CN109470852A - A kind of breast milk polyclonal antibody affinity column and preparation method thereof - Google Patents
A kind of breast milk polyclonal antibody affinity column and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a kind of breast milk polyclonal antibody affinity columns and preparation method thereof, the breast milk polyclonal antibody is to be prepared using the breast milk for removing fat as immunogene, affinity adsorbent is prepared with this breast milk polyclonal antibody and solid phase ligand support, it is added in chromatographic column, breast milk polyclonal antibody affinity column is made after washed, closing, balance.Preparation method of the present invention is simple, can the lower albumen of signal relatively low to abundance in breast milk, in mass spectrum be enriched with, be conducive to the albumen profile that the mankind more correctly recognize breast milk, in terms of the relevant technologies that can apply to breast milk proteins matter group downstream, be worth being widely applied and apply.
Description
Technical field
The invention belongs to immunoaffinity chromatography technical fields.More particularly, to a kind of breast milk polyclonal antibody parent
With column and preparation method thereof.
Background technique
Breast milk is as the ideal natural food of baby, in addition to protein, fat, carbohydrate, minerals, microelement
Deng being provided outside energy and nutriment for infants growth and development, bioactive substance also rich in, enhancing the anti-sense of baby
Dye ability promotes histoorgan development, establishes the various aspects such as self immune system or even adjusting baby's social action and play and focus on
The biological function wanted.
By the research of breast milk proteins matter group, biologically active protein in breast milk is disclosed, in understanding breast milk
The profile of protein and the formula for improving baby milk have key meaning.But these biologically active protein,
Its abundance is relatively low, moreover signal of the low-abundance protein in mass spectrum is lower, is unfavorable for the mankind and more correctly recognizes breast milk
Protein profiles.Therefore high-abundance proteins are removed at present, enrichment low-abundance protein is the main policies for solving research bottleneck.
Traditional Separation of Proteins enrichment method oneself through being unable to satisfy growing technical need, be especially embodied in low rich
Albumen is modified after spending albumen, glycosylation and aspect is analyzed in the separation of high-molecular-weight protein.The research that king waits quietly " utilizes affine in immunity
Chromatographic technique removal breast milk in high-abundance proteins research " in establish it is a kind of removal breast milk in high-abundance proteins method, make
Low-abundance protein is enriched with, but the immune affinity chromatographic column of research preparation can remove 4 in breast milk to a certain extent
Kind high-abundance proteins, elution effect separating effect are poor.
Summary of the invention
The technical problem to be solved by the present invention is to overcome high-abundance proteins removal in existing breast milk and low-abundance protein enrichments
The defect and deficiency of technology provide a kind of breast milk polyclonal antibody, and based on antibody construction immune affinity column, can more have
High-abundance proteins in effect ground removal breast milk, the lower albumen of signal relatively low to abundance in breast milk, in mass spectrum carry out
Enrichment, is conducive to the albumen profile that the mankind more correctly recognize breast milk, can apply to the correlation in breast milk proteins matter group downstream
Technical aspect.
The object of the present invention is to provide a kind of breast milk polyclonal antibodies.
Another object of the present invention is to provide a kind of immune affinity column prepared using above-mentioned breast milk polyclonal antibody.
Above-mentioned purpose of the present invention is achieved through the following technical solutions:
It is to be prepared using the breast milk for removing fat as immunogene the method comprises the steps of firstly, preparing a kind of breast milk polyclonal antibody.
It is preferred that immunizing dose 1.25mg/ is only.
Preferably, antibody-solutions are neutralized with the buffer of meta-alkalescence immediately after by the elution of the solution of slant acidity, avoid resisting
Body loses activity under conditions of slant acidity.
Preferably, the solution of the slant acidity is the Glycine eluent of pH 2.5-3.5.
It is highly preferred that the solution of the slant acidity is the Glycine eluent of pH 3.0.
Preferably, the buffer of the meta-alkalescence is the Tris-HCI of pH8-9.
It is highly preferred that the buffer of the meta-alkalescence is the Tris-HCI of pH8.5.
Preferably, 20-35min is concentrated by ultrafiltration in 0-8 DEG C of antibody after purification, 3000-5000rpm, proposes the concentration of antibody
It is high.
It is highly preferred that 30min is concentrated by ultrafiltration in 4 DEG C of antibody, 4000rpm after purification.
Preferably, gained antibody is purified by Protein A/G agarose.
Specifically, the polyclonal antibody the preparation method is as follows:
S1. breast milk is centrifuged after thawing, and avoids upper layer whey portion, draws lower layer's whey portion；
S2. initial immunity: breast milk and Freund's adjuvant emulsify, and the standard of emulsification is one drop of liquid of drop on liquid level, proof of not scattering
Emulsification is completed；
S3. follow-up immunization: the antigen of equivalent is mixed with non-Freund's complete adjuvant；
S4. the 4th time it is immune after, arterial blood extracting, serum is by Protein A/G agarose purifying.
Preferably, the temperature of defrosting described in step S1 is 0-8 DEG C.
It is highly preferred that the temperature of defrosting described in step S1 is 4 DEG C.
Preferably, the condition of centrifugation described in step S1 is that 1500-2500g is centrifuged 10-30min at 0-8 DEG C.
It is highly preferred that the condition of centrifugation described in step S1 is that 2000g is centrifuged 20min at 4 DEG C.
The immunization method of step S2 and S3: multiple spot subcutaneous injection, immunizing dose 1.25mg/ is only.
The method purified in step S4 is as follows: whole purification process operate on protein purification instrument, by Protein A/G
Agarose fills column, after serum is crossed column repeatedly, first uses 0.15M NaCl, 20mM Na2HPO4, the buffer washing of pH 7.0, to
After baseline is steady, antibody-solutions use the eluent of the Glycine pH 3.0 of 0.1M again, collect the appearance liquid in this stage, then
Antibody is tuned into neutrality with 1M Tris-HCl pH8.5 solution immediately, antibody is avoided to inactivate in acid condition.
Wherein, the solution of antibody elution has been gone certain concentration by super filter tube (4 DEG C, 4000rpm, 30min), is made
The concentration of IgG obtains certain raising.
In addition, being based on above-mentioned breast milk polyclonal antibody, a kind of breast milk polyclonal antibody affinity column is constructed, in column
Fixed is above-mentioned breast milk polyclonal antibody.By the specific binding of antibody and antigen, to remove the high abundance egg in breast milk
It is white.
It preferably, is to be coupled the breast milk polyclonal antibody and solid phase ligand support to obtain affinity adsorbent, it will be close
Breast milk polyclonal antibody affinity column is made in adsorbent filling column.Washed, closing is needed after filling affinity adsorbent, is put down
Breast milk polyclonal antibody affinity column is made in weighing apparatus.
Preferably, the solid phase ligand support is agar polysaccharide 4B.
Preferably, the step of coupling is as follows:
S11. polyclonal antibody is dialysed in coupling buffer；
S12. it is coupled: agar polysaccharide 4B being fitted into chromatographic column, excessive acetone is first washed away with coupling buffer, then by antibody
It is added in chromatographic column, overnight；
S13. it washs: cleaning extra antibody with the coupling buffer of at least 5 times agar polysaccharide 4B volumes；
S14. it closes: being transferred in Block buffer, close the group of any activation；
S15. it balances: first with the 0.2mol/L NaHCO of 5 times of medium volumes3, 0.5mol/L NaCl, pH8.3 buffer it is clear
It washes, then acetic acid/sodium acetate (pH4.0, the NaCl comprising 0.5M) washing of the 0.1M with 5 times of medium volumes, finally with 5 times of media
The 0.1MTris-HCl(pH8.0 of volume includes 0.5MNaCl) washing；The above circulating repetition 3 times；
It S16. include 0.5MNaCl followed by being to use 0.1MTris-HCl(pH8.0)；Obtain agar polysaccharide-antibody coupling
The IAC column of compound.
Wherein preferably, the ingredient of the coupling buffer are as follows: 0.1-0.3mol/L NaHCO3, 0.4-0.6mol/L
NaCl。
It is highly preferred that the ingredient of the coupling buffer are as follows: 0.2mol/L NaHCO3, 0.5mol/L NaCl.
Overnight condition is 4 DEG C, 180-220rpm, 8-12h in step S12.
The method of step S13 washing is: standing, after gel sedimentation, removes the water on upper layer, then is re-poured into coupling buffering
Liquid, repeatedly.
The closed condition of step S14 is: after being transferred in Block buffer, staying overnight at 4 DEG C or is placed at room temperature for 2h, either
It is placed in the Tris-HCl buffer of pH8.0,0.1M and places 2h.
It is further preferred that above-mentioned obtained affinity column is also needed through preservative treatment.
Preferably, method for anticorrosion treatment is impregnated using the PBS buffer solution of 1/10000 Sodium azide.
Furthermore it is preferred that the storage temperature of gained affinity column is 1-10 DEG C.It is preferred that 4 DEG C.
Finally, affinity column of the invention after using, can be reused after regeneration treatment.
Preferably, the method for the regeneration treatment is: being washed with the PBS of 5-10 column volume after use, is finally existed immediately
It is saved with the PBS containing 1/10000 Sodium azide, is saved at 4 DEG C.
The invention has the following advantages:
The invention discloses a kind of breast milk polyclonal antibody affinity columns and preparation method thereof.Preparation method of the present invention is simple,
The high-abundance proteins in breast milk can more effectively be removed, can signal relatively low to abundance in breast milk, in mass spectrum compared with
Low albumen is enriched with, and is conducive to the albumen profile that the mankind more correctly recognize breast milk, be can apply to breast milk proteins matter group
In terms of the relevant technologies for learning downstream, it is worth being widely applied and applies.
Detailed description of the invention
Fig. 1 is the electrophoretogram of purified antibodies；No. 1 swimming lane represents Marker, and No. 2 swimming lanes represent unpurified serum, and No. 3
Swimming lane represents serum after purification.
Fig. 2 is the result of the elution high-abundance proteins of breast milk polyclonal antibody affinity column of the present invention；No. 1 swimming lane represents
Marker, No. 2 swimming lane breast milk samples, the sample (sample of removal high-abundance proteins that No. 3 samples are washed by immune affinity column
This), No. 4 swimming lanes represent the sample (high-abundance proteins) that immune affinity column elutes；2,3 swimming lane applied sample amounts are all 5.45 μ g eggs
It is white.
Specific embodiment
The present invention is further illustrated below in conjunction with Figure of description and specific embodiment, but embodiment is not to the present invention
It limits in any form.Unless stated otherwise, the present invention uses reagent, method and apparatus routinely try for the art
Agent, method and apparatus.
Unless stated otherwise, following embodiment agents useful for same and material are commercially available.
The preparation of 1 breast milk polyclonal antibody of embodiment
1, preparation method
After breast milk removal fat, as immunogene (immunizing dose 1.25mg/ is only), Anti-TNF-α is obtained by immunization method
Body.The specific method is as follows:
4 degree of breast milk defrostings, 2000g is centrifuged 20min under 4 DEG C of degree, avoids upper layer whey portion, draws lower layer's whey portion.It is logical
It crosses BCA and surveys protein concentration.When initial immunity, breast milk and Freund's adjuvant are emulsified, and the standard of emulsification is one drop of liquid of drop on liquid level,
Proof of not scattering emulsification is completed, and follow-up immunization, the antigen of equivalent is mixed with non-Freund's complete adjuvant.Immunization method: multiple spot is subcutaneous
Injection, immunizing dose 1.25mg/ is only.4th time it is immune after take blood, potency is greater than 105, so that it may arterial blood extracting.Obtained blood
It is purified by Protein A/G agarose clearly.
Purification process is as follows: whole purification process operate on protein purification instrument, and Protein A/G agarose is filled
Column after serum is crossed column repeatedly, first uses 0.15M NaCl, 20mM Na2HPO4, the buffer of pH 7.0 washs, steady to baseline
Afterwards, antibody-solutions use the eluent of the Glycine pH 3.0 of 0.1M again, collect the appearance liquid in this stage, use 1M immediately after
Antibody is tuned into neutrality by Tris-HCl pH8.5 solution, and antibody is avoided to inactivate in acid condition.Antibody elution solution passes through super
Chimney filter (4 DEG C, 4000rpm, 30min) is concentrated by ultrafiltration, and the concentration of IgG is made to obtain certain raising.
Antibody purification effect is verified through SDS-PAGE electrophoresis, will be concentrated by ultrafiltration containing antibody-solutions.
2, result
Indirect ELISA surveys potency, as shown in table 1.Four potency exempted from are between 512000 and 1024000, it was demonstrated that immune effect compared with
It is good.
Table 1
After Protein A/G agarose purified blood serum, obtains purified antibodies and carry out electrophoresis, as shown in Figure 1, after purification
There is the heavy chain band of apparent IgG55KDa and the light chain band of 25KDa.
The preparation of 2 breast milk polyclonal antibody affinity column of embodiment
1, a kind of preparation method of breast milk polyclonal antibody IAC is that the breast milk polyclonal antibody for preparing embodiment 1 and agar are more
Sugared gel 4B is coupled to obtain affinity adsorbent, inserts and breast milk polyclonal antibody affinity column is made in column.
Wherein antibody and gel coupling step are as follows:
S11. antibodies buffer is replaced: the polyclonal antibody of purifying in coupling buffer (0.2mol/L NaHCO3, 0.5mol/
The NaCl of L) in dialyse；
S12. it is coupled: agar polysaccharide 4B being fitted into chromatographic column, excessive acetone is first washed away with coupling buffer, then by antibody
It is added in chromatographic column, moves into 4 DEG C of shaking tables and stay overnight；
S13. it washs: cleaning extra antibody with the coupling buffer of at least 5 times gel media volumes, method is: standing, to solidifying
After glue sedimentation, removes the water on gel upper layer, be then re-poured into coupling buffer again, repeatedly；
S14. it closes: the gel of coupled antibody is transferred in Block buffer, overnight or in room temperature condition under the conditions of 4 DEG C
Lower placement 2h, or it is disposed on pH8.0,2h is placed in the Tris-HCl buffer of 0.1M, closes the group of any activation；
S15. it balances: recycling washing medium with the buffer of at least three kinds difference pH, each buffer is at least 5 times of medium
Volume；Specifically first with the 0.2mol/L NaHCO of 5 times of medium volumes3, the buffer solution for cleaning of the NaCl of 0.5mol/L, pH8.3,
It is washed again with acetic acid/sodium acetate of the 0.1M of 5 times of medium volumes (pH4.0, the NaCl comprising 0.5M), finally with 5 times of dielectrics
Long-pending 0.1MTris-HCl(pH8.0 includes 0.5MNaCl) washing；The above circulating repetition 3 times, wash cycles should all wrap each time
Acetic acid/sodium acetate (pH4.0, the NaCl comprising 0.5M) containing 0.1M；
It S16. include 0.5MNaCl followed by being to use 0.1MTris-HCl(pH8.0)；Obtain agar polysaccharide-antibody coupling
The IAC column of compound.
2, subsequent processing
The IAC column that above-mentioned steps S15 is prepared should use the PBS buffer solution of 1/10000 Sodium azide to soak if you need to place a period of time
Bubble carries out preservative treatment.
In addition, IAC column is sure not to freeze, it should be placed in 4 DEG C of refrigerators and save.
Furthermore the regeneration of IAC: after IAC column use, being washed with the PBS of 5-10 column volume immediately, finally with containing 1/
The PBS of 10000 Sodium azides is saved, and is saved in 4 degree of refrigerators.
The use of 3 breast milk polyclonal antibody affinity column of embodiment
1, the application method of the immune affinity column of breast milk polyclonal antibody.Utilize affinity column elution high-abundance proteins, enrichment
The method of low-abundance protein, comprising the following steps:
S21. the sodium azide of remaining is washed away with the PBS solution of 5 times of column volumes；
S22. suitable breast milk is taken, is diluted with PBS solution, loading is carried out, 30 min is incubated at 4 DEG C；
S23. after being incubated for, albumen and other impurity not in conjunction with antibody is cleaned with PBS, is assisted by protein purification instrument
Purifying；
S24. it after baseline stability, is eluted with eluent, collects eluent, use Tris(pH=8.0 of meta-alkalescence at once)
Solution neutralized, the pH of eluent is adjusted to 7.Treated, and eluent is low rich by elution high-abundance proteins, enrichment
The sample for spending albumen, can carry out electrophoresis detection.
Meanwhile the IAC pillar after using, it is cleaned, is reduced acid more to agar with the PBS solution of 5~10 times of column volumes
The degradation of sugar is finally saved using the PBS buffer solution of 1/10000 Sodium azide, and storage temperature is 4 DEG C.At the step
IAC pillar after reason can be with recycling utilization.
2, electrophoresis detection result is as shown in Figure 2
As seen from the figure, No. 2 swimming lanes and No. 3 swimming lanes are compared, and high-abundance proteins have corresponding reduction, while No. 4 swimming lanes have phase
Corresponding high-abundance proteins elute, and low-abundance albumen is enriched with.Prove that affine immune column elutes high abundance well
Albumen, the effect for being enriched with low-abundance protein.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention,
It should be equivalent substitute mode, be included within the scope of the present invention.
Claims (10)
1. a kind of breast milk polyclonal antibody, which is characterized in that the polyclonal antibody be using remove fat breast milk as be immunized
Original is prepared.
2. polyclonal antibody according to claim 1, which is characterized in that antibody-solutions by the solution of slant acidity elution after,
It is neutralized immediately with the buffer of meta-alkalescence.
3. polyclonal antibody according to claim 1, which is characterized in that 0-10 DEG C of antibody, 3000-5000rpm after purification
20-35min is concentrated by ultrafiltration.
4. polyclonal antibody according to claim 1, which is characterized in that gained antibody passes through Protein A/G agarose
Purifying.
5. a kind of breast milk polyclonal antibody affinity column, which is characterized in that is fixed in the column of the immune affinity column is power
Benefit requires any polyclonal antibody of 1-4.
6. breast milk polyclonal antibody affinity column according to claim 5, which is characterized in that be to appoint claim 1-4
The one breast milk polyclonal antibody and solid phase ligand support are coupled to obtain affinity adsorbent, and affinity adsorbent is inserted in column and is made
Obtain breast milk polyclonal antibody affinity column.
7. breast milk polyclonal antibody affinity column according to claim 6, which is characterized in that the solid phase ligand support
For agar polysaccharide 4B.
8. according to any breast milk polyclonal antibody affinity column of claim 5-7, which is characterized in that gained affinity column is also
It need to be through preservative treatment.
9. according to any breast milk polyclonal antibody affinity column of claim 5-7, which is characterized in that gained affinity column
Storage temperature is 1-10 DEG C.
10. according to any breast milk polyclonal antibody affinity column of claim 5-7, which is characterized in that affinity column passes through
After use, it can be reused after regeneration treatment.
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Cited By (1)
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| CN111138538A (en) * | 2020-02-25 | 2020-05-12 | 四川农业大学 | Goose pituitary prolactin protein immunoaffinity chromatography column, preparation method and purification method |
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Application publication date: 20190315 |