CN104292328B - A kind of application of chymostatin and its gene from Urechis uniconctus and its recombinant protein - Google Patents
A kind of application of chymostatin and its gene from Urechis uniconctus and its recombinant protein Download PDFInfo
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- CN104292328B CN104292328B CN201410549248.3A CN201410549248A CN104292328B CN 104292328 B CN104292328 B CN 104292328B CN 201410549248 A CN201410549248 A CN 201410549248A CN 104292328 B CN104292328 B CN 104292328B
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Abstract
The present invention relates to the application of a kind of chymostatin and its gene from Urechis uniconctus and its recombinant protein, belong to biomedical sector.The protein of the chymostatin has amino acid sequence SEQ ID No.1, the Urechis uniconctus chymostatin UCI01 of coding is single chain polypeptide, containing 67 amino acid residues, molecular weight is about 7516Da, isoelectric point is 4.38, without disulfide bond, without obvious signal peptide region.The gene has the base sequence in sequence table SEQ ID No.2, and cDNA sequence overall length is 697bp, wherein the 5` non-translational regions containing 80bp, 413bp 3` non-translational regions and polyA tails, open encoder block are 201bp.The gene is recombinantly expressed using prokaryotic expression technology, the recombinant protein of acquisition has the activity of chymostatin, its IC50 is 53nM.The recombinant protein has preferable pH stability and certain temperature stability, has fungistatic effect to staphylococcus aureus.
Description
Technical field
The present invention relates to a kind of chymostatin and its gene from Urechis uniconctus and its recombinant protein
Application, belong to biomedical sector.
Background technology
Albumen enzyme system in organism is to maintain one of key substance of life, but the abnormal expression or release of protease
Also result in body and produce serious lesion.It is to accumulate necessarily special that organism, which adjusts one of main path of these enzyme activity,
Property protease inhibitors, chymostatin is exactly one of which.
Chymostatin (Chymotrypsin Inhibitor, CI) is widely present in microorganism, pulse family and planted
Thing, grass, arthropod and mammal etc. it is internal, main function is off, prevents or reduced chymotrypsin protein
Enzymatic activity.Chymotrypsin (chymotrypsin) is called chymotrypsin, is a kind of typical serine protease, in biology
Very important effect is played in the Various Tissues and organ of body, is also the important pathogen of some causal organisms or tumour cell
The factor.
According to the difference of structure, molecular weight etc., serpin is divided into different type, wherein potato protein
Enzyme inhibitor I (PI-1) family is a wherein subspecies.The source of potato proteinase inhibitor I families is not limited to potato,
In other biological body (such as：Barley, tomato), animal (such as：Leech) in be also found.Typically there is this proteinoid suppression pancreas to coagulate
In the activity of galactase or elastoser, structure, the molecular weight of this kind of protease is smaller (60~90 amino acid residues),
Disulfide bond is free of in structure.
Urechis uniconctus (Urechis uniconctus) are commonly called as extra large intestines, are a kind of coastal distinctive ocean Yi worms of northern China
Class animal, is the unique class that no pipe Yi mesh is distributed in China coast.It is distributed mainly on China's Bohai and Yellow Seas, Russia, Korea
The ground such as the peninsula, Hokkaido, Japan, Shandong Jiangzhou area is the maximum place of production of the extra large intestines of China.Its individual is thick, and meat tenderness is delicious, body wall
Protein content is up to 22.84%, and rich in a variety of essential amino acids, with high edibility, also known as " nude is extra large
Ginseng ".Meanwhile, Urechis uniconctus are inhabited under coastal hurst intertidal zone in area and subtidal zone phytal zone " U-shaped " tunnel, can be at other
Survive, also have in terms of as Ocean Medicinal active material, the raw material of health products is extracted in the inviable sulfur-rich environment of animal
There is very high potential.
The content of the invention
The purpose of the present invention is to be prepared based on above-mentioned theory and prior art basis there is provided one kind using recombination and expression techniques
, chymostatin and its gene from Urechis uniconctus and the application of its recombinant protein.
The technical scheme is that：
A kind of chymostatin from Urechis uniconctus, the protein tool of the chymostatin
There are amino acid sequence SEQ ID No.1：
MATKEQWPELVGKSGEEAKAAIIAERPELEKVEICNELSPMTMDYRTDRVRIFVNNDGNVVNPPQTG。
The described chymostatin from Urechis uniconctus, the Urechis uniconctus chymotrypsin suppression of coding
Preparation UCI01 is single chain polypeptide, containing 67 amino acid residues, and molecular weight is about 7516Da, and isoelectric point is 4.38, without two sulphur
Key, without obvious signal peptide region.
A kind of chymostatin gene from Urechis uniconctus, the gene has sequence table SEQ ID
Base sequence in No.2.
The described chymostatin gene from Urechis uniconctus, the gene cDNA sequence overall length is
697bp, wherein the 5` non-translational regions containing 80bp, 413bp 3` non-translational regions and polyA tails, opening encoder block is
201bp。
A kind of application of chymostatin gene recombinant protein from Urechis uniconctus, the chymotrypsin protein
The recombination expression engineered strain EUCI01 of enzyme inhibitor gene, realizes the solubility expression of the gene.
The application of the described chymostatin gene recombinant protein from Urechis uniconctus, EUCI01 expression
Recombinant protein, with stronger chymostatin activity, its IC50 is 53nM.
The application of the described chymostatin gene recombinant protein from Urechis uniconctus, chymotrypsin protein
Inhibitor activity, recombinant protein is stable to pH, while having temperature stability.
The application of the described chymostatin gene recombinant protein from Urechis uniconctus, EUCI01 expression
Recombinant protein, can to staphylococcus aureus produce suppression.
Advantages of the present invention and beneficial effect are：
1st, present invention clone obtains the cDNA sequence of a Urechis uniconctus chymostatin, and utilizes restructuring side
Method purifying is prepared for the protein with chymotrypsin protein enzyme inhibition activity.So that, it is that subsequent applications are laid a good foundation.
2nd, the present invention is recombinantly expressed using prokaryotic expression technology to the gene, and the recombinant protein of acquisition has pancreas curdled milk
The activity of protease inhibitors, its IC50 is 53nM.The recombinant protein has preferable pH stability and certain temperature stabilization
Property, there is fungistatic effect to staphylococcus aureus.
Brief description of the drawings
Fig. 1：The SDS-PAGE analysis charts of recombinant protein.Wherein, 1 albumen marker；2 EUCI01 not induced；After 3 inductions
EUC01；4 UCI01 after purification.
Fig. 2：UCI01 is analyzed the inhibition of protease.Wherein, Trypsin is trypsase, and Chymotrypsin is
Chymotrypsin, abscissa Inhibition Concentration represent UCI01 concentration, ordinate Residual
Enzyme activity represent the activity of protease.
Fig. 3：The influence of temperature, pH to UCI01 activity.Wherein, a figures are influences of the pH to UCI01 activity；B is temperature pair
The influence of UCI01 activity, ordinate Residual enzyme activity represent the activity of institute's test proteins enzyme.
Embodiment
It is the embodiment of present invention below, but present disclosure is not limited to this, the specific content of the invention
Including：
1st, Urechis uniconctus chymostatin UCI01 genes
The present invention utilizes the post-stimulatory Urechis uniconctus tissue construction cDNA library of sea-farming pathogen, is surveyed by substantial amounts of
Sequence, obtains the genetic fragment of an albuminoid enzyme inhibitor, its full-length gene is obtained by RACE technologies.By the list of the present invention
Ring, which pierces Yi chymostatins encoding gene and enters line search through NCBI gene databases, to be compared, and does not find to exist any
The report of mutually homogenic or genetic fragment or gene expression product.Found by the comparison analysis to its possible coded product,
Itself and a kind of leech (Helobdella robusta), Anthocidaris crassispina (Strongylocentrotus purpuratus) and one
The similitude for speculating the protein may with protease inhibitors function in a little bacteriums is 42~59%.Pass through protein knot
Fruit analysis finds that it may belong to potato proteinase inhibitor I families, is named as UCI01.
Urechis uniconctus chymostatin UCI01 cDNA sequence has 80bp 5` non-translational regions, 413bp 3`
Non-translational region and polyA tails, 201bp opening encoder block, gene have the base sequence in sequence table SEQ ID No.2
Row.
The Urechis uniconctus chymostatin UCI01 of coding is single chain polypeptide, containing 67 amino acid residues, molecule
Amount is about 7516Da, and isoelectric point is 4.38, without disulfide bond, and the protein of the chymostatin has amino acid sequence
Arrange SEQ ID No.1.
2nd, UCI01 recombinant strains
The present invention designs primer according to the UCI01 genes of acquisition, is subcloned after PCR amplifications to prokaryotic expression plasmid pET25
(a) in, convert into corresponding Host Strains, successfully build engineering strain EUCI01.
According to the operation instruction of pET systems, by optimizing inductive condition, the induced expression of recombinant protein is carried out.Will induction
Ultrasonication after EUC01 ice baths afterwards, 6 based on expression vector institute band histidine-tagged, is carried out using Ni affinity columns
The purifying of recombinant protein.Analyzed through SDS-PAGE, restructuring UCI01 after purification only has a band, and size is about 7.6kDa, it is and pre-
Phase is in the same size.Recombinant protein concentration through coomassie brilliant blue R_250 colorimetric method for determining is 4mg/ml.
3rd, UCI01 application is recombinated
UCI01 protease inhibitory activity determines and uses colorimetric method, using bovine chymotrypsin and people's trypsase,
Wherein chymotryptic substrate is Nsuccinyl-Ala-Ala-Pro-Phe-p-nitroanilide (Sigma, USA), pancreas
The substrate of protease uses BAPNA ((Sigma, USA).After tested, UCI01 shows weaker trypsin inhibition capacity.But
UCI01 has the inhibitory activity of stronger chymotrypsin, and its IC50 is 53nM.PH is subsequently found not have UCI01 activity
There is too big influence, while there is UCI01 preservation 3.5h at certain temperature stability, 80 DEG C can still keep about 20% activity.
Below by drawings and examples, the present invention is described in more detail.
Embodiment 1：Urechis uniconctus chymostatin gene UCI01 acquisition
(1) total tissue RNA is extracted：Take commercially available Urechis uniconctus temporarily to support 2 days in laboratory conditions, select without any illness
Healthy individuals, with about 1 × 106Individual incubated overnight to exponential phase seawater pathogenic bacteria vibrio parahaemolytious (Vibrio
Parahaemdyticus) CGMCC 2164 is injected into Urechis uniconctus body cavity.After 2h, Urechis uniconctus are put to death in anesthesia, take monocyclic thorn
Yi breathing intestines, body lumen wall, body cavity blood cell etc. are organized, and are weighed, and take 200~300mg to organize, and are added 8~10m1 total serum IgEs and are carried
Buffer solution (Trizol solution, TAKARA) is taken, is homogenized in 20m1 glass homogenizers.Isometric phenol/chloroform solution is added, it is acute
Strong to mix, room temperature places 10~15min, and 4~6 DEG C centrifuge 10~15min, reject precipitation in 10000~12000rpm.On reset and add
Enter isometric isopropanol, room temperature places 10~15min, and 4~6 DEG C centrifuge 10~15min, precipitation in 10000~12000rpm
Washed once, dried with 75wt% ethanol, ttom of pipe sediment is total serum IgE.
(2) mRNA purifying：Using PROMEGA companies of the U.S.mRNA Isolation Systems
Kit carries out mRNA and isolated and purified.Concrete operations are：The μ g of total serum IgE 300~500 are taken to be dissolved in 300~500 μ l DEPC water,
65 DEG C of 8~10min of water-bath are put into, 3~5 μ l Oligo (dT) probes and 13~15 μ l 20 × SSC solution (citric acids is added
Sodium buffer solution), mix, place room temperature cooling, referred to as A liquid；Magnetic bead (SA-PMP) is flicked into mixing, to magnetic frame adsorption time 30
~40s, abandons supernatant, plus 0.5 × SSC (sodium citrate buffer solution) 0.3~0.5m1, to magnetic frame 30~40s of adsorption time, most
Plus 0.1 afterwards~0.2ml 0.5 × SSC suspends, referred to as B liquid；A liquid is added in B liquid, room temperature places 10~15min, to magnetic
Power frame adsorbs 30s, abandons supernatant, is washed with 0.1 × SSC 4 times, finally abandon supernatant, plus 0.1~0.2ml DEPC aqueous suspensions, to magnetic
30s is adsorbed on power frame, supernatant is moved to new test tube, the DEPC water for adding 0.15m1 suspends again, to magnetic frame absorption
30s, shifting supernatant to above-mentioned test tube, 1/10 volume 3M (mol/L) sodium acetates of addition and isometric isopropanol, room temperature placement 30~
60min, 4 DEG C, 12000rpm is centrifuged 10~15 minutes, supernatant is abandoned, in the DEPC water for being precipitated and dissolved in 10~20 μ l.
(3) structure of Urechis uniconctus cDNA library：Using CLONTECH companies CreatorTM SMARTTM cDNA
Library Construction Kit.It is specific as follows：
The chain of a, cDNA first synthesizes (mRNA reverse transcriptions)：DEPC processing centrifuge tube in add 1 μ l Urechis uniconctus mRNA,
1 μ l SMART IV oligonucleotides, 1 μ l CDS III/3 ' PCR primers, plus the water of 2 μ l DEPC processing reach cumulative volume
To 5 μ l.The reagent in centrifuge tube and of short duration centrifugation are mixed, 72 DEG C are incubated 2 minutes.By centrifuge tube on ice be incubated 2 minutes, from
The first chain buffer solution for being added in heart pipe in 2.0 μ l kit, 1.0 μ l 20mM (mmol/L) dithiothreitol (DTT), 1.0 μ l
The PowerScript reverse transcriptase of 10mM dNTP mixtures, 1.0 μ l.Reagent and of short duration centrifugation in centrifuge tube are mixed, at 42 DEG C
Insulation 1 hour.Centrifuge tube is placed in the synthesis for stopping the first chain on ice, takes the chains of cDNA first synthesized by 2 μ l standby from centrifuge tube
With.
B, the second chain amplification：Using long end polymeric PCR (LD-PCR) method：By the 2 μ l chains of cDNA first
(mRNA reverse transcriptions), 80 μ l deionized waters, 10 μ l 10 × Advantage 2PCR buffer solutions, 2 μ l 50 × dNTP mixtures,
The Escherichia coli polymerase centrifuge tube of 2 μ l 5 ' PCR primers, 2 μ l CDS III/3 ' PCR primers and 2 μ l is reacted.95
DEG C pre-degeneration 20 seconds, 68 DEG C, 6 minutes, circulates 20 times, the cDNA double-strands of synthesis is placed in into -20 DEG C of preservations by 95 DEG C, 5 seconds.
C, genetic fragment screening：The cDNA library built is connected to18-T Vertor (TAKARA), turn
Change DH5 α, blue hickie screening positive clone uses Applied Biosystems DNA sequencer, model ABI
PRISM 377 carries out nucleotide sequencing.
D, global cDNA sequence acquisition：Primer is designed at the Partial Fragment two ends of the UCI01 genes obtained to sequencing, is utilized
The global cDNA sequence of RACE technical limit spacing genes.Obtaining UCI01 cDNA sequence has 80bp 5` non-translational regions, 413bp 3
` non-translational regions and polyA tails, 201bp opening encoder block.Its DNA sequence dna is SEQ ID No.2：
5`-GCAGTGGTATCAACGCAGAGTACAGGGGACTCTCTGTCCAGCAGCAC
ACGGCGGTGATCAGACTTCGTACAGATATCAAAATGGCAACCAAGGAACAATGGCCCGAGTTGGTTGGCAAGAGTGG CGAGGAAGCCAAGGCTGCTATCATTGCAGAAAGACCTGAGTTGGAAAAGGTTGAAATCTGCAACGAGTTGAGTCCGA TGACCATGGACTACCGCACCGACAGAGTTCGCATTTTTGTCAACAATGACGGCAATGTTGTGAACCCTCCCCAGACC GGTTAGATGTACATCTACATGTAGAATGCTGGAGATGGATTGATTGGAAGTGTCATGTTACTTTAAGAACTGCAATC
TATCTGTGAATGGTGCTGCAAGCGACAAGAAAGTGGTCAATGTTATCTGTTACGTATGTGAACAATGCTGTGAGCCA
GTAATTATTGTGTACTGTCATCTGGATATGTTACCATTCCAACATGTTAACTCAAGTTTGGACCCAATTTTTTTCAA
AATTTCTGGGGGACCCTATCTTCCAACAATTTGATATGGCTGCGTTTCTTATATTTCGATGGAAGTCATCATTTGTC
CAAACAAATTTTGATTGTGTCAGATAAGTTGACGTATGCATTTCTATATGTTGGTAATAACAAAGTAAACTTATATA
AAACTGATTTGTTAAAAAAAAAAAAAAAAAAAAA-3`
E, by being found to gene encoding production analysis, the Urechis uniconctus chymostatin UCI01 of coding is
Single chain polypeptide, containing 67 amino acid residues, molecular weight is about 7516Da, and isoelectric point is 4.38, without disulfide bond.Its amino acid sequence
It is classified as SEQ ID No.1：
MATKEQWPELVGKSGEEAKAAIIAERPELEKVEICNELSPMTMDYRTDRVRIFVNNDGNVVNPPQTG
Embodiment 2：The structure of UCI01 recombinant strains
According to the cDNA sequence of acquisition, design primer carries out the clone of UCI01 genes.Primer sequence is：
Forward primer 5`-CATATGGCAACCAAGGAACAATGGC-3`(NdeI)；
Reverse primer 5`-CTCGAGACCGGTCTGGGGAGGGT-3`(XhoI)。
PCR reactions are carried out under the following conditions：94 DEG C, 2 minutes, 94 DEG C, 30 seconds, 68 DEG C, 30 seconds and 72 DEG C, 40 seconds
Clock, 30 circulations.The recovery of purpose fragment is carried out after the completion of amplification with glue reclaim kit (Tiangeng is biological).By the DNA of recovery
Fragment is transformed into BL21 (DE3) after NdeI and XhoI double digestions after pET25 (a) connections of double digestion processing identical with use
In bacterium, recombinant strains EUCI01 is built.
Embodiment 3：UCI01 induced expression and purifying
(1) induced expression：, will according to the condition of culture (LB culture mediums, 37 DEG C of incubated overnights) of normal intestinal bacteria
EUCI01 is cultivated to logarithmic growth early stage, and final concentration of 0.1~10mmol/L IPTG is added respectively
Glycosides (IPTG) is induced, and bacterium solution is transferred in 5ml centrifuge tubes, 4~6 by 18~37 DEG C, 100~350rpm, 4~12h of induced expression
DEG C, 6000~8000r/min centrifuges 2~5min, will precipitate with the Lysis butter of 2~5ml precoolings flushing 1~2 time, again
After centrifugation, precipitation is resuspended with the Lysis butter of 5ml precoolings, be placed in 0~4 DEG C of ultrasonic disruption (3~5s of working time,
Have a rest 6~8s of time, 350~400W of power, 150~300 circulations).Solution after will be broken is transferred to new 5ml and managed offline
In, 4~6 DEG C, 12000~15000r/min centrifuges 20~30min, and supernatant precipitation, SDS-PAGE analysis purposes are collected respectively
The expression of albumen.As a result find, when the IPTG for adding 0.01~0.2mmol/L makees derivant, 18~25 DEG C, 100~
When 200rpm induces 4~8h, destination protein realizes solubility expression.
(2) prepared by protein purification：The EUCI01 cultivated to logarithmic phase is added to 0.01mmol/L IPTG, in 18 DEG C of inductions
After 8h, ultrasonication takes supernatant.The Ni-NTA pillars that supernatant has been balanced with Lysis Buffer in advance in addition on, with 5~6 times
The Wash Buffer of column volume wash foreign protein off, are eluted fusion protein with the Elution Buffer of 2~3 times of column volumes.Will
The protein molecular that solution after elution PBS dialyzed overnight is 5kDa with minimum molecular weight retains centrifugal column in 4~6
DEG C, 6000~8000rpm centrifuges 20~30min, obtains recombinant protein solution.
(3) protein concentration and purity testing：The concentration for determining recombinant protein using coomassie brilliant blue R250 method is 4mg/ml.
Find that recombinant protein only has a purpose band using SDS-PAGE analyses, size is about 7.6kDa, with expected consistent (Fig. 1).
Embodiment 4：Protein active is determined
(1) rejection ability of protease is tested：The buffer solution of the present embodiment reaction delays for 0.2mmol/L Tris-HCl
Fliud flushing, 0.02mmol/L CaCl2The aqueous solution, pH=8.0.The bovine trypsin or pancreas for taking 300 μ l concentration to be 5.0mg/ml are solidifying
Galactase (BBI), adds isometric sample containing different UCI01 concentration (0,160,320,480,640,800,1000 μ g)
Product, 37 DEG C of water-baths are incubated 30min, add 500 μ l, 1.5mg/ml trypsin substrate BAPNA (Sigma, USA) or pancreas curdled milk
Protease substrate Nsuccinyl-Ala-Ala-Pro-Phe-p-nitroanilide (Sigma, USA).37 DEG C of insulation 5min, plus
Enter 600 μ l, concentration 30wt% acetic acid terminating reaction.10000~12000rpm, centrifuges 3~5min, takes supernatant to determine 405nm
Locate light absorption value.As a result show UCI01 to the activity influence of chymotrypsin less, when UCI01 concentration increase to it is most highly concentrated
When spending, 20% suppression is only produced to tryptic activity, but the activity of chymotrypsin can be significantly inhibited, its IC50 is
53nM (Fig. 2).
(2) influences of the pH to UCI01：The reaction buffer of different pH (4,5,6,7,8,9) is prepared, according in embodiment 4
The method of step 2, determines and changes the influence to UCI01 chymotrypsin rejection ability in pH.As a result as shown in Figure 3 a, pH
It is unobvious to UCI01 activity influence, illustrate that UCI01 has wider pH adaptability.
(3) influence of the temperature to UCI01：UCI01 solution is placed in different temperatures (- 20,4,10,20,30,40,50,70
DEG C) 3.5~4h of middle preservation, followed by the method for the step 2 of embodiment 4, determine the change of UCI01 activity under different heat-retaining conditions
Change.As shown in Figure 3 b, temperature is larger to UCI01 activity influence.Gradually reduced with the rising activity of storage temperature.But 70
DEG C preserve 4h after still have 20% activity.
(4) UCI01 bacteriostasis is determined：UCI01 bacteriostasis is tested using filter paper In Vitro Bacteriostasis method.By laboratory
The test strain of preservation, to exponential phase, takes 10 according to the condition of culture incubated overnight of recommendation7Individual test strain even spread
In on corresponding solid medium flat board, the aseptic filter paper piece containing finite concentration UCI01 is carefully attached at media surface,
37 DEG C are just being put culture 18~24 hours, and observation inhibition zone is formed.As a result show UCI01 to tested Gram-negative bacteria such as lung
Scorching klebsiella (klebsiella pneumoniae), Escherichia coli (Escherichia coil), pseudomonas aeruginosa
(Pseudomonas aeruginosa), first paratyphosum Bacterium (B paratyphoid coil) are without obvious fungistatic effect；
There is weaker bacteriostasis (antibacterial circle diameter to Candida albicans (Monilia albican)<3mm)；To gram-positive bacteria
Staphylococcus aureus (staphylococcus aeruginosa) ATCC25923 fungistatic effects preferably (inhibition zone>10mm).
Claims (4)
1. a kind of chymostatin from Urechis uniconctus, it is characterised in that the chymostatin
Protein amino acid sequence as shown in SEQ ID No.1：
MATKEQWPELVGKSGEEAKAAIIAERPELEKVEICNELSPMTMDYRTDRVRIFVNNDGNVVNPPQTG。
2. a kind of chymostatin gene from Urechis uniconctus, it is characterised in that the base sequence of the gene
As shown in SEQ ID No.2.
3. according to the chymostatin gene from Urechis uniconctus described in claim 2, it is characterised in that should
Gene cDNA sequence overall length is 697bp, wherein the 5` non-translational regions containing 80bp, 413bp 3` non-translational regions and polyA tails
Bar, open encoder block is 201bp.
4. a kind of chymostatin as claimed in claim 1 from Urechis uniconctus is used to prepare golden yellow Portugal
The application of grape coccus inhibitor.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| US6294648B1 (en) * | 1999-07-20 | 2001-09-25 | Bayer Corporation | Protein having proteinase inhibitor activity |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6294648B1 (en) * | 1999-07-20 | 2001-09-25 | Bayer Corporation | Protein having proteinase inhibitor activity |
Non-Patent Citations (3)
| Title |
|---|
| Protease Inhibitors from Plants with Antimicrobial Activity;KimJ.Y.等;《International Journal of Molecular Sciences》;20090723;第4.3节 * |
| 家蚕血液中胰凝乳蛋白酶抑制剂研究――品种间的多态型;赵萍等;《蚕学通讯》;20020930(第03期);全文 * |
| 豆类植物蛋白酶抑制剂研究进展;张莉等;《大豆科学》;20060825(第03期);全文 * |
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