CN109721645A - A kind of albumin A of gene mutation and its application - Google Patents
A kind of albumin A of gene mutation and its application Download PDFInfo
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- CN109721645A CN109721645A CN201711470488.4A CN201711470488A CN109721645A CN 109721645 A CN109721645 A CN 109721645A CN 201711470488 A CN201711470488 A CN 201711470488A CN 109721645 A CN109721645 A CN 109721645A
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- Peptides Or Proteins (AREA)
Abstract
The invention belongs to genetic engineering applied technical field, albumin A and its application of a kind of gene mutation are specifically disclosed.The present invention relates to the business application new paragons of this kind of alkaline-resisting albumin A, utilize the amino or carboxylic group being present in this kind of albumin A, it is the irony microsphere particle surface of core by di-epoxide covalent coupling to metal ferriferous oxide, the hydroxyl magnetic bead particles of alkaline-resisting albumin A have directly been wrapped up using this surface, magnetic field is placed in conjunction with antibody, and magnetic force precipitates quickly, it is simple and quick just to be separated from liquid environment, enormously simplify the operation in laboratory and industrial separation antibody purification.
Description
Technical field
The invention belongs to genetic engineering applied technical fields, and in particular to a kind of albumin A of gene mutation and its application.
Background technique
Antibody drug is a kind of bio-pharmaceutical being concerned in recent years, has huge economic value and social value.
With the development of modern biotechnology, antibody engineering technology is constantly improve, and has become the main force of biotechnology industry, especially
It is occupied an important position in biotechnological pharmaceutics field.Currently, numerous biotech companies and pharmacy corporation progress into
Antibody drug researches and develops field.The antibody developed is mainly used for tumour, immunity disease, organ transplant, infectious diseases, painstaking effort
The treatment of pipe disease etc. and the diagnosis of disease.
As therapeutic antibodies drug is in a large amount of appearance of medical field, production technology also starts the weight by people
Depending on.Large-scale production for antibody drug, it is however generally that, it is that surrounding is entered in the cell or by secretion by cell culture
Culture medium in productive target antibody.Since the process of culture cell needs to be added sugar in the medium, amino acid, growth because
Son, it is therefore necessary to which separation reaches enough purity from culture medium and other cellular components by antibody, can be only used for human body and controls
It treats.The antibody purification mode most generallyd use now is affinity chromatography, it is advantageous that simple and quick, selectivity is by force, early stage is first
The step of carrying out affinity purification, subsequent purification can be significantly reduced.
Staphylococcus aureus protein A (Staphylococcus aureus protein A, SPA) is that one kind can be with
The albumen of immunoglobulin (antibody is also referred to as in the patent application text) specific binding, can be used as aglucon for affine layer
Analysis.Due to protein A affinity chromatography purification efficiency with higher, oneself is through becoming the necessary of industrial production therapeutic monoclonal antibodies
Step.But albumin A affinity media is expensive, causes antibody producing increased costs.If albumin A needed for production is pure
Changing chromatography media may be reused, then can be substantially reduced the production cost of antibody.Since Protein A Chromatography medium purifies every time
Antibody can all leave the albumen not eluted, these residual proteins may include the substance being harmful to the human body, such as virus, endogenous toxic material
Element, so Protein A Chromatography medium must be cleaned when reusing.Present most effective recoverin A chromatography media
Cleaning mode is the basic method referred to as situ cleaning (cleaning-in-place, CIP).Currently, most for CIP step
Effective cleaning agent is NaOH (0.1-0.5mol/L), can efficiently remove the foreign protein combined closely, lipid, nucleic acid, micro-
The pollutants such as biology reuse Protein A Chromatography medium.Impurity can be effectively removed in the cleaning way of this harshness, but
It is to be likely to damage chromatography media.Native protein A not strong basicity resisting is not able to satisfy above-mentioned requirements, therefore is badly in need of establishing alkaline-resisting egg
The technical system of white A, to meet currently the needs of to alkaline-resisting albumin A.The present invention is exactly at us originally to general proteins A gene
On the basis of engineering expression, the mutation of multiple nucleotide sites is carried out to the gene order of albumin A, changes general proteins A amino acid
Sequence becomes alkali resistance albumin A amino acid sequence, and optimizes alkaline-resisting albumin A and express, recycle, isolate and purify process, to meet
Its application in laboratory research and industrial industrialization production.
Summary of the invention
Goal of the invention: the object of the present invention is to provide a kind of albumin A of gene mutation and its applications, by general proteins A base
Because making a kind of rite-directed mutagenesis, high alkali resistance albumin A and its application are become, the albumin A of one side can be firmly bound to antibody
Other regions of molecule in addition to complementary determining region (CDR), can be coupled on the ligand of solid phase carrier, be used for separation antibody,
Albumin A after this kind of mutation can keep chemical stability in strong basicity environment, can be used as the harsh original of chromatography ligand tolerance
Position cleaning condition, another aspect are related to using Protein A ligand progress laboratory and industrial separation antibody purification and with alkali again
Raw process.
The alkali resistance of albumin A of the invention is transformed, and needs to be promoted its IgG-Fc binding using protein engineering method
The alkali resistant stability in region.Albumin A contains the IgG binding structural domain (E, D, A, B and C) there are five very high homology, B knot therein
Structure domain chemical stability is preferable, carries out the mutation of coding nucleotide and amino acid residue to it to construct the better egg of stability
White A analog.B structure domain includes 58 amino acid residues, containing there are two annular region interconnected and three are antiparallel
Helix domain.Its binding ability is mainly that 11 amino acid residues of first and second helix domains work, the
Three helix domains and second annular region do not work.Asparagine residue (Asn) is one and is highly dependent on albumen
The amino acid of matter sequence and space conformation implements the amino that mutation is a common raising protein chemistry stability to it
Sour modifying and decorating.It is selected based on the above idea present invention to three asparagines and other three ammonia in the B structure domain of albumin A
Base acid residue has carried out rite-directed mutagenesis, and the new B structure domain of acquisition is named as RZ structural domain.RZ structural domain is subjected to head and the tail phase
The even polymer target fragment of available alkaline-resisting albumin A.RZ structural domain of the invention is that the genetic engineering in B structure domain is derivative
Object has higher chemical stability and most-often used man-made protein A than natural B structure.
The nucleotide coding of RZ structural domain or its polymer target gene fragment is modified to T4 phage expression preference
Code, and with the pT4-T7 expression plasmid containing T7 promoter or the T4 expression plasmid containing T4 bacteriophage IPIII promoter
PSocN is connected, and is transferred to E.coli host cell, and large-scale culture is expressed, isolated and purified, and obtains alkaline-resisting albumin A.
The purpose of the present invention is achieved through the following technical solutions: the preferred code building of T4 bacteriophage will be used before inventor's several years
B structure domain (58 amino acid residues length) in 5 structural domain E-D-A-B-C genes of general proteins A is implemented to following 6
The coding positional mutation of amino acid residue: A1V, N3A, N6D, N23T, G29A, P57I, new sequence designations are RZ structural domain.
The purpose of the present invention is being directed to the above-mentioned deficiency of the prior art, the egg with high alkaline-resisting characteristic of a kind of mutation is provided
White A.Such albumin A can be used or by RZ- structural domain without using connexon, be combined into multimeric protein, such as dimer,
Tripolymer, the tetramer, pentamer, six aggressiveness and more polymer, or by RZ- structural domain itself or its condensate, connect other
The multifunctional protein compound that albumen is formed.Therefore one belongs to albumin A of the invention itself or is formed with other function albumen
Polymer, also belong to another aspect of the present invention.
The albumin A of a kind of gene mutation, amino acid sequence as shown in SEQ ID NO.1, or with SEQ ID NO.1 institute
That shows that amino acid sequence has 99% an or more homology being capable of all amino acid sequences of binding antibody, wherein described with antibody knot
Conjunction region is 7-57 amino acid sequence.
The nucleotide sequence of the coding albumin A.
Present invention asparagine (Asn) residue poor to alkali tolerance in the B structure domain of albumin A carries out mutation modification,
The strong alkali environment of PH13-14 can be resistant to by making it both, other regions except complementary antibody decision area can also be firmly combined.This
The protein conformation of the antibody binding domain of the modified albumin A of kind is mainly made of 3 bursts of α spirals, and is folded into a kind of three spiral shells
Revolve binding structure.It can be by the region Fc of the surface amino groups acid residue binding antibody of α spiral 1 and 2, or passes through the surface of α spiral 2 and 3
Incomplementarity on the region residue binding antibody Fab determines area.1,2,3 surface hydrophobic residue of α spiral can be in helical bundle simultaneously
It is centrally formed a hydrophobic core, α spiral 1,2,3 can be closely combined together, maintain the rock-steady structure of albumen.With it
He is intolerant to basonuclin the difference is that when this kind of albumin A is placed in the alkaline environment of PH13-14, although maintaining protein stabilized knot
The hydrogen bond of structure disappears, and α spiral 1,2,3 can unfold extension, but strong hydrophobic ability combines α spiral 1,2,3 closely
And guarantee that its relative position does not have significant changes.When this kind of albumin A is placed in the neutral environment of PH7-8 again, α spiral
1,2,3 hydrogen bonds restore, therefore whole conformation can be restored to before being placed in alkaline environment, and other intolerant to basonuclin due to
Lack and maintain the stable hydrophobic core of protein structure, in the alkaline environment for being placed on PH13-14 after, protein structure can be thorough
Bottom is destroyed, and when being placed in the neutral environment of PH7-8 again, albumen entirety conformation can not be restored to being placed in alkaline environment
Before.This albumin A for having alkaline-resisting speciality and its polymer, which are coupled on solid phase carrier, can be used to isolate and purify antibody point
They are coupled upper marker and can be used to detect antibody molecule by son, while if ligand as chromatography media,
Chromatography media can be realized with the clean mode of alkali in situ and be regenerated.
RZ- structural domain list aggressiveness of the invention is made of 58 amino acid, and molecular weight is about 6kD, and 2 polymer molecular amounts are about
For 12kD, 3 polymer molecular amounts are about 18kD, and 4 polymer molecular amounts are about 24kD, and 5 polymer molecular amounts are about 30kD, 6 polymer moleculars
Amount is about 36kD, and single aggressiveness or polymer can be synthesized by chemiluminescent polypeptide or the mode of cell expression obtains.If adopted
The mode expressed with cell, it is necessary to which building carries the cell strain for encoding the gene of protein amino acid sequence first.Therefore it needs
It is carried out according to process below: obtaining the gene of coding albumin A amino acid sequence by way of gene chemical synthesis first, it is next
Step, by encode albumin A amino acid sequence genetic recombination into expression vector, can according to need at this time add in the carrier it is pure
Change label, screening label or indicative label, and then expression vector is steadily transferred in suitable cell, completes entire
The expression system of this kind of albumin A can be produced.Required albumin A (including single aggressiveness or poly with binding antibody molecule
Body) it can be obtained from culture medium or cell.It should be noted that expression needs of the albumin A in cell are closely controlled
System, therefore select extremely important for expressing the carrier of albumin A, it should have following characteristic: 1. expression vectors, which contain, to be opened
Mover or initial transcription site；2. expression vector contains the expression that operon is used to switch gene；3. expression vector contains ribose
Body binding site, 4. expression vectors contain the termination site of transcription and translation, and the stability of transcription and translation product can be improved.
Therefore the carrier for expressing albumin A can be recommended to have PET serial carrier (Novagen), pT4-T7 (Versatile
Bioscience), pSocN (Versatile Bioscience) etc..
The present invention relates to the nucleic acid sequence for encoding above-mentioned albumin A or its polymer, used wantonly million an ancient unit of weight of the inventor as
T4 bacteriophage in the United States Patent (USP) NO.7041441 (issuing on May 2006 time 9) of inventor has a preference for code mode, to nucleic acid
Sequence has carried out codon optimization, and the nucleic acid sequence after optimization avoids the generation of rare codon and the formation of secondary structure,
And it is synthesized in such a way that artificial gene synthesizes.Also use the matter containing T4 bacteriophage IPIII promoter in the patent
Grain expression technology improves the expression efficiency of alkaline-resisting albumin A and reduces production cost.Alkaline-resisting albumin A expression quantity of the invention, can join
See that 1 protein polyacrylamide gel electrophoresis SDS-PAGE of attached drawing schemes.
Joint efficiency of the alkaline-resisting albumin A prepared by the present invention to antibody, hence it is evident that the combination higher than general proteins A to antibody
Rate.Use the micro highly sensitive electrophoresis analyzer of Backman Coulter Inc. company P/ACEMDQ albumen capillary, analysis
With demonstrate the antibody binding capacity of the alkaline-resisting albumin A of the present invention, contrast, find with the antibody binding capacity of general proteins A
Antibody binding capacity average value (42+49)/2=45.5 of the alkaline-resisting albumin A of the present invention, hence it is evident that the antibody knot higher than general proteins A
Conjunction ability average value (30+32)/2=31, is higher by 46.77%, referring to attached drawing 3, Fig. 4, Fig. 5 and Fig. 6.
Using alkaline-resisting albumin A (RZ5) prepared by the present invention, compared with general proteins A (PA) and the combination energy of rabbit antibody IgG
Power effect.Using the different samples of equivalent RZ5 and PA, respectively with enough rabbit antibody IgG gel particles (Sepharose-IgG)
At pH7.4PBS buffered environment hybrid reaction 30 minutes, the albumin A-IgG compound of separation of polymeric was washed, 2.8 acetic acid of pH
Processing, particle external solution neutralize, and are splined on albumen capillary microelectrophoresis analytical column, analyze the egg released from IgG conjugate
White A, is shown in albumen wave spectrum scanning recorder, carries out antibody binding capacity analysis with it.
This kind of albumin A can according to need, and by utilizing its physical property, the various lock out operation of chemical property etc. are carried out
It isolates and purifies.Specifically, for example common protein precipitant processing (salting out method), centrifuge separation, osmotic pressure crush method, ultrasound
Wave is broken, ultrafiltration, gel filtration and adsorption chromatography, ion-exchange chromatography, affinity chromatography, the various liquid such as high-speed liquid chromatography
The combination etc. of phase chromatography, dialysis and these methods.In addition, the protein merged by expressing this albuminoid with affinity labeling,
Affinity purification can be carried out using the label.Affinity labeling described herein, for example, polyhistidine (such as 6 × HIS) label and
FLAG label.The purifying of this kind of albumin A can be carried out using these labels.
The activity of this kind of albumin A binding antibody can pass through ELISA measuring.Such as this kind of albumin A is fixed on
On phase carrier, the antibody that enzyme label is then added is adsorbed on it on albumin A, makes to be formed on solid phase carrier with the method for washing
The complex of antibody and albumin A is separated with other substances in liquid, and the albumin A on such solid phase carrier forms compound with antibody
The amount of body and the amount of enzyme are in one to one ratio.After the reaction substrate of enzyme is added, substrate becomes color products by enzymatic, in this way
The amount for the antibody that albumin A combines can carry out qualitative and quantitative analysis according to the depth of colour generation.
The alkali chemical stability of this kind of albumin A can also easily be confirmed by those skilled in the art.Such as by this
Albuminoid A, which is first immersed in the NaOH of 0.1M concentration, continues 6 hours, albumen capillary microelectrophoresis analysis as described above
Method or ELISA experiment, measure the activity of its binding antibody, how still can be very good the activity for keeping binding antibody, that
Then prove that this kind of albumin A is stable in strong alkali environment.The amino or carboxyl being present in this kind of albumin A are for example utilized again
Group, by di-epoxide, epichlorohydrin, cyanogen bromide, this kind of albumin A is coupled to solid by the coupling agents such as N- hydroxysuccinimide
(Sepharose4B) and chromatographic column is made on phase carrier, then excessive antibody applied molecule, then slow with the phosphate of PH8.0
Fliud flushing washing, is then eluted with the glycine buffer of PH3.0, and the amount for measuring the antibody of elution later carrys out total appearance of measuring column
Amount.Between each circulation, column is handled with the basic method processing of the In-Situ Cleaning of 0.1M NaOH composition, at tens
After circulation, if the total capacity of column does not reduce, then chemical property of this kind of albumin A under strong alkali environment is proved very
Stablize, is highly suitable as the ligand of antibody affinity purification.
The list of albumin A of the present invention, the encoding gene of polymer or albumin A derivative and nucleotides sequence are listed in point
From the application in, purifying and/or detection antibody, such as PCR.
Beneficial outcomes of the invention are as follows: alkaline-resisting albumin A and its polymer and derivative provided by the invention can be firmly combined
To antibody molecule in addition to complementary determining region (CDR) other regions, can be used as and be coupled to the ligand of solid phase carrier for separating
Antibody.This kind of albumin A can keep chemical stability in PH13-14 strong alkali environment, be resistant to as chromatography ligand severe
The situ cleaning condition at quarter, thus increase and applied in purifying and/or detection antibody.
The present invention relates to this kind of albumin As to be chemically modified obtained all energy securely through N-terminal or C-terminal or side-chain radical
The protein derivatives of binding antibody.N- acetyl polypeptide is obtained after being acetylation such as N-terminal amino, and N- rouge is obtained after fatty-acylation
Fat acylating acid polypeptide.For another example C- carboxyl end group is converted into the available polypeptide amide of amide, is converted into ester and obtains polypeptide ester, conversion
Polypeptide thioesters are obtained for thioesters.Derivatization modification is to the acid-base property for adjusting original albumen, stability, dissolubility, reactivity and
Bioactivity plays a significant role.The present invention provides a cysteine residues in the C-terminal of albumin A, and albumin A can pass through sulphur
Ehter bond is mutually coupled with carrier, and this coupling method significantly improves alkaline-resisting albumin A to the binding ability of antibody.
The present invention relates to the commercial applications of this kind of alkaline-resisting albumin A, the detection of purifying and antibody including antibody.Antibody is pure
The application for changing aspect includes a kind of isolated method of the affine analysis of layer, wherein by be adsorbed in albumin A or polymer as described above come
At least one target compound is separated from complex matrices, is a kind of widely used isolation technics.Specific steps include allow containing
The sample solution of antibody flows through Protein A Chromatography medium.In this step, the other components in solution will lead in the clear substantially
It crosses, and antibody is adsorbed on chromatography media.Medium is washed out, such as with phosphate buffer, to remove remaining non-spy
The impurity that the opposite sex combines can also pass through the washing step using alkaline reagent as previously described.In next step,
Allow eluent to flow through chromatography media so that the antibody elution of absorption get off, elution be usually by changing PH, ionic strength or
Emulative substance is added to realize.The application of the context of detection of antibody includes a kind of method for detecting antibody, wherein wrapping
Progress enzyme label, chemiluminescent labeling or isotope labelling first on albumin A are included, antibody is then adsorbed by albumin A
On, it can be realized antibody visualization analysis.
The present invention relates to the business application new paragon of this kind of alkaline-resisting albumin A, using the amino being present in this kind of albumin A or
Carboxylic group is the irony microsphere particle surface of core by di-epoxide covalent coupling to metal ferriferous oxide, directly makes
The hydroxyl magnetic bead particles that alkaline-resisting albumin A has been wrapped up with this surface, magnetic field is placed in conjunction with antibody, and magnetic force precipitates quickly, letter
It is single quickly just to be separated from liquid environment, enormously simplify the operation in laboratory and industrial separation antibody purification.
Detailed description of the invention
Fig. 1 SDS-PAGE detects expression of the albumin A in Escherichia coli, and wherein swimming lane M is molecular weight of albumen Marker；Swimming
Road 1-2, using pT4-T7-PA carrier, the table of 5 structural domains (EDABC) general proteins A (PA) under the IPTG induction of 1 and 0.5mM
It reaches；Swimming lane 3-4 induces the expression of alkaline-resisting albumin A (RZ5) in the IPTG of 1 and 0.5mM using pT4-T7-RZ5 carrier；Swimming lane 5,
The control of plasmid-free host strain；Swimming lane 6, using pSocN-PA carrier, the expression (whole bacterial protein) of PA after placing 1 week；Swimming lane 7, makes
With pSocN-RZ5 carrier, the expression (whole bacterial protein) of RZ5 after placing 1 week；Swimming lane 8 is placed 1 week using pSocN-RZ5 carrier
The expression (supernatant protein) of RZ5 afterwards；Swimming lane 9, the control of plasmid-free host's bacterial lysate；
Fig. 2 SDS-PAGE NI-NTA column purification albumin A, wherein swimming lane M is molecular weight of albumen Marker；Swimming lane 1 uses
PT4-T7-RZ5 carrier, the expression of RZ5 under the IPTG induction of 0.5mM；Swimming lane 2 is not added using pT4-T7--RZ5 carrier
The negative control of IPTG inducing expression；Swimming lane 3, using pSocN-RZ5 carrier, the expression of RZ5 after placing 1 week；Swimming lane 4 uses
PSocN-PA carrier, the expression of PA after placing 1 week；Swimming lane 5 expresses RZ5, NI-NTA column purification using pT4-T7-RZ5 carrier
RZ5；Swimming lane 6 expresses PA, NI-NTA column purification PA using pT4-T7-PA carrier；Swimming lane 7 is expressed using pSocN-RZ5 carrier
RZ5, NI-NTA column purification RZ5；Swimming lane 8 expresses PA, NI-NTA column purification PA using pSocN-PA carrier；
Fig. 3 pT4-T7-PA carrier expresses PA and rabbit antibody binding ability, in conjunction with amount of antibody adsorptive value be 30；
Fig. 4 pSocN-PA carrier expresses PA and rabbit antibody binding ability, in conjunction with amount of antibody adsorptive value be 32；
Fig. 5 pT4-T7-RZ5 carrier expresses RZ5 and rabbit antibody binding ability, in conjunction with amount of antibody adsorptive value be 42；
Fig. 6 pSocN-RZ5 carrier expresses RZ5 and rabbit antibody binding ability, in conjunction with amount of antibody adsorptive value be 49；
Binding ability of Fig. 7 albumin A in conjunction with human antibody, wherein swimming lane M is molecular weight of albumen Marker；Swimming lane 1, no matter
Grain host's bacterial lysate control；Swimming lane 2 has plasmid pT4-T7-RZ5 carrier host strain, expresses without the background of IPTG induction；Swimming
Road 3, using pT4-T7-RZ5 carrier, the expression of RZ5 under the IPTG induction of 0.5mM；Swimming lane 4, Westernblotting is without matter
Grain host's bacterial lysate control；Swimming lane 5, Westernblotting detect the RZ5 and people of pT4-T7-RZ5 vector background expression
The combination situation of IgG；Swimming lane 6, Westernblotting detection using pT4-T7-RZ5 carrier IPTG inducing expression RZ5 with
The combination situation of human IgG；
The test of Fig. 8 albumin A alkali resistance, wherein swimming lane M is molecular weight of albumen Marker；Swimming lane 1, is carried using pSocN-PA
Body, the expression of general proteins A after placing 1 week；Swimming lane 2, using pSocN-RZ5 carrier, the expression of RZ5 after placing 1 week；Swimming lane 3,
Using pT4-T7-PA carrier, in the expression of the IPTG induction PA of 1mM；Swimming lane 4, using pT4-T7-RZ5 carrier, 0.5mM's
The expression of IPTG induction RZ5；Swimming lane 5, using pT4-T7-RZ5 carrier, in the expression of the IPTG induction RZ5 of 1mM；Swimming lane 6, nothing
The control of plasmid host strain；Swimming lane 7, using pT4-T7-RZ5 carrier, not plus IPTG induces the expression of lower RZ5；Swimming lane 8 uses
PSocN-PA carrier is expressed, the PA after NI-NTA column purification, after being handled 6 hours with 0.1MNaOH；Swimming lane 9, uses pT4-T7-
PA carrier is expressed, the PA after NI-NTA column purification, after being handled 6 hours with 0.1MNaOH；Swimming lane 10, is carried using pSocN-RZ5
Body surface reaches, the RZ5 after NI-NTA column purification, after being handled 6 hours with 0.1MNaOH；Swimming lane 11 uses pT4-T7-RZ5 carrier
It expresses, the RZ5 after NI-NTA column purification, after being handled 6 hours with 0.1MNaOH.
Specific embodiment
In the following with reference to the drawings and specific embodiments, the present invention is furture elucidated.
Embodiment 1 constructs alkaline-resisting albumin A (RZ5) and general proteins A (PA) expression vector
The nucleic acid sequence of general proteins A (PA) according to N-terminal with 6 histidine residues, it is close to carry out T4 bacteriophage to it
The preference of numeral sexually revises, and the sequence after change is as shown in SEQ ID NO.5；According to the Preference of T4 phage-code and
The tendency for avoiding the code area mRNA from forming secondary structure designs the RZ that coding N-terminal has the alkaline-resisting albumin A of 6 histidine residues
5 aggressiveness of structural domain (RZ5) gene order, as shown in SEQ ID NO.7；Its corresponding amino acid sequence such as SEQ ID NO.6 institute
Show, referred to as RZ5.According to SEQ ID NO.5 and SEQ ID NO.7 gene order, by Suzhou, Jin Weizhi company synthesizes full length gene,
And sequence verification.
Using the RZ5 DNA after sequence verification as template, respectively with primer 1 (SEQ ID NO.8) and primer 2 (SEQ ID
It NO.9) is primer；Using the PADNA after sequence verification as template, respectively with primer 1 (SEQ ID NO.8) and (the SEQ ID of primer 3
It NO.10) is primer；Pass through the coded sequence of PCR amplification RZ5 and PA using PrimeSTAR archaeal dna polymerase (Takara company),
Reaction cycle is 95 DEG C of 10s, 55 DEG C of 15s, 72 DEG C of 60s, carries out 30 circulations altogether, finally keeps the temperature 10 minutes in 72 DEG C, obtain
PCR reaction solution.Purification and recovery mesh will be carried out with plastic recovery kit (Tiangeng biology) after 1% agarose electrophoresis of PCR reaction solution
DNA fragmentation, target DNA fragment and carrier pSocN or pT4-T7 (Versatile Bioscience) after purification and recovery
NdeI and NotI digestion is used respectively, with method purification and recovery same as described above after 1% agarose electrophoresis.Segment after the recovery is used
T4-DNA ligase (Takara company) 16 DEG C connection overnight, connection product convert bacillus coli DH 5 alpha, be applied to containing
On the plate of 100mg/L ampicillin, 37 DEG C are incubated overnight.After growing clone, with positive gram of colony polymerase chain reaction (PCR) method screening
It is grand.The reaction system of bacterium colony PCR is identical as aforementioned system.Contain RZ5 gene through the positive colony that colony polymerase chain reaction (PCR) method screens
Carrier be respectively designated as pSocN-RZ5 and pT4-T7-RZ5；Carrier containing PA gene be respectively designated as pSocN-PA and
pT4-T7-PA。
2 alkaline-resisting albumin A (RZ5) of embodiment and general proteins A (PA) expression vector
By pSocN-RZ5, pT4-T7-RZ5 carrier containing RZ5 gene and pSocN-PA, pT4-T7- containing PA gene
PA carrier converts Escherichia coli HMS174 competent cell (Novage company) respectively.PSocN-RZ5 and pSocN-PA will be contained
The Escherichia coli HMS174 of carrier be applied to LB plating medium (10g/L tryptone, 5g/L yeast extract, 5g/L NaCl,
12g/L Agar and 100mg/L ampicillin) in 37 DEG C of progress overnight incubations, then it is placed at room temperature for 1 week.It is scraped after 1 week flat
Thallus is resuspended with 1ml PBS in all thallus on plate, with Ultrasonic Cell Disruptor (JY99-IIIBN, the limited public affairs of the new sesame biology in Ningbo
Department) smudge cells, release whole bacterial protein；Part clasmatosis liquid is removed, 12000RPM is centrifuged 10 minutes, draws supernatant protein
Liquid；Whole bacterial protein and supernatant protein carry out the SDS-PAGE of 10-15% gum concentration.As shown in Fig. 1, PA and RZ5 exist
30KD nearby detects a band.
By the HMS174 competent cell containing pT4-T7-RZ5 and pT4-T7-PA carrier.It is put into LB culture medium (10g/L
Tryptone, 5g/L yeast extract, 5g/LNaCl and 100mg/L ampicillin) in, it is cultivated at 37 DEG C, works as large intestine
When bacillus reaches logarithmic growth phase, the IPTG of addition 0.5mM or 1mM carries out inducible protein and expresses 4 hours, is received by centrifugation
Escherichia coli after collection induction.Take a small amount of thallus boil carry out cracking release whole bacterial protein, whole bacterial protein is subjected to 10-15%
The SDS-PAGE of gum concentration.As shown in Fig. 1, PA and RZ5 detects a band near 30KD.
The purifying of the affinity column chromatography of 3 general proteins A of embodiment and alkaline-resisting albumin A
Constant flow pump is rinsed well with distilled water, then glass chromatography column is rinsed well.About 10mlNi- is added in Xiang Zhuzhong
NTA (the raw work in Shanghai) dress column, gets off to column material whole natural sedimentation.Pass through the PBS buffer solution (50mM of constant flow pump 50ml
PH7.4 chromatographic column, flow velocity 2ml/min) are balanced；20ml bacteria breaking liquid (50mM PBS, pH7.4,0.5M NaCl) is used
0.45 μm of membrane filtration, loading, flow velocity 1ml/min；After loading is complete, chromatographic column is rinsed with PBS buffer solution (50mM pH7.4)
It is constant to absorbance, flow velocity 2ml/min；After washing, with elution buffer (10mM NaH2PO4,40mM
Na2HPO4500mM NaCl, 200mM Iminazole) start to elute, flow velocity 2ml/min, eluent is collected, 10- is carried out
The SDS-PAGE of 15% gum concentration is detected.As shown in Fig. 2, after PA and RZ5 are purified, SDS-PAGE electrophoresis showed molecular weight is big
The small single band for 30KD, purity reach 90% or more.
4 alkaline-resisting albumin A (RZ5) of embodiment and general proteins A (PA) and antibody binding capacity are detected
The different samples for making RZ5 and PA prepared with embodiment 3 take RZ5 the and PA protein solution of equivalent (0.5mg), point
Not with enough rabbit antibody IgG gel particles (Sepharose-IgG) at pH7.4PBS buffered environment hybrid reaction 30 minutes,
Albumin A-IgG the compound of separation of polymeric, washing, the processing of pH2.8 acetic acid, particle external solution neutralize, and it is micro- to be splined on albumen capillary
Electrophoretic analysis column is measured, the albumin A released from IgG conjugate is analyzed, is shown in albumen wave spectrum scanning recorder, is carried out with it
Antibody binding capacity analysis.Compare the binding ability of RZ5 and PA Yu rabbit antibody IgG.As a result: such as scheming attached 3, shown in 4,5,6, pT4-
Adsorptive value of the PA of T7-PA carrier expression in conjunction with rabbit antibody is 30；PSocN-PA carrier expresses suction of the PA in conjunction with rabbit antibody
Assignments are 32；It is 42 that pT4-T7-RZ5 carrier, which expresses adsorptive value of the RZ5 in conjunction with rabbit antibody,；PSocN-RZ5 carrier express RZ5 with
The adsorptive value that rabbit antibody combines is 49.Compared with the antibody binding capacity of PA, the antibody binding capacity average value of RZ5 is (42+
49)/2=45.5, hence it is evident that antibody binding capacity average value (30+32)/2=31 higher than PA.
The test of the alkali resistance of 5 alkaline-resisting albumin A (RZ5) of embodiment and general proteins A (PA)
Taking 100 μ L protein concentrations is RZ5 the and PA protein solution of 10mg/ml, is separately added into 100ul 0.1M NaOH,
It is handled under room temperature (25 DEG C) 6 hours, then carries out the SDS-PAGE of 10-15% gum concentration respectively.As a result: as shown in Fig. 8, PA
After 0.1M NaOH is acted on 6 hours, degrade, and RZ5 does not occur obvious degradation.
The binding ability of 6 alkaline-resisting albumin A (RZ5) of embodiment and general proteins A (PA) and human antibody (IgG) detect
By the HMS174 competent cell containing pT4-T7-RZ5 and pT4-T7-PA carrier.It is put into LB culture medium (10g/L
Tryptone, 5g/L yeast extract, 5g/L NaCl and 100mg/L ampicillin) in, it is cultivated at 37 DEG C, works as large intestine
When bacillus reaches logarithmic growth phase, the IPTG for adding 0.5mM carries out inducible protein and expresses 4 hours, by the way that induction is collected by centrifugation
Escherichia coli afterwards.Take a small amount of thallus boil carry out cracking release whole bacterial protein, by whole bacterial protein carry out 10-15% gum concentration
SDS-PAGE.Then be attached to it on nitrocellulose filter albumen progress electrotransfer, with human IgG (doctor moral biology) and
The anti-human secondary antibody of mouse of HRP label marks alkaline-resisting albumin A (RZ5) and general proteins A (PA), Western Blotting display to use
ECL kit (the green skies), concrete operation method refer to ECL kit specification.As a result: as shown in Fig. 7, SDS-PAGE electricity
Swimming display RZ5 detects a band near 30KD, and Western Blotting shows alkaline-resisting albumin A (RZ5) to human antibody
(IgG) it can specifically bind.
Sequence table
SEQ ID NO.1
The amino acid sequence of alkaline-resisting albumin A RZ structural domain
VDAKFDKEQQNAFYEILHLPNLTEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAIK
SEQ ID NO.2
The nucleotide sequence of alkaline-resisting albumin A RZ structural domain
GTTGATGCTAAATTTGATAAAGAACAACAAAATGCTTTTTATGAAATTTTACATTTACCTAATTTAACT
GAAGAACAACGTAATGCTTTTATTCAATCTTTAAAAGATGATCCTTCTCAATCTGCTAATTTATTAGCTGAAGCTAA
AAAATTAAATGATGCTCAAGCTATTAAA
SEQ ID NO.3
The amino acid sequence of the 5 alkaline-resisting albumin As of aggressiveness RZ structural domain (RZ5)
VDAKFDKEQQNAFYEILHLPNLTEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAIKVDAKFDKEQQN
AFYEILHLPNLTEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAIKVDAKFDKEQQNAFYEILHLPNLTEEQRNAF
IQSLKDDPSQSANLLAEAKKLNDAQAIKVDAKFDKEQQNAFYEILHLPNLTEEQRNAFIQSLKDDPSQSANLLAEAK
KLNDAQAIKVDAKFDKEQQNAFYEILHLPNLTEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAIK
SEQ ID NO.4
The nucleotide sequence of the 5 alkaline-resisting albumin As of aggressiveness RZ structural domain (RZ5)
GTTGATGCTAAATTTGATAAAGAACAACAAAATGCTTTTTATGAAATTTTACATTTACCTAATTTAACT
GAAGAACAACGTAATGCTTTTATTCAATCTTTAAAAGATGATCCTTCTCAATCTGCTAATTTATTAGCTGAAGCTAA
AAAATTAAATGATGCTCAAGCTATTAAAGTTGATGCTAAATTTGATAAAGAACAACAAAATGCTTTTTATGAAATTT
TACATTTACCTAATTTAACTGAAGAACAACGTAATGCTTTTATTCAATCTTTAAAAGATGATCCTTCTCAATCTGCT
AATTTATTAGCTGAAGCTAAAAAATTAAATGATGCTCAAGCTATTAAAGTTGATGCTAAATTTGATAAAGAACAACA
AAATGCTTTTTATGAAATTTTACATTTACCTAATTTAACTGAAGAACAACGTAATGCTTTTATTCAATCTTTAAAAG
ATGATCCTTCTCAATCTGCTAATTTATTAGCTGAAGCTAAAAAATTAAATGATGCTCAAGCTATTAAAGTTGATGCT
AAATTTGATAAAGAACAACAAAATGCTTTTTATGAAATTTTACATTTACCTAATTTAACTGAAGAACAACGTAATGC
TTTTATTCAATCTTTAAAAGATGATCCTTCTCAATCTGCTAATTTATTAGCTGAAGCTAAAAAATTAAATGATGCTC
AAGCTATTAAAGTTGATGCTAAATTTGATAAAGAACAACAAAATGCTTTTTATGAAATTTTACATTTACCTAATTTA
ACTGAAGAACAACGTAATGCTTTTATTCAATCTTTAAAAGATGATCCTTCTCAATCTGCTAATTTATTAGCTGAAGC
TAAAAAATTAAATGATGCTCAAGCTATTAAA
SEQ ID NO.5
General proteins A (PA) nucleotide sequence of 6 histidine residues is merged containing N-terminal
ATGCATCATCATCATCATCATGGATCCATGGCTCAACATGATGAAGCACAGCAGAATGCTTTTTATCAA
GTTTTAAATATGCCTAATCTTAATGCTGATCAACGCAATGGTTTTATTCAATCTTTAAAAGATGATCCTTCACAATC
TGCTAATGTTCTTGGTGAAGCTCAAAAACTTAATGACTCTCAAGCTCCGAAAGCTGATAATAACTTTAACAAAGATC
AGCAATCTGCTTTTTATGAAATTCTTAATATGCCGAATTTGAATGAAGCTCAACGTAATGGCTTCATACAATCTCTT
AAGGACGACCCTTCTCAGTCTACTAACGTTCTTGGCGAAGCTAAAAAACTTAACGAGTCTCAAGCTCCTAAAGCCGA
TAACAATTTCAACAAAGAACAGCAAAATGCTTTCTACGAAATTTTGAATATGCCGAACTTAAACGAAGAGCAACGTA
ACGGTTTCATTCAATCATTAAAGGATGACCCTTCACAGTCTGCTAACCTTTTGTCAGAAGCTAAAAAGTTAAATGAA
TCACAAGCACCTAAGGCTGATAACAAATTCAACAAGGAGCAACAGAATGCTTTCTATGAAATTTTACATCTTCCTAA
CTTAAATGAAGAACAACGTAATGGTTTCATACAGTCTCTTAAAGATGACCCGTCTCAGTCTGCTAATCTTTTAGCTG
AGGCTAAAAAGCTTAATGATGCACAAGCACCTAAAGCTGACAACAAATTTAACAAAGAGCAACAGAATGCATTCTAT
GAAATACTTCATTTACCTAACTTAACTGAAGAACAGCGTAACGGCTTTATACAATCACTTAAAGACGATCCTTCTGT
TTCTAAAGAAATTTTAGCAGAAGCTAAGAAGCTTAACGACGCTCAGGCTCCGAAATAA
SEQ ID NO.6
The RZ5 amino acid sequence of 6 histidine residues is merged containing N-terminal
MHHHHHHGSVDAKFDKEQQNAFYEILHLPNLTEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAIKVD
AKFDKEQQNAFYEILHLPNLTEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAIKVDAKFDKEQQNAFYEILHLPN
LTEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAIKVDAKFDKEQQNAFYEILHLPNLTEEQRNAFIQSLKDDPSQ
SANLLAEAKKLNDAQAIKVDAKFDKEQQNAFYEILHLPNLTEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAIKC
SEQ ID NO.7
The RZ5 nucleotide sequence of 6 histidine residues is merged containing N-terminal
ATGCATCATCATCATCATCATGGATCCGTTGATGCTAAATTTGATAAAGAACAACAAAATGCTTTTTAT
GAAATTTTACATTTACCTAATTTAACTGAAGAACAACGTAATGCTTTTATTCAATCTTTAAAAGATGATCCTTCTCA
ATCTGCTAATTTATTAGCTGAAGCTAAAAAATTAAATGATGCTCAAGCTATTAAAGTTGATGCTAAATTTGATAAAG
AACAACAAAATGCTTTTTATGAAATTTTACATTTACCTAATTTAACTGAAGAACAACGTAATGCTTTTATTCAATCT
TTAAAAGATGATCCTTCTCAATCTGCTAATTTATTAGCTGAAGCTAAAAAATTAAATGATGCTCAAGCTATTAAAGT
TGATGCTAAATTTGATAAAGAACAACAAAATGCTTTTTATGAAATTTTACATTTACCTAATTTAACTGAAGAACAAC
GTAATGCTTTTATTCAATCTTTAAAAGATGATCCTTCTCAATCTGCTAATTTATTAGCTGAAGCTAAAAAATTAAAT
GATGCTCAAGCTATTAAAGTTGATGCTAAATTTGATAAAGAACAACAAAATGCTTTTTATGAAATTTTACATTTACC
TAATTTAACTGAAGAACAACGTAATGCTTTTATTCAATCTTTAAAAGATGATCCTTCTCAATCTGCTAATTTATTAG
CTGAAGCTAAAAAATTAAATGATGCTCAAGCTATTAAAGTTGATGCTAAATTTGATAAAGAACAACAAAATGCTTTT
TATGAAATTTTACATTTACCTAATTTAACTGAAGAACAACGTAATGCTTTTATTCAATCTTTAAAAGATGATCCTTC
TCAATCTGCTAATTTATTAGCTGAAGCTAAAAAATTAAATGATGCTCAAGCTATTAAATGCTAA
SEQ ID NO.8
Primer 1
GAATTCCATATGCATCATCATCATCATCATGGAT
SEQ ID NO.9
Primer 2
ATAAGAATGCGGCCGCTTAGCATTTAATAGCTTGAGCATCAT
SEQ ID NO.10
Primer 3
ATAAGAATGCGGCCGCTTAGCATTATTTCGGAGCCTGAGCGT
Claims (9)
1. a kind of albumin A of gene mutation, which is characterized in that its have the amino acid sequence as shown in SEQ ID NO.1, or with
Amino acid sequence shown in SEQ ID NO.1 has all amino acid sequences for capableing of binding antibody of 99% or more homology, wherein
Antibody binding domain is 7-57 amino acid sequence.
2. a kind of gene, which is characterized in that the gene encodes the gene order of albumin A as described in claim 1.
3. a kind of polymer comprising albumin A belonging to claim 1, which is characterized in that polymer is two or more
The polymer that albumin A belonging to claim 1 is formed, or formed for albumin A described in claim 1 and other albumen more
Aggressiveness.
4. a kind of gene, which is characterized in that the gene encodes the gene order of polymer as claimed in claim 3.
5. the N-terminal or C-terminal or side-chain radical of the polymer of the albumin A according to any one of claim 1 or 3 or albumin A
It is chemically modified obtained, all albumin A derivatives that antibody can be firmly combined.
6. albumin A derivative according to claim 5, which is characterized in that the derivative be included in claim 1 or
The N-terminal of Protein A molecules described in any one of 3 is modified obtained by 6 histidine residues.
7. albumin A described in claim 1, albumin A derivative described in polymer as claimed in claim 3 or claim 5
Application in separation, purifying and/or detection antibody.
8. application according to claim 7, which is characterized in that including by the albumin A, the polymer or described
Albumin A derivative, for the ligand in affinity chromatography medium, or for the molecular marked compound in antibody test.
9. anti-in separation, purifying and/or detection according to claim 2, claim 4 or gene order according to any one of claims 8
Application in body.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711470488.4A CN109721645A (en) | 2017-12-29 | 2017-12-29 | A kind of albumin A of gene mutation and its application |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711470488.4A CN109721645A (en) | 2017-12-29 | 2017-12-29 | A kind of albumin A of gene mutation and its application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN109721645A true CN109721645A (en) | 2019-05-07 |
Family
ID=66294309
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201711470488.4A Pending CN109721645A (en) | 2017-12-29 | 2017-12-29 | A kind of albumin A of gene mutation and its application |
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| Country | Link |
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| CN (1) | CN109721645A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114315996A (en) * | 2021-12-31 | 2022-04-12 | 博格隆(浙江)生物技术有限公司 | Preparation method of recombinant Protein A and affinity chromatography medium |
| WO2023046886A1 (en) | 2021-09-24 | 2023-03-30 | Cytiva Bioprocess R&D Ab | Fc binding polypeptides |
| WO2023174900A1 (en) | 2022-03-14 | 2023-09-21 | Cytiva Bioprocess R&D Ab | Vh3 binding polypeptides |
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| WO2023174900A1 (en) | 2022-03-14 | 2023-09-21 | Cytiva Bioprocess R&D Ab | Vh3 binding polypeptides |
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