CN102559721A - Gloydius ussuriensis venom FLE (fibrin olytic enzyme) gene and encoding protein thereof as well as preparation method of eukaryon expression product of Gloydius Ussuriensis venom FLE gene - Google Patents
Gloydius ussuriensis venom FLE (fibrin olytic enzyme) gene and encoding protein thereof as well as preparation method of eukaryon expression product of Gloydius Ussuriensis venom FLE gene Download PDFInfo
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Abstract
The invention discloses a Gloydius ussuriensis venom FLE (fibrin olytic enzyme) gene and an encoding protein thereof as well as a preparation method of a eukaryon expression product of a Gloydius ussuriensis venom FLE gene, for the purpose of providing a Gloydius ussuriensis venom FLE (fibrin olytic enzyme) gene and an encoding protein thereof as well as a preparation method of a eukaryon expression product of a Gloydius ussuriensis venom FLE gene. The gene is one of the following nucleic acid sequences: 1) a DNA (deoxyribonucleic acid) sequence as shown in SEQ ID NO:1 in a sequence table; and 2) a DNA sequence as shown in SEQ ID NO:2 in an encoding sequence table. The amino acid sequence of the encoding protein of the Gloydius ussuriensis venom FLE gene is as shown in SEQ ID NO:2. The gene encoding FLE has fibrinolysis activity, can degrade fibrinogen and fibrin, can be used for dissolving blood clots formed during the periods of myocardial infarction, apoplexy, thrombus and the like and exerts an important effect in the treatment of the symptoms.
Description
Technical field
The present invention relates to the preparation method of agkistrodon halyx pallas venom FLE gene, its proteins encoded and eukaryotic expression product thereof in the Usu.
Background technology
Pallas pit viper in the Usu (Gloydius ussuriensis) is under the jurisdiction of reptilia (Reptilia) Squamata (Squamata) Ophidia (Serpentes) Viperidae (Viperidae) Crotalinae (Crotalinae) Asia Pallas pit viper and belongs to (Gloydius); Mainly be distributed in Heilungkiang, Jilin, Liaoning (the north), the Inner Mongol (to the east of the Daxing'an Mountainrange) in China; Be that China northeast distributes extensively, population quantity is maximum, a kind of poisonous snake of strong toxicity, have important economic value and pharmaceutical use.
The snake venom complicated component contains the multiple bioactive enzyme that has that comprises zymoplasm, Thrombin-like enzyme, Phospholipid hydrolase, phosphodiesterase, Unidasa, arginine esterase etc.Wherein, in snake venom such as Viperinae, Crotalinae and Elapidae, exist one type of direct solution fibrin of ability and fibrinogenic enzyme, (fibrin (ogen) olytic enzyme FLE), is called for short plasmin to be called Tryptase.Nevin fibrinolytic enzyme has fibrin degradation and fibrinogenic effect, can influence the generation of normal zymoplasm through suppressing the VIII thrombin.Research shows that nevin fibrinolytic enzyme has tangible anti thrombotic action, can be used for the treatment of heart and brain thrombus and myocardial infarction, can be used as the powerful antithrombotic agent of a kind of potential, the R&D work of being correlated with in present countries in the world.
Summary of the invention
The present invention provides the preparation method of agkistrodon halyx pallas venom FLE gene, its proteins encoded and eukaryotic expression product thereof in a kind of Usu.
Agkistrodon halyx pallas venom FLE gene is one of following nucleotide sequence in the Usu of the present invention:
1) dna sequence dna of SEQ ID NO:1 in the sequence table;
2) dna sequence dna of SEQ ID NO:2 in the code sequence tabulation.
The aminoacid sequence of the proteins encoded of agkistrodon halyx pallas venom FLE gene is shown in SEQ ID NO:2 in the Usu of the present invention.
The preparation method of agkistrodon halyx pallas venom FLE gene eucaryon expression product in the Usu of the present invention, carry out according to the following steps:
One, makes up the carrier for expression of eukaryon ppFASTBACHTa-FLE recombinant plasmid of agkistrodon halyx pallas venom FLE gene in the Usu; Two, the extraction of ppFASTBACHTa-FLE recombinant plasmid and purifying; Three, recombinant plasmid injection mouse: the ppFASTBACHTa-FLE recombinant plasmid of getting 20 μ g purifying; Be diluted to 1/10 of mouse body weight with saline water, mouse is through the ppFASTBACHTa-FLE recombinant plasmid of tail vein injection dilution, in 5s, injects to finish; Behind the 8h; Mouse is put to death through the cervical vertebra dislocation, take out liver organization, put into saline water; Four, the preparation of eukaryotic expression product: adopting pH is that 7.2 phosphate buffered saline buffer cleans the mouse liver tissue 3～5 times; Ratio according to 200mg tissue/ml adds single stain remover lysate in liver organization then; Homogenate in 4 ℃, the centrifugal 10min of 12000r/min, is got supernatant then; Adopt affinity chromatography to carry out protein purification, promptly accomplish the preparation of agkistrodon halyx pallas venom FLE gene eucaryon expression product in the Usu.
The invention provides agkistrodon halyx pallas venom FLE gene in the Usu, its coding Tryptase FLE has fibrinolytic, can the former and scleroproein of fibrin degradation.Can be used for being dissolved in the blood clot that forms during myocardial infarction, apoplexy, the thrombus etc.,, will in treatment of conditions such as myocardial infarction, apoplexy, thrombus, play a significant role as a kind of strong anti-bolt, thrombolytic drug.
Description of drawings
Fig. 1 is the SDS-PAGE electrophoresis result of eukaryotic expression product in the embodiment three; Fig. 2 is the Western blot detected result of eukaryotic expression product in the embodiment three; Fig. 3 is a control group mice liver organization immunohistochemistry detected result in the embodiment three; Fig. 4 is an experimental mice liver organization immunohistochemistry detected result in the embodiment three; Fig. 5 is the fibrinolytic qualification result of eukaryotic expression product in the embodiment three.
Embodiment
Technical scheme of the present invention is not limited to following cited embodiment, also comprises the arbitrary combination between each embodiment.
Embodiment one: agkistrodon halyx pallas venom FLE gene is one of following nucleotide sequence in this embodiment Usu:
1) dna sequence dna of SEQ ID NO:1 in the sequence table;
2) dna sequence dna of SEQ ID NO:2 in the code sequence tabulation.
Embodiment two: the aminoacid sequence of the proteins encoded of agkistrodon halyx pallas venom FLE gene is shown in SEQ ID NO:2 in this embodiment Usu.
The preparation method of agkistrodon halyx pallas venom FLE gene is following in the Usu:
One, the separation of the total RNA of pallas pit viper poison gland in the Usu
1, pallas pit viper catches in the Mudanjiang City, Heilongjiang Province in the Usu, through the categorizing system inspection, confirms; Break rapidly when 2, drawing materials and get snakehead, use liquid nitrogen flash freezer, be stored in-80 ℃ of refrigerator-freezers again, the time spent places on ice, peels off poison gland rapidly; 3, place liquid nitrogen to be ground into powder rapidly poison gland, in the Eppendorf pipe of the 2ml of the precooling on ice of packing into; 4, the saturated phenol of Tris, the chloroform of 350 μ l, the 2%SDS damping fluid of 700 μ l and the beta-mercaptoethanol of 100 μ l that add 450 μ l, concuss mixing 10min on the vibrator; 5,4 ℃, the centrifugal 10min of 12000r/min; 6, get supernatant in another Eppendorf pipe, add saturated phenol of 350 μ lTris and 350 μ l chloroforms again, behind the vibration 5min, 4 ℃, the centrifugal 7min of 12000r/min; 7, repeating step 6 once; 8, get the 700 μ l chloroforms that supernatant adds precooling, vibration 5min, 4 ℃, the centrifugal 7min of 12000r/min; 9, get supernatant, add the LiCl of 350 μ l absolute ethyl alcohols and 350 μ l 8mol/L, ice bath 10min, 4 ℃, the centrifugal 12min of 12000r/min; 10, abandon supernatant, the instantaneous centrifugal 5S of 12000r/min abandons supernatant; 11, deposition is dissolved in the 10 μ l DEPC water stores, carry out electrophoresis detection simultaneously.
Two, the purifying of the total RNA of pallas pit viper poison gland in the Usu
According to the total RNA of pallas pit viper poison gland in the RNA extraction agent box specification sheets purifying Usu of the living worker's biotechnology in Shanghai ltd.
Three, RT-PCR method amplification snake venom FLE gene
Adopt ThermoScript II AMV test kit (giving birth to worker's biotechnology ltd) operation, the total RNA reverse transcription of pallas pit viper poison gland in the Usu behind the purifying is obtained cDNA available from Shanghai.The gene order of the short-tail pallas pit viper plasmin of having delivered with the GenBank DB (GenBank accession number: AF176679) be foundation, adopt a pair of primer of DNAMAN software design.P1 is the forward primer sequence, for the enzyme that makes things convenient for follow-up test is cut, and peripheral hardware EcoR I restriction enzyme site; P2 is the reverse primer sequence, entrusts Dalian precious (TaKaRa) biotechnology ltd synthetic.
With cDNA is that template is carried out pcr amplification, and reaction system is 50 μ l, is made up of following ingredients:
The pcr amplification condition is: 94 ℃ of preparatory sex change 7min, and 94 ℃ of sex change 90s, 55 ℃ of annealing 60s, 72 ℃ are extended 90s, totally 30 circulations, 72 ℃ are extended 10min, 4 ℃ of preservations again.
Three, the purifying of PCR product and with being connected of carrier
The PCR reaction finishes the back PCR product is carried out the detection of 1% agarose gel electrophoresis; Adopt Shanghai to give birth to worker's biotechnology ltd glue recovery test kit and carry out purifying and recovering; Obtain the purpose fragment of about 777bp, this goal gene is agkistrodon halyx pallas venom FLE gene in the Usu.Agkistrodon halyx pallas venom FLE gene in the Usu is connected on the pMD18-T carrier (available from the precious biotechnology in Dalian ltd), must connects product.
Four, the conversion of recombinant plasmid and evaluation
The JM109 competent cell operation instruction that provides by vast Tyke biotechnology ltd will connect product and be transformed into the JM109 competent cell, and screening positive clone extracts recombinant plasmid.
Five, the Nucleotide of agkistrodon halyx pallas venom FLE gene and determined amino acid sequence and analysis in the Usu
Recombinant plasmid called after pMD18T-FLE; Entrust Shanghai to give birth to the sequencing that worker's biotechnology ltd carries out recombinant plasmid; Sequencing result shows that agkistrodon halyx pallas venom FLE gene in the Usu has the nucleotide sequence of SEQ ID NO:1 in the sequence table; SEQ ID NO:1 in the sequence table is by 777 based compositions, and its encoding sequence is that coding has the protein of the aminoacid sequence of SEQ ID NO:2 in the sequence table from 5 ' end the 1st to 777 bit base.
Embodiment three: the preparation method of agkistrodon halyx pallas venom FLE gene eucaryon expression product in this embodiment Usu, carry out according to the following steps:
One, makes up the carrier for expression of eukaryon ppFASTBACHTa-FLE recombinant plasmid of agkistrodon halyx pallas venom FLE gene in the Usu; Two, the extraction of ppFASTBACHTa-FLE recombinant plasmid and purifying; Three, recombinant plasmid injection mouse: the ppFASTBACHTa-FLE recombinant plasmid of getting 20 μ g purifying; Be diluted to 1/10 of mouse body weight with saline water, mouse is through the ppFASTBACHTa-FLE recombinant plasmid of tail vein injection dilution, in 5s, injects to finish; Behind the 8h; Mouse is put to death through the cervical vertebra dislocation, take out liver organization, put into saline water; Four, the preparation of eukaryotic expression product: adopting pH is that 7.2 phosphate buffered saline buffer cleans the mouse liver tissue 3～5 times; Ratio according to 200mg tissue/ml adds single stain remover lysate in liver organization then; Homogenate in 4 ℃, the centrifugal 10min of 12000r/min, is got supernatant then; Adopt affinity chromatography to carry out protein purification, promptly accomplish the preparation of agkistrodon halyx pallas venom FLE gene eucaryon expression product in the Usu.
Single stain remover lysate is that 8.0 TrisHCl, the NaCl of 150mmol/L, volumetric concentration are that 1% TritonX-100 and zero(ppm) water are formed by 50mmol/L and pH, faces the time spent according to 100: 1 ratio adding Methanesulfonyl chlorides.
The method that makes up the carrier for expression of eukaryon ppFASTBACHTa-FLE recombinant plasmid of agkistrodon halyx pallas venom FLE gene in the Usu in this embodiment step 1 is: a, agkistrodon halyx pallas venom FLE gene in the Usu is connected to the pMD18-T carrier, must connects product pMD18T-FLE; B, pMD18-T-FLE and expression vector plasmid pFASTBACHTa are all adopted EcoR I and Pst I double digestion, reclaim target gene fragment and carrier segments; C, the target gene fragment that reclaims is connected through the T4DNA ligase enzyme with carrier segments; Connect product and be transformed into the JM109 competent cell; Picking male reorganization bacterium colony; Entrust Shanghai to give birth to worker's biotechnology ltd and carry out sequencing, the correct positive strain that checks order promptly contains the ppFASTBACHTa-FLE recombinant plasmid.
Linked system among the step a is following:
| Composition | Consumption |
| 0.3pmol/L FLE gene | 3.5μL |
| pMD18-T | 1μL |
| ddH 2O | 0.5μL |
| Solution?I | 5μL |
Ligation condition: 16 ℃ of water-baths, 8～12h.Said Solution I is the supporting damping fluid of pMD18-T carrier, includes the T4 dna ligase, buys from TaKaRa company.
The system of pMD18-T-FLE double digestion is following among the step b:
| Composition | Consumption |
| pMD?18-T-FLE | 15μL |
| 10×H?buffer | 3μL |
| EcoR?I | 3μL |
| Pst?I | 3μL |
| ddH 2O | 6μL |
Endonuclease reaction condition: 37 ℃ of water-baths, 2h.
The system of expression vector plasmid pFASTBACHTa double digestion is following among the step b:
| Composition | Consumption |
| Expression vector plasmid pFASTBACHTa | 15μL |
| 10×H?buffer | 3μL |
| EcoR?I | 3μL |
| Pst?I | 3μL |
| ddH 2O | 6μL |
Endonuclease reaction condition: 37 ℃ of water-baths, 2h.
Linked system among the step c is following:
| Composition | Consumption |
| Target gene fragment | 7μL |
| Carrier segments | 1μL |
| The T4 dna ligase | 1μL |
| 10×buffer | 1μL |
Ligation condition: 16 ℃ of water-baths, 8～12h.
The process for extracting of ppFASTBACHTa-FLE recombinant plasmid is in this embodiment step 2:
(1) under aseptic condition, the positive strain streak inoculation that order-checking is correct is inverted for 37 ℃ and is cultivated 16h on the LB agar plate that contains 80 μ g/ml Amp; The single bacterium colony of picking; Be inoculated in 50ml and contain in the LB liquid nutrient medium of 80 μ g/ml Amp, 37 ℃ jolt overnight cultures, get bacterium liquid;
(2) get 2ml bacterium liquid in the 2.0ml centrifuge tube, 4 ℃ with the centrifugal 1min of 12000r/min, abandons supernatant, collects thalline, in thalline, adds the solution I of 100 μ l precoolings, concuss mixing on the vortex oscillation device;
(3) add the new solution II of preparing of 200 μ l, cover the tight mouth of pipe, put upside down 0 ℃ of ice bath 5min gently 5 times;
(4) solution III of adding 150 μ l precoolings is covered the tight mouth of pipe, will manage and be inverted the back light shaking to being uniformly dispersed, afterwards 0 ℃ of ice bath 5min.
(5) 4 ℃, the centrifugal 10min of 12000r/min draw supernatant in another centrifuge tube, add isopyknic solution A, the concussion mixing;
(6) 4 ℃, the centrifugal 5min of 12000r/min draw supernatant in another centrifuge tube, add isopyknic solution B, the concussion mixing;
(7) 4 ℃, the centrifugal 5min of 12000r/min draw supernatant in another centrifuge tube, add the absolute ethyl alcohol of the precooling of 2 times of volumes, concussion mixing ,-20 ℃ of held 10min;
(8) 4 ℃, the centrifugal 10min of 12000r/min abandon supernatant, add volumetric concentration again and be 70% ethanol, the piping and druming washing and precipitating;
(9) 4 ℃, the centrifugal 5min of 12000r/min abandon supernatant, centrifuge tube are inverted on the thieving paper seasoning;
(10) in deposition, add 30 μ l ddH
2O (containing 3 μ l RNase), 37 ℃ of water-bath digestion 2h ,-20 ℃ of preservations are subsequent use.
(11) DNA that is extracted 2 μ l are carried out electrophoresis on 1% sepharose, observations under the uv lamp.
Solution I is made up of EDTA and the zero(ppm) water of TrisHCl, 10mmol/L and the pH8.0 of 50mmol/L glucose, 25mmol/L and pH8.0 described in the step (2), once disposes 100ml, behind steam sterilizing 15min under 10 pounds of high pressure, be stored in 4 ℃ subsequent use.
The collocation method of solution II described in the step (3): mother liquor is 10%SDS and 10M NaOH, is diluted to 0.2mol/L NaOH and 1%SDS before the use.
Solution III described in the step (4): configuration 100ml includes 5mol/L potassium acetate 60ml, glacial acetic acid 11.5ml, water 28.5ml.
Solution A was made up of phenol, chloroform and primary isoamyl alcohol in 25: 24: 1 by volume described in the step (5);
Solution B described in the step (6) was by chloroform and primary isoamyl alcohol 24: 1 by volume.
The purification process of ppFASTBACHTa-FLE recombinant plasmid reclaims test kit (worker's biotechnology ltd is given birth in purchase from Shanghai) specification sheets operation according to UNIQ-10 pillar DNA glue in this embodiment step 2.
The method that adopts affinity chromatography to carry out protein purification in this embodiment step 4 is:
A, preparation affinity column: the sepharose that adopts cyanogen bromide-activated; Polyclonal antibody (giving birth to worker's biotechnology ltd by Shanghai synthesizes) coupling with viper venom plasmin in the anti-Usu; The polyclonal antibody affinity column of preparation 3.5 * 20cm adopts balance liquid to carry out the balance of pillar again; Said balance liquid is made up of NaCl and the zero(ppm) water of 0.02mol/L Tris-HCl, 0.5mol/L, and pH is 7.8;
B, go up appearance and cross post: with supernatant, add 3ml in the chromatography column at every turn, place interaction 20min in the incubator, the release effluent adopts balance liquid to carry out balance again, detects liquid until albumen and is in stability number;
C, albumen wash-out: adopt affinity chromatography elutriant wash-out expression product, collect the 1-2ml/ pipe, collect elution peak, dialysis desalting, lyophilize promptly obtains agkistrodon halyx pallas venom FLE gene eucaryon expression product in the Usu of purifying; Said elutriant is made up of Tris-HCl, 1.2mol/LNaCl and the zero(ppm) water of 0.04mol/L, and pH is 7.8.
Adopt following mode to detect the eukaryotic expression product that this embodiment obtains:
Get 20 of body weight 20～25g male mouse of kunming, be divided into 2 groups at random.One group 10 is experimental group, and another 10 of group is a control group.Experimental group adopts the method for this embodiment to handle; Control group mice is through the saline water of tail vein injection body weight 1/10.Behind the 8h, all mouse are adopted and remove eyeball method blood sample collection, put to death through the cervical vertebra dislocation again, and it is subsequent use that the taking-up liver organization is put into saline water.
1, the SDS-PAGE of this embodiment eukaryotic expression product detects
Ratio according to 200mg tissue/ml adds single stain remover lysate respectively in the liver organization of experimental group and control group, homogenate in 4 ℃, the centrifugal 10min of 12000r/min, is got supernatant then; With supernatant and the abundant mixing of isopyknic 2 * SDS sample-loading buffer, boil 5min, again ice bath 2min.Supernatant with mice in control group liver tissue homogenate is contrast, carries out SDS-PAGE (polyacrylamide gel) electrophoresis.Electrophoresis result is as shown in Figure 1, and swimming lane 1 is the molecular weight of albumen standard, and swimming lane 2 is the supernatant of control group mice liver tissue homogenate, and swimming lane 3 is the supernatant of experimental mice liver tissue homogenate, and swimming lane 4 is the supernatant of experimental mice liver tissue homogenate.Compare with control group, all occur a protein expression band obviously and clearly in the sample of experimental group, being illustrated in the mouse liver tissue has expression of recombinant proteins.With reference to molecular weight standard, this proteic molecular weight is about 30kDa, conforms to the expection molecular weight of albumen.
2, the Western blot of this embodiment eukaryotic expression product detects
Recombinant protein places transfering buffering liquid with separation gel behind the SDS-PAGE electrophoresis, soak 30min.Anti-with viper venom plasmin polyclonal antibody in the anti-Usu of rabbit of the living worker's biotechnology in Shanghai ltd preparation as one; SA is the goat anti-rabbit igg of horseradish peroxidase-labeled; Adopt the half dry type transfer method to change film, lucifuge colour developing in the DAB colour developing liquid is when observing clearly brown stripe; Color development stopping reaction immediately, the reservation of taking pictures.Photo is as shown in Figure 2, and swimming lane 1 is the molecular weight of albumen standard, and swimming lane 2 is the hybridization band of murine liver tissue protein sample.Western blot detected result shows, a specific hybridization band occurs at the 30kDa place, shows that the eukaryotic expression product of acquisition is reorganization FLE albumen.
3, the immunohistochemistry of this embodiment eukaryotic expression product detects
With the processing that fixes of the hepatic tissue of experimental group and control group mice; Carry out conventional gradient ethanol dehydration then; It is identical that paraffin embedding, used first antibody and SA and Western blot detect used antibody, brazilwood extract dyeing; The neutral gum mounting, oven dry back is to the tissue slice preservation of taking pictures.Control group mice liver organization immunohistochemistry detected result is as shown in Figure 3; Experimental mice liver organization immunohistochemistry detected result is as shown in Figure 4; Experimental group and control group mice liver tissue slices detect through immunohistochemistry and show; Do not occur the stained positive cell in the control group mice liver organization, it is blue that major part is; Be xanchromatic stained positive cell in a large number and in the experimental mice liver organization, exist, show reorganization FLE albumen high level expression in liver organization, and recombinant protein expressed in tenuigenin mainly.
4, the fibrinolytic of this embodiment eukaryotic expression product is identified
Fibrinolytic is identified the fibrin plate method that adopts.Get 2% agar-agar soln 5ml and (use pH7.4; Contain the damping fluid preparation of 150mM NaCl, 50mM Tris-Cl); In 50 ℃ of water-baths, mix, add the zymoplasm that 1ml contains 10 units before the bed board with 5ml 4% fibrinogen solution (, containing the damping fluid preparation of 150mM NaCl, 50mM Tris-Cl) with pH 7.4; Pour into behind the mixing in the sterile petri dish, room temperature is solidified.After waiting to solidify, on flat board, beat the aperture that diameter is 4mm, every hole adds the supernatant 15 μ l of murine liver tissue homogenate, 37 ℃ of insulation 18～72h, observations.
Do positive control with agkistrodon halyx pallas venom in the natural Usu during evaluation.The snake venom treatment process: fresh snake venom 5 μ l add 45 μ l saline water, mixing, and 4 ℃, the centrifugal 10min of 10000r/min get supernatant, and-20 ℃ of preservations are subsequent use.
Adopt the fibrin plate method that experimental group and control group mice hepatic homogenate have been carried out the fibrinolytic evaluation, and get in the natural Usu viper venom and do positive control.Experimental result is as shown in Figure 5; Can know by figure; Behind 37 ℃ of insulation 18h, tangible fibrinolytic circle has all appearred in experimental group sample ( sample 2 and 3 is the supernatant of experimental mice liver tissue homogenate) and natural snake venom (sample 4), and the fibrinolytic phenomenon does not appear in control sample (sample 1 is the supernatant of control group mice liver tissue homogenate); Be illustrated in the reorganization FLE that expresses in the mouse liver and have fibrinolytic, can the former and scleroproein of fibrin degradation.
Claims (3)
1. agkistrodon halyx pallas venom FLE gene in the Usu is characterized in that agkistrodon halyx pallas venom FLE gene is one of following nucleotide sequence in the Usu:
1) dna sequence dna of SEQ ID NO:1 in the sequence table;
2) dna sequence dna of SEQ ID NO:2 in the code sequence tabulation.
2. the proteins encoded of agkistrodon halyx pallas venom FLE gene in the said Usu of claim 1, the aminoacid sequence that it is characterized in that the proteins encoded of agkistrodon halyx pallas venom FLE gene in the Usu is shown in SEQ ID NO:2.
3. the preparation method of agkistrodon halyx pallas venom FLE gene eucaryon expression product in the said Usu of claim 1 is characterized in that the preparation method of agkistrodon halyx pallas venom FLE gene eucaryon expression product in the Usu carrying out according to the following steps:
One, makes up the carrier for expression of eukaryon ppFASTBACHTa-FLE recombinant plasmid of agkistrodon halyx pallas venom FLE gene in the Usu; Two, the extraction of ppFASTBACHTa-FLE recombinant plasmid and purifying; Three, recombinant plasmid injection mouse: the ppFASTBACHTa-FLE recombinant plasmid of getting 20 μ g purifying; Be diluted to 1/10 of mouse body weight with saline water, mouse is through the ppFASTBACHTa-FLE recombinant plasmid of tail vein injection dilution, in 5s, injects to finish; Behind the 8h; Mouse is put to death through the cervical vertebra dislocation, take out liver organization, put into saline water; Four, the preparation of eukaryotic expression product: adopting pH is that 7.2 phosphate buffered saline buffer cleans the mouse liver tissue 3～5 times; Ratio according to 200mg tissue/ml adds single stain remover lysate in liver organization then; Homogenate in 4 ℃, the centrifugal 10min of 12000r/min, is got supernatant then; Adopt affinity chromatography to carry out protein purification, promptly accomplish the preparation of agkistrodon halyx pallas venom FLE gene eucaryon expression product in the Usu.
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| CN110724678A (en) * | 2019-10-21 | 2020-01-24 | 陕西师范大学 | Medium Agkistrodon halys venom fibrinolytic enzyme and preparation method and application thereof |
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