CN101633929B - Gene for coding FKplase and application thereof - Google Patents

Abstract

The invention discloses a gene for coding FKplase and application thereof. A nucleotide sequence of the gene for coding FKplase is shown as SEQ ID NO.1. A host bacterial strain containing the gene is BL21(DE3)/FK2PmtPA and has the preservation number of CCTCC NO:M209139. The host bacterial strain can efficiently express recombination mutant tPA protein by induction, and the expression content occupies more than 25 percent of the total content of cell protein. The produced recombination tPA protein has the advantages of small molecular weight, high stability and specificity, high in-vitro activity of 4*10<4>IU/mg, long half-life period of 18-20 min, safe use, and the like. The bacterial strain has high genetic stability and production capability and can continuously breed for 30 generations; and the level for producing FK2PmtPA protein reaches more than 800 mg/L.

Description

A kind of gene of coding FKplase and application thereof

Technical field

The invention belongs to biological technical field, be specifically related to a kind of gene and application thereof of coding FKplase.

Background technology

Buman tPA is that (tissue type plasminogen activator tPA), is a kind of serine protease for people's tissue-type plasminogen activator.It contains 527 amino acid, molecular weight 68KD, can become the activation of the Profibrinolysin of non-activity plasmin and bring into play the thrombus dissolving effect, be used for thromboembolism treatments such as Acute Myocardial Infarction, cerebral thrombosis apoplexy, pulmonary infarction clinically, be present good in the world thrombolytic drug, it is fast, safe to have a thrombolysis, more the back disability rate is low, to advantages such as people's no antigens.

TPA is mainly by the vascular endothelial cell expression-secretion, and concentration is very low in the blood plasma, is about 5ng/mL, about 5 minutes of transformation period, and therefore from blood plasma or tissue, directly extract tPA and can not be used for producing, can only adopt genetic engineering technique production.1987, the rtPA that the U.S. takes the lead in ratifying first genetic engineering technique production is used for the clinical treatment Acute Myocardial Infarction, commodity are called alteplase (Activase), and this product belongs to the original tPA of total length, belong to tPA first-generation product, significantly shortcoming is transformation period short (3～6 minutes), active low, to the tPA inhibition PAI non-resistant of higher concentration in patient's blood plasma, thereby using dosage is very big, every pin contains tPA 150～200mg, and the price of every pin is also very expensive.

In order to solve the problem that original tPA exists, on the basis of tPA structure and functional study, the researchist further carries out aminoacid deletion and replaces Study on Variation tPA.TPA structurally can be divided into (finger plot structure territory, F district, 4-49aa), E district (somatomedin functional domain, 50-86aa), (kringle 1 in the K1 district, 87-175aa), K2 district (kringle2,176-261aa) with (proteolytic enzyme district, P district, 262-527aa) five functional domains (naming ﹠ numbering of structural domain is according to gene pool Genbank login number GI 137119 or document Pennica D.et al.Nature 301:214,1983) independently.Each ad hoc structure territory of t-PA and the relation between the various biologic activity of t-PA are illustrated, F district and K2 district all can specificity be incorporated into fibrin clot, K1 district and liver receptor are in conjunction with relevant, P contains serine protease in the district, be responsible for the fiber proenzyme is cut into cellulase, contain the binding site (aa296-299) of the active inhibition PAI of tPA on it.The disappearance in any one district or variation do not influence other regional structure and activity among the tPA, and this provides the foundation for the reorganization of tPA, transformation (being referred to as the tPA mutant).Countries such as the U.S., Germany, Japan, Britain all improve the performance of tPA by reorganization and variation in research, and have separately an intellecture property, as the reteplase of German Pola graceful (BoehringerMannheim) company exploitation (Reteplase, rPA), nPA enzyme (Lanoteplase), the Japan of the TNK enzyme (Tenecteplase) of U.S.'s Genentech (Genentech) company exploitation, U.S.'s Bristol-mayer Si Pubo (Bristol-MyersSpuibb) company exploitation defends the Monteplase (Monteplase) etc. that material (Eisai) is built the exploitation of ripple institute.Reteplase (Reteplase) is a kind of Recomposed tPA product of producing with the intestinal bacteria system expression, form by two structural areas of K2P, disappearance F, E, three zones of K1, contain 355 amino acid (Kohnert U, et al.Protein Engineering 5:93,1992, the patent No.: EP 0 382 174 A1).Its character and natural tPA molecule have had bigger change, stability is improved, transformation period was increased to nearly 15 minutes, but bigger decline is arranged with fibrinous avidity, it is combined into reversible the combination with fibrinous, binding ability is significantly less than natural tPA, and still contains the binding site of the active inhibition PAI of tPA in the reteplase structure, and in vitro tests shows that the PAI-1 of 5IU/mL just can make the reteplase inactivation of 5ng/ml.Its specific activity is 5 * 10 ⁵IU/mg, using dosage 20～40mg, preparation was preserved validity period 2 years at 2 ℃, and the dissolving rear stability is poor, hemorrhage rate height, clinical effectiveness is poor.The TNK enzyme is a kind of total length tPA that produces with Chinese hamster cell (CHO), contain 527 amino acid, variation takes place to replace in totally 6 amino acid three positions, and promptly the Thr103 in K1 district and Asn117 are replaced by Asn and Glu respectively, and the Lys296-His-Arg-Arg299 in P district is replaced by 4 Ala.Compare with original t-PA, the activity of the external anti-PAI of TNK enzyme improves 80 times, and the transformation period prolongs 4 times, and activity has only 82% of original tPA in vivo, and binding fiber albumen ability keeps 87% (Keyt.B.A.et al.PNAS, USA, 91:3670,1994).Monteplase is a kind of total length tPA that is produced by body hamster kidney cell, contain 527 amino acid, the Cys84 in E district is replaced into Ser, its action effect is similar to t-PA, the ability of plasminogen activation is strong 14.9 times when not having than scleroproein when scleroproein exists, but itself and scleroproein combination rate are about 1/3 of original t-PA, and it is descended to some extent to the thrombus site specific.More than three kinds of tPA mutant all go on the market, also has a kind of tPA mutant nPA enzyme (Lanoteplase) that waits for U.S. food and drug administration (FDA) approval in addition, F, E district have been lacked, and modified the glycosylation site in K1 district, compare with original t-PA, its transformation period prolongs 37min, and has improved thrombolysis activity, but reduced fibrinous avidity, and the rate of intracranialing hemorrhage that causes is higher than t-PA.Although various tPA mutant improve the performance of tPA aspect different, s-generation Recomposed tPA still exists poor heat stability, the problem that specificity is not high, using dosage is big and production cost is high.

The expression system that is used to produce tPA at present mainly is animal cell expression system and escherichia expression system.Be used at first production for treating with the cell system of tPA be melanoma cell (Griffiths, JB.et al.Adv.Biochem.Eng.Biotechnol.1987,34:147-166).Subsequently a series of gone on the market and be about to the listing tPA derivative products in, also be to adopt the zooblast system to produce mostly, as the TNK enzyme (Tenecteplase) of U.S.'s Genentech (Genentech) company exploitation, the nPA enzyme (Lanoteplase) of U.S.'s Bristol-mayer Si Pubo (Bristol-MyersSpuibb) company exploitation, be to produce with Chinese hamster cell (CHO), Japan defends material (Eisai) and builds the Monteplase (Monteplase) of ripple institute exploitation and produce with body hamster kidney cell.Since the eukaryotic cell culture systems exist the cell speed of growth slow, produce the yielding poorly of tPA, medium component costliness, culture condition and require characteristics such as height, cause the tPA production cost very high, cause the tPA product price high, patient is difficult to bear economically.

Intestinal bacteria (Escherichia coli) expression system becomes the first-selected host cell of most protein expression study and production owing to its easy and simple to handle, low production cost.Adopt the escherichia expression system production for treating promptly to receive in early days at the tPA drug research that the researchist pays close attention to and achieving success (Harris TJ et al.Mol.Biol.Med.1986,3:279-292 with tPA; Datar RV.et al.Biotechnology.1993,11:349-357), at present in the tPA product of listing, reteplase (Reteplase by the exploitation of German Pola graceful (Boehringer Mannheim) company, rPA) promptly be to produce, but need cotransformation recombinant expression plasmid and helper plasmid to come express recombinant tPA by coli strain K12.

Summary of the invention

Goal of the invention:, the purpose of this invention is to provide a kind of gene of coding FKplase at the deficiencies in the prior art.Contain Recomposed tPA that the host strain of this gene produces and have that molecular weight is little, good stability, activity in vivo height, long half time, high specificity, advantage such as safe in utilization, can be used for treating clinically diseases such as Acute Myocardial Infarction, cerebral thrombosis, pulmonary infarction, medically have important use value.

Another object of the present invention provides the application in producing FKplase of the gene of above-mentioned coding FKplase.

Technical scheme: for achieving the above object, the technical solution used in the present invention is as follows:

A kind of gene of coding FKplase, its nucleotide sequence is shown in SEQ ID NO:1.

A kind of expression vector, the gene of above-mentioned coding FKplase.Described expression vector is recombinant plasmid pETFK2PmtPA.

A kind of host cell contains above-mentioned expression vector.

A kind of host cell contains recombinant plasmid pETFK2PmtPA.

Above-mentioned host cell is for producing the colibacillus engineering of FKplase, this project bacterial strain called after e. coli bl21 (DE3)/pETFK ₂PmtPA (Escherichia coli BL21 (DE3)/pETFK ₂PmtPA), bacterial strain has been submitted Chinese typical culture collection center preservation, preservation address: China to. Wuhan. and Wuhan University, preservation date: on July 2nd, 2009, deposit number is CCTCC NO:M 209139.Contain in the above-mentioned engineering strain cell and be useful on the expression vector of producing FKplase; Contain a kind of recombination mutation buman tPA gene that is useful on the production FKplase on this expression vector, this gene has the nucleotide sequence shown in SEQ ID NO:1.

Above-mentioned expression vector is recombinant plasmid pETFK ₂PmtPA contains in the plasmid just like the described recombination mutation buman tPA of SEQ ID NO:1 gene order.

Engineering strain BL21 (DE3)/pETFK ₂The construction process of PmtPA may further comprise the steps:

(1) construction recombination plasmid pETFK ₂PmtPA:

Is that template amplification goes out Segment A with PCR method with plasmid pETrtPAm-1; Is that template amplification goes out fragment B with PCR method with total length tPAcDNA; Cut A fragment and B fragment respectively with restriction enzyme NheI and KpnI, carry out fragment with the T4 ligase enzyme again and connect, construct recombinant plasmid pETFK2PmtPA; Wherein, contain described nucleotide sequence on the recombinant plasmid pETFK2PmtPA just like SEQ ID NO:1;

This nucleotide sequence is the nucleotide sequence of Recomposed tPA gene, by F, K2, the P district of original tPA gene forming; Wherein, Segment A contains P, F and pET carrier, and the P region sequence is shown in SEQ ID NO:7, and the F region sequence is shown in SEQ ID No:8; Fragment B contains the K2 district, F district, P district and K2 district, and fragment B sequence is shown in SEQ ID NO:9.Identify recombinant plasmid pETFK2PmtPA: with single endonuclease digestion, double digestion recombinant plasmid is carried out preliminary evaluation, carry out finally determining through dna sequencing again.Wherein, plasmid pETrtPAm-1 is Chinese patent ZL 200510019707.8 disclosed plasmids.

(2) amplification recombinant plasmid pETFK ₂PmtPA:

With the recombinant plasmid pETFK2PmtPA transformed into escherichia coli DH5 α that step (1) makes up, obtain coli strain DH5 α/FK2PmtPA; Cultivate coli strain DH5 α/FK ₂PmtPA extracts recombinant plasmid pETFK2PmtPA;

(3) preparation host cell:

Cold CaCl ₂Legal system is equipped with e. coli bl21 (DE3) competent cell; With recombinant plasmid pETFK2PmtPA transformed into escherichia coli BL21 (DE3) competent cell, be coated on the dull and stereotyped last 37 ℃ of growth 14h of the LB that contains 100mg/ml Amp, select single bacterium colony of being of moderate size in 100ml LB substratum, when treating OD600=1, getting 1ml changes the IPTG that 3h in the 20mlLB substratum adds 10mmol/l over to and induces 6h, collect thalline, after SDS-PAGE detects the protein expression situation, obtain engineering strain BL21 (DE3)/K ₂PmtPA is host cell.

Beneficial effect: coli strain Escherichia coli BL21 (the DE3)/FK of gained of the present invention ₂PmtPA has following characteristics:

(1) the used coli strain BL21 of the present invention (DE3), available from gene Dynamic Test Chamber company limited, its genotype is F ^-OmpT hsdSB (rB-mB-) gal (λ cI 857 ind1 Sam7 nin5 lacUV5-T7gene1) dcm (DE3).BL21 is the most widely used host bacteria source of marking protein, and its advantage is to lack lon and ompT proteolytic enzyme, is beneficial to exogenous gene expression protein matter.BL21 (DE3) is a λ DE3 lysogenic bacteria, have the t7 rna polymerase gene on the DE3, the T7 rna polymerase gene in that inductor isopropylthio-(IPTG) or lactose-induced following is arranged, can make target protein obtain high level expression by the control of lacUV5 promotor.

(2) penbritin (Amp) there is resistance, can on the LB-Amp flat board, grows, on flat board, be the typical intestinal bacteria bacterium colony of diameter 1～2mm.The rounded protuberance of bacterium colony, mean diameter are 1.6mm, and appearance is moistening smooth, white.The opticmicroscope hypothallus is a rod-short, the form uniformity.

(3) under isopropylthio-(IPTG) inductive condition, can efficiently express novel recombinant human tPA (FK2PmtPA).The FK2PmtPA molecular weight of albumen of expressing is about 40kD, forms the inclusion body of non-activity in born of the same parents, and expression level reaches more than 25% of cell protein total amount.

(4) passage number is more than 30 times, inheritance stability, stable yield.

(5) the reorganization FK of its expression ₂PmtPA albumen is different from existing other recombination buman tPA (as rt-PA, TNK enzyme, reteplase reteplase etc.).This recombination buman tPA is made up of F, K2, the P-structure district of natural tPA, has removed E and two structural areas of K1 of natural tPA.In addition, compare with natural tPA molecule, contain the replacement sudden change of 3 amino acid codings in the P district, be respectively: S262G (TCC-GGT), R298E (AGG-GAG), R299E (AGG-GAG), there are 3 sudden changes in the F district, is respectively: Q42E (CAG-GAG), H44A (CAC-GCT), disappearance S45 (TCA) (digitized representation is the amino acid position in the natural tPA molecule).

In sum, coli strain Escherichia coli BL21 provided by the invention (DE3)/FK ₂PmtPA can produce FK high-levelly ₂PmtPA albumen utilizes 100 liters of fermentor tanks to carry out pilot scale fermentation production, produces the proteic level of FK2PmtPA and reaches more than the 800mg/L.FK2PmtPA albumen inclusion body detects with molten fine circle method through renaturation and purifying, has the effect that activates the former cellulolytic activity of fibrinolytic enzyme, and activity reaches 4 * 10 ⁴IU/mg reaches international like product level.This bacterial strain genetic stability height, continuous passage still keeps identical throughput more than 30 generations.Further animal experiment shows: FKplase (FK ₂PmtPA) the 18-20 minute activity in vivo transformation period, under the prerequisite that guarantees drug effect, security is high.Colibacillus engineering strain BL21 provided by the invention (DE3)/FK ₂PmtPA, throughput is strong, the output height, the genetic stability height, the novel recombination mutation tPA of production has little, the good stability of molecular weight, activity in vivo height, long half time, high specificity, advantage such as safe in utilization.On every index, meet or exceed international like product level, possess production application and be worth.Appearance has repeatability within the present invention, and those of ordinary skill can obtain this bacterial strain and utilize this bacterial strain to produce recombination mutation tPA according to step provided by the present invention.

The present invention utilizes e. coli bl21 (DE3) to obtain a kind of colibacillus engineering strain BL21 (DE3)/K ₂PmtPA can efficiently express novel reorganization variation tPA albumen FKplase (FK ₂PmtPA), and inheritance stability, going down to posterity still keeps higher tPA output more than 30 times.The reorganization variation tPA albumen (K that expresses ₂PmtPA) the activity in vivo transformation period extends to 18-20 minute; And suddenling change of K2 district and P regioselectivity, kept and fibrin-specific bonded F district and K2 district, strengthened the ability of anti-PAI inhibition, thrombolysis effect two kinds of external tPA products significantly being better than contrasting in animal body, and hemorrhage rate significantly is lower than two kinds of tPA products of contrast.

Description of drawings

Fig. 1 is that plasmid pET FK2PmtPA makes up synoptic diagram.

Fig. 2 is recombinant expression plasmid pET FK ₂The enzyme of PmtPA is cut and is identified 0.7% agarose gel electrophoresis figure.

Fig. 3 is coli strain BL21 (DE3)/FK ₂The PmtPA abduction delivering produces FK2PmtPA protein 10 %SDS-polyacrylamide gel electrophoresis figure.

Fig. 4 detects FK with molten fine circle method ₂The proteic plasminogen activation cellulolytic activity of PmtPA exercising result figure.

Fig. 5 is coli strain BL21 (DE3)/FK ₂The tropina 10%SDS-polyacrylamide gel electrophoresis figure in 30 generations of PmtPA continuous passage.

Embodiment:

The present invention is described in further detail below in conjunction with embodiment and accompanying drawing.Experimental technique among the embodiment, condition is carried out routinely, with reference to as Sa nurse Brooker etc., experimental technique described in the molecular cloning experiment guide (second edition,, Science Press in 1992).

Embodiment 1: plasmid pETFK ₂The structure of PmtPA.

As shown in Figure 1, the structure principle and the process that have shown recombination mutation tPA gene.Natural sophisticated buman tPA contains 527 amino acid, form (F, E, K1, K2 and P) by 5 independent structures districts, the P district contains inhibition PAI land and the enzymic activity district of tPA, K2 contains PAI zone of action and scleroproein land, K1 is relevant with liver removing tPA, F district (referring to the type structural area) is relevant with protein interaction, and when K1K2 lacked, the F district still can interact with scleroproein, Profibrinolysin; The pETrtPAm-1 plasmid contains the P district of F district, G district and the sudden change of expression plasmid pET and tPA gene.The recombination mutation FK2PmtPA gene that the present invention is designed is to consider stability, prolong half-life and the increase of the increase tPA resistance to inhibition PAI, and does not reduce itself and fibrinous bonding force.When the design gene is rebuild and is made a variation, be that template amplification goes out Segment A (containing F, P and pET carrier) with plasmid pETrtPAm-1 at first with PCR method, be that template amplification goes out fragment B (K2) with PCR method with total length tPAcDNA again, be built into plasmid pETFK2PmtPA.Obtain as the gene order as shown in the SED ID NO:1.Concrete implementation step is as follows:

(1) the segmental pcr amplification of A fragment and B

At first synthetic two pairs of primers are used for two segmental amplifications.

Primer 1:5 '-GCC TGT GCT AGCGCA CTC TGC CCT GCC ACT GTT G-3 ' (containing NheI cutting sequence GCTAGC);

Primer 2: 5 '-CC TCC TGC GGT ACCTGC GGC CTG-3 ' (containing KpnI cutting sequence GGTACC);

Primer 3:5 '-GGACA TCG GCT AGCGAG GGA AAC AGT GAC TGC-3 ' (containing NheI cutting sequence GCTAGC);

Primer 4:5 '-GCTCG GAT GGT ACCGCA GGA GGG CAC ATC ACAG-3 ' (containing KpnI cutting sequence GGTACC) °.

With pETrtPAm-1cDNA is amplification template, adds primer 1 and primer 2 in the reaction tubes, the PCR reaction conditions: 94 ℃ of 5min, and 94 ℃ of 1min afterwards, 51 ℃ of 3min, 72 ℃ of 2min totally 33 circulations, last 72 ℃ of 10min are used for amplified fragments A; Another group is an amplification template with total length tPAcDNA, add primer 3 and 4, be used for amplified fragments B, PCR reaction conditions: 94 ℃ of 5min, 94 ℃ of 1min afterwards, 55 ℃ of 1min, 72 ℃ of 1min totally 33 circulations, last 72 ℃ of 10min, pcr amplification product are respectively through the phenol extracting, and 1% low melting-point agarose gel electrophoresis is reclaimed.

(2) recombinant plasmid pETFK ₂The structure of PmtPA

With restriction endonuclease NheI and KpnI difference cutting fragment A and segment B, carry out fragment with the T4 ligase enzyme and connect, make up the pETFK2PmtPA clone, transformed into escherichia coli DH5 α competent cell, positive bacterium colony is obtained in screening on penbritin (Amp)-LB flat board.Through identifying called after DH5 α FK2PmtPA.Plasmid pETFK ₂The evaluation of PmtPA:

Adopt alkaline lysis, with reference to the Sa Samu Brooker etc., molecular cloning experiment guide (second edition,, Science Press in 1992) is from positive bacterium colony DH5 α FK ₂Extract plasmid pETFK in the PmtPA culture ₂PmtPA, with BamHI and HindIII double digestion, 0.7% agarose gel electrophoresis inspection, the result produces 1.23Kb and 2.8Kb two bands, conforms to the expection size, and wherein the band of 1.23kb is represented FK ₂K2 district, F district in the PmtPA gene and P district fragment, the band of 2.8kb then are pET plasmid fragments.Identify that with the EcoRI single endonuclease digestion band of 0.47kb is the part fragment of FK2PmtPA gene, the band of 3.5kD is pET plasmid and part FK ₂The PmtPA gene fragment as shown in Figure 2, conforms to the expection size.Among Fig. 2,1 for the pET carrier through transforming, and removed the 6His coding region, its big or small 2.6kb; 2 is dna molecular quantitative character (Marker); 3 is pETFK ₂The PmtPA plasmid is through the fragment of BamHI and the generation of HindIII double digestion, and wherein the band of 1.23kb is FK ₂F district in the PmtPA gene, K2 district and P district fragment, the band of 2.8kb then are pET plasmid fragments; 4 is with pET FK ₂The segment that the PmtPA plasmid produces through the EcoRI single endonuclease digestion, the band of 0.47kb is the part fragment of FK2PmtPA gene, the band of 3.5kb is pET plasmid and part FK2PmtPA gene fragment.

The order-checking of FK2PmtPA gene is undertaken by handsome (invitrogen) Bioisystech Co., Ltd on the plasmid.Sequencing primer is the t7 rna polymerase sequence universal primer that utilizes on the pET plasmid, and sequencing result shows the FK of acquisition ₂PmtPA gene order and desired design are in full accord, and FK2PmtPA full length gene 1206nt comprises termination codon TGA, 401 amino acid proteins of encoding.With the plasmid called after pETFK2PmtPA that produces.

Embodiment 2: engineering strain BL21 (DE3)/FK ₂The preparation of PmtPA.

(1) recombinant plasmid pETFK ₂The extraction of PmtPA

With DH5 α FK ₂PmtPA is inoculated into the LB substratum, and 37 ℃ of incubated overnight adopt alkaline lysis method of extracting plasmid pETFK2PmtPA.

(2) preparation of BL21 (DE3) competent cell

Use cold CaCl ₂Legal system is equipped with BL21 (DE3) competent cell.BL21 (DE3) bacterial classification inoculation in the 20mlLB liquid nutrient medium, was cultivated 3-5 hour for 37 ℃, culture ice bath 10min, the 4000rpm centrifugal collecting cell adds precooling 0.1M CaCl ₂Re-suspended cell, ice bath 30min, the 4000rpm centrifugal collecting cell adds 500 μ l 0.1M CaCl ₂(by initial incubation volume 20ml) suspension cell places 4 ℃.

(3) recombinant plasmid pETFK ₂PmtPA transforms BL21 (DE3)

The pETFK2PmtPA plasmid is mixed with BL21 (DE3) competent cell, 42 ℃ of heat shock 90s, ice bath 1-2min adds the LB liquid nutrient medium, cultivates 45min for 37 ℃, and coating penbritin (Amp)-LB flat board is cultivated 12-16h, positive bacterium colony can be occurred for 37 ℃.Identify through embodiment 3, can produce FK ₂The proteic bacterial strain called after of PmtPA BL21 (DE3)/FK ₂PmtPA.

Embodiment 3: coli strain BL21 (DE3)/FK ₂PmtPA induces through isopropylthio-(IPTG) and produces FK ₂PmtPA albumen.

From the rapid penbritin of previous step (Amp)-LB flat board, select single bacterium colony, being inoculated into 5ml contains in the LB liquid nutrient medium of 100 μ g/ml penbritins (Amp), cultivate 14h for 37 ℃, press 1: 40 transferred species then in the 2YT substratum, cultivate 2～3h for 37 ℃, adding isopropylthio-(IPTG) is 1mM to final concentration, 37 ℃ of abduction delivering 6h.Centrifugal collection thalline, be suspended in buffer A (20mMTris, 0.5M NaCl, pH8.0), the ultrasonic disruption cell, centrifugal collecting precipitation carries out the SDS-polyacrylamide gel electrophoresis, the analysing protein expression.Fig. 3 is the result of 10%SDS-polyacrylamide gel electrophoresis, and wherein, 1 for cultivating coli strain BL21 (the DE3)/FK before isopropylthio-(IPTG) is induced through LB, 2YT liquid nutrient medium ₂FK does not appear in the tropina of PmtPA ₂PmtPA albumen; 2 induce 6h, ultrasonic disruption coli strain BL21 (DE3)/FK for isopropylthio-(IPTG) ₂PmtPA, the albumen in the centrifugal supernatant that obtains does not have FK2PmtPA albumen; 3 induce 6h, ultrasonic disruption coli strain BL21 (DE3)/FK for isopropylthio-(IPTG) ₂PmtPA, FK2PmtPA albumen has appearred in the albumen in the centrifugal precipitation that obtains.The arrow place shows FK2PmtPA albumen, the FK of expression ₂PmtPA albumen accounts for more than 30% of total cell protein, and its size is about 40kD; 4 for will induce the FK of acquisition ₂The sex change of PmtPA inclusion body is dissolved in 8M urea, and phosphate buffered saline buffer (PBS) the dialyzed overnight renaturation of usefulness pH8.0 utilizes column chromatography DEAE-Sepharose to carry out the FK2PmtPA albumen of purifying again, passes through above-mentioned processing, FK ₂The proteic purity of PmtPA reaches more than 95%; 5 is protein molecule quantitative character (Marker).After process isopropylthio-(IPTG) was induced, coli strain efficiently expressed out FK ₂PmtPA.The expressing protein molecular weight is about 40kD, conforms to the expection size.Expressing protein is the inclusion body form, after cytoclasis, all appears in the precipitation, and expression amount accounts for more than 30% of total protein of cell.

Embodiment 4:100 rises coli strain BL21 (DE3)/FK in the fermentor tank ₂The fermentative production level of PmtPA.

The inclined-plane seed culture: LB inclined-plane solid medium, contain penbritin (Amp) 100 μ g/ml, cultivated 16 hours for 37 ℃.

Shake-flask seed is cultivated: the 2YT liquid nutrient medium, contain penbritin (Amp) 100 μ g/ml, every bottle of 100ml substratum, inoculation one ring, 37 ℃ shaking culture 12-14 hour.

Fermentation culture: used substratum is a fermention medium, and its prescription is: peptone 20g/L, yeast extract 8g/L, NaCl 2g/L, MgSO ₄0.2g/L, bubble enemy 20 μ l/L, pure water preparation, 100 liters of culture volumes.Regulate pH value to 7.0 before the sterilization.Steam sterilizing, cylinder was pressed 0.1mpa (120 ℃) 30 minutes.The control condition of fermentation culture and parameter: 100 liters of culture volumes, pH6.8-7.0; Inoculum size 5%-10%; Stirrer rotating speed 350RPM; Temperature controlling range 35-37 ℃; Tank pressure 0.05-0.07MPa; Air flow 3-5 liter/min (1: 0.3-1: 0.5).By above-mentioned condition and parameter, be cultured to that bacterium amount 6-8 grams per liter, pH value rise to 7.2, DO drops to 20% when following, adds isopropylthio-(IPTG) to final concentration 1mM, 35-37 ℃ is continued cultivation, induced protein expression 6-8 hour.Fermentation whole process is 10-12 hour.

Fermentation can obtain the inclusion body of Recomposed tPA.Continuously ferment with 10 liters of automatic fermenters and to produce 10 batches, thalline output (centrifugation weight in wet base) is stabilized in 8.0-10.0g/L, and tPA crystal (behind the purifying, weight in wet base) is stabilized in more than the 800mg/L.The tPA crystal checks that through the 10%SDS-polyacrylamide gel electrophoresis tPA protein content is about 90%.

Proteinic renaturation of embodiment 5:FK2PmtPA and purifying.

(1) will be through isopropylthio-(IPTG) inductive coli strain BL21 (DE3)/FK ₂The centrifugal collection of PmtPA, be suspended in buffer A (20mM Tris, 0.5M NaCl, pH8.0).

(2) ultrasonic disruption cell, centrifugal collecting precipitation, wash 8 times after, be dissolved in 8M urea, under 4 ℃ of conditions, with phosphate buffered saline buffer (PBS) the dialyzed overnight renaturation of pH8.0.

(3) utilize chromatography column DEAE-Sepharose (available from peace agate West Asia company) to carry out purifying, pillar 10mMTris-Cl (pH8.5) balance, with 10mMTris-Cl (pH8.5)-0.1M-0.5MNacl wash-out, monitor the collecting high-activity peak with nucleic acid protein detector (wavelength 280nm).

Swimming lane 4 among Fig. 3 shows through the FK behind sex change dissolving, renaturation and the purifying ₂PmtPA albumen, behind the DEAE-Sepharose chromatography purification, the proteic purity of FK2PmtPA can reach about 95%.

Embodiment 6: the FK that coli strain BL21 (DE3)/FK2PmtPA expresses ₂Proteic active detection of PmtPA.

Adopt the solusphere method to carry out, step is as follows:

(1) tPA standard substance: available from Beijing pharmaceutical biological product calibrating institute of the Ministry of Health, every bottle contains tPA30000IU, and with the dissolving of 3ml physiological sodium chloride solution, making tPA concentration is 10IU/ul.

(2) human thrombin: be made into 100IU/ml with physiological sodium chloride solution, in-20 ℃ of preservations.

(3) Profibrinolysin:, be made into 0.5mg/ml with physiological sodium chloride solution available from Beijing pharmaceutical biological product calibrating institute.

(4) human fibrinogen: 30mg/ props up, Nat'l Pharmaceutical ﹠ Biological Products Control Institute's preparation standard reagent.Configuration before the experiment, earlier at 37 ℃ of water-bath preheating 15min, physiological sodium chloride solution is preheating simultaneously also before the configuration, and every adds the 5ml physiological sodium chloride solution then, and insulation 30min is left standstill in 37 ℃ of water-baths dissolves it fully, is configured to 6mg/ml solution.

(5) fibrin plate preparation: take by weighing the 125mg agarose, add the 23ml physiological sodium chloride solution, boil dissolving, 60 ℃ of water-bath balances, add 280ul scleroproein (0.5mg/ml), add 14ul zymoplasm (100IU/ml), do not stop to shake up, muddy back is fallen dull and stereotyped, horizontal positioned, and room temperature is solidified.

(6) the active detection: punch on fibrin plate with punch tool, diameter 2mm, in tPA sample and standard substance point hand-hole, 37 ℃ 8 hours, measurement transparent circle diameter.Transparent circle diameter according to standard tPA and FK2PmtPA sample calculates the sample activity.

The result as shown in Figure 4, wherein, 1 negative contrast: physiological sodium chloride solution, molten fine circle does not appear in point sample 2 μ l; 2 is standard control: standard buman tPA sample, and molten fine circle appears in point sample 10 units (10IU); 3 is experimental group: through the FK2PmtPA albumen of renaturation and purifying, molten fine circle appears in point sample 5 μ l.The result shows coli strain BL21 (DE3)/FK of the present invention ₂The FK2PmtPA albumen that PmtPA expresses has the effect of plasminogen activation cellulolytic activity.Can produce molten fine circle by plasminogen activation through the FK2PmtPA albumen behind sex change, renaturation and the purifying, through with the conversion of standard tPA, activity reaches 5 * 10 ⁴IU/mg.

Embodiment 7: coli strain BL21 (DE3)/FK ₂The inheritance stability Journal of Sex Research of PmtPA.

Streak inoculation bacterial strain on penbritin (Amp)-LB solid plate substratum is cultivated 18h for 37 ℃, and picking list bacterium colony continues streak culture, and every biography once is designated as a generation.In going down to posterity every 5 generation picking list colony inoculation 5ml LB liquid nutrient mediums (containing penbritin 100 μ g/ml), cultivate 14h for 37 ℃, press 1: 40 transferred species then in the 2YT substratum, cultivate 3～4h for 37 ℃, adding isopropylthio-(IPTG) is 1mM to final concentration, 37 ℃ of abduction delivering 6h.Centrifugal collection thalline, (20mMTris, 0.5M NaCl pH8.0), carry out SDS-polyacrylamide gel electrophoresis analysing protein expression to be suspended in buffer A.

As shown in Figure 5, wherein, M is protein Marker; 1-7 respectively is starting strain, the 5th, 10,15,20,25,30 generation bacterial strain tropina; The FK that the arrow indication starting strain and the bacterial strain that goes down to posterity are expressed ₂PmtPA albumen.The result shows, after going down to posterity through 30 times, and coli strain BL21 (DE3)/FK ₂PmtPA still can express FK in stability and high efficiency ground after isopropylthio-(IPTG) is induced ₂PmtPA albumen, electrophoretogram and expression amount no significant difference show coli strain BL21 (DE3)/FK ₂PmtPA has good genetic stability.Coli strain BL21 (DE3)/FK ₂PmtPA produces FK ₂The proteic ability of PmtPA does not change.

The proteic animal experiment of embodiment 8:FK2PmtPA.

Transformation period in the proteic body of FK2PmtPA: get three of rabbit, dosage by 200,000 unit/kilograms is injected from ear vein, injection finishes back 5 minutes blood-sample withdrawals for the first time, be added in the tubule of antithrombotics, centrifugal 1500 rev/mins of sedimentation cells, get 5 μ l supernatants and survey the tPA activity, adopted a blood sample and survey the tPA activity, the mean time of the active decline 50% of calculating tPA in later per 5 minutes.Through measuring FK ₂PmtPA was at the intravital transformation period 18-20 of rabbit minute.

SEQUENCE?LISTING

＜110〉Jiangsu Bowen Bio-Technology Co., Ltd.

＜120〉a kind of gene of coding FKplase and application thereof

<130>njsg090804

<160>9

<170>PatentIn?version?3.3

<210>1

<211>1206

<212>DNA

<213>Artificial

<220>

＜223〉Recomposed tPA gene

<220>

<221>CDS

<222>(1)..(1206)

<400>1

atg?gga?aga?tct?tac?caa?gtg?atc?tgc?aga?gat?gaa?aaa?acg?cag?atg 48

Met?Gly?Arg?Ser?Tyr?Gln?Val?Ile?Cys?Arg?Asp?Glu?Lys?Thr?Gln?Met

1 5 10 15

ata?tac?cag?caa?cat?cag?tca?tgg?ctg?cgc?cct?gtg?ctc?aga?agc?aac 96

Ile?Tyr?Gln?Gln?His?Gln?Ser?Trp?Leu?Arg?Pro?Val?Leu?Arg?Ser?Asn

20 25 30

cgg?gtg?gaa?tat?tgc?tgg?tgc?aac?agt?ggc?agg?gca?gag?tgc?gct?agc 144

Arg?Val?Glu?Tyr?Cys?Trp?Cys?Asn?Ser?Gly?Arg?Ala?Glu?Cys?Ala?Ser

35 40 45

gag?gga?aac?agt?gac?tgc?tac?ttt?ggg?aat?ggg?tca?gcc?tac?cgt?ggc 192

Glu?Gly?Asn?Ser?Asp?Cys?Tyr?Phe?Gly?Asn?Gly?Ser?Ala?Tyr?Arg?Gly

50 55 60

acg?cac?agc?ctc?acc?gag?tcg?ggt?gcc?tcc?tgc?ctc?ccg?tgg?aat?tcc 240

Thr?His?Ser?Leu?Thr?Glu?Ser?Gly?Ala?Ser?Cys?Leu?Pro?Trp?Asn?Ser

65 70 75 80

atg?atc?ctg?ata?ggc?aag?gtt?tac?aca?gca?cag?aac?ccc?agt?gcc?cag 288

Met?Ile?Leu?Ile?Gly?Lys?Val?Tyr?Thr?Ala?Gln?Asn?Pro?Ser?Ala?Gln

85 90 95

gca?ctg?ggc?ctg?ggc?aaa?cat?aat?tac?tgc?cgg?aat?cct?gat?ggg?gat 336

Ala?Leu?Gly?Leu?Gly?Lys?His?Asn?Tyr?Cys?Arg?Asn?Pro?Asp?Gly?Asp

100 105 110

gcc?aag?ccc?tgg?tgc?cac?gtg?ctg?aag?aac?cgc?agg?ctg?acg?tgg?gag 384

Ala?Lys?Pro?Trp?Cys?His?Val?Leu?Lys?Asn?Arg?Arg?Leu?Thr?Trp?Glu

115 120 125

tac?tgt?gat?gtg?ccc?tcc?tgc?ggt?acc?tgc?ggc?ctg?aga?cag?tac?agc 432

Tyr?Cys?Asp?Val?Pro?Ser?Cys?Gly?Thr?Cys?Gly?Leu?Arg?Gln?Tyr?Ser

130 135 140

cag?cct?cag?ttt?cgc?atc?aaa?gga?ggg?ctc?ttc?gcc?gac?atc?gcc?tcc 480

Gln?Pro?Gln?Phe?Arg?Ile?Lys?Gly?Gly?Leu?Phe?Ala?Asp?Ile?Ala?Ser

145 150 155 160

cac?ccc?tgg?cag?gct?gcc?atc?ttt?gcc?aag?cac?gag?gag?tcg?ccc?gga 528

His?Pro?Trp?Gln?Ala?Ala?Ile?Phe?Ala?Lys?His?Glu?Glu?Ser?Pro?Gly

165 170 175

gag?cgg?ttc?ctg?tgc?ggg?ggc?ata?ctc?atc?agc?tcc?tgc?tgg?att?ctc 576

Glu?Arg?Phe?Leu?Cys?Gly?Gly?Ile?Leu?Ile?Ser?Ser?Cys?Trp?Ile?Leu

180 185 190

tct?gcc?gcc?cac?tgc?ttc?cag?gag?agg?ttt?ccg?ccc?cac?cac?ctg?acg 624

Ser?Ala?Ala?His?Cys?Phe?Gln?Glu?Arg?Phe?Pro?Pro?His?His?Leu?Thr

195 200 205

gtg?atc?ttg?ggc?aga?aca?tac?cgg?gtg?gtc?cct?ggc?gag?gag?gag?cag 672

Val?Ile?Leu?Gly?Arg?Thr?Tyr?Arg?Val?Val?Pro?Gly?Glu?Glu?Glu?Gln

210 215 220

aaa?ttt?gaa?gtc?gaa?aaa?tac?att?gtc?cat?aag?gaa?ttc?gat?gat?gac 720

Lys?Phe?Glu?Val?Glu?Lys?Tyr?Ile?Val?His?Lys?Glu?Phe?Asp?Asp?Asp

225 230 235 240

act?tac?gac?aat?gac?att?gcg?ctg?ctg?cag?ctg?aaa?tcg?gat?tcg?tcc 768

Thr?Tyr?Asp?Asn?Asp?Ile?Ala?Leu?Leu?Gln?Leu?Lys?Ser?Asp?Ser?Ser

245 250 255

cgc?tgt?gcc?cag?gag?agc?agc?gtg?gtc?cgc?act?gtg?tgc?ctt?ccc?ccg 816

Arg?Cys?Ala?Gln?Glu?Ser?Ser?Val?Val?Arg?Thr?Val?Cys?Leu?Pro?Pro

260 265 270

gcg?gac?ctg?cag?ctg?ccg?gac?tgg?acg?gag?tgt?gag?ctc?tcc?ggc?tac 864

Ala?Asp?Leu?Gln?Leu?Pro?Asp?Trp?Thr?Glu?Cys?Glu?Leu?Ser?Gly?Tyr

275 280 285

ggc?aag?cat?gag?gcc?ttg?tct?cct?ttc?tat?tcg?gag?cgg?ctg?aag?gag 912

Gly?Lys?His?Glu?Ala?Leu?Ser?Pro?Phe?Tyr?Ser?Glu?Arg?Leu?Lys?Glu

290 295 300

gct?cat?gtc?aga?ctg?tac?cca?tcc?agc?cgc?tgc?aca?tca?caa?cat?tta 960

Ala?His?Val?Arg?Leu?Tyr?Pro?Ser?Ser?Arg?Cys?Thr?Ser?Gln?His?Leu

305 310 315 320

ctt?aac?aga?aca?gtc?acc?gac?aac?atg?ctg?tgt?gct?gga?gac?act?cgg 1008

Leu?Asn?Arg?Thr?Val?Thr?Asp?Asn?Met?Leu?Cys?Ala?Gly?Asp?Thr?Arg

325 330 335

agc?ggc?ggg?ccc?cag?gca?aac?ttg?cac?gac?gcc?tgc?cag?ggc?gat?tcg 1056

Ser?Gly?Gly?Pro?Gln?Ala?Asn?Leu?His?Asp?Ala?Cys?Gln?Gly?Asp?Ser

340 345 350

gga?ggc?ccc?ctg?gtg?tgt?ctg?aac?gat?ggc?cgc?atg?act?ttg?gtg?ggc 1104

Gly?Gly?Pro?Leu?Val?Cys?Leu?Asn?Asp?Gly?Arg?Met?Thr?Leu?Val?Gly

355 360 365

atc?atc?agc?tgg?ggc?ctg?ggc?tgt?gga?cag?aag?gat?gtc?ccg?ggt?gtg 1152

Ile?Ile?Ser?Trp?Gly?Leu?Gly?Cys?Gly?Gln?Lys?Asp?Val?Pro?Gly?Val

370 375 380

tac?acc?aag?gtt?acc?aac?tac?cta?gac?tgg?att?cgt?gac?aac?atg?cga 1200

Tyr?Thr?Lys?Val?Thr?Asn?Tyr?Leu?Asp?Trp?Ile?Arg?Asp?Asn?Met?Arg

385 390 395 400

ccg?tga 1206

Pro

<210>2

<211>401

<212>PRT

<213>Artificial

<220>

<223>Synthetic?Construct

<400>2

Met?Gly?Arg?Ser?Tyr?Gln?Val?Ile?Cys?Arg?Asp?Glu?Lys?Thr?Gln?Met

1 5 10 15

Ile?Tyr?Gln?Gln?His?Gln?Ser?Trp?Leu?Arg?Pro?Val?Leu?Arg?Ser?Asn

20 25 30

Arg?Val?Glu?Tyr?Cys?Trp?Cys?Asn?Ser?Gly?Arg?Ala?Glu?Cys?Ala?Ser

35 40 45

Glu?Gly?Asn?Ser?Asp?Cys?Tyr?Phe?Gly?Asn?Gly?Ser?Ala?Tyr?Arg?Gly

50 55 60

Thr?His?Ser?Leu?Thr?Glu?Ser?Gly?Ala?Ser?Cys?Leu?Pro?Trp?Asn?Ser

65 70 75 80

Met?Ile?Leu?Ile?Gly?Lys?Val?Tyr?Thr?Ala?Gln?Asn?Pro?Ser?Ala?Gln

85 90 95

Ala?Leu?Gly?Leu?Gly?Lys?His?Asn?Tyr?Cys?Arg?Asn?Pro?Asp?Gly?Asp

100 105 110

Ala?Lys?Pro?Trp?Cys?His?Val?Leu?Lys?Asn?Arg?Arg?Leu?Thr?Trp?Glu

115 120 125

Tyr?Cys?Asp?Val?Pro?Ser?Cys?Gly?Thr?Cys?Gly?Leu?Arg?Gln?Tyr?Ser

130 135 140

Gln?Pro?Gln?Phe?Arg?Ile?Lys?Gly?Gly?Leu?Phe?Ala?Asp?Ile?Ala?Ser

145 150 155 160

His?Pro?Trp?Gln?Ala?Ala?Ile?Phe?Ala?Lys?His?Glu?Glu?Ser?Pro?Gly

165 170 175

Glu?Arg?Phe?Leu?Cys?Gly?Gly?Ile?Leu?Ile?Ser?Ser?Cys?Trp?Ile?Leu

180 185 190

Ser?Ala?Ala?His?Cys?Phe?Gln?Glu?Arg?Phe?Pro?Pro?His?His?Leu?Thr

195 200 205

Val?Ile?Leu?Gly?Arg?Thr?Tyr?Arg?Val?Val?Pro?Gly?Glu?Glu?Glu?Gln

210 215 220

Lys?Phe?Glu?Val?Glu?Lys?Tyr?Ile?Val?His?Lys?Glu?Phe?Asp?Asp?Asp

225 230 235 240

Thr?Tyr?Asp?Asn?Asp?Ile?Ala?Leu?Leu?Gln?Leu?Lys?Ser?Asp?Ser?Ser

245 250 255

Arg?Cys?Ala?Gln?Glu?Ser?Ser?Val?Val?Arg?Thr?Val?Cys?Leu?Pro?Pro

260 265 270

Ala?Asp?Leu?Gln?Leu?Pro?Asp?Trp?Thr?Glu?Cys?Glu?Leu?Ser?Gly?Tyr

275 280 285

Gly?Lys?His?Glu?Ala?Leu?Ser?Pro?Phe?Tyr?Ser?Glu?Arg?Leu?Lys?Glu

290 295 300

Ala?His?Val?Arg?Leu?Tyr?Pro?Ser?Ser?Arg?Cys?Thr?Ser?Gln?His?Leu

305 310 315 320

Leu?Asn?Arg?Thr?Val?Thr?Asp?Asn?Met?Leu?Cys?Ala?Gly?Asp?Thr?Arg

325 330 335

Ser?Gly?Gly?Pro?Gln?Ala?Asn?Leu?His?Asp?Ala?Cys?Gln?Gly?Asp?Ser

340 345 350

Gly?Gly?Pro?Leu?Val?Cys?Leu?Asn?Asp?Gly?Arg?Met?Thr?Leu?Val?Gly

355 360 365

Ile?Ile?Ser?Trp?Gly?Leu?Gly?Cys?Gly?Gln?Lys?Asp?Val?Pro?Gly?Val

370 375 380

Tyr?Thr?Lys?Val?Thr?Asn?Tyr?Leu?Asp?Trp?Ile?Arg?Asp?Asn?Met?Arg

385 390 395 400

Pro

<210>3

<211>34

<212>DNA

<213>Artificial

<220>

＜223〉contain NheI cutting sequence GCTAGC

<400>3

gcctgtgcta?gcgcactctg?ccctgccact?gttg 34

<210>4

<211>23

<212>DNA

<213>Artificial

<220>

＜223〉contain KpnI cutting sequence GGTACC

<400>4

cctcctgcgg?tacctgcggc?ctg 23

<210>5

<211>32

<212>DNA

<213>Artificial

<220>

＜223〉contain NheI cutting sequence GCTAGC

<400>5

ggacatcggc?tagcgaggga?aacagtgact?gc 32

<210>6

<211>33

<212>DNA

<213>Artificial

<220>

＜223〉contain KpnI cutting sequence GGTACC

<400>6

gctcggatgg?taccgcagga?gggcacatca?cag 33

<210>7

<211>809

<212>DNA

<213>Artificial

<220>

＜223〉5 ' of Segment A end is the 261-527aa in the P district of tPA

<400>7

cctcctgcgg?tacctgcggc?ctgagacagt?acagccagcc?tcagtttcgc?atcaaaggag 60

ggctcttcgc?cgacatcgcc?tcccacccct?ggcaggctgc?catctttgcc?aagcacgagg 120

agtcgcccgg?agagcggttc?ctgtgcgggg?gcatactcat?cagctcctgc?tggattctct 180

ctgccgccca?ctgcttccag?gagaggtttc?cgccccacca?cctgacggtg?atcttgggca 240

gaacataccg?ggtggtccct?ggcgaggagg?agcagaaatt?tgaagtcgaa?aaatacattg 300

tccataagga?attcgatgat?gacacttacg?acaatgacat?tgcgctgctg?cagctgaaat 360

cggattcgtc?ccgctgtgcc?caggagagca?gcgtggtccg?cactgtgtgc?cttcccccgg 420

cggacctgca?gctgccggac?tggacggagt?gtgagctctc?cggctacggc?aagcatgagg 480

ccttgtctcc?tttctattcg?gagcggctga?aggaggctca?tgtcagactg?tacccatcca 540

gccgctgcac?atcacaacat?ttacttaaca?gaacagtcac?cgacaacatg?ctgtgtgctg 600

gagacactcg?gagcggcggg?ccccaggcaa?acttgcacga?cgcctgccag?ggcgattcgg 660

gaggccccct?ggtgtgtctg?aacgatggcc?gcatgacttt?ggtgggcatc?atcagctggg 720

gcctgggctg?tggacagaag?gatgtcccgg?gtgtgtacac?caaggttacc?aactacctag 780

actggattcg?tgacaacatg?cgaccgtga 809

<210>8

<211>150

<212>DNA

<213>Artificial

<220>

＜223〉3 ' of Segment A end is the F region sequence of tPA

<400>8

atgggaagat?cttaccaagt?gatctgcaga?gatgaaaaaa?cgcagatgat?ataccagcaa 60

catcagtcat?ggctgcgccc?tgtgctcaga?agcaaccggg?tggaatattg?ctggtgcaac 120

agtggcaggg?cagagtgcgc?tagcacaggc 150

<210>9

<211>289

<212>DNA

<213>Artificial

<220>

＜223〉fragment B complete sequence

<400>9

ggacatcggc?tagcgaggga?aacagtgact?gctactttgg?gaatgggtca?gcctaccgtg 60

gcacgcacag?cctcaccgag?tcgggtgcct?cctgcctccc?gtggaattcc?atgatcctga 120

taggcaaggt?ttacacagca?cagaacccca?gtgcccaggc?actgggcctg?ggcaaacata 180

attactgccg?gaatcctgat?ggggatgcca?agccctggtg?ccacgtgctg?aagaaccgca 240

ggctgacgtg?ggagtactgt?gatgtgccct?cctgcggtac?catccgagc 289

Claims (8)

1. the gene of a coding FKplase, its nucleotide sequence is shown in SEQ ID NO:1.

2. an expression vector is characterized in that: the gene that contains the described coding FKplase of claim 1.

3. expression vector according to claim 2 is characterized in that: described expression vector is recombinant plasmid pETFK ₂PmtPA.

4. a host cell is characterized in that: contain the described expression vector of claim 2.

5. according to right 4 described host cells, it is characterized in that: contain recombinant plasmid pETFK ₂PmtPA.

6. according to right 4 described host cells, it is characterized in that: described host cell is for producing the colibacillus engineering of FKplase, and its coli strain is BL21 (DE3)/FK ₂PmtPA, its deposit number are CCTCCNO:M 209139.

7. method that makes up the described host cell of claim 6, it is characterized in that: it may further comprise the steps

(1) construction recombination plasmid pETFK ₂PmtPA:

Is that template amplification goes out Segment A with PCR method with plasmid pETrtPAm-1; Is that template amplification goes out fragment B with PCR method with total length tPAcDNA; Cut A fragment and B fragment respectively with restriction enzyme NheI and KpnI, carry out fragment with the T4 ligase enzyme again and connect, construct recombinant plasmid pETFK ₂PmtPA; Wherein, recombinant plasmid pETFK ₂Contain described nucleotide sequence on the PmtPA just like SEQ ID NO:1;

Wherein, 5 ' end of Segment A for the 261-527aa in the P district of tPA shown in SEQ ID No:7,3 ' end of Segment A be the F region sequence of tPA shown in SEQ ID No:8, fragment B complete sequence is shown in SEQ ID No:9;

Wherein, the amplification method of Segment A and fragment B is as follows:

Primer 1:5 '-GCC TGT GCT AGCGCA CTC TGC CCT GCC ACT GTT G-3 ';

Primer 2: 5 '-CC TCC TGC GGT ACCTGC GGC CTG-3 ';

Primer 3:5 '-GGACA TCG GCT AGCGAG GGA AAC AGT GAC TGC-3 ';

Primer 4:5 '-GCTCG GAT GGTACCGCA GGA GGG CAC ATC ACAG-3 ';

With pETrtPAm-1cDNA is amplification template, adds primer 1 and primer 2 in the reaction tubes, the PCR reaction conditions: 94 ℃ of 5min, and 94 ℃ of 1min afterwards, 51 ℃ of 3min, 72 ℃ of 2min totally 33 circulations, last 72 ℃ of 10min are used for amplified fragments A; Another group is an amplification template with total length tPAcDNA, adds primer 3 and 4, is used for amplified fragments B, the PCR reaction conditions: 94 ℃ of 5min, 94 ℃ of 1min afterwards, 55 ℃ of 1min, 72 ℃ of 1min totally 33 circulations, last 72 ℃ of 10min;

(2) amplification recombinant plasmid pETFK ₂PmtPA:

Recombinant plasmid pETFK with step (1) structure ₂PmtPA transformed into escherichia coli DH5 α obtains coli strain DH5 α/FK ₂PmtPA; Cultivate coli strain DH5 α/FK ₂PmtPA extracts recombinant plasmid pETFK ₂PmtPA;

(3) preparation host cell:

Cold CaCl ₂Legal system is equipped with e. coli bl21 (DE3) competent cell; With recombinant plasmid pETFK ₂PmtPA transformed into escherichia coli BL21 (DE3) competent cell obtains engineering strain BL21 (DE3)/K through screening and evaluation ₂PmtPA is host cell.

8. the method for the described host cell of structure claim 6 according to claim 7 is characterized in that: in the step (1), to the recombinant plasmid pETFK that constructs ₂PmtPA identifies, comprises with single endonuclease digestion, double digestion and proving conclusively with dna sequencing after identifying correctly to recombinant plasmid again.

Images