CN107050926A - A kind of coating protein G lemon yellow immune affinity column and preparation method thereof - Google Patents
A kind of coating protein G lemon yellow immune affinity column and preparation method thereof Download PDFInfo
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- CN107050926A CN107050926A CN201710045322.1A CN201710045322A CN107050926A CN 107050926 A CN107050926 A CN 107050926A CN 201710045322 A CN201710045322 A CN 201710045322A CN 107050926 A CN107050926 A CN 107050926A
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- lemon yellow
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
- B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 - B01D15/36
- B01D15/3804—Affinity chromatography
- B01D15/3809—Affinity chromatography of the antigen-antibody type, e.g. protein A, G, L chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54353—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals with ligand attached to the carrier via a chemical coupling agent
Abstract
The invention provides a kind of lemon yellow immune affinity column, including carrier, Protein G and lemon yellow antibody, wherein, the Protein G is coupled on carrier by chemical bond, and the lemon yellow antibody uses cross-linking agents after being combined with Protein G.The invention provides a kind of lemon yellow immune affinity column, including carrier, Protein G and lemon yellow antibody, wherein, the Protein G is coupled on carrier by chemical bond, and the lemon yellow antibody uses cross-linking agents after being combined with Protein G.The lemon yellow affinity column of the present invention, under conditions of certain immune affinity column column capacity, the consumption of antibody is less, so that antibody utilization ratio is higher, and the repeatability in the immune affinity column batch prepared in this way, between batch is more preferably, the difference of intercolumniation is lower.
Description
Technical field
The invention belongs to field of food inspection, and in particular to a kind of coating protein G lemon yellow immune affinity column and its system
Preparation Method.
Background technology
Lemon yellow is a kind of most widely used synthetic food color, for food, medicine, cosmetics, medical apparatus, cigarette
Grass, feed, edible packaging, toy etc., often need quantitatively to detect the lemon yellow in the said goods, because if human body is taken the photograph
Enter amount lemon yellow excessive, injury can be produced.Add lemon yellow in violation of rules and regulations in feed and also have a strong impact on the healthy of people, influence
The normal development of animal husbandry.
At present, the lemon yellow detection method of national Specification is high performance liquid chromatography, thin layer chromatography, enzyme linked immunological
The physico-chemical method such as adsorption test (ELISA) and spectrophotometer method.EUSA (ELISA) has efficient, spirit
It is quick, can sample after the advantages of directly detected through simple process, but there is also the higher shortcoming of false positive rate for this method.
HPLC, LC-MS/MS method are quantitative analysis lemon yellow most common methods.It is miscellaneous to reduce when being detected using liquid phase or liquid matter method
Matter is disturbed, and reduces its influenceing, it is necessary to first carry out purified treatment to the sample of various different substrates to Detection results.It is conventional at present
Purifying treatment method be exactly solid phase extraction, be also column chromatography.It is to purify most popular method at present.Wherein, exempt from
The analyze speed of epidemic disease is affine column method is fast, and sensitivity is high, and separative efficiency and the rate of recovery are high, safe and reliable, thus as the most frequently used
Method.
Method for preparing immunoaffinity purification post at present probably has：The direct coupling method of cyanogen bromide-activated, epoxy chloropropionate
Alkane method, Over-voltage protection etc..The general principle of these methods is all to carry out carrier matrix after chemical activation, and antibody coupling is arrived
On carrier；Protein G (Protein G) is the energy extracted from the cell membrane and culture supernatant of A, C and G groups of many bacterial strains of streptococcus
The albumen combined with IgG Fc fragments specifics, the specific Fc positions with many animals antibody of albumen energy are combined, and
With very high affinity.Although having high-affinity using Protein G, the carrier handled using Protein G can reduce affine layer
The elution speed of post is analysed, so as to reduce the utilization rate and the efficiency of detection of antibody.
The content of the invention
In view of this, the first object of the present invention is to provide a kind of lemon yellow immune affinity column, including carrier, Protein G
With lemon yellow antibody.
Preferably, in lemon yellow immune affinity column of the present invention, the Protein G is included according to GenBank
The Protein G that CAA27638.1 sequence tables reach, and the recombinant protein G Jing Guo sequence optimisation.
Preferably, in lemon yellow immune affinity column of the present invention, the Protein G is based on GenBank
Recombinant protein Gs of the CAA27638.1 Jing Guo sequence optimisation.
Preferably, in lemon yellow immune affinity column of the present invention, the Protein G is coupled at carrier by chemical bond
On, lemon yellow antibody uses cross-linking agents after being combined with Protein G.
Preferably, in lemon yellow immune affinity column of the present invention, the recombinant protein G of described sequence optimisation, including
The effect of carrier-recombinant protein G steric hindrances is reduced, the excellent of recombinant protein G and the binding ability of lemon yellow IgG antibody is added
Change.
Preferably, in lemon yellow immune affinity column of the present invention, the recombinant protein G optimization sites are amputation
GenBank CAA27638.1 sequences 53 amino acid of N-terminal.
Preferably, in lemon yellow immune affinity column of the present invention, described carrier is Ago-Gel；Described fine jade
Sepharose includes Ago-Gel, the Ago-Gel of crosslinking, polyacrylic acid Ago-Gel, the polypropylene of cyanogen bromide-activated
The one or more of acid amides Ago-Gel, sephadex and cellulose；It is highly preferred that the carrier is agarose
Sepharose 4B。
Preferably, in lemon yellow immune affinity column of the present invention, described anti-lemon yellow antibody includes monoclonal IgG
Antibody and polyclonal antibody.
Another object of the present invention is to provide a kind of preparation method of lemon yellow immune affinity column, comprise the following steps：
1) it is support-activated；
2) recombinant protein G is coupled with the carrier activated；
3) handle carrier-recombinant protein G, and with lemon yellow antibody coupling；
4) washing lemon yellow antibody-recombinant protein G- carriers, dress post is to obtain lemon yellow immune affinity column.
Preferably, in the preparation method of lemon yellow immune affinity column of the present invention, comprise the following steps：
1) agarose Sepharose 4B, add 0.8M NaOH, 30% epoxychloropropane, 2mg/ml sodium borohydride
NaBH4, shaking table reaction 8h is activated at 25 DEG C；
2) the Sepharose 4B of every gram of activation add 30~200nmol recombinant protein G, in 0.1M NaHCO3, 0.5M
In NaCl pH8.8 buffer solution, room temperature coupling 2h, or 4 DEG C of couplings are stayed overnight；
3) coupled product reacts at room temperature 2h with 1M TrisHCl pH 8.0；
4) the Sepharose- Protein Gs being coupled, are washed with 20mM pH7.4 PBS；
5) lemon yellow antibody is dissolved in 20mM pH7.4 PBS, every gram of Protein G-Sepharose adds 200nmol
~1.2umol antibody, is reacted at room temperature 30 minutes；
6) lemon yellow antibody-white G-Sepharose carriers are washed with 20mM PBS pH7.4, then adds final concentration
0.2M triethanolamine and 20mM dimethyl pimelate (DMP), pH8.3 react at room temperature 1h；
7) with 20mM pH7.4 monoethanolamine terminating reaction, washed with the 10mMPBS pH7.4 containing 0.01% thimerosal
Lemon yellow antibody-recombinant protein G-Sepharose carriers, fills post, is put in 4 DEG C of preservations；
Wherein, the recombinant protein G is the recombinant protein G based on GenBank CAA27638.1 Jing Guo sequence optimisation.
From the foregoing, it will be observed that the present invention at least has advantages below：
Compared with prior art, the invention has the advantages that：
1) we clone the gene order for having recombinated Protein G (GenBank CAA27638.1), in expression in escherichia coli
The gene recombinant protein G for having high-affinity with antibody is gone out.It make use of the characteristics of Protein G is combined with Ig antibody specificities, Ig G
Antibody is made up of two heavy chains and two light chains, and antibody is divided into Fab areas and Fc areas, wherein, Fab areas are the regions of antigen binding.
Protein G is a kind of special albumen on streptococcal cell wall, has specific binding ability with the Fc regions of antibody.Protein G
After antibody binding, the Fab regions of antibody are free outside, and the antigen binding capacity of antibody is not influenceed.By our gene optimizations
Protein G after restructuring, the Protein G of 1 molecule can combine the Ig G antibody of 3 molecules, with very high affinity of antibody.And
And only antibody can be combined with Protein G, remaining albumen can not be combined with Protein G, and specificity is very high.Based on this Protein G
The immune affinity column of preparation has specificity good, and lemon yellow binding capacity is big, the characteristics of purification efficiency is high.
After the Protein G of genetic recombination is coupled on agarose carrier, then carry out lemon yellow antibody coupling, it is and traditional straight
Connect coupling to compare, after being coupled to using the Protein G of genetic recombination as middle ware arm on carrier, reduce steric hindrance effect, increase
Recombinant protein G and the binding ability of lemon yellow IgG antibody, enable impurity sufficiently to flow out, enhance clean-up effect, so that
Make the Fab ends of antibody towards outside, the non-confrontational former calmodulin binding domain CaM of steric hindrance constitutes interference, after carrier is first handled through Protein G,
Antibody coupling is carried out again, the homogeneity of different purification intercolumniations can be not only improved, and can improve the utilization rate of antibody.
Agarose is pre-processed with the recombinant protein G of the present invention, based on Protein G and IgG special affinity,
After Protein G are combined with antibody Fc fragment, Fab fragments stretch outside, it is easier to capture antigen.Directly use antibody coupling agar
Sugar, bonding position of the antibody on agarose and part are random, and antibody is through the pretreated agaroses of Protein G
On antibody there is consistent directionality, under conditions of certain immune affinity column column capacity, the consumption of antibody is less so that
Antibody utilization ratio is higher, and the repeatability in the immune affinity column batch prepared in this way, between batch is more
Good, the difference of intercolumniation is lower.
Therefore, it is combined with Ig G antibody specificities by designing recombinant protein G, antibody is coupled to by Protein G
On carrier.And the Fab fragments of IgG antibody are sufficiently exposed outside, the capture ability of lemon yellow, lemon is greatly improved
The yellow purification efficiency of lemon also effectively improves.
2) present invention uses the Protein G after genetic modification, the optimization to codon and Ig G combination domain genes are passed through
Optimization.The antibody binding capacity of Protein G is increased substantially, and then improves the purification efficiency of lemon yellow.
3) easy to operate using present invention purifying lemon yellow, several steps can be obtained by the higher lemon yellow of purity, be more convenient
Operator uses.
4) it is very high using obtained lemon yellow purity of the invention, subsequently just can directly it be used without doing other purification process again
In high performance liquid chromatography detection, time and the expense of operator is saved.
Brief description of the drawings
Fig. 1 is the lemon yellow immune affinity column structural representation in one embodiment of the present of invention；
Fig. 2 be one embodiment of the present of invention agarose carrier through recombinant protein G be coupled after further with lemon yellow antibody
It is coupled schematic diagram；
Fig. 3 be agarose carrier directly with lemon yellow antibody coupling schematic diagram；
Fig. 4 be agarose carrier directly with icon implication figure in lemon yellow antibody coupling schematic diagram；
Fig. 5 is influence schematic diagram of the lemon yellow initial concentration in solution to the object adsorption capacity of decontaminating column；
Fig. 6 is lemon yellow immune affinity column bin stability schematic diagram.
Embodiment
There is provided lemon yellow immune affinity column and preparation method thereof in one embodiment of the invention:
1. it is support-activated
Agarose carrier Sepharose 4B are selected, are activated with Epichlorohydrin activation method.
Take 2% preswollen Ago-Gel Sepharose 4B, fully rinsed, washed away with the distilled water of 20 times of volumes
The ethanol of remaining, moisture is filtered out with funnel.
Weigh and filter off 5 grams of wet gel after moisture content, add 7.5 milliliters of 0.8M NaOH, 30% milli of epoxychloropropane 2
Rise, 2mg/ml sodium borohydride NaBH4, 5 milliliters, shaking table reacts 8 hours at 25 DEG C.After reaction, washed with substantial amounts of distillation
Wash, remove the epoxychloropropane mixed in gel.
2. by recombinant protein G and activated carrier Sepharose 4B coupling
By the Ago-Gel Sepharose 4B activated coupling buffer (0.1M NaHCO3, 0.8M NaCl,
PH8.9) wash 3 times.The Sepharose 4B of every gram of activation add 30-200nmol Protein G, and room temperature is coupled 2 hours, or 4
DEG C coupling is stayed overnight.The coupled product lower reaction 2h of 8.0 30 DEG C of nitrogen protections of 1M TrisHCl pH.
The agarose carrier 20mM, pH7.4 that have been coupled phosphate buffer PBS are washed 3 times.Coupling has recombinant protein
G agarose carrier Sepharose is named as recombinant protein G-Sepharose.
Wherein, recombinant protein G is by Protein G sequence (GenBank CAA27638.1, SEQ ID NO：1) N-terminal 53, is amputated
Recombinant protein G sequence (SEQ ID NO2) is obtained after individual amino acid and transfers to the biochemical 's synthesis of gill.
3. lemon yellow antibody is connected with the recombinant protein G on recombinant protein G-Sepharose carriers
Recombinant protein G has specific affinity with antibody, under certain reaction condition, and coupling is had into Protein G
Ago-Gel is attached with antibody, and antibody is connected on agarose.Because the binding site of Protein G and antibody is antibody
Fc regions, the antigen binding domain of antibody is unaffected, has fully ensured that the antigen binding capacity of antibody.
In the PBS that lemon yellow antibody is dissolved in 20mM pH7.4, every gram of Protein G-Sepharose add 150nmol~
1.0umol antibody, is reacted at room temperature 30 minutes.Carrier with reference to after is washed with PBS.Be connected with antibody carrier be named as antibody-
Protein G-Sepharose.
4. carrier is crosslinked
Antibody-protein G-Sepharose is washed 3 times with 20mM PBS pH7.4.Then the three of final concentration 0.2M is added
The dimethyl pimelate (DMP) of monoethanolamine and 20mM, pH8.3, the lower reaction 1h of 30 DEG C of nitrogen protections.
20mM is added, pH9.0 monoethanolamine terminating reaction is washed with the 10mMPBS pH7.4 containing 0.01% thimerosal
Lemon yellow Antibody-protein G-Sepharose carriers.
5. fill post
Lemon yellow Antibody-protein G-Sepharose after crosslinking is fitted into chromatographic column as needed, 4 DEG C of preservations are put in,
The lemon yellow affinity purification post of different capabilities object lemon yellow can be prepared according to actual needs.
Further technical scheme is illustrated below by way of specific embodiment, it should be understood that be only this hair below
Bright exemplary illustration, is not intended to limit the invention scope of the claims.
The preparation of the lemon yellow immunoaffinity purification post of embodiment 1
1. Ago-Gel is activated
Take 2% Ago-Gel Sepharose 4B, fully rinsed with the distilled water of 20 times of volumes, wash away remaining second
Alcohol.Moisture is filtered out with funnel.Weigh and filter off the wet gel 5g after moisture content, add 7.5mL0.8M NaOH, 30% epoxy chlorine
Propane 2mL, 2mg/mL sodium borohydride NaBH4, 5mL, shaking table reacts 8h at 25 DEG C.After reaction, water washing is distilled with 50mL,
Remove the epoxychloropropane mixed in gel.
2. recombinant protein G and the Ago-Gel of activation coupling
The Ago-Gel after 1 gram of activation is taken, with coupling buffer (0.1M NaHCO3, 0.8M NaCl, pH8.9) wash
Wash 3 times.Add 2mg/mL Protein G 20mL, room temperature coupling 4h.
The agarose carrier 20mM, pH7.4 that have been coupled phosphate buffer PBS are washed 3 times.Coupling has recombinant protein
G agarose carrier Sepharose is named as recombinant protein G-Sepharose.
3. anti-lemon yellow IgG monoclonal antibodies are combined with Protein G-Sepharose
Anti- lemon yellow antibody is dissolved in 20mM, pH7.4 PBS, final concentration 2mg/mL, cumulative volume 10mL.
Protein G-Sepharose is washed three times with 20mM pH7.4 PBS, each 30mL.PBS will be dissolved in
In lemon yellow antibody add in washed Protein G-Sepharose, room temperature combination 30min.
Carrier with reference to after is washed three times with 20mM pH7.4 PBS, each 30mL.The carrier for being connected with antibody is named as
Antibody-protein G-Sepharose.
4. the agarose Sepharose for combining IgG is crosslinked
Antibody-Protein G-Sepharose is washed 3 times with 0.1M pH9.0 borate buffer, each 30mL.
By wet gel solid, 0.1M pH9.0 borate buffer 20mL is added, crosslinking agent heptan two is added in buffer solution
Imido dimethyl phthalate (DMP) is to final concentration 20mM.Normal temperature crosslinked 1h.
Add 100mM, pH9.0 monoethanolamine 20mL terminating reactions.And closed 10 minutes with 50mM monoethanolamine 20mL.
The PBS of Antibody-protein G-Sepharose carriers 20mM, pH7.4 after crosslinking are washed 3 times, each 30mL.
5. fill post
Sepharose after crosslinking is resuspended in 10mL 20mM PBS pH7.4, the affinity purification post post of sky is then charged into
In body.
Embodiment 2：The lemon yellow in corn protein powder is purified and detected using lemon yellow immune affinity column
1st, sample treatment
1) 2.0 corn protein powder samples are weighed into 50mL centrifuge tubes；
2) 18mL acetonitrile solutions (V/V 50 is added:50), mixed with vortex instrument or homogenizer vibration, sonic oscillation is extracted
2min, 1500r/min mechanical shaking extraction 3min；
3) 4000g centrifuges 3min at room temperature, and careful 5mL upper liquids of taking out are to a new centrifuge tube, and 50 DEG C of nitrogen dryings are (after dry
Take out immediately)；
4) whole loadings after being redissolved with 10mL PBS (0.01M pH7.4) buffer solution.
2nd, by lemon yellow immunoaffinity purification post equilibrium at room temperature 30min；
3rd, immune affinity column is taken out, injection port is connected with injector syringe, and syringe is linked on gas control crosshead；
4th, affinity column is washed with deionized 3 times, each 10mL adjusts stomata crosshead air pump pressure, makes liquid with 3
Drop/sec flow velocity outflow；
5th, sample is added in purification column, regulation flow is to 1~2 drop/sec.Until sample all flows out purification column.
6th, with distillation water washing purification column 3 times, each 5mL.
7th, 1mL methanol is added, eluted product is collected.
8th, eluted product is detected with high-efficient liquid phase chromatogram HPLC.
1), the preparation of lemon yellow standard items
1.1) lemon yellow standard items are taken, are dissolved with 0.01M pH7.4 PBS, and be diluted to 3ppb；
1.2) the lemon yellow standard items 10mL diluted, whole sample detections are taken；
2), by lemon yellow immunoaffinity purification post equilibrium at room temperature 30min；
3) immune affinity column, is taken out, injection port is connected with injector syringe, and syringe is linked on gas control crosshead；
4) affinity column, is washed with deionized 3 times, each 10mL adjusts stomata crosshead air pump pressure, makes liquid with 3
Drop/sec flow velocity outflow；
5), sample is added in purification column, regulation flow is to 1~2 drop/sec.Until sample all flows out purification column；
6), with distillation water washing purification column 3 times, each 5mL；
7) 1mL methanol, is added, eluted product is collected；
8), eluted product is detected with high-efficient liquid phase chromatogram HPLC.
Liquid-phase condition：
Chromatographic column：Kromasil C18 250mm×4.6mm 5μm
Mobile phase A：Methanol；Mobile phase B：5mmol/L ammonium acetates
Eluent gradient program
Column temperature：35℃；Sample size：10μL；Detection wavelength 254nm；Flow velocity：1.0mL/min.
Embodiment 3：Decontaminating column performance comparision prepared by the two methods of measure of lemon yellow immune affinity column column capacity
The preparation of the decontaminating column first handled through Protein G
Prepared by embodiment 2
The preparation of decontaminating column without Protein G processing
Direct coupling method prepares affinity column
1st, it is support-activated
Agarose carrier is selected, Sepharose 4B are activated with Epichlorohydrin activation method.
Take 2% preswollen Ago-Gel Sepharose 4B, fully rinsed, washed away with the distilled water of 20 times of volumes
The ethanol of remaining, moisture is filtered out with funnel.
Weigh and filter off the wet gel 5g after moisture, add 7.5mL 0.8M NaOH, 30% epoxychloropropane 2mL,
2mg/mL sodium borohydride NaBH4, 5mL, shaking table reacts 8h at 25 DEG C.
After reaction, with substantial amounts of distillation water washing, the epoxychloropropane mixed in gel is removed.
2nd, anti-lemon yellow antibody is connected with Sepharose carriers
Under certain reaction condition, Ago-Gel is attached with antibody, antibody is connected on agarose.
In the PBS that lemon yellow antibody is dissolved in 20mM pH7.4, every gram of Sepharose adds 200nmol~1.2 μm ol
Antibody, reacts at room temperature 30min.
Carrier with reference to after is washed with PBS.The carrier for being connected with antibody is named as antibody-Sepharose.
3rd, carrier is crosslinked
Antibody-Sepharose is washed 3 times with 20mM PBS pH7.4.Then final concentration 0.2M triethanolamine is added
With 20mM dimethyl pimelate (DMP), pH8.3 is reacted at room temperature 1 hour.
20mM is added, pH9.0 monoethanolamine terminating reaction is washed with the 10mM PBS pH7.4 containing 0.01% thimerosal
Lemon yellow antibody-Sepharose carriers.
4. fill post
Antibody-agarose carrier after crosslinking is fitted into chromatographic column as needed, being put in 4 DEG C of preservations can make as needed
The lemon yellow affinity purification post of standby different capabilities.
Column capacity
2mL concentration is 100ng/mL lemon yellow PBS solution loading, then finally uses 5mL eluents with elution pillar
100% methanol elutes object, HPLC detections, according to the following formula, calculates column capacity.
Take by the populated decontaminating column of such as accompanying drawing 1, by the certain density lemon yellow object PBS solutions of 1mL with 1mL/
Min flow velocitys cross post, with the ultrapure washing posts of 10mL, to wash away impurity of the non-specific adsorption on pillar, are eluted with 1mL methanol,
Upper HPLC is quantitatively detected, determines measure of the decontaminating column to " capture " capacity of object.
Fig. 2 show agarose carrier through recombinant protein G be coupled after further with lemon yellow antibody coupling schematic diagram, Fig. 3
Show agarose carrier directly with lemon yellow antibody coupling schematic diagram, wherein, show in Fig. 2, Fig. 3 that each is illustrated in Fig. 4
Agarose, lemon yellow antibody and recombinant protein G are represented respectively.
In purification process, in the case where column volume excessively is certain, with the increase of target concentration in solution to be clean,
The adsorbance for the object being " trapped " in decontaminating column can increase therewith, but its adsorbance will not be with the increase of target concentration
And infinitely increase, antibody has certain adsorption capacity in decontaminating column --- saturated adsorption capacity.This experiment has primarily looked at purification
Relation of the antibody to object initial concentration in the adsorbance and solution of object in post.Fig. 5 is object initial concentration to net
Change the influence curve of antibody adsorbance in post.As shown in Figure 5, generally in decontaminating column antibody to the adsorbance of object with treating
The increase of target concentration in scavenging solution and increase.But this curve can also be divided into Two Areas, region 1：Target in liquid to be clean
When thing initial concentration is less than 180ng/mL, with the increase of object initial concentration in liquid to be clean, adsorbance increase is quickly；Area
Domain 2：When adsorbed material initial concentration is higher than 180ng/mL, with the increase of object initial concentration in liquid to be clean, purification
Antibody is basically unchanged to the adsorbance of object in post.The conformation of albumen after due to being handled in advance through Protein G, makes antibody
It can effectively be effectively combined, cause under the conditions of same amount of purifying carrier with antigen targets, after being handled through Protein G
Absorption carrier the suction without Protein G processing is higher than to the adsorbances of antigen targets (adsorption capacity is about 260ng/mL)
Appendix body (adsorption capacity is about 180ng/mL).
The lemon yellow affine in immunity column stability of embodiment 4
The immune affinity column prepared is preserved through 4 DEG C of refrigerators respectively, when selection 0,20,40,60,90d etc. are different respectively
Between point, immune affinity column and various buffer solutions are balanced to room temperature, by determine column capacity operating method, it is added back
Content of tartrazine in experiment, HPLC detection eluents is received, repeats 5 times, affinity column is evaluated by calculating average recovery rate
Stability.As a result Fig. 6 is seen, with the growth of period of storage, the purifying property of decontaminating column has a certain degree of decline, by 96% drop
It is low to 80%, the requirement of test can be met substantially.
Described above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications also should
It is considered as protection scope of the present invention.
SEQUENCE LISTING
<110>Institute of Feeds,China Academy of Agriculture Sciences
<120>A kind of coating protein G lemon yellow immune affinity column and preparation method thereof
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 480
<212> PRT
<213>Protein G
<400> 1
Glu Phe Asn Lys Tyr Gly Val Ser Asp Tyr Tyr Lys Asn Leu Ile Asn
1 5 10 15
Asn Ala Lys Thr Val Glu Gly Val Lys Asp Leu Gln Ala Gln Val Val
20 25 30
Glu Ser Ala Lys Lys Ala Arg Ile Ser Glu Ala Thr Asp Gly Leu Ser
35 40 45
Asp Phe Leu Lys Ser Gln Thr Pro Ala Glu Asp Thr Val Lys Ser Ile
50 55 60
Glu Leu Ala Glu Ala Lys Val Leu Ala Asn Arg Glu Leu Asp Lys Tyr
65 70 75 80
Gly Val Ser Asp Tyr His Lys Asn Leu Ile Asn Asn Ala Lys Thr Val
85 90 95
Glu Gly Val Lys Asp Leu Gln Ala Gln Val Val Glu Ser Ala Lys Lys
100 105 110
Ala Arg Ile Ser Glu Ala Thr Asp Gly Leu Ser Asp Phe Leu Lys Ser
115 120 125
Gln Thr Pro Ala Glu Asp Thr Val Lys Ser Ile Glu Leu Ala Glu Ala
130 135 140
Lys Val Leu Ala Asn Arg Glu Leu Asp Lys Tyr Gly Val Ser Asp Tyr
145 150 155 160
Tyr Lys Asn Leu Ile Asn Asn Ala Lys Thr Val Glu Gly Val Lys Ala
165 170 175
Leu Ile Asp Glu Ile Leu Ala Ala Leu Pro Lys Thr Asp Thr Tyr Lys
180 185 190
Leu Ile Leu Asn Gly Lys Thr Leu Lys Gly Glu Thr Thr Thr Glu Ala
195 200 205
Val Asp Ala Ala Thr Ala Glu Lys Val Phe Lys Gln Tyr Ala Asn Asp
210 215 220
Asn Gly Val Asp Gly Glu Trp Thr Tyr Asp Asp Ala Thr Lys Thr Phe
225 230 235 240
Thr Val Thr Glu Lys Pro Glu Val Ile Asp Ala Ser Glu Leu Thr Pro
245 250 255
Ala Val Thr Thr Tyr Lys Leu Val Ile Asn Gly Lys Thr Leu Lys Gly
260 265 270
Glu Thr Thr Thr Glu Ala Val Asp Ala Ala Thr Ala Glu Lys Val Phe
275 280 285
Lys Gln Tyr Ala Asn Asp Asn Gly Val Asp Gly Glu Trp Thr Tyr Asp
290 295 300
Asp Ala Thr Lys Thr Phe Thr Val Thr Glu Lys Pro Glu Val Ile Asp
305 310 315 320
Ala Ser Glu Leu Thr Pro Ala Val Thr Thr Tyr Lys Leu Val Ile Asn
325 330 335
Gly Lys Thr Leu Lys Gly Glu Thr Thr Thr Lys Ala Val Asp Ala Glu
340 345 350
Thr Ala Glu Lys Ala Phe Lys Gln Tyr Ala Asn Asp Asn Gly Val Asp
355 360 365
Gly Val Trp Thr Tyr Asp Asp Ala Thr Lys Thr Phe Thr Val Thr Glu
370 375 380
Met Val Thr Glu Val Pro Gly Asp Ala Pro Thr Glu Pro Glu Lys Pro
385 390 395 400
Glu Ala Ser Ile Pro Leu Val Pro Leu Thr Pro Ala Thr Pro Ile Ala
405 410 415
Lys Asp Asp Ala Lys Lys Asp Asp Thr Lys Lys Glu Asp Ala Lys Lys
420 425 430
Pro Glu Ala Lys Lys Glu Asp Ala Lys Lys Ala Glu Thr Leu Pro Thr
435 440 445
Thr Gly Glu Gly Ser Asn Pro Phe Phe Thr Ala Ala Ala Leu Ala Val
450 455 460
Met Ala Gly Ala Gly Ala Leu Ala Val Ala Ser Lys Arg Lys Glu Asp
465 470 475 480
<210> 2
<211> 427
<212> PRT
<213>Recombinant protein G
<400> 2
Gln Thr Pro Ala Glu Asp Thr Val Lys Ser Ile Glu Leu Ala Glu Ala
1 5 10 15
Lys Val Leu Ala Asn Arg Glu Leu Asp Lys Tyr Gly Val Ser Asp Tyr
20 25 30
His Lys Asn Leu Ile Asn Asn Ala Lys Thr Val Glu Gly Val Lys Asp
35 40 45
Leu Gln Ala Gln Val Val Glu Ser Ala Lys Lys Ala Arg Ile Ser Glu
50 55 60
Ala Thr Asp Gly Leu Ser Asp Phe Leu Lys Ser Gln Thr Pro Ala Glu
65 70 75 80
Asp Thr Val Lys Ser Ile Glu Leu Ala Glu Ala Lys Val Leu Ala Asn
85 90 95
Arg Glu Leu Asp Lys Tyr Gly Val Ser Asp Tyr Tyr Lys Asn Leu Ile
100 105 110
Asn Asn Ala Lys Thr Val Glu Gly Val Lys Ala Leu Ile Asp Glu Ile
115 120 125
Leu Ala Ala Leu Pro Lys Thr Asp Thr Tyr Lys Leu Ile Leu Asn Gly
130 135 140
Lys Thr Leu Lys Gly Glu Thr Thr Thr Glu Ala Val Asp Ala Ala Thr
145 150 155 160
Ala Glu Lys Val Phe Lys Gln Tyr Ala Asn Asp Asn Gly Val Asp Gly
165 170 175
Glu Trp Thr Tyr Asp Asp Ala Thr Lys Thr Phe Thr Val Thr Glu Lys
180 185 190
Pro Glu Val Ile Asp Ala Ser Glu Leu Thr Pro Ala Val Thr Thr Tyr
195 200 205
Lys Leu Val Ile Asn Gly Lys Thr Leu Lys Gly Glu Thr Thr Thr Glu
210 215 220
Ala Val Asp Ala Ala Thr Ala Glu Lys Val Phe Lys Gln Tyr Ala Asn
225 230 235 240
Asp Asn Gly Val Asp Gly Glu Trp Thr Tyr Asp Asp Ala Thr Lys Thr
245 250 255
Phe Thr Val Thr Glu Lys Pro Glu Val Ile Asp Ala Ser Glu Leu Thr
260 265 270
Pro Ala Val Thr Thr Tyr Lys Leu Val Ile Asn Gly Lys Thr Leu Lys
275 280 285
Gly Glu Thr Thr Thr Lys Ala Val Asp Ala Glu Thr Ala Glu Lys Ala
290 295 300
Phe Lys Gln Tyr Ala Asn Asp Asn Gly Val Asp Gly Val Trp Thr Tyr
305 310 315 320
Asp Asp Ala Thr Lys Thr Phe Thr Val Thr Glu Met Val Thr Glu Val
325 330 335
Pro Gly Asp Ala Pro Thr Glu Pro Glu Lys Pro Glu Ala Ser Ile Pro
340 345 350
Leu Val Pro Leu Thr Pro Ala Thr Pro Ile Ala Lys Asp Asp Ala Lys
355 360 365
Lys Asp Asp Thr Lys Lys Glu Asp Ala Lys Lys Pro Glu Ala Lys Lys
370 375 380
Glu Asp Ala Lys Lys Ala Glu Thr Leu Pro Thr Thr Gly Glu Gly Ser
385 390 395 400
Asn Pro Phe Phe Thr Ala Ala Ala Leu Ala Val Met Ala Gly Ala Gly
405 410 415
Ala Leu Ala Val Ala Ser Lys Arg Lys Glu Asp
420 425
Claims (10)
1. a kind of lemon yellow immune affinity column, including carrier, Protein G and lemon yellow antibody.
2. lemon yellow immune affinity column according to claim 1, it is characterised in that the Protein G is included according to GenBank
Protein G shown in CAA27638.1 sequences, and the recombinant protein G Jing Guo sequence optimisation.
3. lemon yellow immune affinity column according to claim 2, it is characterised in that the Protein G is based on GenBank
Recombinant protein Gs of the CAA27638.1 Jing Guo sequence optimisation.
4. the lemon yellow immune affinity column according to claims 1 to 3, it is characterised in that the Protein G passes through chemical bond
It is coupled on carrier, lemon yellow antibody uses cross-linking agents after being combined with Protein G.
5. the lemon yellow immune affinity column according to claim 3, it is characterised in that the restructuring egg of described sequence optimisation
White G, including reduced carrier-recombinant protein G steric hindrances effect, and add recombinant protein G and lemon yellow IgG antibody
The optimization of binding ability.
6. the lemon yellow immune affinity column according to claim 3, it is characterised in that the recombinant protein G optimizes site
For amputation GenBank CAA27638.1 sequences 53 amino acid of N-terminal.
7. the lemon yellow immune affinity column according to claim 1, it is characterised in that described carrier is that agarose coagulates
Glue；Described Ago-Gel includes the Ago-Gel, the Ago-Gel of crosslinking, polyacrylic acid agarose of cyanogen bromide-activated
Gel, polyacrylamide Ago-Gel, the one or more of sephadex and cellulose.
8. the lemon yellow immune affinity column according to claim 1, it is characterised in that the lemon yellow antibody includes Dan Ke
Grand IgG antibody and polyclonal antibody.
9. a kind of preparation method of lemon yellow immune affinity column, it is characterised in that comprise the following steps：
1) it is support-activated；
2) recombinant protein G is coupled with the carrier activated；
3) handle carrier-recombinant protein G, and with lemon yellow antibody coupling；
4) washing lemon yellow antibody-recombinant protein G- carriers, dress post is to obtain lemon yellow immune affinity column.
10. preparation method according to claim 9, it is characterised in that comprise the following steps：1) agarose Sepharose
4B, adds 0.8M NaOH, 30% epoxychloropropane, 2mg/ml sodium borohydride NaBH4, shaking table reaction 8h enters at 25 DEG C
Row activation；
2) the Sepharose 4B of every gram of activation add 30~200nmol recombinant protein G, in 0.1MNaHCO3, 0.5M NaCl
In pH8.8 buffer solution, room temperature coupling 2h, or 4 DEG C of couplings are stayed overnight；
3) coupled product reacts at room temperature 2h with 1M TrisHCl pH 8.0；
4) the Sepharose- Protein Gs being coupled, are washed with 20mM pH7.4 PBS；
5) lemon yellow antibody is dissolved in 20mM pH7.4 PBS, every gram of Protein G-Sepharose add 200nmol~
1.2umol antibody, is reacted at room temperature 30 minutes；
6) lemon yellow antibody-white G-Sepharose carriers are washed with 20mM PBS pH7.4, then adds final concentration 0.2M's
The dimethyl pimelate (DMP) of triethanolamine and 20mM, pH8.3 reacts at room temperature 1h；
7) with 20mM pH7.4 monoethanolamine terminating reaction, lemon yellow is washed with the 10mMPBSpH7.4 containing 0.01% thimerosal
Antibody-recombinant protein G-Sepharose carriers, fills post, is put in 4 DEG C of preservations；
Wherein, the recombinant protein G is the recombinant protein G based on GenBank CAA27638.1 Jing Guo sequence optimisation.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114130377A (en) * | 2021-12-14 | 2022-03-04 | 无锡创谱生物科技有限公司 | Affinity chromatography filler, preparation method and application thereof |
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