CN102199615B - cDNA sequence coding fibrinolysin, amino acid sequence and applications of sequences - Google Patents
cDNA sequence coding fibrinolysin, amino acid sequence and applications of sequences Download PDFInfo
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Abstract
The total RNA of Urechis unicinctus, which is used as template, is cloned to obtain the full-length cDNA sequence of the Urechis unicinctus fibrinolysin gene, wherein the open reading frame (ORF) of the sequence has 792 basic bases and is from the initiation codon ATG to the stop codon TAA. The cDNA original coding sequence of the fibrinolysin gene is translated to obtain the amino acid sequence of the Urechis unicinctus fibrinolysin, wherein the amino acid sequence has 263 amino acids in all. Different recombinant plasmid vectors can be constructed to construct fibrinolysin gene engineering bacteria for different receptors such as Escherichia coli and Saccharomyces and ensure that the cDNA sequence of the Urechis unicinctus fibrinolysin gene is expressed in the gene engineering bacteria and prepare fibrinolysin. The prepared fibrinolysin can be utilized to prepare the anticoagulant medicines or antithrombotic medicines for curing cardiovascular diseases and have very good research and development prospects.
Description
Technical field
The present invention relates to a kind of cDNA sequence and aminoacid sequence of the fibrinoclase of encoding, particularly a kind of cDNA sequence of the Urechis uniconctus fibrinoclase of encoding and aminoacid sequence and application thereof belong to biotechnology and biomedicine field.
Background technology
Urechis uniconctus Urechis unicinctuss is the deuterocoel protostomia, be distributed in the coastal zone along the Huanghai Sea and the Bohai Sea of Russia, Japan, Korea and China, live in the zoobenthos of coastal silt substrate, it is categorized as Echiuroidea (Echiurioidea), Yi guiding principle (Echiurida), without pipe Yi order (Xenopneusta), thorn Yi section (Urchidae).At present the research of Urechis uniconctus also is in the fundamental biological knowledge conceptual phase, such as morphological structure, fetal development, the life history, artificial breeding technique and Analysis of Nutritional etc., because it can tolerate high density sulfides, its metabolism research also is an aspect of academia's research.
Be in the patent of invention of ZL200610043371.3 in the patent No., the contriver is take Urechis uniconctus as material, by biochemical extraction, separation, purifying, cryodesiccated method, prepared a kind of thrombus dissolving enzyme, this thrombus dissolving enzyme has anticoagulant active and thrombus is active.In this patent of invention, the contriver does not disclose molecular weight, aminoacid sequence and the associated dna sequence of thrombus dissolving enzyme.
Summary of the invention
The cDNA sequence and the aminoacid sequence thereof that the purpose of this invention is to provide a kind of fibrinoclase of encoding, and carry out the structure of Taka-proteinase genetic engineering bacterium and novel anticoagulation, dissolved thrombus medicine exploitation with it.
A kind of open reading frame of cDNA sequence of the fibrinoclase of encoding is characterized in that:
ATGAAGACACTGTTGCTCATTTCATTGGCCGCTCTTGCCACTGCTGCGCCAGCCACATTC
GATGCTGTGCGTGCACTGAGCGTTGAATCCCGTATCATTGGTGGCTCACCGGCTGACATC
ACCAACTTCCCCTATCAACTGTCTCTGAGATTCATTGGCTCTCATACATGTGGTGCCGTAG
TTGTGTCGGTCAACAGGGCTCTGACAGCTGCCCATTGTACTGATGGACGTGTTATCGGTG
GTTTCACACTTTATGCTGGATCTACAAGTAGACTATCGGATGGTGTACTGTACACCAATTT
GGCAAAGGACGAGCACAGCGACTACAACAATGGTCAATGCACTTTCTGTAATGATATCT
GCATGCTCTCTCTATTAACCAACATGGACACGAGCAATCCCGGTATTGGAGCAATCAGTA
TAGCAGCTACCAGTGATTTTGCAGGAACAACTTGCACTTTGAGTGGATGGGGCCGCACA
TCCAGCAGCATTGATCTCCCCACTAATCTGCAACAGGTCGACGTGGGTGTTATTTCCAAT
ATTGAATGCCAAAGCCGAATGTCAGCAGCTGGAGCCAGTGTAGGAATTCAACATATCTG
CATCTTCAGCAGCAGCCAGGGATCTTGCAATGGTGACAATGGTGGTCCCATGAGATGCA
CAGAGGGCGGCATTGGTGTATTGGCAGGAATCACCTCCTGGGGAATCTCTGTTGGAGGG
GAATGCAGCACTATCTACCCATCCGTCTACGTCAGAGTCACAACCTTTCGGCCATGGATT
GCCGAACGCATG
TAA
A kind of aminoacid sequence of the fibrinoclase of encoding is characterized in that:
MKTLLLISLAALATAAPATFDAVRALSVESR
IIGGSPADITNFPYQLSLRFIGSHTCGAVVVSV
NRALTAAHCTDGRVIGGFTLYAGSTSRLSDGVLYTNLAKDEHSDYNNGQCTFCNDICMLSL
LTNMDTSNPGIGAISIAATSDFAGTTCTLSGWGRTSSSIDLPTNLQQVDVGVISNIECQSRMS
AAGASVGIQHICIFSSSQGSCNGDNGGPMRCTEGGIGVLAGITSWGISVGGECSTIYPSVYVR
VTTFRPWIAERM
The application of the cDNA sequence of above-mentioned coding fibrinoclase in making up the fibrinoclase genetic engineering bacterium.
The aminoacid sequence of above-mentioned coding fibrinoclase is in the anticoagulation medicine of preparation treatment cardiovascular and cerebrovascular diseases or the application in the dissolved thrombus medicine.
Description of drawings:
Fig. 1 fibrin plate detects the scleroproein enzymic activity of supernatant liquor
Wherein: the fermented supernatant fluid of the positive recombinant bacterial strain X-33/pPCZ of 1-6 α AuffeEX, 7 is empty plasmid recombinant bacterial strain X-33/pPICZ α A fermented supernatant fluid
Embodiment
Embodiment 1: the total RNA of Urechis uniconctus extracts
Gather the about 1Kg of fresh and alive Urechis uniconctus from sandy beach, tideland, seashore, Qingdao and (gather people: Bi Qingqing; Phone: 13698668750), support temporarily with fresh seawater, get an amount of Urechis uniconctus body wall and intestinal tissue and extract the total RNA of polypide with guanidine isothiocyanate method, concrete grammar is as follows:
(1) obtain solution D: take by weighing guanidinium isothiocyanate 48g, bay creatine sodium 0.5g puts in the 250ml beaker, add 0.75M sodium citrate solution 3.33ml, add a small amount of DEPC water 20ml, stirring and dissolving, all be transferred in the 100ml volumetric flask, add DEPC water constant volume, get solution D.
[extraction of (2) total RNA: in the 5ml centrifuge tube, add the 1ml solution D, the 225ul chloroform, the 5ul beta-mercaptoethanol, precooling 5min adds 100mg Urechis uniconctus body wall and intestinal tissue, immediately homogenate 2min on ice.Homogenate is in 4 ℃ of centrifugal 10min of lower 12000r.Get centrifuged supernatant 700ul, add the 350ul acidic phenol, the 350ul chloroform, behind the thermal agitation 20s in 4 ℃ of lower 12000r recentrifuge 10min.Get recentrifuge supernatant liquor water 650ul, add isopyknic chloroform, behind the thermal agitation 20s in 4 ℃ of lower 12000r centrifugal 10min for the third time.Get for the third time centrifuged supernatant water 600ul, add the Virahol of isopyknic-20 ℃ of precoolings and the sodium acetate soln (3M of 1/10 volume, pH5.2), precipitate 30min in-20 ℃ behind the mixing, in the 4th centrifugal 30min of 4 ℃ of lower 12000r, abandon supernatant liquor, the aqueous ethanolic solution 1ml with 75% cleans centrifugal precipitation the 4th time, and in the 5th centrifugal 5min of 4 ℃ of lower 13000r, with 10ul RNase-Free water dissolution, add again 5uL buffer, 2uLDNase I after the throw out seasoning that obtains, 0.5ul RNase, 2.5uLRF water behind 37 ℃ of digestion 30min, adds water and complements to 100ul, add 50ul phenol, the extracting of 50ul chloroform once in 4 ℃ of centrifugal 30min of lower 12000r, is got the sodium acetate soln (3M that the supernatant aqueous portion adds isopyknic Virahol and 1/10 volume, pH5.2) precipitation, with the aqueous ethanolic solution washing and precipitating of 75% (weight percent, lower same), the precipitation that obtains 10ul RNase-Free water dissolution, frozen in-80 ℃ of refrigerators, get total RNA sample.
Synthesizing of embodiment 2:cDNA the first chain
Synthetic 3 ' RACE the Adaptor:5 '-TACCGTCGTTCCACTAGTGATTTCACTATAGGTTTTTTTTTTTTTTTT-3 ' of design, and the Oligo dT Primer that substitutes among the PrimeScript lst Strand cDNA Synthesis Kit (TaKaRa company) with 3 ' synthetic RACE Adaptor does the reverse transcription primer, take total RNA of extracting as template, according to the test kit specification sheets, carry out the RT-PCR operation, obtain cDNA the first chain, save backup in-80 ℃.
Embodiment 3: the clone of Urechis uniconctus fibrinoclase gene cDNA
(1) design of primers is synthetic: according to the protein N-terminal sequence ICGGSPADIT of the fibrinoclase of having measured, and the synthetic forward degenerated primer of design: 103,5 '-TCNCCNGCNGAYATHAC-3 '; 104,5 '-AGYCCNGCNGAYATHAC-3 '.According to synthetic reverse primer: the OP of 3 ' RACEAdaptor design, 5 '-TACCGTCGTTCCACTAGTGATTT-3 '
(2) degenerated primer PCR: cDNA the first chain in the embodiment 2 carries out degenerate pcr as template with the combination of primers that design is synthetic.In the PCR of 0.2ml pipe, add successively: cDNA template, 0.5ul; 10 * buffer, 2.5ul; 25mM MgCl
2, 3ul; 2.5mM dNTP, 2ul; 10 μ M forward primers 103/104,2.5ul; 10 μ M reverse primer OP, 1ul; DdH
2O, 13.3ul; The rTaq archaeal dna polymerase, 0.2ul consists of the 25ul reaction system.Above-mentioned reaction system is in the enterprising performing PCR reaction of PCR instrument, and reaction conditions is: 94 ℃ of denaturation 2min; 94 ℃ of sex change 30s, gradient temperature (being respectively 48 ℃, 51 ℃, 54 ℃, 57 ℃, 60 ℃, 63 ℃, 66 ℃) annealing 1min, 72 ℃ are extended 1min, 35 circulations; 72 ℃ are extended 10min, get the degenerate pcr product.
(3) recovery of purpose fragment and clone: get above-mentioned degenerate pcr product in 1.2% agarose gel electrophoresis, cutting purpose band, use sepharose DNA to reclaim test kit (day root biochemical technology company limited) and reclaim the purpose fragment, the purpose fragment that reclaims and pMD18-T carrier (TaKaRa company) are connected 12h in 16 ℃, get connection carrier.Connection carrier with 42 ℃ of heat shock method transformed competence colibacillus E.coli DH5 α, is coated with the LB solid medium that contains penbritin, cultivates for 37 ℃ and select.Mono-clonal on the picking substratum is inoculated respectively in the liquid LB substratum that contains penbritin, cultivate amplification for 37 ℃, carry out bacterium colony PCR checking positive colony with Auele Specific Primer 103/104 with OP, positive strain checks order, and gets the Partial Fragment of Urechis uniconctus goal gene.
5 ' end of (4) 5 ' RACE-PCR amplification purpose fragment: carry out the required primer of 5 ' RACE according to the Partial Fragment design of having checked order: GSP1,5 '-TTTACATGCGTTCGGCAATCCA-3 '; GSP2,5 '-ATTGTTGTAGTCGCTGTGCTCGTC-3 '.Again extract the total RNA of Urechis uniconctus, with 5 '-Full RACE Kit (TaKaRa company), carry out to specifications 5 ' RACE pcr amplification.The PCR product that obtains reclaims test kit through sepharose DNA and reclaims, reclaiming product is connected with 16 ℃ in pMD18-T carrier, and transformed competence colibacillus E.coliDH5 α, carry out bacterium colony PCR checking positive colony through Auele Specific Primer 5 ' RACE Inner Primer and GSP2, positive strain checks order, and gets 5 ' end of Urechis uniconctus goal gene.
(5) Urechis uniconctus fibrinoclase full length gene cDNA amplification: according to RACE PCR result, design forward primer W2seh:5 '-CCTGCATTATGAAGACACTGTTG-3 ', according to degenerated primer PCR consequence devised reverse primer W2anti:5 '-AACGCTCTTAGTGTATTGATGGC-3 ', increase take Urechis uniconctus the first chain cDNA as template, in the PCR of 0.2ml pipe, add successively: cDNA template, 1ul; 10 * Ex Taq Buffer, 5 μ l; 2.5mM dNTP, 4ul; 10 μ M forward primer W2sen, 2ul; 10 μ M reverse primer W2anti, 2ul; DdH
2O, 35.75ul; TaKaRa Ex Taq, 0.25 μ l consists of the 50ul reaction system.Above-mentioned reaction system is in the enterprising performing PCR reaction of PCR instrument, and reaction conditions is: 94 ℃ of denaturation 2min; 94 ℃ of sex change 30s, 55 ℃ of annealing 1min, 72 ℃ are extended 1min, 35 circulations; 72 ℃ are extended 10min, obtain the PCR product.The PCR product is cloned with method described in (3), and the positive strain order-checking obtains following cDNA sequence:
GAAAACTAATTGTGAAAAGACTCCTGCATT
ATGAAGACACTGTTGCTCATTTCATTGGCC
GCTCTTGCCACTGCTGCGCCAGCCACATTCGATGCTGTGCGTGCACTGAGCGTTGAATC
CCGTATCATTGGTGGCTCACCGGCTGACATCACCAACTTCCCCTATCAACTGTCTCTGAG
ATTCATTGGCTCTCATACATGTGGTGCCGTAGTTGTGTCGGTCAACAGGGCTCTGACAGC
TGCCCATTGTACTGATGGACGTGTTATCGGTGGTTTCACACTTTATGCTGGATCTACAAG
TAGACTATCGGATGGTGTACTGTACACCAATTTGGCAAAGGACGAGCACAGCGACTACA
ACAATGGTCAATGCACTTTCTGTAATGATATCTGCATGCTCTCTCTATTAACCAACATGGA
CACGAGCAATCCCGGTATTGGAGCAATCAGTATAGCAGCTACCAGTGATTTTGCAGGAA
CAACTTGCACTTTGAGTGGATGGGGCCGCACATCCAGCAGCATTGATCTCCCCACTAATC
TGCAACAGGTCGACGTGGGTGTTATTTCCAATATTGAATGCCAAAGCCGAATGTCAGCA
GCTGGAGCCAGTGTAGGAATTCAACATATCTGCATCTTCAGCAGCAGCCAGGGATCTTG
CAATGGTGACAATGGTGGTCCCATGAGATGCACAGAGGGCGGCATTGGTGTATTGGCAG
GAATCACCTCCTGGGGAATCTCTGTTGGAGGGGAATGCAGCACTATCTACCCATCCGTCT
ACGTCAGAGTCACAACCTTTCGGCCATGGATTGCCGAACGCATG
TAAACGATGCCATCA
ATACACTAAGAGCGTTTAGATGTTTAACACAATAAATGCCTTCATCATTCAAAAAAAAAA
AAAAAA
Gained Urechis uniconctus fibrinoclase unnamed gene is ufe2, and the open reading frame in the full length cDNA sequence (ORF) is totally 792 bases, originates in the ATG initiator codon that line indicates in the sequence, ends at the TAA terminator codon that line indicates.
According to the cDNA original coding sequence of Urechis uniconctus fibrinoclase gene ufe2, through translating the aminoacid sequence that obtains the Urechis uniconctus fibrinoclase, this sequence is as follows:
MKTLLLISLAALATAAPATFDAVRALSVESR
IIGGSPADITNFPYQLSLRFIGSHTCGAVVVSV
NRALTAAHCTDGRVIGGFTLYAGSTSRLSDGVLYTNLAKDEHSDYNNGQCTFCNDICMLSL
LTNMDTSNPGIGAISIAATSDFAGTTCTLSGWGRTSSSIDLPTNLQQVDVGVISNIECQSRMS
AAGASVGIQHICIFSSSQGSCNGDNGGPMRCTEGGIGVLAGITSWGISVGGECSTIYPSVYVR
VTTFRPWIAERM
In the above-mentioned fibrinoclase aminoacid sequence, line part MKTLLLISLAALATA is the signal peptide sequence of this enzyme, and IIGGSPADIT and Urechis uniconctus fibrinoclase N end sequencing result are basically identical, are the N terminal sequence of this enzyme.
CDNA sequence and the aminoacid sequence of above-mentioned Urechis uniconctus fibrinoclase gene ufe2, inquire about in the NCBI website, compare, with the cDNA sequence of GenBank:HM623463 higher similarity is arranged, but the difference that 56 bases are also arranged, the amino acid comparative result shows, the aminoacid sequence that above-described embodiment obtains and HM623463 aminoacid sequence have 34 different, and few one of amino-acid residue number.
Embodiment 4: the expression of Urechis uniconctus fibrinoclase gene in pichia pastoris phaff
(1) expression vector establishment
CDNA sequence and aminoacid sequence according to the Urechis uniconctus fibrinoclase gene ufe2 among the embodiment 3, remove protein N terminal sequence and terminator codon before, add simultaneously restriction endonuclease XhoI and XbaI enzyme cutting site (shown in the underscore), design primer: EXnativesen:
CTCGAGAAAAGAGAGGCTGAAGCTATCATTGGTGGC;
EXanti:
TCTAGACCCATGCGTTCGGCAATCCATGG; Carry out PCR take the pMD 18-T-ufe2 plasmid that obtains as template.The PCR product is cloned according to method described in the embodiment 3 (3), obtains the positive strain with plasmid pMD18-T-ufeEX, positive strain order-checking, checking gene amplification accuracy.
Use plasmid extraction kit (day root biochemical technology company limited) to extract through the correct plasmid pMD18-T-ufeEX of sequence verification, with XhoI and XbaI enzyme cutting plasmid pMD18-T-ufeEX and carrier pPICZ α A.Enzyme is cut system (20 μ L): pMD18-T-ufeEX/pPICZ α A, 10 μ L; 10 * M buffer, 2 μ L; XhoI, 1 μ L; XbaI, 1 μ L; Distilled water, 6 μ L.37 ℃ of enzymes are cut 4～5h, add 4 μ L, 10 * loading buffer termination reaction, 1% agarose gel electrophoresis, and reclaim enzyme and cut into slices disconnected.
The purpose segment and the plasmid that reclaim are connected.Ligation system (10 μ L): pPICZ α A, 2 μ L; The purpose segment, 2 μ L; T4 DNAligase, 1 μ L; 10 * T4DNAligase buffer, 1 μ L; Distilled water, 4 μ L.16 ℃ are spent the night, with the expression vector called after pPICZ α AufeEX that builds.
Linked system is transformed bacillus coli DH 5 alphas with 42 ℃ of heat shock methods, and be applied to and contain Zeocin
TMOn the LB flat board, must be with plasmid pPICZ α AufeEX positive strain.Select positive colony, extract plasmid, carry out XhoI and XbaI double digestion and detect.The positive strain order-checking, checking gene accuracy.
(2) linearization process of plasmid and recovery
Use plasmid extraction kit (day root biochemical technology company limited) to extract through the plasmid pPICZ of the correct positive strain of sequence verification α AufeEX, and pPICZ α A cut pPICZ α AufeEX and pPICZ α A with BstXI linearizing enzyme.Enzyme is cut system (20 μ L): pPICZ α A ufeEX/pPICZ α A, 10 μ L; 10 * H buffer, 2 μ L; BstXI, 1 μ L; Distilled water, 6 μ L.37 ℃ of enzymes are cut 4～5h, and 1.0% agarose gel electrophoresis detects the linearizing degree, use nucleic acid to reclaim test kit (TakaRa company) and reclaim product, get linearization plasmid pPICZ α AufeEX and pPICZ α A.
(3) the competent preparation of pichia pastoris phaff and electricity transform
Pichia pastoris phaff X-33 (Pichia pastoris X33) is Invitrogen company product, according to EasySelect
TMMethod in the Pichia ExpressionKit specification sheets prepares pichia pastoris phaff X-33 competence, get linearization plasmid pPICZ α A ufeEX and pichia pastoris phaff X-33 competence 80 μ L, mix the electroporation that places the 0.2cm type and transform cup, electric revolving cup is placed 5min on ice.Electroporation transforms the electric shock condition: voltage: 1500V; Resistance: 400 Ω; Electric capacity: 25 μ F; Burst length: 10mS; 1 electric shock.After the electric shock, transform the Sorbitol Solution USP that adds the 1M of 1mL0 ℃ of precooling in the cup in electric shock at once, blow and beat gently evenly with micropipet, place ice bath.Mixed solution in the electric revolving cup is transferred in the new aseptic Eppendorf pipe, 30 ℃, left standstill and cultivate 1-2h.The bacterium liquid of getting after 200 μ l transform is coated on the YPD plate culture medium that contains 100 μ g/mL Zeocin TM.Flat board places 30 ℃ of incubator 3-5d.Select the bacterium colony that grows and be linked on new YPD (the containing 100 μ g/mLZeocin TM) plate culture medium and carry out purifying, get positive recombinant bacterium X-33/pPICZ α AufeEX, and carry out sequence verification.Same method electricity transforms empty plasmid pPICZ α A, gets the recombinant bacterium X-33/pPICZ α A of empty plasmid pPICZ α A.
(4) abduction delivering of pPICZ α A ufeEX in pichia pastoris phaff X-33
The positive recombinant bacterium X-33/pPICZ α A ufeEX that gets the order-checking evaluation is linked in the shaking flask that the 20mLBMGH liquid nutrient medium is housed, 250-300rpm, 28 ℃ of shake-flask culture are to OD600nm=5-6 (about 16h-18h), in 4 ℃ of centrifugal 30min of lower 5000r, the thalline of collecting is transferred in the 100mL BMMH liquid nutrient medium, and 28 ℃ are continued shake-flask culture.Adding 100% methyl alcohol to final concentration every 24h in substratum is 0.5%, cultivates 4d, in 4 ℃ of centrifugal 30min of lower 5000r, gets the fermented supernatant fluid of positive recombinant bacterium X-33/pPICZ α AufeEX.Get simultaneously the recombinant bacterium X-33/pPICZ α A that transforms empty plasmid pPICZ α A, operate according to the same shake-flask culture method of positive recombinant bacterium X-33/pPICZ α AufeEX, nutrient solution is in 4 ℃ of centrifugal 30min of lower 5000r, must transform the fermented supernatant fluid of the recombinant bacterium X-33/pPICZ α A of empty plasmid pPICZ α A, in contrast.Get respectively the fermented supernatant fluid 200 μ L of positive recombinant bacterium X-33/pPICZ α A ufeEX, transform the fermented supernatant fluid 200 μ L of the recombinant bacterium X-33/pPICZ α A of empty plasmid pPICZ α A, add (making method of fibrin plate is seen lower) in the Oxford cup that is placed on the fibrin plate, check the scleroproein enzymic activity with the fibrin plate method.Fibrin plate is put 37 ℃ of insulation 18h, observe the dull and stereotyped upper position generation transparent circle that adds positive recombinant bacterium X-33/pPICZ α AufeEX fermented liquid and (see Fig. 1,1-6), and the fermented supernatant fluid that transforms the recombinant bacterium X-33/pPICZ α A of empty plasmid pPICZ α A can not form transparent circle (seeing Fig. 1,7).Show thus, the fermented supernatant fluid of positive recombinant bacterium X-33/pPICZ α AufeEX has fibrinolytic activity, and the fermented supernatant fluid that transforms the recombinant bacterium X-33/pPICZ α A of empty plasmid pPICZ α A does not have fibrinolytic activity, show that further the expression vector pPICZ α AufeEX with Urechis uniconctus fibrinoclase gene ufe2 obtains expressing in pichia pastoris phaff X-33, have fibrinoclase in the fermented liquid of positive recombinant bacterial strain, be again plasmin.
The fibrin plate method is the classical way of detection fibers protease activity.The making method of fibrin plate: get the 1.2g agarose and add the Tris-HCl damping fluid that 95mL contains NaCl0.15mol/L pH7.4 and boil fully dissolving, after being cooled to 55-60 ℃, the adding concentration expressed in percentage by weight is 1.36% fibrinogen solution 5mL, vibration shakes up gently, gets the agarose gel liquid solution.Getting 2 diameters is the sterilization plate of 120mm, the zymoplasm aqueous solution (50U) and the 0.5mL Profibrinolysin aqueous solution (5U) that add respectively 1mL in each plate, mixing, above-mentioned agarose gel liquid solution is poured in the plate, pour about 35mL in each plate into, rapid mixing, leave standstill 30min after, be solidified as translucent fibrin plate.
The prescription of the YPD plate culture medium among the embodiment 4 is: 10g/L yeast extract paste, 20g/L peptone, 20g/L glucose, 20g/L agar, 100mg/L Zeocin TM, distilled water 1L.The prescription of BMGH liquid nutrient medium is (together lower): 0.1mol/L phosphate buffered saline buffer pH6.0,10g/L ammonium sulfate, 3.4g/L yeast nitrogen base, 4 * 10
-4The g/L vitamin H, 10g/L glycerine.The prescription of BMMH liquid nutrient medium is (together lower): 0.1mol/L phosphate buffered saline buffer pH6.0,10g/L ammonium sulfate, 3.4g/L yeast nitrogen base, 4 * 10
-4The g/L vitamin H, 5.0g/L glycerine.
Embodiment 5: recombinant fiber protein dissolution separation and purification of enzyme
(1) Recombinant Pichia pastoris is induced the product fibrinoclase
Strain X-33/pPICZ α A ufeEX is linked into is equipped with in the 50mL BMGH liquid nutrient medium, 250-300rpm, 28 ℃ are cultured to 48h.The nutrient solution that obtains is through the centrifugal 10min of 4000rpm, abandon supernatant, collecting cell is transferred in the 1000mL BMMH liquid nutrient medium, 28 ℃ of cultivations, and to add 100% methyl alcohol to final concentration every 24h in substratum be 0.5%, is cultured to 72h, it is centrifugal to get fermented liquid, in 4 ℃ of centrifugal 20min of lower 6000rpm, abandon thalline, get fermented supernatant fluid.
(2) fibrinoclase separation and purification
Fermented supernatant fluid is through 90% ammonium sulfate precipitation, get throw out in 4 ℃ of centrifugal 20min of lower 6000rpm, the throw out dissolved in distilled water, the molecular weight cut-off of packing into is in 14000 the dialysis tubing, in 4 ℃ of lower distill water dialysis 24h, liquid is dry through freeze drier in the dialysis tubing, dry thing 15mL sample-loading buffer (0.01ml/LTris-HCl damping fluid, pH7.8) dissolving, with 10,000g high speed frozen centrifugation 30min removes insolubles, get (the 3.5cm * 9.0cm) of Balanced Q-Sepharose Fast Flow post on the supernatant liquor, wash with 0.01mol/L Tris-HCl (pH 7.8) damping fluid, with the sample-loading buffer gradient elution that contains 0-0.5mol/L NaCl, the fraction collection elutriant is measured enzymic activity take casein as substrate Folin-phenol reagent process again, collect each enzyme active component, lyophilize gets the purification of Recombinant fibrinoclase.
Embodiment 6: the thrombolysis activity test of recombinant fiber protein resolvase (plasmin)
20mL puts the small beaker from the new zealand white rabbit heart extracting blood, and its natural coagulation is treated in 4 ℃ of placements, and filter paper blots the thrombus surface, and thrombus is cut into small pieces.
Experiment is established 3 tested group, physiological saline negative control group, Lumbrukinase positive controls and restructuring plasmin experimental group.Get 3 in test tube, each adds the 2g thrombi, be labeled as respectively physiological saline group, Lumbrukinase group, plasmin group, the physiological saline group adds 2mL physiological saline, and the Lumbrukinase group adds 2mL Lumbrukinase normal saline solution (1000U/mL), the plasmin group adds 2mL restructuring plasmin normal saline solution (0.1mg/mL, be equivalent to 500U/mL), after application of sample is complete, place 37 ℃ of insulations, weighing in vitro remains the quality of thrombus behind the 3h, calculates the thrombolysis rate.Experimental result is as shown in table 1, and plasmin group thrombolysis rate when 3h is 76.4%, and Lumbrukinase group thrombolysis rate is 48.4%, and physiological saline group thrombolysis rate is 5.1%.Continue to prolong soaking time 1h, the remaining thrombus of plasmin group is degradable, and Lumbrukinase group and physiological saline group thrombi are without considerable change.Present embodiment proves that this recombinant fiber protein resolvase has the external thrombus solvency action.
Table 1. external thrombus dissolution experiment result
Above embodiment shows, the total RNA that extracts from marine benthos Urechis uniconctus body is template, carry out the RT-PCR operation, obtain cDNA the first chain, and take cDNA the first chain as template, the sequence that the clone obtains is Urechis uniconctus fibrinoclase full length gene cDNA sequence, and the open reading frame of this sequence (ORF) is totally 792 bases, the ATG that line indicates is initiator codon, and the TAA that line indicates is terminator codon.The open reading frame sequence of the full-length cDNA of Urechis uniconctus fibrinoclase gene ufe2 is:
ATGAAGACACTGTTGCTCATTTCATTGGCCGCTCTTGCCACTGCTGCGCCAGCCACATTC
GATGCTGTGCGTGCACTGAGCGTTGAATCCCGTATCATTGGTGGCTCACCGGCTGACATC
ACCAACTTCCCCTATCAACTGTCTCTGAGATTCATTGGCTCTCATACATGTGGTGCCGTAG
TTGTGTCGGTCAACAGGGCTCTGACAGCTGCCCATTGTACTGATGGACGTGTTATCGGTG
GTTTCACACTTTATGCTGGATCTACAAGTAGACTATCGGATGGTGTACTGTACACCAATTT
GGCAAAGGACGAGCACAGCGACTACAACAATGGTCAATGCACTTTCTGTAATGATATCT
GCATGCTCTCTCTATTAACCAACATGGACACGAGCAATCCCGGTATTGGAGCAATCAGTA
TAGCAGCTACCAGTGATTTTGCAGGAACAACTTGCACTTTGAGTGGATGGGGCCGCACA
TCCAGCAGCATTGATCTCCCCACTAATCTGCAACAGGTCGACGTGGGTGTTATTTCCAAT
ATTGAATGCCAAAGCCGAATGTCAGCAGCTGGAGCCAGTGTAGGAATTCAACATATCTG
CATCTTCAGCAGCAGCCAGGGATCTTGCAATGGTGACAATGGTGGTCCCATGAGATGCA
CAGAGGGCGGCATTGGTGTATTGGCAGGAATCACCTCCTGGGGAATCTCTGTTGGAGGG
GAATGCAGCACTATCTACCCATCCGTCTACGTCAGAGTCACAACCTTTCGGCCATGGATT
GCCGAACGCATG
TAA
CDNA original coding sequence by Urechis uniconctus fibrinoclase gene ufe2, translation obtains the aminoacid sequence of Urechis uniconctus fibrinoclase, this aminoacid sequence is totally 263 amino acid, the MKTLLLISLAALATA that line indicates is the signal peptide sequence of this enzyme, and IIGGSPADIT is the N end order-checking of this enzyme.The aminoacid sequence that translation obtains the Urechis uniconctus fibrinoclase is:
MKTLLLISLAALATAAPATFDAVRALSVESR
IIGGAPADITNFPYQLSLRFIGSHTCGAVVVSV
NRALTAAHCTDGRVIGGFTLYAGSTSRLSDGVLYTNLAKDEHSDYNNGQCTFCNDICMLSL
LTNMDTSNPGIGAISIAATSDFAGTTCTLSGWGRTSSSIDLPTNLQQVDVGVISNIECQSRMS
AAGASVGIQHICIFSSSQGSCNGDNGGPMRCTEGGIGVLAGITSWGISVGGECSTIYPSVYVR
VTTFRPWIAERM
The expression fragment ufeEX of Urechis uniconctus fibrinoclase gene is connected with carrier pPICZ α A made up expression vector pPICZ α AufeEX, through electric shock transformed competence colibacillus pichia pastoris phaff X-33, expression vector pPICZ α A ufeEX obtains expressing in the pichia pastoris phaff X-33 of positive restructuring.Have the scleroproein enzymic activity in the fermentation culture of the pichia pastoris phaff X-33 of positive restructuring, and the separation and purification plasmin that goes out to recombinate therefrom.This restructuring plasmin has thrombolytic effect.
The cDNA sequence of above-mentioned Urechis uniconctus fibrinoclase gene, can be by making up different recombinant plasmid vectors, thereby make up such as the fibrinoclase genetic engineering bacterium of isoacceptor not such as intestinal bacteria or yeast, make it in genetic engineering bacterium, to express, thus the preparation fibrinoclase.The fibrinoclase of preparation can be applicable to prepare anticoagulation medicine or the dissolved thrombus medicine for the treatment of cardiovascular and cerebrovascular diseases, has good research and development prospect.
The aminoacid sequence of above-mentioned Urechis uniconctus fibrinoclase can by making up different genetic engineering bacteriums, through the cultivation of genetic engineering bacterium, thereby prepare the fibrinoclase with this aminoacid sequence.The fibrinoclase of preparation can be applicable to prepare anticoagulation medicine or the dissolved thrombus medicine for the treatment of cardiovascular and cerebrovascular diseases, has good research and development prospect.
Claims (4)
1. the open reading frame of the cDNA sequence of the fibrinoclase of encoding is characterized in that:
ATGAAGACACTGTTGCTCATTTCATTGGCCGCTCTTGCCACTGCTGCGCCAGCCACATTCGATGCTGTGCGTGCACTGAGCGTTGAATCCCGTATCATTGGTGGCTCACCGGCTGACATCACCAACTTCCCCTATCAACTGTCTCTGAGATTCATTGGCTCTCATACATGTGGTGCCGTAGTTGTGTCGGTCAACAGGGCTCTGACAGCTGCCCATTGTACTGATGGACGTGTTATCGGTGGTTTCACACTTTATGCTGGATCTACAAGTAGACTATCGGATGGTGTACTGTACACCAATTTGGCAAAGGACGAGCACAGCGACTACAACAATGGTCAATGCACTTTCTGTAATGATATCTGCATGCTCTCTCTATTAACCAACATGGACACGAGCAATCCCGGTATTGGAGCAATCAGTATAGCAGCTACCAGTGATTTTGCAGGAACAACTTGCACTTTGAGTGGATGGGGCCGCACATCCAGCAGCATTGATCTCCCCACTAATCTGCAACAGGTCGACGTGGGTGTTATTTCCAATATTGAATGCCAAAGCCGAATGTCAGCAGCTGGAGCCAGTGTAGGAATTCAACATATCTGCATCTTCAGCAGCAGCCAGGGATCTTGCAATGGTGACAATGGTGGTCCCATGAGATGCACAGAGGGCGGCATTGGTGTATTGGCAGGAATCACCTCCTGGGGAATCTCTGTTGGAGGGGAATGCAGCACTATCTACCCATCCGTCTACGTCAGAGTCACAACCTTTCGGCCATGGATTGCCGAACGCATGTAA。
2. the open reading frame of the cDNA sequence of coding fibrinoclase as claimed in claim 1 is characterized in that this cDNA sequence totally 792 bases, take ATG as initiator codon, TAA is terminator codon.
3. fibrinoclase, it is characterized in that: the aminoacid sequence of above-mentioned fibrinoclase is MKTLLLISLAALATAAPATFDAVRALSVESRIIGGSPADITNFPYQLSLRFIGSHT CGAVVVSVNRALTAAHCTDGRVIGGFTLYAGSTSRLSDGVLYTNLAKDEHSDYNNG QCTFCNDTCMLSLLTNMDTSNPGIGAISIAATSDFAGTTCTLSGWGRTSSSIDLPT NLQQVDVGVISNIECQSRMSAAGASVGIQHICIFSSSQGSCNGDNGGPMRCTEGGI GVLAGITSWGISVGGECSTIYPSVYVRVTTFRPWIAERM.
4. the application of the cDNA sequence of coding fibrinoclase claimed in claim 1 in making up the fibrinoclase genetic engineering bacterium.
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| Title |
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| 初金鑫等.单环刺螠纤溶酶UFE-Ⅱ的分离纯化及其酶学性质.《中国生物制品学杂志》.2010,第23卷(第7期),720-723. |
| 初金鑫等.单环刺螠纤溶酶UFE-I的性质和溶栓活性.《天然产物研究与开发》.2010,第22卷661-664. |
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