CN101962402A

CN101962402A - Astragalus membranaceus glycoprotein as well as preparation method and usage thereof

Info

Publication number: CN101962402A
Application number: CN 200910089580
Authority: CN
Inventors: 冯前进; 薛慧清; 杨向竹; 牛欣; 刘亚明; 赵俊云; 宋强; 王永辉; 张立伟
Original assignee: Shanxi Traditional Chinese Medical College
Current assignee: Shanxi Traditional Chinese Medical College; Shanxi University of Traditional Chinese Mediciine
Priority date: 2009-07-23
Filing date: 2009-07-23
Publication date: 2011-02-02
Anticipated expiration: 2029-07-23
Also published as: CN101962402B

Abstract

The invention discloses an astragalus membranaceus glycoprotein as well as a preparation method and a usage thereof. The astragalus membranaceus glycoprotein of the invention is prepared by the following steps: salting out in astragalus membranaceus water extract by utilizing ammonium sulfate; eliminating free proteins by utilizing a Sevage method; separating and purifying to prepare a new active component by utilizing dialysis and DEAE-C32 anion exchange resin column chromatography; analyzing the new active component to obtain the astragalus membranaceus glycoprotein with the molecular weight of about 53.26kD by utilizing an SDS (Sodium Dodecyl Sulfonate)- polyacrylamide gel electrophoresis method; and further measuring through an experiment and verifying through a drug effect experiment the physicochemical property of the new active component. The astragalus membranaceus glycoprotein of the invention has the advantages of obvious immunosuppression action and wide market prospects, and can be taken as an economical and effective immunosuppressant.

Description

A kind of tragacanthin albumen and its production and use

Invention field

The present invention relates to a kind of Radix Astragali extract, particularly a kind of tragacanthin albumen and its production and use.

Background technology

Glycoprotein (glycoprotein) be a class be connected with covalent linkage with polypeptide or protein by carbohydrate and form conjugated protein, be present in widely in nature animal plant and the certain micro-organisms, be an important class macromole in the organism.The sixties in 20th century, biochemist and chemists have produced interest to the glycoprotein of long-standing neglect, have carried out many-sided research.Discover that more and more glycoprotein has significant medicinal efficacy and nourishing function, can enhancing immunity regulate, suppress tumour, lowering blood glucose, blood fat, anti-oxidant, health care is waited for a long time, and has utility value for human body.Being applied to clinical at present and having had immunocompetent efficiently pharmaceutical protein formulations mostly is glycoprotein.In recent years, the glycoprotein in plant and natural product source is paid much attention in fields such as biological chemistry, physiotechnology, clinical chemistry, pharmacy and food.Particularly be present in the glycoprotein in the plant, have wide utilization prospect aspect new special medicine and the functional foodstuff exploitation.

The Shanxi famous-region drug Radix Astragali is a kind of invigorating QI to consolidate the body surface resistance Chinese medicinal materials commonly used, can enhancing body's immunological function.Since the seventies in 20th century, Chinese scholars has been carried out a large amount of research to Radix Astragali chemical ingredients and pharmacological action, compounds such as the many flavonoids of extraction separation, saponins, polysaccharide therefrom, and its pharmacological action and biological activity studied.But up to now, do not see the proteic research report of relevant tragacanthin as yet.

Summary of the invention

The object of the invention is to disclose a kind of tragacanthin albumen, the present invention also aims to disclose the preparation method of this glycoprotein, the present invention also aims to disclose the purposes of this glycoprotein.

The present invention seeks to be achieved through the following technical solutions:

Tragacanthin albumen of the present invention is prepared by following method:

Get Milkvetch Root, it is ground into Powdered, take by weighing the 200-600 weight part, add water 1200-2000 parts by volume, soaked 8-16 hour, lixiviate 1-3 below 40-60 ℃ hour, filter; Add water 800-1600 parts by volume in the residue again, lixiviate 1-3 below 40-60 ℃ hour, filter, merge filtrate twice, with the centrifugal 20-40 of 2000-4000r/min speed minute, merge supernatant liquor, get crude extract;

Crude extract is concentrated into the 200-400 parts by volume, adds the ammonium sulfate that grinds to form powdery, the limit edged is stirred to saturated, leaves standstill under 2-6 ℃ 12-24 hour, and with the centrifugal 20-40 of 2000-4000r/min speed minute, abandoning supernatant must precipitate;

Precipitation adds the less water dissolving, adds ammonium sulfate again to saturated, leaves standstill 1-3 hour, and with the centrifugal 20-40 of 2000-4000r/min speed minute, abandoning supernatant, precipitation with the less water dissolving, repeated above-mentioned steps again, obtain precipitation, are raw sugar albumen;

Raw sugar albumen is added the less water dissolving, add sevag reagent, violent jolting 10-30 minute, centrifugal, inclining supernatant liquor, removes middle layer metaprotein and lower floor's chloroform, repeats this step and does not almost have metaprotein to the middle layer;

Supernatant liquor is placed dialysis tubing, and pure water dialysis 24-72 hour gets the tragacanthin protein solution, through lyophilize, gets oyster white meal tragacanthin albumen (HQGP).

Tragacanthin albumen of the present invention is preferably as follows the method preparation:

Get Milkvetch Root, it is ground into Powdered, take by weighing 400 weight parts, add water 1600 parts by volume, soaked 12 hours, lixiviate below 55-60 ℃ 2 hours is filtered; Add water 1200 parts by volume in the residue again, lixiviate below 55-60 ℃ 2 hours is filtered, and merges filtrate twice, with 3000r/min speed centrifugal 30 minutes, merge supernatant liquor, crude extract;

Crude extract is concentrated into 300 parts by volume, adds the ammonium sulfate grind to form powdery, the limit edged is stirred to saturated, leaves standstill under 4 ℃ 12 hours, and with 3000r/min speed centrifugal 30 minutes, abandoning supernatant must precipitate;

Precipitation adds the less water dissolving, adds ammonium sulfate again to saturated, leaves standstill 2 hours, and with 3000r/min speed centrifugal 30 minutes, abandoning supernatant, precipitation with the less water dissolving, repeated above-mentioned steps again, obtain precipitation, are raw sugar albumen;

Raw sugar albumen is added the less water dissolving, add sevag reagent, violent jolting 20 minutes, centrifugal, inclining supernatant liquor, removes the yellow colloid degeneration albumen in middle layer and lower floor's chloroform, repeats this step and does not almost have metaprotein to the middle layer;

Supernatant liquor is placed dialysis tubing, on the air constant temperature oscillator,, get the tragacanthin protein solution,, get oyster white meal tragacanthin albumen (HQGP) through lyophilize with pure water dialysis 48 hours.

Get Milkvetch Root, it is ground into Powdered, take by weighing 300 weight parts, add water 1900 parts by volume, soaked 9 hours, lixiviate below 50 ℃ 3 hours is filtered; Add water 1500 parts by volume in the residue again, lixiviate below 50 ℃ 1 hour is filtered, and merges filtrate twice, with 3500r/min speed centrifugal 35 minutes, merge supernatant liquor, crude extract;

Crude extract is concentrated into 250 parts by volume, adds the ammonium sulfate grind to form powdery, the limit edged is stirred to saturated, leaves standstill under 3 ℃ 24 hours, and with 3500r/min speed centrifugal 35 minutes, abandoning supernatant must precipitate;

Precipitation adds the less water dissolving, adds ammonium sulfate again to saturated, leaves standstill 1 hour, and with 2500r/min speed centrifugal 35 minutes, abandoning supernatant, precipitation with the less water dissolving, repeated above-mentioned steps again, obtain precipitation, are raw sugar albumen;

Raw sugar albumen is added the less water dissolving, add sevag reagent, violent jolting 25 minutes, centrifugal, inclining supernatant liquor, removes the yellow colloid degeneration albumen in middle layer and lower floor's chloroform, repeats this step and does not almost have metaprotein to the middle layer;

Supernatant liquor is placed dialysis tubing, on the air constant temperature oscillator,, get the tragacanthin protein solution,, get oyster white meal tragacanthin albumen (HQGP) through lyophilize with pure water dialysis 24 hours.

Get Milkvetch Root, it is ground into Powdered, take by weighing 500 weight parts, add water 1300 parts by volume, soaked 15 hours, lixiviate below 45 ℃ 1 hour is filtered; Add water 900 parts by volume in the residue again, lixiviate below 45 ℃ 3 hours is filtered, and merges filtrate twice, with 2500r/min speed centrifugal 25 minutes, merge supernatant liquor, crude extract;

Crude extract is concentrated into 350 parts by volume, adds the ammonium sulfate grind to form powdery, the limit edged is stirred to saturated, leaves standstill under 5 ℃ 18 hours, and with 2500r/min speed centrifugal 25 minutes, abandoning supernatant must precipitate;

Precipitation adds the less water dissolving, adds ammonium sulfate again to saturated, leaves standstill 3 hours, and with 3500r/min speed centrifugal 25 minutes, abandoning supernatant, precipitation with the less water dissolving, repeated above-mentioned steps again, obtain precipitation, are raw sugar albumen;

Raw sugar albumen is added the less water dissolving, add sevag reagent, violent jolting 15 minutes, centrifugal, inclining supernatant liquor, removes the yellow colloid degeneration albumen in middle layer and lower floor's chloroform, repeats this step and does not almost have metaprotein to the middle layer;

Supernatant liquor is placed dialysis tubing, on the air constant temperature oscillator,, get the tragacanthin protein solution,, get oyster white meal tragacanthin albumen (HQGP) through lyophilize with pure water dialysis 72 hours.

Wherein, described weight part and parts by volume are the relations of g/ml.

The Radix Astragali albumen of the present invention powdery that is creamy white, soluble in water, be insoluble to organic solvents such as methyl alcohol, ethanol, ether, acetone, chloroform; Concentration is that tragacanthin protein solution mensuration pH value under 23 ℃ of 0.096mg/ml is 5.8; Concentration is that the specific rotatory power of the tragacanthin protein solution of 0.08g/100ml is-56.38 °; The charateristic avsorption band of ultraviolet spectrogram is 485nm and 280nm; Infrared spectrogram is shown in 3399.2cm ^-1, 2962.6cm ^-1, 1643.2cm ^-1, 1546.2cm ^-1, 1403.0,1246.5cm ^-1There is obvious absorption peaks at the place; The combination of glycopeptide is the N-glycosidic link in the tragacanthin albumen; The proteic molecular weight of tragacanthin is 50-55kD, preferred 53.26kD.

Description of drawings

Fig. 1: Radix Astragali extract ultraviolet spectrogram

Fig. 2: Radix Astragali extract infrared spectrogram

Fig. 3: the elution curve of extract on DEAE-Cellulose 32 chromatography columns

Fig. 4: Radix Astragali extract is through ultraviolet spectrogram before and after the alkaline purification

Fig. 5: typical curve

Fig. 6: monose compositional analysis figure, Fig. 6 .1:HQGP sample GC figure, Fig. 6 .2: wood sugar (part), Fig. 6 .3: pectinose (part), Fig. 6 .4: seminose (part), Fig. 6 .5: semi-lactosi (part), Fig. 6 .6: glucose (part)

Fig. 7: the SDS-PAGE electrophoretogram, 1,2 is the HQGP sample; 3 is standard protein.

Tragacanthin albumen of the present invention (HQGP) is to adopt ammonium sulfate precipitation, and the Sevage method is removed free protein, again through dialysis and DEAE-C32 anion-exchange resin column chromatography, separates the new active ingredient of a class that obtains from water extraction of astragalus membranaceus.Analyze through the SDS-polyacrylamide gel electrophoresis, get its molecular weight and be about 53.26kD.Further obtain the physico-chemical property of this new active ingredient through experiment, and experimental results show that by the mouse spleen lymphocyte vitro conversion, the tragacanthin albumen (HQGP) with physico-chemical property of the present invention that the inventive method makes all is restraining effect to the propagation of the mouse spleen lymphocyte of vitro culture normal mouse splenic lymphocyte and immunocompromised, various dose HQGP group is carried out statistics relatively with the blank group, utmost point significant difference is all arranged, and restraining effect presents dose-dependently; Tragacanthin albumen of the present invention has significant inhibition activity to mouse boosting cell in-vitro multiplication under the active state simultaneously.Prove thus,

Tragacanthin albumen of the present invention has significant immunoregulatory activity.

Following experimental example and embodiment further specify but are not limited to the present invention

The proteic physico-chemical property research experiment of experimental example 1 tragacanthin of the present invention

1, experiment material

(1) raw material: the Radix Astragali, available from the Huiyuan, Shanxi.

(2) reagent: acrylamide (Acrylamide), bisacrylamide (Bis-Acrylamide), Tutofusin tris (Tris), Tetramethyl Ethylene Diamine (TEMED), beta-mercaptoethanol, Xylene Brilliant Cyanine G R-250, bromjophenol blue (the BBI import is original-pack); Low molecular weight protein (LMWP) standard (LMW-SDS markerkit) (Amersham company); (Ammonium Persulfate AP) (is the import packing of Amresco company) for glycine (Gly), trishydroxymethyl methylglycine (Tricine), sodium lauryl sulphate (SDS), ammonium persulphate; DEAE-Cellulose 32 (Chemical Reagent Co., Ltd., Sinopharm Group); Sodium-chlor, sodium hydroxide, hydrochloric acid, ammonium sulfate, Glacial acetic acid, methyl alcohol, ethanol, propyl carbinol, glycerine, chloroform (being homemade analytical pure); Redistilled water; Dialysis tubing (U.S.).

(3) instrument: AKTA Purifier-10 type bioactive molecules system, U.S. PharmaciaBiotech company; TE612-L type electronic balance, German Sai Duolisi instrument system company limited; The VarianCary50 ultraviolet-visible pectrophotometer; LR4001 type Rotary Evaporators, German HE100LPH company; 98-1-B type electronic thermostatic electric mantle, Heto FD8 vacuum freezing drying oven (Denmark); The limited public LD5-10B type low speed centrifuge of Tianjin Tai Site instrument, Beijing Medical Centrifugal Machine Factory; CA-1111 type cold water circulation device, Shanghai Ai Lang Instr Ltd.; SHB-88 type circulation ability of swimming vacuum pump, Great Wall, Zhengzhou scientific ﹠ trading Co., Ltd.; The desk-top constant temperature oscillator of THE-D (airbath), Taicang experimental installation factory.

2, experimental technique

(1) makes Radix Astragali extract of the present invention by the embodiment of the invention 1 described method through separation, purifying.

(2) states of matter: (1) gained Radix Astragali extract powdery that is creamy white.

(3) solvability is measured: it is a small amount of to get (1) gained Radix Astragali extract, respectively with water, organic solvents such as methyl alcohol, ethanol, ether, acetone, chloroform are tested its solvability, the result shows this extract powdery that is creamy white, soluble in water, be insoluble to organic solvents such as methyl alcohol, ethanol, ether, acetone, chloroform.

(4) pH value is measured: (1) gained Radix Astragali extract is mixed with the aqueous solution that concentration is 0.096mg/ml, measures pH value=5.8 in 23. ℃.

(5) specific rotatory power is measured: (1) gained Radix Astragali extract is configured to the aqueous solution that concentration is 0.08g/100ml, does blank with redistilled water, the glucose solution of 1.002g/100ml is done the specific rotatory power that correcting measuring gets HQGP and is :-56.38 °.

(6) ultraviolet, Infrared spectroscopy

(1) gained Radix Astragali extract is configured to the aqueous solution that concentration is 0.5mg/ml, is blank with water, gets ultraviolet spectrogram in the interscan of 200nm-320nm wavelength region, and as shown in Figure 1, ultraviolet spectrogram indicating characteristic absorption peak is 485nm and 280nm.

It is a small amount of to get the above-mentioned aqueous solution, adopts the KBr pressed disc method, at 4000cm ^-1-500cm ^-1The scope interscan gets infared spectrum, and as shown in Figure 2, absorption peak is positioned at 3500cm ^-1-2800cm ^-1Between, show that extract of the present invention is a saccharide compound, 3399.2,2962.6,1643.2,1546.2,1403.0, there is obvious absorption peaks at the 1246.5cm-1 place, proves that further this sample is a glycoprotein.3399.2cm wherein ^-1About be polysaccharide hydroxyl stretching vibration absorption peak, 2962.6cm ^-1About be sugared C-H stretching vibration absorption peak, 1403cm ^-1Be polysaccharide C-O vibration absorption peak, 1246.5cm ^-1Be the absorption peak of pyranose ring ehter bond C-O-C, 1643.2cm ^-1And 1546.2cm ^-1The place is the N-H vibration absorption peak of amide group.

(7) bioactive molecules systems analysis

Get the Radix Astragali extract of (1) gained, through DEAE-Cellulose 32 column chromatographies, be elutriant wash-out successively with 0.05mol/lLNaCl and 0.1mol/LNaCl respectively, measure its absorbancy at glycoprotein charateristic avsorption band 485nm and 280nm place, with the absorbancy is ordinate zou, draws HQGP absorbancy and elution time graphic representation.As shown in Figure 3, polysaccharide extinction peak, 485nm place is consistent with the time that albumen extinction peak, 280nm place occurs, and illustrates that this extract has the feature of glycoprotein.

(8) mensuration of cardohydrata-peptide linkage type

The Radix Astragali extract of getting (1) gained is made into the aqueous solution of 0.5mg/ml and the NaoH of 0.5mg/ml (0.2mol/L) solution, in 45 ℃ of water bath processing 2h, NaoH solution with water and 0.2mol/L is blank respectively, get ultraviolet spectrogram in the interscan of 200nm-400nm scope, as shown in Figure 4, absorbing does not all appear between 230nm-240nm in the ultraviolet spectrogram of the aqueous solution and alkaline purification sample, and the unsaturated amino acid whose characteristic absorbance of hydroxyl does not appear, determine that thus the combination of glycopeptide in this Radix Astragali extract is the N-cardohydrata-peptide linkage.

(9) total sugar content is measured

With the sulfuric acid-phynol method colour developing, do standard with the glucose solution of 0.02mg/ml, 0.04mg/ml, 0.06mg/ml, 0.08mg/ml, 0.10mg/ml and measure at the 475nm place, getting typical curve is Abs=5.22884*C+0.23032; R=0.98042; (1) gained Radix Astragali extract is mixed with the 0.5mg/ml aqueous solution, under equal conditions measures absorbancy, calculating total sugar content is 6.0%, sees accompanying drawing 5.

(10) monose compositional analysis

Get wood sugar, seminose, pectinose, semi-lactosi, glucose, each 4.6mg of glucuronic acid in 6 ampoules, respectively add the F of 2moL/L ₃Tube sealing behind the CCOOH2mL; 120 ℃ of hydrolysis 3h; residue in the elimination hydrolyzed solution is washed once evaporating water with 1mL behind the evaporate to dryness; each adds 0.13mL anhydrous pyridine and 4mg oxammonium hydrochloride; in 90 ℃ of water-bath 30min, respectively add 0.2mL anhydrous acetic acid acid anhydride after the cooling, in 90 ℃ of acetylize 30min; concentrate, carry out GC and analyze.Get sugared compound sample 4.6mg (containing above-mentioned each about 0.8mg of six kinds of sugar) and the same legal system of this Radix Astragali extract sample of 4.6mg and get trial-product.

GC analysis condition: 19091J-433HP-5 capillary column (30m*0.25mm*0.25um); To be 80 ℃ of beginnings rise to 160 ℃ and keep 2min with 8.0 ℃/min to heating schedule, rises to 200 ℃ and keep 3min with 5.0 ℃/min, rises to 230 ℃ and keep 2.0min with 2.0 ° of c/min again; Fid detector, N ₂Be carrier gas, flow velocity is 0.6mL/s, splits into 10mL/min.

As shown in Figure 6, this extract monose composition mainly contains glucose and pectinose etc.

(11) molecular weight determination

Get (1) gained Radix Astragali extract and carry out the SDS-PAGE electrophoresis, the separation gel massfraction is 15%, and electrode buffer is the 1.5mol/L Tris-Gly-SDS solution of pH=8.3, adopts Xylene Brilliant Cyanine G R-250 dyeing, destainer is 7% glacial acetic acid, applied sample amount 25 μ g.Simultaneously, adopt LMW-SDS markerkit in contrast, tetrabromophenol sulfonphthalein is an indicator, get the SDS-PAGE electrophoretogram, as shown in Figure 7, sample is behind the SDS-PAGE electrophoresis, with Xylene Brilliant Cyanine G R-250 dyeing, get the dyeing zone of a homogeneous, it is pure to show that this extract reaches electrophoresis.Relative mobility per sample records its molecular weight and is about 53.26kD.

(12) amino acid composition analysis: get the Radix Astragali extract of (1) gained, adopt automatic analyzer for amino acids to detect, the amino acid of this extract is formed as shown in table 1:

Amino acid is formed and content in table 1. Radix Astragali extract

The proteic mouse spleen lymphocyte vitro conversion experiment of experimental example 2 tragacanthins of the present invention

1, experiment material

(1) animal: 8～12 the week ages male Balb/c mouse, available from Institute of Experimental Animals, Chinese Academy of Medical Sciences

(2) reagent: Radix Astragali extract (HQGP) Colleges Of Traditional Chinese Medicine Of Shanxi of the embodiment of the invention 1 gained provides;

Astragalus polysaccharides powder injection (Astragalus polysaccharides, APS-P) Pharmagenesis Inc.; Endoxan (CTX) SHANXI POWERDONE PHARMACEUTICAL.,LTD;

Concanavalin A, lipopolysaccharides (LPS), four thiazole salts (MTT) are Sigma company product;

RPMI1640 substratum Hyclone modified form, Sai Mo fly generation that biological chemistry goods (Beijing) company limited;

Calf serum Hangzhou folium ilicis chinensis biomaterial company limited

(3) instrument: CO ₂Incubator (German BINDER); Microplate reader (Switzerland Tecan Safire2); Refrigerated centrifuge (Japanese Sanyo)

2, experimental technique

(1) animal is handled: 10 mouse are divided into two groups at random, intraperitoneal injection.First group is the normal control group, injecting normal saline, and the 0.1ml/10g body weight, second group is model group, injection endoxan, 40ml/10g body weight.Administration 2 days.

(2) mouse boosting cell preparation: mouse is put to death in cervical vertebra dislocation in the 3rd day, 75% alcohol immersion 3 minutes, and aseptic taking-up spleen, by 200 order cells sieve, centrifugal 10 minutes of 1500r/min abandons supernatant, adds 6ml 8.3ml/L Tris-NH ₄CL, room temperature was placed after 10 minutes splitting erythrocyte 1500r/min centrifugal 10 minutes, abandon supernatant, add serum-free RPMI1640 substratum washing 2 times, centrifugal 10 minutes of each 1500r/min abandons supernatant, adds 1～2ml RPMI1640 substratum, regulating cell suspension density is 3 * 106/ml, and detects cell viability＞95% with trypan blue dyeing.

(3) mensuration of splenic lymphocyte vitro conversion: the normal control group splenocyte suspension of preparation is added in the 96 porocyte culture plates, and every hole 100 μ l are divided into 4 groups: 1. blank adds RPMI 1640 substratum 100 μ l; 2. concanavalin A 8 μ l final concentrations are 4 μ g/ml; 3. lipopolysaccharides 30 μ l final concentrations are 15 μ g/m; 4. the Radix Astragali extract of the embodiment of the invention 1 gained of different concns (0.5 μ g/ml, 1 μ g/ml, 2 μ g/ml, 4 μ g/ml, 10 μ g/ml, 30 μ g/ml, 90 μ g/ml).Cumulative volume is 200 μ l.Establish 3 multiple holes for every group.

The model group splenocyte suspension of preparation is added in the 96 porocyte culture plates every hole 100 μ l.Grouping and dosage are with normal control group splenocyte.Establish 3 multiple holes for every group.

Cell plate were put into 37 ℃ of CO2 incubators 68 hours then, inhaled and abandon 100 μ l supernatants, every hole adds 5mg/ml MTT 10 μ l, continues to cultivate 6 hours, and every hole adds the 10%SDS that 100 μ l contain 0.01N hydrochloric acid, fully dissolved cell.On the long microplate reader of Tecan Safire2 all-wave, survey the OD570 light absorption value, with the size of expression lymphocyte transformation propagation.

3, statistical procedures

Data information is used Expression is used SPSS13.0 and is carried out statistical procedures, and each is organized and relatively carries out one-way ANOVA check between the mean.

4, experimental result

MTT the results are shown in Table 2, table 3.Radix Astragali extract all is restraining effect to the propagation of the mouse spleen lymphocyte of vitro culture normal mouse splenic lymphocyte and immunocompromised, various dose HQGP group is carried out statistics relatively with the blank group, utmost point significant difference is all arranged, and restraining effect presents dose-dependently, and this Radix Astragali extract has significant immunoregulatory activity.

The influence that table 2 Radix Astragali extract reacts normal mice spleen lymphocytes proliferation (n=3,

Compare * P＜0.01 with the blank group

The influence that table 3 Radix Astragali extract reacts the immunocompromised mice spleen lymphocytes proliferation (n=3,

Compare * P＜0.01 with the blank group

Experimental example 3 tragacanthin albumen of the present invention suppress activity experiment to mouse boosting cell in-vitro multiplication under the active state

1, experiment material

(1) animal: 8～12 the week ages male Balb/c mouse, the B57BL/6J mouse is available from Institute of Experimental Animals, Chinese Academy of Medical Sciences.Animal lot number: SCXK (capital) 2004-0001

(2) reagent: the Radix Astragali extract Colleges Of Traditional Chinese Medicine Of Shanxi of the embodiment of the invention 2 preparations provides; Concanavalin A (ConA), lipopolysaccharides (LPS), four thiazole salts (MTT), RNase-A are Sigma company product; Propidium iodide (PI); RPMI1640 substratum Hyclone modified form, Sai Mo fly generation that biological chemistry goods (Beijing) company limited; Calf serum Hangzhou folium ilicis chinensis biomaterial company limited

(3) instrument: CO2 incubator (German BINDER), microplate reader (Switzerland Tecan Safire2), refrigerated centrifuge (Japanese Sanyo), flow cytometer, constant water bath box

2, experimental technique

(1) mouse boosting cell preparation: with experimental example 1.

(2) HQGP of different concns is to the influence of mouse spleen lymphocyte vitro conversion

ConA stimulates following influence to T cell proliferation: every hole adds splenocyte suspension 100 μ l and the ConA6 μ l (final concentration 3 μ g/ml) for preparing in the flat Tissue Culture Plate in 96 holes.The HQGP that adds different concns.The concentration of HQGP is made as 9 experimental group (n=3), is respectively 0,0.01,0.05,0.1,0.25,0.5,1.0,3.0,10.0 μ g/ml; Every hole adds complete 1640 substratum again, makes final volume be 200 μ l.

LPS stimulates down the influence to B cell proliferation: experimental technique and HQGP drug level, experimental group are the same, and the LPS that every hole adds is 30 μ l (final concentration 15 μ g/ml).

Cell plate are placed contain 5%CO ₂And the incubator under the saturated humidity condition, hatched 72 hours for 37 ℃.Inhale and abandon 100 μ l nutrient solution supernatants in stopping hatching preceding 6 hours every holes, add 5mg/ml MTT 10 μ l, continue to hatch 6 hours after, every hole adds the 10%SDS that 100 μ l contain 0.01N hydrochloric acid, fully dissolved cell.On the long microplate reader of Tecan Safire2 all-wave, survey OD ₅₇₀Light absorption value is with the size of expression lymphocyte transformation propagation.

(3) different time gives HQGP influence to mouse T lymphocyte propagation

Every hole adds splenocyte suspension 100 μ l and the ConA6 μ l (final concentration 3 μ g/ml) for preparing in the flat Tissue Culture Plate in 96 holes.Add same concentration HQGP (10 μ g/ml) respectively at different time.Add HPGP when cell is hatched beginning and be designated as 0h, hatch and be designated as 12h in 12 hours, by that analogy.Experiment is divided into 7 groups (n=4), is respectively 0,12,24,46,48,60h, control group only adds complete RPMI-1640 substratum, does not add HQGP.Final volume is 200 μ l.Hatch and detection method same (2).

(4) cells were tested by flow cytometry HQGP is to the influence in mouse T lymphocyte cycle

Every hole adds splenocyte suspension 1ml, ConA50 μ l (final concentration 2.5 μ g/ml), HQGP20 μ l (final concentration 1 μ g/ml) or Tri 20 μ l (final concentration 0.01 μ g/ml) and the complete RPMI-1640 substratum 950 μ l that prepare in the flat Tissue Culture Plate in 6 holes.Final volume is 2ml.In containing 5%CO ₂And the incubator under the saturated humidity condition, hatched 72 hours for 37 ℃.If three multiple holes.

Cell harvesting in the cell plate is centrifugal in centrifuge tube, 200g, 10 minutes.With PBS washed cell 2 times, add 0.5mL PBS and blow evenly, must dispel.With the 1mL syringe cell is picked up, firmly squeeze in 1mL 70% (precooling) ethanol, seal film and seal.Fix more than 24 hours for 4 ℃.Get 300g, centrifugal 10 minutes, collect fixed cell, PBS washes 2 times; With 0.2mL PBS re-suspended cell and go in the 1.5ml centrifuge tube piping and druming (preventing cytoclasis) gently; Add the about 10 μ L of RNase-A and be about 50 μ g/mL to final concentration, 37 ℃ of water-bath digestion 30min; Add the about 10 μ L of PI after the cooling and be about 50 μ g/mL to final concentration, lucifuge dyeing 30min in ice bath.With 300 orders (40～50 microns in aperture) nylon net filter, flow cytometer detects.

(5) HQGP suppresses the relation of mice spleen lymphocytes proliferation and lymphocyte activation

Splenocyte suspension 100 μ l, the HQGP (5 μ g/ml) that the adding of every hole prepares in the flat Tissue Culture Plate in 96 holes and the ConA of different concns.Experiment is divided into 6 groups (n=3), and ConA concentration is respectively 0,0.5,1.0,2.0,4.0,6.0 μ g/ml.Control group adds the ConA of above different concns respectively, but does not add HQGP.Final volume is 200 μ l.Hatch and detection method same (2).Calculate stimulation index SI=experimental group OD ₅₇₀Value/control group OD ₅₇₀The proliferative response of value representation cell.

(6) restraining effect of HQGP in the two-way mixing lymph reaction of mouse allogeneic

Get C57BL/6J respectively and the Balb/C mouse spleen prepares cell suspension, method is adjusted cell density and is 2 * 10 with (1) ⁶/ ml.In the flat Tissue Culture Plate in 96 holes, add the HQGP of each 100 μ l of two kinds of splenocyte suspensions, different concns in every hole respectively.If 7 of experimental group (n=3), the concentration of HQGP are respectively 0,0.05,0.1,0.5,1.0,5.0,10.0 μ g/ml.Other establishes independent C57BL cell hole and independent Balb/C cell hole, and positive drug Tri (0.05 μ g/ml) hole and ConA stimulation hole, is three multiple holes.CO ₂Incubating feather cockscomb cultivated 4 days.Cultivation and microplate reader measuring method are with (2).Calculate inhibiting rate according to following formula:

Inhibiting rate=(mix blank hole OD ₅₇₀Value-experiment medicine cell hole OD ₅₇₀Value)/mixing blank hole OD570 value.

3, statistical procedures

4, experimental result

(1) HQGP of different concns is to the influence of mouse spleen lymphocyte vitro conversion.See Table 4, table 5.HQGP can suppress ConA, LPS inductive mouse T cell, B cell proliferation, and the propagation of T, bone-marrow-derived lymphocyte is suppressed to show tangible dose-dependently.But HQGP is more remarkable to the T cytosis, and the effect of B cell is only just displayed when the high dosage.

Table 4HQGP stimulates the lymphopoietic influence of mice spleen T down to conA (3 μ g/ml)

Compare * P＜0.05 with the blank group

Table 5HQGP stimulates the influence of mice spleen bone-marrow-derived lymphocyte propagation down to LPS (15 μ g/ml)

Compare * P＜0.05 with the blank group

(2) different time gives HQGP influence to mouse T lymphocyte propagation.See Table 6, HQGP all plays restraining effect at different time to the T lymphopoiesis, but has only 0h, 12h, 24h administration and control group that significant difference (P＜0.05) is arranged, and the restraining effect that produces at 12h adding HQGP is the strongest.48h, 60h administration also show restraining effect but do not have statistical significance.

The administration of table 6 different time is to lymphopoietic influence

Compare * P＜0.05 with the blank group

(3) HQGP is to the influence in mouse T lymphocyte cycle.See Table 7, after HQGP (the 1 μ g/ml) effect, G0/G1 phase cell is compared with control group and is increased (P＜0.05), and G2/M phase cell also increases (P＜0.05) than control group, but S phase cell obviously reduces (P＜0.05).After positive drug Tri (the 0.01 μ g/ml) effect simultaneously, G0/G1 phase cell is compared with control group and is increased obviously (P＜0.05), and G2/M phase cell is not compared with control group and changed, and S phase cell reduces very obviously (P＜0.05).

Table 7HQGP to the influence (72h) of mouse T lymphocyte cell cycle (

N=3)

Compare * P＜0.05 with the blank group

(4) HQGP suppresses the relation of mice spleen lymphocytes proliferation and lymphocyte activation.See Table 8, after HQGP (the 5 μ g/ml) effect, the propagation of splenic lymphocyte all is suppressed under the different ConA concentration.From stimulation index, the restraining effect of lymphocyte activation degree and HQGP does not have positive correlation.When there not being ConA to do the time spent (lymphocyte remains static), HQGP also shows restraining effect.

Table 8HQGP suppresses the relation of mice spleen lymphocytes proliferation and lymphocyte activation

(5) restraining effect of HQGP in the two-way mixing lymph reaction of mouse allogeneic.See Table 9, reaction all has restraining effect to the HQGP of different concns to allogeneic mixing lymph, but the restraining effect of high density is more remarkable, and inhibiting rate is 74.77%.Positive control drug Tri also has significant inhibitory effect, and relatively there were significant differences (P＜0.05) with GP (10 μ g/m), and inhibiting rate is 85.17%.

Table 9GP is to the restraining effect of mouse allogeneic mixed lymphocyte reacion

Compare * P＜0.05 with the blank group

Compare a.P＜0.05 with GP (10 μ g/ml) group.

Experimental example 4 tragacanthin albumen are in the intravital immunocompetence experiment of mouse

1, experiment material

(1) animal: 8～12 the week ages male Balb/c mouse, available from Institute of Experimental Animals, Chinese Academy of Medical Sciences, lot number: SCXK (capital) 2004-0001

(2) reagent: the embodiment of the invention 1 described Radix Astragali extract (HQGP)

Triptolide (Triptoli de, Tri) Nat'l Pharmaceutical ﹠ Biological Products Control Institute's lot number: 111567-200502

The fluorescein-labeled anti-mouse CD3 antibody of FITC U.S. eBioscience company product

The anti-mouse CD8 antibody U.S. eBioscience company product of PE mark

The anti-mouse CD4 antibody U.S. company BD product of PerCP mark

The anti-mouse CD19 antibody U.S. eBioscience company product of APC mark

The anti-mouse CD49b antibody U.S. eBioscience company product of APC mark

Lysate

Catalog number (Cat.No.) is the mouse regulatory T cells staining kit of 88-8115-40, comprises following component:

1.FITC the anti-mouse CD4 antibody of mark: 50 μ g

2.PE the anti-mouse CD25 antibody of mark: 50 μ g

3.PE-Cy5 the anti-mouse of mark/rat Foxp3 antibody: 25 μ g

4.PE-Cy5 mark rat IgG2a homotype contrast: 25 μ g

5. the anti-mouse CD16/32 antibody of affinity purification: 50 μ g

6. flow cytometer dyeing damping fluid: 200ml U.S. eBioscience company product

7. fix/rupture of membranes concentrated solution 30ml U.S. eBioscience company product

8. fix/the rupture of membranes diluent: 100ml U.S. eBioscience company product

9. rupture of membranes damping fluid (10 times of concentration) 100ml U.S. eBioscience company product

(3) instrument: refrigerated centrifuge (Japanese Sanyo); Flow cytometer FACSCalibur (U.S. BD); The spiral vibrator; Heparin sodium anti-freezing vacuum test tube.

2, experimental technique

(1) animal is handled

Mouse adapts to be raised a week, freely drinks water, takes food.According to the mouse body weight, with CHISS software 10 mouse are divided into 5 groups at random, 8 every group.

Grouping: normal control group, the basic, normal, high dosage group of HQGP, triptolide group.

Administration: every group of mouse peritoneal drug administration by injection, press 0.1ml/kg.d, successive administration 7 days was plucked eyeball and is got blood on the 8th day.It is as follows that each organizes dosage:

Normal control group physiological saline

HQGP low dose group 1mg/kg.d

Dosage group 5mg/kg.d among the HQGP

HQGP high dose group 10mg/kg.d

Triptolide group 40 μ g/kg.d

(2) the expression mouse lymphocyte subpopulation flow cytometry step of flow cytometry analysis mouse peripheral blood medium size lymphocyte subgroup and FoxP3

1) on each streaming, adds 100ul anti-freezing mouse peripheral blood in the sample pipe.

2) add recommendation optimal dose fluorescent mark surface antigen antibody and whole blood uniform mixing, experimental design is 2 testing tubes:

a)CD3FITC/CD8PE/CD4PerCP/CD19?APC

b)CD3FITC/CD49b?APC

3) reacting at normal temperature without light is 20 minutes, adds 2ml erythrocyte splitting working fluid in every pipe, cracking 10 minutes.Centrifugal 5 minutes of 300g abandons supernatant.

4) add 2ml precooling PBS re-suspended cell, centrifugal 5 minutes of 300g abandons supernatant.

5) add 500ul PBS re-suspended cell, last machine testing.

Mouse regulatory T cells FoxP3 expresses the flow cytometry step

1) on each streaming, adds 100ul anti-freezing mouse eyeground blood in the sample pipe.

2) add to recommend optimal dose fluorescent mark surface antigen antibody (CD4, CD25).

3) reacting at normal temperature without light is 20 minutes, and every pipe adds 2ml erythrocyte splitting working fluid, cracking 10 minutes, and centrifugal 5 minutes of 300g abandons supernatant, 2ml PBS re-suspended cell, washed cell is once.

4) with the Flow Cytometry Staining Buffer of precooling or the PBS washed cell of precooling.

5) add fixing/rupture of membranes working fluid (Fixation/Permeabilization) of 1ml behind the vortex concussion re-suspended cell, and vortex mixing once more.

6) lucifuge is 4 ℃, hatches 30 minutes.

7) add 2ml Permeabilization Buffer (Cat.No 00-8333) working fluid centrifuge washing cell and abandoning supernatant.

8) repeat the 5th step operated wash cell.

9) add an amount of Affinity Purified anti-mouse CD16/32 (Fc Block) (diluting), hatched 15 minutes 4 ℃ of lucifuges with Permeabilization Buffer working fluid.

10) confining liquid need not flush away, (dilute with Permeabilization Buffer working fluid, dilute according to recommended density, suggestion is diluted to 10ul/Test directly to add an amount of fluorescent mark Foxp3 antibody, the antibody that contains recommended density), lucifuge was hatched 30 minutes for 4 ℃ at least.

11) add 2ml Permeabilization Buffer working fluid, centrifuge washing cell and abandoning supernatant.

12) repeat the previous step washed cell.

13), and go up the machine testing analysis with the Flow Cytometry Staining Buffer re-suspended cell of an amount of volume.

Owing to used cell fixation and rupture of membranes in the dyeing course, on form, having certain variation, so need do certain adjustment during FSC/SSC figure centre circle door compared with viable cell.

(3) data processing

The CellQuest software analysis is adopted in the flow cytometer data analysis.Each is organized cell subsets ratio data data and uses

Expression is used SPSS13.0 and is carried out statistical procedures, and each is organized and relatively carries out one-way ANOVA check between the mean.

(4) experimental result

1) generalized case of mouse

Abdominal injection tragacanthin albumen is after three days, and the high dose group mouse begins to show and the control group evident difference.Few moving, slow in reacting, the weight loss of hair color tarnish, dispirited, the back of a bow occurs and phenomenon such as chilly is arranged.The situation of middle dosage group is better slightly than high dose group, but also shows and the tangible difference of control group.Control group mice but shows well, is difficult symptom.

2) respectively organize the mouse body weight change

Each changes obviously before and after organizing the administration of mouse mean body weight, sees Table 10, especially tragacanthin albumen high dosage and middle dosage group.

Respectively organize the mouse body weight before and after table 10 administration

In three dosage of HQGP, high dosage (10mg/kg.d) is the most obvious to the influence of mouse body weight, and mean body weight reduces by 3.75 grams, in the effect of (5mg/kg.d), low dosage (1mg/kg.d) group weaken successively.

3) mouse peripheral blood total lymphocyte ratio

Total lymphocyte accounts for leukocyte count purpose ratio such as table 11 in the mouse peripheral blood.

Table 11HQGP accounts for the white corpuscle ratio to total lymphocyte influence

Compare * * .P＜0.01 with the blank group; Compare * .P＜0.05 with the blank group

After the administration, the total lymphocyte number has all reduced in each group of HQGP and the Tri group peripheral blood.The total lymphocyte of the high, medium and low dosage group of HQGP accounts for leukocytic ratio significantly to be reduced, and compares P＜0.01 with normal group.The Tri group more also has statistical significance, P＜0.05 with normal group.

4) peripheral T lymphocyte subsets of mice

Total T cell, CD3+CD4+CD8-T cell, CD3+CD4-CD8+T cell account for lymphocytic ratio and CD3+CD4+/CD3+CD8+ ratio such as table 12 in the mouse peripheral blood.

Table 12HQGP to the influence of T cell and T cell subsets (

N=8)

Compare * .P＜0.05 with the blank group

Among the HQGP, the total T cell of low dose group accounts for lymphocytic ratio and descends, and with the normal group contrast significant difference, P＜0.05 arranged.

Table 12, dosage makes T cell subsets CD4+CD8-T cell account for lymphocytic ratio obviously to reduce among the HQGP, with normal group P＜0.05 relatively; Obviously raise and make the CD4-CD8+T cell account for lymphocytic ratio, compare P＜0.05 with normal group.CD4+/CD8+ ratio significantly descends, and compares P＜0.05 with normal group.Simultaneously, Tri makes the CD3+CD8+T cell account for lymphocytic ratio and obviously raises, and compares P＜0.05 with normal group.CD4+/CD8+ ratio significantly descends, and compares P＜0.05 with normal group.

In parallel laboratory test, the dosage group is identical to the influence of mouse peripheral blood T cells subgroup with the Tri group among the HQGP.

5) mouse peripheral blood regulatory T cells foxP3 expresses

HQGP is as shown in table 13 to the influence that mouse regulatory T cells foxP3 expresses.

The influence that table 13HQGP expresses mouse regulatory T cells foxP3 ( N=8)

Compare * .P＜0.05 with the blank group; Compare * * .P＜0.01 with the blank group

By above data as can be known, HQGP is high, medium and low respectively organizes mouse peripheral blood regulatory T lymphocyte (CD4+CD25+) and expresses the ratio that the cell of FoxP3 accounts for CD4+ and with normal group difference extremely significantly, P＜0.01 are arranged relatively; Also compared the CD4+CD25+FoxP3+ cell simultaneously and accounted for lymphocytic ratio, with normal group contrast, HQGP height, middle dosage group difference are extremely remarkable, P＜0.01.The low dose group significant difference, P＜0.05.In an experiment group, the cell proportion of expressing FoxP3 in the CD4+CD25+T cell does not have significant difference, P＞0.05.

6) mouse peripheral blood NK cell ratio

HQGP accounts for lymphocytic scale effect to NK, NKT cell in the mouse peripheral blood, as table 14.

Table 14HQGP to the influence of NK, NKT cell proportion (

N=8)

High, medium and low dosage HQGP is improved to NK cell proportion in the mouse peripheral blood, but has only high dose group and normal group that significant difference is relatively arranged, P＜0.05.Behind but very obvious, high, the middle dosage HQGP effect of the influence mouse to the NKT cell, the NKT cell proportion significantly increases in the peripheral blood, and is dose-dependently.High, middle dosage group and normal group relatively have utmost point significant difference, P＜0.01.HQGP small dose group and normal group compare the reduction of NKT cell proportion and there were significant differences, P＜0.05.The Tri group is but showing utmost point significant difference aspect the propagation inhibition of NKT cell simultaneously.

4.4.7 mouse peripheral blood B cell ratio

HQGP sees Table 15 to the influence of mouse peripheral blood B cell ratio.

Table 15HQGP is to the influence of mouse peripheral blood B cell ratio

Compare * .P＜0.05 with the blank group;

As shown in Table 15, HQGP height, middle dosage can improve the mouse peripheral blood B cell and account for lymphocytic ratio, with normal group significant difference are arranged relatively, P＜0.05, and show dose-dependently.The Tri group does not relatively have difference with normal group.

Following embodiment all can realize the described effect of above-mentioned experimental example

Embodiment

Embodiment 1: the proteic preparation of tragacanthin

Get Milkvetch Root, it is ground into Powdered, take by weighing 400g, add water 1600ml, soaked 12 hours, lixiviate below 55-60 ℃ 2 hours is filtered; Add water 1200ml in the residue again, lixiviate below 55-60 ℃ 2 hours is filtered, and merges filtrate twice, with 3000r/min speed centrifugal 30 minutes, merge supernatant liquor, crude extract;

Crude extract is concentrated into 300ml, adds the ammonium sulfate grind to form powdery, the limit edged is stirred to saturated, leaves standstill under 4 ℃ 12 hours, and with 3000r/min speed centrifugal 30 minutes, abandoning supernatant must precipitate;

Supernatant liquor is placed dialysis tubing, on the air constant temperature oscillator,, get the tragacanthin protein solution, through lyophilize with pure water dialysis 48 hours, get oyster white meal tragacanthin albumen (HQGP), recording molecular weight through the analysis of SDS-polyacrylamide gel electrophoresis is 53.26kD.

Embodiment 2: the proteic preparation of tragacanthin

Get Milkvetch Root, it is ground into Powdered, take by weighing 300g, add water 1900ml, soaked 9 hours, lixiviate below 50 ℃ 3 hours is filtered; Add water 1500ml in the residue again, lixiviate below 50 ℃ 1 hour is filtered, and merges filtrate twice, with 3500r/min speed centrifugal 35 minutes, merge supernatant liquor, crude extract;

Crude extract is concentrated into 250ml, adds the ammonium sulfate grind to form powdery, the limit edged is stirred to saturated, leaves standstill under 3 ℃ 24 hours, and with 3500r/min speed centrifugal 35 minutes, abandoning supernatant must precipitate;

Supernatant liquor is placed dialysis tubing, on the air constant temperature oscillator, dialysed 24 hours with pure water, get the tragacanthin protein solution, through lyophilize, get oyster white meal tragacanthin albumen (HQGP), recording molecular weight through the analysis of SDS-polyacrylamide gel electrophoresis is 53.26kD, and the charateristic avsorption band of ultraviolet spectrogram is 485nm and 280nm.

Embodiment 3: the proteic preparation of tragacanthin

Get Milkvetch Root, it is ground into Powdered, take by weighing 500g, add water 1300ml, soaked 15 hours, lixiviate below 45 ℃ 1 hour is filtered; Add water 900ml in the residue again, lixiviate below 45 ℃ 3 hours is filtered, and merges filtrate twice, with 2500r/min speed centrifugal 25 minutes, merge supernatant liquor, crude extract;

Crude extract is concentrated into 350ml, adds the ammonium sulfate grind to form powdery, the limit edged is stirred to saturated, leaves standstill under 5 ℃ 18 hours, and with 2500r/min speed centrifugal 25 minutes, abandoning supernatant must precipitate;

Supernatant liquor is placed dialysis tubing, on the air constant temperature oscillator, dialysed 72 hours with pure water, get the tragacanthin protein solution, through lyophilize, get oyster white meal tragacanthin albumen (HQGP), recording molecular weight through the analysis of SDS-polyacrylamide gel electrophoresis is 53.26kD, and infrared spectrogram is shown in 3399.2cm ^-1, 2962.6cm ^-1, 1643.2cm ^-1, 1546.2cm ^-1, 1403.0,1246.5cm ^-1There is obvious absorption peaks at the place.

Claims

1. a tragacanthin albumen (HQGP), the molecular weight that it is characterized in that this glycoprotein is 50-55kD.

2. tragacanthin albumen as claimed in claim 1, the molecular weight that it is characterized in that this glycoprotein is 53.26kD.

3. tragacanthin albumen as claimed in claim 1 or 2 is characterized in that this glycoprotein powdery that is creamy white, and is soluble in water, is insoluble to organic solvent methyl alcohol, ethanol, ether, acetone and chloroform; Concentration is that tragacanthin protein solution mensuration pH value under 23 ℃ of 0.096mg/ml is 5.8; Concentration is that the specific rotatory power of the tragacanthin protein solution of 0.08g/100ml is-56.38 °.

4. tragacanthin albumen as claimed in claim 1 or 2, it is characterized in that this tragacanthin albumen is configured to the aqueous solution that concentration is 0.5mg/ml, with water is blank, gets ultraviolet spectrogram in the interscan of 200nm-320nm wavelength region, and the indicating characteristic absorption peak is 485nm and 280nm.

5. tragacanthin albumen as claimed in claim 1 or 2 is characterized in that this tragacanthin albumen is configured to the aqueous solution that concentration is 0.5mg/ml, adopts the KBr pressed disc method, at 4000cm ^-1-500cm ^-1The scope interscan gets infared spectrum, and absorption peak is positioned at 3500cm ^-1-2800cm ^-1Between, 3399.2,2962.6,1643.2,1546.2,1403.0, there is obvious absorption peaks at the 1246.5cm-1 place; 3399.2cm wherein ^-1Be polysaccharide hydroxyl stretching vibration absorption peak, 2962.6cm ^-1Sugar C-H stretching vibration absorption peak, 1403cm ^-1Be polysaccharide C-O vibration absorption peak, 1246.5cm ^-1Be the absorption peak of pyranose ring ehter bond C-O-C, 1643.2cm ^-1And 1546.2cm ^-1The place is the N-H vibration absorption peak of amide group.

6. tragacanthin albumen as claimed in claim 1 or 2, the combination that it is characterized in that the glycopeptide of this glycoprotein is the N-cardohydrata-peptide linkage.

7. tragacanthin albumen as claimed in claim 1 or 2, it is characterized in that in this glycoprotein that amino acid is formed and content is: every 100g glycoprotein contains total amino acid content 57.18g, aspartic acid 4.50g wherein, Threonine 3.20g, Serine 2.33g, L-glutamic acid 6.49g, glycine 3.83g, L-Ala 1.68g, Gelucystine 1.42g, Xie Ansuan 5.99g, methionine(Met) 0.92g, Isoleucine 2.31g, leucine 2.34g, trorsine 14 .39g, phenylalanine 1.93g, Methionin 6.50g, Histidine 2.00g, arginine 1.44g, proline(Pro) 5.91g.

8. as the arbitrary described tragacanthin albumen of claim 1 to 7, it is characterized in that this glycoprotein is prepared from by following method:

Raw sugar albumen is added the less water dissolving, add sevag reagent, violent jolting 10-30 minute, centrifugal, inclining supernatant liquor, removed the yellow colloid degeneration albumen in middle layer and lower floor's chloroform, repeated this step and did not almost have metaprotein to the middle layer;

Supernatant liquor is placed dialysis tubing, and pure water dialysis 24-72 hour gets the tragacanthin protein solution, through lyophilize, gets oyster white meal tragacanthin albumen.

9. tragacanthin albumen as claimed in claim 8 is characterized in that this glycoprotein is prepared from by following method:

Supernatant liquor is placed dialysis tubing, on the air constant temperature oscillator,, get the tragacanthin protein solution,, get oyster white meal tragacanthin albumen through lyophilize with pure water dialysis 48 hours.

10. tragacanthin albumen as claimed in claim 8 is characterized in that this glycoprotein is prepared from by following method:

Supernatant liquor is placed dialysis tubing, on the air constant temperature oscillator,, get the tragacanthin protein solution,, get oyster white meal tragacanthin albumen through lyophilize with pure water dialysis 24 hours.

11. tragacanthin albumen as claimed in claim 8 is characterized in that this glycoprotein is prepared from by following method:

Supernatant liquor is placed dialysis tubing, on the air constant temperature oscillator,, get the tragacanthin protein solution,, get oyster white meal tragacanthin albumen through lyophilize with pure water dialysis 72 hours.

12. as the application of the arbitrary described tragacanthin albumen of claim 1 to 7 in the preparation immunosuppressive drug.

13. application as claimed in claim 12 is characterized in that wherein said immunosuppression is meant that the propagation to the splenic lymphocyte of normal splenic lymphocyte of vitro culture and immunocompromised all is restraining effect.

14. application as claimed in claim 12 is characterized in that wherein said immunosuppression is meant suppressor T cell and B cell proliferation.