CN103848914B - A kind of the Bufrudin polypeptide and preparation method thereof and purposes of tool anticoagulating active - Google Patents
A kind of the Bufrudin polypeptide and preparation method thereof and purposes of tool anticoagulating active Download PDFInfo
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Abstract
The present invention relates to biological field, a kind of Bufrudin polypeptide with anticoagulating active is disclosed and containing the clinical preparation of the polypeptide.The amino acid sequence of Bufrudin polypeptide of the present invention is as shown in SEQ ID No.1.The present invention also provides the preparation methods and purposes of the Bufrudin active peptide.
Description
Technical field
The present invention relates to biotechnology, more particularly to the Bufrudin polypeptide of a kind of tool anticoagulating active and its preparation side
Method and purposes.
Background technology
Hirudin（hirudin）The small molecular weight protein being made of more than 60 a amino acid, relative molecular weight 7kDa
Left and right, is the strongest thrombin inhibitor found so far.Natural hirudin is from Hirudo medicinalis saliva earliest
It is separated in gland, at least 20 various mutations bodies, wherein the hirudin extracted by whole body is known as HV1, by head extraction
Hirudin is known as HV2, and the two amino acid sequence homology is 86%.
The N-terminal of natural hirudin is made of hydrophobic amino acid, the stable conformation of 3 disulfide bond compositions.It is negative that C-terminal is rich in band
The acidic amino acid residue of charge can be combined with the positive charge position of fibrin ferment, and fibrin ferment and fiber egg are prevented with electrostatic interaction
White former recognition site combines.The Pro46-Lys47-Pro48 at intermediate position is combined with the Arg rings of fibrin ferment, be hirudin with
The catalytic site that fibrin ferment combines.In addition, the sulphation of 63 Try residues improves the binding ability of it and fibrin ferment, enhancing
The specificity of hirudin anticoagulant blood effect.
Since doctor's leech feeding habits are more miscellaneous, people gradually turn to attention in the Southeast Asia with mammal for object of mainly sucking blood
Hiruto（Hirudinariamanillensis）, it is desirable to obtain the anticoagulative substance for being more suitable for the mankind.Steiner in 1992 etc.
2 kinds of hirudins are extracted from the hiruto for originate from Manila for the first time.Scacheri in 1993 etc. has isolated 2 from hiruto
Kind hirudin variant（Bufrudin HM1 and HM2）, and its amino acid sequence, cDNA clone and expression are measured, expression product has
Anticoagulating active.Bufrudin（HM1 and HM2）Activity and doctor leech source hirudin（HV1 and HV2）It is similar, but the two is same
Source property is less than 60%, and sulfation sites are not present in the former.
Natural hirudin is extracted due to leech limited source, in career in medicine leech and can not meet clinical wilderness demand, continues to seek
Seek the solution of a large amount of polypeptides of the synthesis with anticoagulating active.
Invention content
The present invention expands Bufrudin gene piece using China Guangxi hiruto as raw material, by the method for genetic recombination
Section, and activity expression is carried out in escherichia expression system, institute of the present invention is measured using the thrombin-antithrombin III complex of Chinese Pharmacopoeia
The anticoagulant active of Bufrudin active peptide is stated, activity is up to 20080ATU/g.
Bufrudin active peptide provided by the invention, amino acid sequence is as shown in SEQ ID No.1.
The present invention also provides the genes for encoding the Bufrudin active peptide.Preferably, the gene nucleotide series are such as
Shown in SEQ ID No.2.
It is a further object to provide the preparation methods of the Bufrudin active peptide.
In a specific embodiment, preparation method of the present invention comprises the steps of：
Step 1：To encode the cDNA of the Bufrudin active peptide as template, design primer carries out PCR amplification, and upstream is drawn
Object is as shown in SEQ ID No.3, and downstream primer is as shown in SEQ IDNo.4；
Step 2：Recombination to construct prokaryotic expression carrier is carried out to amplified production and plasmid；Gained prokaryotic expression carrier is transferred to
Host cell E.coliBL21（DE3）Carry out protein expression；
Step 3：Thalline is collected, precipitation is abandoned in centrifugation, receives supernatant；Into supernatant plus ammonium sulfate reaches ammonium sulfate saturation degree, stands
Supernatant is abandoned in centrifugation afterwards, and precipitation pH 7.4PBS is taken to dissolve；
Step 4：Step 3 gained lysate ultrafiltration is cleaned, film is washed with 0.1M NaCl solutions, collects across liquid；Across liquid
With G-25 column desalinations, collect in chromatography collection of illustrative plates first absorption peak to get.
The template nucleotide sequence is as shown in SEQ ID No.2.The pcr amplification reaction condition is：95℃5min；94
DEG C 30s, 55 DEG C of 60s, 72 DEG C of 45s, 30 times；72℃10min.
Preferably, the plasmid is Pet22b.It is highly preferred that protein expression described in step 2 is induced through IPTG.
Preferably, the ultrafiltration is with after 0.45 μm of acetate fiber membrane filtration, with 30kDa super filter tubes, in 4 DEG C,
4000rpm ultrafiltration.In the specific implementation mode of the present invention, it is that will dissolve supernatant 13000g centrifugation 10min, receives supernatant,
With 0.45 μ μm of acetate fiber membrane filtration supernatant；30kDa MilliPore super filter tubes, in 4 DEG C, 4000rpm ultrafiltration are used afterwards.
The present invention also provides purposes of the Bufrudin active peptide in preparing anticoagulant.
The present invention also provides clinical preparations, including the Bufrudin active peptide and pharmaceutically acceptable auxiliary material.
The clinical preparation includes oral preparation or ejection preparation, and oral preparation is preferably capsule or tablet, particle
Agent；Ejection preparation is preferably injection or freeze drying powder injection.
Inventor is by many experiments discovery, the culture medium after the control group induction of the Escherichia coli containing pET22b plasmids
The supernatant of component and smudge cells is not or almost without activity.And the training of the recombination bacillus coli containing 4 kinds of isomers
The supernatant for supporting base component and smudge cells shows different degrees of anticoagulant active, the amino acid sequence of 4 kinds of isomers,
Compare two-by-two and is only had differences there are one amino acid sequence.Recombination bacillus coli induction containing pET22b-HMb-1 recombinant plasmids
Nutrient media components afterwards show highest anticoagulant active, are 300ATU/mL.And use the thrombin-antithrombin III complex of Chinese Pharmacopoeia
To the Bufrudin active peptide in the nutrient media components（The peaks I）Anticoagulant active be measured, activity be up to 20080ATU/g,
Compared with natural Bufrudin dry powder activity 1300ATU/g.
Zoopery is shown, after white rabbit gavage gives Bufrudin active peptide FNZ of the present invention, before administration and is administered
Different time points are taken a blood sample afterwards, observe the anticoagulant effect of FNZ.The result shows that oral 4mg/kg dosage Bufrudin of the present invention
Active peptide FNZ can significantly extend TT, APTT, but be acted on without extension PT, illustrate that FNZ mainly acts on intrinsic coagulation access,
And exogenous Coagulation pathways are without effect.FNZ is shorter to the extension time of TT, and the range of linearity of dose-effect relationship is relatively narrow, and FNZ pairs
The APTT extension times are longer and the range of linearity is wider.It these results suggest that Bufrudin active peptide FNZ of the present invention in vivo
With anticoagulating active, there is good potential applicability in clinical practice.
Description of the drawings
Fig. 1 is the building process of plasmid vector of the present invention；
Fig. 2 is pET22b-HMb-1 induced expression results；Wherein M：marker（Red arrow is expressed as 7.8kDa）；1：
Full bacterium before pET22b-HMb-1-1 inductions；2：Full bacterium after pET22b-HMb-1-1 inductions；3：It is broken after pET22b-HMb-1-1 inductions
Broken supernatant；4：It is crushed precipitation after pET22b-HMb-1-1 inductions；5：Full bacterium before pET22b-HMb-1-2 inductions；6：pET22b-
Full bacterium after HMb-1-2 inductions；7：It is crushed supernatant after pET22b-HMb-1-2 inductions；8：It is heavy to be crushed after pET22b-HMb-1-2 inductions
It forms sediment.
Fig. 3 is that Bufrudin G-25 column desalting processings chromatograph collection of illustrative plates；
When Fig. 4 is oral various dose FNZ extended to rabbit PT-effect curve；
When Fig. 5 is oral various dose FNZ extended to rabbit APTT-effect curve；
When Fig. 6 is oral various dose FNZ extended to rabbit TT-effect curve.
Specific implementation mode
The invention discloses Bufrudin polypeptide of a kind of anticoagulating active and preparation method thereof and purposes, people in the art
Member can use for reference present disclosure, be suitably modified technological parameter realization.In particular, it should be pointed out that all similar substitutions and modifications
Apparent to those skilled in the art, they are considered as including within the present invention.The product of the present invention, method
And application is described by preferred embodiment, related personnel can obviously not depart from the content of present invention, spirit and model
Enclose it is interior to method described herein and application be modified or suitably change and combine, to realize and apply the technology of the present invention.
In order to make those skilled in the art more fully understand technical scheme of the present invention, with reference to specific embodiment pair
The present invention is described in further detail.
Embodiment 1：RT-PCR expands Bufrudin genetic fragment
Guangxi hiruto live body is taken, hiruto head tissue is cut（About 1-2cm）, using TRIzol（Invitrogen）
Method extracts the total serum IgE of tissue.Using the total serum IgE of extraction as template, reverse transcription is carried out first and obtains cDNA.Design sense primer Hmgx
up（As shown in SEQ ID No.3）（ATGTTCTCTCTCAAGTTGTTCG）With downstream primer Hmgxdn（Such as SEQ ID No.4 institutes
Show）（TTAATTCAATATATCTTCATCTGGG）, using cDNA as template, PCR amplification Bufrudin gene, reaction condition is：95
℃5min；94 DEG C of 30s, 55 DEG C of 60s, 72 DEG C of 45s（30 times）；72℃10min.Amplified production is as shown in SEQ ID No.2.
Embodiment 2：Expression plasmid is built
1 gained gene of embodiment is transformed, upstream adds initiation codon ATG, and carries Msc I recognition sites,
Downstream carries Xho I recognition sites.In order to avoid recombinantly expressing the active influence of sequence pair Bufrudin introduced in the process, institute
Not add any label in construction of expression vector, while removing the signal peptide series of Bufrudin itself.Genetic fragment is adopted
With restriction enzyme Msc I and Xho I double digestions, same plasmid Pet22b is carried out double using identical restriction enzyme
Digestion, T4 ligases connect linearization plasmid and genetic fragment, obtain pet22b-hmb-1, vector construction process such as Fig. 1.
Embodiment 3：Protein expression and purification and detection
3.1 protein expression
The Bufrudin prokaryotic expression carrier pET22b-HM conversions BL21 built（DE3）, and carry out induced expression.It lures
Conducting bar part is 0.5mM IPTG, 37 DEG C of 5h.The electrophoresis result See Figure 2 of induced expression, is showed no apparent purpose band after induction.
Determination of activity finds that the activity of nutrient media components is 300ATU/mL.The anticoagulant active of nutrient media components is than on smudge cells
The anticoagulant active of clear liquid is high, thus it is speculated that possible Bufrudin molecular weight is smaller, is largely secreted into culture medium.
By RT-PCR product gel extractions, carrier T is connected, the different transformant of picking is sequenced, and is screened different different
Structure body.10 transformants of picking, finally obtain 4 kinds of isomers（HMb-1, HMb-2, HMb-3, HMb-4）.Compare this 4 kinds of isomeries
The amino acid sequence of body, it can be seen that compare only had differences there are one amino acid sequence two-by-two.
With reference to embodiment 1, respectively using the gene of 4 kinds of isomers as template, PCR amplification is carried out, reaction condition is 95 DEG C
5min；94 DEG C of 30s, 55 DEG C of 60s, 72 DEG C of 45s（30 times)；72℃10min.Carrier pET22b is connected after digestion, obtains recon
pET22b-HMb-1、pET22b-HMb-2、pET22b-HMb-3、pET22b-HMb-4。
Since the expression quantity of recombination Bufrudin is relatively low, can not be arrived by electrophoresis detection, and consider naturally
The content of Bufrudin is also relatively fewer, and the phenomenon that activity is not low, so the different component to recombinant bacterium carries out fibrin ferment
Anticoagulating activity is verified, to determine whether Bufrudin successful expression and positions recombinant polypeptide.
Sample be containing pET22b plasmids and containing pET22b-HMb-1, pET22b-HMb-2, pET22b-HMb-3,
The supernatant of the front and back nutrient media components and smudge cells of recombination bacillus coli induction of pET22b-HMb-4 recombinant plasmids.Recombination
The determination of activity of Bufrudin is measured using the thrombin-antithrombin III complex of Chinese Pharmacopoeia.Natural Bufrudin dry powder activity is
The activity data of 1300ATU/g, recombinant bacterium are shown in Table 1.
Table 1 recombinates the verification of Bufrudin fibrin ferment anticoagulating activity
Preliminary qualitative test finds the nutrient media components after the control group induction of the Escherichia coli containing pET22b plasmids
Supernatant with smudge cells is not or almost without activity.And the culture medium of the recombination bacillus coli containing 4 kinds of isomers
The supernatant of component and smudge cells shows different degrees of anticoagulant active.
Conclusion：The anticoagulant active that different isomers is showed also has very big difference.Contain pET22b-HMb-1 weights
Nutrient media components after the recombination bacillus coli induction of group plasmid show very high anticoagulant active, are 300ATU/mL.
3.2pET22b-HMb-1 recombinates Bufrudin purifying
A. thalline culture
3L LB culture mediums, inoculum concentration 5%, 37 DEG C, 0.5Mm IPTG inductions are added when OD6000.6 for 160rpm cultures,
Continue to cultivate 6h receipts bacterium.4000rpm 4 DEG C, centrifuges 1h, abandons precipitation, receives supernatant.
B. viability examination
In culture supernatant can qualitative detection to anticoagulant active.
C. ammonium sulfate precipitation
It is slowly added to ammonium sulfate solids after crushing into culture supernatant, reaches the final concentration of 65%-80% of ammonium sulfate（That is 65-
80g/100ml）, preferably 70%.4 DEG C of gentle agitations stand 1h.8000rpm, centrifuges 30min, abandons supernatant, take precipitation by 4 DEG C.With
pH 7.4PB S（20mM PB, 150mM NaCl）Dissolving, final volume 30mL.
D. ultrafiltration cleans
Dissolving supernatant is centrifuged into 10min with 13000g, supernatant is received, with 0.45 μm of acetate fiber membrane filtration supernatant.Afterwards
With 30kDa MilliPore super filter tubes, in 4 DEG C, 4000rpm ultrafiltration.It is primary that film is washed with 0.1M NaCl, is collected across liquid.
F.G-25 column desalinations
Across liquid G-25 column desalinations, chromatographs in collection of illustrative plates and occur 3 absorption peaks altogether, see Fig. 3, collect respectively, measured concentration,
It the results are shown in Table 2.The analysis of III peak is salt peak.
2 Bufrudin purification process sample record of table
G. it is freeze-dried
By I peaks sample G-25, II peaks G-25 pre-freeze 12h in -80 DEG C of refrigerators is lyophilized sample with freeze dryer, is tied after 48h
Beam is lyophilized, and freeze-dried powder is dispensed respectively, 4 DEG C save backup.
H. determination of activity：It is measured using the thrombin-antithrombin III complex of Chinese Pharmacopoeia, it is as a result as follows：I peak activities：20080ATU/
g；II peak activities：≤100ATU/g
The peaks I ingredient is analyzed, is polypeptide, amino acid sequence is as shown in SEQ ID No.1.
Embodiment 4：Anti-freezing experimental study in polypeptide rabbit body of the present invention
After this experiment large ear rabbit gavage gives FNZ, different time points are taken a blood sample before administration and after administration, observe FNZ's
Anticoagulant effect, to be exploitation FNZ oral administration preparation based theoreticals.
1 experiment material
1.1 test sample
1.1.1 title：FNZ（Bufrudin active peptide, amino acid sequence is as shown in SEQ ID No.1）.Test sample number：
TN-1203。
1.1.2 physicochemical property：White powder.
1.1.3 main component and content：External antithrombin activity 21000ATU/g.
1.1.4 intend clinical indication：Lipid-loweringing, anti-inflammatory, anti-freezing.
1.1.5 source and lot number：Shijiazhuang Yiling Pharmaceutical Co., Ltd, 20120101.
1.1.6 test sample is taken care of：Freezen protective.
1.2 instrument medicines and main agents
Partial thromboplastin time（APTT）, prothrombin time（PT）And thrombin time（TT）Kit：
French Stago companies.
Sodium citrate：Beijing Chemical Plant.
1.3 experimental system
1.3.1 animal germline：Large ear rabbit.
1.3.2 animal rank：Regular grade.
1.3.3 Animal Sex and quantity：15, male.
1.3.4 animal age：About 5 ~ 6 monthly ages.
1.3.5 the weight of animals：1.0kg.
1.3.6 animal origin：It is purchased from Hebei province's Experimental Animal Center, credit number：SCXK（Ji）2008-1-003 is qualified
Card number：1207107.
1.3.7 rearing conditions：Animal feeding is in Hebei province combination of Chinese tradiational and Western medicine Medicine Research Academy new drug evaluation center.Illumination 12
Hour/day, 16~26 DEG C of temperature, relative humidity 40~70%, 20~24 DEG C of observed temperature survey humidity 40~68%.Experiment is set
Apply interior continuous ventilating, timing sterilization.White rabbit cage, 1/cage.
1.3.8 quarantine procedures：3 days animal quarantine phases newly received.Quarantine observe drinking water for animals, ingest and health
Situation, and whether there is disease and intimations of mortality.
1.3.9 feed：Experimental animal full-valence pellet feed.
1.3.10 drinking-water：The filling drinking bottle of water is commonly used, is freely drunk for animal.It is daily to rinse drinking bottle and change water one
It is secondary.
1.3.11 mark：Cage card marks.
2 experimental methods
2.1 experimental design foundations
Using standard：What State Food and Drug Administration promulgated《Chinese medicine registration management is replenished regulations》、《Chinese medicine, day
Right drug legislation classification and declaration material requirement》With《Drug non-clinical research quality management practices》.
The measuring principle of anti-freezing method is can to inhibit Coagulation test caused by fibrin ferment according to Bufrudin, to make portion
Divide the factor I time（APTT）, prothrombin time（PT）And thrombin time（TT）Extend.Wherein antithrombase
（Antithrombin, AT）It is internal most important anticoagulant substances, 80% anticoagulation is completed by AT, AT shortages or low
Under inactivation of thrombin can be caused to reduce, coagulation function is hyperfunction.The effect of AT neutralization activity coagulation factors with blood coagulation activity carry out and
Enhancing, therefore the consumption level of AT is the important indicator for reflecting fibrin ferment and generating.
Maxima solubility experiment shows FNZ maxima solubilities be 166.7mg/ml, in this experiment height, middle dose group FNZ with
400mg/ml, 200mg/ml, which are suspended, to be administered.
2.2 dosage and grouping
Rabbit is randomly divided into 3 groups by weight.
Anti-freezing experiment grouping and dosage in table 3FNZ rabbit bodies
2.4 medication
Gastric infusion.
2.4 test samples are prepared and are preserved
The preparation of FNZ sample solutions：Precision weighs the solution that FNZ N.S. prepare 400mg/ml, as FNZ high doses；
The solution for preparing 200mg/ml, as FNZ middle dosages；The solution for preparing 100mg/ml, as FNZ low dosages.
2.5 test samples are given
Liquid is extracted with syringe and carries out gastric infusion, and administered volume is 10ml/kg.
2.6 experimental procedure
Rabbit is randomly divided into 3 groups, i.e. FNZ high dose groups, FNZ middle dose groups, FNZ low dose groups by weight, is filled respectively
Stomach is administered once.1h, 2h, 3h, 4h, 6h, 8h auricular vein take blood 2ml before administration and after administration, anti-with 3.8% sodium citrate
Solidifying, separated plasma refrigerates to be measured.
2.7 Testing index
Using STA Compact (CT) full-automatic blood coagulation analyzer, measures rabbit and part thrombokinase after preceding and administration is administered
The Clotogen time（APTT）, prothrombin time（PT）And thrombin time（TT）.
2.8 key instrument
SL2001N electronic balances：Shanghai Min Qiao precision scientific instruments Services Co., Ltd.
BT224S Sai Duolisi analytical precision balances：Sai Duolisi scientific instrument（Beijing）Co., Ltd.
STA Compact (CT) full-automatic blood coagulation analyzer：French Stago companies.
2.9 statistical method
Experimental data carries out analyzing processing, statistical result mean ± standard deviation using SPSS11.5 statistical softwares
It indicates, group difference, which compares, carries out One-Way ANOVA Dunnettt inspections.
3 results
The result shows that FNZ（Bufrudin active peptide, amino acid sequence is as shown in SEQ ID No.1）Can significantly it extend
TT, APTT, but to PT without effect is obviously prolonged, illustrate that FNZ mainly acts on intrinsic coagulation access, and exogenous blood coagulation is logical
Road is without effect.FNZ is shorter to the extension time of TT, and the range of linearity of dose-effect relationship is relatively narrow, and FNZ is longer to the APTT extension times
And the range of linearity is wider.Since limited number of animals is administered, high dose group APTT and TT value standard deviation is larger, statistical result showed
No difference of science of statistics removes 1h and more significant difference before administration after abnormal high outlier statistical result showed administration（p
□0.05）.It the results are shown in Table 4 and Fig. 4-6.
Table 4 takes orally influence of the different time to rabbit PT, APTT, TT after FNZ
Note：* the p compared with before administration<0.05
4 conclusions
FNZ does not show anticoagulant effect with 1g/kg, 2g/kg dosage.FNZ is shown with 4g/kg dosages
Anticoagulant effect is shown.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Claims (14)
1. a kind of Bufrudin active peptide, amino acid sequence is as shown in SEQ ID No.1.
2. encoding the gene of Bufrudin active peptide described in claim 1.
3. gene according to claim 2, which is characterized in that its nucleotide sequence is as shown in SEQ ID No.2.
4. the preparation method of Bufrudin active peptide described in claim 1.
5. preparation method according to claim 4, comprises the steps of：
Step 1：To encode the cDNA of Bufrudin active peptide described in claim 1 as template；Design primer carries out PCR amplification,
Sense primer is as shown in SEQ ID No.3, and downstream primer is as shown in SEQ ID No.4；
Step 2：Recombination to construct prokaryotic expression carrier is carried out to amplified production and plasmid；Gained prokaryotic expression carrier is transferred to host
CellE.coliBL21（DE3）Carry out protein expression；
Step 3：Thalline is collected, precipitation is abandoned in centrifugation, receives supernatant；Into supernatant plus ammonium sulfate reaches ammonium sulfate concentrations 65-80%, stands
Supernatant is abandoned in centrifugation afterwards, and 7.4 PBS of precipitation pH is taken to dissolve；
Step 4：Step 3 gained lysate ultrafiltration is cleaned, film is washed with 0.1M NaCl solutions, collects across liquid；Across liquid G-
25 column desalinations, collect chromatography collection of illustrative plates in first absorption peak to get.
6. preparation method according to claim 5, which is characterized in that the nucleotide sequence of the template such as SEQ ID
Shown in No.2.
7. preparation method according to claim 5, which is characterized in that the plasmid is pET22b.
8. preparation method according to claim 5, which is characterized in that the protein expression is induced through IPTG.
9. preparation method according to claim 5, which is characterized in that the pcr amplification reaction condition is：95℃ 5min；
94 DEG C of 30 s, 55 DEG C of 60 s, 72 DEG C of 45 s, 30 times；72℃ 10min.
10. preparation method according to claim 5, which is characterized in that the ultrafiltration is with 0.45 μm of acetate fiber filter membrane
After filtering, with 30 kDa super filter tubes, ultrafiltration is carried out in 4 DEG C, 4000 rpm.
11. clinical preparation, including Bufrudin active peptide described in claim 1 and pharmaceutically acceptable auxiliary material.
12. clinical preparation according to claim 11, which is characterized in that it is oral preparation or ejection preparation.
13. clinical preparation according to claim 12, which is characterized in that the oral preparation be capsule or tablet,
Granula；The ejection preparation is injection or freeze drying powder injection.
14. purposes of the Bufrudin active peptide in preparing anticoagulant described in claim 1.
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| CN105175510B (en) * | 2015-10-22 | 2018-06-01 | 江苏大学 | The polypeptide with anticoagulant active of display technique of bacteriophage screening |
| CN108866205A (en) * | 2018-07-25 | 2018-11-23 | 中国食品药品检定研究院 | Identify the specific primer of hiruto based on molecular biology method |
| CN111454352B (en) * | 2020-03-05 | 2021-07-23 | 中国科学院昆明动物研究所 | Active protein HMEI-A, encoding gene of active protein HMEI-A and application |
| CN115572329B (en) * | 2021-06-21 | 2024-02-06 | 王大勇 | Poecilobdella manillensis gene recombinant hirudin with slower activity enhancement metabolism and preparation method thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1055010A (en) * | 1990-03-22 | 1991-10-02 | 联合电化学工业股份有限公司 | The secretory product of hirudin derivatives |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1055010A (en) * | 1990-03-22 | 1991-10-02 | 联合电化学工业股份有限公司 | The secretory product of hirudin derivatives |
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| Novel hirudin variants from the leech Hirudinaria manillensis amino acid sequence，cDNA cloning and genomic organization;Emanuela Scacheri et al.;《European Journal of Biochemistry》;19931231;第214卷;第297页左栏第3段-第298页左栏第1段，第299页左栏第2段，图1 * |
| 在大肠杆菌中信号肽介导水蛭素III分泌表达研究;谭树华等;《第四届中国新医药博士论坛论文集》;19991101;第438-442页 * |
| 广西菲牛蛭水蛭素基因cDNA的克隆与序列分析;黎渊弘;《中国生化药物杂志》;20111231;第32卷(第2期);第92页右栏第1段，第92-93页1-2.5，第94页3.4 * |
| 菲牛蛭素体内外抗凝实验研究;黄爱民等;《广西医科大学学报》;20060228;第23卷(第1期);第30-32页 * |
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| CN103848914A (en) | 2014-06-11 |
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