CN1259411C - Strain of Pichia yeast genetic engineering, preparation method and application - Google Patents
Strain of Pichia yeast genetic engineering, preparation method and application Download PDFInfo
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Abstract
The present invention discloses a genetic engineering strain of Pichia pastoris, a preparing method thereof and applications thereof. The strain is Pichia pastoris X-33(pPIC6A-EFE), CCTCC M204004. The preparing method comprises the following steps: firstly, carrying out PCR amplification to earthworm fibrinolysin; secondly, cloning a PCR product to a T carrier; thirdly, constructing shuttle expression plasmid; fourthly, constructing a genetic engineering yeast strain for expressing fibrinolysin genes. The present invention has the advantages of simple operation, clear lysis zone, easy observation and good repeatability.
Description
Technical field
The present invention relates to a kind of pichia pastoris gene engineering bacterial strain of expressing the earthworm fibrinolysin gene, also relate to the preparation method for preparing pichia pastoris gene engineering bacterial strain and the application in drug development simultaneously.
Background technology
Earthworm is commonly called as earthworm, and at the applicating history in existing thousands of years of China, but heat-clearing, flat liver, Zhichuan, vein relaxing cure mainly illnesss such as high fanatical hot-tempered, convulsion with spasms.Nineteen eighty-three, Japan H.Mihara found that first Lumbricidae (Lumbrici dae) earthworm aqueous extract has direct solution fibrin and Profibrinolysin activation, its activeconstituents is called plasmin (earthworm fibrinolytic enzyme, EFE), claim Lumbrukinase again.
At present existing multiple earthworm fibrinolysin preparation is applied to clinical, is mainly various capsule preparations, all extracts from earthworm enteric cavity and body fluid,, hundred Austria multiple, Bo Luoke as general grace, and thrombolysis capsules etc. are used in the cardiovascular and cerebrovascular diseases aspect more.
Traditional earthworm fibrinolysin capsule preparations is applied to clinical certain curative effect is arranged, but also has obvious defects, at first is complicated component, can not intravenous injection, and the oral administration effect is slow, thereby can not be applied to treat the acute heart, cerebral vessels embolism disease; Secondly capsule complex manufacturing technology is produced the restriction that is subjected to vermiculture season, cycle, product production, quality instability; Separation and purification plasmin technology is loaded down with trivial details from polypide, may contain various micromolecule polypeptides or other chemical substance in the product, may cause the anaphylaxis of body.
Summary of the invention
The purpose of this invention is to provide a kind of Pichi strain Pichia pastoris X-33 (pPIC6A-EFE), this bacterial strain can be expressed and be produced the genetically engineered plasmin, and this bacterium cost is low, and large scale fermentation is convenient for production, is easy to realize industrialization.
Another object of the present invention provides the method for preparing Pichi strain, and this method is easy and simple to handle, and the dissolving circle is clear, is easy to observe good reproducibility.
The invention still further relates to the purposes of genetically engineered plasmin in thrombolytic drug, can be used for preparing the medicinal preparation for oral administration of treatment embolic cardiovascular disorder, also can be used for producing healthcare products with prevention thrombus disease, useful cardiovascular health.
Earthworm fibrinolysin involved in the present invention is from Lumbricidae powder Lumbricus, nucleotide sequence of a kind of Lumbricidae powder Lumbricus earthworm fibrinolysin gene and preparation method thereof is provided in one embodiment of the invention, this method is to be template with the total RNA of Lumbricidae powder Lumbricus earthworm, use the specific oligonucleotide primer, obtain fibrinolytic enzyme gene cDNA through the reverse transcription PCR method.The positive earthworm of powder is purchased in Hua Zhong Agriculture University, and gene sequencing result shows that cDNA is made up of 747 Nucleotide, and 5 ' end to 3 ' terminal sequence is:
The nucleotide sequence (SEQ ID No.1) of Lumbricidae powder Lumbricus earthworm fibrinolysin gene
acatggaacttcctcccggaacaaaaattgtcggaggaattgaagctagaccatacgagttcccatggcaggtgtccgtccgaaggaaatctt
ccgattcccatttctgcggaggtagcatcatcaacgatcgttgggttgtctgcgctgctcactgcatgcagggagaggcccccgctctggtttca
ttggtcgtgggtgagcacgacaggagtgcagcgagtacagtacgtcagactcatgacgttgatagcatcttcgttcacgaggactacaacaca
aataccctagagaacgacgtttctgtcatcaagacatctgttgccatcactttcgacatcaacgttggtccaatctgcgccccagatccggctaa
cgactacgtctaccgtaagagccagtgttccggatggggaactatcaattcaggtggaatctgctgtcccaacgttctgcgatacgtgacgctg
aatgacacaaccaaccaatactgcgaagatgtatacccactaaattcaatctacgacgatatgatttgcgcgtcggacaacactgggggtaac
gacagagactcctgccagggtgactccggcggccctctgagcgtcaaggatggtagtggaatcttcagcctgattggtattgtgtcttgggga
attggttgcgcttctggctatccaggagtctactcccgcgtcggattccatgctgcatggatcaccgacatcatcaccaacaactaaaccg
Wherein 3-740 position Nucleotide is the mature peptide sequence of genes encoding, i.e. plasmin protein sequence, and the 741-743 position is terminator codon TAA.The protein amino acid sequence of this genes encoding is:
The aminoacid sequence (SEQ ID No.2) of Lumbricidae powder Lumbricus earthworm fibrinolysin gene
MELPPGTKIVGGIEARPYEFPWQVSVRRKSSDSHFCGGSIINDRWVVCAAHCMQGEAPAL
VSLVVGEHDRSAASTVRQTHDVDSIFVHEDYNTNTLENDVSVIKTSVAITFDINVGPICAP
DPANDYVYRKSQCSGWGTINSGGICCPNVLRYVTLNDTTNQYCEDVYPLNSIYDDMICAS
DNTGGNDRDSCQGDSGGPLSVKDGSGIFSLIGIVSWGIGCASGYPGVYSRVGFHAAWITDI
ITNN
The plasmin molecular weight of albumen that the present invention relates to is 26.4kD, and iso-electric point is 4.5, is rich in glycine, Serine, Xie Ansuan and aspartic acid.Its biochemical characteristic also has: 1. thermostability: 60 ℃ of heating of plasmin are after 30 minutes, enzymic activity does not obviously reduce, enzymic activity begins to descend more than 65 ℃, 30 minutes enzymic activitys of 85 ℃ of heating completely lose, 2. acid acceptance: plasmin does not have considerable change in pH1～11 enzymic activity, and pH is lower than 1 or be higher than at 11 o'clock, and enzymic activity obviously descends, best fibrinolytic reaction pH value a wider range is between 7～10.
A kind of preparation of Lumbricidae powder Lumbricus earthworm fibrinolysin gene is provided in one embodiment of the invention and has been cloned into the method for intestinal bacteria (E.coli), this method comprises: amplimer is synthetic, the total RNA of earthworm extracts, the reverse transcription of mRNA, the pcr amplification of goal gene, the recovery of target gene fragment, target gene fragment are cloned into carrier, earthworm fibrinolysin cDNA sequencing.
(1) amplimer is synthetic: according to the earthworm fibrinolysin cDNA sequences Design PCR oligonucleotide amplimer of having delivered among the GeneBank.Long 18 Nucleotide: the ACATGGAACTTCCTCCCG of upstream primer (P1), long 19 Nucleotide: the ATCACCAACAACTAAACCG of downstream primer (P2).
(2) the total RNA of earthworm extracts: get 3～5 earthworm adults, after the aqua sterilisa rinsing of handling with DEPC, be placed in the plate that is covered with the filter paper that wets hunger and spend the night, make it tell intravital to the greatest extent silt as much as possible.After cleaning up with DEPC water once more, in liquid nitrogen, be ground into powder, extract total RNA according to the product description of the RNeasyR Mini Kit of QIAGEN company.The RNA solution that extracts is stored in-80 ℃, with standby.
(3) the synthetic cDNA article one chain of reverse transcription: the ReverseTranscription Reaction test kit specification sheets according to Promega company carries out, and is synthetic cDNA first chain of primer with Oligo (dT) 20.Reaction conditions is: 42 ℃ 1 hour; 95 ℃ 5 minutes; 3 ℃ 5 minutes.
(4) pcr amplification of goal gene: carry out the PCR reaction according to the PCR reaction system parameter that provides in the Reverse Transcription Reaction test kit.Its reaction parameter is: 95 ℃ of pre-sex change 4 minutes; 94 ℃ of sex change 1 minute, 55 ℃ of annealing 1.5 minutes, 72 ℃ were extended 20 circulations 2 minutes; 94 ℃ of sex change 1 minute, 55 ℃ of annealing 1.5 minutes, 72 ℃ were extended 10 circulations 2 minutes; 72 ℃ were extended 10 minutes again.
(5) recovery of target gene fragment: reclaim the test kit specification sheets according to Shanghai China Shun PCR of bio-engineering corporation product and reclaim target gene fragment.
(6) target gene fragment is cloned into carrier: the PCR product of recovery is connected with the T carrier, transformed competence colibacillus cell intestinal bacteria (E.coli) JM109, picking colony rapid extraction plasmid carries out enzyme and cuts evaluation, resulting positive colony is to comprise the intestinal bacteria that the present invention relates to gene, is used for the propagation and the preservation of gene.
(7) earthworm fibrinolysin cDNA sequencing
Extract the plasmid DNA dideoxy method and measure nucleotide sequence, sequencing primer is the M13 promoter primer.The sequencing result of Lumbricidae powder Lumbricus earthworm fibrinolysin gene nucleotide: long 747 Nucleotide of earthworm fibrinolysin cDNA sequence, adenosine (A), cytidine(C (C), the fast content of holding in the mouth or the eyes nucleosides (G) and thymidine (T) of bird are respectively: A-185, C-197, G-189; T-176.Gene mature peptide sequence is a 3-740 position Nucleotide, 246 amino acid of encoding, and all the other nucleotide sequences are non-coding region.This protein cDNA mature peptide terminator codon is positioned at gene 741-743 position, and termination codon is TAA.
One of technical essential of the present invention is the structure and the abduction delivering of expressing the pichia pastoris gene engineering bacterial strain of fibrinolytic enzyme gene.
Finishing red (Pichia) yeast expression system is a kind of eukaryotic expression system efficiently that development in recent years is got up, it has the following advantages: (1) expression amount height, because pichia spp fast growth, and the expression vector use is strong promoter---alcohol oxidase promoter, the exogenous protein expression amount is very high.(2) good stability does not exist because the expression vector of this system is not plasmid with self-replicating in yeast, but by stable being incorporated on the yeast chromosomal of homologous recombination, so the engineering strain that makes up is very stable.(3) active high.The largest benefit of foreign protein secreting, expressing in pichia spp can be carried out the protein translation post-treatment exactly, comprises formation, protein folding and the glycosylation etc. of disulfide linkage.Compare with some other eukaryotic expression systems such as cereuisiae fermentum, pichia spp more approaches mammiferous glycosylation to the foreign protein glycosylation, so the glycoprotein of Pichia anomala expression is considered to the human body that is used for preferably.(4) be convenient to suitability for industrialized production.That pichia spp has is ready-made, sophisticated fermentation technique carries out enlarged culturing, and pichia yeast expression system can utilize methyl alcohol to grow as sole carbon source, and large scale fermentation is convenient for production, and cost is low, is easy to realize industrialization.
The earthworm fibrinolysin that the present invention relates to derives from the positive earthworm of powder (Lumbricus rubellus), and this fragment has 741bp, 246 amino acid of encoding.The present invention has made up a strain pichia pastoris gene engineering bacterial strain, obtains genetically engineered plasmin albumen by the method production of fermenting, and the fibrin plate method has proved that this albumen has fibrinolytic biological activity.
It is pichia spp Pichia pastoris X-33 (pPIC 6A-EFE) that a kind of structure engineering strain, bacterial strain are provided in one embodiment of the invention, CCTCC M204004.Express the method for earthworm fibrinolysin gene in Pichia yeast, this method is to express the earthworm fibrinolysin gene with pPIC 6A carrier (Invitrogen company) in pichia spp X-33, comprising: the pcr amplification of (1) earthworm fibrinolysin gene; (2) the PCR product cloning is to the T carrier; (3) structure of shuttle expression plasmid; (4) structure of the Yeast gene engineering bacterial strain of expression fibrinolytic enzyme gene, its step is as follows:
(1) pcr amplification of earthworm fibrinolysin gene: with the T vector plasmid that contains fibrinolytic enzyme gene is template, according to the design of the cloning site on EFE mature peptide and expression vector pPIC6A primer, and upstream primer P1:
GAATTCGCATGGAACTTCCTCCCG (the underscore place is restriction endonuclease EcoRI site), downstream primer P2:
TCTAGACGGTTTAGTTGTTGGTGAT (the underscore place is restriction endonuclease Xba I site).
(2) the PCR product cloning is to the T carrier: reclaim the gene fragment in the PCR product, and clone the carrier in pGEM-T, (pGEM-T-EFE2) changes in the e. coli jm109 with this recombinant vectors.
(3) structure of shuttle expression plasmid: extract recombinant vectors pGEM-T-EFE2, with EcoR I and Xba I double digestion to obtain purpose fragment EFE; Same enzyme is cut the pPIC6A empty carrier and make it dephosphorylation under the CIAP effect.With the EFE that obtains behind linearizing pPIC6A empty carrier behind the dephosphorylation and the double digestion under the effect of T4DNA ligase enzyme 4 ℃ spend the night, to obtain recombinant expression vector pPIC6A-EFE, this recombinant vectors is changed in the e. coli jm109 increases.
(4) structure of the Yeast gene engineering bacterial strain of expression fibrinolytic enzyme gene;
A. linearization of shuttle expression plasmid: extract recombinant plasmid pPIC6A-EFE, be cut into linearity with the SacI enzyme, enzyme is cut empty plasmid pPIC6A in contrast equally.
B. transform: method is with reference to Invitrogen company expression vector specification sheets: utilize biochemical method that linearizing recombinant plasmid pPIC6A-EFE and empty plasmid pPIC6A are transformed the X-33 yeast strain, on the YPD that contains blasticidin (Blasticidin) 300mg/ml (1% yeast extract, 2% peptone, 2% glucose) solid medium, cultivated 2-3 days, up to there being single bacterium colony to occur.
C. screen positive transformant: picking 10-20 single bacterium colony 30C in the YPD liquid nutrient medium, 250rpm shaking table cultivated until logarithmic growth late period, extracted the PCR that genome carries out recon and identified.The universal primer that primer provides for Invitrogen company expression vector, its sequence is upstream: GACTGGTTCCAATTGACAAGC, downstream: GCAAATGGCATTCTGACATCC, the positive colony called after Pichia pastoris X-33 (pPIC6A-EFE) that identifies, typical culture collection center deposit number is CCTCC M204004 in China
Two of technical essential of the present invention is genetically engineered plasmin induction expression of protein and determination of activity.
The gene A OX1 and the AOX2 of two coding alcohol oxidases are arranged in pichia spp, can utilize methyl alcohol to grow fast, and when having other carbon sources in the substratum, the AOX gene is not expressed as sole carbon source.In pichia pastoris engineered strain, foreign gene is cloned into the control of AOX1 promotor down, must also be that sole carbon source is realized efficiently expressing of foreign protein simultaneously with methyl alcohol as inductor therefore, when abduction delivering, should replenish methyl alcohol to keep its content of 0.5%.
A kind of method of pichia pastoris engineered strain abduction delivering is provided in an embodiment of the present invention, the substratum that can be used for the pichia pastoris engineered strain abduction delivering includes but are not limited to following substratum: MGY, MM, BMM, BMMY, the collocation method of various substratum is the method that present technique field personnel are familiar with very much, can obtain detailed information by the reference pertinent literature.
The pichia pastoris engineered strain derivational expression method is as follows: single bacterium colony of picking reorganization bacterium is to the 20mlYPD liquid nutrient medium, and 30 ℃, 250rpm cultivation activate; 30 ℃ of the bacterium liquid switching 500ml MGY liquid nutrient mediums, the 250rpm that get after 16 hours after 10ml activates cultivate enrichment; The bacterium liquid of enrichment after 24 hours is through centrifugal collection thalline (1500g, 10 minutes, 4 ℃); The thalline of collecting is resuspended in 1000ml MM liquid nutrient medium carries out abduction delivering, it is 0.5% that every 24h adds methyl alcohol to final concentration; Induce centrifugal collection thalline behind the 96h (30000g, 20 minutes, 4 ℃), thalline adopts the granulated glass sphere crush method to prepare the protein extract to be used for determination of activity.
The current national standard of still not having the earthworm fibrinolysin determination of activity, current measuring method can only be with reference to the activity determination method of other thrombolytic drug.Such as the bubble rise method of urokinase activity and arginine esterase method (TAME) method of mensuration Ahylysantinfarctase, but all inapplicable mensuration earthworm fibrinolysin of these two kinds of methods, because plasmin has the effect of plasminogen activator and proteolytic ferment, when thrombus, can bring into play this two kinds of functions, and the antiplasmin activity of this enzyme is significantly greater than kinase activity.
The fibrin plate method can be expressed the ability of plasmin thrombolysis objectively.Contain proplasmin in the fibrin plate, under the zymogenesis of plasmin, change Tryptase into.Answer the lyase effect of plasmin in addition, make fibrinogen generation hydrolytic action, become soluble small molecular peptide and amino acid, the application of sample place is dull and stereotyped to form translucent solusphere thereby make, and determines the activity unit of enzyme according to the size of circle.Within the specific limits, dull and stereotyped solusphere size and concentration journey linear dependence, the activity of therefore measuring fibrin plate can be regarded the dual function of plasmin and activating enzyme as.Use engineered method then can obtain plasmins in a large number by simple fermentation, separation purifying technique is simple, and the product purity height is at the bottom of the cost.This method has simple to operate, and dissolving circle is clear, be easy to observe advantages such as good reproducibility.
The method that provides a kind of fibrin plate method to measure the biologic activity of plasmin in one embodiment of the invention, by determination of activity, the activity unit of engineering strain fermented liquid of the present invention surpasses 2,900,000U/L.
Engineered protein involved in the present invention has purposes widely in the research and development field of medical treatment, health, health-related medicine, can be used for preparing the tablet, suppository of treatment embolic cardiovascular disorder etc., also can be used for producing healthcare products with prevention thrombus disease, useful cardiovascular health.
Compare with traditional earthworm fibrinolysin capsule preparations, genetically engineered drug involved in the present invention has following advantage: (1) capsule complex manufacturing technology, need artificial a large amount of breeding earthworm, separation and purification plasmin technology is loaded down with trivial details from polypide, various micromolecule polypeptides or other chemical substance may be contained in the product, the anaphylaxis of body may be caused.Use engineered method then can obtain plasmins in a large number by simple fermentation, separation purifying technique is simple, and the product purity height is at the bottom of the cost; (2) the thrombolysis class medicine of biological extraction needs charcoal absorption in process of production, mainly is to be used for decolouring, to adsorb thermal source, the removal of impurity and to help filter.Engineered protein purity height involved in the present invention, pure, the pyrogen-free of quality avoids taking off the pollution to environment of charcoal process and gac, and production cost is low.
The genetically engineered plasmin that the present invention produces can mix with acceptable carrier (as vehicle, weighting agent, absorption enhancer etc.) pharmaceutically, perhaps mixes use with other medicines, can be made into required formulation according to the ordinary method of pharmaceutical field.
Above-mentioned is medicine or other biological products of main active ingredient with the genetically engineered plasmin, its making processes or method are familiar with by this technical field personnel, production process should be carried out according to country relevant law, rules, comprise the Pharmacopoeia of the People's Republic of China (Chinese Pharmacopoeia Commission, 2000) and " Chinese biological goods rules " (the Chinese biological standard of articles council, 2000).
In an embodiment of the present invention, a kind of method for preparing genetically engineered plasmin thrombolytic drug preparation is provided, and in animal model, carried out the test of thrombolysis, the result shows that genetically engineered plasmin involved in the present invention has good thrombolytic effect, and these can say the purposes of genetically engineered plasmin in thrombolytic drug that relates to as illustrations the present invention.
A kind of engineering strain, pichia spp Pichia pastoris X-33 (pPIC6A-EFE) depositary institution: Chinese typical culture collection center, preservation date: on January 12nd, 2004 deposit number: CCTCC No:M204004.
Description of drawings
The pcr amplification result of accompanying drawing 1 fibrinolytic enzyme gene
The enzyme of accompanying drawing 2 recombinant plasmid pUCm-T-EFE is cut qualification result
The enzyme of accompanying drawing 3 recombinant expression vector pPIC6A-EFE is cut qualification result
Accompanying drawing 4 fibrin plate methods are measured the biologic activity of plasmin
A is a genetically engineered plasmin protein method fermented supernatant fluid; B is 10ul plasmin standard substance (10000U/ml); C is 10ul contrast bacterium pichia pastoris X-33 (pPIC6A) tunning.
Embodiment
The invention will be further described below in conjunction with drawings and Examples, substratum among all embodiment and molecular biology working method are familiar with by those skilled in the art, can " " (laboratory manual, cold spring port, 1989) etc. obtain detailed information molecular cloning with reference to Sambrook etc.
The gene PCR amplification and the clone of embodiment 1. earthworm fibrinolysins
(1) amplimer is synthetic
According to the earthworm fibrinolysin cDNA sequences Design PCR oligonucleotide amplimer of having delivered among the GeneBank.Long 18 the Nucleotide P1:ACATGGAACTTCCTCCCG of upstream primer, long 19 the Nucleotide P2:ATCACCAACAACTAAACCG of downstream primer.Primer is given birth to worker's biotechnology company limited by Shanghai and is synthesized, through the PAGE purifying.
(2) the total RNA of earthworm extracts
Get 3～5 earthworm adults, after the aqua sterilisa rinsing of handling with DEPC, be placed in the plate that is covered with wet filter paper hunger and spend the night, make it tell intravital to the greatest extent silt as much as possible.After cleaning up with DEPC water once more, in liquid nitrogen, be ground into powder, extract total RNA according to the product description of the RNeasyRMini Kit of QIAGEN company.The RNA solution that extracts is stored in-80 ℃, with standby.
(3) the synthetic cDNA article one chain of reverse transcription
Reverse Transcription Reaction test kit specification sheets according to Promega company carries out, and is synthetic cDNA first chain of primer with Oligo (dT) 20.The concrete operations step is as follows: following reagent is added one in the PCR reaction tubes that DEPC soaks and sterilising treatment is crossed: 25m,MMg,Cl2 12 μ l; Damping fluid 6 μ l; 10mM dNTP 6 μ l; Recombinant RNasinRibonuclease Inhibitor 2 μ l; AVM ThermoScript II 4 μ l; Oligo (dT) 20 6 μ l; The total RNA 15 μ l of earthworm; Sterilization distilled water 9 μ l.Reaction conditions is: 42 ℃ 1 hour; 95 ℃ 5 minutes; 3 ℃ 5 minutes.
(4) pcr amplification of goal gene
Get 8 μ L reverse transcription reaction products, carry out the PCR reaction according to the PCR reaction system parameter that provides in the Reverse Transcription Reaction test kit.Reactive component is: damping fluid 2.5 μ l; 25mM MgCl2 1.5 μ l; 10mM dNTP 2 μ l; Upstream primer 1 μ l; Downstream primer 1 μ l; MRNA reverse transcription product 15 μ l; RT-PCR enzyme mixture 2 μ l; Sterilization distilled water 5 μ l.Its reaction parameter is: 95 ℃ of pre-sex change 4min; 94 ℃ of sex change 1min, 55 ℃ of annealing 1.5min, 72 ℃ are extended 2min, 20 circulations; 94 ℃ of sex change 1min, 55 ℃ of annealing 1.5min, 72 ℃ are extended 2min, 10 circulations; 72 ℃ are extended 10min again.1% agarose detects PCR result, and electrophoresis result as shown in Figure 1.
(5) recovery of target gene fragment
Reclaim the test kit specification sheets according to Shanghai China Shun PCR of bio-engineering corporation product and reclaim target gene fragment, its process is as follows: get 30 μ lPCR reaction product and add 1.4mlPB liquid (test kit provides), mixture moved in the adsorption column centrifugal 15 seconds, abandoned waste liquid; In adsorption column, add 400 μ lPB liquid, leave standstill 1 minute after, centrifugal 15 seconds, abandon waste liquid; Add 500 μ lW1 liquid (test kit provides) again, centrifugal 15 seconds, abandon waste liquid, repeat once; In adsorption column, add 300 μ l T1 liquid (test kit provides), leave standstill 1 minute after, centrifugal 30 seconds, collect centrifugate, be stored in-20 ℃.
(6) target gene fragment is cloned into carrier
Target gene fragment is cloned into the pUCm-T carrier, and this carrier is given birth to worker bio-engineering corporation available from Shanghai.Reactive component is as follows: PCR product 5 μ l; PUCm-T carrier 1 μ l; 10 * T4DNA ligase enzyme damping fluid, 1 μ l; T4DNA ligase enzyme 1 μ l; Deionized water 2 μ l; Cumulative volume is 10 μ l, and 4 ℃ of connections are spent the night.
The preparation of competent cell: the single JM109 bacterium colony of picking, being inoculated in 3ml does not contain in the LB substratum of penbritin, 37 ℃ of overnight incubation, get above-mentioned bacterium liquid next day is inoculated in the 50mlLB nutrient solution in proportion at 1: 100,37 ℃ of vibrations 3 hours treat that bacterium liquid OD value is at 0.6 o'clock, centrifugal 8 minutes of 4 ℃ of 5000rpm, abandon supernatant, precipitation 0.1MCaCl
2Suspend, 5000rpm is centrifugal 8 minutes again, abandons supernatant, and precipitation is with an amount of 0.1M CaCl
2Resuspended, in the rearmounted ice bath of packing 6 hours standby.
Connect the conversion of product: get above-mentioned connection product 5 μ l and added 100 μ l competent cell ice baths 60 minutes, 42 ℃ of heat-shockeds 90 seconds, put ice bath again 2 minutes, the LB substratum 0.9ml that adds no penbritin, cultivated 1 hour for 37 ℃, getting 200 μ l bacterium liquid adds to be coated with behind 40 μ l 20mg/ml 5-bromo-4 chloro-, 3 indoles (D-galactoside (X-gal)) and 4 μ l 0.1M isopropylthiogalactoside (IPTG) mixings and contains penbritin LB plate, cultivated 16 hours for 37 ℃, picking white colony rapid extraction plasmid carries out enzyme and cuts evaluation, enzyme is cut product through 1% agarose detected result as shown in Figure 2, its positive colony called after pUCm-T-EFE.
The quick a small amount of preparation of plasmid DNA: picking list colony inoculation contains the LB substratum of penbritin in 4ml, and 37 ℃ of shaking culture are spent the night.Bacterium liquid adds in the 1.5ml centrifuge tube, centrifugal 8 minutes of 5000rpm, abandon supernatant, precipitation is suspended in 15 μ l suspension (50mM glucose, 25mMTris-HCl, 10mMEDTA, PH8.0), add 200 μ l lysate (0.2MNaOH, 1%SDS), put ice bath after 5 minutes, add 150 μ l neutralizer (3.0MKAc, PH4.8), ice bath 10 minutes, centrifugal 10 minutes of 12000rpm abandons precipitation, on reset and add that equal-volume phenol/the chloroform extracting once, on reset and add 2 times of volume dehydrated alcohols, centrifugal 10 minutes of 12000rpm, throw out is washed 1 time with 70% ethanol, vacuum is drained and is added 20 μ l TE (10mMTris-HCl, 1mM EDTA, PH8.0 and 1 μ l RNaseA 10mg/ml) ,-20 ℃ are frozen standby.
(7) earthworm fibrinolysin cDNA sequencing
Extract the plasmid DNA dideoxy method and measure nucleotide sequence, sequencing is given birth to worker's biotechnology company limited by Shanghai and is finished, and sequencing primer is the M13 promoter primer.
Measurement result: long 747 Nucleotide of earthworm fibrinolysin cDNA sequence, the content of adenosine (A), cytidine(C (C), guanosine-(G) and thymidine (T) is respectively: A-185, C-197, G-189; T-176 (sequence No.1), 246 amino acid (sequence No.2) of encoding.Gene mature peptide sequence is a 3-740 position Nucleotide, 246 amino acid of encoding, and all the other nucleotide sequences are non-coding region.This protein cDNA mature peptide terminator codon is positioned at gene 741-743 position, and termination codon is TAA.This isoelectric points of proteins (IP) of inferring according to earthworm fibrinolytic enzyme cDNA sequence is 4.15, and protein is acid.
(1) pcr amplification of earthworm fibrinolysin gene
With the pUCm-T-EFE plasmid is template, according to the design of the cloning site on EFE mature peptide and expression vector pPIC6A primer, upstream primer P1:GAATTC GCA TGG AAC TTCCTC CCG (the underscore place is restriction endonuclease EcoR I site), downstream primer P2:TCT AGACGG TTT AGT TGT TGG TGA T (the underscore place is restriction endonuclease Xba I site).Primer is synthetic by Shanghai Sangon bio-engineering corporation, and synthetic product is through the PAGE purifying.Reactive component is: damping fluid 2.5 μ l; 25mM MgCl
21.5 μ l; 10mM dNTP 2 μ l; Upstream primer 1 μ l; Downstream primer 1 μ l; PUCm-T-EFE plasmid 1 μ l; LA Taq archaeal dna polymerase 2 μ l; Sterilization distilled water 19 μ l.Its reaction parameter is: 95 ℃ of pre-sex change 4min; 94 ℃ of sex change 1min, 55 ℃ of annealing 1.5min, 72 ℃ are extended 2min, 30 circulations; 72 ℃ are extended 10min again.
(2) the PCR product cloning is to the T carrier
Reclaim the test kit specification sheets according to Shanghai China Shun PCR of bio-engineering corporation product and reclaim target gene fragment, extension amplification outcome obtains recombinant vectors pGEM-T-EFE2 in pGEM-T carrier (Promega company), and this recombinant vectors is changed in the e. coli jm109.
(3) structure of shuttle expression plasmid
Extract recombinant vectors pGEM-T-EFE2, with EcoR I and Xba I double digestion to obtain purpose fragment EFE; Same enzyme is cut the pPIC6A empty carrier and make it dephosphorylation under the CIAP effect.With the EFE that obtains behind linearizing pPIC6A empty carrier behind the dephosphorylation and the double digestion under the effect of T4DNA ligase enzyme 4 ℃ spend the night, to obtain recombinant expression vector pPIC6A-EFE, this recombinant vectors is changed in the e. coli jm109 increases.Picking white colony rapid extraction plasmid carries out enzyme and cuts evaluation, and enzyme is cut product through 1% agarose detected result as shown in Figure 3.
(4) transform pichia spp X-33
Method is with reference to Invitrogen company expression vector specification sheets (Pichia expressionvectors for selection on blasticidin and purification of recombinant proteinsmanual).
The single bacterium colony of picking pichia spp X-33 is in 5 milliliters of YPD substratum of inoculation, 30 ℃ of 250rpm shaking table overnight incubation, by 1% inoculum size inoculation 200mlYPD liquid nutrient medium, 30 ℃ of 250rpm shaking tables were cultivated 3-5 hour, cell concentration reaches 0.8-1.0, centrifugal 10 minutes of 1500g, precipitation is resuspended with 10ml sterilization distilled water, centrifugal 10 minutes of 1500g, precipitation is resuspended among the LiCl of 0.4ml 100mM and is transferred in the 1.5ml centrifuge tube of sterilization, and centrifugal 15 seconds of 12000g abandons supernatant, the LiCl that adds 0.16ml 0.1M is resuspended, standby.
Extract recombinant plasmid pPIC6A-EFE, be cut into linearity with the SacI enzyme, same enzyme is cut empty plasmid pPIC6A in contrast.
Get the above-mentioned competent cell of 50 μ l, centrifugal 15 seconds of 12000g carefully draws supernatant with pipettor, adds following component in order successively: 240 μ l 50%PEG; 36 μ l 1M LiCl; 25 μ l 2mg/ml salmon sperm dnas; 50 μ l (5-10 μ g) linearization recombinant plasmid pPIC6A-EFE.With linearization empty plasmid pPIC6A in contrast.Concuss made complete mixing in one minute, 30 ℃ left standstill 30 minutes, 42 ℃ heat shock 20-25 minute, centrifugal 15 minutes of 6000-8000rpm, precipitation is resuspended with the 1mlYPD substratum, cultivates 1-4 hour, and gets 25-100 μ l figure and be distributed in the YPD solid medium that contains 300 μ g/ml blasticidins (Blasticidin) for 30 ℃, cultivated 2-3 days for 30 ℃, up to there being single bacterium colony to occur.
Utilize biochemical method that linearizing recombinant plasmid pPIC6A-EFE and empty plasmid pPIC6A are transformed the X-33 yeast strain (with reference to Pichia expression vectors for selection onblasticidin and purification of recombinant proteins manual, Invitrogen).On the YPD solid medium that contains Blasticidins Hcl (300mg/ml), cultivated 2-3 days, up to there being single bacterium colony to occur.
(5) screening positive transformant
Picking 10-20 single bacterium colony in the YPD liquid nutrient medium 30 ℃, 250rpm shaking table were cultivated until logarithmic growth late period, extracted the PCR that genome carries out recon and identified.Resulting positive recombinant is Mut+.
Pastoris genomic dna preparation method: get the centrifugal 10min of 1.5ml nutrient solution 4000r/min.Thalline is with the liquid nitrogen broken wall of milling.Add 7ml DNA damping fluid (100m mol/L Tris-HCl, pH8.0,10m mol/L EDTA, 1%SDS).Mixing, 65 ℃ of insulation 1h.The centrifugal 15min of 10000r/min.Supernatant phenol extracting 2 times, each centrifugal 7min of 5000r/min.Use the chloroform extracting again 1 time, the centrifugal 7min of 5000r/min.With 2.5 times of ethanol sedimentations (add the 0.3mol/L sodium-acetate, pH5.2), the centrifugal 10min of 10000r/min.70% ethanol is washed, and hair dryer dries up.Add TE or aqueous suspension, add RNase.
The PCR authentication method: the universal primer that primer provides for Invitrogen company expression vector, its sequence are the upstream: GACTGG TTCCAATTGACAAGC; Downstream: GCAAATGGCATTC TGACATCC.PCR carries out according to a conventional method, and amplification condition is: 94 ℃ of 45s, 54 ℃ of 1min, 72 ℃ of 1min, 40 circulations.Whether amplified production is correct with detecting its size with 0.8% agarose.
(1) single bacterium colony of picking reorganization bacterium is gone into 20mlYPD liquid nutrient medium (1% yeast extract, 2% peptone, 2% glucose), and 30C, 250rpm cultivate and activate;
30 ℃ of the bacterium liquid switching 500ml MGY liquid nutrient mediums (1.34%YNB, 4x 10-5% vitamin H, 1% glycerine), the 250rpm that get after (2) 16 hours after 10ml activates cultivate enrichment;
(3) the bacterium liquid of enrichment after 24 hours is through centrifugal collection thalline (1500g, 10min, 4 ℃);
(4) thalline of collecting is carried out abduction delivering with being resuspended in 1000ml MM liquid nutrient medium (1.34%YNB, 4x 10-5% vitamin H, 1% methyl alcohol), it is 0.5% that every 24h adds methyl alcohol to final concentration;
(5) induce centrifugal collection thalline after 96 hours (30000g, 20min, 4 ℃).
(6) before the resuspended bacterial sediment of yeast zymolase damping fluid, measure the volume of packed cell, below institute all carry out in steps at 4 ℃;
(7) granulated glass sphere with 1 volume breaks bacterium damping fluid (20mmol/L Tris-Cl, pH7.9,10mmol/L MgCl
2, 1m mol/L EDTA, 5% glycerine, 1m mol/L DTT, 0.3m mol/L ammonium sulfate, 1m mol/L PMSF) and re-suspended cell;
(8) with the broken bacterium damping fluid of granulated glass sphere and the cytomixis of 2 volumes, add the ice-cold pickling glass pearl of 4 volumes;
(9) cell suspension is transferred in the centrifuge tube of band screw socket of suitable size (suspension accounts for the volume of centrifuge tube≤60%-70%), in 4 ℃ with the 30～60s that vibrates under the top speed, pipe placed place 1～2min on ice, repeat 3～5 times, in the degree of test under microscope broken cell, continue step 10;
(10) the precipitation granulated glass sphere decants supernatant, adds the broken bacterium damping fluid of granulated glass sphere of 2～4 volumes, puts upside down pipe 5～10 times, waits the granulated glass sphere post precipitation to decant supernatant, merges supernatant liquor;
(11) supernatant liquor of He Binging is in 4 ℃, and the centrifugal 60min of 12000g collects supernatant and is extract.During long storage, be divided into aliquot quick freezing in liquid nitrogen ,-80 ℃ of storages.
The proteic determination of activity of embodiment 4. genetically engineered plasmins
Adopt the fibrin plate method to measure the biologic activity of genetically engineered plasmin, by determination of activity, the activity unit of fermented liquid surpasses 2.9E+06U/L.Fig. 4 is for surveying periodic solusphere, and detection method can be with reference to the method [Hao Suli, Shen Jia, the research of Lumbrukinase biological activity assay method, 1996 the 10th the 6th phases of volume of Chinese Pharmaceutical Affairs] of Hao Suli etc., and specific operation process is as follows.
(1) bed board: get scleroproein stoste (it is an amount of to take by weighing Fibrinogen. contain the solution of 4.2mg coagulable protein in being made into every milliliter with the Tris-HCl damping fluid) 12.7ml, proplasmin liquid (getting the solution that proplasmin contains lu in being made into every milliliter with the Tris-HCl damping fluid) 0.67ml, agar solution (takes by weighing agar 0.8g and adds Tris-HCl damping fluid 100ml, heating for dissolving filters) 13.4ml.Zymoplasm liquid (get zymoplasm and be made into the solution that contains 70B.PU with the Tris-HCl damping fluid) 0.67ml.Mixing is poured in the synthetic glass dish of diameter 9cm, and room temperature is placed half an hour.Coagulating good plate does not evenly have bubble, is creamy white.
(2) point sample: it is an amount of to take by weighing the plasmin standard substance.Contain 2200,110,550,270 four concentration in being made into every milliliter with the Trios-HCI damping fluid.Draw 10 μ l reference liquid points on plate with microsyringe.Should leave a determining deviation between point and point, cover glass then and pull.Putting 37 ℃ of constant temperature and cultivated 18 hours, remove lid, with the perpendicular diameter of its solusphere of vernier caliper measurement, is ordinate with two diameter products.Standard substance unit is an abscissa, mapping drawing standard curve or ask regression equation on log-log graph paper.The concentration of trial-product is taken in the standard curve range, pushes away at the plasmin mark with the vertical two diameter products of trial-product solusphere and looks into unit of activity on the curve.
(3) detected result: two diameter products are ordinate, and standard substance unit is an abscissa, and regression equation is lgY=-1.334+0.479lgX, and Y is that the perpendicular diameter of solusphere is long-pending, and unit is cm
2, X is an activity unit.The result shows that the activity unit of fermented liquid is 2.9E+06U/L.
With the fragmentation of genetically engineered tunning (method is seen embodiment 2), collect supernatant then, damping fluid (23 the gram glycine/liter, 1.6 the gram Sodium phosphate dibasic/liter, 0.55 the gram SODIUM PHOSPHATE, MONOBASIC/liter, pH7.0) dialysis in contains 1 milligram of concentration with ultrafiltration and concentration to every milliliter at last, sterilizes with the membrane filtration in 0.22 μ m aperture.Filtrate can be taken by directly oral aqua patients, perhaps about 1 milliliter of filtrate (about 300000 unit of activity) bottling, and the pulvis finished product is made in lyophilize, and the warm water dissolving is oral during use.
Application---the thrombolytic effect of embodiment 6. genetically engineered plasmin protein formulations
Rabbit external jugular vein thrombus method is easy and simple to handle, does not need specific apparatus, all has the research department to adopt at home and abroad, is the feasible method of research medicine thrombus dissolving effect.This method can be with reference to [Zhao Weiming, Xu Chaoqian, He Shuzhuan, Wang Ling, the experimental study of genetically engineered plasmin thrombolytic effect: Harbin Medical University's journal, 2002 (36) 3:183-185] [Xu Shuyun, Bian Rulian, the Chen Xiu chief editor. pharmacological experimental methodology. Beijing: People's Health Publisher, 1982.] [Zhu Yan, what is fair and unbiased, Sun Kedi, Wang Shaofei, the genetically engineered plasmin is to the influence of experimental thrombosis, China Medicine University's journal, 2000,31 (1): 50～52.]
(1) makes neck artery-vein bypass experimental model
Experimental rabbit with 20% urethane 5ml/kg auricular vein injecting anesthetic, is carried on the back the position and fixed, peel off tracheae, insert a plastic casing (tracheal secretion can pass through this sleeve pipe sucking-off for a long time), separate left side external jugular vein and right carotid.Put into 4 trumpeter's art silk threads of a segment length 6cm in three sections polyethylene tube stage casings, be full of heparin-saline solution (50U/ml) in the polyethylene tube: after inserting left external jugular vein with an end of polyethylene tube, accurately inject heparin-saline solution (50U/ml) anti-freezing by polyethylene tube, and then the other end of polyethylene tube inserted right common carotid artery, open bulldog clamp, blood flow in the polyethylene tube from right common carotid artery, return left external jugular vein, form the bypass of neck artery-vein, after keeping 3h, get blood, middle Herba Clinopodii takes out silk thread rapidly and weighs.
(2) experiment grouping and route of administration
36 of rabbit, body weight 2.5 ± 0.2kg is divided into 6 groups at random, is respectively dosage group in model group, physiological saline control group, genetically engineered plasmin small dose group, the genetically engineered plasmin, the heavy dose of group of genetically engineered plasmin, urokinase positive controls.
It is as follows that each organizes concrete route of administration: model group: the neck artery-vein bypass model of manufacturing; Physiological saline control group: auricular vein equal-volume injecting normal saline; Genetically engineered plasmin small dose group: auricular vein injection genetically engineered plasmin 1250U/kg; Dosage group in the genetically engineered plasmin: auricular vein injection genetically engineered plasmin 2500U/kg; The heavy dose of group of genetically engineered plasmin: auricular vein injection genetically engineered plasmin 5000U/kg; Urokinase positive controls: auricular vein injection urokinase 2 * 10
4U/kg.
Medication: after the neck artery-vein bypass modelling, physiological saline control group, genetically engineered plasmin small dose group, middle dosage group, heavy dose of group and urokinase positive controls are set up back 15min administration respectively at bypass, observe 3h.
(3) thrombolytic effect
Adopt rabbit artery-vein bypass thrombotic model, measure the thrombolytic effect (wet weight of thrombus, dry weight) of earthworm fibrinolysin; Heart extracting blood is measured the influence of earthworm fibrinolysin to plasma fibrin enzyme (FIB), blood plasma euglobulin lysis time (ELT), fibrin split product biochemical indicators such as (FDP).
Experimental result sees Table 1 and table 2.In this experiment in the genetically engineered plasmin dosage group and heavy dose of group ELT is significantly shortened, illustrate that the genetically engineered plasmin has activated fibrinolytic system.Genetically engineered plasmin small dose group, middle dosage group are compared with model group with heavy dose of group in this experiment can obviously increase FDP content, illustrates that the genetically engineered plasmin can make the fibrinolytic of blood system strengthen.In this experiment, FIB does not have obvious variation, shows that the genetically engineered plasmin is difficult for producing hemorrhage side reaction.
Table 1 genetically engineered plasmin is to the influence of ETL FDP numerical value and FIB content
| Group | ELT(min) | FDP(ng·L -1) | FIB(g·L -1) | |||
| ＜120 | ≥130 | ＜5 | 5-20 | ≥20 | ||
| The heavy dose of group of dosage group (2500U/kg) genetic engineering fibrinolysin (5000U/kg) urokinase group in model group physiological saline group genetic engineering fibrinolysin small dose group (1250U/kg) the genetic engineering fibrinolysin | 0 0 0 4.1 * 5 * 5 * | 7.9 7.9 7.8 4.1 * 3 * 3 * | 8.1 8.1 4.9 1.1 * 1 * 0 * | 0 0 0 4 * 1 * 1 * | 0 0 2.5 3 * 6 * 7 * | 4.60±2.89 5.90±2.98 5.34±2.80 ☆ 3.75±1.84 ☆ 3.00±1.93 ☆ 2.60±1.02 ☆ |
ELT, FDP are all in routine number, and through rank test, * and physiological saline group relatively are ρ＜0.01; FIB content is checked through q, and ☆ and physiological saline group be ρ＜0.05 relatively.
The influence that table 2 genetically engineered plasmin forms the rabbit thrombus in vivo (x ± S)
| Group | The example number | Dosage | Weight in wet base (ng) | Dry weight (ng) | Inhibiting rate (%) |
| Model group is heavily managed the heavy dose of group of dosage group genetic engineering fibrinolysin urokinase group in the salt solution group genetic engineering fibrinolysin small dose group genetic engineering fibrinolysin | 8 8 8 8 8 8 | 1250U/kg 2500U/kg 5000U/ | 70.25±2.04 85.31±3.61 63.58±9.60 △△ 39.00±5.9 **△△☆ 29.49±4.58 **△△ 21.11±5.48 **△△ | 22.01±1.83 28.25±1.02 19.55±3.67 △△ 14.26±1.05 **△△ ☆ 12.21±2.01 **△△ 8.91±2.07 **△△ | 24.5 53.6 65.3 75.1 |
* and model group compare, ρ＜0.01; △ △ and physiological saline group compare, ρ＜0.01; The large, medium and small dosage group of ☆ is ρ＜0.05 relatively.
Sequence table
NO：1
＜110〉Green Life Lab Co Ltd
＜120〉a kind of pichia pastoris gene engineering bacterial strain and preparation method and application
＜130〉Lumbricidae powder Lumbricus earthworm (Lumbricus rubellus) fibrinolytic enzyme gene Nucleotide
Sequence
<160>1
<170>PatentIn version3.1
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<213>Lumbricus rubellus
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acatggaact tcctcccgga acaaaaattg tcggaggaat tgaagctaga ccatacgagt 60
tcccatggca ggtgtccgtc cgaaggaaat cttccgattc ccatttctgc ggaggtagca 120
tcatcaacga tcgttgggtt gtctgcgctg ctcactgcat gcagggagag gcccccgctc 180
tggtttcatt ggtcgtgggt gagcacgaca ggagtgcagc gagtacagta cgtcagactc 240
atgacgttga tagcatcttc gttcacgagg actacaacac aaatacccta gagaacgacg 300
tttctgtcat caagacatct gttgccatca ctttcgacat caacgttggt ccaatctgcg 360
ccccagatcc ggctaacgac tacgtctacc gtaagagcca gtgttccgga tggggaacta 420
tcaattcagg tggaatctgc tgtcccaacg ttctgcgata cgtgacgctg aatgacacaa 480
ccaaccaata ctgcgaagat gtatacccac taaattcaat ctacgacgat atgatttgcg 540
cgtcggacaa cactgggggt aacgacagag actcctgcca gggtgactcc ggcggccctc 600
tgagcgtcaa ggatggtagt ggaatcttca gcctgattgg tattgtgtct tggggaattg 660
gttgcgcttc tggctatcca ggagtctact cccgcgtcgg attccatgct gcatggatca 720
ccgacatcat caccaacaac taaaccg 747
NO：2
＜110〉Green Life Lab Co Ltd
＜120〉a kind of pichia pastoris gene engineering bacterial strain and preparation method and application
＜130〉Lumbricidae powder Lumbricus earthworm (Lumbricus rubellus) fibrinolytic enzyme gene amino acid
Sequence
<160>1
<170>PatentIn version 3.1
<210>1
<211>246
<212>PRT<213>Lumbricus rubellus
<220><221>PEPTIDE
<222>(1)..(246)
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<400>1
Met Glu Leu Pro Pro Gly Thr Lys Ile Val Gly Gly Ile Glu Ala Arg
1 5 10 15
Pro Tyr Glu Phe Pro Trp Gln Val Ser Val Arg Arg Lys Ser Ser Asp
20 25 30
Ser His Phe Cys Gly Gly Ser Ile Ile Asn Asp Arg Trp Val Val Cys
35 40 45
Ala Ala His Cys Met Gln Gly Glu Ala Pro Ala Leu Val Ser Leu Val
50 55 60
Val Gly Glu His Asp Arg Ser Ala Ala Ser Thr Val Arg Gln Thr His
65 70 75 80
Asp Val Asp Ser Ile Phe Val His Glu Asp Tyr Asn Thr Asn Thr Leu
85 90 95
Glu Asn Asp Val Ser Val Ile Lys Thr Ser Val Ala Ile Thr Phe Asp
100 105 110
Ile Asn Val Gly Pro Ile Cys Ala Pro Asp Pro Ala Asn Asp Tyr Val
115 120 125
Tyr Arg Lys Ser Gln Cys Ser Gly Trp Gly Thr Ile Asn Ser Gly Gly
130 135 140
Ile Cys Cys Pro Asn Val Leu Arg Tyr Val Thr Leu Asn Asp Thr Thr
145 150 155 160
Asn Gln Tyr Cys Glu Asp Val Tyr Pro Leu Asn Ser Ile Tyr Asp Asp
165 170 175
Met Ile Cys Ala Ser Asp Asn Thr Gly Gly Asn Asp Arg Asp Ser Cys
180 185 190
Gln Gly Asp Ser Gly Gly Pro Leu Ser Val Lys Asp Gly Ser Gly Ile
195 200 205
Phe Ser Leu Ile Gly Ile Val Ser Trp Gly Ile Gly Cys Ala Ser Gly
210 215 220
Tyr Pro Gly Val Tyr Ser Arg Val Gly Phe His Ala Ala Trp Ile Thr
225 230 235 240
Asp Ile Ile Thr Asn Asn
245
Claims (5)
1, a kind of pichia pastoris gene engineering bacterial strain is characterized in that, this bacterial strain is pichia spp Pichia pastoris X-33 (pPIC 6A-EFE), CCTCC M204004.
2, a kind of preparation method who is used for the described engineering strain of claim 1, this method comprises the following steps:
The pcr amplification of A, earthworm fibrinolysin gene: with the T vector plasmid that contains fibrinolytic enzyme gene is template, according to the design of the cloning site on EFE mature peptide and expression vector pPIC6A primer;
B, PCR product cloning are to the T carrier: reclaim the gene fragment in the PCR product, and clone the carrier in pGEM-T, pGEM-T-EFE2 changes in the e. coli jm109 with this recombinant vectors;
The structure of C, shuttle expression plasmid: extract recombinant vectors pGEM-T-EFE2, with EcoR I and Xba I double digestion to obtain purpose fragment EFE; Same enzyme is cut the pPIC6A empty carrier and make it dephosphorylation under the CIAP effect, with the EFE that obtains behind linearizing pPIC6A empty carrier behind the dephosphorylation and the double digestion under the effect of T4DNA ligase enzyme 4 ℃ spend the night, to obtain recombinant expression vector pPIC6A-EFE, this recombinant vectors is changed in the e. coli jm109 increase;
The structure of the Yeast gene engineering bacterial strain of D, expression fibrinolytic enzyme gene, extract recombinant plasmid pPIC6aA-EFE, be cut into linearity with the SacI enzyme, same enzyme is cut empty plasmid pPIC6A in contrast, utilize biochemical method that linearizing recombinant plasmid pPIC6A-EFE and empty plasmid pPIC6A are transformed the X-33 yeast strain, cultivated 2-3 days containing on the YPD solid medium of blasticidin 300mg/ml, picking 10-20 single bacterium colony in the YPD liquid nutrient medium 30 ℃, the 250rpm shaking table was cultivated until logarithmic growth late period, extracted the PCR that genome carries out recon and identified.
3, the preparation method of engineering strain according to claim 2, it is characterized in that: the cloning site design primer on the expression vector pPIC 6A is, upstream primer: GAATTC GCATGG AAC TTC CTC CCG, downstream primer: TCT AGA CGG TTT AGT TGTTGG TGA T.
4, the application of the genetically engineered earthworm fibrinolysin of the described pichia pastoris gene engineering bacterial strain expression of a kind of claim 1 in the medicinal preparation for oral administration of preparation treatment embolic cardiovascular disorder.
5, the described pichia pastoris gene engineering bacterial strain of a kind of claim 1 genetically engineered earthworm fibrinolysin of expressing has application in the healthcare products of prevention thrombus disease, useful cardiovascular health in production.
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