then, stir, put in the refrigerator and spend the night, next day, taking-up added 10mg NaBH 4Mixing, add equivalent saturated ammonium sulphate enzyme conjugates after 3 hours, centrifugal, remove supernatant, precipitation is with the washing of 50% saturated ammonium sulphate once, centrifugal, remove supernatant again, precipitation is put in the dialysis tubing with ice-cold normal saline dialysis desalination (need change liquid 5 times) with 0.01mol/L PBS 3ml dissolving, mat is centrifugal to be removed if any precipitating, and supernatant is light brown and is enzyme conjugates.Add 60% glycerine PBS, packing is preserved standby.
2, the LD5 molecule ELISA with the HRP mark detects whose anti-HCV antibody
Buy commercial whose anti-HCV ELISA antibody assay kit (Shanghai Kehua Bio-technology Co., Ltd), the serum of 383 parts of hemodialysis patients is carried out the ELISA detection of whose anti-HCV antibody according to the specification sheets of test kit, different is that the mark of the enzyme in test kit diluent is reconfigured, and original enzyme complex is changed into the LD5 molecule of HRP mark.Be specially: add 100 μ L diluents in the detection plate hole of envelope antigen, add the detection serum of 10 μ L again, build the plate lid, put in 37 ℃ of water-baths, insulation 45min.Get rid of the serum in the plate hole, wash five times, get rid of raffinate in the clear opening, on filter paper, pat again and blot with washing lotion.The LD5 molecule (1mg/ml) of HRP mark was added in the enzymic-labelled antibody diluent (the healthy bovine serum of 0.02mol/L pH7.2PBS-0.05% tween 20-0.1% gelatinum-10%) by 1: 500, behind the mixing, every empty 100 μ L that add put in 37 ℃ of water-baths insulation 45min.Get rid of the enzyme complex liquid in the plate hole, wash five times, get rid of raffinate in the clear opening, on filter paper, pat again and blot with washing lotion.With 100 μ L micro suction dispensers, the TMB A liquid of every Kong Jiaxin preparation and each 100 μ L of B liquid, at room temperature lucifuge is reacted about 5～10min, with 2mol/L sulfuric acid termination reaction.Mark tester with enzyme, under wavelength 450nm, measure each hole OD value.All samples are measured according to the specification sheets of test kit with whose anti-HCV ELISA antibody assay kit (Shanghai Kehua Bio-technology Co., Ltd), and the measurement result of changing into the LD5 molecule of HRP mark with enzyme complex compares.The results are shown in Table 1, as calculated, the LD5 molecule ELISA detection whose anti-HCV detection of antibodies result who uses the HRP mark compares susceptibility with the detected result of commercial test kit and reaches 99.3%, specificity reaches 100%, total coincidence rate reaches 99.7%, illustrates that the LD5 molecule of HRP mark has reached the detection requirement of commercialization fully.
The LD5 molecule ELISA of table 1 HRP mark detects the comparison of whose anti-HCV antibody and commercial test kit detected result
Positive (section China) Negative (section China)
Positive (LD5) 145 0 145
Negative (LD5) 1 237 238
146 237 383
Embodiment 6 uses novel evolved immunoglobulin binding molecule immunochromatographyassay assay specific antibody
1, trisodium citrate reduction method prepares gold sol
Get 0.01% aqueous solution of chloraurate 100ml and be heated to and boil, stir and accurately add 1% trisodium citrate aqueous solution 0.7ml down, flavous aqueous solution of chloraurate became red-purple in 2 minutes, continued to boil 15 minutes, and the cooling back returns to original volume with distilled water.
2, LD5 mark Radioactive colloidal gold
1. regulate gold sol to required pH6.0 with 0.1mol/L K2CO3 or 0.1mol/L HCl.
2. in the 100ml gold sol, add 2～3ml LD5 albumen (10mg/ml), stirred 2～3 minutes.
3. add 5ml 1%PEG20000 solution.
4. in centrifugal 30～60 minutes of 10000～100000g (selecting different centrifugal conditions according to size), the careful suction removed supernatant liquor (must guard against and topple over).
5. precipitation is suspended in certain volume and contains in the damping fluid of 0.2～0.5mg/ml PEG20000, after the centrifugation, recover with same damping fluid again, concentration is advisable about with A1cm/540nm=1.5, and is anticorrosion with the 0.5mg/ml sodium azide, puts 4 ℃ of preservations.
3, the preparation of immune chromatograph testing strip
Test strip is made up of water-absorption fiber, nitrocellulose filter and absorbent filter three parts.Water-absorption fiber partly is attached with the colloid gold immune mixture; On nitrocellulose filter, use HCV antigen (1.5mg/L) and human IgG (1mg/L) line (1mm is wide), respectively as detection line and control line, dry, with 50g/L BSA sealing 2h, with 0.01mol/L PBS washing 3 times, stick at successively after the drying on the white plastic sheet (upholder), be cut into the strip of 0.5cm * 10cm, add the siccative sealing and preserve.The whose anti-HCV immune chromatograph testing strip of preparation is seen Figure 19.
4, anti-HCV detects
0.1～0.2mL serum point is added in the electric sample hole that test strip contains an end of golden marker to get final product.The result judges: two red stripes to occur on the test strip nitrocellulose filter is the anti-Hp-CagA IgG positive; A red stripes (near the absorbent filter end, control line) only appears negative. and if losing efficacy appears then is considered as reagent in the redfree band.The result determines the time: the strong positive sample can be seen red colloid gold particle at the detection line place and assemble in 5min; Weak positive sample needs 10min to determine approximately.With this whose anti-HCV immune chromatograph testing strip the serum of 383 parts of hemodialysis patients is carried out the whose anti-HCV antibody test, and compare with commercial whose anti-HCV ELISA antibody test reagent (Shanghai Kehua Bio-technology Co., Ltd), the results are shown in Table 2, as calculated, immune chromatograph testing strip detects whose anti-HCV detection of antibodies result and compares with the detected result of commercial test kit, susceptibility reaches 94.5%, specificity reaches 99.2%, total coincidence rate reaches 97.4%, illustrates that the whose anti-HCV immune chromatograph testing strip of LD5 preparation has reached the detection requirement of practicability fully.
The whose anti-HCV immune chromatograph testing strip of table 2 LD5 preparation and the comparison of commercial test kit detected result
Positive (section China) Negative (section China)
Positive (LD5 chromatography strip) 138 2 140
Negative (LD5 chromatography strip) 8 235 243
146 237 383
Sequence table
＜110〉Shanghai Runlong Biology Science and Technology Co., Ltd
＜120〉a kind of evolved immunoglobulin binding molecule, its preparation method and application
<130> PCNRL060599
<160> 2
<170> PatentIn version 3.1
<210> 1
<211> 346
<212> PRT
<213> Artificial sequence
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<223> misc_feature
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Lys Glu Lys Thr Pro Glu Glu Pro Lys Glu Glu Val Thr Ile Lys Ala
1 5 10 15
Asn Leu Ile Tyr Ala Asp Gly Lys Thr Gln Thr Ala Glu Phe Lys Gly
20 25 30
Thr Phe Glu Glu Ala Thr Ala Glu Ala Tyr Arg Tyr Ala Asp Leu Leu
35 40 45
Ala Lys Glu Asn Gly Lys Tyr Thr Val Asp Val Ala Asp Lys Gly Tyr
50 55 60
Thr Leu Asn Ile Lys Phe Ala Gly Glu Leu Ala Asp Ala Gln Gln Asn
65 70 75 80
Asn Phe Asn Lys Asp Gln Gln Ser Ala Phe Tyr Glu Ile Leu Asn Met
85 90 95
Pro Asn Leu Asn Glu Ala Gln Arg Asn Gly Phe Ile Gln Ser Leu Lys
100 105 1l0
Asp Asp Pro Ser Gln Ser Thr Asn Val Leu Gly Glu Ala Lys Lys Leu
115 120 125
Asn Glu Ser Gln Ala Pro Lys Glu Leu Lys Glu Lys Thr Pro Glu Glu
130 135 140
Pro Lys Glu Glu Val Thr Ile Lys Ala Asn Leu Ile Tyr Ala Asp Gly
145 150 155 160
Lys Thr Gln Thr Ala Glu Phe Lys Gly Thr Phe Glu Glu Ala Thr Ala
165 170 175
Glu Ala Tyr Arg Tyr Ala Asp Leu Leu Ala Lys Glu Asn Gly Lys Tyr
180 185 190
Thr Val Asp Val Ala Asp Lys Gly Tyr Thr Leu Asn Ile Lys Phe Ala
195 200 205
Gly Glu Leu Ala Asp Ala Gln Gln Asn Asn Phe Asn Lys Asp Gln Gln
210 215 220
Ser Ala Phe Tyr Glu Ile Leu Asn Met Pro Asn Leu Asn Glu Ala Gln
225 230 235 24O
Arg Asn Gly Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Thr
245 250 255
Asn Val Leu Gly Glu Ala Lys Lys Leu Asn Glu Ser Gln Ala Pro Lys
260 265 270
Glu Leu Lys Glu Lys Thr Pro Glu Glu Pro Lys Glu Glu Val Thr Ile
275 280 285
Lys Ala Asn Leu Ile Tyr Ala Asp Gly Lys Thr Gln Thr Ala Glu Phe
290 295 300
Lys Gly Thr Phe Glu Glu Ala Thr Ala Glu Ala Tyr Arg Tyr Ala Asp
305 310 315 320
Leu Leu Ala Lys Glu Asn Gly Lys Tyr Thr Val Asp Val Ala Asp Lys
325 330 335
Gly Tyr Thr Leu Asn Ile Lys Phe Ala Gly
340 345
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aaagaaaaaa caccagaaga accaaaagaa gaagttacta ttaaagcaaa cttaatctat 60
gcagatggaa aaacacaaac agcagaattc aaaggaacat ttgaagaagc aacagcagaa 120
gcatacagat atgctgactt attagcaaaa gaaaatggta aatatacagt agacgttgca 180
gataaaggtt atactttaaa tattaaattt gctggagagc tcgctgatgc gcaacaaaat 240
aacttcaaca aagatcaaca aagcgccttc tatgaaattt tgaacatgcc taacttaaac 300
gaagcgcaac gcaatggttt cattcaaagt cttaaagacg atccaagcca aagcactaac 360
gttttaggtg aagctaaaaa attaaacgaa tctcaagcac cgaaagagct caaagaaaaa 420
acaccagaag aaccaaaaga agaagttact attaaagcaa acttaatcta tgcagatgga 480
aaaacacaaa cagcagaatt caaaggaaca tttgaagaag caacagcaga agcatacaga 540
tatgctgact tattagcaaa agaaaatggt aaatatacag tagacgttgc agataaaggt 600
tatactttaa atattaaatt tgctggagag ctcgctgatg cgcaacaaaa taacttcaac 660
aaagatcaac aaagcgcctt ctatgaaatt ttgaacatgc ctaacttaaa cgaagcgcaa 720
cgcaatggtt tcattcaaag tcttaaagac gatccaagcc aaagcactaa cgttttaggt 780
gaagctaaaa aattaaacga atctcaagca ccgaaagagc tcaaagaaaa aacaccagaa 840
gaaccaaaag aagaagttac tattaaagca aacttaatct atgcagatgg aaaaacacaa 900
acagcagaat tcaaaggaac atttgaagaa gcaacagcag aagcatacag atatgctgac 960
ttattagcaa aagaaaatgg taaatataca gtagacgttg cagataaagg ttatacttta 1020
aatattaaat ttgctgga 1038

Claims (28)

1. the evolved immunoglobulin binding molecule of a reorganization is characterized in that, it is albumen or its conservative property variant protein with the aminoacid sequence shown in the SEQ ID NO:1.
2. evolved immunoglobulin binding molecule as claimed in claim 1 is characterized in that, it has the aminoacid sequence shown in the SEQ ID NO:1.
3. isolating polynucleotide is characterized in that, its coding claim 1 or 2 described albumen.
4. polynucleotide as claimed in claim 3 is characterized in that, it has the nucleotide sequence shown in the SEQ ID NO:2.
5. an expression vector is characterized in that, the expression regulation sequence that it contains claim 3 or 4 described polynucleotide and links to each other with this polynucleotide sequence operability.
6. carrier as claimed in claim 5 is characterized in that, described carrier is a prokaryotic vector.
7. carrier as claimed in claim 5 is characterized in that, described carrier is PET-32a (+).
8. a host cell is characterized in that, it is transformed by the described expression vector of arbitrary claim among the claim 5-7.
9. host cell as claimed in claim 8 is characterized in that described host cell is a prokaryotic cell prokaryocyte.
10. host cell as claimed in claim 9 is characterized in that described prokaryotic cell prokaryocyte is intestinal bacteria.
11. host cell as claimed in claim 10 is characterized in that described intestinal bacteria are BL21-TRXB.
12. the preparation method of the evolved immunoglobulin binding molecule of a reorganization, it is characterized in that, this method comprises: (1) cultivates the described host cell of arbitrary claim among the claim 8-11 being fit to express under the condition of evolved immunoglobulin binding molecule; (b) from culture, isolate and have immunoglobulin (Ig) in conjunction with active polypeptide.
13. claim 1 or 2 described evolved immunoglobulin binding molecules are used for external binding domain-immunoglobulin.
14. evolved immunoglobulin binding molecule purposes as claimed in claim 13 is characterized in that, evolved immunoglobulin binding molecule and external binding domain-immunoglobulin bonded mode are dibit point binding pattern.
15. evolved immunoglobulin binding molecule purposes as claimed in claim 14 is characterized in that, described dibit point binding pattern is the binding pattern in evolved immunoglobulin binding molecule and immunoglobulin kappa light chain and these two sites of VH3 heavy chain.
16. as the described application of the arbitrary claim of claim 13-15, wherein said immunoglobulin (Ig) is selected from one or more among IgG, IgM, IgA, IgG Fab or the IgG Fc.
17. application as claimed in claim 16, wherein said IgG, IgM, IgA, IgG Fab or IgG Fc derive from the people.
18. as the described application of the arbitrary claim of claim 13-15, wherein said immunoglobulin (Ig) is scFv.
19. as the described application of arbitrary claim in claim 13-15 or the claim 17, it is characterized in that, evolved immunoglobulin binding molecule be used for the external immunology detection of antibody.
20. application as claimed in claim 16 is characterized in that, evolved immunoglobulin binding molecule is used for the external immunology detection of antibody.
21. application as claimed in claim 18 is characterized in that, evolved immunoglobulin binding molecule is used for the external immunology detection of antibody.
22. application as claimed in claim 19 is characterized in that described immunology detection is enzyme linked immunological absorption or immunochromatography or immunohistochemical methods.
23., it is characterized in that described immunology detection is enzyme linked immunological absorption or immunochromatography or immunohistochemical methods as claim 20 or 21 described application.
24., it is characterized in that evolved immunoglobulin binding molecule is used for antibody purification as the described application of arbitrary claim in claim 13-15 or the claim 17.
25. application as claimed in claim 16 is characterized in that evolved immunoglobulin binding molecule is used for antibody purification.
26. application as claimed in claim 18 is characterized in that evolved immunoglobulin binding molecule is used for antibody purification.
27. application as claimed in claim 24, wherein said antibody are genetic engineering antibody or natural antibody or monoclonal antibody.
28. as claim 25 or 26 described application, wherein said antibody is genetic engineering antibody or natural antibody or monoclonal antibody.
CNB2006101186944A 2006-11-23 2006-11-23 Evolved immunoglobulin binding molecule, and its preparation method and uses Expired - Fee Related CN100486993C (en)

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102115497A (en) * 2009-12-30 2011-07-06 中国人民解放军第二军医大学 Human IgA immunoglobulin combination molecule having intramolecular affinity effect
CN105153309A (en) * 2014-06-16 2015-12-16 中国人民解放军第二军医大学 Evolved immunoglobulin binding molecule D-C-G3 and preparation method and application thereof
CN105524173A (en) * 2016-01-28 2016-04-27 东南大学 VHH (variable domain of heavy chain of heavy-chain) antibody for humanized antibody Fc fragment and application of VHH antibody
CN109402036A (en) * 2018-11-07 2019-03-01 河北科技师范学院 The application of ox source Salmonella enteritidis trxB gene delection
CN110907436A (en) * 2019-12-04 2020-03-24 浙江李子园食品股份有限公司 Chemiluminescence immunoassay kit and method for milk allergen
CN113302198A (en) * 2019-01-24 2021-08-24 日本特殊陶业株式会社 Immunoglobulin purification method and immunoglobulin purification apparatus, and immunoglobulin production method and immunoglobulin production apparatus
CN115820704A (en) * 2022-12-22 2023-03-21 武汉爱博泰克生物科技有限公司 Recombinant plasmid for expressing protein L, application thereof and expression method of recombinant protein L

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102115497A (en) * 2009-12-30 2011-07-06 中国人民解放军第二军医大学 Human IgA immunoglobulin combination molecule having intramolecular affinity effect
CN105153309A (en) * 2014-06-16 2015-12-16 中国人民解放军第二军医大学 Evolved immunoglobulin binding molecule D-C-G3 and preparation method and application thereof
CN105524173A (en) * 2016-01-28 2016-04-27 东南大学 VHH (variable domain of heavy chain of heavy-chain) antibody for humanized antibody Fc fragment and application of VHH antibody
CN109402036A (en) * 2018-11-07 2019-03-01 河北科技师范学院 The application of ox source Salmonella enteritidis trxB gene delection
CN113302198A (en) * 2019-01-24 2021-08-24 日本特殊陶业株式会社 Immunoglobulin purification method and immunoglobulin purification apparatus, and immunoglobulin production method and immunoglobulin production apparatus
CN110907436A (en) * 2019-12-04 2020-03-24 浙江李子园食品股份有限公司 Chemiluminescence immunoassay kit and method for milk allergen
CN115820704A (en) * 2022-12-22 2023-03-21 武汉爱博泰克生物科技有限公司 Recombinant plasmid for expressing protein L, application thereof and expression method of recombinant protein L
CN115820704B (en) * 2022-12-22 2023-06-16 武汉爱博泰克生物科技有限公司 Recombinant plasmid for expressing protein L, application thereof and expression method of recombinant protein L

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