CN101081862B - Rapid purification and for beta trichosanthin protein and crystallization technology - Google Patents
Rapid purification and for beta trichosanthin protein and crystallization technology Download PDFInfo
- Publication number
- CN101081862B CN101081862B CN2006100849606A CN200610084960A CN101081862B CN 101081862 B CN101081862 B CN 101081862B CN 2006100849606 A CN2006100849606 A CN 2006100849606A CN 200610084960 A CN200610084960 A CN 200610084960A CN 101081862 B CN101081862 B CN 101081862B
- Authority
- CN
- China
- Prior art keywords
- trichosanthin
- beta
- buffer solution
- protein
- acetate buffer
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Images
Landscapes
- Peptides Or Proteins (AREA)
Abstract
The present invention relates to fast purifying and crystallizing technology for beta-trichosanthin. Beta-trichosanthin is prepared with trichosanthes root as material, and through peeling, washing, crushing, extracting with buffering liquid, and twice gradient elution with cation exchange resin. Inside buffering liquid of citric acid with 2M concentration of KCl as precipitant, beta-trichosanthin may grow into orthogonal crystal of P2<1>2<1>2<1> space group and with cell parameters a=38.244 A, b=86.934 A and c=135.785 A. The beta-trichosanthin purifying and crystallizing process is simple, fast and effective.
Description
Technical field
The present invention relates to biochemical field, particularly relate to the fast purifying and the crystallization technique thereof of beta trichosanthin protein.
Background technology
(Trichosanthin TCS) is the piece root of drawing together building (Trichosantheskirilowii) from cucurbitaceous plant---a kind of basic protein that separation and purification obtains the former green material of Chinese medicine Snakegourd Root to Trichosanthin.The chemical nature of Trichosanthin be a kind of molecular weight be 27kDa not sacchariferous single chain polypeptide, belong to 1 type ribosome inactivating protein (Ribosome Inactivating Protein; RIP); Have RNAN-glycosidase activity and multiple zymetology and pharmacologically active (people such as Pan Kezhen., Structure-FunctionRelationship of Trichosanthin, Pure and Appl.Chem.66; 57-64,1994; Shaw PC, et al, Resent advances in trichosanthin, Toxicon, 45,683-689,2005).1989; People such as McGrath find that Trichosanthin can duplicate (McGrath et al. by HIV inhibiting (HIV) in the T of acute infection lymphocyte and chronically infected scavenger cell; Proc.Natl.Acad.Sci.USA, 86:2844-2848,1989); Soon, the drugs approved by FDA Trichosanthin carries out clinical trial.The induced labor of Trichosanthin is active, and is antitumor, antiviral, and particularly the anti-AIDS cytotoxic activity has caused attention widely.
The Chinese science man at first separation and purification and crystallization Trichosanthin (people such as Wang Yahui, the purifying of Trichosanthin and the preliminary study of character thereof, animal journal, 22,117-143,1976; People such as Jin Shanwei, the preparation and the physicochemical property thereof of the chemical I crystal trichosanthin of Trichosanthin, chemical journal, 39,917-925,1981; Fujian Inst. of Matter Structure, Chinese Academy of Sciences etc., the crystal of Trichosanthin is cultivated and the structure cell basic parameter, Science Bulletin, 176-178,1977; People such as Wang Jiahuai, a kind of new crystal Trichosanthin crystal, Science Bulletin; 943-944,1985), and abroad; People such as Maraganore ability in 1987 reported first is separated to Trichosanthin (Maraganore etal.J.Biol.Chem.262,116280,1987).Simultaneously; Report successively to isolate from smallpox powder agglomates root kind and draw together building albumen (people such as Jin Shanwei; Chemistry wall bulletin 15160-1681997); TAP29 (Lee-Huang et al, Proc.Natl.Acad.Sci.USA 88 6570-65741991) and α β γ-Trichosanthin (Narayanna etal., Plant Science162 79-85 2002).These work show that these albumen all belong to 1 type ribosome inactivating protein, have the RNA-N glycosidase activity, and antitumor, antiviral, particularly the anti-AIDS cytotoxic activity.
Prior art is the method according to Jin Shanwei, with the method for acetone fractional precipitation, through the refining Trichosanthin of lyophilize preparation; Refining Trichosanthin is in barbitol buffer solution; With NaNO3 is that precipitation agent grows monoclinic crystal (C2 spacer, a=75.60 dust, b=75.44 dust; The c=88.36 dust, β=99.50 °; And P2
1Spacer, a=73.4 dust, b=74.7 dust, c=87.9 dust, β=97.7 °); In citrate buffer solution, be precipitation agent with KCl, grow quadrature crystal (P2
12
12
1Spacer, a=38.305 dust, b=76.225 dust, c=79.213 dust).The Trichosanthin injection liquid commodity that Kingsoft, Shanghai pharmaceutical factory produces are exactly crystal trichosanthin solution.
People such as Jin Shanwei go the smallpox powder agglomates root of juice from squeezing; After saline water extracting, ammonium sulfate precipitation, dialysis, lyophilize; Separate through Sephadex G-75; Obtain to draw together building albumen (trichobitacin) through the cationite gradient elution again, the molecular weight that mass spectrum (ES-MS) is measured is 27228, and amino acid is formed similar with Trichosanthin with the part order.People such as Narayanan are from smallpox powder agglomates root; Through the PBS extracting; After the lyophilize, obtain Trichosanthin bullion (lyophilized powder), and then go up the Mono-S post and isolate α β gamma trichosanthin on a small quantity through the cationite gradient elution; Beta trichosanthin protein is at molecular weight (26kDa), and amino acid is formed similar with Trichosanthin with the part order.People such as Lee-Huang from smallpox powder agglomates root through the PBS extracting; Dialysis; Behind the cationite gradient elution, dialysis also concentrates after Sephadex G-75 separates acquisition TAP29, and it is variant with Trichosanthin on molecular weight (29kDa) and partial amino-acid order.
Summary of the invention
The objective of the invention is based on above-mentioned prior art basis; From Trichosanthin injection liquid and Trichosanthin crystal, isolate 2 equipotential albumen through high-resolution cation exchange column chromatography; What and custom according to content; Difference called after α Trichosanthin and beta trichosanthin protein, according to the chromatography behavior of beta trichosanthin protein on ion exchange column, the present invention provides from the method and the crystal technique thereof of the primary material prepn beta trichosanthin protein of Snakegourd Root simultaneously.This albumen is under the precipitation agent crystallization condition with 2M KCl in citrate buffer solution, grows into the quadrature crystal, and through X-XRD X with use the synchrotron radiation data analysis, this crystal belongs to P2
12
12
1Spacer, unit cell parameters are the a=38.244 dust, b=86.934 dust, c=135.785 dust.Different with the quadrature crystal unit cell parameters of original Trichosanthin.
The Trichosanthin injection liquid that Kingsoft, Shanghai pharmaceutical factory produces shows as a band in SDS-PAGE, molecular weight is 27kDa, on FPLC; Analyze with MonoS HR5/5 post (available from Pharmacia company), when condition setting is 10mM phosphate buffered saline buffer pH7.5 for A liquid, B liquid is A liquid+1M NaCl, flow velocity 1ml/min; The Mono-S post with A liquid balance after, application of sample, A liquid is washed post 5ml; 0-50%B liquidus property gradient elution 10ml after 100%B liquid is washed 5ml then, uses A liquid balance 5ml again.It is that NaCl concentration goes out the peak (see figure 1) during for 0.183M that Trichosanthin reaches 8.67ml at elutriant.
But we notice that an acromion that is about 8.5ml is arranged at this peak before.So we fall slow gradient, other conditions are constant, and only gradient is set to 0-10%B liquidus property gradient elution 10ml, and the acromion of first precondition just goes out the peak at the 11.29ml place, and major ingredients goes out the peak at the 12.07ml place.11.29ml the peak is exactly a beta trichosanthin protein, the 12.07ml peak is exactly the α Trichosanthin, and both peak area ratios are the 1:4.5 (see figure 2).
After the refining Trichosanthin that lyophilize obtains in the 0.75M citrate buffer solution, is a precipitation agent with 2M KCl, grow up to the Trichosanthin crystal with acetone fractional precipitation; After this albumin crystal cleaned, smash to pieces, dissolving, an appearance Mono-S post is gone up in centrifugal back; Condition setting is 10mM phosphate buffered saline buffer pH7.5 for A liquid, and B liquid is A liquid+1M NaCl, flow velocity 1ml/min; The Mono-S post with A liquid balance after, application of sample, A liquid is washed post 5ml; 0-10%B liquidus property gradient elution 10ml after 100%B liquid is washed 5ml then, uses A liquid balance 5ml again.(with above-mentioned the same, but because the A liquid of configuration, B liquid concentration is difference slightly; So appearance time changes slightly), when 10.91ml and 11.84ml, go out the peak, the 10.91ml peak is exactly a beta trichosanthin protein; 11.84ml the peak is exactly the α Trichosanthin, both peak area ratios are the 1:7.6 (see figure 3).
Based on above-mentioned 2 work, the present invention also provides from the technology of the primary material prepn beta trichosanthin protein of Snakegourd Root,
Technical scheme of the present invention is: the former green material peeling of Snakegourd Root is cleaned, and chopping adds 5mM, the pH4.5 acetate buffer solution; Extracting, homogenate adds 50% Glacial acetic acid min. 99.5 and transfers pH4.0; Then under 4 ℃ of conditions, the centrifugal 30min of 16000 * g, the supernatant application of sample is to 20mM; On the post of pH4.5 acetate buffer solution equilibrated strong cation-exchanging resin (S-Sepharose Fast Flow or SP-Sepharose Fast Flow), column length requires more than 20cm, and 280nm detects in real time; Use 20mM, the pH4.5 acetate buffer solution is washed till baseline, uses 20mM again; The pH6.5 phosphoric acid buffer is washed till baseline, with the slow gradient elution of 15-20 times of column volume damping fluid, generally can go out 3 peak (see figure 4)s then.Collect the 2nd peak, transfer pH4.0 with 50% Glacial acetic acid min. 99.5, add the acetate buffer solution of two volumes, carry out gradient elution with Zeo-karb again, main peak is exactly the beta trichosanthin protein (see figure 5).
The beta trichosanthin protein of institute of the present invention separation and purification; On SDS-PAGE, present a band; Molecular weight is 27kDa; Show as a peak (see figure 6) at strong cation prepacked column Mono-S post (available from Pharmacia company), on gel-filtration prepacked column Superdex-75 (available from Pharmacia company), show as a peak (see figure 7).Analyze through mass spectrum (ES-MS), the beta trichosanthin protein molecular weight is 27163.0 and 27102.0.The molecular weight that records the α Trichosanthin under the same terms is 27173.0 and 27103.0, can think that the molecular weight of the two is identical.
The prepared beta trichosanthin protein of the present invention adopts the sessile drop method technology, and mother liquid concentration is 20mg/ml, at 0.075M, in the pH5.4 citrate buffer solution, is precipitation agent with 2M KCl, just can grow beta trichosanthin protein quadrature crystal.Through X-ray diffraction analysis or synchrotron radiation diffraction analysis, crystal belongs to P2
12
12
1Spacer, its unit cell parameters are the a=38.244 dust, the b=86.934 dust, and the c=135.785 dust, neither with the crystal formation (unit cell parameters) of the crystallization Snakegourd Root of former report with (P2
12
12
1Spacer, a=38.305 dust, b=76.225 dust, c=79.213 dust; The C2 spacer, a=75.60 dust, b=75.44 dust, c=88.36 dust, β=99.50 °; And P2
1Spacer, a=73.4 dust, b=74.7 dust, c=87.9 dust, β=97.7 °).We have collected the data of a cover 1.6 dusts.
The present invention compared with prior art has following positively effect:
1. the present invention confirms to contain in Trichosanthin injection liquid and the Trichosanthin crystal two equipotential albumen: α Trichosanthin and beta trichosanthin protein; They are all identical on molecular weight, amino acid composition and order; All have N-glycosidase activity and HIV-resistant activity; Only variant slightly in highly sensitive, the technical appearance time of high resolving power chromatography, the unit cell parameters of the protein crystal of generation is different.
2. the present invention adopts 2 step Zeo-karb elution techniques; Without acetone or ammonium sulfate precipitation and dialysis; Steps such as lyophilize have been avoided many protein-denatured troublesome operation that cause, can from the former green material of Trichosanthin, go out beta trichosanthin protein by sharp separation.
3. the present invention is preferred for buffer concentration, the pH value of separation and purification, and gradient has improved separating effect.
4. the beta trichosanthin protein that the present invention drew is that precipitation agent can be turned out the beta trichosanthin protein crystal with 2M KCl in citrate buffer solution, and crystal belongs to P2
12
12
1Spacer, its unit cell parameters are the a=38.244 dust, b=86.934 dust, c=135.785 dust.For this proteic 26S Proteasome Structure and Function research provides important structured data.
Description of drawings
Now accompanying drawing is made brief description:
Fig. 1. the Trichosanthin injection liquid is elution profile on Mono-S HR5/5 post.X-coordinate is an elutriant ml number, and ordinate zou is mAU, and Trichosanthin reaches the 8.76ml place at elutriant and promptly goes out the peak when 0.188M NaCl (pH7.5,10mM phosphate buffered saline buffer), and the figure middle polyline is a salt concn, and full scale is 1MNaCl.
Fig. 2. the Trichosanthin injection liquid is elution profile on Mono-S HR5/5 post.X-coordinate is an elutriant ml number, and ordinate zou is mAU, and beta trichosanthin protein reaches the 11.29ml place promptly at 0.629MNaCl (pH7.5 at elutriant; The 10mM phosphate buffered saline buffer) goes out the peak time; The α Trichosanthin goes out the peak at the 12.07ml place, and the figure middle polyline is a salt concn, and full scale is 1M NaCl.
Fig. 3. the Trichosanthin crystal is elution profile on Mono-S HR5/5 post.X-coordinate is an elutriant ml number, and ordinate zou is mAU, and beta trichosanthin protein reaches the 10.91ml place promptly at 0.591MNaCl (pH7.5 at elutriant; The 10mM phosphate buffered saline buffer) goes out the peak time; The α Trichosanthin goes out the peak at the 11.84ml place, and the figure middle polyline is a salt concn, and full scale is 1M NaCl.
Fig. 4. beta trichosanthin protein is elution profile on S-Sepharose Fast Flow post.X-coordinate is an elutriant ml number, and ordinate zou is AU, i.e. A
280Detected value, dotted line is at 20mM among the figure, under the pH6.5 phosphoric acid buffer condition, the linear gradient line of NaCl concentration, beta trichosanthin protein goes out the peak about 0.1M NaCl place, and the α Trichosanthin goes out the peak about 0.15M NaCl place.
Fig. 5. beta trichosanthin protein elution profile again on S-Sepharose Fast Flow post.X-coordinate is an elutriant ml number, and ordinate zou is AU, i.e. A
280Detected value, dotted line is at 10mM among the figure, under the pH7.5 phosphoric acid buffer condition, the linear gradient line of NaCl concentration, beta trichosanthin protein goes out the peak about the 0.175MNaCl place.
Fig. 6. beta trichosanthin protein is elution profile on Mono-S HR5/5 post.X-coordinate is an elutriant ml number, and ordinate zou is mAU, and beta trichosanthin protein reaches the 10.91ml place at elutriant and promptly goes out the peak when 0.0591M NaCl (pH7.5,10mM phosphate buffered saline buffer), and the α Trichosanthin goes out the peak at the 11.89ml place.The figure middle polyline is a salt concn, and full scale is 1M NaCl.
Fig. 7. Trichosanthin is elution profile on the Supedex-75 post.X-coordinate is an elutriant ml number, and ordinate zou is mAU, and beta trichosanthin protein reaches the 13.34ml place at elutriant and goes out the peak.
Embodiment
Below in conjunction with accompanying drawing, further describe the present invention through embodiment.
Embodiment 1: the fast separating and purifying beta trichosanthin protein
Remove the Snakegourd Root that skin is cleaned, add 100ml pH4.5, the 10mM acetate buffer solution soaks, homogenate secondary on refiner; Transfer pH4.0 with 50% Glacial acetic acid min. 99.5 then, 16000 * g under 4 ℃ of conditions again, centrifugal 30 ', collect supernatant; On the Sp-Sepharose Fast Flow post of supernatant application of sample to 1.6 * 23cm (this post has been used pH4.5,20mM acetate buffer solution balance), after continuing to use pH4.5,20mM acetate buffer solution to wash out an assorted peak; Use pH6.5, the 20mM phosphate buffered saline buffer continues to wash post, can wash out an assorted peak again; Use 0-0.3M NaCl (at pH6.5, in the 20mM phosphate buffered saline buffer) gradient elution then, collect the 2nd peak (see figure 4).
Merge the 2nd peak, add 50% Glacial acetic acid min. 99.5 and transfer pH4, add the pH4.5 of 2 times of volumes again, the 20mM acetate buffer solution; Last appearance continues to use pH4.5 to using on the identical acetate buffer solution equilibrated Sp-Sepharose Fact Flow post, and the 20mM acetate buffer solution is washed post; Use pH7.5 again, the 10mM phosphoric acid buffer is washed post, uses 00.4M NaCl (at pH7.5 then; In the 10mM phosphoric acid buffer) gradient elution, collect main peak, this peak is exactly the beta trichosanthin protein (see figure 5).
Embodiment 2: strong cation prepacked column Mono-S analyzes
Beta trichosanthin protein elution profile (see figure 6) on Mono-S HR5/5 post of the present invention's preparation: A liquid is 10mM phosphate buffered saline buffer pH7.5, and B liquid is A liquid+1M NaCl, flow velocity 1ml/min; The Mono-S post with A liquid balance after; Application of sample, A liquid is washed post 5ml, 0-10%B liquidus property gradient elution 10ml; After 100%B liquid is washed 5ml then, use A liquid balance 5ml again.Beta trichosanthin protein reaches 10.91ml at elutriant, and to be that NaCl concentration goes out during for 0.0591M one unimodal.
Embodiment 3: gel-filtration prepacked column Superdex-75 analyzes
Fig. 7 is beta trichosanthin protein elution profile on the Superdex-75 post that the present invention prepares, and elution buffer is 0.1M NH
4HCO
3, it is one unimodal that pH8, flow velocity 0.4ml/min, beta trichosanthin protein go out when elutriant reaches 13.34ml.
Embodiment 4: the crystal growth of beta trichosanthin protein
The beta trichosanthin protein of the present invention preparation is as mother liquor, protein concentration 20mg/ml, and pond liquid is used 0.075M; The pH5.4 citrate buffer solution, 2M KCl is a precipitation agent, cultivates protein crystal with sessile drop method; Visible crystals after 2 days; 1 all left and right sides crystal is grown up, and through X-ray diffraction analysis or synchrotron radiation diffraction analysis, crystal belongs to P2
12
12
1Spacer, its brilliant full parameter is the a=38.244 dust, b=86.934 dust, c=135.785 dust.
Claims (1)
1. the method for the fast purifying of a beta trichosanthin protein is cleaned the former green material peeling of Snakegourd Root, and chopping adds 5mM; The extracting of pH4.5 acetate buffer solution, homogenate adds 50% Glacial acetic acid min. 99.5 and transfers pH4.0; Then under 4 ℃ of conditions, the centrifugal 30min of 16000 * g, the supernatant application of sample is to 20mM; On the post of pH4.5 acetate buffer solution equilibrated strong cation-exchanging resin S-Sepharose Fast Flow or SP-Sepharose Fast Flow, column length requires more than 20cm, and 280nm detects in real time; Use 20mM, the pH4.5 acetate buffer solution is washed till baseline, uses 20mM again; The pH6.5 phosphoric acid buffer is washed till baseline, with 15-20 times of column volume damping fluid 0-0.3M NaCl gradient elution, generally can go out 3 peaks then; Collect the 2nd peak, transfer pH4.0, add the acetate buffer solution of two volumes with 50% Glacial acetic acid min. 99.5; Last appearance is used pH4.5 to the post of using identical acetate buffer solution equilibrated strong cation-exchanging resin Sp-Sepharose Fast Flow, the 20mM acetate buffer solution is washed post; Use pH7.5 again, the 10mM phosphoric acid buffer is washed till baseline, uses 0-0.4M NaCl gradient elution then; Can obtain beta trichosanthin protein; It is characterized in that: after the damping fluid extracting,, just can obtain beta trichosanthin protein through 2 step strong cat ion exchange column gradient elutions.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2006100849606A CN101081862B (en) | 2006-05-31 | 2006-05-31 | Rapid purification and for beta trichosanthin protein and crystallization technology |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2006100849606A CN101081862B (en) | 2006-05-31 | 2006-05-31 | Rapid purification and for beta trichosanthin protein and crystallization technology |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101081862A CN101081862A (en) | 2007-12-05 |
| CN101081862B true CN101081862B (en) | 2012-02-22 |
Family
ID=38911676
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2006100849606A Expired - Fee Related CN101081862B (en) | 2006-05-31 | 2006-05-31 | Rapid purification and for beta trichosanthin protein and crystallization technology |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101081862B (en) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1424323A (en) * | 2001-12-14 | 2003-06-18 | 中国科学院福建物质结构研究所 | Preparation and use of secondary crystallized super purified radix trichosanthis protein |
-
2006
- 2006-05-31 CN CN2006100849606A patent/CN101081862B/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1424323A (en) * | 2001-12-14 | 2003-06-18 | 中国科学院福建物质结构研究所 | Preparation and use of secondary crystallized super purified radix trichosanthis protein |
Non-Patent Citations (4)
| Title |
|---|
| Pushpa Narayanan 等.Isolation and characterization of new isoforms of trichosanthin from Trichosanthes kirilowii.<Plant Science>.2002, * |
| 孙建忠等.简便快速分离天花粉病毒蛋白的一种方法》.《生物化学杂志》.1994, * |
| 袁惠东等.用Blue Sepharose CL-6B快速纯化天花粉蛋白.<生物化学与生物物理进展》.1997, * |
| 金善炜等.天花粉蛋白的化学.《化学学报》.1981, * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101081862A (en) | 2007-12-05 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Mishkind et al. | Distribution of wheat germ agglutinin in young wheat plants | |
| CN102370671B (en) | Active fraction in lucid ganoderma fruiting body, extracting method, application thereof and preparation | |
| JP2004505925A (en) | Chlorella preparations exhibiting immunomodulatory properties | |
| Mann et al. | Solubilization of human leucocyte membrane isoantigens | |
| HUYNH et al. | Isolation and characterization of a 30 kDa protein with antifungal activity from leaves of Engelmannia pinnatifida | |
| CN101081862B (en) | Rapid purification and for beta trichosanthin protein and crystallization technology | |
| CN104086664A (en) | Gracilaria lemaneiformis polysaccharide extract and preparation method and application thereof | |
| Huizing et al. | Xylan and mannan as cell wall constituents of different stages in the life-histories of some siphoneous green algae | |
| US4522814A (en) | Composition of matter from Cryptosiphonia woodii useful for the treatment of herpes simplex virus | |
| CN101709083B (en) | Fibrinolytic protein from scorpion tails, preparation method and application thereof | |
| CN101386644A (en) | Pumpkin protein 2 and preparation method and application thereof | |
| CN103134714A (en) | Method for extracting sugar cane protein used for dimensional electrophoresis | |
| JPS5815920A (en) | Improving agent for biophylactic function | |
| JP4581082B2 (en) | Autoimmunity enhancer, method for producing the same, and cosmetics using the same | |
| CN101724029A (en) | Anti-tumor protein and preparation method and application thereof | |
| CN1379088A (en) | Radish chitin bindin and its preparing process | |
| Al-Sohaimy et al. | Anti-HCV lectin from Egyptian Pisum sativum | |
| JP4604240B2 (en) | Photoinhibitory immunity recovery agent and method for producing the same | |
| CN1102959C (en) | Technological process of extracting nutritious acetasa SOD | |
| KR920017663A (en) | Refined tick allergens | |
| CN1982335A (en) | Production of trichosanthin by one-step method | |
| JPH0236124A (en) | Antiviral agent | |
| CN101274097B (en) | Atomizing grippe vaccine composition and preparation thereof | |
| Atanasov et al. | Anti-platelet fraction from Galega officinalis L. Inhibits platelet aggregation | |
| CN1062563C (en) | Anti-fungus protein extracted from tuber of elevated gastrodia and preparation method therefor |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C17 | Cessation of patent right | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20120222 Termination date: 20140531 |