CN104117227B - A kind of deoxynivalenol immune affinity column and its production and use - Google Patents
A kind of deoxynivalenol immune affinity column and its production and use Download PDFInfo
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- CN104117227B CN104117227B CN201310153241.5A CN201310153241A CN104117227B CN 104117227 B CN104117227 B CN 104117227B CN 201310153241 A CN201310153241 A CN 201310153241A CN 104117227 B CN104117227 B CN 104117227B
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Abstract
The present invention relates to a kind of deoxynivalenol immune affinity column and its production and use.This immunoaffinity purification post utilizes Protein G to be coupled on agarose gel carrier, then with the antibody of anti-deoxynivalenol and the Protein G coupling on agarose.Then recycling cross-linking agent loads affinity column after cross-linking deoxynivalenol antibody protein G agarose gel carrier.This immune affinity column is mainly useful for the purification of the deoxynivalenol in food, feedstuff, flavoring agent, wine and beverage and other multiple samples.
Description
Technical field:
The present invention relates to immune affinity column of a kind of deoxynivalenol and its production and use.
Belong to field of detection of food safety.
Background technology:
Deoxynivalenol (deoxynivalenol, DON) has another name called vomitoxin
(vomitoxin, VT), a kind of single-ended spore of one of secondary metabolite mainly produced by some Fusarium spp.
Mei Xi race toxin.DON is modal one in Trichothecenes toxin, although research shows that DON does not has
The most carcinogenic, mutagenicity, but there is toxicity widely, particularly immunologic function is had significantly impact,
Dosage according to DON is different with open-assembly time causes immunosuppressant or immunostimulation.Feed intake is with mould
The feedstuff of verticillium toxin all can be poisoned, and the pig effect of telling the cause of DON is most sensitive, and per os minimum vomiting amount is
0.1～0.2mg/kg, main toxigenic bacterium is Fusarium graminearum, Fusarium nivale etc..DON is whole world feedstuff
One of contaminated mold toxin with grain, Ye Shi China malignant tumor district occurred frequently resident's main grain wants contaminated mold
One of toxin.In standard GB/T 2761-2005 " mycotoxin limitation in food ", in Semen Tritici aestivi and Semen Maydis
The limitation of DON is 1000 μ g/kg.The state compulsory standard GB20833-that on March 1st, 2007 implements
Allowance≤the 1mg/kg of DON in swine feed in 2007.
The detection method that deoxynivalenol is conventional at present has thin layer chromatography (TLC), enzyme linked immunological
Adsorption test (ELISA), gas chromatography (GC) and high performance liquid chromatography (HPLC) etc.
3.1 Tlc Determination methods (thin layerchromatography, TLC) this be set up the earliest
Kind of detection method, have simplicity, economy, to features such as equipment and reviewer are less demanding.It it is country of China
One of standard detecting method [7].But owing to the degree of accuracy of TLC method is low, operating process is complicated, analysis result
Repeatable poor with repeatability, this method is still at present except North America and outside Europe other countries, especially develops
The conventional method of mycotoxin in Chinese Home detection food and feedstuff.
3.2 elisa (ELISA) elisa is also referred to as immunoassay,
It is the immunoassay technology based on immune antibody, is class analysis method quick, sensitive and simple to operate,
The detection of the micromolecular compound such as toxin and toxic compounds is applied the most quite varied, had inspection
Survey ELISA method and the product of vomitoxin.But the major defect of the ELISA method of detection DON is at present
Cause its detection value higher owing to there is antibody used with the cross reaction of the acetylation analog of DON, be easier to
False positive occurs.Additionally, ELISA method is used to be difficult in pure organic solution agent detect DON.
Gas chromatography (gaschromatography GC) is owing to containing 3 hydroxyls, easy shape in DON molecule
Become stable hydrogen bond, reduce volatility, therefore must perform the derivatization before carrying out gas chromatographic analysis.Will
DON is derivative forms trimethyl silane thing.But derivative reagent such as heptafluorobutyric anhydride (HFBA) is expensive, and
Volatile.GC has the advantages such as selectivity sensitive, high, accuracy and accuracy.Additionally, can also with GC
Realize detecting while multiple mycotoxin.But in GC chromatograph, there are the following problems: standard curve is linear
Relation is bad, response drift, the delay of last sample introduction sample and repeat sample introduction when memory effect, MS detection
The coefficient of variation is big and there is the problems such as matrix interference.
High performance liquid chromatography (high performanceliquid chromatography, HPLC) is efficient
Liquid-phase chromatography method (HPLC) high performance liquid chromatography is wide to the suitability of sample, by analyze object volatility and
The restriction of heat stability, thus compensate for the deficiency of gas chromatography.It is that quantitative analysis mycotoxin is the most frequently used
Method.
When using high performance liquid chromatography detection deoxynivalenol, need first by deoxynivalenol
Bacterium enol sample carries out purified treatment.The most conventional purifying treatment method is exactly solid phase extraction, is also post layer
Analysis method.It is that current mycotoxin purifies most popular method.Wherein, the analysis speed of immune affinity column method
Degree is fast, highly sensitive, and separation efficiency and the response rate are high, it is not necessary to the mycotoxin reference material of severe toxicity is demarcated,
Safe and reliable.Thus become most common method.MacDonald SJ, waits and takes off immune affinity column purified treatment
Oxygen nivalenol sample, the method then carrying out HPLC detection is described in detail.
[MacDonald SJ J AOAC Int, 2005,88 (4): 1197～1204]
Probably have for preparing the method for immunoaffinity purification post at present: the direct coupling method of cyanogen bromide-activated, ring
Oxygen chloropropane method, Over-voltage protection etc..The ultimate principle of these methods is all that carrier matrix is carried out chemical activation
After, by antibody coupling to carrier.But owing to coupling is nonspecific, any position of antibody has can
Energy and carrier conjugation, directivity is uncontrollable.Affinity purification column purification deoxynivalenol bacterium certainly will be affected
The efficiency of enol.
Summary of the invention:
It is an object of the invention to provide a kind of deoxynivalenol immune affinity column and preparation method thereof
And purposes
In order to achieve the above object, in the present invention, make use of the function of Protein G, Protein G is a kind of G
Albumen on type streptococcal cell wall, can combine at the specific Fc position with many animals antibody.And
There is the highest affinity.We have cloned the gene order of Protein G, and have carried out excellent to its codon
Change, after restructuring, gone out at expression in escherichia coli and had the gene recombinant protein G of high-affinity with antibody.By base
After being coupled on carrier because of the Protein G of restructuring, then by deoxynivalenol antibody coupling to Protein G
On, prepare deoxynivalenol Protein G carrier, then with cross-linking agent, carrier has been cross-linked.
The deoxynivalenol immune affinity column of high-affinity it is the formation of after carrier dress post after crosslinking.This parent
Easy with column operation, purification deoxynivalenol efficiency is high.Sample is the most permissible after simple process
It is purified, obtains the deoxynivalenol that purity is the highest.For high performance liquid chromatography detection and fluorescence
Instrument detects.
Concrete operations are as follows:
The advantage of present invention maximum is exactly the feature that make use of Protein G to be combined with Ig antibody specificity, and IgG resists
Body is made up of two heavy chains and two light chains, and antibody is divided into Fab district and Fc district, and wherein, Fab district is antigen
In conjunction with region.
Protein G is a kind of special albumen on streptococcal cell wall, has specific with the Fc region of antibody
Binding ability.After Protein G and antibodies, the Fab region of antibody is free outside, does not affect the antigen of antibody
Binding ability.The Protein G of the present invention includes the Protein G expressed according to GenBank CAA27638.1 sequence,
Protein G after our gene optimization restructuring, the Protein G of 1 molecule can be in conjunction with the IgG of 3 molecules
Antibody, has the highest affinity of antibody.And only antibody can be combined with Protein G, remaining albumen all can not
Being combined with Protein G, specificity is the highest.It is good that the immune affinity column prepared based on this Protein G has specificity,
Deoxynivalenol binding capacity is big, the feature that purification efficiency is high.
Deoxynivalenol immune affinity column and preparation method thereof is described as follows:
The most support-activated
Select agarose carrier, sepharose4B, activate with Epichlorohydrin activation method.
Take the preswollen agarose gel sepharose4B of 2%, fully rinse with the distilled water of 20 times of volumes,
Wash away the ethanol of remaining, filter out moisture with funnel.
Weigh the wet gel 5 grams after filtering off moisture content, add the NaOH of 7.5 milliliters of 0.8M, the epoxy chlorine of 30%
2 milliliters of propane, the sodium borohydride NaBH of 2mg/ml4, 5 milliliters, at 25 DEG C, shaking table reacts 8 hours.
After reaction, use substantial amounts of distilled water wash, remove the epoxychloropropane mixed in gel.
2. by the coupling of Protein G Yu activated carrier sepharose4B
The agarose gel sepharose4B coupling buffer (NaHCO of 0.1M that will have activated3, 0.8M
NaCl, pH8.9) wash 3 times.Add the Protein G of 2mg/ml, room temperature coupling 4 hours.
The phosphate buffer PBS of good for coupling agarose carrier 20mM, pH7.4 is washed 3 times.Coupling
There is the agarose carrier sepharose named Protein G-sepharose of Protein G.
The most anti-deoxynivalenol antibody is connected with the Protein G on Protein G-sepharose carrier
Protein G and antibody have specific affinity, under certain reaction condition, coupling are had Protein G
Agarose gel be attached with antibody.Antibody is connected on agarose.Knot due to Protein G Yu antibody
Closing the Fc region that position is antibody, the antigen binding domain of antibody is unaffected, has fully ensured that the antigen of antibody
Binding ability.
Anti-deoxynivalenol antibody is dissolved in the PBS of 20mM, pH7.4, final concentration 2mg/ml.
By in the PBS washed Protein G-sepharose of antibody addition 20mM pH7.4.Room temperature is tied
Close 30 minutes.
Carrier PBS after in conjunction with washs.Connect the carrier named antibody protein G having antibody
sepharose。
4. carrier crosslinking
The antibody protein G sepharose borate buffer of 0.1M pH9.0 is washed 3 times.Then
Add the borate buffer of 0.1M pH9.0, buffer adds cross-linking agent such as imidic acid dimethyl ester in heptan two
(DMP) is to final concentration 20mM.Normal temperature crosslinked 1 hour.
The ethanolamine adding 50mM, pH9.0 terminates reaction.And close 10 minutes with the ethanolamine of 50mM.
The PBS of antibody protein G sepharose carrier 20mM, pH7.4 after crosslinking washs 3 times.
5. dress post
Antibody-agarose carrier after crosslinking is loaded in chromatographic column as required, can prepare not as required
Deoxynivalenol affinity purification post with capacity.The structure of chromatographic column is as shown in Figure 1.
Compared with prior art, present invention have the advantage that
1) sufficiently make use of the characteristic that Protein G and IgG antibody are specific binding, make antibody pass through Protein G
It is coupled on carrier.And outside the Fab fragment of IgG antibody is sufficiently exposed to, increase substantially
The capturing ability of deoxynivalenol, the purification efficiency of deoxynivalenol is also
Effectively improve.
2) present invention uses the Protein G after genetic modification, by the optimization of codon and IgG binding domain
The optimization of gene.The antibody binding capacity making Protein G increases substantially, and then improves deoxidation snow
The purification efficiency of rotten Fusarium spp. enol.
3) using purification deoxynivalenol of the present invention easy and simple to handle, several steps can be obtained by purity relatively
High deoxynivalenol, more convenient operator uses.
4) use the deoxynivalenol purity that obtains of the present invention the highest, follow-up need not do again other pure
Change processes and is just used directly for high performance liquid chromatography detection or fluoroscopic examination, saves operator
Time and expense.
Accompanying drawing explanation
Fig. 1: deoxynivalenol immune affinity column structural representation
A: injection port；B: cylinder；C: sieve plate；D: deoxynivalenol Antibody-protein G-sepharose
Filler E: sieve plate；F: outlet
Fig. 2: deoxynivalenol content detection HPLC collection of illustrative plates in flour
DON: deoxynivalenol detection peak；
Detailed description of the invention
Embodiment 1: the preparation of deoxynivalenol immunoaffinity purification post
The present invention prepares a preferred embodiment of deoxynivalenol immunoaffinity purification post such as
Under:
1. agarose gel activation
Take the agarose gel sepharose2B of 2%, fully rinse with the distilled water of 20 times of volumes, wash away residual
The ethanol deposited.Moisture is filtered out with funnel.Weigh the wet gel 5 grams after filtering off moisture content, add 7.5 milliliters
The NaOH of 0.8M, the epoxychloropropane of 30% 2 milliliters, the sodium borohydride NaBH of 2mg/ml4, 5 milliliters,
At 25 DEG C, shaking table reacts 8 hours.
After reaction, with 50 milliliters of distilled water washs, remove the epoxychloropropane mixed in gel.
2. the coupling of the agarose gel of Protein G and activation
Take the agarose gel after 1 gram of activation, with the coupling buffer (NaHCO of 0.1M3, 0.8M NaCl,
PH8.9) washing 3 times.Add Protein G 20ml of 2mg/ml, room temperature coupling 4 hours.
The phosphate buffer PBS of good for coupling agarose carrier 20mM, pH7.4 is washed 3 times.Coupling
There is the agarose carrier sepharose named Protein G-sepharose of Protein G.
The most anti-deoxynivalenol IgG monoclonal antibody is combined with Protein G-sepharose
Anti-deoxynivalenol antibody is dissolved in the PBS of 20mM, pH7.4, final concentration 2mg/ml,
Cumulative volume 10ml.
Protein G-sepharose the PBS of 20mM PH7.4 is washed three times, each 30ml.Will
The deoxynivalenol antibody being dissolved in PBS adds in washed Protein G-sepharose.Room
Temperature combines 30 minutes.
Carrier after in conjunction with the PBS of 20mM pH7.4 washs three times, each 30ml.Connect the load having antibody
Body named antibody protein G sepharose.
4. combine the agarose sepharose crosslinking of IgG
Antibody protein G sepharose is washed 3 times with the borate buffer of 0.1M pH9.0, every time
30ml。
By wet gel solid, add the borate buffer 20ml of 0.1M pH9.0, buffer adds crosslinking
Agent imidic acid dimethyl ester in heptan two (DMP) is to final concentration 20mM.Normal temperature crosslinked 1 hour.
The ethanolamine 20ml adding 100mM, pH9.0 terminates reaction.And seal with the ethanolamine 20ml of 50mM
Close 10 minutes.
The PBS of antibody protein G sepharose carrier 20mM, pH7.4 after crosslinking washs 3 times,
30ml every time.
5. dress post
By resuspended with 10 milliliters of 20mM PBS pH7.4 for the sepharose after crosslinking, it is then charged into the parent of sky
With in purification column cylinder.
Embodiment 2: utilize the rotten sickle of deoxidation snow in deoxynivalenol immune affinity column purification and detection flour
Cutter bacterium enol
1. flour sample processes:
----weigh flour sample 25g, 5g Polyethylene Glycol, add 100ml distilled water；
----shaking table acutely vibrates 20min so that it is mix completely；
----filter with microfibre filter paper, collects filtrate；
----take 1mL filtrate is for sample detection.
2. by deoxynivalenol immunoaffinity purification post equilibrium at room temperature 30 minutes.
3. taking out immune affinity column, injection port is connected with injector syringe, and syringe is linked on gas control crosshead.
4. it is washed with deionized affinity column 3 times, each 10 milliliters, regulates pore crosshead air pump pressure, make liquid
Body flows out with the flow velocity of 3 drops/sec.
5. adding in purification column by sample, regulation flow is to 1-2 drop/sec.Until sample all flows out purification column.
6. use distilled water wash purification column 3 times, each 5 milliliters.
7. add 1ml methanol, collect eluted product.
8. eluted product high-efficient liquid phase chromatogram HPLC detects.
9. high performance liquid chromatography testing result is shown in Table 1, deoxynivalenol chromatogram such as accompanying drawing 2:
From detection collection of illustrative plates find out, flour sample through deoxynivalenol immune affinity column after purification, with height
Effect liquid phase chromatogram (HPLC) detection can detect deoxynivalenol (vomitoxin), its content
For 819.78972ng/ul.
Claims (1)
1. the preparation method of the immune affinity column of a purification deoxynivalenol, it is characterised in that:
(1) support-activated
Select agarose carrier sepharose4B, activate with Epichlorohydrin activation method, take the preswollen agarose gel sepharose4B of 2%, fully rinse with the distilled water of 20 times of volumes, weigh the wet gel 5 grams after filtering off moisture, add the NaOH of 7.5 milliliters of 0.8M, the epoxychloropropane of 30% 2 milliliters, the sodium borohydride NaBH of 5 milliliters of 2mg/ml4, after reaction, use distilled water wash；
(2) by the coupling of Protein G Yu activated carrier sepharose4B
The agarose gel sepharose4B 0.1M NaHCO that will have activated3, 0.8M NaCl, pH8.9 coupling buffer washs, and adds the Protein G of 2mg/ml, room temperature coupling, is washed 3 times the phosphate buffer PBS of good for coupling agarose carrier 20mM, pH7.4, make Protein G-sepharose；
(3) anti-deoxynivalenol antibody is connected with the Protein G on Protein G-sepharose carrier
Anti-deoxynivalenol antibody is dissolved in the PBS of 20mM, pH7.4, final concentration 2mg/ml, by in the PBS washed Protein G-sepharose of antibody addition 20mM, pH7.4, room temperature combines, carrier after in conjunction with PBS washs, and connects the carrier named Antibody-protein G-sepharose having antibody；
(4) carrier crosslinking
The Antibody-protein G-sepharose borate buffer of 0.1M pH9.0 is washed 3 times, it is subsequently adding the borate buffer of 0.1M pH9.0, imidic acid dimethyl ester DMP to final concentration 20mM in heptan two is added in buffer, normal temperature crosslinked 1 hour, the ethanolamine adding 50mM, pH9.0 terminates reaction, closing 10 minutes with the ethanolamine of 50mM, the PBS of 20mM, pH7.4 of the Antibody-protein G-sepharose carrier after crosslinking washs 3 times；
(5) dress post
Antibody-agarose carrier after crosslinking is loaded in chromatographic column as required.
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| CN104645666B (en) * | 2014-11-15 | 2016-09-28 | 于秋香 | A kind of nitric acid oxidation processing method of nano-graphite carbon black |
| CN104707362A (en) * | 2015-01-05 | 2015-06-17 | 重庆出入境检验检疫局检验检疫技术中心 | Composite affinity column for purifying 3-acetyl deoxynivalenol, aflatoxin, ochratoxin A and zearalenone |
| CN105842449A (en) * | 2015-01-14 | 2016-08-10 | 北京康诺生物科技有限公司 | Deoxynivalenol immunomagnetic bead, preparation method and application thereof |
| CN105136915B (en) * | 2015-07-08 | 2017-05-10 | 浙江省海洋水产研究所 | Immunoaffinity method |
| CN107262074A (en) * | 2017-01-23 | 2017-10-20 | 北京美正生物科技有限公司 | A kind of deoxynivalenol aptamers affinity column and its production and use |
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| CN101846685A (en) * | 2010-06-03 | 2010-09-29 | 中国海洋大学 | Enzyme-linked immunoassay kit for chlamys ferreri blood cells and preparation method thereof |
| CN102115493A (en) * | 2009-12-30 | 2011-07-06 | 本元正阳基因技术有限公司 | Preparation method for recombinant protein G and application of same |
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| CN102115493A (en) * | 2009-12-30 | 2011-07-06 | 本元正阳基因技术有限公司 | Preparation method for recombinant protein G and application of same |
| CN101846685A (en) * | 2010-06-03 | 2010-09-29 | 中国海洋大学 | Enzyme-linked immunoassay kit for chlamys ferreri blood cells and preparation method thereof |
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