CN102199584B - Antithrombotic enzyme Eupolytinl from Eupolyphaga sinensis Walker and gene and application thereof - Google Patents
Antithrombotic enzyme Eupolytinl from Eupolyphaga sinensis Walker and gene and application thereof Download PDFInfo
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Abstract
The invention relates to antithrombotic enzyme Eupolytinl from Eupolyphaga sinensis Walker, a gene and application thereof, and belongs to the field of biomedicine. In the invention, the natural antithrombotic enzyme Eupolytinl is obtained from intestines in alimentary tracts of the Eupolyphaga sinensis Walker through separation and purification, an encoding gene of the natural antithrombotic enzyme Eupolytinl is cloned, and a genetic engineering product of which the activity is equivalent to that of a natural protein is obtained by utilizing an Escherichia coli expression system. A precursor of the antithrombotic enzyme Eupolytinl from the Eupolyphaga sinensis Walker consists of 254 amino acids, wherein 1th to 28th amino acids are a signal peptide part; 29th to 254th amino acids are mature Eupolytinl proteins; and a primary structure of an amino acid sequence of the precursor is shown as SEQ ID NO: 1. The encoding gene of the antithrombotic enzyme Eupolytinl consists of 888 nucleotide sequences, wherein 85th to 765th nucleotides encode the mature Eupolytinl proteins; and a nucleotide sequence of the gene is GenBank accession Gu187070. Through animal experiments, the recombinant expression antithrombotic enzyme Eupolytinl from the Eupolyphaga sinensis Walker which has a natural resource can be applied to the preparation of medicaments for treating thrombotic diseases.
Description
Technical field:
The present invention provides a kind of Eupolyphaga Seu Steleophaga antithrombotic enzyme Eupolytin1 and gene and application, belongs to field of biomedicine technology.
Background technology:
The standard of living of Along with people's improves constantly, and rhythm of life is more and more faster, busy work, and irrational dietary structure, erratic daily life system, the numerous inducements such as excessive drinking and shortage sports of smoking cause increasing people to suffer from various cardiovascular disordeies.World Health Organization points out that the whole world has 1,800 ten thousand people to die from heart trouble and other cardiovascular disordeies every year at present, accounts for 1/3rd of global death toll, expects this numeral of the year two thousand twenty and might break through 20,000,000.Thrombotic disease has a strong impact on human beings'health, is to cause one of the highest cause of disease of mortality ratio.In decades in the past, the medicine for treating thrombus owner who has developed will comprise thrombolysis medicine, anti-freezing medicine and antiplatelet drug.But all antithrombotic reagents at present on the market often are accompanied by ypotension, systematic spinoff such as hemorrhage, therefore, are badly in need of the safe antithrombotic reagent of development of new.
In the social production practical activity that long-term and natural condition are struggled, China people utilize various Animal resources preventions and treatment various diseases that long history is arranged, and have accumulated abundant medicinal knowledge and experience.Record many prescriptions promoting blood circulation and removing blood stasis in Compendium of Material Medica and the elswhere medicine ancient books and records.Chinese medicine is the important component part of motherland's medicine and pharmacology, and its effect is affirmed by history and modern clinical efficacy in several thousand.Eupolyphaga Seu Steleophaga (Eupolyphaga sinensis Walker) is under the jurisdiction of Arthropoda Blattodea Corydiidae, is a kind of important Chinese medicinal materials, has the effect of removing blood stasis, reunion of fractured tendons and bones, is widely used more than 1,000 year by China people of all nationalities.From traditional Chinese medicine, excavating the medicine guide molecule of prevention and treatment thrombus disease, is that very important meaning is arranged for human beings'health.
Summary of the invention:
The object of the present invention is to provide a kind of Eupolyphaga Seu Steleophaga antithrombotic enzyme and gene and application.
Technical scheme of the present invention comprises preparation and the determined amino acid sequence of Eupolyphaga Seu Steleophaga antithrombotic enzyme Eupolytin1; The clone of Eupolyphaga Seu Steleophaga antithrombotic enzyme Eupolytin1 gene, pharmacological evaluation four partial contents of the in-vitro recombination expression of Eupolytin1 and Eupolyphaga Seu Steleophaga antithrombotic enzyme Eupolytin1.
Eupolyphaga Seu Steleophaga antithrombotic enzyme Eupolytin1 is that we separate a kind of active protein that obtains, the about 24KDa of molecular weight first from Eupolyphaga Seu Steleophaga digestive tube midgut.Antithrombotic enzyme Eupolytin1 precursor is made up of 254 amino acid; Wherein 1-28 amino acids (italicized item) is the signal peptide part; The 29-254 amino acids is sophisticated Eupolytin1 albumen, and this precursor complete sequence primary structure is (aminoacid sequence is SEQ ID NO:1):
IVGGSDANIEDLPYQLSFEYGGSHMCGASIIS 60
ENWVVTAAHCVDGVSASSARFRAGSSVRGSGGSLHQATQLIANPQYDYYTIDYDVAVARV 120
ATPFSYGSGVQPISLISVEPSAGQTATVSGWGTFFSGGSLPRQLQVVSVPIVSPQQCNND 180
YASYGGITENMICAAEEQGGKDPCQGDSSGPLTVGGQLAGIVSWGAGCAQRGYPGVYSNV 240
ATLRKFITDETGVN- 254
The clone of Eupolyphaga Seu Steleophaga antithrombotic enzyme Eupolytin1 gene comprises: the total RNA of Eupolyphaga Seu Steleophaga digestive tube midgut extracts, the mRNA purifying, and mRNA reverse transcription and cDNA library construction, the design primer utilizes PCR method amplification coding gene.Gene sequencing result shows that the encoding sox (GenBankaccession Gu187070) of Eupolyphaga Seu Steleophaga antithrombotic enzyme Eupolytin1 is made up of 888 Nucleotide; The sophisticated Eupolytin1 albumen of 85-765 position nucleotide coding, 5 ' end to 3 ' terminal sequence of this gene is (nucleotides sequence is classified SEQ ID NO:2 as):
atgcttcgtctgctggttctgacctgtcttgttgcctcctgcttcggcgcccgcctacct 60
aggcgtcctcgtcttgatggacgcattgttggtggttctgatgccaatattgaagatctt 120
ccctaccagctctcttttgagtacggtggcagtcacatgtgcggtgcctctatcatcagt 180
gagaactgggtagtgaccgcagcccactgtgtggatggtgtttcagcaagcagtgccagg 240
ttccgtgcgggaagcagcgtcagaggcagtggcggttccctacaccaagcaacccaacta 300
attgccaacccccaatacgactactacaccattgattacgatgtcgctgttgctagggta 360
gctactccattcagctacggaagcggagttcagcccattagccttatctcagtagaacct 420
tctgctggacaaactgcaacagtaagtggctggggaacttttttttctggcggatcactt 480
cccagacaactgcaagtggtgtccgtccctattgttagcccccagcaatgcaacaatgac 540
tatgcttcatacggtggaattacagagaacatgatttgcgctgctgaggagcaaggaggc 600
aaggacccatgccaaggtgactcaagcggccctctcactgtcgggggacaactcgccgga 660
attgtatcttggggcgcaggttgtgcgcagagaggttacccaggtgtttactccaatgtt 720
gcaaccctaaggaagttcatcaccgacgagactggagtcaactaaagtttaatatgtttg 780
atctttatgtaagtttgtgaattgtgtgacatttacaatgctgtacatacgttcaaatat 840
attaaaatttttaaaagcaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa 888
Natural origin of the present invention and recombinant expressed Eupolyphaga Seu Steleophaga antithrombotic enzyme Eupolytin1 external, have significant hydrolysis of fibrin/plasminogen and the directly effect of plasminogen activation.In vivo, concentration is that the Eupolytin1 of 0.01-0.06 μ mol handles after 48 hours and can remove X 5189 inductive caudal vein thrombus fully; When rat arteriovenous shut thrombosis was tested, concentration was that the Eupolytin1 of 0.06 μ mol/kg can make thrombus weight reduce to 1 ± 0.2mg, and the urokinase of same concentrations can only make thrombus weight reduce to 1.7 ± 0.3mg; When the intravenous injection sample concentration rose to 0.12 μ mol/kg, rabbit did not have bleeding.So Eupolytin1 can being employed as preparation treatment thrombotic diseases medicine.
Description of drawings:
Fig. 1 .A is the CM-Sephadex-C25 ion exchange chromatography of Eupolyphaga Seu Steleophaga digestive tube midgut centrifuged supernatant; Fig. 1 .B is the protein-active peak further Sephadex of the mistake G-75 gel permeation chromatography that obtains behind the CM-Sephadex-C25 ion exchange chromatography of Eupolyphaga Seu Steleophaga digestive tube midgut centrifuged supernatant; Fig. 1 .C is that the active peak of Sephadex G-75 gel permeation chromatography is after fast liquid chromatography Resource Q; Fig. 1 .D is that the active peak of fast liquid chromatography Resource Q is after fast liquid chromatography Mono Q; Obtain the Eupolytin1 of purifying at last, arrow is depicted as the target protein of purifying in wherein scheming.Reduction and the non-reduced SDS-PAGE electrophorogram of also having the Eupolytin1 of purifying among Fig. 1 .D
Fig. 2 .A is the former effect of Eupolytin1 hydrolysis of fibrin of natural among the present invention and reorganization, and wherein NE represents natural Eupolytin1, the Eupolytin1 of RE representative reorganization.Fig. 2 .B is the influence of metals ion to Eupolytin1 hydrolysis of fibrin former (fibrinogen) effect, and Fig. 2 .C is the influence of proteinase inhibitor to the former effect of Eupolytin1 hydrolysis of fibrin.
Fig. 3 .A is the dose-effect relationship of Eupolytin1 hydrolysis of fibrin former (Fibrinogen) effect natural among the present invention, and Fig. 3 .B is the timing relationship of the natural former effect of Eupolytin1 hydrolysis of fibrin.
Fig. 4. be the effect of Eupolytin1 plasminogen activation and hydrolysis of fibrin; Fig. 4 .A is Profibrinolysin (P); Fig. 4 .B is Eupolytin1 (E4), and Fig. 4 .C is that Eupolytin1 and Profibrinolysin are hatched (E+P) jointly, and Fig. 4 .D is that urokinase and Profibrinolysin are hatched (U+P) jointly.
Fig. 5. be antithrombotic Function detection in the Eupolytin1 body.Fig. 5 .A is the anti thrombotic action of Eupolytin1 in the experiment of rat arteriovenous shut thrombosis, and Fig. 5 .B is the anti thrombotic action of Eupolytin1 in the experiment of X 5189 inductive caudal vein thrombus.
Embodiment:
Embodiment one: preparation and the determined amino acid sequence of Eupolyphaga Seu Steleophaga antithrombotic enzyme Eupolytin1
One, the dissection of Eupolyphaga Seu Steleophaga digestive tube midgut, homogenate and processing
The method that Eupolyphaga Seu Steleophaga (Eupolyphaga sinensis) is dissected is following: with little scissors polypide is cut off along dorsomeson; Choose digestive tube with tweezers; Cut the midgut part, put into acetic acid-sodium-acetate buffer of 0.05M pH5.0, after all Eupolyphaga Seu Steleophaga samples are dissected and finished; The centrifugal 10min of 5000g, supernatant is in-80 ℃ of preservations.
Two, the separation and purification of Eupolyphaga Seu Steleophaga antithrombotic enzyme Eupolytin1
Supernatant with above-mentioned collection is a raw material, and according to following program purifying Eupolytin1, each step of separation and purification is all carried out the Fibrinogen hydrolytic activity and detected, and concrete grammar is following:
A.CM-Sephadex-C25 ion exchange chromatography: Eupolyphaga Seu Steleophaga digestive tube midgut centrifuged supernatant; Be splined on the CM-Sephadex-C25 ion exchange chromatography (U.S. GE Health product, 2.6 * 40cm) posts are with acetic acid-sodium-acetate buffer wash-out of 0.05M pH5.0; Automatically Fraction Collector is collected; Flow velocity is 3.0ml/10min, and shown in Fig. 1 .A, each merges, and the peak part detects the little peptide substrates activity of serine stretch protein enzymic hydrolysis respectively, blood coagulation is active and the Fibrinogen hydrolytic activity.
B.Sephadex G-75 gel permeation chromatography: the active peak that the CM-Sephadex-C25 ion exchange chromatography obtains; Freeze-drying is dissolved in distilled water; Be splined on Sephadex G-75 gel permeation chromatography (GE Health product; 2.6 * 100cm) post, shown in Fig. 1 .B, each merges, and the peak part detects platelet aggregation activity respectively, the little peptide substrates of serine stretch protein enzymic hydrolysis is active, blood coagulation is active and the Fibrinogen hydrolytic activity.C.AKTA FPLC Resource Q:Sephadex G-75 gel permeation chromatography active part is in the 20mmol/L of pH 7.8 Tris-HCl damping fluid dialysis 16hr; This step operates in
Purifier system carries out; Linear gradient with 0-1mol/L NaCl is carried out wash-out; Flow velocity is 1ml/min; Shown in Fig. 1 .C, each merges, and the peak part detects platelet aggregation activity respectively, the little peptide substrates of serine stretch protein enzymic hydrolysis is active, blood coagulation is active and the Fibrinogen hydrolytic activity.
D.AKTA FPLC Mono Q:FPLC Resource Q active part is in the 20mmol/LTris-HCl of pH 8.3 damping fluid dialysis 16hr; This step operates in
Purifier system carries out; Linear gradient with 0-1mol/L NaCl is carried out wash-out; Flow velocity is 1ml/min; Shown in Fig. 1 .D, obtain the Eupolyphaga Seu Steleophaga antithrombotic enzyme of purifying, called after Eupolytin1.
E. the determined amino acid sequence of Eupolyphaga Seu Steleophaga antithrombotic enzyme Eupolytin1: the Eupolyphaga Seu Steleophaga antithrombotic enzyme Eupolytin1 after the FPLC that learns from else's experience separates carries out the SDS-PAGE electrophoresis, dyes with Xylene Brilliant Cyanine G R-250, obtains the homogeneous band behind the purifying, shown in Fig. 1 .D.The antithrombotic enzyme Eupolytin1 of purifying is carried out the order-checking of Edman edman degradation Edman, and recording its N terminal sequence is IVGGSDANIEDLPYQLSFE-.For obtaining more peptide segment information, with pancreatin antithrombotic enzyme Eupolytin1 is digested, measure the interior peptide sequence after the hydrolysis, obtained the aminoacid sequence of 5 peptide fragment altogether.Peptide section 1:TIDYDVAVAR; Peptide section 2:VATPFSYGSGVQ; Peptide section 3:QLQVVSVPIVSPQQCNNDYAS; Peptide section 4:DPCQGDSSGPLTVG; Peptide section 5:GYPGVYSNVATLR.
Embodiment two: the clone of Eupolyphaga Seu Steleophaga antithrombotic enzyme Eupolytin1 gene
One, the total RNA of Eupolyphaga Seu Steleophaga digestive tube midgut extracts
A. the vivisection Eupolyphaga Seu Steleophaga is got 60 Eupolyphaga Seu Steleophaga digestive tube midgut tissues, adds the 1mLTrizol extracting solution (American I nvitrogen company) of precooling, places homogenate on ice 15 minutes.
B. the chloroform that adds Trizol 1/5 volume, about 15 seconds of violent mixing, room temperature was placed 5 minutes, and 4 ℃, centrifugal 10 minutes of 12000rpm gets supernatant.
C. supernatant adds isopyknic Virahol, and room temperature was placed 10 minutes, and 4 ℃, centrifugal 10 minutes of 12000rpm, deposition is washed once with 75% ethanol, dries, and pipe end throw out is the total RNA of Eupolyphaga Seu Steleophaga digestive tube midgut.
Two, the purifying of Eupolyphaga Seu Steleophaga digestive tube midgut mRNA
PolyATtract
the mRNA Isolation Systems test kit of U.S. PROMEGA company is adopted in Eupolyphaga Seu Steleophaga digestive tube midgut mRNA separation and purification.
A. get the total RNA 500 μ g of Eupolyphaga Seu Steleophaga digestive tube midgut and be dissolved in the 500 μ L DEPC water, 65 ℃ of water-baths 10 minutes add Oligo (dT) probe and 13 μ L, the 20 * SSC solution of people 3 μ L, and mixing is placed the room temperature cooling, is called A liquid.
B. the washing of magnetic bead (SA-PMP): magnetic bead is flicked mixing,, abandon supernatant, add 0.5 * SSC, 300 μ L,, add 100 μ L, 0.5 * SSC at last and suspend, be called B liquid to magnetic force frame absorption 30 seconds to magnetic force frame absorption 30 seconds.
C. A liquid is added in the B liquid, room temperature was placed 10 minutes, to magnetic force frame absorption 30 seconds, abandoned supernatant; With 0.1 * SSC washing 4 times, abandon supernatant at last, add 100 μ L DEPC aqueous suspensions; Absorption is 30 seconds to the magnetic force frame, and supernatant is moved to new test tube, and it is resuspended to add 150 μ L DEPC water again; To magnetic force frame absorption 30 seconds, move supernatant to above-mentioned test tube, be the Eupolyphaga Seu Steleophaga digestive tube midgut mRNA of purifying.
D. the pH5.2 that adds 1/10 volume, the sodium acetate solution of 3M, the equal-volume Virahol, in-70 ℃ of placements 30 minutes, 4 ℃, centrifugal 10 minutes of 12000rpm abandoned supernatant, and resolution of precipitate is in 10 μ L DEPC water.
Three, Eupolyphaga Seu Steleophaga digestive tube midgut cDNA library construction
Adopt the CLONTECH Creator of company
TMSMART
TMCDNA Library Construction Kit makes up Eupolyphaga Seu Steleophaga digestive tube midgut cDNA library.
A.cDNA first chain is synthetic
Add 2 μ l Eupolyphaga Seu Steleophaga digestive tube midgut mRNA, 1 μ l SMART IV primer, 1 μ l CDSIII/3 ' PCR primer, 1 μ l RNA-free water in aseptic PCR pipe, make TV reach 5 μ l, mixing is also of short duration centrifugal.
1.72 ℃ insulation 2min is hatched 2min on ice.
2. in above-mentioned PCR pipe, add 2 μ l, 5 * the first chain buffer, 1 μ l 20mmol/L DTT, 1 μ l 10mmol/L dNTP mixture, 1 μ l PowerScript reversed transcriptive enzyme, mixing is also of short duration centrifugal.
3. after placing 42 ℃ of insulations of PCR appearance 1hr, ice bath stops the synthetic of first chain.
B. adopt long terminal polymerase chain reaction (LD-PCR) method amplification cDNA second chain
1. with 1 μ l cDNA, first chain, 40 μ l deionized waters, 5 μ l, 10 * Advantage 2PCR damping fluid, 1 μ l, 50 * dNTP mixture, 1 μ l, 5 ' PCR primer, 1 μ l CDS III/3 ' PCR primer and 1 μ l polysaccharase mixing in the PCR pipe.
2. in the PCR appearance, increase by following program: 95 ℃, 20sec; 22 circulations: 95 ℃, 5sec; 68 ℃, 6min.
3. after amplification finished, double-stranded cDNA placed-70 ℃ of preservations with synthetic.
C. the enzyme conversion cutting, connect and connect product:
1. in Eppendorf tube, add 1 μ L pMD19-T carrier (Japanese Takara company), the double-stranded solution of 4 μ L Eupolyphaga Seu Steleophaga digestive tube midgut cDNA, full dose is 5 μ L.
2. the ligase enzyme buffer mixture that adds 5 μ L (equivalent).
3.16 ℃ reaction 2 hours.
4. full dose (10 μ L) is added in the 100 μ L DH5 α competent cells (sky, Beijing root biochemical technology ltd), ice bath 30 minutes.
5.42 after 90 seconds of ℃ heating, in ice, placed 1 minute again.
6. add 37 ℃ of LB substratum 900 μ L of bathing of temperature, 37 ℃ of slow shaking culture 60 minutes.
7. get 200 μ L and coat on the LB substratum that contains X-Gal, IPTG, Amp 37 ℃ and cultivated 16 hours, form single bacterium colony.
8. each LB plate is with 5mL LB liquid nutrient medium washing bacterium colony, and it is frozen to add 30% glycerine.The cDNA that makes up approximately contains 2 * 10
6Individual independent clone.
Four, amplification of Eupolyphaga Seu Steleophaga antithrombotic enzyme Eupolytin1 gene order and mensuration:
Aminoacid sequence according to separation and purification gained Eupolyphaga Seu Steleophaga embolism-resisting enzyme Eupolytin1 among the embodiment one; We have designed a degenerated primer 1810-R; Employed joint primer 5 ' primer pairing during with structure Eupolyphaga Seu Steleophaga digestive tube midgut cDNA library, forward and reverse primer sequence is:
5’primer：5’-aag?cag?tgg?tat?caa?cgc?aga?gt-3’
1810-R：5’-at(a/t/c)gt(c/t)gg(c/t)ggt(a/t)(g/c)(c/t)gatgc-3’
Wherein, two bases in the bracket are represented degenerated primer.
1810-R and joint primer 5 ' primer pairing are used, and cDNA is a template with Eupolyphaga Seu Steleophaga digestive tube midgut, carries out PCR.Its reaction conditions is: 95 ℃ of preparatory sex change 4min, then carry out 35 in following condition and take turns circulation, 94 ℃ of 30sec, 55 ℃ of 30sec, 72 ℃ of 40sec, 72 ℃ of 10min then.The PCR product screens the upstream portion of the encoding sequence that has inserted the Eupolytin1 that comprises the signal peptide encoding sox through sequencing.
According to the nucleotide sequence design forward signal peptide primer 1810-F of above-mentioned acquisition, with 3 ' end connector primer CDS III/3 ' the PCR pairing that use in constructed cDNA library, forward and reverse primer sequence is respectively again:
1810-F：5’-atg?ctt?cgt?ctg?ctg?gtt?ctg?acc-3’；
CDS?III/3’PCR：5’-att?cta?gag?gcc?gag?gcg?gcc?atg-3’
1810-F and the pairing of CDS III/3 ' PCR primer are template with the Eupolyphaga Seu Steleophaga digestive tube midgut cDNA that makes up, and carry out pcr amplification.Its reaction conditions is: 95 ℃ of preparatory sex change 4min; 94 ℃ of 30sec then, 57 ℃ of 30sec, 72 ℃ of 60sec carry out 30 and take turns circulation; Last 72 ℃ of 10min.The PCR product is through sequencing; Can mate fully with 6 sections aminoacid sequences of acquisition among the embodiment one after the nucleotide sequence translation that obtains; Screened the global cDNA sequence of coding Eupolytin1 thus; The result shows that the cDNA sequence of the Eupolytin1 that encodes is made up of 888 Nucleotide, and 5 ' end to 3 ' terminal sequence is:
Wherein, italicized item is a signal peptide, and the Eupolytin1 that the part that adds frame and separation and purification obtain carries out the sequence that amino acid sequencing obtains and is complementary.
Embodiment three: the in-vitro recombination expression of Eupolytin1
A. the structure of recombinant plasmid pET-Eupolytin1
The first step: the primer with the band restriction enzyme site carries out gene amplification to goal gene
Utilization contain the primer 5 of enteropeptidase restriction enzyme site '-BD1810-1:5 '-
ATTGTTGGTGGTT-3 ' with contain the primer 3 of Hind III restriction enzyme site with the protection base '-BD1810-1:5 '-CCCCC
CTAGTTGACTCCAG-3 '; With Eupolyphaga Seu Steleophaga digestive tube midgut cDNA library is that template increases, and the PCR reaction conditions is: 95 ℃ of preparatory sex change 4min; 94 ℃ of 30 sec then, 57 ℃ of 30 sec, 72 ℃ of 60 sec carries out 30 and takes turns circulation; Last 72 ℃ of 10min.The PCR product will add the enteropeptidase restriction enzyme site at 5 ' end of goal gene, and ' end adds Hind III restriction enzyme site and protection base 3.
With last round of PCR product is template; Primer 5 '-BD1810-2:5 '-CGG
-3 ' that utilization contains KPN1 restriction enzyme site and protection base cooperates 3 '-the BDl810-1 primer uses; Carry out pcr amplification, the PCR reaction conditions is the same.The PCR product will add KPN1 restriction enzyme site and protection base at last round of product 5 ' end.
The PCR product is carried out 1% agarose gel electrophoresis, cut the purpose band, utilize gel DNA to reclaim test kit (sky, Beijing root biochemical technology ltd) and reclaim goal gene.
Second step: the preparation of DNA
The escherichia coli vector bacterial strain (U.S. Novagen company) of inoculation pET-32a plasmid contains in the LB substratum of ammonia benzyl in 5ml; Overnight cultures in 37 ℃ of shaking tables; Use plasmid extraction kit (sky, Beijing root biochemical technology ltd) to extract plasmid, concrete steps are following:
1) 5ml pET-32a overnight culture, 12000rpm, centrifugal 1 minute, complete supernatant discarded was in microorganism collection to a pipe.
2) column equilibration: adsorption column is put into collection tube, adds 500 μ l balance liquids, and centrifugal 1 minute of 12000rpm discards the liquid in the collection tube.
3) add 250 μ l solution P1 (2-8 ℃ of preservation), suspension thalline in the first thalline.Add 250 μ l solution P2, put upside down mixing 6-8 time, at this moment, Guan Zhongying is limpid liquid.Otherwise insufficient for cracking, should reduce biomass.In pipe, add 350 μ l solution P3 again, put upside down mixing 6-8 time, occur white precipitate in the pipe, centrifugal 10 minutes of 12000rpm.Draw supernatant to adsorption column, do not draw white precipitate.
4) 12000rpm is centrifugal 1 minute, discards the liquid in the collection tube.
5) in adsorption column, add 500 μ l protein liquid removal PD, centrifugal 1 minute of 12000rpm discards the liquid in the collection tube.
6) add 700 μ l rinsing liquid PW, 12000rpm centrifugal 30 seconds, abandon waste liquid.Add 500 μ l rinsing liquid PW again, 12000rpm centrifugal 30 seconds, abandon waste liquid.Centrifugal 2 minutes of 12000rpm.
7) adsorption column is put into a clean centrifuge tube, uncaps and dries.
8) elutriant needs preheating in 65-70 ℃ of water-bath in advance.Central authorities add 50 μ l elutriant EB to adsorption column.Room temperature held 2 minutes.Centrifugal 2 minutes of 12000rpm collects the liquid in the centrifuge tube, is institute's upgrading grain ,-20 ℃ of preservations.
The 3rd step: double digestion DNA and goal gene
At 37 ℃ of while double digestion (KPN1 and HindIII, TaKaRa company) pET-32a
+Plasmid with the goal gene of going up restriction enzyme site, the double digestion system is 20 μ l.Enzyme is cut product behind 1% agarose gel electrophoresis, cuts the purpose band, utilizes gel DNA to reclaim test kit (Beijing hundred Tyke bio tech ltds) and reclaims the double digestion product, and it is subsequent use to put-20 ℃ of preservations.
The 4th step: enzyme is cut the connection of product
With the linear pET-32a of the double digestion product that is recovered to
+Plasmid and goal gene T4DNA ligase enzyme (T4DNA Ligase, Takara) effect connects down, and 16 ℃ of connections are spent the night.
B. the conversion of recombinant plasmid
To connect product is converted into and uses CaCl
2-MgCl
2E. coli bl21 (DE3) competent cell of method preparation; Getting an amount of converted product is applied on the LB culture medium flat plate that contains 100 μ g/ml Amp; Cultivate 16hr through 37 ℃; Detect with PCR, positive colony is sent biotech firm to check order and confirms to include digestive tube midgut antithrombotic enzyme Eupolytin1 gene order, and contain the enteropeptidase restriction enzyme site sequence of joint and reorganization.
C. recombinant protein abduction delivering
The picking recombinant clone is seeded to the liquid LB substratum that contains 100 μ g/ml Amp, and 37 ℃ of overnight cultures are got above-mentioned bacterium liquid next day and are seeded at 1: 100 in proportion in the fresh LB substratum of 1000ml, and 37 ℃ of about 3hr of shaking culture make OD
600Reach 0.6, adding IPTG is 0.4mmol/L to final concentration, and 28 ℃ are continued cultivation and induce 3hr.
D. the separation and purification of recombination expression product rEupolytin1 (recombinated Eupolytin1)
The first step: the His-Tag fusion rotein that contains rEupolytin1 separates
Every liter of induced product is in 8000rpm, and centrifugal 10min gathers in the crops thalline.With thalline with 100ml Binding Buffer (pH 8.0,0.1mol/L PBS) resuspended after, ultrasonic degradation (ultra 3sec; Stop 3sec, altogether 10min), the centrifugal 10min of 5000g; Abandon supernatant; The induced precipitation thing of collecting is resuspended in (every liter of tunning adds 50ml Binding Buffer) among the Binding Buffer, and ultrasonic then, centrifugal, condition is the same.The ultrasonic degradation deposition is resuspended with the Binding Buffer that contains 6mol/L urea; Ice bath 60min; 4 ℃, the centrifugal 30min of 16000rpm is through 0.45 μ m membrane filtration; Be splined on the His Bind Resin that uses sex change Binding Buffer balance good (German Merck company) affinity column, collect the His-Tag fusion rotein.
Second step: the renaturation of inclusion body
Method with gradient dilution is carried out renaturation to inclusion body, and dialysis buffer liquid is followed successively by the Tris-HCl of the 20mmol/L that contains 6mol/L urea, and pH 6.0; The Tris-HCl that contains the 20mmol/L of 4mol/L urea, pH 6.5; The Tris-HCl that contains the 20mmol/L of 3mol/L urea, pH 6.8; The Tris-HCl that contains the 20mmol/L of 2mol/L urea, pH 7.2; The Tris-HCl that contains the 20mmol/L of 1mol/L urea, pH 7.4; The Tris-HCl of urea-containing 20mmol/L not, pH7.4, damping fluid is changed in each dialysis approximately twice, each about 5hr of the timed interval.
The 3rd step: the enteropeptidase cutting discharges rEupolytin1
Carry out in the Tris-HCL damping fluid of the 20mmol/L that enzyme is cut at pH7.4.The ratio of enteropeptidase and target protein is 1: 250 (w/w), hatches 8hr for 23 ℃.
The 4th step: the separation and purification of rEupolytin1
Enzyme is cut product behind 20mmol/L Tris-HCl pH 8.3 damping fluids dialysis 12hr; Be splined on AKTA FPLC Mono Q post; Linear gradient with 0-1mol/L NaCl is carried out wash-out; Remove enteropeptidase and fusion rotein, obtain recombinant expressed rEupolytin1, and detect it and have the former activity of hydrolysis of fibrin.
Embodiment four: the pharmacological evaluation of Eupolyphaga Seu Steleophaga antithrombotic enzyme Eupolytin1
The Eupolyphaga Seu Steleophaga antithrombotic enzyme Eupolytin1 that gets the separation and purification acquisition carries out following pharmacologically active and detects.
One, the Fibrinogen hydrolytic activity detects
Get the Fibrinogen that 10 μ L concentration are 2mg/mL (saline water preparation) (U.S. Sigma company), after 1 hour, carry out the 12%SDS-PAGE electrophoretic analysis in 37 ℃ of insulations then with Eupolytin1 (natural purifying protein and the recombinant expression protein) sample of 1 μ g.Experimental result show antithrombotic enzyme Eupolytin1 can be fast the former α-chain of hydrolysis of fibrin up hill and dale, beta chain and γ chain are shown in Fig. 2 .A.
1) proteinase inhibitor and metals ion are to the influence of the former effect of Eupolytin1 hydrolysis of fibrin
The result shows Ca
2+And Mg
2+The effect that minimal effect Eupolytin1 hydrolysis of fibrin is former, Zn
2+And Cu
2+Part suppresses the former effect of Eupolytin1 hydrolysis of fibrin, shown in Fig. 2 .B; Proteinase inhibitor PMSF can significantly suppress the activity of Eupolytin1, yet inhibitors of metalloproteinase EDTA does not have influence to Eupolytin1, shown in Fig. 2 .C.
2) dose-effect relationship of the former effect of Eupolytin1 hydrolysis of fibrin and timing relationship
Dosage is 0.2 μ g, 0.4 μ g, 0.6 μ g; 0.8 μ g; 1.0 the Eupolytin1 of μ g and 1.2 μ g is 37 ℃ of common incubations of Fibrinogen (U.S. Sigma company) of 2mg/mL (saline water preparation) after 1 hour with 10 μ L concentration respectively, the SDS-PAGE electrophoretic analysis is shown in Fig. 3 .A.Dosage is that Eupolytin1 and the 10 μ L concentration of 0.4 μ g are the common incubation 5min of Fibrinogen of 2mg/mL (saline water preparation), 10min, and 15min, 20min, 30min, 40min, 50min, behind the 90min, the SDS-PAGE electrophoretic analysis is shown in Fig. 3 .B.The Eupolytin1 that the result shows 1 μ g just can complete hydrolysis in 90min falls α-chain, beta chain and the γ chain of the plasminogen of 20 μ g.
Two, the effect of Eupolytin1 plasminogen activation and hydrolysis of fibrin
1ml plasminogen (5mg/ml); 1ml contains 1% agarose solution of 1 unit zymoplasm (by 20mM Tris-HCl; PH 7.6 and contain the damping fluid preparation of 0.1M NaCl), said mixture adds in 12 orifice plates, room temperature was placed after 1 hour; Add a, Profibrinolysin b, sample c, sample+Profibrinolysin d, urokinase+Profibrinolysin respectively, as shown in Figure 4.Experimental result shows that Eupolytin1 has the effect of plasminogen activation and hydrolysis of fibrin.
Three, Eupolytin1 anti-thrombus function in animal body detects
1) anti thrombotic action of Eupolytin1 in the experiment of rat arteriovenous shut thrombosis
180-220g rat (9 every group) intraperitoneal injection of ketamine (50mg/kg) and xylazine (15mgkg); Scratch skin of neck, peel off left jugular vein and RCCA, (wherein two are the long polyethylene tube (PE-60) of 10cm to insert a trident shunt catheter; Connect left jugular vein and RCCA respectively; Middle one is the long polyethylene tube (PE-160) of 3cm), place the coarse nylon wire (60mm is long, and diameter is 0.26mm) that is folded into 2 sections in the intermediary polyethylene tube.The sample of vena femoralis injection different concns.The result shows that Eupolytin1 concentration is that the Eupolytin1 of 0.06 μ mol/kg can make thrombus weight reduce to 1 ± 0.2mg, and the urokinase of same concentrations can only make thrombus weight reduce to 1.7 ± 0.3mg.
2) anti thrombotic action of Eupolytin1 in the experiment of X 5189 inductive caudal vein thrombus
X 5189 causes the anti-thrombus function that mouse tail thrombus model detects Eupolyphaga Seu Steleophaga embolism-resisting enzyme Eupolytin1.Get 30 Male Kunming strain mice (body weight 17-22g, unming Medical College's Experimental Animal Center provides) and be divided into 3 groups (n=10) at random, the 1st group is physiology saline control group; 2nd, 3 groups give 0.01-0.06 μ mol sample respectively, behind the processing 30min, with physiological saline solution X 5189 (Carrageenan; Type I; U.S. Sigma company), concentration is 1%, the mouse peritoneal injection.After 6 hours, the tail vein is injected the sample with dosage once more.After 48 hours, judge thrombotic occurrence rate and mean length according to the tail skin colour-change.The result shows that Eupolytin1 can obviously suppress the mouse tail thrombosis in the animal model of selecting for use.
Pharmacological evaluation shows in the body of Eupolyphaga Seu Steleophaga antithrombotic enzyme: concentration is that the Eupolytin1 processing of 0.01-0.06 μ mol can be removed X 5189 inductive caudal vein thrombus after 48 hours fully; When rat arteriovenous shut thrombosis was tested, concentration was that the Eupolytin1 of 0.06 μ mol/kg can make thrombus weight reduce to 1 ± 0.2mg, and the urokinase of same concentrations can only make thrombus weight reduce to 1.7 ± 0.3mg.Eupolyphaga Seu Steleophaga antithrombotic enzyme Eupolytin1 can being employed as preparation treatment thrombotic diseases medicine.
Claims (4)
1. Eupolyphaga Seu Steleophaga antithrombotic enzyme Eupolytin1 is characterized in that this albumen is from Eupolyphaga Seu Steleophaga digestive tube midgut, to separate a kind of active protein that obtains, and is made up of 226 amino acid, and molecular weight 26KDa, its aminoacid sequence are shown in the SEQ ID NO:1.
2. the gene of coding Eupolyphaga Seu Steleophaga antithrombotic enzyme Eupolytin1 is characterized in that GenBank accession Gu187070 is made up of 888 nucleotide sequences, and its nucleotides sequence is classified as shown in the SEQ ID NO:2.
3. like the said gene of claim 2, the sophisticated Eupolytin1 albumen of 85-765 position nucleotide coding in its SEQ ID NO:2 sequence.
4. the application of the described Eupolyphaga Seu Steleophaga antithrombotic of claim 1 enzyme Eupolytin1 in preparation treatment thrombotic diseases medicine.
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