CN104624176A - 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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CN104624176A
CN104624176A CN201510075253.XA CN201510075253A CN104624176A CN 104624176 A CN104624176 A CN 104624176A CN 201510075253 A CN201510075253 A CN 201510075253A CN 104624176 A CN104624176 A CN 104624176A
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bmps
compound
agglutinin
crosslinking agent
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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 compound and preparation method thereof and application
Technical field
The present invention relates to a kind of bacillus nanometer magnetosome-agglutinin and preparation method thereof and application, belong to separating and purifying technology field.
Background technology
Sugar can form the saccharide complexes such as glycoprotein, proteoglycans and glycolipid with the covalent bond such as protein or lipid, is informational molecule important in human body, in much life and lysis, plays specific identification and mediation.The minor variations of its structure or quantity just may cause the change of its function, thus causes the generation of various disease, which results in the very big enthusiasm that people study sugar group.Along with progress and the biological development of science and technology, increasing technology is applied among the research of sugar group, as lectin affinity chromatography technology, forward position affinity chromatography technology, high performance liquid chromatography, mass spectrum, carbohydrate chip technology etc.In the research of sugar group, be mainly 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 capture technique conventional is at present lectin affinity chromatography technology.Agglutinin is a kind of nonimmune source, non-enzyme material of separating-purifying from each Plants, invertebrate or higher mammal, the protein that can be combined with a class glycosyl specificity, extremely important in the research process of glycoproteomics, be widely used in the type of glycoprotein and glycolipid sugar chain in identification of cell.Lectin affinity chromatography technology is exactly the compatibility utilizing agglutinin and sugar chain, thus be applied in affinity chromatography and be separated saccharide complex, and due to the affinity of agglutinin and sugar chain relatively low, so more easily can be reached reclaim the object of saccharide complex by wash-out.
Although currently reported, agglutinin is fixed on Prof. Du Yucang epoxidation magnetic nano particle to capture the method for saccharide complex.But the preparation of Prof. Du Yucang epoxidation magnetic nano particle is comparatively loaded down with trivial details and the uniform particle diameter of nano particle is poor.And bacillus nanometer magnetosome (Bacterial Magnetic Particles, BMP) BMPs surface has native amino group, and the coupling of agglutinin can be used for by derivatization further.In particle diameter, the less about 40nm of BMPs particle diameter, and synthesized by magnetotactic bacteria because of it, particle diameter is comparatively homogeneous.
The present invention can be the research that sugar group learns and to provide convenience feasible research material, facilitates the acquisition of saccharide complex, to study sugar group better.The more artificial magnetic nano particle of preparation method of the present invention is simple, and magnetic nano particle homogeneity is better, and uniform particle diameter can improve the coupling efficiency of particle and agglutinin and the efficiency be combined with each other with glycoprotein thereof.The BMPs-agglutinin compound of preparation of the present invention is faster than traditional chromatographic column with the step and time being separated specific glycoprotein in enrichment, and be separated glycoprotein step compared with traditional affinity chromatography few, the time is short.
Summary of the invention
The invention provides a kind of bacillus nanometer magnetosome-agglutinin compound and preparation method thereof and application, agglutinin is coupled on BMPs by the present invention, for enrichment associated glycoprotein, and utilize removal of surfactant BMPs surface protein, avoid introducing unrelated protein, interpolation PE etc. are to increase primary amino radical quantity simultaneously, improve coupling efficiency.
First object of the present invention is to provide a kind of bacillus nanometer magnetosome-agglutinin compound, and described compound generates carrying out coupling through surperficial deproteinized, the bacillus nanometer magnetosome adding primary amino radical process and agglutinin.
Described surperficial deproteinized is with surfactant process bacillus nanometer magnetosome; Described interpolation primary amino radical modifies bacillus nanometer magnetosome surface with phosphatide; Described coupling is completed by crosslinking agent.
Second object of the present invention is to provide a kind of method preparing compound described in claim 1.
Described method comprises: (1) BMPs finishing: weigh BMPs, adds surfactant and process, to remove the albumen on BMPs surface, then collect BMPs, be resuspended in phospholipid solution, ultrasonic, hatch, obtain the BMPs of finishing, be i.e. BMPs-phosphatide complexes; (2) preparation of BMPs-crosslinking agent compound: BMPs-phosphatide complexes obtained in the previous step is suspended, broken up, then adds cross-linking agent solution and react, collects, namely obtains BMPs-crosslinking agent compound; (3) preparation of BMPs-crosslinking agent-agglutinin compound: BMPs-crosslinking agent compound obtained in the previous step is joined in agglutinin solution, ultrasonicly to break up, reaction, collect, namely BMPs-crosslinking agent-agglutinin compound is obtained, i.e. BMPs-agglutinin compound.
Surfactant in described step 1 processes, and in one embodiment of the invention, is at 60-100 DEG C of heating 5-20min or room temperature concussion 1-3h, wherein adopts the method for heating, fast faster, better effects if.Remove the albumen in the little surface of magnetic own, can avoid introducing unrelated protein in follow-up test.
Phosphatide in described step 1, in one embodiment of the invention, is phosphatidyl-ethanolamine (PE) and phosphatidylserine (PS), can provides more primary amino radical for BMPs surface, for next step coupling agglutinin provides more multidigit point.
Described step 1 is in metastable condition by the form of the phosphatide parcel magnetic particle that ultrasonic method is formed, and can ensure that the efficiency of magnetic particle coupling lipid is higher.
Hatching in described step 1 in one embodiment of the invention, is that lucifuge is hatched, and can ensure that the efficiency of magnetic particle coupling lipid is higher.
Described step 2, in one embodiment of the invention, BMPs-phosphatide complexes is suspended in PB buffer solution (namely only to add Na 2hPO 4and KH 2pO 4) in, use ultrasonic breaing up.
Described step 2, in one embodiment of the invention, be select concentration below 10mM, the PB buffer solution of pH 7-9.Described buffer solution can regulate ion concentration, and solution is in comparatively under low ion concns, and now BMPs colloidal solution is more stable, and BMPs particle disperses more, not easily assembles, thus improves coupling efficiency.
After each step of described step 1, step 2, step 3 collects BMPs or its compound, in one embodiment of the invention, also comprise the BMPs of collection or its compound buffer solution or aqueous suspension, ultrasonicly to break up, collect, get supernatant, repeated washing 3 times.During cleaning, preferably adopt the buffer solution of high concentration, the stability of colloidal solution can be destroyed, BMPs is assembled faster, can the time be shortened.Described cleaning buffer solution, in one embodiment of the invention, adopts the PB buffer solution of more than 40mM.
Described collection in one embodiment of the invention, is use magnet or magnetic frame to collect.BMPs solution is colloidal solution actually, in the collection process of each step, needs first to be adsorbed by BMPs by external force, namely destroys the stability of colloid.
Described method, in one embodiment of the invention, specifically comprise: (1) BMPs finishing: weigh BMPs, by every 1mg BMPs, the concentration adding 50 μ L-100mL is that the surfactant solution of 0.5-2% carries out heating or shake process, to remove the albumen on BMPs surface, then BMPs is collected, in the phosphatidyl-ethanolamine PE being resuspended in 1mg-100mg/mL after cleaning or phosphatidylserine PS solution, ultrasonic, hatch, obtain the BMPs of finishing, be i.e. BMPs-phosphatide complexes; (2) preparation of BMPs-crosslinking agent compound: BMPs-phosphatide complexes obtained in the previous step suspends, breaks up by buffer solution with being 10:1-1:10 with BMPs mass volume ratio, then crosslinking agent quality is added and 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, collect, namely obtain BMPs-crosslinking agent compound; (3) preparation of BMPs-crosslinking agent-agglutinin compound: preparation 0.1-10mg/ml agglutinin solution, by the mass ratio 1:10 to 10:1 of agglutinin and compound obtained in the previous step, compound obtained in the previous step is joined in agglutinin solution, ultrasonicly to break up, in 4-40 DEG C of concussion reaction 10-120min, collect, obtain BMPs-agglutinin compound.
Described crosslinking agent and BMPs-phosphatide complexes mass ratio in one embodiment of the invention, are 1:2-2:1.
Described BMPs-crosslinking agent compound 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: lauryl sodium sulfate (SDS), softex kw (CTAB), 3-[3-(courage amido propyl) dimethylamino] propane sulfonic acid inner salt (CHAPS), Triton X-100 (Triton X-100).
Described crosslinking agent, in one embodiment of the invention, can be following any one: glutaraldehyde, butanedial, N, N '-two succinimidyl carbonates, 3,3 '-two sulphur two sulfosuccinimide propionic ester, dimethyl adipimide.
Described crosslinking agent in one embodiment of the invention, is double amber imide suberate sodium salt (BS 3).
Described agglutinin can be any one agglutinin.
Described agglutinin, in one embodiment of the invention, for following any one: ConA (conA), wheat germ agglutinin (WGA), sophora japonica lectin (SJA), LCA (LCA).
The described BMPs-agglutinin compound prepared, in one embodiment of the invention, closed, preservation in bovine serum albumin(BSA) (BSA).
The described BMPs-agglutinin compound prepared, in one embodiment of the invention, add confining liquid (10mM PBS containing 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, adopt 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, 2007, prepared by the method described in 45 (1): 75-81.
3rd object of the present invention is to provide the application of described compound in separation and purification field.
Described application, in one embodiment of the invention, is used to be separated glycoprotein.
Described application, in one embodiment of the invention, that glycoprotein mixture and BMPs-agglutinin compound are reacted, combine, make Glycoprotein binding on this magnetic particle compound, clean this magnetic particle compound with cleaning buffer solution again, finally with elution buffer, the glycoprotein of specific binding on this compound is eluted.
Beneficial effect of the present invention:
(1) utilize BMPs surface to have native amino group, and the character of coupling can be used for by derivatization further, and the advantage that particle diameter is less, particle diameter is comparatively homogeneous, prepare BMPs-agglutinin compound.By modifying BMPs surface, remove BMPs oneself protein to impact during subsequent applications, with the addition of PE or PS is that coupling agglutinin provides more primary amino radical simultaneously, improves coupling efficiency, improves the dispersiveness of BMPs simultaneously;
(2) utilize crosslinking agent, as linking arm, BMPs and agglutinin have been carried out covalent coupling, and ensure that agglutinin identification sugar chain region is not damaged;
(3) by regulating the concentration of buffer solution, shortening preparation time, improve coupling efficiency.During coupling reaction, carry out in low-concentration buffer, to maintain colloidal stability, make not easily to assemble between BMPs more to disperse, thus reach better coupling efficiency.And when cleaning, adopt the buffer solution of high concentration can destroy the stability of colloidal solution, make it assemble faster, in test, need repeatedly BMPs be cleaned, adsorb under high concentration buffer solution, can the time be shortened.
(4) the compound particle diameter prepared is little, homogeneity and good dispersion, can improve the coupling efficiency of particle and agglutinin and the efficiency be combined with each other with glycoprotein thereof.
Accompanying drawing explanation
Fig. 1: different B MPs and compound FT-IR figure; A: original BMPs, b: with the BMPs after surfactant process, c:BMPs-PE compound, d:BMPs-conA compound;
Fig. 2: different B MPs and the scanning electron microscope (SEM) photograph of compound; (a) (b): original BMPs, (c) (d): with the BMPs after surfactant process, (e) (f): BMPs-PE compound, (g) (h): BMPs-conA compound;
Fig. 3: the SDS-PAGE figure after the BMPs coupling agglutinin conA of different disposal method; Each swimming lane is: 1, maker, 2, conA marks product, 3, original BMPs is through BS 3after coupling conA, supernatant after 5%SDS boiling water boiling 5min; 4, BMPs-PE compound is through BS 3after coupling conA, supernatant after 5%SDS boiling water boiling 5min; 5, BMPs-PE compound is with after conA mixing 2h, supernatant after 5%SDS boiling water boiling 5min;
Fig. 4: BMPs-WGA compound is separated the SDS-PAGE figure of chicken egg white; Each swimming lane is: 1, maker, 2, BSA marks product, 3, WGA marks product, 4, be separated before the clear and conA albumen composition of ovum gallinaceum, 5, be separated after the clear and conA albumen composition of ovum gallinaceum, 6, eluent.
Detailed description of the invention
The preparation of embodiment 1 BMPs-conA compound
BMPs finishing: 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 by 100mM phosphate (PB, pH=7.5) buffer solution for cleaning 3 times; After processing, BMPs Eddy diffusion is in 20mg/mL PE solution, the ultrasonic 10min of 100W; After lucifuge hatches 2h, clean BMPs tri-times as stated above.Namely BMPs-PE compound is obtained.
BMPs-BS 3the preparation of compound: draw 900 μ L 4mM PB buffer solution suspension 1mg previous step gained compounds, 80W is ultrasonic to be broken up; Weigh BS 30.572mg, add BMPs suspension, 37 DEG C after fully dissolving with 100 μ L distilled water, 200rpm reacts 0.5h; The reacted BMPs compound of magnetic frame collector, removes supernatant, and 100mM PB buffer solution suspends, and after 80w ultrasonic wave is broken up, collect with magnet, remove supernatant, repeated washing three times, namely obtains BMPs-BS 3compound.
BMPs-BS 3the preparation of-conA compound: weigh 1mg agglutinin concanavalin (conA) and be dissolved in 1mL 4mM PB buffer solution, be mixed with 1mg/mL agglutinin solution; Previous step gained BMPs-BS3 compound 1mg is suspended in agglutinin conA solution, and 80W is ultrasonic to be broken up, 37 DEG C, and 200rpm reacts 1h; The reacted BMPs compound of magnetic frame collector, removes supernatant, and 100mM PB buffer solution suspends, and after 80w ultrasonic wave is broken up, collect with magnet, remove supernatant, repeated washing three times, obtains BMPs-BS 3-conA compound, i.e. BMPs-conA compound.
The magnetic composite of synthesis is characterized by infrared, Electronic Speculum and protein adhesive.
Infrared: as can be seen from Figure 1: original BMPs is at 1636cm -1and 1575cm -1place has two peaks to be respectively amide Ⅰ and acid amides II band, shows that original BMPs contains protein matter; After surfactant process, this peak disappears, and shows that BMPs surface protein is removed; BMPs-PE compound is at 880cm -1place have peak be C-N face outside wave, show that PE is coupled in BMPs; 1631cm after coupling agglutinin -1and 1568cm -1place has two peaks to be respectively amide Ⅰ and acid amides II band, shows that conA is coupled to BMPs.
Electronic Speculum: as can be seen from Figure 2: original BMPs dispersiveness is poor, and surface can be observed lipid film; After surfactant process, BMPs dispersiveness is deteriorated, and face grease plasma membrane is thinning, shows that surface mass is partially removed; After forming compound with PE, dispersiveness improves, and lipid film is thickening, unclear boundaries, and form is comparatively loose, shows that PE is successfully coupled to BMPs; Connect the BMPs dispersiveness after agglutinin still better, skin covering of the surface is thinning, and boundary is obvious, and form is comparatively tight, infers by after BS3 coupling protein, through protein encapsulation, causes form to become tight.The BMPs-conA compound prepared, particle diameter is little, homogeneity good, good dispersion.
Protein adhesive: with get original BMPs and the BMPs-PE compound of 1mg under the same conditions with conA coupling, after 5%SDS boiling water boiling 5min, carry out SDS-PAGE.Result as shown in Figure 3.Experimental result shows, conA is successfully coupled to magnetic corpusculum.Through contrast known BMPs-PE compound carrying conA amount much larger than original BMPs bearing capacity, and avoid the impact that the albumen of original BMPs own brings, illustrate that PE can improve more functional group, thus effectively improve coupling efficiency.
The preparation of embodiment 2 BMPs-wheat germ agglutinin (WGA) compound
Prepare BMPs-wheat germ agglutinin (WGA) compound by the following method:
(1) BMPs finishing: weigh 1mg BMPs, add the CTAB solution that 50uL concentration is 2%, 20min is heated in 60 DEG C, to remove the albumen on BMPs surface, then collected by centrifugation BMPs, be resuspended in the phosphatidylserine PS solution of 1mg/mL after cleaning, the ultrasonic 1h of 80W, lucifuge hatches 24h, and with 40mM phosphate (PB, pH=7) buffer solution for cleaning 3 times, obtains BMPs-PS compound;
(2) preparation of BMPs-glutaraldehyde compound: get 1mg BMPs-PS compound, suspend with the buffer solution of 100uL concentration 10mM, ultrasonicly to break up, then the glutaraldehyde solution containing 0.2mg glutaraldehyde is added, in 4-40 DEG C of concussion reaction 10-60min, collect, namely obtain BMPs-glutaraldehyde compound;
(3) preparation of BMPs-WGA compound: preparation 0.1mg/ml wheat germ lectin cellulose solution, 1mg BMPs-glutaraldehyde compound is joined in 100uL wheat germ lectin cellulose solution, suspend, ultrasonicly to break up, in 4-40 DEG C of concussion reaction 10-120min, collect with magnet, remove supernatant, and by 60mM phosphate (PB, pH=7) buffer solution for cleaning 3 times, obtain BMPs-WGA compound.
Embodiment 3 BMPs-LCA (LCA)
Prepare BMPs-LCA compound by the following method:
BMPs finishing: weigh 1mg BMPs, add the CHAPS solution that 100mL concentration is 0.5%, concussion process 2h, to remove the albumen on BMPs surface, then collected by centrifugation BMPs, be resuspended in the PE solution of 100mg/mL after cleaning, the ultrasonic 30min of 100W, lucifuge is hatched, and with 120mM phosphate (PB, pH=9) buffer solution for cleaning 3 times, obtains BMPs-PE compound;
The preparation of BMPs-dimethyl adipimide compound: get 1mg BMPs-PE compound, suspend with the PB buffer solution of the pH7 of 10mL concentration 6mM, ultrasonicly to break up, then the dimethyl adipimide solution containing 10mg dimethyl adipimide is added, in 4-40 DEG C of concussion reaction, collected by centrifugation, namely obtains BMPs-dimethyl adipimide compound;
The preparation of BMPs-LCA compound: preparation 10mg/ml lentil-lectin cellulose solution, 1mg BMPs-dimethyl adipimide compound is joined in 10mL wheat germ lectin cellulose solution, suspend, ultrasonicly to break up, in 4-40 DEG C of concussion reaction 10-120min, collected by centrifugation, remove supernatant, and by 100mM phosphate (PB, pH=8) buffer solution for cleaning 3 times, obtain BMPs-LCA compound.
The BMPs-LCA compound prepared, particle diameter is little, homogeneity good, good dispersion, high with the efficiency that be combined with each other of glycoprotein.
Embodiment 4 BMPs-conA compound is used for the separation of high mannose type glycoprotein in NmUMG cell
(1) mammary gland of mouse epithelial cell NMuMG cell chulture and protein extraction
With containing 10% hyclone, the DMEM of 100IU/mL penicillin cultivates based on 37 DEG C, 5%CO 2cultivate NMuMG cell under condition, be paved with after at the bottom of ware until cell, by culture dish as on ice, with 4 DEG C of precooling PBS (137mM NaCL, 2.7mM KCL, 10mM Na 2hPO 4, 2mM KH 2pO 4) at the bottom of buffer solution for cleaning ware 3 times, remove culture medium, separately get PBS 1.5ml and add at the bottom of ware, with spatula cell scraped and remove supernatant in the centrifugal 5min of 2ml centrifuge tube 1000rpm, add 1ml RIPA lysate (1%Triton X-100,150mM NaCl, 25mM Tris pH7.4,5mM EDTA, 0.5% NaTDC, 0.1%SDS, 5mM tetrasodium pyrophosphate, 50mM sodium fluoride, 1mM Na 3vO 4, 2mM phenylmethylsulfonyl fluoride, 0.076U/ml Aprotinin), and 10mM phenylmethylsulfonyl fluoride (PMSF) is is fully blown and beaten, as after 30min on ice in 4 DEG C of centrifugal 15min of 12000rpm, get supernatant and protein liquid is stand-by in-80 DEG C of preservations.
(2) in NMuMG cell protein, high mannose type glycoprotein is separated: NMuMG cell protein is dissolved in (20mM Tris-HCl (pH 7.4), 0.15M NaCl, 1M CaCl in binding buffer liquid 2, 1M MgCl 2, 1mM MnCl 2), make its final concentration of protein at about 1mg/ml.Clean BMPs-ConA compound 3 times with binding buffer liquid, in BMPs-Con A compound, then add the NMuMG cell protein of 900 μ L, concussion reaction 1h in 25 DEG C of shaking tables, reaction terminates the unconjugated albumen of rear collection.Magnetic particle BMPs-ConA compound is cleaned 3-5 time, until cleaning fluid OD levels off to zero again with cleaning buffer solution.With elution buffer (20mM Tris-HCl (pH 7.4), 0.15M NaCl, 1M CaCl 2, 1M MgCl 2, 1mM MnCl 2, 500mM Alpha-Methyl mannoside) and wash-out agglutinin magnetic particle complex BMPs-ConA compound 1h, in order to the glycoprotein of wash-out specific binding on complex BMPs-ConA compound.
(3) eluted protein checking
Utilize SDS-PAGE, and agglutinin decoration method checking eluted protein.
Embodiment 5 BMPs-WGA compound is used for the separation of chicken egg white
WGA can specific recognition chicken egg white, and nonrecognition conA.
(1) get chicken egg white and each 1mg of conA, be dissolved in (20mM Tris-HCl (pH 7.4), 0.15M NaCl, 1M CaCl in binding buffer liquid 2, 1M MgCl 2, 1mM MnCl 2).Clean BMPs-WGA compound 3 times with binding buffer liquid, in BMPs-WGA compound, then add chicken egg white and the conA mixed liquid of protein of 900 μ L, concussion reaction 1h in 25 DEG C of shaking tables, reaction terminates the unconjugated albumen of rear collection.Magnetic particle BMPs-WGA compound is cleaned 3-5 time, until cleaning fluid OD levels off to zero again with cleaning buffer solution.With elution buffer (20mM Tris-HCl (pH 7.4), 0.15MNaCl, 1M CaCl 2, 1M MgCl 2, 1mM MnCl 2, 100mM N-Acetyl-D-glucosamine) and wash-out agglutinin magnetic particle complex BMPs-WGA compound 1h, in order to the chicken egg white of wash-out specific binding on BMPs-WGA compound.
(2) SDS-PAGE checking: get the clear and each 1 μ L of conA mixed liquor of ovum gallinaceum before and after being separated, separately get eluent 20 μ L loading, carry out electrophoretic separation in 10%SDS-PAGE, after coomassie brilliant blue staining, decolouring, development.As shown in Figure 4, in swimming lane 5, chicken egg white content comparatively obviously reduces in swimming lane 4 result, and con A content there is no significant change, shows that synthesized BMPs-WGA compound specificly can settle accounts conjunction with ovum gallinaceum.Only containing chicken egg white in eluent, not containing con A, BSA and WGA.Show that synthesized BMPs-WGA compound can specific separation chicken egg white.
Although the present invention with preferred embodiment openly as above; but it is also not used to limit the present invention, any person skilled in the art, without departing from the spirit and scope of the present invention; all can do various changes and modification, what therefore protection scope of the present invention should define with claims is as the criterion.

Claims (10)

1. bacillus nanometer magnetosome-agglutinin compound, is characterized in that, the bacillus nanometer magnetosome and agglutinin that add primary amino radical process through surperficial deproteinized, surface are carried out coupling and generate by described compound.
2. compound according to claim 1, is characterized in that, described surperficial deproteinized is with surfactant process bacillus nanometer magnetosome; Described interpolation primary amino radical modifies bacillus nanometer magnetosome surface with phosphatide; Described coupling is completed by crosslinking agent.
3. prepare the method for compound described in claim 1 for one kind, it is characterized in that, described method comprises: (1) BMPs finishing: weigh BMPs, adds surfactant and process, to remove the albumen on BMPs surface, then BMPs is collected, be resuspended in phospholipid solution, ultrasonic, hatch, obtain the BMPs of finishing, i.e. BMPs-phosphatide complexes; (2) preparation of BMPs-crosslinking agent compound: BMPs-phosphatide complexes obtained in the previous step is suspended, broken up, then adds cross-linking agent solution and react, collects, namely obtains BMPs-crosslinking agent compound; (3) preparation of BMPs-crosslinking agent-agglutinin compound: BMPs-crosslinking agent compound obtained in the previous step is joined in agglutinin solution, ultrasonicly to break up, reaction, collect, namely BMPs-crosslinking agent-agglutinin compound is obtained, i.e. BMPs-agglutinin compound.
4. method according to claim 3, it is characterized in that, described method specifically comprises: (1) BMPs finishing: weigh BMPs, and by every 1mg BMPs, the concentration adding 50 μ L-100mL is that the surfactant solution of 0.5-2% carries out heating or shake process, to remove the albumen on BMPs surface, then BMPs is collected, in the phosphatidyl-ethanolamine PE being resuspended in 1mg-100mg/mL after cleaning or phosphatidylserine PS solution, ultrasonic, hatch, obtain BMPs-phosphatide complexes; (2) preparation of BMPs-crosslinking agent compound: BMPs-phosphatide complexes obtained in the previous step suspends, breaks up by buffer solution with being 10:1-1:10 with BMPs mass volume ratio, then crosslinking agent quality is added and 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, collect, namely obtain BMPs-crosslinking agent compound; (3) preparation of BMPs-crosslinking agent-agglutinin compound: preparation 0.1-10mg/ml agglutinin solution, by the mass ratio 1:10 to 10:1 of agglutinin and compound obtained in the previous step, compound obtained in the previous step is joined in agglutinin solution, ultrasonicly to break up, in 4-40 DEG C of concussion reaction 10-120min, collect, obtain BMPs-agglutinin compound.
5. the method according to claim 3 or 4, is characterized in that, the compound buffer solution that described step 2, step 3 obtain or aqueous suspension, ultrasonicly to break up, and repeated washing 3 times, regathers.
6. the method according to claim 3 or 4, it is characterized in that, described surfactant be following any one: lauryl sodium sulfate, softex kw, 3-[3-(courage amido propyl) dimethylamino] propane sulfonic acid inner salt, Triton X-100.
7. the method according to claim 3 or 4, it is characterized in that, described crosslinking agent be following any one: glutaraldehyde, butanedial, N, N '-two succinimidyl carbonate, 3,3 '-two sulphur two sulfosuccinimide propionic ester, dimethyl adipimide, double amber imide suberate sodium salt.
8. the method according to claim 3 or 4, is characterized in that, described agglutinin be following any one: ConA, wheat germ agglutinin, sophora japonica lectin, LCA.
9. compound described in claim 1 or 2 is in the application in separation and purification field.
10. application according to claim 9, it is characterized in that, will be that glycoprotein mixture and BMPs-agglutinin compound are reacted, combine, make Glycoprotein binding on this magnetic particle compound, clean this magnetic particle compound with cleaning buffer solution again, finally with elution buffer, the glycoprotein of specific binding on this compound is eluted.
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