WO2023274091A1 - 一种以基因工程水稻表达和制备重组瑞替普酶的方法 - Google Patents

一种以基因工程水稻表达和制备重组瑞替普酶的方法 Download PDF

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WO2023274091A1
WO2023274091A1 PCT/CN2022/101339 CN2022101339W WO2023274091A1 WO 2023274091 A1 WO2023274091 A1 WO 2023274091A1 CN 2022101339 W CN2022101339 W CN 2022101339W WO 2023274091 A1 WO2023274091 A1 WO 2023274091A1
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reteplase
recombinant reteplase
recombinant
rice
phosphate
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PCT/CN2022/101339
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French (fr)
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杨代常
余文卉
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武汉禾元生物科技股份有限公司
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/6456—Plasminogen activators
    • C12N9/6459—Plasminogen activators t-plasminogen activator (3.4.21.68), i.e. tPA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
    • C12N15/8205—Agrobacterium mediated transformation
    • C—CHEMISTRY; METALLURGY
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8257—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits for the production of primary gene products, e.g. pharmaceutical products, interferon
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21068—Tissue plasminogen activator (3.4.21.68), i.e. tPA

Definitions

  • the invention belongs to the field of biotechnology, and in particular relates to a method for expressing, separating and purifying recombinant reteplase using genetically engineered rice as a bioreactor.
  • Reteplase is a third-generation thrombolytic drug with a molecular weight of about 39.6kDa and a composition of 355 amino acids. It is a recombinant human tissue-type plasminogen activator (t-PA) ) derivatives can activate plasminogen to active plasminase to degrade fibrin in thrombus and play a thrombolytic effect.
  • t-PA tissue-type plasminogen activator
  • r-PA is suitable for thrombolytic therapy in adults with acute myocardial infarction caused by coronary artery infarction, and can improve ventricular function after myocardial infarction.
  • Cardiovascular and cerebrovascular diseases are one of the major diseases that endanger human life and health, and the morbidity, disability and mortality of thrombotic diseases are much higher than other diseases.
  • According to data from the Ministry of Health of my country there are more than 10 million patients with thrombosis in China, and more than 2 million people die from thrombosis every year, accounting for more than 50% of the total death toll, and thrombosis has shown an obvious upward trend in my country.
  • the domestic thrombolytic drug market is dominated by first-generation and second-generation products, of which alteplase (t-PA), lumbrokinase and plasmin accounted for 30%, 29% and 26% of the revenue respectively, ranking
  • t-PA alteplase
  • the second-generation alteplase is the most widely used thrombolytic agent in foreign countries.
  • the alteplase in the Chinese market is completely dependent on imports, and Boehringer Ingelheim's Aitongli has a 100% market share.
  • a major disadvantage of alteplase is its short half-life of 4-6 minutes, requiring continuous intravenous administration.
  • the first-generation thrombolytics have been withdrawn from the European and American markets, but due to their low price, they are still widely used in primary hospitals in my country and occupy a huge market share.
  • the third-generation thrombolytic drug r-PA is the application of genetic engineering technology to the natural human tissue plasminogen activator t-PA Fg (finger region), EGF (growth factor region), K1 (ring domain) region Removed, a non-glycosylated recombinant tissue plasminogen activator that retains amino acids 1-3 and 176-527, that is, the intermediate deletion of t-PA, and deleted the structure related to inactivation in the liver area. Therefore, it has a long half-life (15-18min), has no immunogenicity, can antagonize some inhibitors, has strong thrombolytic ability, and is convenient to administer.
  • Thrombolysis compared with the second-generation thrombolytic drug t-PA, has the advantages of improving specific thrombolytic efficiency, prolonging half-life, reducing systemic bleeding, and has a high rate of vascular recanalization, no allergic reactions, and no toxic side effects. Therefore, r-PA has greater application prospects.
  • r-PA has been successfully expressed in Escherichia coli, yeast, mammalian cells and filamentous fungi, but so far there has been no report on using rice endosperm to produce recombinant reteplase on a large scale, and the purification involved Methods: (1) Inactive inclusion bodies were formed after expression in Escherichia coli, and disulfide bond pairing refolding was required in vitro.
  • ETI-Sepharose Erythrina trypsin inhibitor
  • ETI-Sepharose 4B Affinity chromatography column, to separate r-PA
  • One object of the present invention is to provide a large-scale method of using rice endosperm cell bioreactor to efficiently express recombinant reteplase, extract and separate and purify recombinant reteplase (OsrPA) from genetically engineered rice seeds.
  • the present invention provides a method for extracting and isolating and purifying recombinant reteplase from recombinant reteplase genetically engineered rice, comprising the following steps:
  • step (2) Genetically transform rice with the expression vector described in step (1), obtain genetically engineered rice, and obtain recombinant reteplase genetically engineered rice seeds after cultivation;
  • step (4) The primary product I obtained in step (4) is subjected to hydrophobic chromatography to obtain an intermediate product II containing recombinant reteplase;
  • step (5) The intermediate product II obtained in step (5) is subjected to benzamidine affinity chromatography to obtain purified recombinant reteplase.
  • step (3) the recombinant reteplase genetically engineered rice is dried and shelled, processed into semi-polished rice, and ground into 80-100 mesh rice flour; the ratio of rice flour and extraction buffer to 1:5 (weight/volume, kg/L ), and extract at 24-26°C for 2-3 hours to obtain a recombinant reteplase extraction mixture; add 2%-5% perlite to the extraction mixture and filter the filtrate through a 0.22 ⁇ m filter membrane It is the crude extract of recombinant reteplase, wherein the composition of the extraction buffer is: 20mM phosphate buffer, 500mM sodium chloride, pH7.5;
  • the ion exchange chromatography medium is used for primary separation and purification.
  • the ion exchange chromatography packing materials include NanoGel 50sp, NanoQ 30L, NanoGel 50Q, UniGel 30Q, UniGel 80Q, Unigel MMC 50s and Unigel MMA 50s (Suzhou Nanomicro Technology Co., Ltd. Co., Ltd.), preferably using NanoGel 50sp filler for primary separation and purification.
  • the best conductivity of the sample solution is 14-16mS/cm, where 6.4 ⁇ 6.6, the sample volume does not exceed 221CV; use 20mM phosphate and 180mM sodium chloride buffer solution with pH 6.5, and carry out 40CV impurity protein elution at a flow rate of 200 ⁇ 250cm/h, the best conductivity of the washing buffer 20mS/cm; 20mM phosphate and 280mM sodium chloride buffer solution with pH 6.5 was used to elute 50CV recombinant reteplase at a flow rate of 200-250cm/h, and the best elution buffer conductance was 28- 32mS/cm, collect the eluate rich in recombinant reteplase, and obtain the primary product I containing recombinant reteplase;
  • step (5) use hydrophobic chromatography media for intermediate separation and purification.
  • the hydrophobic chromatography packing materials include UniHR Phenyl 80L LS (Suzhou Nanovitas Technology Co., Ltd.), UniHR Phenyl 30L (Suzhou Nanovitas Technology Co., Ltd.), Octyl 4FF (Borglon (Shanghai) Biotechnology Co., Ltd.), Phenyl low sub (Borglon (Shanghai) Biotechnology Co., Ltd.), preferably use UniHR Phenyl 30L (Suzhou Nanomicro Technology Co., Ltd.) packing column for intermediate Isolation and Purification.
  • step (6) the final stage of separation and purification is carried out using affinity chromatography packing materials, including Benzamidine 4FF (072J low ligand and 191J high ligand, Suzhou Nanomicro Technology Co., Ltd.) and Benzamidine Bestarose 4FF (Bo Gelong (Shanghai) Biotechnology Co., Ltd.), preferably use Benzamidine Bestarose 4FF (Burgeron (Shanghai) Biotechnology Co., Ltd.) filler.
  • affinity chromatography packing materials including Benzamidine 4FF (072J low ligand and 191J high ligand, Suzhou Nanomicro Technology Co., Ltd.) and Benzamidine Bestarose 4FF (Bo Gelong (Shanghai) Biotechnology Co., Ltd.), preferably use Benzamidine Bestarose 4FF (Burgeron (Shanghai) Biotechnology Co., Ltd.) filler.
  • the pH of the buffer solution is 5.0, and the eluate rich in recombinant reteplase is collected to obtain purified recombinant reteplase.
  • a kind of plant expression vector comprising:
  • step 2) transforming the vector obtained in step 2) into the rice callus, and obtaining the genetically engineered rice plants transformed with recombinant reteplase through culturing, screening and induction;
  • the recombinant reteplase expression vector preferably has a structure as shown in FIG. 2 .
  • the invention constructs a recombinant reteplase expression carrier 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 require in vitro renaturation, has high safety and is easy to be scaled up, and the obtained recombinant reteplase has an HPLC purity of more than 98%.
  • FIG. 1 Schematic diagram of the plasmid structure of pOsPMP773.
  • FIG. 1 Schematic diagram of the plasmid structure of pOsPMP774.
  • FIG. 1 Schematic diagram of the plasmid structure of pOsPMP775.
  • Figure 4 PCR detection of target genes in some plants of genetically engineered rice.
  • M is DNA standard molecular weight marker
  • 1-24 are different plants of T1 generation transgenic materials.
  • Fig. 6 Detection results of circle-dissolving activity of crude extract of recombinant reteplase under different extraction conditions. Among them, in fibrin plate 1-5, numbers 1-6 are standard curve detection, and 7-12 are sequentially 20-fold and 40-fold dilution detection of extracts under different extraction conditions from 1 to 15.
  • FIG. 7 SDS-PAGE and WB detection results of crude extract of recombinant reteplase with different extraction pH, temperature and time.
  • M is the standard molecular weight marker
  • 7.0-10.0 represent different extraction pHs
  • LGC is the background plant.
  • the red arrows correspond to the target band and the degradation band, respectively.
  • 1 to 6 in B represent extraction time (hours), overnight represents overnight extraction, 007 represents batch number 775-214-49-007 rice noodles, and 006 represents batch number 775-214-49-006 rice noodles.
  • FIG. 8 Selection of ion-exchange chromatography media for primary purification.
  • M in A is the standard molecular weight marker; load means the sample solution, FT means the penetration solution, 10-80 means different conductance (mS/cm), 2M means 2M sodium chloride;
  • the upper picture in B is containing reteplase
  • FT1 indicates the penetration solution after loading 10CV
  • FT2 indicates the penetration solution after loading 20CV
  • load indicates the loading solution
  • the figure below shows the 4-fold bold solution containing reteplase Diluted sample detection
  • FT1 indicates the penetration solution after loading 5CV
  • FT2 indicates the penetration solution after loading 20CV
  • load indicates the loading solution
  • 20, 30, 50, 70 and 80 in C indicate different conductivity ( mS/cm)
  • 2M means 2M sodium chloride.
  • FIG. 9 Chromatographic optimization of NanoGel 50sp cation exchange media for primary purification.
  • L represents the sample solution
  • FT represents the penetration solution
  • 30, 40 and 70 represent different conductivities (mS/cm)
  • 2M represents 2M sodium chloride
  • E represents the eluent
  • L represents the sample solution
  • FT stands for penetration fluid
  • 20, 25 and 32 represent different conductivity (mS/cm)
  • 1M represents 1M sodium chloride
  • L in C represents loading liquid
  • FT represents penetration liquid
  • 18, 32 and 40 represent different Conductivity (mS/cm)
  • 1M means 1M sodium chloride
  • 2M means 2M sodium chloride.
  • FIG. 10 Chromatographic capacity confirmation of NanoGel 50sp cation exchange media for primary purification.
  • L represents the loading liquid
  • FT represents the penetration liquid
  • W represents the eluent
  • E1 is the eluent of 25mS/cm
  • E2 is the eluent of 28mS/cm
  • E3 is the eluent of 32mS/cm
  • E1- Torr indicates the tailing of E1
  • 1M indicates 1M NaCl.
  • FIG. 12 Optimization of chromatographic conditions for UniHR Phenyl 30L, an intermediate purification hydrophobic packing material.
  • M is the standard molecular weight marker
  • L is the sample solution
  • 1 ⁇ 6 is the gradient elution sample number
  • Elu is the final elution sample
  • M is the standard molecular weight marker
  • L is the sample solution
  • FT is the breakthrough solution
  • W represents the eluent
  • E1 represents the 15 mS/cm eluent containing 0.5% glycerol
  • E2 represents the 16 mS/cm eluate containing 10% ethanol.
  • FIG. 13 Chromatography optimization of final affinity purification medium BGL Benzamidine 4FF.
  • M is the standard molecular weight Marker
  • 6.5, 6.0, 5.5, and 3.0 are different pH values
  • 2, 4, 10 and 15 are different conductivities (mS/cm)
  • L is the sample solution
  • FT is the penetration solution
  • W is the washing solution
  • E is the eluent
  • B L is the sample solution
  • FT is the penetration solution
  • W is the washing solution
  • 1 ⁇ 4 are the samples collected in sections
  • NaK is disodium hydrogen phosphate and phosphoric acid Potassium dihydrogen buffer
  • NaAc is sodium acetate buffer
  • E is the eluent
  • Econcentrated represents the concentrated solution of the eluent.
  • Fig. 15 Electrophoresis detection results of 3 batches of recombinant reteplase samples in small test process, where M is the standard molecular weight Marker.
  • the rice-specific promoter Gt13a and its signal peptide were selected to mediate the expression of the recombinant reteplase gene in rice endosperm cells.
  • the reteplase gene sequence (Genbank registration number: KU053049.1), Nanjing GenScript Biotechnology Co., Ltd. was entrusted to synthesize it according to the rice-preferred genetic codon, as shown in SEQ ID NO.1, through the rice-preferred codon After optimization, 23.2% of nucleotides and 32.3% of codons were changed, but the corresponding amino acid sequence remained unchanged, and the constructed plasmid was pOsPMP773 (Fig. 1).
  • the synthetic codon-optimized reteplase gene (SEQ ID NO.1) was digested with MlyI and XhoI and cloned into pOsPMP003 digested with NaeI and XhoI, and an intermediate vector plasmid was constructed by T4 ligase pOsPMP774 ( Figure 2), the entire expression cassette containing the Gt13a promoter, signal peptide sequence, codon-optimized reteplase gene and Nos terminator with a length of 2386bp was inserted into the binary expression vector pcl300 digested by HindIII and EcoRI , construct the Agrobacterium-mediated bacteria plasmid, named as pOsPMP775 ( Figure 3)
  • the pOsPMP775 plasmid was transformed into Agrobacterium tumefaciens EHA105 (Invitrogen, USA), and pOsPMP775 was transformed into the callus of rice variety LGC through Agrobacterium tumefaciens, and a complete plant was formed after cultivation, screening and induction.
  • the specific method is as follows:
  • the Agrobacterium containing the expression vector pOsPMP775 was expanded and cultured, smeared on a karimycin-resistant plate, and cultivated in a 28°C incubator for 2 to 3 days; a single colony of Agrobacterium was inoculated into the suspension medium (AAM liquid) with an inoculation loop culture medium), shake the bacteria at 28°C.
  • AAM liquid suspension medium
  • AAM loop culture medium shake the bacteria at 28°C.
  • AS acetosyringone, 250 mg/ml
  • Transfer the co-cultured callus to a sterilized Erlenmeyer flask wash the callus 5-7 times with sterile water; soak the infected callus with sterile water containing 0.5g/L cephalosporin for about 30 minutes, then shake at 28°C, 180-200rpm for 20-30 minutes; pour off the sterilized water containing antibiotics, put the triangular flask upside down in the sterilized medium containing filter paper for about 15 minutes to dry, then transfer the callus Cultivate on the selection medium containing HPT antibiotics for 20-30 days.
  • the callus with HPT resistance was transferred to a differentiation medium (N 6 medium), and cultured at 26° C. under light for 20-30 days.
  • the differentiated seedlings were selected from the differentiation medium, transferred to the MS medium containing 1/2 for rooting culture, and after 30 days of light culture at 28°C, they were transferred to the field for growth.
  • the identification results showed that 87 strains of positive recombinant reteplase transgenic rice were obtained through Agrobacterium tumefaciens mediated transformation, and the PCR identification results of some target genes in genetically engineered rice are shown in FIG. 4 .
  • the obtained recombinant reteplase-positive seedlings were transplanted to room temperature and grown to maturity, and single plants capable of normal fruiting were harvested.
  • the identification results showed that 42 individual plants of recombinant reteplase-positive transgenic rice expressed reteplase protein, with expression levels ranging from 1.9 ⁇ g/g to 62.5 ⁇ g/g, among which there were 5 individual plants with higher expression levels, expressing The amount ranges from 41.2 to 62.5 ⁇ g/g.
  • the results showed that as the extraction pH increased, the r-PA degradation band gradually increased, and when the pH of the extract was higher than 8.5, the degradation band Therefore, the pH of the extraction buffer can be controlled to be 7.5-8.0, which can reduce the generation of degradation and reduce the difficulty of the subsequent purification process.
  • the results of Elisa expression and lytic activity showed that at pH 7.5, the protein in the extract of recombinant reteplase The specific activity was the highest, therefore, the extraction pH of recombinant reteplase was determined to be 7.5.
  • the above-mentioned ion-exchange chromatography packing includes NanoGel 50sp, NanoQ 30L , NanoGel 50Q, UniGel 30Q, UniGel 80Q, Unigel MMC 50s and Unigel MMA 50s; by designing different phosphate buffer loading conductance and pH, and NaCl salt concentration gradient elution, the results show that NanoGel 50sp packing (pH 6.0 and 6.5, conductance 15.56ms/cm) can be combined with recombinant reteplase, WB shows that recombinant reteplase can be eluted at pH6.0 ⁇ 6.5 30 ⁇ 40ms/cm and pH7.0 ⁇ 7.5 20 ⁇ 30ms/cm (Fig.
  • the recombinant reteplase primary product I was prepared, and ammonium sulfate was added to the primary product I so that the concentration of ammonium sulfate in the sample solution was 0.6M, 0.7M and 0.8M, 5mL UniHR Phenyl 80L LS, UniHR Phenyl 30L, BGL Octyl 4FF, BGL Phenyl low sub packing chromatography were carried out respectively, the results showed that the separation effect of BGL Phenyl low sub and Octyl 4FF was poor, and the separation effect of UniHR Phenyl 30L was comparable to that of UniHR Phenyl 80L LS, but UniHR Phenyl The NaOH eluted fractions of 80L LS and UniHRPhenyl 30L all had a band suspected of the target protein (Figure 11).
  • the primary product I of recombinant reteplase was prepared, and ammonium sulfate was added to the primary product I so that the concentration of ammonium sulfate in the sample solution was 0.4M, and carried out in a 0.5% glycerol system Salt concentration gradient elution, the results showed that under the 0.5% glycerol system, the degradation band of reteplase, amino acid-deficient protein, glycosylated protein and non-glycosylated protein had a certain degree of separation ( Figure 12A), and finally determined 0.5 Under the % glycerol system, the conductivity of the phosphate buffer is 40-45mS/cm, which is better for washing impurities, and 15mS/cm is better for elution (Fig. 12B).
  • Example 2 According to the extraction determined in Example 2, 3 and 4, the conditions of cation exchange chromatography and hydrophobic chromatography were used to prepare the intermediate product II of recombinant reteplase, and the pH of the intermediate product II was adjusted to 7.5, adding different concentrations of sodium chloride (0.1 M, 0.5M, 1M), respectively carried out 5mL nano-microbenzamidine-4FF (072J) low ligand, (191J) high ligand, BGL Benzamidine Bestarose 4FF affinity packing chromatography, the results are as follows:
  • BGL Benzamidine Bestarose 4FF has the strongest binding ability to reteplase, and BGL Benzamidine Bestarose 4FF is selected for subsequent chromatography optimization.
  • the conditions of cation exchange chromatography and hydrophobic chromatography were used to prepare the intermediate product II of recombinant reteplase, adjust the pH of the intermediate product II to 7.5, and use it as the sample solution of BGL Benzamidine Beatarose 4FF 20mM phosphate buffer with different pH (6.5, 6.2, 6.0) and different conductivities were optimized for impurity washing.

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Abstract

一种经水稻密码子优化的重组瑞替普酶基因，相关载体及制备基因工程水稻种子的方法和重组瑞替普酶分离纯化方法。将基因工程稻谷加工粉碎后与提取缓冲液混合，提取过滤后得到含重组瑞替普酶的粗提取物；分离纯化步骤依次包括：1)将含重组瑞替普酶的粗提取物进行阳离子交换层析，得到初级产物I；2)将初级产物I进行疏水层析，得到中间产物II；3)将中间产物II进行苯甲脒亲和层析，得到纯化的重组瑞替普酶。

Description

一种以基因工程水稻表达和制备重组瑞替普酶的方法 技术领域
本发明属于生物技术领域，具体涉及以基因工程水稻为生物反应器，表达和分离纯化重组瑞替普酶的方法。
背景技术
瑞替普酶(Reteplase，简称r-PA)为第三代溶栓药物，分子量约39.6kDa，355个氨基酸组成，是重组人组织型纤溶酶原激酶(Tissue-type plasminogen activator，t-PA)的衍生物，可以使纤维蛋白溶解酶原激活为有活性的纤溶蛋白溶解酶，以降解血栓中的纤维蛋白，发挥溶栓作用。r-PA适用于成人由冠状动脉梗塞引起的急性心肌梗塞的溶栓疗法，能够改善心肌梗塞后的心室功能。
心脑血管疾病是危害人类生命和健康的主要疾病之一，其中血栓类疾病的发病率、致残率和死亡率均远远高于其他疾病。据我国卫生部资料显示，我国有超过千万的血栓病患者，每年死于血栓疾病的人数就超过200万，占总死亡人数的50％以上，且血栓疾病在我国已呈现明显上升趋势。目前国内溶栓药物市场以第一代、第二代产品为主，其中阿替普酶(t-PA)、蚓激酶和纤溶酶收入占比分别为30％、29％、26％，排名前三位，均为国家医保乙类产品。第二代的阿替普酶是国外使用最多的溶栓剂，我国市场上的阿替普酶则完全依赖进口，勃林格殷格翰的爱通立占有100％的市场份额。阿替普酶的一个主要劣势是半衰期短，仅为4-6分钟，需要持续静脉给药。第一代溶栓剂已经退出欧美市场，但是由于价格便宜，仍然在我国基层医院广泛使用，占有极大的市场份额。第三代溶栓药物r-PA是应用基因工程技术将天然人组织纤溶酶原激活剂t-PA的Fg(指型区)，EGF(生长因子区)，K1(环状结构域)区去除，保留了第1～3和176～527位氨基酸的一种非糖基化重组组织纤溶酶原激活剂，也就是t-PA的中间缺失体，删除了与肝内灭活相关的结构区域。因此，具有较长的半衰期(15～18min)，无免疫原性，能拮抗一些抑制剂，溶栓能力强，给药方便，不需要按个体给药，可短时间，小剂量给药，快速溶栓，相比于第二代溶栓药物t-PA具有提高特异性溶栓效率、延长半衰期，减少全身系统性出血，且具有血管再通率高、无过敏反应、无毒副作用等优点，故r-PA具有更大的应用前景。
目前已成功在大肠杆菌、酵母菌、哺乳动物细胞以及丝状真菌中进行了r-PA的表达，但是至今未见有关使用水稻胚乳来大规模生产重组瑞替普酶的报道，涉及到的纯化方法：(1)大肠杆菌表达后形成无活性的包涵体，体外需要进行二硫键配对复性，利用刺桐胰蛋白酶抑制剂(ETI)能够特异性结合r-PA的特点，用ETI-Sepharose 4B亲和层析柱，分离得到r-PA；(2)利用转基因兔乳表达r-PA，通过硫酸铵沉淀、酸碱沉淀、超滤、亲和层析(Lysine HyperD、Blue Sepharose 6FF)、离子交换(SP BestaroseTM FF)、凝胶过滤(Surpdex G75)等方法分离纯化r-PA，未见有关从水稻种子中分离纯化重组瑞替普酶方法的报道。国内市售药派通欣在大肠杆菌中表达，但是大肠杆菌表达后无活性，需要体外复性等操作，而利用水稻胚乳细胞表达系统来高效表达重组r-PA克服了需体外复性等操作，具有绿色、安全、容易规模化等优点。
发明内容
本发明的一个目的是提供一种规模化的利用水稻胚乳细胞生物反应器来高效表达重组瑞替普酶，从基因工程水稻种子中提取和分离纯化重组瑞替普酶(OsrPA)的方法。
为实现水稻胚乳细胞高效表达重组瑞替普酶目的，本发明提供一种从重组瑞替普酶基因工程水稻中提取和分离纯化重组瑞替普酶的方法，包括下述步骤：
(1)构建在水稻种子中表达重组瑞替普酶的植物表达载体，所述载体含有经水稻密码子优化的瑞替普酶基因；
(2)用步骤(1)所述的表达载体遗传转化水稻，获得基因工程水稻，栽培后获得重组瑞替普酶基因工程水稻种子；
(3)从步骤(2)所述的重组瑞替普酶基因工程水稻种子中提取含重组瑞替普酶的粗提取物；
(4)将步骤(3)获得的含重组瑞替普酶的粗提取物经阳离子交换层析，得到初级产物I；
(5)将步骤(4)获得的初级产物I经疏水层析，得到含重组瑞替普酶的中级产物II；
(6)将步骤(5)获得中级产物II经苯甲脒亲和层析，得到纯化的重组瑞替普酶。
优选的技术方案为：
(1)构建包含如SEQ ID NO.1所示的经水稻密码子优化的瑞替普酶基因的植物表达载体；
(2)用上述植物表达载体遗传转化水稻获得基因工程水稻，栽培后获得重组瑞替普酶基因工程水稻种子；
(3)从重组瑞替普酶基因工程水稻种子中提取含有重组瑞替普酶的粗提取物；
(4)将含有重组瑞替普酶的粗提取物经阳离子交换层析，得到初级产物I，其中所述阳离子交换层析的介质为NanoGel 50sp；
(5)采用疏水层析对初级产物I进行中级纯化，获得中级产物II，其中所述疏水层析的介质为UniHR Phenyl 30L；
(6)采用亲和层析对中级产物II进行末级纯化，获得纯化的重组瑞替普酶，其中所述的亲和层析介质为博格隆Benzamidine Bestarose 4FF。
根据本发明的一个优选实施方式：
步骤(3)中，将重组瑞替普酶基因工程水稻晒干脱壳，加工成半精米，研磨成80～100目的米粉；米粉与提取缓冲液按照1：5(重量/体积，kg/L)的比例混匀，在24～26℃提取2～3小时，获得重组瑞替普酶提取混合物；提取混合物中加入2％-5％的珍珠岩压滤，滤液再经0.22μm滤膜过滤后即为重组瑞替普酶的粗提取物，其中所述提取缓冲液的成分为：20mM磷酸盐缓冲液，500mM氯化钠，pH7.5；
步骤(4)中，采用离子交换层析介质进行初级分离纯化，离子交换层析填料包括NanoGel 50sp，NanoQ 30L，NanoGel 50Q，UniGel 30Q，UniGel 80Q，Unigel MMC 50s和Unigel MMA 50s(苏州纳微科技股份有限公司)，优选使用NanoGel 50sp填料进行初级分离纯化。用4-6倍柱体积(CV)的pH为6.5的20mM磷酸盐和130mM氯化钠缓冲液，以200～250cm/h的线性流速平衡层析柱；将上述的重组瑞替普酶粗提取物与pH6.5的20mM磷酸盐缓冲液按照体积比1：2.5混匀稀释，0.22um滤膜过滤后作为上样液，最佳上样液电导为14～16mS/cm，其中上样液pH为6.4～6.6，上样体积不超过221CV；采用pH为6.5的20mM磷酸盐和180mM氯化钠缓冲液，以200～250cm/h的流速进行40CV杂质蛋白洗脱，最佳洗杂缓冲液电导为20mS/cm；采用pH为6.5的20mM磷 酸盐和280mM氯化钠缓冲液，以200～250cm/h的流速进行50CV重组瑞替普酶的洗脱，最佳洗脱缓冲液电导为28～32mS/cm，收集富含重组瑞替普酶的洗脱液，获得含重组瑞替普酶的初级产物I；
步骤(5)中，采用疏水层析介质进行中级分离纯化，疏水层析填料包括UniHR Phenyl 80L LS(苏州纳微科技股份有限公司)，UniHR Phenyl 30L(苏州纳微科技股份有限公司)，Octyl 4FF(博格隆(上海)生物技术有限公司)，Phenyl low sub(博格隆(上海)生物技术有限公司)，优选使用UniHR Phenyl 30L(苏州纳微科技股份有限公司)填料的层析柱进行中级分离纯化。用5～10CV的pH6.5，20mM磷酸盐和0.37M硫酸铵缓冲液，以350～400cm/h的流速平衡层析柱；用3M硫酸铵调节重组瑞替普酶初级产物I的电导为59～61mS/cm，pH调整为6.5，作为上样液，最佳上样液电导为60mS/cm，全部上样；采用pH6.5，含0.5％甘油的20mM磷酸盐和260mM硫酸铵的缓冲液进行20CV杂质蛋白洗脱，最佳洗杂缓冲液电导为45mS/cm；采用pH6.5，含0.5％甘油的20mM磷酸盐和68mM硫酸铵的缓冲液进行20CV重组瑞替普酶的洗脱，最佳洗脱缓冲液电导为15mS/cm，收集富含重组瑞替普酶的洗脱液，获得含重组瑞替普酶的中级产物II；
步骤(6)中，采用亲和层析填料进行末级分离纯化，亲和层析填料包括Benzamidine 4FF(072J低配基和191J高配基，苏州纳微科技股份有限公司)及Benzamidine Bestarose 4FF(博格隆(上海)生物技术有限公司)，优选使用Benzamidine Bestarose 4FF(博格隆(上海)生物技术有限公司)填料。用5～10CV的pH7.5，20mM磷酸盐和68mM硫酸铵缓冲液，以350～400cm/h的流速平衡层析柱；将中级产物II的pH调整为7.5，作为上样液，全部上样；采用pH6.0的20mM磷酸盐缓冲液进行10CV杂质蛋白洗脱；采用pH4.8～5.2的20mM磷酸盐和20mM醋酸盐缓冲液进行5CV重组瑞替普酶的洗脱，最佳洗脱缓冲液的pH为5.0，收集富含重组瑞替普酶的洗脱液，获得纯化的重组瑞替普酶。
根据本发明的另一方面，提供一种植物表达载体，包括：
1)合成如SED ID NO.1所示的经水稻密码子优化的瑞替普酶基因序列；
2)构建水稻胚乳细胞特异性表达的重组瑞替普酶表达载体；
3)将步骤2)中获得的载体转化水稻愈伤组织中，经培养，筛选和诱导获得转重组瑞替普酶基因工程水稻植株；
其中，所述重组瑞替普酶表达载体优选具有如图2所示的结构。
本发明构建了在水稻胚乳细胞中表达的重组瑞替普酶表达载体，在水稻中成功地表达了重组瑞替普酶，建立了提取并纯化该瑞替普酶的方法。本发明的方法无需体外复性，安全性高，容易规模化，所获得的重组瑞替普酶达到98％以上的HPLC纯度。
附图说明
图1.pOsPMP773质粒结构示意图。
图2.pOsPMP774质粒结构示意图。
图3.pOsPMP775质粒结构示意图。
图4.基因工程水稻部分植株目的基因的PCR检测。其中M为DNA标准分子量Marker；1～24分别为T1代转基因材料的不同植株。
图5.不同提取条件下重组瑞替普酶粗提物SDS-PAGE和WB检测结果。其中M为标准分子量Marker；1-15分别表示表1中1～15不同提取条件，LGC为背景植株。
图6.不同提取条件下重组瑞替普酶粗提物溶圈活性检测结果。其中纤维蛋白平板1～5中，编号1～6为标曲检测，7～12依次为1～15不同提取条件下提取液进行20倍和40倍稀释检测。
图7.不同提取pH，温度和时间对重组瑞替普酶粗提物SDS-PAGE和WB检测结果。A中M为标准分子量Marker；7.0～10.0分别表示不同提取pH，LGC为背景植株。红色箭头分别对应目的条带和降解带。B中1～6表示提取时间(小时)，过夜表示过夜提取，007表示775-214-49-007批号米粉，006表示775-214-49-006批号米粉。
图8.初级纯化离子交换层析介质的选择。A中M为标准分子量Marker；load表示上样液，FT表示穿透液，10～80表示不同的电导(mS/cm)，2M表示2M氯化钠；B中上图为含瑞替普酶粗体液1倍稀释后的样品检测，FT1表示上样10CV后的穿透液，FT2表示上样20CV后的穿透液，load表示上样液；下图为含瑞替普酶粗体液4倍稀释后的样品检测，FT1表示上样5CV后的穿透液，FT2表示上样20CV后的穿透液，load表示上样液；C中20，30，50，70和80表示不同的电导(mS/cm)，2M表示2M氯化钠。
图9.初级纯化阳离子交换填料NanoGel 50sp层析优化。A中L表示上样 液，FT表示穿透液，30，40和70表示不同的电导(mS/cm)，2M表示2M氯化钠，E表示洗脱液；B中L表示上样液，FT表示穿透液，20，25和32表示不同的电导(mS/cm)，1M表示1M氯化钠；C中L表示上样液，FT表示穿透液，18，32和40表示不同的电导(mS/cm)，1M表示1M氯化钠，2M表示2M氯化钠。
图10.初级纯化阳离子交换填料NanoGel 50sp层析载量确认。L表示上样液，FT表示穿透液，W表示洗杂液，E1为25mS/cm的洗脱液，E2为28mS/cm的洗脱液，E3为32mS/cm的洗脱液，E1-托表示E1的拖尾，1M表示1M氯化钠。
图11.中级纯化疏水层析介质的选择。L表示上样液，FT表示穿透液，1～8表示梯度洗脱样品编号。
图12.中级纯化疏水填料UniHR Phenyl 30L层析条件优化。A中M为标准分子量Marker，L表示上样液，1～6表示梯度洗脱样品编号，Elu表示最终洗脱样品；B中M为标准分子量Marker，L表示上样液，FT表示穿透液，W表示洗杂液，E1表示含0.5％甘油的15mS/cm洗脱液，E2表示含10％乙醇的16mS/cm洗脱液。
图13.末级亲和纯化介质BGL Benzamidine 4FF层析优化。A中M为标准分子量Marker，6.5，6.0，5.5，3.0为不同的pH值，2，4，10和15为不同的电导(mS/cm)，L为上样液，FT为穿透液，W为洗杂液，E为洗脱液；B中L为上样液，FT为穿透液，W为洗杂液，1～4为分段收集的样品，NaK为磷酸氢二钠和磷酸二氢钾缓冲液，NaAc为醋酸钠缓冲液，E为洗脱液，E浓代表洗脱液浓缩液。
图14.3批小试工艺层析图谱。
图15.3批小试工艺重组瑞替普酶样品电泳检测结果，其中M为标准分子量Marker。
图16.3批小试工艺重组瑞替普酶RP-HPLC检测结果。
具体实施方式
以下将通过实施例和附图以详细说明本发明的技术方案，从而更好地阐述本发明的特点和优势。所提供的实施例应被解释为对本发明方法的举例说明， 而不以任何方式限制本发明揭示的技术方案。
实施例中使用的试剂和仪器，除特殊说明以外，均为普通市售。
【实施例1】高效表达重组瑞替普酶基因工程水稻的制备
1.重组瑞替普酶基因表达载体的构建
本实施例选用水稻特异性启动子Gt13a及其信号肽来介导重组瑞替普酶基因在水稻胚乳细胞中表达。根据瑞替普酶基因序列(Genbank登记号：KU053049.1)，委托南京金斯瑞生物科技有限公司根据水稻偏好的遗传密码子合成，具体如SEQ ID NO.1所示，经水稻偏好密码子优化后的核苷酸改变了23.2％，密码子改变了32.3％，但其对应的氨基酸序列没有变化，构建的质粒为pOsPMP773(图1)。将所述合成的经密码子优化的瑞替普酶基因(SEQ ID NO.1)用MlyI和XhoI酶切后克隆到经NaeI和XhoI酶切的pOsPMP003中，经T4连接酶构建一个中间载体质粒pOsPMP774(图2)，将长度为2386bp的含Gt13a启动子，信号肽序列及密码子优化的瑞替普酶基因和Nos终止子的整个表达盒插入到经HindIII和EcoRI酶切双元表达载体pcl300，构建农杆介导菌质粒，命名为pOsPMP775(图3)
2.基因工程水稻遗传转化
将pOsPMP775质粒转化到根癌农杆菌EHA105(美国Invitrogen公司)，通过根癌农杆菌介导将pOsPMP775转化到水稻品种LGC的愈伤组织中，经培养、筛选和诱导后形成完整的植株。具体方法如下：
(1)愈伤组织诱导
将成熟的水稻种子脱壳后用70％乙醇浸泡灭菌1分钟，20％次氯酸钠再处理30分钟；用无菌水清洗5～7次后，将种子接种到诱导培养基(N 6培养基)上，每皿接种6～8粒，在32℃光照处理大约5～7天。
(2)农杆菌制备
将含有表达载体pOsPMP775的农杆菌进行扩大培养，在卡拉霉素抗性平皿中涂菌，28℃培养箱中培养2～3天；用接种环取农杆菌单菌落接种到悬浮培养基(AAM液体培养基)中，在28℃摇菌培养。
(3)农杆菌侵染(共培养)
将愈伤组织转移到灭菌的三角瓶中，调整农杆菌悬浮液OD 600值于0.05～0.1；将种子悬浮在AAM培养基中，侵染1.5分钟，期间持续摇晃；弃菌液，用无菌滤纸吸干多余的菌液，将愈伤组织取出置于无菌滤纸上沥干30～45分钟；将无菌滤纸放在2N 6-AS培养基上，再将含有AS(乙酰丁香酮，250mg/ml)的500μl AAM滴在直径9cm的无菌滤纸上，将侵染过的愈伤组织放在滤纸上，25℃黑暗共培养3天。
(4)水洗和筛选
将共培养的愈伤组织转移到灭菌的三角瓶中，用无菌水清洗愈伤组织5～7次；用含0.5g/L浓度的头孢霉素灭菌水浸泡感染的愈伤组织约30分钟，然后在28℃，180～200rpm摇20～30分钟；倒掉含有抗生素的灭菌水，将三角瓶倒置于含滤纸的灭菌培养基中大约15分钟晾干后，转移愈伤组织到含有HPT抗生素的筛选培养基上培养20～30天。
(5)愈伤组织分化
将经过20～30天选择后具有HPT抗性的愈伤组织转移到分化培养基(N 6培养基)上，在26℃光照培养20～30天。
(6)生根
从分化培养基中挑选出分化后的小苗，转移到含有1/2的MS培养基上进行生根培养，在28℃光照培养30天后，转移到田间生长。
3.基因工程水稻鉴定
(1)水稻基因组DNA提取
取T0代HPT阳性再生苗的叶片约2cm，分别放入离心管中，编号；加入600μl CTAB提取缓冲液(2％CTAB，1.38M NaCl，0.1M Tris-HCl，20mM EDTA，pH8.0)，在震荡破碎机震荡，65℃保温60分钟，期间不时摇动；加入等体积的氯仿/异丙醇，轻缓颠倒离心管混匀，室温下，12000rpm离心10分钟；将上清液转入另一新的1.5ml离心管中，加入等体积的异丙醇，缓慢颠倒离心管混匀，室温下放置60分钟；12000rpm离心10分钟，去上清，70％乙醇漂洗，沉淀风干；风干后的DNA加入80μl的TE缓冲液溶解DNA，-20℃保存备用。
(2)目的基因PCR扩增检测
取1μl水稻基因组DNA作为模板，设置阳性(质粒DNA)和阴性(无菌水)对照；利用重组瑞替普酶正向引物GT13a-F(SED ID NO.2： 5’-AGCTACCAGGGCAACAGCGA-3’)和反向引物R-PA-R(SED ID NO.3：5’-AGCTGCTGATGAGGATGCCG-3’)进行PCR扩增，产物理论大小为670bp；PCR扩增反应体系(25μl体系)：2.5μl 10x Buffer，0.15μl rTaq酶(5U/μl)，4μl dNTP(2.5mM)，引物各0.5μl；加ddH 2O至25μl；PCR扩增条件：94℃预变性5分钟，94℃变性30秒，59.8℃退火反应30秒，72℃延伸45秒，35个循环，最后72℃延伸10分钟；扩增产物经1％琼脂胶，EB染色后150V，200mA电泳15分钟，凝胶成像仪中观察结果。
鉴定结果表明，通过根癌农杆菌介导转化获得87株阳性的重组瑞替普酶转基因水稻，部分基因工程水稻目的基因PCR鉴定结果如图4所示。
4.重组瑞替普酶表达量的鉴定
将获得的重组瑞替普酶阳性苗移栽到室温中生长至成熟，收种能够正常结实的单株。采用DY7449-05(R&D)试剂盒进行阳性单株的定量检测，捕获抗体包板后，取10μl的蛋白提取液稀释1000倍，加入酶标板中，室温孵育2小时；采用含0.05％吐温20的磷酸钠缓冲液洗板后，加入100μl检测抗体，室温孵育2小时；洗板后加入100μl HRP-链霉亲和素，室温反应20min后洗板，每孔加入100μlTMB室温避光显色15～20分钟，加入50μl 2M硫酸终止反应，OD450读数。
鉴定结果表明，重组瑞替普酶阳性转基因水稻有42个单株表达瑞替普酶蛋白，表达量从1.9μg/g到62.5μg/g，其中有5个表达量较高的单株，表达量范围在41.2～62.5μg/g。
SEQ ID NO.1：
Figure PCTCN2022101339-appb-000001
Figure PCTCN2022101339-appb-000002
SEQ ID NO.2：
5’-AGCTACCAGGGCAACAGCGA-3’
SEQ ID NO.3：
5’-AGCTGCTGATGAGGATGCCG-3’
【实施例2】从基因工程水稻中提取重组瑞替普酶
1.重组瑞替普酶提取条件初步筛选
将基因工程水稻脱壳后加工成半精米，研磨成80～100目的米粉，利用JMP软件选取提取pH(4-10)、提取盐浓度(0-0.5M)、提取温度(8-37℃)三个因素，以r-PA提取蛋白比活性(IU/mg)为响应值进行响应曲面设计，制表如下：
表1不同提取条件设计
实验编号 提取pH 提取盐浓度(mM) 提取温度(℃)
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
称取15份米粉，每份1g于10ml离心管中，按照上表实验设计进行实验，每管加入5ml提取液，提取1h，10000g离心5min，取上清进行SDS-PAGE/WB检测(图5)，Elisa表达量检测和溶圈法活性检测(图6)。另称取1g LGC米粉用20mM PB，250mM NaCl，pH7.0在27℃提取1h后同上处理，作为阴性对照。结果显示，重组瑞替普酶提取蛋白比活性对提取缓冲液的pH最敏感，其次是提取缓冲液的盐浓度。利用预测刻画器分析，在pH8.4，盐浓度为476mM，提取温度为18℃时，重组瑞替普酶提取的蛋白比活性最高。
2.重组瑞替普酶提取条件确定
(1)不同提取缓冲液pH对重组瑞替普酶提取的影响
称取7份775-214-49-007米粉，每份1g于10ml离心管中，然后向各管中依次加入5ml pH为7.0、7.5、8.0、8.5、9.0、9.5、10.0的磷酸盐提取液，25℃提取2h，10000g离心5min，取上清进行SDS-PAGE/WB检测(图7A)，Elisa表达量和溶圈活性检测。另称取1g LGC米粉用pH10.0提取液在25℃提取2h后同上处理，结果显示，随着提取pH的升高，r-PA降解带逐渐增多，提取液的pH高于8.5时降解带开始出现，因此可控制提取缓冲液的pH为7.5-8.0，减少降解产生，降低后续纯化工艺难度，Elisa表达量和溶圈活性检测结果表明pH7.5时，重组瑞替普酶提取液中蛋白比活性最高，因此，确定重组瑞替普酶的提取pH为7.5。
(2)不同提取温度和提取时间对重组瑞替普酶提取的影响
称取10份1g 775-214-49-007r-PA米粉和2份1g 775-214-49-006r-PA米粉，按照W/V＝1：5的比例向其中加入5ml 20mM PB，500mM NaCl，pH7.5提取液，分别按照25℃提取1h，2h，4h，6h和过夜(21h)，4℃提取1h，2h，4h，6h和过夜(21h)；提取完后，10000g离心10min，取上清进行SDS-PAGE/WB检测(图7B)，Elisa表达量和溶圈活性检测。结果显示，重组瑞替普酶在4℃下提取的含量和活性都低于25℃提取，过夜提取后，重组瑞替普酶含量和活性都降低，提示不能过夜提取，确定重组瑞替普酶提取温度为25℃，提取时间为2h。因此，确定重组瑞替普酶的提取条件：20mM PB，500mM NaCl，pH7.5，25℃，提取2h。
【实施例3】重组瑞替普酶的离子交换层析介质初级纯化
1.初级纯化离子交换层析介质的选择
根据实施例2中确定的提取条件制备含重组瑞替普酶的粗提物，然后采用Ep管离心捕获法进行离子交换层析填料的初筛，上述离子交换层析填料包括NanoGel 50sp，NanoQ 30L，NanoGel 50Q，UniGel 30Q，UniGel 80Q，Unigel MMC 50s和Unigel MMA 50s；通过设计不同的磷酸盐缓冲液上样电导和pH，以及NaCl盐浓度梯度洗脱，结果显示，NanoGel 50sp填料(pH 6.0和6.5，电导15.56ms/cm)可以结合重组瑞替普酶，WB显示重组瑞替普酶在pH6.0～6.5 30～40ms/cm和pH7.0～7.5 20～30ms/cm可以洗脱(图8A)；阴离子交换填料NanoQ 30L，NanoGel 50Q，UniGel 30Q，UniGel 80Q在重组瑞替普酶的粗提物稀释1倍后上样10CV和20CV都穿透，稀释4倍后上样5CV部分穿透，上样20CV大量穿透(图8B)，说明阴离子交换填料对于提取液的载量小于5CV，不适合作为第一步捕获层析填料；Unigel MMC 50s在pH6.5 100～500mM NaCl上样没有穿透，但是重组瑞替普酶主要在0.5M NaOH中洗脱，Unigel MMA 50s填料在pH7.5和pH8.0含250mM NaCl磷酸盐缓冲液上样部分穿透，含100mM NaCl磷酸盐缓冲液上样没有穿透，重组瑞替普酶在pH6.5 20mM PB 30～80mS/cm可以洗脱(图8C)。
2.初级纯化阳离子交换填料NanoGel 50sp层析优化
根据实施例2中确定的提取条件制备含重组瑞替普酶的粗提物，利用20mM磷酸盐缓冲液，加入不同浓度的氯化钠，设计pH为6.5的不同的上样电导16，17和18mS/cm，洗杂电导18，20和25mS/cm及洗脱电导32和40mS/cm，检测结果显示，Nanogel 50sp上样电导为18mS/cm时，穿透液检测到微量瑞替普酶(图9A)，电导在16～17mS/cm时瑞替普酶未穿透(图9B)，说明上样电导需要控制在17mS/cm以下；洗杂电导由18mS/cm增加至20mS/cm时，洗杂液检测到轻微瑞替普酶，当洗杂电导增加25mS/cm时，大部分瑞替普酶洗脱下来(图9C)，表明洗杂电导不能超过20mS/cm；瑞替普酶在25mS/cm基本可以完全洗脱，32mS/cm，40mS/cm，1M NaCl均能检测到少量的瑞替普酶(图9D)，确定洗脱电导范围为25～40mS/cm。
3.初级纯化阳离子交换填料NanoGel 50sp层析载量确认
根据实施例2中确定的提取条件制备含重组瑞替普酶的粗提物，加入20mM磷酸盐缓冲液，使上样缓冲液的电导为16mS/cm后，分别上样100CV，75CV和60CV(稀释前的体积)，检测结果显示Nanogel 50sp上样100CV，穿透液中检测到微量的瑞替普酶，洗杂液中也检测到部分瑞替普酶(图10A)，需降低上 样量；上样75CV，穿透液中没有瑞替普酶，但是洗杂液中仍有少量瑞替普酶(图10B)，继续降低上样载量至60CV，无目的蛋白穿透，洗脱液E1(25.3ms/cm)，E2(28.1ms/cm)，E3(32.5ms/cm)中都能检测到瑞替普酶，但E3洗脱组分的杂蛋白(50kD杂带)占主要成分(图10C)，因此，确定Nanogel 50sp上样载量为60CV，优化洗脱条件为pH6.5，电导为28～30ms/cm。
【实施例4】重组瑞替普酶的疏水层析介质中级纯化
1.中级纯化疏水层析介质的选择
根据实施例2和3确定的提取和阳离子层析条件制备重组瑞替普酶初级产物I，初级产物I中添加硫酸铵，使上样液的硫酸铵浓度为0.6M，0.7M和0.8M，分别进行5mL UniHR Phenyl 80L LS，UniHR Phenyl 30L，BGL Octyl 4FF，BGL Phenyl low sub填料层析，结果显示BGL Phenyl low sub以及Octyl 4FF分离效果较差，UniHRPhenyl 30L分离效果与UniHRPhenyl 80L LS相当，但UniHRPhenyl 80L LS和UniHRPhenyl 30L的NaOH洗脱组分均有一点疑似目的蛋白的条带(图11)。
2.中级纯化疏水填料UniHR Phenyl 30L层析条件优化
根据实施例2和3确定的提取和阳离子层析条件制备重组瑞替普酶初级产物I，初级产物I中添加硫酸铵，使上样液的硫酸铵浓度为0.4M，在0.5％甘油体系进行盐浓度梯度洗脱，结果显示在0.5％甘油体系下，瑞替普酶的降解带，缺氨基酸蛋白，糖基化蛋白和非糖基化蛋白具有一定的分离度(图12A)，最终确定0.5％甘油体系下，磷酸盐缓冲液电导为40～45mS/cm洗杂效果较好，15mS/cm洗脱效果较好(图12B)。
【实施例5】重组瑞替普酶的亲和层析介质末级纯化
1.末级亲和纯化层析介质的选择
根据实施例2，3和4确定的提取，阳离子交换层析和疏水层析条件制备重组瑞替普酶中级产物II，将中级产物II的pH调整为7.5，加入不同浓度的氯化钠(0.1M，0.5M，1M)，分别进行5mL纳微苯甲脒-4FF(072J)低配基，(191J)高配基，BGL Benzamidine Bestarose 4FF亲和填料层析，结果如下：
  0.1M NaCl 0.5M NaCl 1M NaCl
纳微苯甲脒-4FF(072J) 不结合 不结合 部分结合
纳微苯甲脒-4FF(191J) 不结合 结合 结合
BGL Benzamidine Bestarose4FF 结合 结合 结合
说明BGL Benzamidine Bestarose 4FF与瑞替普酶结合力最强，选择BGL Benzamidine Bestarose 4FF进行后续层析优化。
2.末级亲和纯化介质BGL Benzamidine Bestarose 4FF层析条件优化
根据实施例2～4确定的提取，阳离子交换层析和疏水层析条件制备重组瑞替普酶中级产物II，将中级产物II的pH调整为7.5，作为BGL Benzamidine Beatarose 4FF的上样液，采用20mM磷酸缓冲液不同的pH(6.5，6.2，6.0)和不同的电导进行洗杂优化，结果显示20mM磷酸缓冲液，pH6.0，电导1.6mS/cm具有较好的洗杂效果(图13A)；采用不同pH(5.5，5.0)的缓冲液和电导进行洗脱优化，结果显示20mM磷酸缓冲液，20mM醋酸盐缓冲液，pH5.0，电导3.0mS/cm具有较好的洗脱效果(图13B)
【实施例6】重组瑞替普酶3批小试工艺验证
根据实施例2～5确定的最优提取、初级产物I纯化，中级产物II纯化及末级产物III纯化条件，进行3批小试工艺验证，验证过程及验证结果如下所述。1.重组瑞替普酶粗提物制备(提取)
称取350g重组瑞替普酶米粉(775-214-17-3)，加入1.75L 20mM PB，500mM NaCl，pH 7.5提取液后，25℃提取2h；提取完成后，加入2％～5％(w/v)助滤剂，采用正压过滤的方式进行压滤。滤液加入2.5倍体积的20mM PB pH6.5样品稀释液，调整pH为6.5，0.22μm滤膜过滤后即为重组瑞替普酶粗提物(NanoGel50sp层析上样液)。
2.重组瑞替普酶初级产物I纯化
采用4-6倍柱体积(CV)的pH为6.5的20mM磷酸盐和130mM氯化钠缓冲液，以200～250cm/h的线性流速平衡填充有NanoGel 50sp，装柱高度10cm的XK16/20层析柱，以上述重组瑞替普酶粗提物作为上样液进行上样，上样体积为221CV；采用pH为6.5的20mM磷酸盐和180mM氯化钠缓冲液，以200～250cm/h的流速进行40CV杂质蛋白洗脱；采用pH为6.5的20mM磷酸盐和280mM氯化钠缓冲液，以200～250cm/h的流速进行50CV重组瑞替普酶的洗脱，收集富含重组瑞替普酶的洗脱液，获得含重组瑞替普酶的初级产物I；
3.重组瑞替普酶中级产物II纯化
采用5～10CV的pH6.5，20mM磷酸盐和0.37M硫酸铵缓冲液，以350～400cm/h的流速平衡填充有UniHR Phenyl 30L，装柱高度20cm的XK16/40 层析柱；用3M硫酸铵调节重组瑞替普酶初级产物I的电导为59～61mS/cm，pH调整为6.5，作为上样液，全部上样；采用pH6.5，含0.5％甘油的20mM磷酸盐和260mM硫酸铵的缓冲液进行20CV杂质蛋白洗脱；采用pH6.5，含0.5％甘油的20mM磷酸盐和68mM硫酸铵的缓冲液进行20CV重组瑞替普酶的洗脱，收集富含重组瑞替普酶的洗脱液，获得含重组瑞替普酶的中级产物II；
4.重组瑞替普酶末级产物III纯化
采用5～10CV的pH7.5，20mM磷酸盐和68mM硫酸铵缓冲液，以350～400cm/h的流速平衡填充有BGL Benzamidine 4FF，装柱高度20cm的C10/40层析柱；将中级产物II的pH调整为7.5，作为上样液，全部上样；采用pH6.0的20mM磷酸盐缓冲液进行10CV杂质蛋白洗脱；采用pH5.0的20mM磷酸盐和20mM醋酸盐缓冲液进行5CV重组瑞替普酶的洗脱，收集富含重组瑞替普酶的洗脱液，获得纯化的重组瑞替普酶(末级产物III)。
结果显示，层析图谱如图14所示，3批小试层析图谱基本一致，SDS-PAGE纯度检测结果如图15所示，非还原样品纯度大于99％，RP-HPLC纯度检测结果如图16所示，纯度大于98％，蛋白平均得率为21.5mg/kg米粉，蛋白平均比活性为4.92x10 5IU/mg。

Claims (15)

  1. 一种以基因工程水稻表达和制备重组瑞替普酶的方法，依次包括以下步骤：
    (1)构建在水稻种子中表达重组瑞替普酶的植物表达载体，所述载体含有经水稻密码子优化的瑞替普酶基因；
    (2)用步骤(1)所述的表达载体遗传转化水稻，获得基因工程水稻，栽培后获得重组瑞替普酶基因工程水稻种子；
    (3)从步骤(2)所述的重组瑞替普酶基因工程水稻种子中提取含重组瑞替普酶的粗提取物；
    (4)将步骤(3)获得的含重组瑞替普酶的粗提取物经阳离子交换层析，得到初级产物I；
    (5)将步骤(4)获得的初级产物I经疏水层析，得到含重组瑞替普酶的中级产物II；
    (6)将步骤(5)获得中级产物II经苯甲脒亲和层析，得到纯化的重组瑞替普酶。
  2. 根据权利要求1所述的方法，其中所述植物表达载体是将如SEQ ID NO.1所示的经水稻密码子优化的瑞替普酶基因，与水稻特异性启动子Gt13a及其信号肽导入质粒载体构建。
  3. 根据权利要求1所述的方法，其特征在于，步骤(3)所述重组瑞替普酶粗提物的制备方法包括：
    3a)将步骤(2)获得的重组瑞替普酶基因工程水稻种子晒干脱壳，加工成半精米，研磨成80～100目的米粉；
    3b)将上述米粉与提取液按照1：5(重量/体积，kg/L)的比例混匀，在24～26℃提取2～3小时，获得提取混合物；所述提取缓冲液的成分为：20mM磷酸盐缓冲液，500mM氯化钠，pH7.5；
    3c)将步骤(2)的提取混合物加入2％-5％的珍珠岩压滤，滤液再经0.22um滤膜过滤后即为重组瑞替普酶的粗提取物。
  4. 根据权利要求1所述的方法，其特征在于，步骤(3)所述重组瑞替普酶初级产物I离子交换层析介质选自NanoGel 50sp，NanoQ 30L，NanoGel 50Q，UniGel 30Q，UniGel 80Q，Unigel MMC 50s和Unigel MMA 50s。
  5. 根据权利要求4所述的方法，其特征在于所述离子交换层析的介质为NanoGel 50sp。
  6. 根据权利要求1所述的方法，其特征在于，步骤(4)所述重组瑞替普酶初级产物I通过下述方法制备：
    4a)采用4-6倍柱体积(CV)的pH为6.5的20mM磷酸盐和130mM氯化钠缓冲液，以200～250cm/h的线性流速平衡NanoGel 50sp层析柱；
    4b)将步骤(3)获得的重组瑞替普酶粗提取物与pH6.5的20mM磷酸盐缓冲液按照体积比1：2.5混匀稀释，0.22μm滤膜过滤后作为上样液，上样液电导为14～16mS/cm，其中上样液pH为6.4～6.6，上样体积不超过221CV；
    4c)采用pH为6.5的20mM磷酸盐和180mM氯化钠缓冲液，以200～250cm/h的流速进行40CV杂质蛋白洗脱，洗杂缓冲液电导为20mS/cm；
    4d)采用pH为6.5的20mM磷酸盐和280mM氯化钠缓冲液，以200～250cm/h的流速进行50CV重组瑞替普酶的洗脱，洗脱缓冲液电导为28～32mS/cm，收集富含重组瑞替普酶的洗脱液，获得含重组瑞替普酶的初级产物I。
  7. 根据权利要求1所述的方法，其特征在于，步骤(5)所述重组瑞替普酶中级产物II疏水层析介质选自UniHR Phenyl 80L LS，UniHR Phenyl 30L，BGL Octyl 4FF和BGL Phenyl low sub。
  8. 根据权利要求7所述的方法，其特征在于所述疏水层析介质为UniHR Phenyl 30L。
  9. 根据权利要求1所述的方法，其特征在于，步骤(5)所述重组瑞替普酶中级产物II是通过下述方法制备：
    5a)采用5～10CV的pH6.5，20mM磷酸盐和0.37M硫酸铵缓冲液，以350～400cm/h的流速平衡UniHR Phenyl 30L层析柱；
    5b)用3M硫酸铵调节重组瑞替普酶初级产物I的电导为59～61mS/cm，pH调整为6.5，作为上样液，上样液电导为60mS/cm，全部上样；
    5c)采用pH6.5，含0.5％甘油的20mM磷酸盐和260mM硫酸铵的缓冲液进行20CV杂质蛋白洗脱，洗杂缓冲液电导为45mS/cm；
    5d)采用pH6.5，含0.5％甘油的20mM磷酸盐和68mM硫酸铵的缓冲液进行20CV重组瑞替普酶的洗脱，洗脱缓冲液电导为15mS/cm，收集富含重组瑞 替普酶的洗脱液，获得含重组瑞替普酶的中级产物II。
  10. 根据权利要求1所述的方法，其特征在于，步骤(6)所述苯甲脒亲和层析的层析介质选自纳微Benzamidine 4FF(072J低配基和191J高配基)和博格隆Benzamidine Bestarose 4FF。
  11. 根据权利要求10所述的方法，其特征在于所述苯甲脒亲和层析介质为博格隆Benzamidine Bestarose 4FF。
  12. 根据权利要求1所述的方法，其特征在于，步骤(6)所述纯化的重组瑞替普酶是通过下述方法制备：
    6a)采用5～10CV的pH7.5，20mM磷酸盐和68mM硫酸铵缓冲液，以350～400cm/h的流速平衡博格隆Benzamidine Bestarose 4FF层析柱；
    6b)将中级产物II的pH调整为7.5，作为上样液，全部上样；
    6c)采用pH6.0的20mM磷酸盐缓冲液进行10CV杂质蛋白洗脱；
    6d)采用pH4.8～5.2的20mM磷酸盐和20mM醋酸盐缓冲液进行5CV重组瑞替普酶的洗脱，洗脱缓冲液的pH为5.0，收集富含重组瑞替普酶的洗脱液，获得纯化的重组瑞替普酶。
  13. 一种重组植物表达载体，通过将经水稻密码子优化的瑞替普酶基因，水稻特异性启动子Gt13a及其信号肽导入质粒载体构建。
  14. 根据权利要求13所述的植物表达载体，其特征在于所述经水稻密码子优化的瑞替普酶基因的核苷酸序列如SEQ ID NO.1所示。
  15. 一种根据权利要求1～12任一项所述方法制备的重组瑞替普酶。
PCT/CN2022/101339 2021-06-30 2022-06-24 一种以基因工程水稻表达和制备重组瑞替普酶的方法 WO2023274091A1 (zh)

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