CN1626661A - Phytohemagglutinin and application - Google Patents
Phytohemagglutinin and application Download PDFInfo
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- CN1626661A CN1626661A CNA2004100740271A CN200410074027A CN1626661A CN 1626661 A CN1626661 A CN 1626661A CN A2004100740271 A CNA2004100740271 A CN A2004100740271A CN 200410074027 A CN200410074027 A CN 200410074027A CN 1626661 A CN1626661 A CN 1626661A
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Abstract
The present invention provides a gene and encoded protein thereof capable of maintaining the survival of the hemopoietic stem/progenitor cell and self renewing. The gene provided according to the invention is DNA sequence of sequence 1 in the sequence label; the protein has aminophenol sequence of sequence 2 or sequence 3 in the sequence label. The gene and encoded protein is desired to be important maintain factor or protein medicament for amplification in vitro and induced directional differentiation of the hemopoietic stem/progenitor cell clinical production, which supplies adequate target cell capable of maintaining, adjusting and controlling cycle process of cell for gene curing etc. research, and supplies help to the research of blood production control and adjustment, and research of renewing the stem cell, and supplies new theory and method for maintain and clinic application of other stem cell.
Description
Technical field
The present invention relates to biomedicine field, specifically relate to a kind of phytohemagglutinin the work of external long term maintenance hematopoietic stem deposit with self in application.
Background technology
Hematopoietic stem cell transplantation has been present various pernicious, the non-pernicious neoplastic hematologic disorder disease commonly used and the treatment means of heavy dose of cancer chemotherapy patient marrow-reconstitution, be the required cell concentration that obtains medical treatment, many people attempt the means expanding hemopoietic ancestral cells by exsomatize (exvivo), comprise using feeder cell and various cytokine couplings etc.But have contradiction between the self of cell proliferation and cell and the maintenance of multipotency, amplification often causes the forfeiture of hematopoietic stem versatility.On the contrary; if can for making the cell cycle synchronization be beneficial to next step gene therapy, remove remaining tumour cell with the hematopoietic stem long term maintenance in stationary phase; the protection hemopoietic stem cell, and adjust and carry out cell amplification suitable opportunity or transplant all very favourable.
Lectin is intravital class protein of animals and plants or glycoprotein, the binding ability that has high special with monose in cell surface protein or the composite of lipid or oligosaccharides.Many phytohemagglutinins can both be as the exogenous stimulation factor of zooblast signal transduction, in various physiological responses, play a role, since Stillmark in 1988 found that the legume-seeds extract has agglutination to zooblast, increasing lectin obtained development and use.Utilize some lectin of beans can stimulate the propagation of lymphocyte populations, as ConA, PHA, dyers' grapes etc.; The research field relevant with tumour, immunity and hematopoiesis at lectin also has new progress in recent years.
Cultivate hematopoietic stem with lectin long term maintenance under isolated condition and will solve the many problems that occur in the hematopoietic stem cell transplantation, expand the scope of its clinical application.
Summary of the invention
The purpose of this invention is to provide a kind of have keep hematopoietic stem work and deposit gene and encoded protein matter thereof with the phytohemagglutinin of self ability.
A kind of phytohemagglutinin gene, it is one of following nucleotide sequences:
1) dna sequence dna of sequence 1 in the sequence table;
2) with sequence table in the dna sequence dna that limits of sequence 1 have 90% above homology, and the identical function protein DNA sequence of encoding.
The complete opening code-reading frame of described phytohemagglutinin gene is the 1st to 819 totally 819 bases of sequence 1 in the sequence table.
A kind of phytohemagglutinin precursor protein matter, it is one of following amino acid sequences:
1) aminoacid sequence of sequence 2 in the sequence table;
2) aminoacid sequence of sequence 2 passes through replacement, disappearance or the interpolation of one or several amino-acid residue and has identical active by sequence 2 deutero-protein with the aminoacid sequence of sequence 2 in the sequence table.
The proteinic two class subunit main body sequences of described phytohemagglutinin are respectively the 17th to 38 totally 22 amino acid and the 124th to 152 totally 29 amino acid of sequence 2 in the sequence table.
The proteinic formation subunit of a kind of phytohemagglutinin, it is one of following aminoacid sequence:
1) one or more sequences of the aminoacid sequence group of sequence 3 in the sequence table;
2) one or more sequences of the aminoacid sequence group of sequence 3 are passed through replacement, disappearance, interpolation or the combination of one or several amino-acid residues and are had identical active by sequence 3 deutero-protein with the aminoacid sequence group of sequence 3 in the sequence table.
Proteinic three small molecular weights of described phytohemagglutinin constitute β, α 1, α 2 amino-acid sequences that subunit N end homing sequence is respectively sequence 3 in the sequence table.
Utilize the Genbank database of NCBI that gene of the present invention is retrieved, the result shows do not have gene in the database gene order is identical therewith.
Because protein provided by the present invention adds CD34 separately to
+In the substratum of hematopoietic stem, just can keep CD34
+The work of hematopoietic stem is deposited and is reached more than 28 days, phase when keeping most of cell all to be in the G0/G1 of cell cycle simultaneously, and kept very a high proportion of colony forming cell, therefore gene of the present invention and encoded protein matter thereof are expected to become clinical marrow, peripheral blood, the umbilical cord blood hematopoietic ancestral cells is transplanted the important factor or the protein medicaments kept, for expanding marrow, peripheral blood, researchs such as the cell therapy of umbilical cord blood hematopoietic ancestral cells and gene therapy provide enough energy long term maintenance, the desirable target cell of adjustable cell cycle progression, and impel the relevant gene therapy of hematopoietic cell more effectively and be widely used in clinically, will bring huge social benefit and economic benefit.Gene of the present invention and encoded protein matter thereof can be the research that aspects such as the conduction of hematopoietic stem response cytokine generation signal, cycle regulating, propagation, differentiation change in the hematopoiesis regulation process new approaches are provided, for self mechanism, the clear and definite leukemic origin cause of formation of inquiring into stem cell and treat and offer help, also long term maintenance and the clinical application for other stem cell provides new theory and method; And the variant production for the different conceptual phases of protein effect are developed comprises the target gene of new antibodies, biochip, the new drug that filters out etc., also will promote to form emerging Human genome industry.
Description of drawings
Fig. 1 is the proteic 15%SDS-PAGE electrophoresis result of the present invention.
Fig. 2 is the results of hybridization of lectin of the present invention to the special acceptor of different cell surfaces.Wherein show respectively: A human cord blood positive findings, B human cord blood negative control, C CD34
+The cell positive result, D CD34
+Negative control, E mononuclearcell, F human erythroleukemia cell K562, G human erythroleukemia cell HEL, H human erythroleukemia cell U937, I human T lymphocyte leukemia cell Jurkat, J human B lymphocyte leukemia cell Raji, K human myeloid leukemia cell KG1, L people's haematogonium KG1a, M transitional cell bladder carcinoma cell line EJ1, N people's tire nephrocyte HEK293, O liver cancer cell HepG2, P human cervical carcinoma cell Hela, Q human bone marrow substrate cell HFCL, R human breast cancer cell MCF-7.
Fig. 3 for lectin of the present invention and FL separately and applied in any combination to CD34
+The result of hematopoietic stem propagation influence.
Fig. 4 for lectin of the present invention and FL to CD34
+The exercising result of the cell cycle of hematopoietic stem.
All represent lectin of the present invention among each figure with FRIL.
Embodiment
The clone of embodiment 1, agglutinin gene
Gene cDNA clone of the present invention is from the newborn bean seedlings cDNA library of cow gram Dolichos labab, and the condition of primer sequence and polymerase chain reaction thereof (PCR) is as follows:
primer1：5’CGTCACAGAACGCCTCTTGTAA?3’
primer2：5’CGCATGTTTCCCAGTAAGGTTA?3’
50 μ l reaction systems comprise: 4 μ l cDNA (100ng/ μ l), primer1 and the primer2 of each 1 μ l, 10 μ M, 4 μ l dNTP (2.5mM), 1 μ l Ex Taq archaeal dna polymerase (TakaRa company, 5u/ μ l), 5 μ l, 10 * PCRBuffer, 38 μ l aqua sterilisas.Reaction conditions is: 94 ℃ of 30s; 59 ℃ of 30s, 72 ℃ of 45s, 35 circulations, 72 ℃ of 7min.Reaction product is cloned into pMD-18T carrier (TakaRa company).With the ordinary method order-checking, the result shows that the gene that the present invention is cloned into is the dna sequence dna of sequence 1 in the sequence table.
Embodiment 2, proteinic affinitive layer purification
Protein source of the present invention is in cow gram Dolichos lablab dry seeds, utilize lectin to the characteristic of specific glycosyl high-affinity affinitive layer purification in addition, process is as follows: cow gram is pulverized, with 5 times of volume Tris damping fluids (50mmol/L Tris-HCl, 1mmol/L MgCl
2, 1mmol/L CaCl
2, pH8.0) homogenate, 4 ℃ of balances are more than 4 hours; 4 ℃ of 20000g high speed centrifugation 20min get supernatant; Transfer pH to 4.0 with Glacial acetic acid, the same high speed centrifugation is got supernatant; 40%NaOH transfers pH to 8.0, and the same high speed centrifugation is got supernatant, i.e. the lectin crude extract.Affinity chromatography medium (mannose-Sepharose matrix after crude extract and the Tris damping fluid balance, Pharmacia company) combination, 4 ℃ of reactions are more than 4 hours, then wash to there not being albumen to flow out, obtain the pure solution of lectin FRIL with 200mmol/L Alpha-Methyl α-D-mannoside wash-out again with the Tris damping fluid.15%SDS-PAGE result as can be seen from the figure has 5 tangible bands as shown in Figure 1 in the 10kD-25kD scope, be 5 subunits of isolating phytohemagglutinin.
The N terminal amino acid order-checking of embodiment 3, protein portion subunit
15%SDS-PAGE separates each subunit band of purified lectin of beans, be transferred on the pvdf membrane by half-dried transfer method, measure N with 491A sequential analysis of protein instrument through automatic edman degradation and hold preceding 5 aminoacid sequences, the result shows that it is the aminoacid sequence of sequence 3 in the sequence table that its part of the purified protein of the present invention constitutes subunit N terminal sequence.As can be seen from the table, there are two components in proteic α 2 subunits of the present invention, and one of them is that one of N end disappearance is amino acid whose.
The mensuration of embodiment 4, activity of lectin
Extract rabbit auricular vein blood, handle the back and be made into 2% red cell suspension with physiological saline.On V-type blood coagulation plate, with lectin of the present invention PBS (137mmol/L NaCl, 2.7mmol/L KCl, 4.3mmol/L Na
2HPO
47H
2O, 1.4mmol/L KH
2PO
4, pH=7.3) doubling dilution (1: 2), every hole adds 25 μ L, adds 2% fresh blood cell suspension 25 μ L again, after vibration shakes up, places 1～2h, the generation of visual inspection agglutination phenomenon for 25 ℃.The result shows, the lectin of purifying still has agglutination activity being diluted to about 300ng/ml, and protein of the present invention is a kind of phytohemagglutinin with aggegation hemocyte ability.
The sugared inhibition test of embodiment 5, lectin
Sucrose, seminose, α-D seminose, 3-O α-D seminose, glucose, Alpha-Methyl α-D-mannoside are made into 1mol/L concentration with distilled water respectively, on V-type blood coagulation plate with PBS by 1: 10 gradient doubling dilution, every hole adds 25 μ L, add the lectin of the present invention 25 μ L of 900ng/ml again toward each hole, and 2% fresh blood cell suspension 50 μ L, place 1～2h for 25 ℃, visual inspection suppresses the result, and the sugar that can combine lectin of the present invention with the red corpuscle competition will cause erythrocytic deposition.Sugar suppresses experimental result and sees Table 1.By finding out in the table, the main and 3-O α-D seminose of lectin of the present invention has very high affinity, and lectin of the present invention is a kind of lectin at 3-O α-D seminose specific combination.
Table 1 lens culinaris agglutinin sugar suppresses experiment
The sugar effective weaker concn of competing reaction (mol/L)
Sucrose 10
-2
Seminose 10
-2
α-D seminose 10
-3
glucose 10
-1
Alpha-Methyl α-D-mannoside-
3-Oa-D seminose 10
-5
The histological chemistry of the special acceptor of cell surface of embodiment 6, lectin is detected
The protein that purifying of the present invention is obtained with ordinary method with horseradish peroxidase HRP mark, the probe that detects as histological chemistry.
Utilize histochemical method to detect the expression of protein acceptor of the present invention in multiple hematopoietic cell system and tissue lines, experimental result shows, the proteinic acceptor of the present invention does not all have expression on human cervical carcinoma, mammary cancer, bladder cancer, liver cancer, tire nephrocyte, marrow stromal cell, in multiple hematopoietic cell, the human cord blood mononuclearcell MNC and the CD34 of human erythroleukemia cell HEL, people's haematogonium KG1a and separation and purification only arranged also
+There is special acceptor in cell surface, does not also have acceptor on all the other human myeloid leukemia cell KG1, U937, human erythroleukemia cell K562, the human T lymphocyte leukemia cell Jurkat, and CD34
+In the cell, exist the cell of the special acceptor of protein of the present invention also only to account for wherein a part.The result as shown in Figure 2.Fig. 2 demonstrates, and the proteinic acceptor of the present invention is that the part hematopoietic stem is peculiar, and albumen mass-energy specificity of the present invention plays a role at hematopoietic stem.
The acceptor of lectin of the present invention is flt3, and another part of flt3 is FL (flt3 ligand), and it is one of the cytokine of the amplification in vitro hematopoietic stem of present widespread use, contrast lectin of the present invention and FL external to CD34
+The effect of keeping of hematopoietic stem, method is as follows: according to CD34
+Cellular segregation test kit (miniMACS magnetic cell sorting system, Miltenyi BiotechGmbH company, Germany) operation steps separation and purification cord blood CD 34
+Cell, cell is with 4～8 * 10
5The concentration of/mL is incubated in RPMI 1640 substratum that contain 10%FBS, 0.5%BSA, add FRIL, FL, (FRIL+FL) cultivation respectively, the final concentration that FRIL, FL add is 300ng/mL, do not change substratum, in certain hour counting cells amount, and collecting cell is used for PI dyeing, flow cytometer detects the cell cycle, and (2000～8000 cell inoculations contain 5ng/mL GM-CSF, 5ng/mL IL-3,50ng/mL SCF, 2U/mL EPO, 5 * 10 in 0.5mL to the cultivation of part cell mixing colony in addition
-5The methylcellulose gum premix substratum MethoCult of mol/L 2-ME, 10%FBS, 10%HS
TMAmong the SF, add, be cultured to the various colonies that the 16d counting forms by every hole 0.5mL in 24 orifice plates.Colony density is per 10
4Individual plastidogenetic colony number.) the cell proliferation curve sees Fig. 3, the cell cycle detected result is seen Fig. 4, mixes colony and cultivates count results and see Table 2.Fig. 3, Fig. 4 show, with the CD34 of lectin cultivation of the present invention
+Cell G
0/ G
1The ratio of phase exceeds the exposure level of FL always more than 80%, compares the difference on the cell count simultaneously, infers that lectin of the present invention has prolonged the cell cycle of hemopoietic stem cell, has reduced the cell count that enters division and breeding.Table 2 shows, with lectin long-term cultivation CD34 of the present invention
+The relative FL of cell shows higher colony and forms ability, and visible lectin of the present invention more is applicable to the versatility of prolonged preservation hematopoietic stem.Above presentation of results, lectin of the present invention have the stronger external CD34 of keeping
+The ability with the self potentiality is deposited in the work of hematopoietic stem, mainly is by with CD34
+Hematopoietic stem is trapped in the G of cell cycle
0/ G
1Phase realizes.
Table 2 lens culinaris agglutinin and FL various combination are to CD34
+The hematopoietic stem colony forms the influence of ability
Day Medium Myeloid Erythroid Mix
0 824±141 8±3 83±10
3 FL 1001±165 10±7 17±21
FRIL 790±202
* 21±17 8±6
FL+FRIL 863±392 0 16±15
Blank 795±28 35±14 17±8
7 FL 902±252 34±30 107±95
FRIL 818±376 35±22 90±84
FL+FRIL 836±240 119±97 107±91
Blank 432±107 0 0
14 FL 260±137 11±10 15±13
FRIL 326±121
* 21±13
* 23±9
*
FL+FRIL 272±147
*** 0
*** 5±5
***
21 FL 130±126 1±1 4±3
FRIL 295±117
** 6±4
* 12±9
*
FL+FRIL 347±303
**** 0
**** 0
***
28 FL 120±64 0 1±1
FRIL 172±60
* 0 1±2
FL+FRIL 180±74 2±2 1±1
*P＜0.05 lectin of the present invention is compared with the FL group,
*P＜0.01 lectin of the present invention is compared with the FL group,
* *There is interaction between P＜0.001 lectin of the present invention and the FL,
* * *There is interaction between P＜0.05 lectin of the present invention and the FL.
Represent lectin of the present invention with FRIL in the table.
Sequence table
<160>2
<210>1
<211>819
<212>DNA
＜213〉Dolichos French beans kind (Dolichos lablab L.[Lablab vulgaris Savi])
<400>1
atgtttccca?gtaaggttaa?gtcagcacag?tcattgtcat?ttagtttcac 50
caagtttgat?cctaaccaag?aggatcttat?cttccaaggt?catgccactt 100
ctacaaacaa?tgtcttacaa?ctcaccaagt?tagacagtgc?aggaaaccct 150
gtgagttcta?gtgcgggaag?agtgttatat?tctgcaccat?tgcgcctttg 200
ggaagactct?gcggtattga?caagctttga?caccattatc?aactttgaaa 250
tctcaacacc?ttacacttct?cgtatagctg?atggcttggc?cttcttcatt 300
gcaccacctg?actctgtcat?cagttatcat?ggtggttttc?ttggactctt 350
tcccaacgca?aacactctca?acaactcttc?cacctctgaa?aaccaaacca 400
ccactaaggc?tgcatcaagc?aacgttgttg?ctgttgaatt?tgacacctat 450
cttaatcccg?attatggtga?tccaaactac?atacacatcg?gaattgacgt 500
caactctatt?agatccaagg?taactgctaa?gtgggactgg?caaaatggga 550
aaatagccac?tgcacacatt?agctataact?ctgtctctaa?aagactatct 600
gttactactt?attatcctgg?gagtaaacct?gcgactctct?cctatgatat 650
tgagttacat?acagtgcttc?ctgaatgggt?cagagtaggg?ttatctgctt 700
caactggaca?agataaagaa?agaaataccg?ttcactcatg?gtctttcact 750
tcaagcttgt?ggaccaatgt?ggcgaagaag?gagaatgaaa?acaagtatat 800
tacaagaggc?gttctgtga 819
<210>2
<211>272
<212>PRT
＜213〉Dolichos French beans kind (Dolichos lablab L.[Lablab vulgaris Savi])
<400>2
Met?Phe?Pro?Ser?Lys?Val?Lys?Ser?Ala?Gln?Ser?Leu?Ser?Phe?Ser
1 5 10 15
Phe?Thr?Lys?Phe?Asp?Pro?Asn?Gln?Glu?Asp?Leu?Ile?Phe?Gln?Gly
20 25 30
His?Ala?Thr?Ser?Thr?Asn?Asn?Val?Leu?Gln?Leu?Thr?Lys?Leu?Asp
35 40 45
Ser?Ala?Gly?Asn?Pro?Val?Ser?Ser?Ser?Ala?Gly?Arg?Val?Leu?Tyr
50 55 60
Ser?Ala?Pro?Leu?Arg?Leu?Trp?Glu?Asp?Ser?Ala?Val?Leu?Thr?Ser
65 70 75
Phe?Asp?Thr?Ile?Ile?Asn?Phe?Glu?Ile?Ser?Thr?Pro?Tyr?Thr?Ser
80 85 90
Arg?Ile?Ala?Asp?Gly?Leu?Ala?Phe?Phe?Ile?Ala?Pro?Pro?Asp?Ser
95 100 105
Val?Ile?Ser?Tyr?His?Gly?Gly?Phe?Leu?Gly?Leu?Phe?Pro?Asn?Ala
110 115 120
Asn?Thr?Leu?Asn?Asn?Ser?Ser?Thr?Ser?Glu?Asn?Gln?Thr?Thr?Thr
125 130 135
Lys?Ala?Ala?Ser?Ser?Asn?Val?Val?Ala?Val?Glu?Phe?Asp?Thr?Tyr
140 145 150
Leu?Asn?Pro?Asp?Tyr?Gly?Asp?Pro?Asn?Tyr?Ile?His?Ile?Gly?Ile
155 160 165
Asp?Val?Asn?Ser?Ile?Arg?Ser?Lys?Val?Thr?Ala?Lys?Trp?Asp?Trp
170 175 180
Gln?Asn?Gly?Lys?Ile?Ala?Thr?Ala?His?Ile?Ser?Tyr?Asn?Ser?Val
185 190 195
Ser?Lys?Arg?Leu?Ser?Val?Thr?Thr?Tyr?Tyr?Pro?Gly?Ser?Lys?Pro
200 205 210
Ala?Thr?Leu?Ser?Tyr?Asp?Ile?Glu?Leu?His?Thr?Val?Leu?Pro?Glu
215 220 225
Trp?Val?Arg?Val?Gly?Leu?Ser?Ala?Ser?Thr?Gly?Gln?Asp?Lys?Glu
230 235 240
Arg?Asn?Thr?Val?His?Ser?Trp?Ser?Phe?Thr?Ser?Ser?Leu?Trp?Thr
245 250 255
Asn?Val?Ala?Lys?Lys?Glu?Asn?Glu?Asn?Lys?Tyr?Ile?Thr?Arg?Gly
260 265 270
Val?Leu
272
<210>3
Lectin part subunit N terminal amino acid sequence of the present invention
subunit AA?sequence?of?N-terminal
β Ala?Gln?Ser?Leu?Ser?Phe
α1 Thr?Thr?Thr?Lys?Ala?Ala
α2 Asp?Ser?Ser?Thr?Ser/Ser?Ser?Thr?Ser?Gln
Claims (6)
1, a kind of have the work of external long term maintenance hematopoietic stem and deposit lectin of beans gene with the self ability, and it is one of following nucleotide sequences:
1) dna sequence dna of sequence 1 in the sequence table;
2) with sequence table in the dna sequence dna that limits of sequence 1 have 90% above homology, and the identical function protein DNA sequence of encoding.
2, gene according to claim 1 is characterized in that: described to have the gene that the work of external long term maintenance hematopoietic stem deposits with the self ability be the dna sequence dna of sequence 1 in the sequence table.
3, gene according to claim 1 and 2 is characterized in that: described have the work of external long term maintenance hematopoietic stem and deposit the 1st to 819 totally 819 bases that complete opening code-reading frame with self ability gene is a sequence 1 in the sequence table.
4, a kind of have the work of external long term maintenance hematopoietic stem and deposit protein with the self ability, its precursor protein have the aminoacid sequence of sequence 2 in the sequence table or with the aminoacid sequence of sequence 2 through replacement, disappearance or the interpolation of one or several amino-acid residue and have identical active by sequence 2 deutero-protein with the aminoacid sequence of sequence 2.
5, protein according to claim 4 is characterized in that: described proteinic precursor has the aminoacid sequence of sequence 2 in the sequence table.
6, according to claim 3 or 4 described protein, it is characterized in that: described have the work of external long term maintenance hematopoietic stem and deposit that to constitute subunit N end with the proteinic part of self ability be one or more groups aminoacid sequence of sequence 3 in the sequence table.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2004100740271A CN1626661A (en) | 2003-10-31 | 2004-09-01 | Phytohemagglutinin and application |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200310103392.6 | 2003-10-31 | ||
| CN200310103392 | 2003-10-31 | ||
| CNA2004100740271A CN1626661A (en) | 2003-10-31 | 2004-09-01 | Phytohemagglutinin and application |
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| Publication Number | Publication Date |
|---|---|
| CN1626661A true CN1626661A (en) | 2005-06-15 |
Family
ID=34796279
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|---|---|---|---|
| CNA2004100740271A Pending CN1626661A (en) | 2003-10-31 | 2004-09-01 | Phytohemagglutinin and application |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101013135B (en) * | 2007-02-12 | 2012-01-25 | 安徽农业大学 | Method for sifting functional fungoid having specific affinity with non-leguminous plant by using agglutinin |
| CN101382543B (en) * | 2008-07-09 | 2012-04-11 | 中国农业大学 | Method for detecting activity of lectin of beans |
| CN101508730B (en) * | 2009-03-18 | 2012-05-23 | 王敏康 | Extract method for lectin of leguminous plants |
-
2004
- 2004-09-01 CN CNA2004100740271A patent/CN1626661A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101013135B (en) * | 2007-02-12 | 2012-01-25 | 安徽农业大学 | Method for sifting functional fungoid having specific affinity with non-leguminous plant by using agglutinin |
| CN101382543B (en) * | 2008-07-09 | 2012-04-11 | 中国农业大学 | Method for detecting activity of lectin of beans |
| CN101508730B (en) * | 2009-03-18 | 2012-05-23 | 王敏康 | Extract method for lectin of leguminous plants |
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