CN113337493A

CN113337493A - Method for expressing and preparing recombinant reteplase by using genetically engineered rice

Info

Publication number: CN113337493A
Application number: CN202110735731.0A
Authority: CN
Inventors: 杨代常; 余文卉
Original assignee: Wuhan Healthgen Biotechnology Co Ltd
Current assignee: Wuhan Healthgen Biotechnology Co Ltd
Priority date: 2021-06-30
Filing date: 2021-06-30
Publication date: 2021-09-03
Anticipated expiration: 2041-06-30
Also published as: CN113337493B; WO2023274091A1

Abstract

The invention discloses a rice codon optimized recombinant reteplase gene, a related vector, a method for preparing a genetic engineering rice seed and a method for separating and purifying the recombinant reteplase. Processing and crushing genetically engineered rice, mixing the crushed genetically engineered rice with an extraction buffer solution, and extracting and filtering to obtain a crude extract containing recombinant reteplase; the separation and purification steps sequentially comprise: 1) carrying out cation exchange chromatography on the crude extract containing the recombinant reteplase to obtain a primary product I; 2) carrying out hydrophobic chromatography on the primary product I to obtain an intermediate product II; 3) and (3) performing benzamidine affinity chromatography on the intermediate product II to obtain the purified recombinant reteplase. The method has the advantages of no need of in vitro renaturation, high safety and easy scale production, and the obtained recombinant reteplase has the HPLC purity of more than 98 percent.

Description

Method for expressing and preparing recombinant reteplase by using genetically engineered rice

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a method for expressing, separating and purifying recombinant reteplase by taking genetically engineered rice as a bioreactor.

Background

Reteplase (r-PA) is a third-generation thrombolytic drug, has a molecular weight of about 39.6kDa and is composed of 355 amino acids, is a derivative of recombinant human Tissue-type plasminogen kinase (t-PA), and can activate plasminogen to active fibrinolytic enzyme so as to degrade fibrin in thrombus and play a role in thrombolysis. r-PA is suitable for thrombolytic therapy of acute myocardial infarction caused by coronary artery infarction in adults, and can improve ventricular function after myocardial infarction.

Cardiovascular and cerebrovascular diseases are one of the main diseases which endanger human life and health, wherein the morbidity, disability rate and mortality of thrombotic diseases are far higher than those of other diseases. According to the data of the Ministry of health of China, more than ten million patients with thrombus disease in China are shown, the number of the patients dying of thrombus disease is more than 200 thousands every year, the number of the patients dying of thrombus disease accounts for more than 50% of the total number of the patients dying of thrombus disease, and the thrombus disease has a remarkable rising trend in China. At present, the domestic thrombolytic drug market mainly comprises first-generation and second-generation products, wherein the income percentages of alteplase (t-PA), lumbrukinase and plasmin are respectively 30%, 29% and 26%, and the first three are national medical insurance type B products. The alteplase of the second generation is the most used thrombolytic agent abroad, the alteplase in the market of China completely depends on import, and the lotto of the Boringer Yiger occupies 100% of the market share. One major disadvantage of alteplase is the short half-life of only 4-6 minutes, requiring continuous intravenous administration. The first generation of thrombolytic agent has already exited the European and American market, but is still widely used in the primary hospitals in China due to low price, and has a great market share. The third generation thrombolytic drug r-PA is a non-glycosylated recombinant tissue plasminogen activator which is obtained by removing Fg (finger region), EGF (growth factor region) and K1 (ring structure region) of natural human tissue plasminogen activator t-PA by applying genetic engineering technology and retaining amino acids at 1-3 and 176-527, namely the middle deletion body of t-PA and deleting the structure region related to the inactivation in the liver. Therefore, the thrombolytic drug has a longer half-life period (15-18 min), is free from immunogenicity, can antagonize a plurality of inhibitors, has strong thrombolytic capacity, is convenient to administer, does not need to be administered according to individuals, can be administered in a short time and in a small dose, can be used for quickly dissolving thrombus, has the advantages of improving specific thrombolytic efficiency, prolonging half-life period, reducing systemic bleeding of the whole body, having high vascular recanalization rate, no anaphylactic reaction, no toxic or side effect and the like compared with a second generation thrombolytic drug t-PA, and has a wider application prospect.

The expression of r-PA in Escherichia coli, yeast, mammalian cells and filamentous fungi has been successfully carried out at present, but reports on the large-scale production of recombinant reteplase by using rice endosperm are not found so far, and the purification method is involved: (1) forming an inactive inclusion body after escherichia coli expression, performing disulfide bond pairing renaturation in vitro, and separating by using an erythrina indica trypsin inhibitor (ETI) through an ETI-Sepharose 4B affinity chromatography column by utilizing the characteristic that the ETI can be specifically combined with r-PA to obtain the r-PA; (2) the r-PA is expressed by transgenic rabbit milk, and is separated and purified by methods such as ammonium sulfate precipitation, acid-base precipitation, ultrafiltration, affinity chromatography (Lysine HyperD, Blue Sepharose 6FF), ion exchange (SP BestaroseTM FF), gel filtration (Surdex G75) and the like, and no report about a method for separating and purifying recombinant reteplase from rice seeds is found. The medicine paitongxin sold in the domestic market is expressed in escherichia coli, but the escherichia coli is inactive after expression, and needs operations such as in vitro renaturation, and the like, while the rice endosperm cell expression system is used for efficiently expressing recombinant r-PA, so that the operations such as in vitro renaturation are overcome, and the rice endosperm cell expression system has the advantages of being green, safe, easy to scale and the like.

Disclosure of Invention

The invention aims to provide a method for extracting, separating and purifying recombinant reteplase (OsrPA) from genetically engineered rice seeds by utilizing a rice endosperm cell bioreactor in a large scale to efficiently express the recombinant reteplase.

In order to realize the purpose of efficiently expressing recombinant reteplase in rice endosperm cells, the invention provides a method for extracting, separating and purifying recombinant reteplase from recombinant reteplase genetic engineering rice, which comprises the following steps:

(1) constructing a plant expression vector for expressing recombinant reteplase in rice seeds, wherein the vector contains a reteplase gene optimized by rice codons;

(2) genetically transforming rice by using the expression vector in the step (1) to obtain genetically engineered rice, and cultivating to obtain recombinant reteplase genetically engineered rice seeds;

(3) extracting a crude extract containing recombinant reteplase from the recombinant reteplase gene engineering rice seeds in the step (2);

(4) subjecting the crude extract containing the recombinant reteplase obtained in the step (3) to cation exchange chromatography to obtain a primary product I;

(5) carrying out hydrophobic chromatography on the primary product I obtained in the step (4) to obtain a middle-grade product II containing the recombinant reteplase;

(6) and (5) performing benzamidine affinity chromatography on the intermediate product II obtained in the step (5) to obtain the purified recombinant reteplase.

The preferable technical scheme is as follows:

(1) constructing a plant expression vector containing a rice codon-optimized reteplase gene shown as SEQ ID No. 1;

(2) genetically transforming rice by using the plant expression vector to obtain genetically engineered rice, and cultivating to obtain recombinant reteplase genetically engineered rice seeds;

(3) extracting a crude extract containing recombinant reteplase from the recombinant reteplase gene engineering rice seeds;

(4) carrying out cation exchange chromatography on a crude extract containing the recombinant reteplase to obtain a primary product I, wherein a medium of the cation exchange chromatography is NanoGel50 sp;

(5) performing intermediate purification on the primary product I by adopting hydrophobic chromatography to obtain an intermediate product II, wherein the medium of the hydrophobic chromatography is UniHR Phenyl 30L;

(6) and (3) carrying out final-stage purification on the intermediate-stage product II by adopting affinity chromatography to obtain the purified recombinant reteplase, wherein the affinity chromatography medium is the bocglong Benzamidine Bestarose 4 FF.

According to a preferred embodiment of the invention:

in the step (3), the recombinant reteplase genetically engineered rice is dried in the sun and shelled, processed into semi-polished rice, and ground into rice flour of 80-100 meshes; rice flour and extraction buffer were mixed according to 1: 5 (weight/volume, kg/L) and extracting for 2-3 hours at 24-26 ℃ to obtain a recombinant reteplase extraction mixture; adding 2-5% of perlite into the extraction mixture for filter pressing, and filtering the filtrate by a 0.22 mu m filter membrane to obtain a crude extract of the recombinant reteplase, wherein the components of the extraction buffer solution are as follows: 20mM phosphate buffer, 500mM sodium chloride, pH 7.5;

in the step (4), ion exchange chromatography media are used for primary separation and purification, and the ion exchange chromatography packing comprises NanoGel50sp, NanoQ 30L, NanoGel 50Q, UniGel 30Q, UniGel 80Q, Unigel MMC 50s and Unigel MMA 50s (Suzhou Na micro science and technology Co., Ltd.), and the NanoGel50sp packing is preferably used for primary separation and purification. Equilibrating the chromatographic column with 4-6 Column Volumes (CV) of 20mM phosphate and 130mM sodium chloride buffer at pH6.5 at a linear flow rate of 200-250 cm/h; mixing the crude extract of the recombinant reteplase with 20mM phosphate buffer solution with pH6.5 according to a volume ratio of 1: 2.5, uniformly mixing and diluting, filtering with a 0.22um filter membrane to obtain a sample loading solution, wherein the optimal sample loading solution conductivity is 14-16 mS/cm, the pH of the sample loading solution is 6.4-6.6, and the sample loading volume is not more than 221 CV; eluting 40CV impurity protein by adopting 20mM phosphate and 180mM sodium chloride buffer solution with the pH value of 6.5 at the flow rate of 200-250 cm/h, wherein the electrical conductance of the optimal impurity washing buffer solution is 20 mS/cm; eluting 50CV recombinant reteplase by adopting 20mM phosphate with the pH value of 6.5 and 280mM sodium chloride buffer solution at the flow rate of 200-250 cm/h, wherein the electrical conductance of the optimal elution buffer solution is 28-32 mS/cm, and collecting eluent rich in the recombinant reteplase to obtain a primary product I containing the recombinant reteplase;

in the step (5), a hydrophobic chromatography medium is adopted for medium-level separation and purification, and the hydrophobic chromatography filler comprises UniHR Phenyl 80L LS (suzhou nano micro technology corporation), UniHR Phenyl 30L (suzhou nano micro technology corporation), Octyl 4FF (bocglong (shanghai) biotechnology limited), Phenyl low sub (bocglong (shanghai) biotechnology limited), and preferably a chromatography column using UniHR Phenyl 30L (suzhou nano micro technology corporation) filler for medium-level separation and purification. Balancing the chromatographic column by using 5-10 CV of pH6.5, 20mM phosphate and 0.37M ammonium sulfate buffer solution at the flow rate of 350-400 cm/h; adjusting the conductance of the recombinant reteplase primary product I to 59-61 mS/cm by using 3M ammonium sulfate, adjusting the pH to 6.5, using the product as a sample loading solution, wherein the optimum sample loading solution conductance is 60mS/cm, and completely loading; eluting 20CV impurity protein with buffer solution of 20mM phosphate containing 0.5% glycerol and 260mM ammonium sulfate at pH6.5, wherein the optimum conductivity of the impurity-washing buffer solution is 45 mS/cm; eluting 20CV recombinant reteplase by using a buffer solution with pH6.5, 20mM phosphate containing 0.5% of glycerol and 68mM ammonium sulfate, wherein the electric conductance of the optimal elution buffer solution is 15mS/cm, and collecting an eluent rich in the recombinant reteplase to obtain a middle-grade product II containing the recombinant reteplase;

in the step (6), the final-stage separation and purification is performed by using affinity chromatography packing, wherein the affinity chromatography packing comprises Benzamidine 4FF (072J low ligand and 191J high ligand, Suzhou Na micro-technology GmbH) and Benzamidine Bestarose 4FF (Boglong (Shanghai) Biotechnology GmbH), and preferably, the Benzamidine Bestarose 4FF (Boglong (Shanghai) Biotechnology GmbH) packing is used. Balancing the chromatographic column by using 5-10 CV of pH7.5, 20mM phosphate and 68mM ammonium sulfate buffer solution at the flow rate of 350-400 cm/h; adjusting the pH value of the intermediate product II to 7.5 to be used as a sample loading solution, and completely loading the sample; eluting 10CV impurity protein by using 20mM phosphate buffer solution with the pH value of 6.0; and (3) eluting the 5CV recombinant reteplase by using 20mM phosphate and 20mM acetate buffer solution with the pH value of 4.8-5.2, wherein the pH value of the optimal elution buffer solution is 5.0, and collecting eluent rich in the recombinant reteplase to obtain the purified recombinant reteplase.

According to another aspect of the present invention, there is provided a plant expression vector comprising:

1) synthesizing a rice codon-optimized reteplase gene sequence shown as SED ID NO. 1;

2) constructing a recombinant reteplase expression vector for rice endosperm cell specific expression;

3) transforming the vector obtained in the step 2) into rice callus, and obtaining a transgenic recombinant reteplase gene engineering rice plant through culture, screening and induction;

wherein, the recombinant reteplase expression vector preferably has a structure as shown in figure 2.

The invention constructs a recombinant reteplase expression vector expressed in rice endosperm cells, successfully expresses the recombinant reteplase in rice and establishes a method for extracting and purifying the reteplase. The method of the invention does not need in vitro renaturation, has high safety and is easy for large scale production, and the obtained recombinant reteplase reaches the HPLC purity of more than 98 percent.

Drawings

FIG. 1 shows the structure of pOsPMP773 plasmid.

FIG. 2 shows the structure of pOsPMP774 plasmid.

FIG. 3 shows the plasmid structure of pOsPMP 775.

FIG. 4 shows PCR detection of target gene of rice plant of genetic engineering. Wherein M is a DNA standard molecular weight Marker; 1-24 are different plants of T1 generation transgenic material.

FIG. 5 shows the SDS-PAGE and WB detection results of the recombinant reteplase crude extract under different extraction conditions. Wherein M is a standard molecular weight Marker; 1-15 respectively represent 1-15 different extraction conditions in Table 1, and LGC is a background plant.

FIG. 6 shows the results of the detection of the activity of the recombinant crude reteplase extract in the form of a lysoloop under different extraction conditions. In the fibrin plate 1-5, the number 1-6 is used as standard yeast detection, and the number 7-12 is used for 20-fold and 40-fold dilution detection of the extracting solution under different extracting conditions of 1-15 in sequence.

FIG. 7 shows the results of SDS-PAGE and WB detection of recombinant crude reteplase extracts at different extraction pH, temperature and time. M in A is a standard molecular weight Marker; 7.0-10.0 respectively represent different extraction pH values, and LGC is taken as a background plant. The red arrows correspond to the destination and degradation bands, respectively. In B, 1 to 6 represent extraction time (hours), overnight represents overnight extraction, 007 represents 775-214-49-007 batch rice flour, and 006 represents 775-214-49-006 batch rice flour.

FIG. 8 selection of primary purification ion exchange chromatography media. M in A is a standard molecular weight Marker; load represents sample liquid, FT represents penetration liquid, 10-80 represents different conductances (mS/cm), and 2M represents 2M sodium chloride; the upper graph in B is the detection of a sample after 1-fold dilution of crude body fluid containing reteplase, FT1 represents the penetration liquid after 10CV loading, FT2 represents the penetration liquid after 20CV loading, and load represents the sample liquid; the lower graph shows the detection of a sample after 4-fold dilution of a crude body fluid containing reteplase, FT1 shows the transudation solution after sample application of 5CV, FT2 shows the transudation solution after sample application of 20CV, and load shows the sample application solution; in C, 20, 30, 50, 70 and 80 represent different conductances (mS/cm), and 2M represents 2M sodium chloride.

FIG. 9. Primary purification cation exchange packing NanoGel50sp chromatography optimization. In A, L represents the sample, FT represents the permeate, 30, 40 and 70 represent different conductances (mS/cm), 2M represents 2M sodium chloride, and E represents the eluent; in B, L represents a sample solution, FT represents a transudate solution, 20, 25 and 32 represent different conductances (mS/cm), and 1M represents 1M sodium chloride; in C, L represents the sample solution, FT represents the transudate solution, 18, 32 and 40 represent different conductances (mS/cm), 1M represents 1M sodium chloride, and 2M represents 2M sodium chloride.

FIG. 10. primary purified cation exchange packing NanoGel50sp chromatographic load confirmation. L represents the sample, FT represents the permeate, W represents the eluate, E1 is an eluate at 25mS/cm, E2 is an eluate at 28mS/cm, E3 is an eluate at 32mS/cm, E1-torr represents the tail of E1, and 1M represents 1M sodium chloride.

FIG. 11. selection of medium grade purified hydrophobic chromatography media. L represents a sample liquid, FT represents a penetration liquid, and 1 to 8 represent gradient elution sample numbers.

FIG. 12. medium grade purified hydrophobic filler UniHR Phenyl 30L chromatography condition optimization. In the A, M is a standard molecular weight Marker, L represents a sample loading solution, 1-6 represents the number of a gradient elution sample, and Elu represents a final elution sample; in B, M is a standard molecular weight Marker, L is a sample solution, FT is a permeate solution, W is a wash solution, E1 is a 15mS/cm eluate containing 0.5% glycerol, and E2 is a 16mS/cm eluate containing 10% ethanol.

FIG. 13 final stage affinity purification medium BGL Benzamidine 4FF chromatography optimization. In A, M is a standard molecular weight Marker, 6.5, 6.0, 5.5 and 3.0 are different pH values, 2, 4, 10 and 15 are different conductances (mS/cm), L is a sample loading solution, FT is a penetrating solution, W is a washing impurity solution, and E is an eluent; and in the step B, L is a sample loading solution, FT is a penetrating solution, W is a washing impurity solution, 1-4 are samples collected in sections, NaK is a buffer solution of disodium hydrogen phosphate and potassium dihydrogen phosphate, NaAc is a buffer solution of sodium acetate, E is an eluent, and E is a concentrated solution of the eluent.

FIG. 14.3 batch pilot process chromatogram.

FIG. 15.3 shows the results of electrophoretic detection of a small batch of technical recombinant reteplase samples, where M is a standard molecular weight Marker.

FIG. 16.3 batch of small test technical recombinant reteplase RP-HPLC detection results.

Detailed Description

The technical solutions of the present invention will be described in detail below by way of examples and figures, so as to better illustrate the features and advantages of the present invention. The examples provided should be construed as illustrative of the method of the invention and not limiting the technical solutions disclosed in the invention in any way.

The reagents and instruments used in the examples were all those generally commercially available except for the specific instructions.

[ example 1 ] preparation of genetically engineered Rice expressing recombinant reteplase with high efficiency

1. Construction of recombinant reteplase gene expression vector

In the embodiment, a rice specific promoter Gt13a and a signal peptide thereof are selected to mediate the expression of the recombinant reteplase gene in rice endosperm cells. According to the sequence of the reteplase gene (Genbank accession number: KU053049.1), the Nanjing Kingsler Biotech limited company is entrusted to synthesize the gene according to the preferred genetic codon of rice, specifically as shown in SEQ ID NO.1, the nucleotide is changed by 23.2% after the codon is optimized by the rice preferred codon, the codon is changed by 32.3%, but the corresponding amino acid sequence is not changed, and the constructed plasmid is pOsPMP773 (figure 1). The synthesized codon-optimized reteplase gene (SEQ ID NO.1) is cut by MlyI and XhoI and then cloned into NaeI and XhoI cut pOsPMP003, an intermediate vector plasmid pOsPMP774 (figure 2) is constructed by T4 ligase, the whole expression cassette which is 2386bp in length and contains a Gt13a promoter, a signal peptide sequence, a codon-optimized reteplase gene and a Nos terminator is inserted into a binary expression vector pcl300 cut by HindIII and EcoRI, and an agrobacterium-mediated bacterium plasmid is constructed and named as pOsPMP775 (figure 3)

2. Genetic engineering of rice

The pOsPMP775 plasmid is transformed into Agrobacterium tumefaciens EHA105 (Invitrogen, USA), the pOsPMP775 is transformed into the callus of the rice variety LGC through the mediation of Agrobacterium tumefaciens, and a complete plant is formed after the culture, the screening and the induction. The specific method comprises the following steps:

(1) callus induction

Husking mature rice seeds, soaking the rice seeds in 70% ethanol for sterilization for 1 minute, and treating the rice seeds for 30 minutes by using 20% sodium hypochlorite; washing with sterile water for 5-7 times, and inoculating the seeds to an induction culture medium (N)₆A culture medium) is added to the culture medium,6-8 grains are inoculated on each dish and the dish is irradiated with light at 32 ℃ for about 5-7 days.

(2) Preparation of Agrobacterium

Carrying out amplification culture on agrobacterium containing an expression vector pOsPMP775, coating the agrobacterium in a kanamycin-resistant plate, and culturing for 2-3 days in an incubator at 28 ℃; a single colony of the agrobacterium tumefaciens is inoculated into a suspension medium (AAM liquid medium) by using an inoculating loop, and is shake-cultured at 28 ℃.

(3) Agrobacterium infection (Co-culture)

Transferring the callus into a sterilized Erlenmeyer flask, and adjusting the OD of the Agrobacterium suspension₆₀₀The value is 0.05 to 0.1; suspending the seeds in AAM medium, infecting for 1.5 minutes, and continuously shaking; discarding the bacterial liquid, sucking the redundant bacterial liquid by using sterile filter paper, taking out the callus, placing the callus on the sterile filter paper, and draining for 30-45 minutes; placing sterile filter paper in 2N₆On AS medium, 500. mu.l of AAM containing AS (acetosyringone, 250mg/ml) was dropped on sterile filter paper with a diameter of 9cm, and the infected calli were placed on the filter paper and cultured in the dark at 25 ℃ for 3 days.

(4) Washing and screening

Transferring the co-cultured callus into a sterilized triangular flask, and cleaning the callus with sterile water for 5-7 times; soaking the infected callus in sterile water containing 0.5g/L of cefuroxime axetil for about 30 minutes, and then shaking at the temperature of 28 ℃ and the speed of 180-200 rpm for 20-30 minutes; pouring out the sterilized water containing the antibiotics, pouring the triangular flask into a sterilized culture medium containing filter paper for about 15 minutes, airing, and transferring the callus to a screening culture medium containing the HPT antibiotics for culturing for 20-30 days.

(5) Callus differentiation

Transferring the callus with HPT resistance after 20-30 days selection to a differentiation medium (N)₆Culture medium), and culturing at 26 deg.C for 20-30 days.

(6) Rooting

Selecting differentiated plantlets from the differentiation culture medium, transferring the plantlets to an MS culture medium containing 1/2 for rooting culture, performing illumination culture at 28 ℃ for 30 days, and transferring the plantlets to a field for growth.

3. Genetic engineering rice identification

(1) Extraction of rice genome DNA

Taking leaves of T0 HPT positive regeneration seedlings about 2cm, respectively putting the leaves into a centrifuge tube, and numbering; adding 600 μ l CTAB extraction buffer (2% CTAB, 1.38M NaCl, 0.1M Tris-HCl, 20mM EDTA, pH8.0), shaking in a shaking crusher, and holding at 65 deg.C for 60 min while shaking; adding chloroform/isopropanol with the same volume, slightly reversing the centrifuge tube, mixing uniformly, and centrifuging at 12000rpm for 10 minutes at room temperature; transferring the supernatant into another new 1.5ml centrifuge tube, adding isopropanol with the same volume, slowly turning the centrifuge tube upside down, mixing, and standing at room temperature for 60 minutes; centrifuging at 12000rpm for 10min, removing supernatant, rinsing with 70% ethanol, and air drying the precipitate; the air-dried DNA was dissolved in 80. mu.l of TE buffer and stored at-20 ℃ until use.

(2) PCR amplification detection of target gene

Taking 1 mul of rice genome DNA as a template, and setting positive (plasmid DNA) and negative (sterile water) controls; PCR amplification is carried out by utilizing a forward primer GT13a-F (SED ID NO. 2: 5'-AGCTACCAGGGCAACAGCGA-3') and a reverse primer R-PA-R (SED ID NO. 3: 5'-AGCTGCTGATGAGGATGCCG-3') of the recombined reteplase, and the theoretical size of a product is 670 bp; PCR amplification reaction system (25. mu.l system): 2.5. mu.l 10 Xbuffer, 0.15. mu.l rTaq enzyme (5U/. mu.l), 4. mu.l dNTP (2.5mM), 0.5. mu.l each of primers; add ddH₂O to 25 μ l; PCR amplification conditions: pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 30 sec, annealing at 59.8 ℃ for 30 sec, extension at 72 ℃ for 45 sec, 35 cycles, and final extension at 72 ℃ for 10 min; the amplification product is subjected to 150V electrophoresis and 200mA electrophoresis for 15 minutes after EB staining by 1% agar gel, and the result is observed in a gel imager.

The identification result shows that 87 positive recombinant reteplase transgenic rice strains are obtained through agrobacterium tumefaciens mediated transformation, and the PCR identification result of the target gene of part of the genetically engineered rice is shown in figure 4.

4. Identification of expression level of recombinant reteplase

Transplanting the obtained recombinant reteplase positive seedling to a room temperature to grow to be mature, and harvesting a single plant which can normally fruit. Carrying out quantitative detection on positive single strains by adopting a DY7449-05(R & D) kit, after capturing an antibody coated plate, diluting 10 mu l of protein extracting solution by 1000 times, adding the diluted protein extracting solution into an ELISA plate, and incubating for 2 hours at room temperature; washing the plate with sodium phosphate buffer solution containing 0.05% Tween 20, adding 100. mu.l of detection antibody, and incubating at room temperature for 2 hours; and (3) adding 100 mu l of HRP-streptavidin after washing the plate, reacting at room temperature for 20min, washing the plate, adding 100 mu l of TMB into each hole, developing for 15-20 min in a dark place at room temperature, adding 50 mu l of 2M sulfuric acid to terminate the reaction, and reading OD 450.

The identification result shows that 42 individuals of the recombinant reteplase positive transgenic rice express reteplase protein, the expression amount is from 1.9 mu g/g to 62.5 mu g/g, 5 individuals with higher expression amount are available, and the expression amount ranges from 41.2 mu g/g to 62.5 mu g/g.

SEQ ID NO.1：

1 AGCTACCAGG GCAACAGCGA CTGCTACTTC GGCAACGGCA GCGCCTACCG CGGCACCCAC

61 AGCCTCACCG AGAGCGGCGC CAGCTGCCTC CCGTGGAACA GCATGATCCT CATCGGCAAG

121 GTGTACACCG CCCAGAACCC GAGCGCCCAG GCCCTCGGCC TCGGCAAGCA CAACTACTGC

181 CGCAACCCGG ACGGCGACGC CAAGCCGTGG TGCCACGTGC TCAAGAACCG CCGCCTCACC

241 TGGGAGTACT GCGACGTGCC GAGCTGCAGC ACCTGCGGCC TCCGCCAGTA CAGCCAGCCG

301 CAGTTCCGCA TCAAGGGCGG CCTCTTCGCC GACATCGCCA GCCACCCGTG GCAGGCCGCC

361 ATCTTCGCCA AGCACCGCCG CAGCCCGGGC GAGCGCTTCC TCTGCGGCGG CATCCTCATC

421 AGCAGCTGCT GGATCCTCAG CGCCGCCCAC TGCTTCCAGG AGCGCTTCCC GCCGCACCAC

481 CTCACCGTGA TCCTCGGCCG CACCTACCGC GTGGTGCCGG GCGAGGAGGA GCAGAAGTTC

541 GAGGTGGAGA AGTACATCGT GCACAAGGAG TTCGACGACG ACACCTACGA CAACGACATC

601 GCCCTCCTCC AGCTCAAGAG CGACAGCAGC CGCTGCGCCC AGGAGAGCAG CGTGGTGCGC

661 ACCGTGTGCC TCCCGCCGGC CGACCTCCAG CTCCCGGACT GGACCGAGTG CGAGCTCAGC

721 GGCTACGGCA AGCACGAGGC CCTCAGCCCG TTCTACAGCG AGCGCCTCAA GGAGGCCCAC

781 GTGCGCCTCT ACCCGAGCAG CCGCTGCACC AGCCAGCACC TCCTCAACCG CACCGTGACC

841 GACAACATGC TCTGCGCCGG CGACACCCGC AGCGGCGGCC CGCAGGCCAA CCTCCACGAC

901 GCCTGCCAGG GCGACAGCGG CGGCCCGCTC GTGTGCCTCA ACGACGGCCG CATGACCCTC

961 GTGGGCATCA TCAGCTGGGG CCTCGGCTGC GGCCAGAAGG ACGTGCCGGG CGTGTACACC

1021 AAGGTGACCA ACTACCTCGA CTGGATCCGC GACAACATGC GCCCGTGA

SEQ ID NO.2：

5’-AGCTACCAGGGCAACAGCGA-3’

SEQ ID NO.3：

5’-AGCTGCTGATGAGGATGCCG-3’

[ example 2 ] extraction of recombinant reteplase from genetically engineered Rice

1. Preliminary screening of recombinant reteplase extraction conditions

Hulling genetically engineered rice, processing the hulled genetically engineered rice into semi-polished rice, grinding the semi-polished rice into rice flour of 80-100 meshes, selecting three factors of extraction pH (4-10), extraction salt concentration (0-0.5M) and extraction temperature (8-37 ℃) by using JMP software, and performing response surface design by taking specific activity (IU/mg) of r-PA extracted protein as a response value, wherein the table is prepared as follows:

TABLE 1 design of different extraction conditions

Experiment number	pH of extraction	Concentration of extraction salt (mM)	Extraction temperature (. degree.C.)
				1	10	250	7.6
2	7	250	27
				3	4	250	37
4	10	500	27
				5	4	0	27
6	7	0	37
				7	7	250	27
8	4	500	27
				9	7	0	7.6
10	10	0	27
				11	4	250	7.6
12	7	500	7.6
				13	10	250	37
14	7	500	37
				15	7	250	27

Weighing 15 parts of rice flour, putting 1g of rice flour into a10 ml centrifuge tube, carrying out experiments according to the experimental design shown in the table above, adding 5ml of extracting solution into each tube, extracting for 1h, centrifuging for 5min at 10000g, taking supernatant, and carrying out SDS-PAGE/WB detection (figure 5), Elisa expression amount detection and activity detection by a ring-dissolving method (figure 6). Another 1g of LGC rice flour was weighed and extracted with 20mM PB, 250mM NaCl, pH7.0 at 27 ℃ for 1h and treated as above as a negative control. The results show that the specific activity of the recombinant reteplase extracted protein is most sensitive to the pH of the extraction buffer, followed by the salt concentration of the extraction buffer. The specific activity of the protein extracted by the recombinant reteplase was the highest at pH8.4, a salt concentration of 476mM and an extraction temperature of 18 ℃ as analyzed by a predictive carver.

2. Determination of extraction conditions of recombinant reteplase

(1) Influence of different extraction buffer pH on extraction of recombinant reteplase

Weighing 7 parts of 775-214-49-007 rice flour, putting 1g of each part into a10 ml centrifuge tube, then adding 5ml of phosphate extract with pH of 7.0, 7.5, 8.0, 8.5, 9.0, 9.5 and 10.0 into each tube in sequence, extracting for 2h at 25 ℃, centrifuging for 5min at 10000g, taking supernatant to carry out SDS-PAGE/WB detection (figure 7A), and detecting Elisa expression amount and lysozyme. And (2) extracting 1g of LGC rice flour by using an extracting solution with the pH value of 10.0 for 2 hours at 25 ℃, and then treating the rice flour in the same way, wherein the results show that r-PA degradation zones are gradually increased along with the increase of the extraction pH value, and the degradation zones begin to appear when the pH value of the extracting solution is higher than 8.5, so that the pH value of an extraction buffer solution can be controlled to be 7.5-8.0, the degradation is reduced, the difficulty of the subsequent purification process is reduced, and Elisa expression quantity and stability detection results show that the specific activity of protein in the recombinant reteplase extracting solution is highest when the pH value is 7.5, so that the extraction pH value of the recombinant reteplase is determined to be 7.5.

(2) Influence of different extraction temperatures and extraction times on extraction of recombinant reteplase

Weighing 10 parts of 1g 775-214-49-007r-PA rice flour and 2 parts of 1g 775-214-49-006r-PA rice flour, and performing the following steps: 5 to the mixture, 5ml of 20mM PB, 500mM NaCl, pH7.5 extract was added, and extracted at 25 ℃ for 1h, 2h, 4h, 6h and overnight (21h), respectively, at 4 ℃ for 1h, 2h, 4h, 6h and overnight (21 h); after extraction, 10000g was centrifuged for 10min, and the supernatant was subjected to SDS-PAGE/WB detection (FIG. 7B), and Elisa expression level and lysoloop activity detection. The result shows that the content and the activity of the recombinant reteplase extracted at 4 ℃ are lower than those extracted at 25 ℃, after overnight extraction, the content and the activity of the recombinant reteplase are reduced, which indicates that the overnight extraction cannot be performed, and the extraction temperature of the recombinant reteplase is determined to be 25 ℃, and the extraction time is 2 hours. Thus, the extraction conditions of the recombinant reteplase were determined: 20mM PB, 500mM NaCl, pH7.5, 25 ℃, extract for 2 h.

Example 3 Primary purification of recombinant reteplase Using ion exchange chromatography Medium

1. Selection of primary purification ion exchange chromatography media

Preparing a crude extract containing recombinant reteplase according to the extraction conditions determined in example 2, and then carrying out preliminary screening of ion exchange chromatography packing including NanoGel50sp, NanoGel 30L, NanoGel 50Q, UniGel 30Q, UniGel 80Q, UniGel MMC 50s and UniGel MMA 50s by using an Ep tube centrifugal trapping method; through designing different phosphate buffer solution loading conductances and pH values and NaCl salt concentration gradient elution, the result shows that NanoGel50sp filler (pH values of 6.0 and 6.5 and the conductances of 15.56ms/cm) can be combined with the recombinant reteplase, and WB shows that the recombinant reteplase can be eluted at the pH values of 6.0-6.530-40 ms/cm and 7.0-7.520-30 ms/cm (fig. 8A); anion exchange fillers NanoQ 30L, NanoGel 50Q, UniGel 30Q and UniGel 80Q are subjected to sample loading of 10CV and 20CV penetration after being diluted by 1 time of a crude extract of the recombinant reteplase, and are subjected to sample loading of 5CV penetration after being diluted by 4 times, wherein the sample loading of 20CV penetration is large (figure 8B), which indicates that the anion exchange fillers are less than 5CV for the loading of an extracting solution and are not suitable for being used as first-step capture chromatography fillers; unigel MMC 50s did not penetrate at pH 6.5100-500 mM NaCl, but recombinant reteplase was eluted mainly in 0.5M NaOH, Unigel MMA 50s packing penetrated at pH7.5 and pH8.0 with 250mM NaCl phosphate buffer loading, with 100mM NaCl phosphate buffer loading, and recombinant reteplase eluted at pH 6.520mM PB 30-80 mS/cm (FIG. 8C).

2. Optimization of primary purification cation exchange packing NanoGel50sp chromatography

Preparing a crude extract containing recombinant reteplase according to the extraction conditions determined in example 2, adding sodium chloride with different concentrations by using 20mM phosphate buffer solution, designing different loading conductances of 16, 17 and 18mS/cm at pH6.5, washing conductances of 18, 20 and 25mS/cm and elution conductances of 32 and 40mS/cm, and detecting results show that trace reteplase is detected in a penetrating fluid when the loading conductances of Nanogel50sp are 18mS/cm (figure 9A) and the reteplase is not penetrated when the conductances are 16-17 mS/cm (figure 9B), thereby indicating that the loading conductances need to be controlled below 17 mS/cm; when the impurity washing conductivity is increased from 18mS/cm to 20mS/cm, slight reteplase is detected in the impurity washing liquid, and when the impurity washing conductivity is increased by 25mS/cm, most reteplase is eluted (figure 9C), which indicates that the impurity washing conductivity cannot exceed 20 mS/cm; the reteplase can be completely eluted at 25mS/cm basically, a small amount of reteplase can be detected at 32mS/cm, 40mS/cm and 1M NaCl (figure 9D), and the elution conductance range is determined to be 25-40 mS/cm.

3. Primary purification cation exchange filler NanoGel50sp chromatography loading confirmation

Preparing a crude extract containing recombinant reteplase according to the extraction conditions determined in example 2, adding 20mM phosphate buffer to make the conductivity of the loading buffer 16mS/cm, then loading 100CV, 75CV and 60CV (volume before dilution), respectively, and the detection result shows that Nanogel50sp is loaded with 100CV, trace reteplase is detected in the penetration solution, and part of reteplase is also detected in the impurity washing solution (FIG. 10A), and the loading amount needs to be reduced; and (3) loading 75CV, wherein the penetration solution does not contain reteplase, but the impurity washing solution still contains a small amount of reteplase (figure 10B), the loading capacity is continuously reduced to 60CV, the target protein is not penetrated, and the reteplase can be detected in the eluent E1(25.3ms/cm), E2(28.1ms/cm) and E3(32.5ms/cm), but the impurity protein (50kD impurity band) of the E3 elution component accounts for the main component (figure 10C), so that the loading capacity of Nanogel50sp is determined to be 60CV, the optimized elution condition is pH6.5, and the conductivity is 28-30 ms/cm.

Example 4 Medium purification of recombinant reteplase in hydrophobic chromatography Medium

1. Selection of Medium purification hydrophobic chromatography Medium

The recombinant reteplase primary product I was prepared according to the extraction and cation chromatography conditions determined in examples 2 and 3, and ammonium sulfate was added to the primary product I so that the ammonium sulfate concentration of the sample solution was 0.6M, 0.7M and 0.8M, and 5mL of UniHR Phenyl 80L LS, UniHR Phenyl 30L, BGL Octyl 4FF and BGL Phenyl low sub filler chromatography were performed, respectively, and it was found that BGL Phenyl low sub and Octyl 4FF were less effective in separation, and that UniHRPhenyl 30L was comparable to UniHRPhenyl 80L LS, but NaOH-eluting fractions of UniHRPhenyl 80L LS and UniHRPhenyl 30L had a band with a suspected target protein (fig. 11).

2. Medium-grade purified hydrophobic filler UniHR Phenyl 30L chromatographic condition optimization

The method comprises the steps of preparing a recombinant reteplase primary product I according to the extraction and cation chromatography conditions determined in examples 2 and 3, adding ammonium sulfate into the primary product I to enable the concentration of the ammonium sulfate in a sample solution to be 0.4M, and performing salt concentration gradient elution in a 0.5% glycerol system, wherein the results show that the degradation zone, the amino acid-deficient protein, the glycosylated protein and the non-glycosylated protein of the reteplase have certain separation degrees in the 0.5% glycerol system (figure 12A), and finally, the impurity washing effect is better when the conductivity of a phosphate buffer solution is 40-45 mS/cm and the elution effect is better when the conductivity of the phosphate buffer solution is 15mS/cm under the 0.5% glycerol system (figure 12B).

Example 5 Final purification of recombinant reteplase with affinity chromatography Medium

1. Selection of final affinity purification chromatography media

The recombinant reteplase intermediate product II was prepared according to the extraction, cation exchange and hydrophobic chromatography conditions determined in examples 2, 3 and 4, the pH of the intermediate product II was adjusted to 7.5, sodium chloride (0.1M, 0.5M, 1M) was added at different concentrations, 5mL of the nalmefene formamidine-4 FF (072J) low ligand, (191J) high ligand, BGL Benzamidine Bestarose 4FF affinity packing chromatography was performed, and the results were as follows:

	0.1M NaCl	0.5M NaCl	1M NaCl
				nanobenzamidine-4 FF (072J)	Is not combined with	Is not combined with	Partial bonding
Nano micro benzamidine-4 FF (191J)	Is not combined with	Bonding of	Bonding of
				BGL Benzamidine Bestarose4FF	Bonding of	Bonding of	Bonding of

The BGL Benzamidine Bestarose 4FF and the reteplase have the strongest binding force, and the BGL Benzamidine Bestarose 4FF is selected for subsequent chromatography optimization.

2. Final-stage affinity purification medium BGLBenzamidine Bestarose 4FF chromatography condition optimization

Preparing a recombinant reteplase intermediate product II according to the conditions of extraction, cation exchange chromatography and hydrophobic chromatography determined in the embodiment 2-4, adjusting the pH of the intermediate product II to 7.5, using the intermediate product II as a sample loading solution of BGL Benzamidine Beatarose 4FF, and performing impurity washing optimization by adopting different pH (6.5, 6.2 and 6.0) and different conductance of a 20mM phosphate buffer solution, wherein the result shows that the 20mM phosphate buffer solution, the pH6.0 and the conductance 1.6mS/cm have a better impurity washing effect (FIG. 13A); the results of the elution optimization using buffers of different pH (5.5, 5.0) and conductance showed that 20mM phosphate buffer, 20mM acetate buffer, pH5.0, and conductance 3.0mS/cm had better elution performance (FIG. 13B)

[ example 6 ] verification of recombinant reteplase 3 batches of bench test Process

According to the conditions of optimal extraction, primary product I purification, intermediate product II purification and final product III purification determined in the embodiments 2-5, 3 batches of small-scale process verification are performed, and the verification process and the verification results are as follows.

1. Preparation (extraction) of recombinant reteplase crude extract

Weighing 350g of recombinant reteplase rice flour (775-214-17-3), adding 1.75L of 20mM PB, 500mM NaCl and pH7.5 extracting solution, and extracting at 25 ℃ for 2 h; after extraction, 2-5% (w/v) of filter aid is added, and filter pressing is carried out by adopting a positive pressure filtration mode. The filtrate was added with 2.5 volumes of 20mM PB pH6.5 sample diluent, pH was adjusted to 6.5, and the crude recombinant reteplase extract (NanoGel50sp chromatography sample) was obtained after 0.22 μm filtration.

2. Purification of primary product I of recombinant reteplase

Adopting 20mM phosphate with pH of 6.5 and 130mM sodium chloride buffer solution with 4-6 times of Column Volume (CV), filling NanoGel50sp at a linear flow rate of 200-250 cm/h in a balanced manner, filling an XK16/20 chromatographic column with the column height of 10cm, and taking the recombinant reteplase crude extract as a sample solution for sampling, wherein the sampling volume is 221 CV; eluting 40CV impurity protein by adopting 20mM phosphate with pH of 6.5 and 180mM sodium chloride buffer solution at the flow rate of 200-250 cm/h; eluting 50CV recombinant reteplase by adopting 20mM phosphate with the pH value of 6.5 and 280mM sodium chloride buffer solution at the flow rate of 200-250 cm/h, and collecting eluent rich in the recombinant reteplase to obtain a primary product I containing the recombinant reteplase;

3. purification of intermediate product II of recombinant reteplase

Adopting 5-10 CV pH6.5, 20mM phosphate and 0.37M ammonium sulfate buffer solution, filling UniHR Phenyl 30L at the flow rate of 350-400 cm/h in a balanced manner, and filling an XK16/40 chromatographic column with the column height of 20 cm; adjusting the conductance of the recombinant reteplase primary product I to 59-61 mS/cm by using 3M ammonium sulfate, adjusting the pH to 6.5, using the product as a loading solution, and completely loading; eluting 20CV impurity protein with buffer solution of 20mM phosphate containing 0.5% glycerol and 260mM ammonium sulfate, and pH 6.5; eluting 20CV recombinant reteplase by using a buffer solution with pH6.5, containing 20mM phosphate of 0.5% glycerol and 68mM ammonium sulfate, and collecting an eluent rich in the recombinant reteplase to obtain a middle-grade product II containing the recombinant reteplase;

4. purification of final product III of recombinant reteplase

The method comprises the steps of filling BGL Benzamidine 4FF in a buffer solution of 5-10 CV, pH7.5, 20mM phosphate and 68mM ammonium sulfate at a flow rate of 350-400 cm/h in a balanced manner, and filling a C10/40 chromatographic column with the column height of 20 cm; adjusting the pH value of the intermediate product II to 7.5 to be used as a sample loading solution, and completely loading the sample; eluting 10CV impurity protein by using 20mM phosphate buffer solution with the pH value of 6.0; eluting the 5CV recombinant reteplase by using 20mM phosphate and 20mM acetate buffer solution with pH5.0, and collecting the eluate rich in the recombinant reteplase to obtain purified recombinant reteplase (final product III).

The result shows that the chromatogram is shown in figure 14, the chromatograms of the 3 batches of the small tests are basically consistent, the purity detection result of SDS-PAGE is shown in figure 15, the purity of the non-reduced sample is more than 99%, the purity detection result of RP-HPLC is shown in figure 16, the purity is more than 98%, the average protein yield is 21.5mg/kg of rice flour, and the average specific activity of the protein is 4.92x10⁵IU/mg。

SEQUENCE LISTING

<110> Wuhanhe grass element Biotechnology Ltd

<120> a method for expressing and preparing recombinant reteplase by using genetically engineered rice

<130> WH1190-21P150388

<160> 3

<170> PatentIn version 3.5

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<211> 1068

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cagttccgca tcaagggcgg cctcttcgcc gacatcgcca gccacccgtg gcaggccgcc 360

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gccctcctcc agctcaagag cgacagcagc cgctgcgccc aggagagcag cgtggtgcgc 660

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gtgggcatca tcagctgggg cctcggctgc ggccagaagg acgtgccggg cgtgtacacc 1020

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Claims

1. A method for expressing and preparing recombinant reteplase by using genetically engineered rice sequentially comprises the following steps:

2. The method according to claim 1, wherein the plant expression vector is constructed by introducing a rice codon-optimized reteplase gene represented by SEQ ID No.1, a rice specific promoter Gt13a, and a signal peptide thereof into a plasmid vector.

3. The method according to claim 1, wherein the crude recombinant reteplase extract obtained in step (3) is prepared by a method comprising:

3a) drying and shelling the recombinant reteplase gene engineering rice seeds obtained in the step (2), processing into semi-polished rice, and grinding into rice flour of 80-100 meshes;

3b) mixing the rice flour and the extracting solution according to the proportion of 1: 5 (weight/volume, kg/L) and extracting for 2-3 hours at 24-26 ℃ to obtain an extraction mixture; the extraction buffer solution comprises the following components: 20mM phosphate buffer, 500mM sodium chloride, pH 7.5;

3c) and (3) adding the extraction mixture obtained in the step (2) into 2-5% of perlite for pressure filtration, and filtering the filtrate by using a 0.22um filter membrane to obtain a crude extract of the recombinant reteplase.

4. The method according to claim 1, wherein the ion exchange chromatography medium for the primary product I of the recombinant reteplase of step (3) is selected from the group consisting of NanoGel50sp, NanoQ 30L, NanoGel 50Q, UniGel 30Q, UniGel 80Q, Unigel MMC 50s and Unigel MMA 50 s.

5. The method according to claim 4, wherein the medium for ion exchange chromatography is NanoGel50 sp.

6. The process according to claim 1, characterized in that the recombinant reteplase primary product I of step (4) is prepared by:

4a) balancing a NanoGel50sp chromatographic column at a linear flow rate of 200-250 cm/h by using 20mM phosphate and 130mM sodium chloride buffer solution with pH of 6.5 and with the volume (CV) of the column being 4-6 times that of the column;

4b) mixing the crude recombinant reteplase extract obtained in the step (3) with 20mM phosphate buffer solution with pH6.5 according to a volume ratio of 1: 2.5, uniformly mixing and diluting, filtering by using a 0.22-micron filter membrane to obtain a sample loading solution, wherein the conductivity of the sample loading solution is 14-16 mS/cm, the pH of the sample loading solution is 6.4-6.6, and the sample loading volume is not more than 221 CV;

4c) eluting 40CV impurity protein by adopting 20mM phosphate and 180mM sodium chloride buffer solution with the pH value of 6.5 at the flow rate of 200-250 cm/h, wherein the conductivity of the impurity washing buffer solution is 20 mS/cm;

4d) eluting 50CV recombinant reteplase by adopting 20mM phosphate with the pH value of 6.5 and 280mM sodium chloride buffer solution at the flow rate of 200-250 cm/h, wherein the electric conductance of the elution buffer solution is 28-32 mS/cm, collecting eluent rich in the recombinant reteplase, and obtaining a primary product I containing the recombinant reteplase.

7. The method of claim 1, wherein the recombinant reteplase intermediate product II hydrophobic chromatography medium of step (5) is selected from the group consisting of UniHR Phenyl 80L LS, UniHR Phenyl 30L, BGL Octyl 4FF and BGL Phenyl low sub.

8. The method according to claim 7, characterized in that the hydrophobic chromatography medium is UniHR Phenyl 30L.

9. The method according to claim 1, wherein the intermediate product II of the recombinant reteplase of step (5) is prepared by:

5a) 5-10 CV of pH6.5, 20mM phosphate and 0.37M ammonium sulfate buffer solution are adopted to balance a UniHR Phenyl 30L chromatographic column at the flow rate of 350-400 cm/h;

5b) adjusting the conductance of the recombinant reteplase primary product I to 59-61 mS/cm by using 3M ammonium sulfate, adjusting the pH to 6.5, using the product as a sample loading solution, wherein the conductance of the sample loading solution is 60mS/cm, and completely loading the sample;

5c) eluting 20CV impurity protein by using a buffer solution with pH6.5, 20mM phosphate containing 0.5% of glycerol and 260mM ammonium sulfate, wherein the conductivity of the impurity washing buffer solution is 45 mS/cm;

5d) eluting 20CV recombinant reteplase by using a buffer solution with pH6.5, 20mM phosphate containing 0.5% of glycerol and 68mM ammonium sulfate, wherein the electric conductance of the elution buffer solution is 15mS/cm, and collecting an eluent rich in the recombinant reteplase to obtain a middle-grade product II containing the recombinant reteplase.

10. The method of claim 1, wherein the chromatography medium for Benzamidine affinity chromatography in step (6) is selected from the group consisting of nano Benzamidine 4FF (072J low ligand and 191J high ligand) and bociclone Benzamidine Bestarose 4 FF.

11. The method of claim 10, wherein the Benzamidine affinity chromatography medium is bociclone Benzamidine Bestarose 4 FF.

12. The method according to claim 1, wherein the purified recombinant reteplase of step (6) is prepared by:

6a) 5-10 CV pH7.5, 20mM phosphate and 68mM ammonium sulfate buffer solution are adopted to balance the bocolong Benzamidine Bestarose 4FF chromatographic column at the flow rate of 350-400 cm/h;

6b) adjusting the pH value of the intermediate product II to 7.5 to be used as a sample loading solution, and completely loading the sample;

6c) eluting 10CV impurity protein by using 20mM phosphate buffer solution with the pH value of 6.0;

6d) and (3) eluting the 5CV recombinant reteplase by using 20mM phosphate and 20mM acetate buffer solution with the pH value of 4.8-5.2, wherein the pH value of the elution buffer solution is 5.0, and collecting eluent rich in the recombinant reteplase to obtain the purified recombinant reteplase.

13. A recombinant plant expression vector is constructed by introducing a rice codon-optimized reteplase gene, a rice specific promoter Gt13a and a signal peptide thereof into a plasmid vector.

14. The plant expression vector according to claim 13, wherein the rice codon-optimized reteplase gene has a nucleotide sequence shown in SEQ ID No. 1.

15. A recombinant reteplase prepared according to the method of any one of claims 1 to 12.