CN106279419A - A kind of antibody binding proteins Protein L and preparation method thereof - Google Patents
A kind of antibody binding proteins Protein L and preparation method thereof Download PDFInfo
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- CN106279419A CN106279419A CN201610640471.8A CN201610640471A CN106279419A CN 106279419 A CN106279419 A CN 106279419A CN 201610640471 A CN201610640471 A CN 201610640471A CN 106279419 A CN106279419 A CN 106279419A
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- C07—ORGANIC CHEMISTRY
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- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/42—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins
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- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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- C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
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Abstract
The present invention provides a kind of antibody binding proteins Protein L and preparation method thereof, described antibody binding proteins Protein L includes the B2 domain being repeated 8 times, a kind of preparation method of the antibody binding proteins Protein L of the present invention, including the step utilizing isocaudarner technology to repeat copy structure territory, obtain antibody binding proteins Protein L can be the most special the large-scale antibody of capture and antibody fragment, reduce non-specific binding, and improve the purity of antibody fragment；The high Dynamic binding capacities utilizing antibody binding proteins Protein L shortens purification time, and reduces cost；The antibody binding proteins Protein L pH tolerance range of the present invention is wide, and application simplicity, steady quality is reliable；The antibody binding proteins Protein L of the present invention, has high-affinity, it is possible to specific binding antibody and antibody fragment.
Description
Technical field
The present invention relates to protein purification filler field, be specifically related to a kind of antibody binding proteins, refer more particularly to one
Antibody binding proteins for protein purification and preparation method thereof.
Background technology
Antibody is a kind of baroque biomacromolecule, is made up of multiple aminoacid.The IgG used in antibody drug
(immunoglobulin G) cultivate at cell, isolated and purified during easily there is the change of inhomogeneity, these inhomogeneities make to resist
The drug effect of body, safety aspect are affected.In existing system, expression antibody fragment and sample to be purified often impurity are relatively
Many, bring challenge to purification work.
The antibody binding proteins Protein L of efficiently purifying albumen is thin from Anaerobic Bacteria peptostreptococcus magnus first
Cell wall is separated, is a kind of energy and specific for immunoglobulin combines widely protein, is possible not only to combine institute
Some Ig classes (IgG, IgM, IgA, IgE and IgD), it is also possible to combine single-chain antibody and Fab fragment, Protein L can not do
In the case of disturbing antigen binding site, with the K chain combination of antibody.Protein L can combine various source and subclass widely
Antibody, including people, mice, the immunoglobulin in rat source.At present, Protein L market sold has: Jin Sirui's
The Recombinant Protein L etc. of Protein L Resin, Biovision, but the Protein L of these reagent is close
The highest with power, purification time is longer and relatively costly.
Because above-mentioned defect, the design people, the most in addition research and innovation, to founding a kind of antibody binding proteins
Protein L and preparation method thereof so that it is have more the value in industry.
Summary of the invention
For solving above-mentioned technical problem, it is an object of the invention to provide a kind of antibody binding proteins Protein L and system thereof
Preparation Method, this albumen has higher affinity and wider array of Ig, Ig hypotype incorporation range.
A kind of antibody binding proteins Protein L of the present invention, including repetitive structure territory.
Further, described repetitive structure territory is B2 domain.
Further, described antibody binding proteins Protein L includes the B2 domain being repeated 8 times, and has such as SEQ
Aminoacid sequence shown in ID NO:1, the DNA molecular of coding aminoacid sequence shown in SEQ ID NO:1 has such as SEQ ID
Nucleotide sequence shown in NO:2, described B2 domain has the aminoacid sequence as shown in SEQ ID NO:3, encodes SEQ ID
The DNA molecular of the aminoacid sequence shown in NO:3 has the nucleotide sequence as shown in SEQ ID NO:4.
The preparation method of a kind of antibody binding proteins Protein L of the present invention, repeats to copy including utilizing isocaudarner technology
The step of shellfish domain.
Further, utilize isocaudarner technology to repeat copy structure territory to comprise the following steps:
(1) according to the gene order of Protein L*8, design primer Pr-1 and Pr-2 carries out PCR amplification, and at primer two
End is provided with the restriction enzyme site of isocaudarner, purified pcr product, then utilizes DNA ligase to connect；Primer sequence is as follows:
Pr-1:5'-AAAAGCTTGAAGGAGATATACATATG-3'；
Pr-2:5'-TGCTCGAGACAGCAACAACTGCCGCCACCACC-3'；
(2) it is repeated 3 times above-mentioned PCR amplification according to step (1), obtains being repeated 8 times the genetic fragment of B2 domain, described base
Because fragment is inverted, order-checking obtains expression plasmid.
Further, proceed to expression vector is cultivated and obtains expression strain by the expression plasmid obtained in step (2).
Further, the expression strain obtained is passed through cultivation, purification, obtains described antibody binding proteins Protein L.
Further, in step (1), described DNA ligase is T4DNA ligase.
Further, in step (1), described isocaudarner is BamH I and Bg III, in Xba I and Spe I, Sph I and Nsp I
A kind of.
Further, the one during described expression vector is pET series and pGEX series.
By such scheme, the present invention at least has the advantage that
The invention discloses a kind of antibody binding proteins Protein L for efficiently purifying albumen, research finds
Protein L contains five high homology repetitive structure territories of B1-B5, and repetitive structure territory is generally deposited in immunity chou hop protein
, the combination activity of antibody binding proteins Protein L has close relationship with B repetitive structure territory；Research shows containing super
Crossing the antibody binding proteins in a B repetitive structure territory, have a higher affinity, wherein the affinity of B1-B4 domain can be with
Complete antibody associated proteins Protein L compares favourably, and B5 domain does not increase the Protein L affinity to Kappa light chain；This
The antibody binding proteins Protein L of invention chooses the B2 domain in protein structure, utilizes isocaudarner technology to repeat through 8 times
Copy, it is thus achieved that the antibody binding proteins that a kind of antagonist affinity is higher；It can be efficiently applied to detection, purification and quantization and resist
Body and antibody/antigen complex, have higher affinity to people, mice, rat, the immunoglobulin in pig source, have wider
Ig and Ig hypotype incorporation range.
The invention provides the preparation method of this antibody binding proteins, utilize isocaudarner technical antagonism binding protein precursor
The B2 domain of Protein L carries out 8 times repeating copy, it is thus achieved that antibody binding proteins Protein L can be the most special catch
Obtain large-scale antibody and antibody fragment, reduce non-specific binding, and improve the purity of antibody fragment；Utilize antibodies
The high Dynamic binding capacities of albumen Protein L shortens purification time, and reduces cost；The antibody binding proteins of the present invention
Protein LpH tolerance range is wide, and application simplicity, steady quality is reliable；The antibody binding proteins Protein L of the present invention, tool
There is high-affinity, it is possible to specific binding antibody and antibody fragment.
Described above is only the general introduction of technical solution of the present invention, in order to better understand the technological means of the present invention,
And can be practiced according to the content of description, below with presently preferred embodiments of the present invention and coordinate accompanying drawing describe in detail as after.
Accompanying drawing explanation
Fig. 1 is expression strain sds polyacrylamide gel electrophoresis detection figure in embodiments of the invention 1；
Fig. 2 is that the sds polyacrylamide of the antibody binding proteins Protein L obtained in embodiments of the invention 1 coagulates
Gel electrophoresis detection figure；
Fig. 3 is expression strain sds polyacrylamide gel electrophoresis detection figure in embodiments of the invention 2；
Fig. 4 is that the sds polyacrylamide of the antibody binding proteins Protein L obtained in embodiments of the invention 2 coagulates
Gel electrophoresis detection figure.
Detailed description of the invention
Below in conjunction with the accompanying drawings and embodiment, the detailed description of the invention of the present invention is described in further detail.Hereinafter implement
Example is used for illustrating the present invention, but is not limited to the scope of the present invention.
Embodiment 1
(1) structure of antibody binding proteins Protein L expression plasmid
(1.1) according to known genes of interest sequence, primer Pr-1 and Pr-2 of design synthesis is utilized to carry out PCR amplification,
And isocaudarner BamH I and Bg III restriction enzyme site, purified pcr product it is provided with at the two ends of primer, PCR reaction condition is:
Step one, 94 DEG C, 5 minutes；Step 2,94 DEG C, 20 seconds, 55 DEG C, 20 seconds, 72 DEG C, 30 seconds, 30 circulations；Step 3,72 DEG C, 5
Minute；Reclaim PCR primer, utilize isocaudarner BamH I and Bg III enzyme action domain, utilize T4 ligase to be connected on carrier；Draw
Thing sequence is as follows:
Pr-1:5'-AAAAGCTTGAAGGAGATATACATATG-3'；
Pr-2:5'-TGCTCGAGACAGCAACAACTGCCGCCACCACC-3'；
(1.2) being repeated 3 times above-mentioned PCR amplification experiment according to above-mentioned steps, obtain expression plasmid, this plasmid contains 8 B2
The genetic fragment of domain, is transformed into the genetic fragment of 8 B2 domains in escherichia coli, extracts plasmid, and order-checking is just obtaining
The true positive expression plasmid containing 8 B2 domain copies；
(2) expression of antibody binding proteins Protein L
(2.1) screening of expression strain: by the positive expression Plastid transformation of acquisition in step (1) to bacillus coli DH 5 alpha
In, cultivate screening high-expression clone in a small amount；6 equipped with the container of 5ml LB culture medium in inoculation convert after monoclonal bacterium
Body, cultivates 4 hours in the shaking table of 37 DEG C, is subsequently adding 0.5mM IPTG inducing culture and collects thalline after 3 hours；Take a small amount of
Thalline, add 100 μ l electrophoresis sample-loading buffers and boil 5 minutes, after cooling, 12000rpm is centrifuged 5min, takes supernatant and carries out
Sds polyacrylamide gel electrophoresis detects, as it is shown in figure 1, as can be seen from Figure 1: destination protein is expressed good, and band is clear
Clear.
(2.2) the extensive expression of fusion protein: in selecting step (2.1), screening obtains the expression strain that expression is the highest
As extensive expression strain, it is inoculated in the LB culture medium of 100ml, incubated overnight in 37 DEG C of shaking tables, is then used
100ml strain overnight is inoculated in the LB culture medium of 10L, and cultivating at 37 DEG C to OD is 0.5-0.6, adds 0.5mM's
IPTG inducing culture 3 hours, now bacterium solution OD is 2.2, centrifugal collection thalline 10g；
(3) purification of antibody binding proteins Protein L
(3.1) 10g getting collection expresses the escherichia coli of destination protein, fully dissolves with 100ml balance Buff, and stirring is all
Fully crack with Ultrasonic Cell Disruptor after even, then cracking supernatant 12000rpm, centrifugal 20min are taken supernatant；Use 0.22um filter
Filtration sterilization, the removal of impurity, make sample fully clarify；
(3.2) chromatography purification
(3.2.1) capture: Ni Sepharose 6 Fast Flow (FF) column connects AKTA Purification,；
The 0.2M NaOH using 5CV rinses tomographic system and soaks 3h, in then going heat source water flushing tomographic system extremely with at least 10CV
Property；The control thermal source 0.2M NiSO4 using 5CV rinses tomographic system, then goes heat source water to rinse tomographic system with 5CV；Use
5CV balance Buff 1 (20mM Tris, 250mM NaCl, 10mM Imidazole, pH8.0) rinses tomographic system, by pretreatment
After sample carry out loading with the flow velocity of 5ml/min, then rinse tomographic system with 5CV balance Buff 1；3CV is used to balance Buff
1 and eluting Buff 1 (20mM Tris, 250mM NaCl, 500mM Imidazole, pH8.0), uses step level type of elution (to divide
Three steps: E50, E100, E250, E500) protein of interest, take the albumen of 50mM, 100mM and 250mM purification, desalination after merging
After carry out SP High Performance moderate purification；
(3.2.2) moderate purification: AKTA Purification in SP High Performance column connection, makes
Rinse tomographic system with the 0.2M NaOH of 5CV and soak 3h, in then going heat source water flushing tomographic system extremely with at least 10CV
Property；The control thermal source 0.2M NiSO4 using 5CV rinses tomographic system, goes heat source water to rinse tomographic system with 5CV；5CV is used to put down
Weighing apparatus Buff 2 (20mM PB, PH7.0) rinses tomographic system, then pretreated sample is carried out with the flow velocity of 5ml/min on
Sample, then rinse tomographic system with 5CV balance Buff 2；Use 3CV balance Buff 2 and eluting Buff 2 (20mM PB, 1M
NaCl, pH7.0), use one-step elution mode protein of interest；
(3.2.3) polishing purification: Superdex 200 connects upper AKTA Purification, uses the control heat source water of 3CV
Fully rinse tomographic system,；Using 3CV Equilibration buffer wash tomographic system, the sample then capture reclaimed is with 2ml/min
Flow velocity carry out loading, then with 3CV level pad eluting；Sample fraction is received according to purification collection of illustrative plates.
(3.3) post processing
Merging fraction, 100 times of dialysis are to preserving system；Taking sample after dialysis ,-80 DEG C are done freeze thawing test, choose freeze thawing and close
The sample of lattice, makes quality inspection, warehouse-in, and sds polyacrylamide gel electrophoresis detection figure is as in figure 2 it is shown, can from Fig. 2
Go out: destination protein is expressed clear, and stripe size meets.
Embodiment 2
The present embodiment is identical with embodiment 1 step, and its difference is: in step (2.2), cultivates to OD be at 37 DEG C
0.5-0.6, adds IPTG inducing culture 24 hours under the conditions of 16 DEG C of 0.1mM, and now bacterium solution OD is 2.1, centrifugal collection bacterium
Body 10g；Sds polyacrylamide gel electrophoresis detection figure during the screening of expression strain is as it is shown on figure 3, as can be seen from Figure 3:
Destination protein is expressed good, and band is clear.；Sds polyacrylamide gel electrophoresis detection figure is as it can be seen, can from Fig. 4
Go out: destination protein is expressed clear, and stripe size meets.
The above is only the preferred embodiment of the present invention, is not limited to the present invention, it is noted that for this skill
For the those of ordinary skill in art field, on the premise of without departing from the technology of the present invention principle, it is also possible to make some improvement and
Modification, these improve and modification also should be regarded as protection scope of the present invention.
Claims (10)
1. an antibody binding proteins Protein L, it is characterised in that: described antibody binding proteins Protein L includes weight
Complex structure territory.
Antibody binding proteins Protein L the most according to claim 1, it is characterised in that: described repetitive structure territory is B2
Domain.
Antibody binding proteins Protein L the most according to claim 1 and 2, it is characterised in that: described antibody binding proteins
Protein L includes the B2 domain being repeated 8 times, and has the aminoacid sequence as shown in SEQ ID NO:1, encodes SEQ
The DNA molecular of the aminoacid sequence shown in ID NO:1 has the nucleotide sequence as shown in SEQ ID NO:2, described B2 structure
Territory has the aminoacid sequence as shown in SEQ ID NO:3, the DNA molecular of coding aminoacid sequence shown in SEQ ID NO:3
There is the nucleotide sequence as shown in SEQ ID NO:4.
4. the preparation method of the arbitrary antibody binding proteins Protein L as described in claims 1 to 3, it is characterised in that:
Including the step utilizing isocaudarner technology to repeat copy structure territory.
Preparation method the most according to claim 4, it is characterised in that utilize isocaudarner technology to repeat copy structure territory and include
Following steps:
(1) according to the gene order of Protein L*8, design primer Pr-1 and Pr-2 carries out PCR amplification, and sets at primer two ends
There are the restriction enzyme site of isocaudarner, purified pcr product, then utilize DNA ligase to connect；Primer sequence is as follows:
Pr-1:5'-AAAAGCTTGAAGGAGATATACATATG-3'；
Pr-2:5'-TGCTCGAGACAGCAACAACTGCCGCCACCACC-3'；
(2) it is repeated 3 times above-mentioned PCR amplification according to step (1), obtains the genetic fragment of the B2 domain being repeated 8 times, described gene
Fragment is inverted, order-checking obtains expression plasmid.
Preparation method the most according to claim 5, it is characterised in that: the expression plasmid obtained in step (2) is proceeded to table
Reach and carrier is cultivated and obtains expression strain.
Preparation method the most according to claim 6, it is characterised in that: the expression strain obtained is passed through cultivation, purification,
To described antibody binding proteins Protein L.
Preparation method the most according to claim 5, it is characterised in that: in step (1), described DNA ligase is that T4DNA connects
Connect enzyme.
Preparation method the most according to claim 5, it is characterised in that: in step (1), described isocaudarner is BamH I and Bg
III, the one in Xba I and Spe I, Sph I and Nsp I.
Preparation method the most according to claim 6, it is characterised in that: described expression vector is pET series and pGEX series
In one.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1634990A (en) * | 2004-10-14 | 2005-07-06 | 上海润龙生物科技有限公司 | High affinity immune globulin binding molecule and method for preparation |
| WO2011161420A2 (en) * | 2010-06-23 | 2011-12-29 | King's College London | Coincidence detection |
| CN102787125A (en) * | 2011-08-05 | 2012-11-21 | 北京大学 | Method for building TALE (transcription activator-like effector) repeated sequences |
| CN105504042A (en) * | 2016-02-04 | 2016-04-20 | 北京艺妙神州医疗科技有限公司 | Capture probe used for detecting CAR-T (Chimeric Antigen Receptor-T) cells, method for detecting content of cells, and application |
| WO2016096643A1 (en) * | 2014-12-17 | 2016-06-23 | Ge Healthcare Bioprocess R&D Ab | Modified kappa light chain-binding polypeptides |
-
2016
- 2016-08-08 CN CN201610640471.8A patent/CN106279419A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1634990A (en) * | 2004-10-14 | 2005-07-06 | 上海润龙生物科技有限公司 | High affinity immune globulin binding molecule and method for preparation |
| WO2011161420A2 (en) * | 2010-06-23 | 2011-12-29 | King's College London | Coincidence detection |
| CN102787125A (en) * | 2011-08-05 | 2012-11-21 | 北京大学 | Method for building TALE (transcription activator-like effector) repeated sequences |
| WO2016096643A1 (en) * | 2014-12-17 | 2016-06-23 | Ge Healthcare Bioprocess R&D Ab | Modified kappa light chain-binding polypeptides |
| CN105504042A (en) * | 2016-02-04 | 2016-04-20 | 北京艺妙神州医疗科技有限公司 | Capture probe used for detecting CAR-T (Chimeric Antigen Receptor-T) cells, method for detecting content of cells, and application |
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Application publication date: 20170104 |