CN106669638A - Protein G coated Sundan red I immunoaffinity column and preparation method thereof - Google Patents
Protein G coated Sundan red I immunoaffinity column and preparation method thereof Download PDFInfo
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- CN106669638A CN106669638A CN201710045321.7A CN201710045321A CN106669638A CN 106669638 A CN106669638 A CN 106669638A CN 201710045321 A CN201710045321 A CN 201710045321A CN 106669638 A CN106669638 A CN 106669638A
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
- B01J20/286—Phases chemically bonded to a substrate, e.g. to silica or to polymers
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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
- B01D15/10—Selective adsorption, e.g. chromatography characterised by constructional or operational features
- B01D15/20—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to the conditioning of the sorbent material
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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
- B01D15/10—Selective adsorption, e.g. chromatography characterised by constructional or operational features
- B01D15/22—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to the construction of the column
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Abstract
The invention provides a Sundan red I immunoaffinity column which comprises a carrier, protein G and a Sundan red I antibody, wherein the protein G is coupled with the carrier through chemical bonds; the Sundan red I antibody is in cross-link with the protein G by using a cross-linking agent after being joined; the protein G is recombinant protein G which is based on GenBank CAA27638.1 and is subjected to gene recombinant expression. Under the condition that detection properties are not affected, the use amount of the Sundan red I antibody of a sepharose gel carrier pretreated with the protein G is about 3/4 of that of an antibody for preparing purification column packing by using a direct coupling method. The Sundan red I provided by the invention is very high in purity, can be directly used in high performance liquid chromatography detection without need of extra subsequent purification treatment, therefore, the time and the cost of operators can be saved.
Description
Technical field
The invention belongs to field of food inspection, and in particular to a kind of Sudan red immune affinity column and its system of coating protein G
Preparation Method.
Background technology
Tonyred is a kind of azo of synthetic, oil-soluble chemical industry stain, scientist Da Di in 1896 by its
It is named as tonyred and uses till today.Tonyred is largely used in the fields such as biology, chemistry, for machine oil, car wax and footwear
The industrial products such as oil.As people are to tonyred structure and causing toxicity progressively understanding, international cancer research institution (IARC) will
Tonyred be classified as the 3rd class can carcinogen, though this kind of material lack it is enough directly make the carcinogenic evidence of the mankind, with latent
Carcinogenic danger.
Tonyred is lipotropy azo-compound, and outward appearance is insoluble in water in kermesinus or buff flat crystal, main bag
Include I, II, III and IV 4 types.Wherein II, III and IV are the chemical derivative of I.The metabolites in vivo of Sudan I
For aniline and 1- amino-beta naphthal.Aniline is the raw material for manufacturing dyestuff, rubber accelerator and antioxidant etc., is a kind of important
Organic synthesis intermediate.But once aniline contacts human body skin or into after digestive system, on the one hand because aniline can be straight
Connect and act on hepatocyte, cause toxic hepatic disease, it is also possible to induce liver cell gene and morph, increased human canceration
Probability；On the other hand, it is possible to because the Fe (II) that aniline combines hemoglobin is oxidized to Fe (III), cause hemoglobin
Methemoglobinemia cannot be suffered from reference to oxygen.According to another report, long-term intake aniline can also cause the nervous system of human body to damage
Evil.Add tonyred in violation of rules and regulations in feedstuff and also have a strong impact on the healthy of people, affect the normal development of animal husbandry.
At present the conventional detection method of tonyred has thin layer chromatography (TLC), elisa (ELISA), height
Effect liquid phase chromatogram method (HPLC) and Liquid Chromatography-Mass Spectrometry (LC-MS/MS) etc..Elisa (ELISA) has
Have the advantages that it is efficient, sensitive, can sample after Jing simple process directly detected, but the method to there is also false positive rate higher
Shortcoming.HPLC, LC-MS/MS method is quantitative analyses tonyred most common method.When being detected using liquid phase or liquid matter method, it is
Impurity interference is reduced, its impact to Detection results is reduced, needs first carry out purified treatment to the sample of various different substrates.Mesh
Front conventional purifying treatment method is exactly solid phase extraction, is also column chromatography.It is to purify most popular method at present.Its
In, the analyze speed of affine in immunity column method is fast, and sensitivity is high, and separation efficiency and the response rate are high, safe and reliable, thus become most
Conventional 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 ultimate principle of these methods is all to carry out carrier matrix after chemical activation, antibody coupling to be arrived
On carrier；Protein G (Protein G) is the energy extracted from the cell wall and culture supernatant of many bacterial strains of streptococcus A, C and G group
The albumen combined with IgG Fc fragments specifics, the albumen energy specificity in combination with the Fc positions of many animals antibody, and
With very high affinity.Although there is high-affinity using Protein G, affine layer can be reduced using the carrier of Protein G process
The elution speed of analysis post, so as to reduce the utilization rate of antibody and the efficiency of detection.
The content of the invention
In view of this, the first object of the present invention is to provide a kind of Sudan red immune affinity column, including carrier, Protein G
With Sudan red antibody, wherein, the Protein G is coupled on carrier by chemical bond, and the Sudan red antibody is combined with Protein G
After use cross-linking agents.
Preferably, in Sudan red 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 through the recombiant protein G of DNA recombinant expression.
Preferably, in Sudan red immune affinity column of the present invention, the Protein G is based on GenBank
Recombiant protein Gs of the CAA27638.1 through DNA recombinant expression.
Preferably, in Sudan red immune affinity column of the present invention, the Protein G is coupled at carrier by chemical bond
On, Sudan red antibody uses cross-linking agents after being combined with Protein G.
Preferably, in Sudan red immune affinity column of the present invention, the recombiant protein G of described DNA recombinant expression,
Carrier-sterically hindered effects of recombiant protein G are including reduced, the combination energy of recombiant protein G and Sudan red IgG antibody is increased
The optimization of power.
Preferably, in Sudan red immune affinity column of the present invention, the recombiant protein G optimization sites are amputation
GenBank CAA27638.1 sequences 53 aminoacid of N-terminal.
Preferably, in Sudan red immune affinity column of the present invention, described carrier is agarose gel；Described
The agarose gel of agarose gel including cyanogen bromide-activated, the agarose gel of crosslinking, polyacrylic acid agarose gel, poly- third
One or more of acrylamide agarose gel, polydextran gel and cellulose；It is highly preferred that the carrier is agarose
Sepharose 4B。
Preferably, in Sudan red immune affinity column of the present invention, described anti-Sudan red antibody includes monoclonal
IgG antibody and polyclonal antibody.
Another object of the present invention is to provide a kind of preparation method of Sudan red immune affinity column, comprise the following steps：
1) it is support-activated；
2) recombiant protein G is coupled with the carrier of activation；
3) process carrier-recombiant protein G, and with Sudan red antibody coupling；
4) Sudan red antibody-recombiant protein G- carriers are washed, dress post obtains Sudan red immune affinity column.
Preferably, in the preparation method of Sudan red immune affinity column of the present invention, comprise the following steps：
1) agarose Sepharose 4B, add the NaOH of 0.8M, 30% epoxychloropropane, the sodium borohydride of 2mg/ml
NaBH4, shaking table reaction 8h is activated at 25 DEG C；
2) the Sepharose 4B of per gram of activation add the recombiant protein G of 30~200nmol, in 0.1M NaHCO3, 0.5M
In the buffer of NaCl pH8.8, room temperature is coupled 2h, or 4 DEG C are coupled overnight；
3) the coupled product room temperature reaction 2h of 1M TrisHCl pH 8.0；
4) the Sepharose- Protein Gs being coupled, are washed with the PBS of 20mM pH7.4；
5) Sudan red antibody is dissolved in the PBS of 20mM pH7.4, every gram of Protein G-Sepharose adds 200nmol
~1.2umol antibody, room temperature reaction 30 minutes；
6) Sudan red Antibody-protein G-Sepharose carriers are washed with 20mM PBS pH7.4, is subsequently adding dense eventually
The triethanolamine of degree 0.2M and the dimethyl pimelate (DMP) of 20mM, pH8.3, room temperature reaction 1h；
7) with the ethanolamine terminating reaction of 20mM pH7.4, washed with the 10mMPBS pH7.4 containing 0.01% thimerosal
Sudan red Antibody-protein G-Sepharose carriers, fill post, are put in 4 DEG C of preservations；
Wherein, the recombiant protein G is the recombiant protein based on GenBank CAA27638.1 through DNA recombinant expression
G。
From the foregoing, it will be observed that the present invention at least has advantages below：
1) we clone the gene order of Protein G of having recombinated (GenBank CAA27638.1), in expression in escherichia coli
The gene recombinant protein G for having high-affinity with antibody is gone out.The characteristics of make use of Protein G to be 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 the binding ability of specificity with the Fc regions of antibody.Protein G
After antibodies, the Fab regions of antibody are free outside, do not affect the antigen binding capacity of antibody.Through our gene optimizations
Protein G after restructuring, the Protein G of 1 molecule can be with reference to 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 Sudan red binding capacity is big, the characteristics of purification efficiency is high.
After the Protein G of gene recombinaton is coupled on agarose carrier, then Sudan red antibody coupling is carried out, it is and traditional
Directly it is coupled and compares, after being coupled on carrier as middle ware arm with the Protein G of gene recombinaton, reduce sterically hindered effect, increases
Add the binding ability of recombiant protein G and Sudan red IgG antibody, impurity is sufficiently flowed out, enhance clean-up effect,
So that the Fab ends of antibody are towards outside, sterically hindered non-confrontational former calmodulin binding domain CaM constitutes interference, at carrier elder generation Jing Protein Gs
After reason, then antibody coupling is carried out, can not only improve the homogeneity of different purification intercolumniations, and the utilization rate of antibody can be improved.
Therefore, it is combined with Ig G antibody specificities by designing recombiant protein G, antibody is coupled to by Protein G
On carrier.And the Fab fragments for causing IgG antibody sufficiently expose outside, and Sudan red capture ability is greatly improved,
Sudan red purification efficiency also effectively improves.Test shows that under conditions of detection performance is not affected, first Jing Protein Gs are pre-
The expense of the Sudan red antibody of agarose gel carrier of process is about the need that direct coupling method prepares the antibody of purification column packing
3/4ths of consumption.
2) present invention uses the Protein G after genetic modification, by the optimization to codon and Ig G domain gene is combined
Optimization.The antibody binding capacity for making Protein G is increased substantially, and then improves Sudan red purification efficiency.
3) Sudan red easy to operate using purification of the present invention, several steps can be obtained by higher Sudan red of purity, more square
Convenient to operate person uses.
4) the Sudan red purity for being obtained using the present invention is very high, subsequently just can be direct without doing other purification process again
For high performance liquid chromatography detection, time and the expense of operator are saved.
Description of the drawings
Fig. 1 is the Sudan red 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 Jing recombiant protein G coupling after further with Sudan red antibody
It is coupled schematic diagram；
Fig. 3 be agarose carrier directly with Sudan red antibody coupling schematic diagram；
Fig. 4 be agarose carrier directly with icon meaning figure in Sudan red antibody coupling schematic diagram；
Fig. 5 is impact schematic diagram of the Sudan red initial concentration to the object adsorption capacity of decontaminating column in solution；
Fig. 6 is Sudan red immune affinity column bin stability schematic diagram.
Specific embodiment
In one embodiment of the invention, there is provided Sudan red immune affinity column and preparation method thereof:
1st, it is support-activated
Agarose carrier, Sepharose 4B is selected to be activated with Epichlorohydrin activation method.
2% preswollen agarose gel Sepharose 4B are taken, is fully rinsed with the distilled water of 20 times of volumes, washed away
The ethanol of remaining, with funnel moisture is filtered out.
Weigh and filter off the wet gel 5g after moisture, add NaOH, the 30% epoxychloropropane 2mL of 7.5mL 0.8M,
Sodium borohydride NaBH4, the 5mL of 2mg/mL, the shaking table reaction 8h at 25 DEG C.
After reaction, substantial amounts of distilled water wash is used, remove the epoxychloropropane mixed in gel.
2nd, by the coupling of Protein G and activated carrier Sepharose 4B
By the agarose gel Sepharose 4B for the having activated coupling buffer (NaHCO of 0.1M3, 0.8M NaCl,
PH8.9) wash 3 times.The Sepharose 4B of per gram of activation add the Protein G of 30~200nmol, room temperature to be coupled 2h, or 4 DEG C
It is coupled overnight.The coupled product room temperature reaction 2h of 1M TrisHCl pH 8.0.
The agarose carrier 20mM being coupled, the phosphate buffer PBS of pH7.4 are washed 3 times.Coupling has recombiant protein
The agarose carrier Sepharose of G is named as Protein G-Sepharose.
Wherein, recombiant protein G is by Protein G sequence ((GenBank CAA27638.1, SEQ ID NO：1), N-terminal is amputated
Recombiant protein G sequence (SEQ ID NO2) is obtained after 41 aminoacid and transfers to the synthesis of gill biochemistry (Shanghai) Co., Ltd..
3rd, Sudan red antibody is resisted to be connected with the recombiant protein G on Protein G-Sepharose carriers
Protein G has the affinity of specificity with antibody, under certain reaction condition, has recombiant protein G's by being coupled
Agarose gel is attached with antibody, and antibody is connected on agarose.Because recombiant protein G is with the binding site of antibody
The Fc regions of antibody, the antigen binding domain of antibody is unaffected, has fully ensured that the antigen binding capacity of antibody.
Sudan red antibody is dissolved in the PBS of 20mM pH7.4, every gram of Protein G-Sepharose add 200nmol~
1.2 μm of ol antibody, room temperature reaction 30min.
Carrier with reference to after is washed with PBS.The carrier for being connected with antibody is named as antibody-recombiant protein G-Sepharose.
4th, carrier crosslinking
Antibody-recombiant protein G-Sepharose is washed into 3 times with 20mM PBS pH7.4.It is subsequently adding final concentration 0.2M
Triethanolamine and 20mM dimethyl pimelate (DMP), pH8.3, room temperature reaction 1 hour.
20mM, the ethanolamine terminating reaction of pH9.0 is added to be washed with the 10mM PBS pH7.4 containing 0.01% thimerosal
Sudan red antibody-recombiant protein G-Sepharose carriers.
5th, post is filled
Antibody-agarose carrier after crosslinking is fitted into as needed in chromatographic column, being put in 4 DEG C of preservations can make as needed
The Sudan red affinity purification post of standby different capabilities.
Further technical scheme is illustrated below by way of specific embodiment, it should be understood that be only below this
Bright exemplary illustration, is not limited to the protection domain of the claims in the present invention.
The preparation of the Sudan red immunoaffinity purification post of embodiment 1
1. agarose gel activation
2% agarose gel Sepharose 4B are taken, is fully rinsed with the distilled water of 20 times of volumes, wash away the second of remaining
Alcohol.Moisture is filtered out with funnel.Weigh and filter off the wet gel 5g after moisture content, add the NaOH of 7.5mL0.8M, 30% epoxy chlorine
The sodium borohydride NaBH of propane 2mL, 2mg/mL4, 5mL, the shaking table reaction 8h at 25 DEG C.After reaction, 50mL distilled water washs are used,
Remove the epoxychloropropane mixed in gel.
2. the coupling of recombiant protein G and the agarose gel of activation
The agarose gel after 1 gram of activation is taken, with the coupling buffer (NaHCO of 0.1M3, 0.8M NaCl, pH8.9) wash
Wash 3 times.Protein G 20mL of 2mg/mL, room temperature is added to be coupled 4h.
The agarose carrier 20mM being coupled, the phosphate buffer PBS of pH7.4 are washed 3 times.Coupling has recombiant protein
The agarose carrier Sepharose of G is named as recombiant protein G-Sepharose.
3. resist Sudan red IgG monoclonal antibodies to be combined with Protein G-Sepharose
Anti- Sudan red antibody is dissolved in the PBS of 20mM, pH7.4, final concentration 2mg/mL, cumulative volume 10mL.
Protein G-Sepharose is washed three times with the PBS of 20mM pH7.4, each 30mL.PBS will be dissolved in
In Sudan red antibody add in washed Protein G-Sepharose, room temperature combines 30min.
Carrier with reference to after the PBS of 20mM pH7.4 is washed three times, each 30mL.The carrier for being connected with antibody is named as
Antibody-protein G-Sepharose.
4. it is crosslinked with reference to the agarose Sepharose of IgG
Antibody-Protein G-Sepharose is washed 3 times with the borate buffer of 0.1M pH9.0, each 30mL.
By wet gel solid, the borate buffer 20mL of 0.1M pH9.0 is added, cross-linking agent heptan two is added in buffer
Imido dimethyl phthalate (DMP) is to final concentration 20mM.Normal temperature crosslinked 1h.
Add 100mM, the ethanolamine 20mL terminating reactions of pH9.0.And closed 10 minutes with the ethanolamine 20mL of 50mM.
The PBS of the Antibody-protein G-Sepharose carrier 20mM after crosslinking, pH7.4 is washed 3 times, each 30mL.
5. post is filled
Sepharose after crosslinking is resuspended in into 10mL 20mM PBS pH7.4, the affinity purification post post of sky is then charged into
In body.
The Sudan red immunoaffinity purification post recovery test of embodiment 2
Recovery of standard addition measurement result (n=3)
Three kinds of sample recovery rate experimental results see the table below：
Embodiment 3：The measure of Sudan red immune affinity column column capacity --- the inventive method is net with prepared by conventional method
Change column performance to compare
The preparation of the decontaminating column that first Jing recombiant proteins G is processed is prepared by embodiment 1
Affinity column is prepared without the direct coupling method for preparing of the decontaminating column of recombiant protein G process
1st, it is support-activated
Agarose carrier, Sepharose 4B is selected to be activated with Epichlorohydrin activation method.
2% preswollen agarose gel Sepharose 4B are taken, is fully rinsed with the distilled water of 20 times of volumes, washed away
The ethanol of remaining, with funnel moisture is filtered out.
Weigh and filter off the wet gel 5g after moisture, add the NaOH, 30% epoxychloropropane 2mL, 2mg/ of 7.5mL0.8M
The sodium borohydride NaBH of mL4, 5mL, the shaking table reaction 8h at 25 DEG C.
After reaction, substantial amounts of distilled water wash is used, remove the epoxychloropropane mixed in gel.
2nd, Sudan red antibody is resisted to be connected with Sepharose carriers
Under certain reaction condition, agarose gel is attached with antibody, antibody is connected on agarose.
Sudan red antibody is dissolved in the PBS of 20mM pH7.4, every gram of Sepharose adds 200nmol~1.2 μ
Mol antibody, room temperature reaction 30min.
Carrier with reference to after is washed with PBS.The carrier for being connected with antibody is named as antibody-Sepharose.
3rd, carrier crosslinking
Antibody-Sepharose is washed into 3 times with 20mM PBS pH7.4.It is subsequently adding the triethanolamine of final concentration 0.2M
With the dimethyl pimelate (DMP) of 20mM, pH8.3, room temperature reaction 1 hour.
20mM, the ethanolamine terminating reaction of pH9.0 is added to be washed with the 10mM PBS pH7.4 containing 0.01% thimerosal
Sudan red antibody-Sepharose carriers.
4. post is filled
Antibody-agarose carrier after crosslinking is fitted into as needed in chromatographic column, being put in 4 DEG C of preservations can make as needed
The Sudan red affinity purification post of standby different capabilities.
10mL concentration for 100ng/mL Sudan red PBS solution with 1mL/min flow velocity loadings, then use 10mL ultra-pure waters
Drip washing pillar is Sudan red on pillar to wash away non-specific adsorption, finally with 5mL eluents by object eluting, HPLC
Detection, according to the following formula, calculates column capacity.
Take by the populated decontaminating column of such as accompanying drawing 1, the certain density object solution of 1mL crossed into post, with distillation post is washed,
Use 1mL methanol-eluted fractions, upper HPLC to carry out detection by quantitative, determine measure of the decontaminating column to " capture " capacity of object.
Fig. 2 show agarose carrier Jing recombiant protein G coupling after further with Sudan red antibody coupling schematic diagram；Fig. 3
Show agarose carrier directly with Sudan red antibody coupling schematic diagram.Wherein, each diagram in Fig. 2, Fig. 3 is shown in Fig. 4
Agarose, Sudan red antibody and recombiant 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 of 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
Antibody is to the adsorbance of object and the relation of object initial concentration in solution 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 200ng/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 200ng/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.Due to the conformation of the advance albumen Jing after Protein G process, antibody is made
Effectively can be effectively combined with antigen targets, cause under the conditions of same amount of purifying carrier, Jing after Protein G process
Absorption carrier to the adsorbance (adsorption capacity is about 280ng/mL) of antigen targets higher than the suction without Protein G process
Appendix body (adsorption capacity is about 200ng/mL).
The Sudan red affine in immunity column stability of embodiment 4
The immune affinity column for preparing is distinguished into 4 DEG C of Refrigerator stores of Jing, when respectively selection 0,20,40,60,90d etc. is different
Between point, immune affinity column and various buffer are balanced to room temperature, by determine column capacity operational approach, it is added back
Experiment is received, Sudan red content in HPLC detection eluents, repetitive operation 5 times evaluates affinity column 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 93% drop
It is low to 83%, the requirement of test can be met substantially.
The 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 Sudan red immune affinity column of coating protein G 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>Recombiant 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 Sudan red immune affinity column, including carrier, Protein G and Sudan red antibody, it is characterised in that the Protein G
It is coupled on carrier by chemical bond, the Sudan red antibody uses cross-linking agents after being combined with Protein G.
2. Sudan red immune affinity column according to claim 1, it is characterised in that the Protein G include according to
The Protein G that GenBank CAA27638.1 sequence tables reach, and through the recombiant protein G of DNA recombinant expression.
3. Sudan red immune affinity column according to claim 2, it is characterised in that the Protein G is based on GenBank
Recombiant protein Gs of the CAA27638.1 through DNA recombinant expression.
4. according to the Sudan red immune affinity column described in claims 1 to 3, it is characterised in that the Protein G is by chemistry
Key is coupled on carrier, and Sudan red antibody uses cross-linking agents after being combined with Protein G.
5. according to the Sudan red immune affinity column described in claim 3, it is characterised in that described DNA recombinant expression
Recombiant protein G, including reduced carrier-sterically hindered effects of recombiant protein G, increased recombiant protein G with Sudan red antibody
The optimization of the binding ability of IgG.
6. according to the Sudan red immune affinity column described in claim 3, it is characterised in that the recombiant protein G optimizes site
To amputate GenBank CAA27638.1 sequences 53 aminoacid of N-terminal.
7. according to the Sudan red immune affinity column described in claim 1, it is characterised in that described carrier is that agarose coagulates
Glue；Described agarose gel includes the agarose gel of cyanogen bromide-activated, agarose gel, the polyacrylic acid agarose of crosslinking
One or more of gel, polyacrylamide agarose gel, polydextran gel and cellulose.
8. according to the Sudan red immune affinity column described in claim 1, it is characterised in that described anti-Sudan red antibody bag
Include monoclonal IgG antibody and polyclonal antibody.
9. a kind of preparation method of Sudan red immune affinity column, it is characterised in that comprise the following steps：
1) it is support-activated；
2) recombiant protein G is coupled with the carrier of activation；
3) process carrier-recombiant protein G, and with Sudan red antibody coupling；
4) Sudan red antibody-recombiant protein G- carriers are washed, dress post obtains Sudan red immune affinity column.
10. preparation method according to claim 9, it is characterised in that comprise the following steps：1) agarose Sepharose
4B, adds the NaOH of 0.8M, 30% epoxychloropropane, the sodium borohydride NaBH of 2mg/ml4, shaking table reaction 8h enters at 25 DEG C
Row activation；
2) the Sepharose 4B of per gram of activation add the recombiant protein G of 30~200nmol, in 0.1M NaHCO3, 0.5M NaCl
In the buffer of pH8.8, room temperature is coupled 2h, or 4 DEG C are coupled overnight；
3) the coupled product room temperature reaction 2h of 1M TrisHCl pH 8.0；
4) the Sepharose- Protein Gs being coupled, are washed with the PBS of 20mM pH7.4；
5) Sudan red antibody is dissolved in the PBS of 20mM pH7.4, every gram of Protein G-Sepharose add 200nmol~
1.2umol antibody, room temperature reaction 30 minutes；
6) Sudan red Antibody-protein G-Sepharose carriers are washed with 20mM PBS pH7.4, is subsequently adding final concentration
The triethanolamine of 0.2M and the dimethyl pimelate (DMP) of 20mM, pH8.3, room temperature reaction 1h；
7) with the ethanolamine terminating reaction of 20mM pH7.4, with the 10mM PBS pH7.4 containing 0.01% thimerosal the Sudan is washed
Red I Antibody-protein G-Sepharose carriers, fill post, are put in 4 DEG C of preservations；
Wherein, the recombiant protein G is the recombiant protein G based on GenBank CAA27638.1 through DNA recombinant expression, institute
Recombiant protein G optimization sites are stated for amputation GenBank CAA27638.1 sequences 53 aminoacid of N-terminal.
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| CN108479114A (en) * | 2018-03-29 | 2018-09-04 | 山东美正生物科技有限公司 | A kind of research of diarrhetic shellfish poisons immune affinity column and its preparation method and application |
| CN108479113A (en) * | 2018-03-29 | 2018-09-04 | 山东美正生物科技有限公司 | A kind of aminoglycoside antibiotics immune affinity column and its preparation method and application |
| CN108553940A (en) * | 2018-03-29 | 2018-09-21 | 山东美正生物科技有限公司 | A kind of tetracycline antibiotics immune affinity column and its preparation method and application |
| CN108761059A (en) * | 2018-03-29 | 2018-11-06 | 山东美正生物科技有限公司 | A kind of diethylstilbestrol class antibiotic immune affinity column and its preparation method and application |
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| CN108325510A (en) * | 2018-03-29 | 2018-07-27 | 山东美正生物科技有限公司 | A kind of memory loss saxitoxin immune affinity column and its preparation method and application |
| CN108479114A (en) * | 2018-03-29 | 2018-09-04 | 山东美正生物科技有限公司 | A kind of research of diarrhetic shellfish poisons immune affinity column and its preparation method and application |
| CN108479113A (en) * | 2018-03-29 | 2018-09-04 | 山东美正生物科技有限公司 | A kind of aminoglycoside antibiotics immune affinity column and its preparation method and application |
| CN108553940A (en) * | 2018-03-29 | 2018-09-21 | 山东美正生物科技有限公司 | A kind of tetracycline antibiotics immune affinity column and its preparation method and application |
| CN108761059A (en) * | 2018-03-29 | 2018-11-06 | 山东美正生物科技有限公司 | A kind of diethylstilbestrol class antibiotic immune affinity column and its preparation method and application |
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