CN110819613A

CN110819613A - Cell strain and method for expressing reteplase rPA

Info

Publication number: CN110819613A
Application number: CN201910711006.2A
Authority: CN
Inventors: 谢伟全
Original assignee: Individual
Current assignee: Individual
Priority date: 2019-08-02
Filing date: 2019-08-02
Publication date: 2020-02-21
Anticipated expiration: 2039-08-02
Also published as: CN110819613B

Abstract

The invention belongs to the field of biological pharmacy, and particularly relates to a method for expressing reteplase by human cells. The method comprises the following specific steps: synthesizing a cDNA sequence containing a secretion signal and encoding reteplase by a gene, inserting a lentivirus expression vector, constructing a lentivirus vector capable of secreting and expressing reteplase, packaging lentivirus particles, infecting human cells 293F with the lentivirus particles, screening a monoclonal cell strain with high copy and stable expression of the reteplase by puromycin resistance carried in the lentivirus vector, testing the expression content and the enzyme activity of the reteplase, and verifying the established expression method. The invention has the advantages that: the reteplase expression cell strain established by the method is a human 293F cell and does not contain pyrogenic endotoxin; and the expression cell strain is suspended and stably expresses the reteplase, is easy to amplify in a large scale, secretes and expresses the reteplase, has few impurities and is easy to purify.

Description

Cell strain and method for expressing reteplase rPA

Technical Field

The invention relates to the field of biological pharmacy, in particular to a novel method for expressing reteplase rPA by human cell secretion.

Background

Currently, thromboembolism is a common cardiovascular disease that seriously harms human health, placing a heavy burden on the patient's individuals, families, and society. Thrombolytic therapy is currently the most effective means of treating thromboembolic disorders. The rPA is a deletion mutant of human tissue plasminogen activator tPA, is a single-chain non-glycosylated protein, has the molecular weight of 39kDa and contains 9 pairs of disulfide bonds. Clinically, rPA is easier to act on the inside of thrombus, and has strong fibrinolysis effect and quick response.

In view of the annual increase in embolic patients, there is a very high clinical demand for reteplase. However, the current process for preparing reteplase is mainly based on transient expression in E.coli. The reteplase produced by the process exists in the form of inclusion bodies, and the inclusion bodies have no biological activity and only have activity after renaturation. However, inclusion bodies lost a large amount of renaturation and the molecules were heterogeneous. In addition, the Escherichia coli expression product is often mixed with pyrogen substances such as endotoxin, and clinical application requires a specific process for removing the pyrogen. In addition, the application of yeast and mammalian cells to produce the reteplase has high cost, long period and lower yield.

According to the invention, a lentivirus vector is adopted to integrate a reteplase coding gene sequence containing a secretion signal into a human cell 293F, a cell strain capable of stably secreting and expressing reteplase is screened, so that the obtained reteplase is soluble and expressed, does not contain endotoxin, the subsequent purification process is simplified through secretion and expression, one-step purification is really realized, and activity identification also shows that the reteplase prepared by the method has better biological activity.

Disclosure of Invention

In one aspect, the present invention provides a method for expressing reteplase rPA, comprising the steps of: infecting a host cell with a lentiviral particle comprising a gene encoding reteplase; culturing the host cell for expression of reteplase; separating to obtain the reteplase.

In one aspect, the host cell is a human cell.

In one or more embodiments, the human cell is a 293 cell, preferably, a 293F cell.

In one embodiment, the lentiviral particles are packaged from a plenti-rPA-puro plasmid.

In one or more embodiments, the plasmid carries a secretion signal and a cDNA sequence encoding reteplase.

In one embodiment, the cDNA sequence is: SEQ ID No. 2.

In one embodiment, wherein the amino acid sequence of reteplase is: SEQ ID No. 1.

In one aspect, the invention also provides a cell strain for stably and efficiently expressing reteplase rPA, which is obtained by infecting host cells with lentiviral particles containing a reteplase gene encoding reteplase, wherein the host cells are human cells, preferably 293 cells, and more preferably 293F cells.

In one embodiment, the amino acid sequence of the reteplase is: SEQ ID No. 1.

In one embodiment, the cDNA sequence is: SEQ ID No. 2.

In another aspect, the present invention also provides a method for expressing reteplase rPA, comprising the steps of: infecting human embryo kidney cells 293F with lentivirus particles lenti-rPA-puro, and screening to obtain a 293F cell strain with high resistance; screening a high-resistance monoclonal cell strain 293F-rPA for expressing reteplase; detecting the expression quantity of reteplase; a monoclonal cell line expressing high concentration and high activity rPA was verified.

In one or more embodiments, the plasmid carries a secretion signal selected from a short (5-30 amino acids in length) peptide chain that directs the transfer of the newly synthesized protein to the secretory pathway, and a cDNA sequence encoding reteplase.

In one embodiment, the cDNA sequence is: SEQ ID No. 2.

In one embodiment, the highly resistant 293F cell line is selected by puromycin at a graded concentration.

In one embodiment, the highly resistant monoclonal cell line 293F-rPA expressing reteplase is screened in a double dilution culture.

In one embodiment, the expression level of reteplase is detected by an enzyme-linked immunosorbent assay.

In one embodiment, reteplase activity is assayed by thrombolytic methods, and monoclonal cell lines expressing high concentrations and high activity rPA are validated.

Drawings

FIG. 1 is an immunoblot identification of rPA in the supernatant. 1: the 293F-rPA1# cell line expresses supernatant; 2: the 293F-rPA2# cell line expresses supernatant; 3: the 293F-rPA3# cell line expresses supernatant; 4: the supernatant was expressed from 293F cells infected with the empty virus.

FIG. 2 is a thrombolytic activity assay of rPA. 1: a tPA standard; 2: the 293F-rPA1# cell line expresses supernatant; 3: the 293F-rPA2# cell line expresses supernatant; 4: the 293F-rPA3# cell line expresses supernatant; 5: the supernatant was expressed from 293F cells infected with the empty virus.

Detailed Description

The technical solution of the present invention is further explained by the following embodiments. The examples are given solely to assist in understanding the invention and are not to be construed as limiting the invention in any way.

Definition of

The thrombo-embolism is a common cardiovascular disease seriously harming human health, and brings heavy burden to patients, families and society, and the thrombolysis treatment is the most effective means for treating the thromboembolic disease at present.

"treatment" can be effected in a number of different ways, including cure, palliation, and as prophylaxis (prophyxiases). Curative treatment is generally intended to cure a clinical condition, such as a disease or disorder, already present in the treated individual. Palliative therapy generally refers to therapy aimed at ameliorating a clinical condition already present in an individual without the need to cure the disease or disorder. Prophylactic treatment is generally intended to prevent the onset or worsening of clinical symptoms, i.e. to prevent them from progressing to a more severe stage.

The rPA is a deletion mutant of human tissue plasminogen activator tPA, is a single-chain non-glycosylated protein, has the molecular weight of 39kDa and contains 9 pairs of disulfide bonds. Clinically, rPA is easier to act on the inside of thrombus, and has strong fibrinolysis effect and quick response.

Detailed Description

In some embodiments, a method of expressing reteplase rPA comprises the steps of: infecting host cells with lentivirus particles containing a reteplase gene, culturing the host cells to express reteplase, and separating to obtain the reteplase.

In at least one embodiment, a lentivirus vector is adopted to integrate a reteplase coding gene sequence containing a secretion signal into a human cell 293F, a cell strain capable of stably secreting and expressing reteplase is screened, so that the obtained reteplase is soluble and expressed, does not contain endotoxin, the subsequent purification process is simplified through secretion expression, one-step purification is really achieved, and activity identification also shows that the reteplase prepared by the method has better biological activity.

Lentiviral particles

As used herein, a "lentiviral particle" is a replication-defective lentiviral particle. Such lentiviral particles can be produced from a lentiviral vector comprising the following elements: a 5 'lentiviral LTR, a tRNA binding site, a packaging signal, a promoter operably linked to a polynucleotide signal encoding the fusion protein, a second strand DNA synthesis origin, and a 3' lentiviral LTR.

In at least one embodiment, the lentiviral particles are gene therapy vectors developed based on HIV-1 (human immunodeficiency virus type I).

In at least one example, lentiviral particles were packaged according to a3 plasmid system (pMD2.G, psPAX2, plenti-rPA-puro), 3 plasmids were introduced into host cells in a specified ratio (pMD2.G: psPAX2: plenti-rPA-puro ═ 5:10: 15. mu.g) by lipofection, and the host cells were then placed in CO₂Culturing in an incubator.

In at least one embodiment, the rPA-encoding DNA sequence (SEQ. ID No.1) is obtained by in vitro whole gene synthesis by a chemical synthesis method, EcoRI enzyme sites and NotI enzyme sites are respectively introduced at the 5 end and the 3 end of the rPA, then the rPA is recombined into a lentiviral expression vector plenti-puro by a restriction endonuclease method, and the correct rPA expression plasmid plenti-rPA-puro is obtained by sequencing.

Host cell

As used herein, a "host cell" is a cell of human origin. In at least one embodiment, the cells used to package the virus include HEK293, CHO cells, and the like, and preferably, 293F cells. In at least one embodiment, a lentivirus vector is used to integrate a reteplase-encoding gene sequence comprising a secretion signal into human cells 293F, and cell lines stably expressing reteplase are selected, whereby the resulting reteplase is endotoxin-free.

In at least one example, lentiviral particles were packaged in accordance with a 3-plasmid system (pMD2.G, psPAX2, plenti-rPA-puro), 3 plasmids were introduced into 10 by lipofection in a specified ratio (pMD2.G: psPAX2: plenti-rPA-puro ═ 5:10: 15. mu.g)⁶Human 293T cells, then the 293T cells were placed in CO₂Culturing in an incubator.

Secretion signal

"secretion signal" as used herein refers to a short (5-30 amino acids in length) peptide chain that directs the transfer of the newly synthesized protein to the secretory pathway. Extracellular proteins play an important role in the formation, differentiation, maintenance, etc. of multicellular organisms. The fate of many individual cells, such as growth including survival, proliferation, migration, differentiation or interaction with other cells, is typically controlled by information received from other cells and/or the immediate environment (immediatate environment). This information is often transmitted by secreted polypeptides (e.g., mitotic factors, survival factors, cytotoxic factors, differentiation factors, neuropeptides, and hormones) which are received and interpreted by various cell receptors or membrane-bound proteins. These secreted polypeptides or signal molecules typically reach their site of action in the extracellular environment via the cellular secretory pathway.

In at least one embodiment, a method of expressing reteplase rPA, comprising the steps of: infecting human embryo kidney cells 293F with lentivirus particles lenti-rPA-puro, and screening to obtain a 293F cell strain with high resistance; screening a high-resistance monoclonal cell strain 293F-rPA for expressing reteplase; detecting the expression quantity of reteplase; a monoclonal cell line expressing high concentration and high activity rPA was verified.

In at least one embodiment, the lentiviral particles are packaged from a plenti-rPA-puro plasmid.

In at least one embodiment, the plasmid carries a secretion signal and a cDNA sequence encoding reteplase.

In at least one embodiment, the cDNA sequence is: SEQ ID No. 2.

In at least one embodiment, the highly resistant 293F cell line is selected by puromycin at a graded concentration.

In at least one embodiment, the highly resistant monoclonal cell line 293F-rPA expressing reteplase is screened in a multiple dilution culture.

In at least one embodiment, the expression level of reteplase is detected by an enzyme-linked immunosorbent assay.

In at least one example, reteplase activity was assayed by thrombolytic assay, and monoclonal cell lines expressing high concentrations and high activity rPA were verified.

In at least one embodiment, wherein the amino acid sequence of the reteplase is: SEQ ID No. 1.

In at least one embodiment, a reteplase coding DNA sequence (rPA) containing a secretion signal is synthesized by a chemical synthesis method, EcoRI enzyme sites and NotI enzyme sites are respectively introduced into two ends of the sequence, and then the sequence is subcloned into a lentiviral expression vector plenti-puro to obtain a recombinant plasmid plenti-rPA-puro. Packaging with a 4-plasmid system to obtain lentivirus particles lenti-rPA, wherein the lentivirus particles carry a gene sequence for encoding rPA. Human cells 293F were infected with lentivirus particles lenti-rPA and the mol value was set to 10. The cell line 293F-rPA containing high copy virus particles is screened by puromycin, and the puromycin gradient concentration is 0.5, 1.0, 1.5 and 2.0 mg/ml. The cell strain 293F-rPA obtained by screening is cultured in a suspension culture medium by a small test way for 5 days continuously, 100ul of supernatant is reserved every day, and the rPA content in the cell expression supernatant is measured by an enzyme-linked immunosorbent assay method. rPA thrombolytic activity was determined by thrombolytic assay.

In at least one embodiment, the rPA expression method provided by the invention is simple and feasible, can stably secrete expression, is easy to scale and is convenient for product purification.

In at least one embodiment, the lentivirus particles containing the gene coding the reteplase are infected into host cells, the host cells are cultured for expressing the reteplase, and the constructed monoclonal cell strain secreting and expressing the rPA can secrete the rPA with better biological activity.

Examples

Example 1

Construction of rPA Lentiviral expression plasmid

1. The complete DNA sequence of rPA is obtained by literature data retrieval and analysis, the DNA sequence (SEQ ID No.2) for coding the rPA is obtained by in vitro whole gene synthesis by a chemical synthesis method, EcoRI (CAS: 1040S, Takara bioengineering Co., Ltd.) and NotI enzyme (CAS: 1166S, Takara bioengineering Co., Ltd.) sites are respectively introduced into the 5 end and the 3 end of the rPA, then the rPA is recombined into a lentivirus expression vector plenti-puro (CAS: 39481, Addgene) by a restriction endonuclease method, and the correct expression plasmid plenti-rPA-puro of the rPA is obtained by sequencing.

Example 2

Construction of rPA-expressing cell line

1. Lentiviral particles expressing rPA were prepared.

(1) Lentiviral particles were packaged according to the 3 plasmid system (pMD2.G, psPAX2, plenti-rPA-puro) (Addgene), and 3 plasmids were introduced into 10 cells by the lipofection method (CAS:40802ES02, san Hai Sheng Biotech Co., Ltd.) in a specific ratio (pMD2.G: psPAX2: plenti-rPA-puro ═ 5:10: 15. mu.g)⁶Human 293T cells (ATCC) and then exposing 293T cells to CO₂Culturing in an incubator;

(2) centrifuging for 48h, collecting culture supernatant, 1200rpm for 5min, adding equal amount of culture medium into cells, and continuing culturing;

(3) after further culturing for 24h, centrifugally collecting culture supernatant at 1200rpm for 5 min;

(4) mixing the cell culture supernatants, adding 10% PEG8000/NaCL solution (CAS: A100159-0500, biological engineering Co., Ltd.), mixing, and ice-cooling for 1 hr;

(5)15000g of centrifugal ice bath solution for 15 min;

(6) the supernatant was discarded, and the pellet on the walls and bottom was resuspended in an appropriate amount of PBS (CAS: E607008-0500, Biotech Co., Ltd.) and dispensed in 100. mu.l portions to obtain rPA-expressing lentivirus particles lenti-rPA, which were stored at low temperature.

2.293F cell preparation.

(1) Thawing frozen cell 293F cell 1 tube in 37 deg.C water bath, adding 1ml fresh DMEM medium (CAS:11965092, ThermoFisher Scientific), and centrifuging at 1200rpm for 5 min;

(2) pouring out the supernatant, resuspending the cell sediment by using a fresh DMEM medium, transferring the cell sediment into a 250ml shake flask, adding the DMEM medium to 50ml, and then placing the shake flask into a CO2 incubator for shake culture at the rotation speed of 100 rpm;

(3) after culturing for 48h, when the cells are in logarithmic growth phase, centrifuging at 1200rpm for 5min to collect the cells;

(4) the cell pellet was resuspended in fresh DMEM medium and the cell density was adjusted to 107/1ml for use.

3. Lenti-rPA of lentivirus particles infected 293F cells.

(1) Adding the lentivirus particles lenti-rPA prepared in the step 1 into the 293F cells prepared in the step 2 according to the MOI values (virus titer/cell number) of 10, 20, 30 and 40, gently mixing uniformly, and then placing in a CO2 incubator for standing for 30 min;

(2) transferring the standing 293F cell mixture into a 250ml shake flask, adding DMEM medium to 50ml, and placing the mixture in a CO2 incubator for shaking culture at 100 rpm;

(3) after culturing for 72h, centrifuging at 1200rpm for 5min, and collecting cell precipitate;

(4) total RNA in cells is extracted by an RNA extraction kit (CAS:9767, Takara Bio Inc.), then the full-length expression of rPA is detected by an RT-PCR method, the relative amount of rPA is detected by a qPCR method, and the rPA is verified to be successfully integrated into the genome of 293F cells, so that mixed cells carrying rPA genes are obtained.

4. Screening monoclonal cell lines carrying high copy rPA.

(1) Culturing the mixed 293F cells verified to be correct in the step 3 in a 24-well plate at 4000/1 well;

(2) designing puromycin (CAS: A610593-0025, biological engineering Co., Ltd.) gradient concentration 0.5, 1.0, 1.5, 2.0mg/ml, adding into 24-pore plate containing cell, continuing culturing;

(3) observing the growth state of the cells every day, and supplementing a proper amount of DMEM culture medium and puromycin;

(4) selecting 293F cells with good growth state in the highest concentration puromycin (2.0mg/ml), then diluting and culturing the cells in the holes according to a 10-fold ratio, and screening monoclonal cell strains;

(5) the diluted cultured cells were observed daily and supplemented with DMEM medium and puromycin (2mg/ml) in appropriate amounts;

(6) observing the number of cells in each diluted hole under a microscope, and taking the hole with only 1 cell for amplification culture;

(7) after the well cells were continuously expanded and cultured to 107 total numbers, the cells were frozen and stored for later use, and the single-particle cell line 293F-rPA containing high-copy rPA was obtained as 3 lines, which were respectively designated as S1, S2, and S3.

rPA expression assay.

(1) Placing the monoclonal cell strain 293F-rPA obtained by screening in the step 4 in a 5ml DMEM medium for shaking culture at 100 rpm;

(2) designing time points including 4h, 8h, 12h, 16h, 20h, 24h, 36h and 48h, taking 0.2ml of supernatant at each time point, centrifuging at 1200rpm for 5min, and collecting the supernatant;

(3) detecting the rPA content in the supernatant by using a BCA quantitative kit (CAS: C503021, biological engineering Co., Ltd.) to confirm that the rPA content secreted by the S1 cell strain for 24 hours is up to 60 mu g/ml;

(4) SDS-PAGE gel (CAS: C631100-0100, Biotechnology, Inc.) with a concentration of 15% was prepared, rPA expression was identified by immunoblotting technique, and the results showed that all 3 selected cell lines secreted rPA.

Example 3

rPA thrombolytic Activity assay

1. Preparing a fibrin plate: weighing 0.02g fibrin (CAS:9001-32-5, Sigma-Aldrich), adding 5ml PBS, mixing, placing in 37 deg.C water bath to preheat until fully dissolving, simultaneously adding appropriate amount thrombin (CAS:9002-04-4, Sigma-Aldrich) into 1ml PBS solution, gently mixing, placing in 37 deg.C water bath to preheat; weighing 0.07g of agarose (CAS:9012-36-6, Sigma-Aldrich), adding 7ml of PBS solution, heating to dissolve, and cooling to about 50 ℃ to obtain agarose solution; and uniformly mixing the prepared thrombin solution and the fibrin solution, immediately adding the mixture into an agarose solution, uniformly mixing, pouring the mixture into a plate with the diameter of 10cm, and cooling at room temperature to obtain the fibrin plate.

2. The fibrin plate prepared in step 1 was punched with a tip, 5. mu.l of the rPA sample prepared in example 2 was added, and the resulting mixture was incubated in an incubator at 37 ℃ for 12 hours, followed by observation of the size of the thrombolytic ring. The expression supernatant of 293F cells infected with empty lentiviral vector was used as a negative control, and human tissue plasminogen activator tPA (CAS: T0831, Sigma-Aldrich) was used as a positive control.

3. And (4) evaluating the results: the result shows that the constructed monoclonal cell strain secreting and expressing rPA can secrete rPA with better biological activity, and the activity of the rPA is equivalent to that of positive control tPA.

Sequence listing

<110> thansfer

<120> cell strain for expressing reteplase rPA and method thereof

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<170>SIPOSequenceListing 1.0

<210>1

<211>377

<212>PRT

<213>Artificial Sequence

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Met Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly

1 5 10 15

Ala Val Phe Val Ser Pro Ser Tyr Gln Gly Asn Ser Asp Cys Tyr Phe

20 25 30

Gly Asn Gly Ser Ala Tyr Arg Gly Thr His Ser Leu Thr Glu Ser Gly

35 40 45

Ala Ser Cys Leu Pro Trp Asn Ser Met Ile Leu Ile Gly Lys Val Tyr

50 55 60

Thr Ala Gln Asn Pro Ser Ala Gln Ala Leu Gly Leu Gly Lys His Asn

65 70 75 80

Tyr Cys Arg Asn Pro Asp Gly Asp Ala Lys Pro Trp Cys His Val Leu

85 90 95

Lys Asn Arg Arg Leu Thr Trp Glu Tyr Cys Asp Val Pro Ser Cys Ser

100 105 110

Thr Cys Gly Leu Arg Gln Tyr Ser Gln Pro Gln Phe Arg Ile Lys Gly

115 120 125

Gly Leu Phe Ala Asp Ile Ala Ser His Pro Trp Gln Ala Ala Ile Phe

130 135 140

Ala Lys His Arg Arg Ser Pro Gly Glu Arg Phe Leu Cys Gly Gly Ile

145 150 155 160

Leu Ile Ser Ser Cys Trp Ile Leu Ser Ala Ala His Cys Phe Gln Glu

165 170 175

Arg Phe Pro Pro His His Leu Thr Val Ile Leu Gly Arg Thr Tyr Arg

180 185 190

Val Val Pro Gly Glu Glu Glu Gln Lys Phe Glu Val Glu Lys Tyr Ile

195 200 205

Val His Lys Glu Phe Asp Asp Asp Thr Tyr Asp Asn Asp Ile Ala Leu

210 215 220

Leu Gln Leu Lys Ser Asp Ser Ser Arg Cys Ala Gln Glu Ser Ser Val

225 230 235 240

Val Arg Thr Val Cys Leu Pro Pro Ala Asp Leu Gln Leu Pro Asp Trp

245 250 255

Thr Glu Cys Glu Leu Ser Gly Tyr Gly Lys His Glu Ala Leu Ser Pro

260 265 270

Phe Tyr Ser Glu Arg Leu Lys Glu Ala His Val Arg Leu Tyr Pro Ser

275 280 285

Ser Arg Cys Thr Ser Gln His Leu Leu Asn Arg Thr Val Thr Asp Asn

290 295 300

Met Leu Cys Ala Gly Asp Thr Arg Ser Gly Gly Pro Gln Ala Asn Leu

305 310 315 320

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

325 330 335

Asp Gly Arg Met Thr Leu Val Gly Ile Ile Ser Trp Gly Leu Gly Cys

340 345 350

Gly Gln Lys Asp Val Pro Gly Val Tyr Thr Lys Val Thr Asn Tyr Leu

355 360 365

Asp Trp Ile Arg Asp Asn Met Arg Pro

370 375

<210>2

<211>1132

<212>DNA

<213>Artificial Sequence

<400>2

atggatgcaa tgaagagagg gctctgctgt gtgctgctgc tgtgtggagc agtcttcgtt 60

tcgccctctt accaaaaaca gtgactgcta ctttgggaat gggtcagcct accgtggcac 120

gcacagcctc accgagtcgg gtgcctcctg cctcccgtgg aattccatga tcctgatagg 180

caaggtttac acagcacaga accccagtgc ccaggcactg ggcctgggca aacataatta 240

ctgccggaat cctgatgggg atgccaagcc ctggtgccac gtgctgaaga accgcaggct 300

gacgtgggag tactgtgatg tgccctcctg ctccacctgc ggcctgagac agtacagcca 360

gcctcagttt cgcatcaaag gagggctctt cgccgacatc gcctcccacc cctggcaggc 420

tgccatcttt gccaagcaca ggaggtcgcc cggagagcgg ttcctgtgcg ggggcatact 480

catcagctcc tgctggattc tctctgccgc ccactgcttc caggagaggt ttccgcccca 540

ccacctgacg gtgatcttgg gcagaacata ccgggtggtc cctggcgagg aggagcagaa 600

atttgaagtc gaaaaataca ttgtccataa ggaattcgat gatgacactt acgacaatga 660

cattgcgctg ctgcagctga aatcggattc gtcccgctgt gcccaggaga gcagcgtggt 720

ccgcactgtg tgccttcccc cggcggacct gcagctgccg gactggacgg agtgtgagct 780

ctccggctac ggcaagcatg aggccttgtc tcctttctat tcggagcggc tgaaggaggc 840

tcatgtcaga ctgtacccat ccagccgctg cacatcacaa catttactta acagaacagt 900

caccgacaac atgctgtgtg ctggagacac tcggagcggc gggccccagg caaacttgca 960

cgacgcctgc cagggcgatt cgggaggccc cctggtgtgt ctgaacgatg gccgcatgac 1020

tttggtgggc atcatcagct ggggcctggg ctgtggacag aaggatgtcc cgggtgtgta 1080

caccaaggtt accaactacc tagactggat tcgtgacaac atgcgaccgt ga 1132

Claims

1. A method for expressing reteplase rPA, comprising the steps of:

infecting a host cell with a lentiviral particle comprising a gene encoding reteplase;

culturing the host cell for expression of reteplase;

separating to obtain the reteplase;

preferably, the amino acid sequence of the reteplase is: SEQ ID No. 1.

2. The method of claim 1, wherein the host cell is a human cell.

3. The method of claim 2, wherein the human cell is a 293 cell; preferably, it is 293F cells.

4. The method of claim 1, wherein the lentiviral particle is packaged from a plenti-rPA-puro plasmid.

5. The method of claim 4, wherein the plasmid carries a secretion signal and a cDNA sequence encoding reteplase; preferably, the cDNA sequence is: SEQ ID No. 2.

6. A cell strain for expressing reteplase rPA is obtained by infecting host cells with lentiviral particles containing a reteplase gene; preferably, the amino acid sequence of the reteplase is: ID No.1, wherein said host cell is a human cell, preferably a 293 cell, more preferably a 293F cell.

7. The cell strain of claim 6, wherein the lentiviral particle is packaged from a plenti-rPA-puro plasmid.

8. The cell strain of claim 7, wherein the plasmid carries a secretion signal and a cDNA sequence encoding reteplase; preferably, the cDNA sequence is: SEQ ID No. 2.

9. A method for expressing reteplase rPA, comprising the steps of:

infecting human embryo kidney cells 293F with lentivirus particles lenti-rPA-puro, and screening to obtain a 293F cell strain with high resistance; preferably, the 293F cell strain with high resistance is obtained by screening puromycin with gradient concentration;

screening a high-resistance monoclonal cell strain 293F-rPA for expressing reteplase, preferably screening the high-resistance monoclonal cell strain 293F-rPA for expressing reteplase by a multiple ratio dilution culture method;

detecting the expression quantity of the reteplase, preferably detecting the expression quantity of the reteplase by an enzyme-linked immunosorbent assay.

The monoclonal cell line expressing high-concentration and high-activity rPA is verified, preferably, the activity of reteplase is detected by a thrombolytic method, and the monoclonal cell line expressing high-concentration and high-activity rPA is verified.

10. The method of claim 9, wherein the amino acid sequence of reteplase is: SEQ ID No. 1.