CN1370833A - Pit viper venom batroxobin gene cDNA sequence of Dalian Snake Island in Liaoning Prov. of China and its cloning - Google Patents
Pit viper venom batroxobin gene cDNA sequence of Dalian Snake Island in Liaoning Prov. of China and its cloning Download PDFInfo
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Abstract
The present invention relates to the field of bioengineering, and provides venom batroxibin gene cDNA sequence and corresponding amino acid sequence of Gloydius Shedaoensis is Dalian Liaoning provices; Pichia pastoris secretion type expression vector pPIC9K containing the gene and strain containing the vector; and colibacillus JM109/pPIC9K and Pichia pastors cell strain GS115/pPIC9K. The present invention may be used in gene method to recombine batroxobin and to diagnose and treat thrombus diseases.
Description
The invention belongs to bioengineering field.Specially refer to the acquisition and the cloning process thereof of the gene complete sequence of coding Thrombin-like enzyme in the agkistrodon shedaoensis venom of Dalian.
On our earth, more than 200 kind of poisonous snake approximately living.They are divided into 4 big classes usually: 1) Hydrophiae, 2) Elapidae, 3) Viperdae and 4) be rich in trace in the Crotalidae. snake venom but the kind enzyme of proteolysis of kind more than 50 nearly.Up to the present, the different proteolytic ferment of nearly kind more than 150 is separated, and wherein has the structure of 1/3rd proteolytic ferment definite.These enzyme of proteolysis are divided into two types according to its hydrolysis mechanism usually: silk-protein enzyme and metalloprotease.Thrombin-like enzyme is a kind of as the silk-protein enzyme, mainly is distributed in the venom of Viperdae and Crotalidae.Different with the mechanism of action of zymoplasm is, Thrombin-like enzyme only discharges the A chain in the Fibrinogen, thereby and zymoplasm is that Fibrinogen is converted into is fibrinous by A chain and B chain are all discharged.Because Thrombin-like enzyme does not activate factor XIII, therefore formed fibrin blood clot is a non-crosslinked, and is easy to be the blood plasma degraded, thereby eliminates the thrombus in the microcirculation system rapidly.This shows that Thrombin-like enzyme can be used as a kind of suitable perhaps better alternative medicine, as treatment thrombotic diseases such as heparin.Why Thrombin-like enzyme is studied widely, and its reason just is that it can be used for treating the medical science prospect of cardiovascular disorder and thrombus disease.
Yet though the separation and Extraction Thrombin-like enzyme is used for the history of clinical existing many decades from natural snake venom, its use still is subjected to some restrictions.These restrictions mainly are to come from following three aspects: 1) immune response appears in patient, and tracing it to its cause mainly is that the resulting product of i.e. separation and purification is impure owing to contain the impurity of trace in the commodity Thrombin-like enzyme preparation; 2) resource-constrained and to be present in Thrombin-like enzyme content in the natural poisonous snake venom few thereby limited output; 3) separation and purification difficulty makes that production cost is too high owing to there is the biological activity enzyme more than 50 kinds in the snake venom, the content of each enzyme all seldom and similar performance, the unusual difficulty of separation and purification thus.
As everyone knows, produce protein with engineered method and have many remarkable advantages, as: the microbial fermentation cost is low; Expression amount often will exceed 10-100 times of natural expression amount, and it is easy that separation and purification becomes; Other active protein expression amounts except that target protein matter are low.This shows, thrombase-like gene is cloned in the microbe, particularly in eukaryotic cell, express the production Thrombin-like enzyme and will overcome above-mentioned restriction, make Thrombin-like enzyme be used for the clinical possibility that becomes as antithrombotic reagent greater amount ground by engineered method.
The objective of the invention is to obtain in the agkistrodon shedaoensis venom of Dalian, Liaoning Province thrombase-like gene cDNA sequence first and be cloned in the expression vector by bionic method, make that producing the recombination classes zymoplasm with gene engineering method becomes possibility, finally reaches the purpose of diagnosis and treatment and prevention thrombus disease.
The present invention carries out homology analysis research by the thrombase-like gene to other poisonous snake poison gland of known nucleotide sequence, according to synthetic four oligonucleotide primers of the high zone design of homology, the Dalian total RNA of agkistrodon shedaoensis poison gland with extraction is a template, the synthetic amplification of RT-PCR method thrombase-like gene sequence wherein, inboard primer with design is a primer, carry out secondary PCR, and to this PCR product order-checking.The gene of these references is: acutin is from Agkistrodon acutus, flavoxobin is from Trimeresurus flavoviridis (habu snake, crotalinae) crotalinae, ancrod is from calloselasma rhodostoma, batroxobin from Bothrios atrix andCalobin from agkistrodon caliginosus.Obtained the complete sequence of thrombase-like gene cDNA in Dalian agkistrodon shedaoensis (Gloydius Shedaoensis) venom thus.CDNA complete sequence according to Thrombin-like enzyme, the design primer, the ripe thrombase-like gene of thrombase-like gene coding is cloned among the expression vector pPIC9K, and by the thermal shock method this carrier is converted in the escherichia coli jm109 competent cell, by electric shocking method this expression vector is incorporated in the genome of pichia spp cell strain GS115.
( Gloydius Shedaoensis ) cDNA：ATGGTGCTGATCAGAGTGCTAGCAAACCTTCTGATACTACAGCTGTCTTACGCACAAAAGTCTTCTGAACTGATCATTGGAGGTGATGAATGTAACATAAATGAACATCGGTTCCTTGTAGCCTTATACACCTCTAGATCTAGGAGGTTTTATTGCGGTGGGACTTTGATCAACCAGGAATGGGTGCTCACCGCTGCACACTGCGACAGGAAAAATATCCGGATAAAGCTTGGTATGCATAGCGAAAAGGTACCAAATGAGGATGCAGAGACAAGAGTCCCAAAGGAGAAGTTCTTTTGTCTCAGTAGCAAAACCTACACCAAATGGGACAAGGACATCATGTTGATGAGCTGAAAAGACCTGTTAACAACAGTACACACATCGCGCCTGTCAGCTTGCCTTCCAACCCTCCCAGTGTGGACTCAGTTTGCCGTGTTATGGGATGGGGTACAATCACATCTCCTCAAGAGACTTATCCCGATGTCCCCCATTGTGCTAACATTAACATACTTGATTATGAGGTGTGCAAGCAGCTCACGGAGGGTTGCCAGCAACAAGCAGAACATTGTGTGCAGGTATCCTGGGAGGCAAAGATTCATGTAAGGGTGACTCTGGGGGACCCCTCATCTGTAAGTCTTAAGCCTGGTGTCTACACCAAGGTCTTCGATTATACTGAGTGGATCCAGAGCAATTGCAGGAAATACAGATGCAACCTGCCCCCCATGAaaacttttgaaaagttaagaggagaaaatgtaacatattactacatctcttcctatccctaaccatatccaactacattggaatctattcccaggcagtaagcttttttaagactcaaataggactgcctttgaagtaagaaatgctcaaaatagtgctgcagggatcatgtcccatttaatttcagtataaaacaatctcagttaaatggaggcctgttttagggtgaggtgcaatattttctgactctaaaatgcaccattccaaatattttaacctctgaatatctttccatttctgtccacttctgggacagcggggtccttgatgctctttgagcttgtcttcttgcagacgtttcattacccagctaggtaacatcatcagtgctagaatattctcttctattggtacttctgtggcatttacaatacgctcatatggagtcatgcagtcaccctacaaacatatccatatacctggtcccactggtgcctaaaaaggaccccagattaacccgcactttccaatcctaaatagaatcttttgagaatcgtgttttcatgtaaattctcaggtatccacagc
Tgcataaatcggcaaaaaaaaaaaaaaaaaaa
Thus, thrombase-like gene includes the Nucleotide of 1.4kb in Dalian, Liaoning Province agkistrodon shedaoensis (Gloydius Shedaoensis) venom.The initiator codon of translation is ATG (Nucleotide 1-3), and terminator codon is TGA (Nucleotide 781-783).16 poly VITAMIN B4 sequence (poly (A) of non-coding region distance at 3 ' end
+) on the position of 15 Nucleotide in upstream, exist to add poly (A)
+AATAAAA box (Nucleotide 1373-1378) signal.
According to its nucleotide sequence, it is as follows to derive the coded prlmary structure of protein of thrombase-like gene in Dalian, Liaoning Province agkistrodon shedaoensis (Gloydius Shedaoensis) venom:
Its open reading frame of IIGGDECNINEHRFLVALYTSRSRRFYCGGTLINQEWVLTAAHCDRKNIRIKLGMH SEKVPNEDAETRVPKEKFFCLSSKTYTKWDKDIMLMRLKRPVNNSTHIAPVSLPSN PPSVDSVCRVMGWGTITSPQETYPDVPHCANINILDYEVCQAAHGGLPATSRTLCA GILKGGKDSCKGDSGGPLICNGQFQGIASWGAHPCGQSLKPGVYTKVFDYTEWIQS IIAGNTDATCPPE comprises 780 Nucleotide, 260 amino acid of encoding.Wherein, 24 amino acid (adding the square frame part) of front are signal peptide sequence, Thrombin-like enzyme formed the former precursor of Thrombin-like enzyme earlier in this explanation Dalian, Liaoning Province agkistrodon shedaoensis (GloydiusShedaoensis) venom before forming mature protein, was restriction enzyme site after the A position wherein.Compare with the Thrombin-like enzyme in other sources, this sequence has the conservative property of height.The complete sequence that contains ripe thrombase-like gene among the expression vector pPIC9K.
Based on above narration, significant advantage of the present invention and effect are: 1. extract first, check order and clone thrombase-like gene in Dalian, Liaoning Province agkistrodon shedaoensis (Gloydius Shedaoensis) venom; 2. constructed expression vector and bacterial strain can be used for gene engineering method and produce the medicine of Thrombin-like enzyme as treatment and pre-preventing thrombosis.Because of the Thrombin-like enzyme of this coded by said gene does not activate factor XIII, therefore formed fibrin blood clot is a non-crosslinked, and to be easy to be the blood plasma degraded, thereby eliminates the thrombus in the microcirculation system rapidly.So Thrombin-like enzyme can be used as a kind of suitable perhaps better alternative medicine and is used for the treatment of cardiovascular disorder and thrombus disease; Because produce the Thrombin-like enzyme medicine, overcome the restriction of originating again with engineered method, the restriction of content, limiting factor that patient's immune side reaction and separation and purification cost are too high or the like, its commercial value is inestimable.Simultaneously, Chinese and worldwide aging trend makes paralysis have patient's number of cardiovascular disorder and thrombus disease to increase, and therefore producing the Thrombin-like enzyme medicine with gene engineering method has far-reaching social effect again.
Below be described in detail most preferred embodiment of the present invention:
Embodiment 1:
In Dalian agkistrodon shedaoensis (Gloydius Shedaoensis) venom acquisition of thrombase-like gene 1., the extraction of the total RNA of pallas pit viper poison gland
Get the pallas pit viper alive of prey on autumn phase, clamp its head, make it bite cleaning with the cleaning tweezers
Watch-glass to venom flows out, and puts back in the cage.Feeding not.After five days, chat method toxin expelling again before complying with
Once its head is cut with the sterilization scissors, taken out poison gland, put in the liquid nitrogen rapidly and grind.
Extract total RNA and adopt TRIZOL test kit (GIBICOL company, the U.S.).All operations is all pressed
The operational guidance that provides according to test kit production firm carries out.2., the synthetic amplification of RT-PCR method pallas pit viper poison gland thrombase-like gene
The synthetic amplification of RT-PCR method pallas pit viper poison gland thrombase-like gene adopts RT-PCR test kit------5-full RACE Core Set (TAKARA company, Japan).RT reaction conditions following (20 μ l system):
The total RNA+8.5 μ of 1 μ l l RNA free H
2O
↓
65℃，10min
↓
Place on ice
↓
Add the RT reaction solution:
MgCl
2 4μl
RNase?Inhibitor 0.5μl
AMV?Reverse?Transcriptase 1μl
10X?RNA?PCR?Buffer 2μl
d?NTP 2μl
Oligo?dT-adapter?Primer 1μl
↓
42℃，30min
↓
95 ℃, the 5minPCR reaction:
According to homology, the synthetic 5 end oligonucleotide degenerate primer TLE-F1 of design, 3 end primers are that TLE-M13M4 is provided by test kit.Primer is as follows: TLE-F15 ' ATGGTGCTGATCAGMGTG 3 ' (M=A+T) TLE-M13 M4 5 ' CGCCAGGGTTTTCCCAGTCACGAC 3 ' PCR reactant is composed as follows:
MgCl
2 10μl
10X?LA?Taq?Buffer 10μl
Sterile distilled water 66.5 μ l
cDNA 1μl
dNTP 10μl
TLE-F1(20pmol) 1μl
TLE-M13M4(20pmol) 1μl
LA?Taq?ploymerase(125U)?0.5μl
100μl
The concrete reaction conditions of PCR is as follows:
72℃ 10min
4 ℃ of 5min 3., class in secondary PCR amplification Dalian agkistrodon shedaoensis (Gloydius Shedaoensis) venom
Batroxobin gene
The step of getting 10 times of 1 μ l dilutions 2. in the RT-PCR product as template, with according to homology
The inboard primer TLE-F2 step of replacing of design 2. TLE-F1 carries out pcr amplification.The PCR reactant
Composed as follows:
10X?Ex?Taq?Buffer 5μl
Sterile distilled water 37.75 μ l
1/10?cDNA 1μl
dNTP(2.5mmol/L) 4μl
TLE-F2(20pmol) 1μl
TLE-M13M4(20pmol) 1μl
Ex?Taq?ploymerase(125U)?0.25μl
The concrete reaction conditions of 50 μ lPCR is as follows:
94℃ 2min
72℃ 1min
4 ℃ of 5min 4., order-checking is with the preparation of template
With 200 μ l secondary PCR products electrophoresis in 1% sepharose, under ultraviolet lamp with special
Amplified band cuts out, and SUPREC packs into
TMIn-01 centrifuge tube (Millipore company, the U.S.),
Placed 15 minutes for-80 ℃, after dissolving under the room temperature, in 10, the centrifugal 5min of 000g.To flow out
The liquid ethanol sedimentation, with 20 μ l sterile distilled water dissolving DNAs ,-20 ℃ of preservations are standby.5., the sequential analysis of secondary PCR product
The secondary PCR product that reclaims is done sequencing reaction with TLE-F2, at ABI377 type automatic sequencer
On carry out sequential analysis (all examining orders all entrust TAKARA Dalian company to finish).First
Design on the result that subsequence is analyzed (with program SEQUENCE 3.0) sequencing primer TLE-F3,
Do sequencing reaction with TLE-F3 again and surveyed remaining sequence.The sequence of primer TLE-F2 and TLE-F3 is as follows: thrombase-like gene cDNA in TLE-F2 5 ' GTCATTGGAGGTGATGAATG 3 ' TLE-F3 5 ' CATCCCTGTGGTCAAAGTCT 3 ' 6., Dalian agkistrodon shedaoensis (Gloydius Shedaoensis) venom
Determining of complete sequence
The 2. middle RT-PCR product of step of getting 10 times of 1 μ l dilutions is used primer TLE-F1 and TLE-R1 respectively as template; TLE-F1 and TLE-R3; TLE-F2 and TLE-M13M4 carry out pcr amplification.Corresponding PCR product is called after template I respectively, template II, template III.The sequence of reverse primer is as follows: TLE-R1 5 ' TTTGGGACTCTTGTTTGTGC 3 ' TLE-R3 5 ' CCATAACACGGCAAACTAAG 3 ' TLR-R4 5 ' CTTACTGCCTGGGAATAGAT 3 ' PCR reactant is composed as follows:
10X?Ex?Taq?Buffer 10μl
Sterile distilled water 76.5 μ l
1/10?cDNA 1μl
dNTP(2.5mmol/L) 8μl
TLE-F(20pmol) 2μl
TLE-M13M4/R(20pmol) 2μl
Ex?Taq?ploymerase(125U) 0.5μl
100μl
The concrete reaction conditions of PCR is as follows:
94℃ 2min
72℃ 1min
4℃ 5min
Get 5 μ l PCR products, detect through 3% agarose gel electrophoresis.Reclaim the PCR product as the order-checking template.Method is 4. identical with step.React order-checking with template I-primer TLE-R1, template II-primer TLE-R3, template III-primer TLE-R4 respectively.Thus, obtain thrombase-like gene cDNA complete sequence in Dalian agkistrodon shedaoensis (GloydiusShedaoensis) venom.
Embodiment 2:
Clone and the transformed into escherichia coli JM109 of thrombase-like gene in expression vector pPIC9K in Dalian agkistrodon shedaoensis (Gloydius Shedaoensis) venom
1. PCR synthesizes at 5 ' end and 3 ' and holds the ripe thrombase-like gene contain two restriction enzyme sites respectively
According to 6. sequencing result of step among the embodiment 1, design the ripe thrombase-like genes of two primer amplifications.Wherein 5 ' of forward primer LIGF1 end includes the Ste13 restriction enzyme site in expression vector pPIC9k α-factor signal sequence; 5 ' the end of reverse primer TDLR1 includes the restriction enzyme site of EcoR1.With step among the embodiment 1 2. the RT-PCR product produce at 5 ' end and 3 ' by PCR reaction and to hold the ripe thrombase-like gene that contains two restriction enzyme sites respectively as template.Article two, the sequence of primer is as follows: LIGF:5 ' GAAAAGAGAGGCTGAAGCTATCATTGGAGGTGATGAATG3 ' TDLR1:5 ' CCGAATTCTTATCATGGGGGGCAGGTTGCA3 ' PCR reactant is composed as follows:
10X?Pyrobest?Buffer 5μl
Sterile distilled water 37.75 μ l
1/10?cDNA 1μl
dNTP(2.5mmol/L) 4μl
LIGF1(10pmol) 1μl
TDLR1(10pmol) 1μl
Pyrobest?ploymerase(125U)?0.25μl
50μl
The concrete reaction conditions of PCR is as follows:
94℃ 1min
4℃ 5min
2. PCR method is synthesized the α-factor signal sequence that contains ripe thrombase-like gene 5 ' end parts sequence
According to expression vector pPIC9k α-factor signal nucleotide sequence and ripe thrombase-like gene sequence, design two primers.Wherein forward primer LIGF2 is α-factor signal 5 ' terminal sequence, and reverse primer LIGR1 is that 5 ' end carries ripe thrombase-like gene 5 ' terminal sequence and 3 ' end contains the sequence of α-factor signal sequence Ste13 restriction enzyme site.Article two, the sequence of primer is as follows: LIGF2:5 ' CAAACGATGAGATTTCCTTC3 ' LIGR1:5 ' ATTCATCACCTCCAATGATAGCTTCAGCCTCTCTTTTC3 ' PCR reactant is composed as follows:
10X?Pyrobest?Buffer 5μl
Sterile distilled water 37.75 μ l
pPIC9K(1ng/μl) 1μl
dNTP(2.5mmol/L) 4μl
LIGF2(10pmol) 1μl
LIGR1(10pmol) 1μl
Pyrobest?ploymerase(125U)?0.25μl
50μl
The concrete reaction conditions of PCR is as follows:
94℃ 1min
4℃ 5min
3. PCR method is inserted ripe thrombase-like gene in expression vector pPIC9K α-factor signal sequence
With step 1. and the PCR product 2. respectively get 0.5 μ l as template, with forward primer LIGF2 and oppositely TDLR1 carry out the PCR ligation as primer.Other reactants compositions and reaction conditions are with step 1..
4. the 3. processing of middle PCR product of step
At first, the PCR product is carried out purifying.Purification process is as follows:
100 μ l PCR products
↓ adding 10 μ l 3M NaAc and 300 μ l, 100% ethanol
Mixing ,-80 ℃ of freezing 10min
↓
12, the centrifugal 10min of 000rpm, abandons supernatant by 4 ℃
↓ adding 70% ethanol 1ml, mixing
12, the centrifugal 5min of 000rpm, abandons supernatant by 4 ℃
↓
Centrifugal drying, 3min
Then, purified product is used the BamH1 enzymic digestion.The enzymic digestion reaction conditions is as follows:
Purified product
K?buffer 10μl
BamH1 (TAKARA, Dalian) 10 μ l
Sterile distilled water 80 μ l
100 μ l, 37 ℃, 2 hours
Afterwards, with the enzymic digestion product purification.Product behind the purifying is done the flush end processing.Flush end processing reaction condition is as follows:
Blunting buffer 1μl
T
4Dna ligase 1 μ l
H
2O 8μl
10μl，37℃，10min
Then, after above-mentioned flush end product purification, use the EcoR1 enzymic digestion.The enzymic digestion reaction conditions is as follows:
Flush end product behind the previous step purifying
10x?buffer 10μl
EcoR1 (TAKARA, Dalian) 10 μ l
Sterile distilled water 80 μ l
100 μ l, 37 ℃, 2 hours
At last, repeat purification step to remove EcoR1.
5. the processing of expression vector pPIC9K
The processing of expression vector pPIC9K also will be through BamH1 digestion, purifying, flush endization, purifying, EcoR1 digestion, the processing that purifying etc. are 4. identical with step.
6. the reorganization of ripe thrombase-like gene in expression vector pPIC9K
The ligation of carrier and goal gene:
PPIC9K after the processing (50ng/ μ l) 1 μ l
Goal gene after the processing (25ng/ μ l) 5 μ l
Solution I (TAKARA, Dalian) 6 μ l
12 μ l, 16 ℃, 4 hours
7. recon transformed into escherichia coli JM109
Get 10 μ l recombinant plasmids and 100 μ l competence JM109 cell mixings, ice bath is the thermal shock conversion after half an hour, and 42 ℃, 45 seconds.In ice, place after 2 minutes, add 0.94ml SOC liquid nutrient medium, 37 ℃ of concussions (160-225rpm) 1 hour.Get 100 μ l and be coated with the LB flat board, 37 ℃ of incubated overnight.The PCR method detects positive colony.Picking positive colony upgrading grain.Order-checking, consistent with cDNA sequence among the embodiment 1.
Embodiment 3:
The expression vector pPIC9K that contains thrombase-like gene in Dalian agkistrodon shedaoensis (Gloydius Shedaoensis) venom is integrated in the pichia spp cell GS115 genome
1. linearization of recombinant plasmid
Recombinant plasmid becomes shape material grain after the SacI enzymic digestion.The digestion reaction condition is as follows:
Recombinant plasmid 480 μ l
10x?L?buffer 60μl
SacI(10U/μl) 60μl
600 μ l, spend the night by 37 ℃
2. enzyme is cut the purifying of product
Enzyme is cut product 600 μ l ↓+200 μ l H
2O800 μ l ↓+800 μ l phenol: chloroform: 25 ℃ of primary isoamyl alcohol (25: 24: 1), 11,000rpm is centrifugal, 25 ℃ of 8min ↓ get supernatants ↓+800 μ l chloroform/primary isoamyl alcohol, 11,000rpm is centrifugal, 8min ↓ get supernatant ↓+800 μ l 100% ice-cold ethanol mixing ↓-80 ℃, 10min ↓ 4 ℃, 11,000rpm is centrifugal, 8min ↓ abandon supernatant, stay precipitation ↓ centrifugal drying, it is 2 μ g/ μ l ↓-20 ℃ of preservations conversions of 3. shocking by electricity that 3min ↓ add TE is dissolved to final concentration
80 μ l fresh culture GS115 cell+10 μ l steps are recombinant plasmid 2.
↓
Insert behind the mixing in the ice-cold electric shock tank of 0.2cm
↓
Insulation is 5 minutes in ice
↓
Electric shock transforms (electric shock instrument: 1500V, Bio-Rad E.coli Pulser, the U.S.)
↓
Add 1M Sorbitol 1ml immediately
↓
Be transferred in the eppendorf centrifuge tube
↓
Be coated with the MD flat board, 30 ℃ of cultivations
The positive bacterium colony of picking carries out PCR inspection bacterium.So far,------pichia spp cell GS115 that obtains containing the expression strain of thrombase-like gene in Dalian agkistrodon shedaoensis (GloydiusShedaoensis) venom.
Claims (5)
1. thrombase-like gene cDNA sequence and clone thereof in the agkistrodon shedaoensis venom of LiaoNing, China province Dalian, it is characterized in that it is: carry out homology analysis research by thrombase-like gene to other poisonous snake venom of known nucleotide sequence, according to synthetic four oligonucleotide primers of the high zone design of homology, the Dalian total RNA of agkistrodon shedaoensis poison gland with extraction is a template, the synthetic amplification of RT-PCR method thrombase-like gene sequence wherein, inboard primer with design is a primer, carry out secondary PCR, and, obtained the complete sequence of thrombase-like gene cDNA in Dalian agkistrodon shedaoensis (Gloydius Shedaoensis) venom thus to this PCR product order-checking; Simultaneously, cDNA complete sequence according to Thrombin-like enzyme, the design primer, the ripe thrombase-like gene of thrombase-like gene coding is cloned among the expression vector pPIC9K, and by the thermal shock method this carrier is converted in the escherichia coli jm109 competent cell, by electric shocking method this expression vector is incorporated in the genome of pichia spp cell strain GS115.
2.1cDNA,：ATGGTGCTGATCAGAGTGCTAGCAAACCTTCTGATACTACAGCTGTCTTACGCACAAAAGTCTTCTGAACTGATCATTGGAGGTGATGAATGTAACATAAATGAACATCGGTTCCTTGTAGCCTTATACACCTCTAGATCTAGGAGGTTTTATTGCGGTGGGACTTTGATCAACCAGGAATGGGTGCTCACCGCTGCACACTGCGACAGGAAAAATATCCGGATAAAGCTTGGTATGCATAGCGAAAAGGTACCAAATGAGGATGCAGAGACAAGAGTCCCAAAGGAGAAGTTCTTTTGTCTCAGTAGCAAAACCTACACCAAATGGGACAAGGACATCATGTTGATGAGCTGAAAAGACCTGTTAACAACAGTACACACATCGCGCCTGTCAGCTTGCCTTCCAACCCTCCCAGTGTGGACTCAGTTTGCCGTGTTATGGGATGGGGTACAATCACATCTCCTCAAGAGACTTATCCCGATGTCCCCCATTGTGCTAACATTAACATACTTGATTATGAGGTGTGCAAGCAGCTCACGGAGGGTTGCCAGCAACAAGCAGAACATTGTGTGCAGGTATCCTGGGAGGCAAAGATTCATGTAAGGGTGACTCTGGGGGACCCCTCATCTGTAAGTCTTAAGCCTGGTGTCTACACCAAGGTCTTCGATTATACTGAGTGGATCCAGAGCAATTGCAGGAAATACAGATGCAACCTGCCCCCCATGAaaacttttgaaaagttaagaggagaaaatgtaacatattactacatctcttcctatccctaaccatatccaactacattggaatctattcccaggcagtaagcttttttaagactcaaataggactgcctttgaagtaagaaatgctcaaaatagtgctgcagggatcatgtcccatttaatttcagtataaaacaatctcagttaaatggaggcctgttttagggtgaggtgcaatattttctgactctaaaatgcaccattccaaatattttaacctctgaatatctttccatttctgtccacttctgggacagcggggtccttgatgctctttgagcttgtcttcttgcagacgtttcattacccagctaggtaacatcatcagtgctagaatattctcttctattggtacttctgtggcatttacaatacgctcatatggagtcatgcagtcaccctacaaacatatccatatacctggtcccactggtgcctaaaaaggaccccagattaacccgcactttccaatcctaaatagaatcttttgagaatcgtgttttcatgtaaattctcaggtatccacagc
Tgcataaatcggcaaaaaaaaaaaaaaaaaa
3. thrombase-like gene cDNA sequence and clone thereof in the agkistrodon shedaoensis venom of LiaoNing, China according to claim 1 province Dalian, it is characterized in that: the expression vector that contains this thrombase-like gene is the pPIC9K/ thrombase-like gene.
4. thrombase-like gene cDNA sequence and clone thereof in the agkistrodon shedaoensis venom of LiaoNing, China according to claim 1 province Dalian is characterized in that: intestinal bacteria (E.coli) recipient cell that contains the expression vector pPIC9K/ thrombase-like gene of this thrombase-like gene is the JM109/ thrombase-like gene.
5. thrombase-like gene cDNA sequence and clone thereof in the agkistrodon shedaoensis venom of LiaoNing, China according to claim 1 province Dalian is characterized in that: pichia spp (Pichia pasotris) recipient cell that contains the expression vector pPIC9K/ thrombase-like gene of this thrombase-like gene is the GS115/pPIC9K/ thrombase-like gene.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005045022A1 (en) * | 2003-10-31 | 2005-05-19 | Biobud Co., Ltd. | Thrombin-like recombinant batroxobin expressed by pichia sp.and production method thereof |
| CN1324132C (en) * | 2004-10-19 | 2007-07-04 | 北京大学 | Snake venom thrombin-like enzyme and its encoding gene and application |
| CN100351381C (en) * | 2005-09-02 | 2007-11-28 | 中山大学 | Novel thrombase-like gene and its use |
| WO2008119203A1 (en) * | 2007-03-30 | 2008-10-09 | Shanghai Wanxing Biopharmaceuticals, Co., Ltd. | A purified recombinant batroxobin with high specific activity |
| CN100462436C (en) * | 2005-10-12 | 2009-02-18 | 益生生技开发股份有限公司 | Fast conversion method of competence cell |
| CN100564532C (en) * | 2006-12-18 | 2009-12-02 | 中国人民解放军军事医学科学院生物工程研究所 | A kind of batroxobin and preparation method thereof and own coding gene |
| CN102839145A (en) * | 2012-05-16 | 2012-12-26 | 中国农业大学 | Genetic engineering bacterium expressing snake venom kininogenase as well as constructing method and application thereof |
| WO2022109758A1 (en) * | 2020-11-26 | 2022-06-02 | 沈喆景 | Application of proteolytic enzyme of pit viper in treatment of alzheimer's disease |
-
2001
- 2001-02-27 CN CN01106262A patent/CN1370833A/en active Pending
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005045022A1 (en) * | 2003-10-31 | 2005-05-19 | Biobud Co., Ltd. | Thrombin-like recombinant batroxobin expressed by pichia sp.and production method thereof |
| CN1324132C (en) * | 2004-10-19 | 2007-07-04 | 北京大学 | Snake venom thrombin-like enzyme and its encoding gene and application |
| CN100351381C (en) * | 2005-09-02 | 2007-11-28 | 中山大学 | Novel thrombase-like gene and its use |
| CN100462436C (en) * | 2005-10-12 | 2009-02-18 | 益生生技开发股份有限公司 | Fast conversion method of competence cell |
| CN100564532C (en) * | 2006-12-18 | 2009-12-02 | 中国人民解放军军事医学科学院生物工程研究所 | A kind of batroxobin and preparation method thereof and own coding gene |
| WO2008119203A1 (en) * | 2007-03-30 | 2008-10-09 | Shanghai Wanxing Biopharmaceuticals, Co., Ltd. | A purified recombinant batroxobin with high specific activity |
| US7939067B2 (en) | 2007-03-30 | 2011-05-10 | Shanghai Wanxing Biopharmaceuticals, Co., Ltd. | Purified recombinant batroxobin with high specific activity |
| CN102839145A (en) * | 2012-05-16 | 2012-12-26 | 中国农业大学 | Genetic engineering bacterium expressing snake venom kininogenase as well as constructing method and application thereof |
| WO2022109758A1 (en) * | 2020-11-26 | 2022-06-02 | 沈喆景 | Application of proteolytic enzyme of pit viper in treatment of alzheimer's disease |
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