CN104624176B - Bacterial magnetic particle (BMP)-agglutinin complex and preparation method and application thereof - Google Patents

Bacterial magnetic particle (BMP)-agglutinin complex and preparation method and application thereof Download PDF

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CN104624176B
CN104624176B CN201510075253.XA CN201510075253A CN104624176B CN 104624176 B CN104624176 B CN 104624176B CN 201510075253 A CN201510075253 A CN 201510075253A CN 104624176 B CN104624176 B CN 104624176B
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bmps
complex
agglutinin
linking agent
cross
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关锋
李想
杨刚龙
郭佳
刘昌梅
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Jiangnan University
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Abstract

The invention discloses a bacterial magnetic particle (BMP)-agglutinin complex and a preparation method and application thereof, belonging to the technical field of separation and purification. The preparation method comprises the following steps: performing surface modification and transformation on BMPs, and adding a cross-linking agent and agglutinin for coupling, thereby obtaining the BMP-agglutinin complex. The surfaces of the BMPs are modified, the influence of self-proteins of the BMPs on subsequent application is eliminated, more primary amino is provided for agglutinin coupling due to the addition of PE, the coupling efficiency is improved, and the dispersity of the BMPs is improved; the cross-linking agent (BS3) serves as a connecting arm, so that covalent coupling is realized between the BMPs and the agglutinin, and an agglutinin recognition sugar chain area is not damaged; and moreover, the concentration of a buffer solution is regulated, the preparation time is shortened, and the coupling efficiency is improved. The complex disclosed by the invention is used for enriching and separating specific glycoproteins, and compared with traditional affinity chromatography, the method disclosed by the invention has the advantages of fewer steps, short time and the like.

Description

A kind of bacillus nanometer magnetosome-agglutinin complex and preparation method and application
Technical field
The present invention relates to a kind of bacillus nanometer magnetosome-agglutinin and preparation method and application, belong to and isolate and purify skill Art field.
Background technology
Sugar can form the saccharide complexes such as glycoprotein, Dan Baiduotang proteoglycan PG and glycolipid with covalent bond such as protein or lipids, is Important informational molecule in human body, plays specific identification and mediation in many life and lysis.Its structure Or the minor variations of quantity may cause the change of its function, thus leading to the generation of various diseases, which results in people couple Sugar group learns the very big enthusiasm of research.With scientific and technological progress and biological development, increasing technology is applied to sugared group Among the research learned, such as lectin affinity chromatography technology, forward position affinity chromatograph technology, high performance liquid chromatography, mass spectrum, carbohydrate chip skill Art etc..In the research that sugared group is learned, it is broadly divided into three parts: structure sugar group, functional sugar group and Information Biology.
The acquisition of saccharide complex and separation are the bases that sugar group learns research, and conventional capture technique is that agglutinin is affine at present Chromatographic technique.Agglutinin be a kind of nonimmune source of separating-purifying from various plants, invertebratess or higher mammal, Non-enzyme material, the protein being combined with a class glycosyl specificity, extremely important in the research process of glycoproteomics, It is widely used in the type of glycoprotein and glycolipid sugar chain in identification of cell.Lectin affinity chromatography technology is exactly to utilize agglutinin With the affinity of sugar chain, thus being applied in affinity chromatography separate saccharide complex, and affine due to agglutinin and sugar chain Power is relatively low, so being easier to be eluted such that it is able to reach the purpose reclaiming saccharide complex.
Although currently reported, agglutinin is fixed on synthetic epoxidation magnetic nano particle to capture saccharide complex Method.But synthetic epoxidation magnetic nano particle to prepare relatively complicated and nano-particle uniform particle diameter poor.And Bacillus nanometer magnetosome (bacterial magnetic particles, bmp) bmps surface has native amino group, and can Derivatization is used for the coupling of agglutinin further.In terms of particle diameter, the less about 40nm of bmps particle diameter, and because it is closed by magnetotactic bacteria Become, particle diameter is more homogeneous.
The present invention can be the feasible research material of the research provides convenient of sugar group, facilitates the acquisition of saccharide complex, with more Well sugar group is studied.The more artificial magnetic nano particle of preparation method of the present invention is simple, and magnetic nano particle homogeneity is more Good, uniform particle diameter can improve granule and the coupling efficiency of agglutinin and its efficiency be combineding with each other with glycoprotein.The present invention Preparation bmps- agglutinin complex being enriched with and to separate the step of specific glycoprotein and time faster than traditional chromatographic column, Few compared with traditional affinity chromatography separation glycoprotein step, the time is short.
Content of the invention
The invention provides a kind of bacillus nanometer magnetosome-agglutinin complex and preparation method and application, the present invention Agglutinin is coupled on bmps, for being enriched with associated glycoprotein, and utilizes removal of surfactant bmps surface protein, it is to avoid Introduce unrelated protein, add pe etc. to increase primary amino radical quantity simultaneously, improve coupling efficiency.
First purpose of the present invention is to provide a kind of bacillus nanometer magnetosome-agglutinin complex, and described complex is The bacillus nanometer magnetosome through surface Deproteinization, adding primary amino radical process and agglutinin are coupled and are generated.
Described surface Deproteinization is to process bacillus nanometer magnetosome with surfactant；Described interpolation primary amino radical is to use phospholipid Bacillus nanometer magnetosome surface is modified；Described coupling is to be completed by cross-linking agent.
Second object of the present invention is to provide a kind of method preparing complex described in claim 1.
Methods described includes: (1) bmps surface modification: weighs bmps, adds surfactant to be processed, to remove The albumen on bmps surface, then collects bmps, is resuspended in phospholipid solution, ultrasonic, and incubation obtains the bmps of surface modification, that is, Bmps- phosphatide complexes；(2) preparation of bmps- cross-linking agent complex: bmps- phosphatide complexes obtained in the previous step are suspended, Break up, be subsequently adding cross-linking agent solution and reacted, collect, that is, obtain bmps- cross-linking agent complex；(3) bmps- cross-linking agent- The preparation of agglutinin complex: bmps- cross-linking agent complex obtained in the previous step is added in agglutinin solution, ultrasonic beats Dissipate, reaction, collect, that is, obtain bmps- cross-linking agent-agglutinin complex, i.e. bmps- agglutinin complex.
Surfactant in described step 1 is processed, and in one embodiment of the invention, is at 60-100 DEG C Heating 5-20min or room temperature concussion 1-3h, wherein using heating method, quickly faster, effect more preferable.Remove the little body surface of magnetic Face albumen itself, can avoid introducing unrelated protein in follow-up test.
Phospholipid in described step 1, in one embodiment of the invention, is PHOSPHATIDYL ETHANOLAMINE (pe) and phosphatidyl silk Propylhomoserin (ps), can provide more primary amino radicals for bmps surface, and being coupled agglutinin for next step provides more sites.
The form that the phospholipid that described step 1 is formed by ultrasonic method wraps up magnetic granule is in metastable condition it is ensured that magnetic Granule is coupled the in hgher efficiency of lipid.
Incubation in described step 1, in one embodiment of the invention, is lucifuge incubation it is ensured that magnetic granule is even Join the in hgher efficiency of lipid.
Described step 2, in one embodiment of the invention, bmps- phosphatide complexes are suspended in pb buffer (i.e. Only add na2hpo4And kh2po4) in, broken up using ultrasonic.
Described step 2, in one embodiment of the invention, is in below 10mm, ph 7-9 pb from concentration Buffer.Described buffering liquid energy adjusts ion concentration, so that solution is in compared with low ion concns, and now bmps colloid solution is more Stable, bmps granule more disperses, and is difficult to assemble, thus improving coupling efficiency.
After described step 1, step 2, each step of step 3 collect bmps or its complex, in one kind enforcement of the present invention In mode, also include by the bmps of collection or its complex buffer or aqueous suspension, ultrasonic break up, collect, take supernatant, repeat Cleaning 3 times.During cleaning, it is preferred to use the buffer of high concentration, the stability of colloid solution can be destroyed, so that bmps is gathered faster Collection, can shorten the time.Described cleaning buffer, in one embodiment of the invention, using the pb buffering of more than 40mm Liquid.
Described collection, in one embodiment of the invention, is to be collected using Magnet or magnetic frame.Bmps is molten Liquid is colloid solution actually, during the collection of each step, needs first to be adsorbed bmps with external force, that is, destroys stablizing of colloid Property.
Methods described, in one embodiment of the invention, specifically includes: (1) bmps surface modification: weigh bmps, By every 1mg bmps, add 50 μ l-100ml concentration be 0.5-2% surfactant solution heated or concussion at Reason, to remove the albumen on bmps surface, then collects bmps, is resuspended in PHOSPHATIDYL ETHANOLAMINE pe of 1mg-100mg/ml after cleaning Or in Phosphatidylserine ps solution, ultrasonic, incubation, obtain the bmps of surface modification, i.e. bmps- phosphatide complexes；(2) The preparation of bmps- cross-linking agent complex: be 10:1-1:10 with bmps mass volume ratio buffer will be obtained in the previous step Bmps- phosphatide complexes suspend, break up, and being subsequently adding cross-linking agent quality with bmps- phosphatide complexes mass ratio is 1:10 to 5:1 Cross-linking agent solution, in 4-40 DEG C of concussion reaction 10-60min, collect, that is, obtain bmps- cross-linking agent complex；(3) bmps- hands over The preparation of connection agent-agglutinin complex: prepare 0.1-10mg/ml agglutinin solution, compound with obtained in the previous step by agglutinin The mass ratio 1:10 to 10:1 of thing, complex obtained in the previous step is added in agglutinin solution, ultrasonic breaks up, in 4-40 DEG C Concussion reaction 10-120min, collects, obtains final product bmps- agglutinin complex.
Described cross-linking agent and bmps- phosphatide complexes mass ratio, in one embodiment of the invention, are 1:2-2:1.
Described bmps- cross-linking agent complex and agglutinin mass ratio, in one embodiment of the invention, are 1:2-2: 1.
Described surfactant, in one embodiment of the invention, can be following any one: dodecyl sulfur Sour sodium (sds), cetyl trimethylammonium bromide (ctab), 3- [3- (gallbladder amido propyl) dimethylamino] propane sulfonic acid inner salt (chaps), Triton X-100 (triton x-100).
Described cross-linking agent, in one embodiment of the invention, can be following any one: glutaraldehyde, butanedial, N, n '-two succinimidyl carbonate, 3,3 '-two sulfur double sulfosuccinimide propionic ester, dimethyl adipimide.
Described cross-linking agent, in one embodiment of the invention, is double amber imide suberate sodium salt (bs3).
Described agglutinin can be any one agglutinin.
Described agglutinin, in one embodiment of the invention, be following any one: ConA (cona), Wheat germ agglutinin (wga), sophora japonica lectin (sja), lens culinaris agglutinin (lca).
The described bmps- agglutinin complex preparing, in one embodiment of the invention, in bovine serum albumin Closing, preservation in (bsa) in vain.
The described bmps- agglutinin complex preparing, in one embodiment of the invention, adds confining liquid (10mm pbs contains 1%bsa, and contains 2%peg 4000,2%peg 6000), 4 DEG C save backup.
The preparation method of described bmps, in one embodiment of the invention, is using document xiang l, wei j, jianbo s et al.purified and sterilized magnetosomes from magnetospirillum gryphiswaldense msr-1were not toxic to mouse fibroblasts in vitro[j].letters In applied microbiology, the method described in 2007,45 (1): 75-81 is preparing.
Third object of the present invention is to provide described complex isolating and purifying the application in field.
Described application, in one embodiment of the invention, is used to separate glycoprotein.
Described application, in one embodiment of the invention, is by glycoprotein mixture and bmps- agglutinin complex Reaction, combination, make Glycoprotein binding on this magnetic particle complex, then clean this magnetic particle with cleaning buffer solution compound Thing, is finally eluted specifically binding to the glycoprotein on this complex with elution buffer.
Beneficial effects of the present invention:
(1) have native amino group using bmps surface, and the property being coupled can be used for by derivatization further, and grain The advantage that footpath is less, particle diameter is more homogeneous, is prepared for bmps- agglutinin complex.By modifying to bmps surface, remove To impact during subsequent applications, with the addition of pe or ps is to be coupled agglutinin to provide more primaquines to bmps oneself protein simultaneously Base, improves coupling efficiency, improves the dispersibility of bmps simultaneously；
(2) as linking arm, bmps has been carried out covalent coupling with agglutinin by the use of cross-linking agent, and ensured that agglutinin identifies Sugar chain region is not damaged；
(3) pass through to adjust the concentration of buffer, shorten preparation time, improve coupling efficiency.During coupling reaction, low Carrying out in concentration buffer liquid, more being disperseed so that being difficult between bmps to assemble with maintaining colloidal stability, thus reaching more preferably even Connection efficiency.And when cleaning, the stability of colloid solution can be destroyed using the buffer of high concentration so as to faster assemble, test In, need repeatedly bmps to be carried out, adsorbed under high concentration buffer, the time can be shortened.
(4) the complex particle diameter preparing is little, homogeneity and good dispersion, can improve the coupling of granule and agglutinin Efficiency and its efficiency be combineding with each other with glycoprotein.
Brief description
The ft-ir figure of Fig. 1: different bmps and its complex；A: original bmps, b: after being processed with surfactant Bmps, c:bmps-pe complex, d:bmps-cona complex；
Fig. 2: the scanning electron microscope (SEM) photograph of different bmps and its complex；(a) (b): original bmps, (c) (d): use surface activity Bmps after agent process, (e) (f): bmps-pe complex, (g) (h): bmps-cona complex；
Sds-page after the bmps of Fig. 3: different disposal method is coupled agglutinin cona schemes；Each swimming lane is: 1, maker, 2nd, cona mark product, 3, original bmps is through bs3After being coupled cona, supernatant after 5%sds boiling water boiling 5min；4th, bmps-pe complex Through bs3After being coupled cona, supernatant after 5%sds boiling water boiling 5min；5th, bmps-pe complex with after cona mixing 2h, 5%sds Supernatant after boiling water boiling 5min；
The sds-page figure of Fig. 4: bmps-wga complex separation chicken egg white；Each swimming lane is: 1, maker, 2, bsa mark Product, 3, wga mark product, 4, separate before ovum gallinaceum is clear and cona albumen composition, 5, separate after ovum gallinaceum clear and cona albumen is combined Thing, 6, eluent.
Specific embodiment
The preparation of embodiment 1 bmps-cona complex
Bmps surface modification: weigh 1mg bmps 100 DEG C of heating 5min in 100 μ l 1%sds solution, to remove bmps Surface protein；Magnetic frame collects bmps, and with 100mm phosphate (pb, ph=7.5) buffer solution for cleaning 3 times；Bmps after processing It is resuspended in 20mg/ml pe solution, the ultrasonic 10min of 100w；After lucifuge incubation 2h, cleaning bmps tri- times as stated above.I.e. Obtain bmps-pe complex.
bmps-bs3The preparation of complex: draw 900 μ l 4mm pb buffer suspension 1mg previous step gained complex, 80w is ultrasonic to be broken up；Weigh bs30.572mg, adds bmps suspension after fully being dissolved with 100 μ l distilled water, 37 DEG C, 200rpm is anti- Answer 0.5h；Magnetic frame catcher reacted bmps complex, removes supernatant, and 100mm pb buffer suspends, and 80w ultrasound wave is broken up Afterwards, collected with Magnet, go supernatant, repeated washing three times, that is, obtain bmps-bs3Complex.
bmps-bs3The preparation of-cona complex: weigh 1mg agglutinin Con A Concanavalin (cona) and be dissolved in 1ml 4mm Pb buffer, is configured to 1mg/ml agglutinin solution；Previous step gained bmps-bs3 complex 1mg is suspended in agglutinin cona Solution, 80w is ultrasonic to be broken up, 37 DEG C, and 200rpm reacts 1h；Magnetic frame catcher reacted bmps complex, removes supernatant, 100mm pb buffer suspends, and after 80w ultrasound wave is broken up, is collected with Magnet, goes supernatant, repeated washing three times, obtain bmps- bs3- cona complex, i.e. bmps-cona complex.
The magnetic composite of synthesis is characterized by infrared, Electronic Speculum and albumin glue.
Infrared: as can be seen from Figure 1: original bmps is in 1636cm-1And 1575cm-1Place has two peaks to be respectively amide band and amide Band, shows that original bmps contains protein matter；After processing through surfactant, this peak disappears, and has shown bmps surface protein Remove；Bmps-pe complex is in 880cm-1It is to wave outside the face of c-n that there is peak at place, shows that pe is coupled in bmps；It is coupled agglutinin 1631cm afterwards-1And 1568cm-1Place has two peaks to be respectively amide band and amide band, shows that cona is coupled to bmps.
Electronic Speculum: as can be seen from Figure 2: original bmps dispersibility is poor, and surface can be observed lipid film；Process through surfactant Bmps dispersibility is deteriorated afterwards, and face grease plasma membrane is thinning, shows that surface mass is partially removed；Formed after complex with pe, dispersion Property improve, lipid film is thickening, boundary obscure, form is more loose, shows that pe is successfully coupled to bmps；After connecting agglutinin Bmps dispersibility is still preferable, and skin covering of the surface is thinning, boundary substantially, form more closely thus it is speculated that after bs3 coupling protein, warp Protein encapsulation, causes form to become tight.The bmps-cona complex preparing, particle diameter is little, homogeneity is good, good dispersion.
Albumin glue: with taking 1mg original bmps and bmps-pe complex to be coupled with cona under the same conditions, 5%sds boils After decocting in water 5min, carry out sds-page.Result is as shown in Figure 3.Test result indicate that, cona has successfully been coupled to magnetic corpusculum.Warp Cross contrast and understand that bmps-pe complex carries cona amount and is much larger than original bmps bearing capacity, and avoid original bmps egg itself The impact that leucorrhea comes, illustrates that pe can improve more functional groups, thus effectively increasing coupling efficiency.
The preparation of embodiment 2 bmps- wheat germ agglutinin (wga) complex
Bmps- wheat germ agglutinin (wga) complex be prepared as follows:
(1) bmps surface modification: weigh 1mg bmps, add the ctab solution that 50ul concentration is 2%, at 60 DEG C of heating Reason 20min, to remove the albumen on bmps surface, is then centrifuged for collecting bmps, is resuspended in the phosphatidyl silk ammonia of 1mg/ml after cleaning In sour ps solution, the ultrasonic 1h of 80w, lucifuge is incubated 24h, and with 40mm phosphate (pb, ph=7) buffer solution for cleaning 3 times, obtains Bmps-ps complex；
(2) preparation of bmps- glutaraldehyde complex: take 1mg bmps-ps complex, with the buffering of 100ul concentration 10mm Liquid suspend, ultrasonic break up, be subsequently adding the glutaraldehyde solution containing 0.2mg glutaraldehyde, in 4-40 DEG C of concussion reaction 10-60min, Collect, that is, obtain bmps- glutaraldehyde complex；
(3) preparation of bmps-wga complex: prepare 0.1mg/ml wheat germ agglutinin solution, by 1mg bmps- glutaraldehyde Complex is added in 100ul wheat germ agglutinin solution, suspends, ultrasonic break up, in 4-40 DEG C of concussion reaction 10-120min, use Magnet is collected, and removes supernatant, and with 60mm phosphate (pb, ph=7) buffer solution for cleaning 3 times, obtains final product bmps-wga complex.
Embodiment 3 bmps- lens culinaris agglutinin (lca)
Bmps-lca complex be prepared as follows:
Bmps surface modification: weigh 1mg bmps, add the chaps solution that 100ml concentration is 0.5%, concussion processes 2h, To remove the albumen on bmps surface, it is then centrifuged for collecting bmps, is resuspended in after cleaning in the pe solution of 100mg/ml, 100w is ultrasonic 30min, lucifuge is incubated, and with 120mm phosphate (pb, ph=9) buffer solution for cleaning 3 times, obtains bmps-pe complex；
The preparation of bmps- dimethyl adipimide complex: take 1mg bmps-pe complex, with 10ml concentration 6mm The pb buffer of ph7 suspends, ultrasonic break up, be subsequently adding the dimethyl adipimide containing 10mg dimethyl adipimide Solution, in 4-40 DEG C of concussion reaction, is collected by centrifugation, that is, obtain bmps- dimethyl adipimide complex；
The preparation of bmps-lca complex: prepare 10mg/ml lens culinaris agglutinin solution, by 1mg bmps- dimethyl oneself two Acid imide complex is added in 10ml wheat germ agglutinin solution, suspends, ultrasonic breaks up, in 4-40 DEG C of concussion reaction 10- 120min, is collected by centrifugation, and removes supernatant, and with 100mm phosphate (pb, ph=8) buffer solution for cleaning 3 times, obtains final product bmps-lca multiple Compound.
The bmps-lca complex preparing, particle diameter is little, homogeneity is good, good dispersion, be combineding with each other of glycoprotein Efficiency high.
Embodiment 4 bmps-cona complex is used for the separation of high mannose type glycoprotein in nmumg cell
(1) mammary gland of mouse epithelial cell nmumg cell culture and protein extraction
With containing 10% hyclone, the dmem culture of 100iu/ml penicillin is based on 37 DEG C, 5%co2Under the conditions of cultivate Nmumg cell, is paved with behind ware bottom after cell, by culture dish as on ice, with 4 DEG C of pre-cooling pbs (137mm nacl, 2.7mm kcl,10mm na2hpo4,2mm kh2po4) buffer solution for cleaning ware bottom 3 times, remove culture medium, separately take pbs 1.5ml to add ware Cell is scraped with spatula and removes supernatant in 2ml centrifuge tube 1000rpm centrifugation 5min, adds 1ml ripa lysate (1% by bottom Triton x-100,150mm nacl, 25mm tris ph7.4,5mm edta, 0.5% NaTDC, 0.1%sds, 5mm Tetrasodium pyrophosphate, 50mm sodium fluoride, 1mm na3vo4, 2mm Phenylmethanesulfonyl fluoride, 0.076u/ml aprotinin), and 10mm benzyl Sulfuryl fluoride (pmsf) is fully blown and beaten, and is centrifuged 15min as 30min on ice after 4 DEG C of 12000rpm, take supernatant be protein liquid in- 80 DEG C of preservations are stand-by.
(2) in nmumg cell protein, high mannose type glycoprotein separates: nmumg cell protein is dissolved in reference to buffering (20mm tris-hcl (ph 7.4), 0.15m nacl, 1m cacl in liquid2, 1m mgcl2, 1mm mncl2) so as to albumen is eventually Concentration is in 1mg/ml.Clean bmps-cona complex 3 times with combination buffer, then in bmps-con a complex Add the nmumg cell protein of 900 μ l, concussion reaction 1h in 25 DEG C of shaking tables, reaction collects unconjugated albumen after terminating.Use again Cleaning buffer solution cleaning magnetic particle bmps-cona complex 3-5 time, until cleanout fluid od levels off to zero.Use elution buffer (20mm tris-hcl (ph 7.4), 0.15m nacl, 1m cacl2, 1m mgcl2, 1mm mncl2, 500mm Alpha-Methyl manna Glucosides) eluting agglutinin magnetic particle complex bmps-cona complex 1h, specifically bind to complex in order to eluting Glycoprotein on bmps-cona complex.
(3) wash-out protein checking
Using sds-page, and agglutinin staining checking wash-out protein.
Embodiment 5 bmps-wga complex is used for the separation of chicken egg white
Wga can specific recognition chicken egg white, and nonrecognition cona.
(1) chicken egg white and each 1mg of cona are taken, be dissolved in combination buffer (20mm tris-hcl (ph 7.4), 0.15m nacl, 1m cacl2, 1m mgcl2, 1mm mncl2).Clean bmps-wga complex 3 times with combination buffer, then Chicken egg white and the cona mixed liquid of protein of 900 μ l is added in bmps-wga complex, concussion reaction 1h in 25 DEG C of shaking tables, Reaction collects unconjugated albumen after terminating.Clean magnetic particle bmps-wga complex 3-5 time again with cleaning buffer solution, until Cleanout fluid od levels off to zero.With elution buffer (20mm tris-hcl (ph 7.4), 0.15mnacl, 1m cacl2, 1m mgcl2, 1mm mncl2, 100mm n- acetylglucosamine) and eluting agglutinin magnetic particle complex bmps-wga complex 1h, Specifically bind to the chicken egg white on bmps-wga complex in order to eluting.
(2) sds-page checking: take the ovum gallinaceum before and after separating clear and each 1 μ l of cona mixed liquor, separately take on eluent 20 μ l Sample, is separated by electrophoresis in 10%sds-page, after coomassie brilliant blue staining, decolouring, development.Result is as shown in figure 4, swimming lane In 5, chicken egg white content significantly reduces compared with swimming lane 4, and con a content has no significant change, shows synthesized bmps- Wga complex specific can settle accounts conjunction with ovum gallinaceum.Only chicken egg white is contained, without con a, bsa and wga in eluent. Show that synthesized bmps-wga complex specific can separate chicken egg white.
Although the present invention is open as above with preferred embodiment, it is not limited to the present invention, any is familiar with this skill The people of art, without departing from the spirit and scope of the present invention, can do various changes and modification, therefore the protection model of the present invention Enclosing should be by being defined that claims are defined.

Claims (9)

1. a kind of bacillus nanometer magnetosome-agglutinin complex it is characterised in that described complex be by through surface Deproteinization, The bacillus nanometer magnetosome that primary amino radical process is added on surface is coupled and is generated with agglutinin；Described surface Deproteinization is to use Surfactant processes bacillus nanometer magnetosome；Described interpolation primary amino radical is to bacillus nanometer magnetosome with the phospholipid containing primary amino radical Surface is modified；Described coupling is to be completed by cross-linking agent.
2. a kind of method preparing complex described in claim 1 is it is characterised in that methods described includes: (1) bacteria nano magnetic Corpusculum bmps surface modification: weigh bmps, add surfactant to be processed, to remove the albumen on bmps surface, Ran Houshou Collection bmps, is resuspended in phospholipid solution, ultrasonic, and incubation obtains the bmps of surface modification, i.e. bmps- phosphatide complexes；(2) The preparation of bmps- cross-linking agent complex: bmps- phosphatide complexes obtained in the previous step are suspended, break up, is subsequently adding cross-linking agent Solution is reacted, and collects, that is, obtains bmps- cross-linking agent complex；(3) preparation of bmps- cross-linking agent-agglutinin complex: Bmps- cross-linking agent complex obtained in the previous step is added in agglutinin solution, ultrasonic break up, reaction, collect, that is, obtain Bmps- cross-linking agent-agglutinin complex, i.e. bmps- agglutinin complex.
3. method according to claim 2 is it is characterised in that methods described specifically includes: (1) bmps surface modification: scale Take bmps, by every 1mgbmps, the concentration adding 50 μ l-100ml is that the surfactant solution of 0.5-2% enters heating or shakes Swing process, to remove the albumen on bmps surface, then collect bmps, after cleaning, be resuspended in the PHOSPHATIDYL ETHANOLAMINE of 1-100mg/ml In pe or Phosphatidylserine ps solution, ultrasonic, incubation, obtain bmps- phosphatide complexes；(2) bmps- cross-linking agent complex Preparation: with bmps volume mass than the buffer for 10:1-1:10ml/mg by bmps- phosphatide complexes obtained in the previous step Suspend, break up, being subsequently adding cross-linking agent quality with bmps- phosphatide complexes mass ratio is the cross-linking agent solution of 1:10 to 5:1, in 4-40 DEG C of concussion reaction 10-60min, collects, that is, obtains bmps- cross-linking agent complex；(3) bmps- cross-linking agent-agglutinin is combined The preparation of thing: prepare 0.1-10mg/ml agglutinin solution, by agglutinin and complex obtained in the previous step mass ratio 1:10 extremely 10:1, complex obtained in the previous step is added in agglutinin solution, ultrasonic breaks up, in 4-40 DEG C of concussion reaction 10- 120min, collects, obtains final product bmps- agglutinin complex.
4. according to the method in claim 2 or 3 it is characterised in that the complex that described step 2, step 3 obtain buffers Liquid or aqueous suspension, ultrasonic break up, repeated washing 3 times, regather.
5. according to the method in claim 2 or 3 it is characterised in that described surfactant be following any one: 12 Alkyl sodium sulfate, cetyl trimethylammonium bromide, 3- [3- (gallbladder amido propyl) dimethylamino] propane sulfonic acid inner salt, Polyethylene Glycol Octyl phenyl ether.
6. according to the method in claim 2 or 3 it is characterised in that described cross-linking agent be following any one: glutaraldehyde, Butanedial, n, n '-two succinimidyl carbonate, 3,3 '-two sulfur double sulfosuccinimide propionic ester, dimethyl adipyl Imines, double amber imide suberate sodium salt.
7. according to the method in claim 2 or 3 it is characterised in that described agglutinin be following any one: Semen Canavaliae coagulation Element, wheat germ agglutinin, sophora japonica lectin, lens culinaris agglutinin.
8. complex described in claim 1 is isolating and purifying the application in field.
9. application according to claim 8 is it is characterised in that be by glycoprotein mixture and bmps- agglutinin complex Reaction, combination, make Glycoprotein binding on bacillus nanometer magnetosome-agglutinin complex, then clean antibacterial with cleaning buffer solution Nano magnetic corpusculum-agglutinin complex, finally will specifically bind to the glycoprotein eluting on this complex with elution buffer Get off.
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