Enzyme coding gene as well as expression and application thereof

Abstract

The reteplase is used for thrombolysis, and has good thrombus penetrability, higher thrombolysis efficiency and longer half-life period. The medicine is clinically used for emergency treatment of myocardial infarction, ischemic stroke and pulmonary embolism. The invention discloses a nucleotide sequence for preparing soluble reteplase by a cell-free protein expression system. The soluble reteplase can be efficiently produced with low complexity.

Description

Technical Field

[0001] The present application belongs to the field of biotechnology, and specifically relates to a gene encoding reteplase, and an expression template for cell-free protein expression constructed with the participation of the encoding gene, and its application. Background Art

[0002] Thrombotic diseases are common clinical diseases, including acute myocardial infarction (AMI) and acute ischemic stroke (AIS). They are characterized by high disability and mortality rates, and seriously endanger human health. Thrombolytic drugs play a very critical role in the treatment of thrombotic diseases. According to research progress, thrombolytic drugs can be divided into three generations: the first generation of thrombolytic drugs are non-specific plasminogen activators, such as streptokinase and urokinase. The thrombolysis rate is slow and the risk of bleeding is high, so they have been gradually eliminated. The second generation of thrombolytic drugs is represented by tissue plasminogen activator (tPA), such as alteplase and urokinase. The second generation of thrombolytic drugs has high fibrin specificity, high selectivity, high recanalization rate, and low bleeding risk. However, they have a short half-life, high short-term administration dose, and are expensive. The third generation of thrombolytic drugs includes reteplase, tenecteplase, ranteplase, recombinant human urokinase, etc. Compared with the second generation, the third generation of thrombolytic drugs has a long half-life, increased fibrin specificity, and less consumption of fibrinogen.

[0003] Reteplase (rPA) is a third-generation thrombolytic drug with the advantages of good specificity and long half-life. It is modified from the second-generation thrombolytic drug tPA, containing 355 of the 527 amino acids of natural tPA (amino acids 1-3 and 176-527), retaining the activity-related kringle-2 and serine protease domains, with better thrombus penetration, higher thrombolytic efficiency, and longer half-life. It is clinically used for the emergency treatment of myocardial infarction, ischemic stroke, and pulmonary embolism. Reteplase has a complex molecular structure and contains 9 pairs of disulfide bonds. It easily forms inactive inclusion bodies when expressed in Escherichia coli, and the production process is complicated. Figure 2 As shown, Reteplase contains more cysteine ​​and proline, and protein expression is difficult. And the spatial structure is complex. Since Reteplase is very difficult to directly express soluble active proteins, since it was launched in the United States in 1996, all commercially available Reteplase preparations are obtained by renaturation from inclusion bodies. The production process requires inclusion body denaturation and renaturation, which is complex, time-consuming, and inefficient, which seriously affects the production efficiency of Reteplase. Summary of the invention

[0004] The present application discloses a nucleic acid molecule for encoding reteplase, and a plasmid containing a gene fragment of the nucleotide sequence of the nucleic acid molecule. The present application also discloses an expression template for preparing reteplase by cell-free protein expression, a cell-free protein expression system, a corresponding method for preparing reteplase, and the use of the corresponding plasmid and expression target in the cell-free protein expression system to express reteplase.

[0016] The present application provides a nucleic acid molecule, the nucleotide sequence of which is shown in SEQ ID NO.1, SEQ ID NO.6 or SEQ ID NO.7, which is used to encode reteplase.

[0017] In some embodiments, the nucleotide sequence of the nucleic acid molecule can be obtained by using the amino acid sequence of Reteplase (for example, determined by querying the Drugbank database) and transforming it according to the codon preference of Escherichia coli. The nucleotide sequence can also be obtained by design optimization (for example, a combination of manual design and experimental verification, or codon optimization design software, etc.).

[0018] The present application also provides a plasmid, which comprises a gene fragment containing the nucleotide sequence of the nucleic acid molecule as described above.

[0019] In some embodiments, the nucleotide sequence of the plasmid is shown as SEQ ID NO.3, SEQ ID NO.8 or SEQ ID NO.9.

[0020] The plasmid can be obtained by inserting a gene fragment containing the nucleotide sequence of the nucleic acid molecule described above into a target plasmid based on homologous recombination. The nucleotide sequence of the target plasmid is shown in SEQ ID NO.2.

[0021] The present application also provides a linear DNA template for preparing reteplase through cell-free protein expression, wherein the nucleotide sequence of the linear DNA template is shown in SEQ ID NO.10 or SEQ ID NO.11.

[0022] In some embodiments, the linear DNA template can be obtained by adding protein expression elements to the upstream and downstream (5' end and 3' end) of the nucleotide sequence of the aforementioned reteplase gene. The protein expression element can be a regulatory sequence such as a promoter, an enhancer, a terminator, etc. Existing gene splicing methods such as PCR using primers can be used to connect the nucleotide sequence of the above-mentioned nucleic acid molecule and the protein expression element.

[0023] In some embodiments, the nucleotide sequence of the protein expression element upstream of the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO. 4, and the nucleotide sequence of the protein expression element downstream of the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO. 5. The nucleotide sequence SEQ ID NO. 4 may include a promoter and a ribosome binding site, and the nucleotide sequence SEQ ID NO. 5 may include a terminator.

[0024] The present application also provides a cell-free protein expression system. The system is used to express reteplase and may include a cell-free expression system and an expression template. In some embodiments, the cell-free expression system may include a cell extract and a supplement system. The cell extract may be selected from any one or any combination of Escherichia coli cell extract, yeast cell extract, wheat germ extract, insect cell extract, rabbit reticulocyte extract, and CHO cell extract. Alternatively or preferably, the cell extract may be an Escherichia coli cell extract. The supplement system may be selected from any one or any combination of phosphate buffer or its functional equivalent, magnesium salt or its functional equivalent, potassium salt or its functional equivalent, oxalate or its functional equivalent, amino acid or amino acid mixture or its functional equivalent, nucleotide mixture or its functional equivalent. Exemplarily, the phosphate buffer or its functional equivalent may be a phosphate buffer, Tris buffer, HEPES buffer, etc. The magnesium salt or its functional equivalent may be magnesium acetate, magnesium glutamate, etc. The potassium salt or its functional equivalent may be potassium acetate, potassium glutamate, etc. The oxalate or its functional equivalent may be sodium oxalate, potassium oxalate, etc. The amino acid or amino acid mixture or its functional equivalent can be glycine, alanine, valine, leucine, isoleucine, phenylalanine, proline, tryptophan as shown in NO.7, which is used to encode reteplase.

[0025] In some embodiments, the nucleotide sequence of the nucleic acid molecule can be obtained by using the amino acid sequence of Reteplase (for example, determined by querying the Drugbank database) and transforming it according to the codon preference of Escherichia coli. The nucleotide sequence can also be obtained by design optimization (for example, a combination of manual design and experimental verification, or codon optimization design software, etc.).

[0026] The present application also provides a plasmid, which comprises a gene fragment containing the nucleotide sequence of the nucleic acid molecule as described above.

[0027] In some embodiments, the nucleotide sequence of the plasmid is shown as SEQ ID NO.3, SEQ ID NO.8 or SEQ ID NO.9.

[0028] The plasmid can be obtained by inserting a gene fragment containing the nucleotide sequence of the nucleic acid molecule described above into a target plasmid based on homologous recombination. The nucleotide sequence of the target plasmid is shown in SEQ ID NO.2.

[0029] The present application also provides a linear DNA template for preparing reteplase through cell-free protein expression, wherein the nucleotide sequence of the linear DNA template is shown in SEQ ID NO.10 or SEQ ID NO.11.

[0030] In some embodiments, the linear DNA template can be obtained by adding protein expression elements to the upstream and downstream (5' end and 3' end) of the nucleotide sequence of the aforementioned reteplase gene. The protein expression element can be a regulatory sequence such as a promoter, an enhancer, a terminator, etc. Existing gene splicing methods such as PCR using primers can be used to connect the nucleotide sequence of the above-mentioned nucleic acid molecule and the protein expression element.

[0031] In some embodiments, the nucleotide sequence of the protein expression element upstream of the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO. 4, and the nucleotide sequence of the protein expression element downstream of the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO. 5. The nucleotide sequence SEQ ID NO. 4 may include a promoter and a ribosome binding site, and the nucleotide sequence SEQ ID NO. 5 may include a terminator.

[0032] The present application also provides a cell-free protein expression system. The system is used to express reteplase and may include a cell-free expression system and an expression template. In some embodiments, the cell-free expression system may include a cell extract and a supplement system. The cell extract may be selected from any one or any combination of Escherichia coli cell extract, yeast cell extract, wheat germ extract, insect cell extract, rabbit reticulocyte extract, and CHO cell extract. Alternatively or preferably, the cell extract may be an Escherichia coli cell extract. The supplement system may be selected from any one or any combination of phosphate buffer or its functional equivalent, magnesium salt or its functional equivalent, potassium salt or its functional equivalent, oxalate or its functional equivalent, amino acid or amino acid mixture or its functional equivalent, nucleotide mixture or its functional equivalent. Exemplarily, the phosphate buffer or its functional equivalent may be a phosphate buffer, Tris buffer, HEPES buffer, etc. The magnesium salt or its functional equivalent may be magnesium acetate, magnesium glutamate, etc. The potassium salt or its functional equivalent may be potassium acetate, potassium glutamate, etc. The oxalate or its functional equivalent may be sodium oxalate, potassium oxalate, etc. The amino acid or amino acid mixture or its functional equivalent can be a mixture of one or more of the twenty basic amino acids or non-natural amino acids such as glycine, alanine, valine, leucine, isoleucine, phenylalanine, proline, tryptophan, serine, tyrosine, cysteine, methionine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine and histidine. The nucleotide mixture or its functional equivalent can be a combination of one or more of ATP, AMP, CTP, CMP, GTP, GMP, UTP, UMP, etc. Alternatively or preferably, the supplement system can include phosphate buffer, AMP, UMP, CMP, GMP, potassium glutamate, magnesium glutamate, potassium oxalate and an amino acid mixture. For example, 10mM phosphate buffer, 1.2mMAMP, 0.85mM UMP, 0.85mM CMP, 0.85mM GMP, 300mM potassium glutamate, 8mM magnesium glutamate, 2.7mM potassium oxalate, and 2mM amino acid mixture.

[0033] In some embodiments, the volume ratio of the cell extract to the supplement system can be 1:1 to 1:2, for example, 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2.

[0034] In some embodiments, the expression template can be a plasmid or a linear DNA template as described above. In the entire cell-free protein expression system, the concentration of the expression template in the cell-free protein expression system is 5-50 ng / μL. For example, 5 ng / μL, 10 ng / μL, 15 ng / μL, 20 ng / μL, 25 ng / μL, 30 ng / μL, 35 ng / μL, 40 ng / μL, 45 ng / μL, 50 ng / μL, etc.

[0035] The present application also provides a method for preparing reteplase. The method uses the above-mentioned cell-free protein expression system to achieve the expression of reteplase. Exemplarily, the expression is completed after incubation at 15-37° C. for 2-24 hours.

[0036] The present application also provides an application of the above plasmid or linear DNA template to express reteplase in a cell-free protein expression system. When applied to a cell-free protein expression system, soluble reteplase can be directly prepared.

[0037] The optimized reteplase gene disclosed in the present application can be used to prepare soluble reteplase in a cell-free protein expression system with a high expression level. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 It is a protein electrophoresis diagram of preparing soluble reteplase using a cell-free expression system according to some embodiments of the present application;

[0039] Figure 2 It is the amino acid number analysis and spatial structure diagram of Reteplase protein;

[0040] Figure 3 It is a schematic diagram of protein production of different optimized sequences of Reteplase genes according to some embodiments of the present application;

[0041] Figure 4 It is a schematic diagram of protein yield when different concentrations of potassium glutamate are added to the reteplase expression system shown in some embodiments of the present application;

[0042] Figure 5 This is a protein electrophoresis diagram of Reteplase expressed in cells according to some embodiments of the present application. DETAILED DESCRIPTION

[0043] In order to more thoroughly understand the invention, some definitions are listed below. The above definitions are intended to include grammatical equivalents.

[0044] " nucleic acid " used in the present application means the polymer composed of nucleotide units (ribonucleotides, deoxyribonucleotides, related naturally occurring structural variants and non-naturally occurring analogs thereof) by phosphodiester bonds. Therefore, the term includes nucleotide polymers, wherein nucleotides and the bonds therebetween include non-naturally occurring synthetic analogs, such as but not limited to thiophosphates, phosphoramidates, methyl phosphonates, chiral methyl phosphonates, 2'-O-methyl ribonucleotides, peptide nucleic acids (PNA) etc. For example, automatic DNA synthesizers can be used to synthesize these polynucleotides. It should be understood that when nucleotide sequence is represented by DNA sequence (i.e. A, T, G, C), this also includes RNA sequences (i.e. A, U, G, C) in which "U" replaces "T".

[0045] "Coding" used in this application means the inherent properties of a specific nucleotide sequence in a polynucleotide, such as a gene, cDNA or mRNA, used as a template for synthesizing other polymers and macromolecules in a biological process with a determined nucleotide sequence, or a determined amino acid sequence and the resulting biological properties. Therefore, if the transcription and translation of the mRNA produced by the gene produces protein in a cell or other biological system, the gene encodes a protein. The coding strand (its nucleotide sequence is identical to the mRNA sequence and is generally provided in a sequence table) and the non-coding strand (used as a transcription template, gene or cDNA) can be referred to as a coded protein. Or other products of the gene or cDNA. Unless otherwise indicated, "nucleotide sequences encoding amino acid sequences" include all nucleotide sequences that are degenerate forms of each other and encode the same amino acid sequence. The nucleotide sequences of coded proteins and RNA may include introns.

[0046] "Plasmid" as used in this application means a plasmid artificially constructed on the basis of a natural plasmid to adapt to laboratory operations. A nucleic acid molecule can be introduced into a host cell to produce a transformed host cell. A vector may include a nucleic acid sequence that allows it to replicate in a host cell, such as an origin of replication, and may also include one or more selectable marker genes and other genetic elements known in the art.

[0047] As used in this application, "cell-free protein expression" means that cell lysates containing the components necessary for protein synthesis (ribosomes, transfer RNA, aminoacyl synthetases, initiation / elongation / termination factors, guanosine triphosphate, ATP, Mg2+ and K+) are synthesized in vitro. "Linear DNA template" can synthesize the target protein through transcription, translation, etc. during cell-free protein expression.

[0048] Unless otherwise stated, all technical and scientific terms used in this application have the same meaning as those generally understood by those of ordinary skill in the art to which this disclosure belongs. Unless the context clearly indicates otherwise, the singular terms "a", "an" and "the" include plural indicators. It should also be understood that all base sizes or amino acid sizes given for nucleic acids or polypeptides, and all molecular weights or molecular weight values ​​are approximate and are provided for description. Although methods and materials similar or equivalent to those described in this application can be used for the practice or testing of the present disclosure, suitable methods and materials are described below. The term "comprising" means "including". All publications, patent applications, patents and other references mentioned in this application are incorporated by reference as a whole. In the event of a conflict, this specification (including term explanations) shall prevail. In addition, materials, methods and embodiments are illustrative only and not restrictive.

[0049] In order to make the purpose, technical solution and advantages of the present application clearer, the technical solution of the present application will be clearly and completely described below in combination with the specific embodiments of the present application and the corresponding drawings. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present application.

[0050] Example 1 Preparation of soluble reteplase using a cell-free expression system

[0051] 1> Obtain the target protein coding sequence

[0052] The amino acid sequence of Reteplase (DB00015) was searched through Drugbank and converted into a nucleotide sequence according to the codon preference of Escherichia coli: SEQ ID NO.1, as shown below:

[0053] SEQ ID NO.1:

[0054]

[0055] 2>Construction of reteplase expression plasmid

[0056] The target gene sequence SEQ ID NO.1 was inserted into the plasmid SEQ ID NO.2 containing the protein expression element by homologous recombination to obtain the complete expression plasmid SEQ ID NO.3. As shown below:

[0057] SEQ ID NO.2:

[0058]

[0059] SEQ ID NO.3:

[0060]

[0061] 3> Preparation of soluble reteplase using a cell-free expression system

[0062] The components of the cell-free expression system are as follows: 10mM phosphate buffer, 1.2mM AMP, 0.85mM UMP, 0.85mM CMP, 0.85mM GMP, 260mM potassium glutamate, 8mM magnesium glutamate, 2.7mM potassium oxalate, 2mM amino acid mixture, 30% by volume of Escherichia coli cell extract, and 20ng / μL of the above expression plasmid; each component is added to a 48-well cell culture plate, mixed evenly, placed in a constant temperature mixer, reacted at 25°C and 600rpm for 16 hours, then taken out and the expression of reteplase is identified by protein electrophoresis.

[0063] The experimental results are as follows Figure 1 As shown, soluble reteplase was prepared using a cell-free expression system with a yield of 400 μg / mL;

[0064] Example 2 Preparation of soluble reteplase using optimized template

[0065] The cell-free expression system has the ability to directly use a linear expression template, without the need to construct the target gene into a plasmid, which significantly simplifies the workflow of protein expression identification. In this example, a linear expression template was used to screen the reteplase expression sequence.

[0066] 1> Obtain the target protein coding sequence

[0067] Based on SEQ ID NO.1, a series of sequences S1 to S15 were designed through a combination of manual design, AI optimization and experimental verification.

[0068] 2>Construct linear expression template

[0069] Add protein expression elements, SEQ ID NO.4 and SEQ ID NO.5, before and after each target gene sequence to obtain a complete linear expression template, as shown below:

[0070] SEQ ID NO.4:

[0071] tggagcggatcggggattgtctttcttcagctcgctgatgatatgctgacgctcaatgccgtttggcctccgactaacgaaaatcccgcatttggacggctgatccgattggcacggcggacggcgaatggcggagcagacgctcgtccgggggcaatgagatatgaaaaagcctgaactcaccgcgacgtatcgggccctggccaggatctgatattaatacgactcactataggggagctgtcaccggatgtgctttccggtctgatgagtccgtgaggacgaaacagCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACC

[0072] SEQ ID NO.5:

[0073] GGTTCTTCTCATCATCATCATCATCACTAAGCTAGCCTGtaaGGATCCGAATTCAAGCTTtgaccggctgcagcccgtacctctagtagagtcgagggggaacccggccgcgtccggcgcccccgccgccttcgacgagatcccgcaaaagcggcctttgactccctgcaagcctcagcgaccgaatatatcggttatgcgtgggcgatggttgttgtcattgtcggcgcaactatcggtatcaagctgtttaagaaattcacctcgaaagcaagctgataaaccgatacaattaaaggctccttttggagcctttttttttggagattttcaacgtgaaaaaattattattcgcaattcctttagttgttcctttctattctcactccgctgaaactgttgaaagttgtttagcaaaacctcatacagaaaattcatttactaacgtctggaaagacgacaaaactttagatcctctagctagagtcgaccgg

[0074] 3>Preparation of Soluble Retaplase Using a Cell-Free Expression System

[0075] The components of the cell-free expression system are as follows: 10 mM phosphate buffer, 1.2 mM AMP, 0.85 mM UMP, 0.85 mM CMP, 0.85 mM GMP, 260 mM potassium glutamate, 8 mM magnesium glutamate, 2.7 mM potassium oxalate, 2 mM amino acid mixture, 30% by volume of Escherichia coli cell extract, 2 μM Gams protein, and 10 ng / μL of the above linear expression template; each component is added to a 48-well cell culture plate, mixed evenly, placed in a constant temperature mixer, reacted at 25° C. and 600 rpm for 16 hours, then taken out, and the expression of reteplase is identified by protein electrophoresis;

[0076] The experimental results are as follows Figure 3 As shown, a cell-free expression system is used to prepare soluble reteplase, with a yield of 400-800 μg / mL; wherein sequences S2 and S5 have higher yields. The nucleotide sequence of S2 is shown in SEQ ID NO.6, and the nucleotide sequence of the corresponding linear expression template is shown in SEQ ID NO.10, and the nucleotide sequence of S5 is shown in SEQ ID NO.7. The nucleotide sequence of the corresponding linear expression template is shown in SEQ ID NO.11 as follows:

[0077] SEQ ID NO.6:

[0078]

[0079] SEQ ID NO.7:

[0080]

[0081] SEQ ID NO.10:

[0082]

[0083] SEQ ID NO.11:

[0084]

[0085] Example 3 Optimizing the reaction system to increase soluble reteplase

[0086] 1> Obtain the target protein coding sequence

[0087] Same as S2 and S5 in Example 2.

[0088] 2>Build expression template

[0089] The target gene sequences SEQ ID NO.6 and SEQ ID NO.7 were inserted into the plasmid SEQ ID NO.2 containing the protein expression element by homologous recombination to obtain complete expression plasmids SEQ ID NO.8 and SEQ ID NO.9, respectively. As shown below:

[0090] SEQ ID NO.8:

[0091]

[0092] SEQ ID NO.9:

[0093]

[0094] 3> Preparation of soluble reteplase using a cell-free expression system

[0095] The components of the cell-free expression system are as follows: 10 mM phosphate buffer, 1.2 mM AMP, 0.85 mM UMP, 0.85 mM CMP, 0.85 mM GMP, 0-400 mM potassium glutamate, 8 mM magnesium glutamate, 2.7 mM potassium oxalate, 2 mM amino acid mixture, 30% by volume of Escherichia coli cell extract, and 20 ng / μL of the above expression plasmid; each component is added to a 48-well cell culture plate, mixed evenly, placed in a constant temperature mixer, reacted at 25° C. and 600 rpm for 16 hours, then taken out, and the expression of reteplase is identified by protein electrophoresis;

[0096] The experimental results are as follows Figure 4 As shown, the cell-free expression system was used to prepare soluble reteplase, with a yield of 400-1200 μg / mL; the yield of soluble reteplase was highest when the potassium glutamate concentration was 300 mM. The protein activity was tested by the plate dissolution circle method and was 5.7*10^5U / mg, which was higher than the commercial standard of 5.0*10^5U / mg.

[0097] Example 4: Cell expression system for preparing soluble reteplase system scale-up experiment

[0098] 1> Obtain the target protein coding sequence

[0099] Same as S5 in Example 2;

[0100] 2>Build expression template

[0101] The same as the target gene sequence SEQ ID NO.9 in Example 3.

[0102] 3> Preparation of soluble reteplase using a cell-free expression system

[0103] The components of the cell-free expression system are as follows: 10 mM phosphate buffer, 1.2 mM AMP, 0.85 mM UMP, 0.85 mM CMP, 0.85 mM GMP, 300 mM potassium glutamate, 8 mM magnesium glutamate, 2.7 mM potassium oxalate, 2 mM amino acid mixture, 30% by volume of Escherichia coli cell extract, and 20 ng / μL of the above expression plasmid; each component is added to the corresponding reaction container (0.1 mL in a 48-well cell culture plate, 1 mL in a 6-well cell culture plate, 10 mL in a 250 mL conical flask, and 100 mL in a 2 L conical flask), mixed evenly, placed in a constant temperature shaker, reacted at 25° C. and 180 rpm for 16 hours, then taken out, and the expression of reteplase was identified by protein electrophoresis;

[0104] The experimental results showed that the yield of soluble reteplase prepared by cell-free expression systems of different volumes was about 1 mg / mL.

[0105] Comparative Example 1 Expression of Reteplase in Escherichia coli Cells

[0106] 1>Construction of expression strain

[0107] Take the expression plasmid in Example 1, transform E. coli BL21 (DE3), spread on LB plates with corresponding antibiotics, and culture at 37°C for 16 hours. Pick a single clone on the plate and transfer it into 5 mL LB liquid culture medium containing corresponding antibiotics, and culture at 37°C overnight with shaking.

[0108] 2>Expression, purification and identification of Reteplase

[0109] Inoculate the above overnight culture into 50mL of fresh LB liquid medium containing the corresponding antibiotics at a ratio of 1:100, culture at 37℃ with shaking until OD600 reaches 0.6, add IPTG with a final concentration of 0.5mM for induction, and change the culture temperature to 25℃ for overnight culture for about 16h. Collect the culture, centrifuge at 4℃, 5000g for 5min, collect the bacteria, add 10mL 1×PBS to resuspend, place the bacterial suspension in an ice bath for ultrasonic lysis for 10min (3s working, 3s rest) until the bacterial liquid is clear and transparent, centrifuge at 4℃, 12000g for 5min, and transfer the lysis supernatant to a new 15mL centrifuge tube for use. Take 200μL nickel beads, centrifuge at 6000g for 30s, remove the supernatant; resuspend the nickel beads with 200μL 1×PBS, centrifuge at 6000g for 30s, repeat the wash twice, add the washed nickel beads to the supernatant of the lysate, slowly shake at 4℃ for 1h; centrifuge at 6000g for 15min, and discard the supernatant. Add 400μL 5mM imidazole washing solution (50mM NaH2PO4, 300mM NaCl, 5mM imidazole) to resuspend the gel, centrifuge and discard the supernatant, repeat the wash 5 times; add 200μL 500mM imidazole elution solution (50mM NaH2PO4, 300mM NaCl, 500mM imidazole), gently resuspend the gel, centrifuge and collect the supernatant. The supernatant is the purified His-tagged target protein.

[0110] The experimental results are as follows Figure 5 As shown, there was no soluble expression of reteplase in E. coli cells.

[0111] The nucleic acid molecule encoding reteplase disclosed in the present application and the expression template for preparing soluble reteplase in a cell-free protein expression system can obtain high-yield expression of reteplase. At the same time, the preparation of reteplase using a cell-free protein expression system has low cost, simple operation, good repeatability, and can directly prepare soluble reteplase.

[0112] Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0113] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

[0114] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. A nucleic acid molecule, characterized in that The nucleotide sequence is shown as SEQ ID NO.1, SEQ ID NO.6 or SEQ ID NO.7, which is used to encode reteplase.

2. A plasmid, characterized in that The plasmid comprises a gene fragment comprising the nucleotide sequence of claim 1.

3. The plasmid according to claim 2, characterized in that The nucleotide sequence of the plasmid is shown as SEQ ID NO.3, SEQ ID NO.8 or SEQ ID NO.

9.

4. The plasmid according to claim 2, characterized in that The plasmid is obtained by inserting the gene fragment into a target plasmid based on homologous recombination. The nucleotide sequence of the target plasmid is shown in SEQ ID NO.

2.

5. A linear DNA template for preparing reteplase by cell-free protein expression, characterized in that: The nucleotide sequence of the linear DNA template is shown in SEQ ID NO.10 or SEQ ID NO.

11.

6. The linear DNA template according to claim 5, characterized in that The linear DNA template is obtained based on the following operations: Providing the nucleic acid molecule according to claim 1; Protein expression elements are added upstream and downstream of the nucleotide sequence of the nucleic acid molecule to obtain the linear DNA template; the nucleotide sequences of the protein expression elements are shown in SEQ ID NO.4 and SEQ ID NO.

5.

7. A cell-free protein expression system for expressing reteplase, characterized in that: The system includes a cell-free expression system and an expression template; The cell-free expression system includes a cell extract and a supplement system; The expression template comprises the plasmid according to any one of claims 2 to 4, or the linear DNA template according to claim 5 or 6.

8. The cell-free protein expression system according to claim 7, characterized in that: The concentration of the expression template in the cell-free protein expression system is 5-50 ng / μL.

9. The cell-free protein expression system according to claim 7, characterized in that: The cell extract is selected from any one or any combination of Escherichia coli cell extract, yeast cell extract, wheat germ extract, insect cell extract, rabbit reticulocyte extract, and CHO cell extract.

10. The cell-free protein expression system according to claim 7, characterized in that: The supplement system is selected from any one or any combination of phosphate buffer or its functional equivalent, magnesium salt or its functional equivalent, potassium salt or its functional equivalent, oxalate or its functional equivalent, amino acids or amino acid mixtures or their functional equivalents, nucleotide mixtures or their functional equivalents.

11. A method for preparing reteplase, characterized in that: The method comprises: Obtain the cell-free protein expression system according to any one of claims 7 to 10; Cell-free protein expression is performed based on the cell-free protein expression system to obtain reteplase.

12. The method according to claim 11, characterized in that The expression temperature of the cell-free protein expression is 15-37°C and the expression time is 2-24h.

13. Use of the plasmid according to any one of claims 2 to 4, or the linear DNA template according to claim 5 or 6, in a cell-free protein expression system to express reteplase.

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