阅读说明：本技术 一种表达瑞替普酶rPA的细胞株及方法 (Cell strain and method for expressing reteplase rPA ) 是由 谢伟全 于 2019-08-02 设计创作，主要内容包括：本发明属于生物制药领域,具体涉及一种人源细胞表达瑞替普酶的方法细胞株。该方法具体步骤为：基因合成含有分泌信号的编码瑞替普酶的cDNA序列并插入慢病毒表达载体,构建可以分泌表达瑞替普酶的慢病毒载体,并包装慢病毒颗粒,然后以此慢病毒颗粒感染人源细胞293F,以慢病毒载体内携带的嘌呤霉素抗性筛选高拷贝、稳定表达瑞替普酶的单克隆细胞株,测试瑞替普酶表达含量及酶活性,验证建立的表达方法。本发明的优势在于：本方法建立的瑞替普酶表达细胞株是人源293F细胞,不含有致热源内毒素；并且表达细胞株是悬浮、稳定表达瑞替普酶,易于规模化放大,同时是分泌表达瑞替普酶,杂质少,易于纯化。(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.)

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.

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

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.

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

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.