WO2022089340A1 - 一种利用基因工程水稻表达人糜蛋白酶原和制备重组人糜蛋白酶的方法 - Google Patents

一种利用基因工程水稻表达人糜蛋白酶原和制备重组人糜蛋白酶的方法 Download PDF

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WO2022089340A1
WO2022089340A1 PCT/CN2021/125969 CN2021125969W WO2022089340A1 WO 2022089340 A1 WO2022089340 A1 WO 2022089340A1 CN 2021125969 W CN2021125969 W CN 2021125969W WO 2022089340 A1 WO2022089340 A1 WO 2022089340A1
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recombinant human
chymotrypsinogen
rice
chymotrypsin
genetically engineered
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French (fr)
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杨代常
董亮亮
李坤鹏
尹恒
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武汉禾元生物科技股份有限公司
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    • 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)
    • C12N9/6427—Chymotrypsins (3.4.21.1; 3.4.21.2); Trypsin (3.4.21.4)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
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    • 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
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    • C12N15/09—Recombinant DNA-technology
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    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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    • 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
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    • C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host

Definitions

  • the invention relates to the field of biotechnology, in particular to a method for expressing human chymotrypsinogen and preparing recombinant human chymotrypsin by using genetically engineered rice as a bioreactor.
  • Chymotrypsin also called chymotrypsin, belongs to the family of serine proteases and can specifically hydrolyze the peptide bonds formed by the carboxyl groups of aromatic amino acids. Chymotrypsinogen is secreted by the pancreas, and after entering the small intestine with pancreatic juice, it is cleaved by trypsin between Arg15 and Ile16 (numbered by Bovine chymotrypsinA sequence) into two parts connected by disulfide bonds, and then chymotrypsin cleaves the short peptide Serl4 at Leu13 -Argl5, cleaves the short peptide Tyr147-Asn148 at Tyrl46 and Asn148 to form three polypeptide chains linked by disulfide bonds and has biological activity.
  • Chymotrypsin has a wide range of clinical uses. Its pharmacological action is to decompose proteins and promote the removal of blood clots, purulent secretions and necrotic tissue. It is mainly used in ophthalmic surgery to relax the ciliary ligament and relieve traumatic iridocyclitis. ; Can also be used for wounds or local inflammation to reduce local secretion and edema.
  • chymotrypsin can digest various proteins, when expressed in other expression systems, it will cause the degradation of the life-sustaining proteins of the host cells, resulting in low expression levels of the existing expression systems. Therefore, the existing chymotrypsin preparation is still extracted from the pancreas of bovine or porcine. After graded salting out, chymotrypsinogen is first prepared, and after activation by trypsin, it is obtained by recrystallization and purification with ammonium sulfate and ethanol. This process has poor extraction specificity, long operation time and low yield. The amino acid sequence of chymotrypsin extracted from bovine or porcine pancreas is different from that of human chymotrypsin.
  • the amino acid sequence of bovine chymotrypsin is 82% homologous to human chymotrypsin, and the amino acid sequence of porcine chymotrypsin is the same as that of human chymotrypsin.
  • the homology of proteases is only 43%.
  • the existing animal-derived chymotrypsin not only has the risk of introducing animal-derived viruses, but also may cause an immune response after the heterologous protein enters the human body, which greatly increases the safety risk of drug use.
  • Human chymotrypsinogen is a peptide chain containing 263 amino acids. During the activation process in vivo, it first reaches the extracellular space through the signal peptide. After the signal peptide is hydrolyzed by peptidase, the number of amino acids contained in the protein molecule becomes 245, namely chymotrypsinogen. When chymotrypsinogen is transported to the small intestine, it is activated by trypsin to form the active form.
  • Animal-free and humanized biological products are the current development trend in the biopharmaceutical industry. From the first animal-free recombinant human insulin extracted from animal pancreas in 1982, to the first plasma-free recombinant thrombin in 2008, to the first animal-free plant-derived recombinant in 2020 Human serum albumin has completed a phase 1 clinical study in the United States. The development of animal-free and humanized chymotrypsin is in line with the development trend of biological drugs.
  • Patent CN104342423A discloses a preparation method and application of a highly active recombinant human chymotrypsin, the method is to use Escherichia coli to express the inclusion body of recombinant human chymotrypsin. Pure recombinant human chymotrypsin.
  • the invention utilizes a rice endosperm cell bioreactor to express recombinant human chymotrypsin efficiently, extracts and purifies recombinant human chymotrypsin from genetically engineered rice, and obtains biologically active recombinant human chymotrypsin through activation.
  • An object of the present invention is to provide a method for expressing recombinant human chymotrypsinogen in genetically engineered rice, comprising the following steps:
  • step (3) transforming the vector obtained in step (2) into the callus regeneration tissue of a rice variety
  • the recombinant human chymotrypsinogen expression vector preferably has the structure shown in FIG. 3 .
  • Another object of the present invention is to provide a method for preparing recombinant human chymotrypsin from genetically engineered rice of recombinant human chymotrypsinogen, comprising the following steps:
  • the method for extracting and purifying recombinant human chymotrypsinogen comprises the following steps:
  • the purified recombinant human chymotrypsinogen is activated by trypsin and freeze-dried to obtain recombinant human chymotrypsin.
  • the method for extracting and purifying recombinant human chymotrypsinogen comprises the following steps:
  • the genetically engineered rice of recombinant human chymotrypsinogen is dehulled and processed into semi-finished rice, which is ground into 80-100 mesh rice flour.
  • the components of the extraction buffer are: 4.44-69.66 g/L disodium hydrogen phosphate dodecahydrate, 0.58-19.71 g/L citric acid monohydrate, pH 2.5-8.0.
  • the cationic chromatography medium includes SPBestrose FF (Borgron (Shanghai) Biotechnology Co., Ltd.), Unigel 80SP (Suzhou Nano Micro Technology Co., Ltd.), Unigel 80CM (Suzhou Nano Technology Co., Ltd.) Micro Technology Co., Ltd.), Unigel MMC 50S (Suzhou Nano Micro Technology Co., Ltd.).
  • SPBestrose FF Boss Land (Borgron (Shanghai) Biotechnology Co., Ltd.)
  • Unigel 80SP Sazhou Nano Micro Technology Co., Ltd.
  • Unigel 80CM Suzhou Nano Technology Co., Ltd.
  • Micro Technology Co., Ltd. Micro Technology Co.
  • Unigel MMC 50S Suzhou Nano Micro Technology Co., Ltd.
  • Unigel 80SP chromatography medium is used.
  • the chromatographic column is equilibrated with a linear flow rate of 300-600 cm/h using 4-6 column volumes (CV) of a buffer solution of disodium hydrogen phosphate-citric acid with a pH of 3.0-3.5;
  • the crude extract in step 1) is a sample solution, wherein the pH of the sample solution is 3.0-3.5, and the sample volume does not exceed 74 CV; disodium hydrogen phosphate-citric acid buffer solution with 5-10 CV and pH of 3.5-4.0 is used, Carry out the elution of impurity protein at a linear flow rate of 300-600cm/h; use disodium hydrogen phosphate-citric acid buffer solution containing 100-200mM sodium chloride with pH 3.9-4.1 at a linear flow rate of 300-600cm/h Elution of recombinant human chymotrypsinogen is performed, and the eluate containing recombinant human chymotrypsinogen is collected to obtain a primary product containing recombin
  • Anion chromatography media include Unigel80Q (Suzhou Nano Micro Technology Co., Ltd.), NanoGel 50Q (Suzhou Nano Micro Technology Co., Ltd.), MMA-50S (Suzhou Nano Micro Technology Co., Ltd.), MMA-50S (Suzhou Nano Micro Technology Co., Ltd.) Co., Ltd.), Q Bestrose FF (Borgron (Shanghai) Biotechnology Co., Ltd.), Unigel 30Q (Suzhou Nanowei Technology Co., Ltd.), Q Bestrose HP (Borgron (Shanghai) Biotechnology Co., Ltd.).
  • Unigel 80Q chromatography medium is used.
  • the method for activating recombinant human chymotrypsinogen comprises the following steps:
  • step 2 The recombinant human chymotrypsin in step 2 is appropriately concentrated with a 5-10kDa ultrafiltration membrane package (polyethersulfone material), and dialyzed with ultrapure water until the conductance is reduced to below 0.5mS/cm, and the pH is adjusted to 5.5-6.5. Plated and freeze-dried.
  • a 5-10kDa ultrafiltration membrane package polyethersulfone material
  • the present invention also provides recombinant human chymotrypsinogen expressed by rice endosperm cells according to the method of the present invention.
  • the present invention relies on two dominant platforms—rice endosperm cell protein expression platform (Oryz HiExp ) and protein purification platform (Oryz Pur ), uses rice endosperm cells to efficiently express recombinant human chymotrypsinogen, and can obtain SEC after extraction and two-step chromatography - Recombinant human chymotrypsinogen with HPLC purity higher than 99%. After the recombinant human chymotrypsinogen is activated, the recombinant human chymotrypsin is obtained, and its activity can reach more than 1500U/mg, which is higher than the standard of 1000U/mg in the 2020 edition of the Chinese Pharmacopoeia. Compared with the traditional production process, the production process of the present invention has no animal-derived materials, simpler operation, shorter production time, higher safety, and environmental friendliness, has important application prospects, and conforms to the development trend of biopharmaceuticals in the future.
  • Figure 1 Schematic diagram of the pOsPMP799 plasmid structure.
  • Figure 2 Schematic diagram of the pOsPMP800 plasmid structure.
  • FIG. 1 Schematic diagram of the pOsPMP801 plasmid structure.
  • Figure 4 PCR detection of target genes in some genetically engineered rice plants.
  • M is the DNA standard molecular weight Marker
  • 1-23 are different plants of the T1 generation genetically engineered materials
  • P is the positive control plasmid
  • the red arrow points to the target gene.
  • Figure 5 Detection of rice expression in some lines of genetically engineered rice.
  • M is the DNA standard molecular weight Marker
  • 2, 7, 8, 9, 16, and 20 represent different lines in the breeding process of genetically engineered rice, respectively
  • the red arrow points to the target protein.
  • FIG. 8 SDS-PAGE detection results of Unigel 80SP, Unigel 80CM and Unigel MMC 50S chromatographic elution fractions.
  • A is the SDS-PAGE detection result of Unigel80SP chromatography elution fraction;
  • B is the SDS-PAGE detection result of Unigel80CM chromatography elution fraction;
  • C is the SDS-PAGE detection result of Unigel50S chromatography elution fraction;
  • M is the standard molecular weight Marker; Wash means washing impurities; CIP1 and CIP2 mean regeneration 1 and regeneration 2, respectively;
  • FT2 is the permeate of the trailing part during re-equilibration.
  • Fig. 10 SDS-PAGE detection results of different salt concentration washing chromatography components at pH 4.0.
  • M is the standard molecular weight Marker
  • W1-1, W1-2 are the wash-off peaks collected in sections
  • Elu is the elution collection solution
  • CIP is the regeneration solution.
  • FT is the permeate; 25, 50, 75, 100, and 150 represent different elution fractions containing mM sodium chloride, respectively; the red arrows indicate the target protein.
  • M is the standard molecular weight marker
  • L is the loading solution
  • FT is the penetration solution
  • W is the impurity washing solution
  • Elu is the elution collection solution
  • CIP is the regeneration solution
  • Ethanol non-reduction means that ⁇ -mercaptoethanol is not added during sample preparation.
  • FIG. 13 Unigel 80Q chromatography different elution conditions chromatographic collection liquid electrophoresis detection. Among them, 1 represents the elution collection solution of pH 6.5 and 50 mM sodium chloride in the reducing state; 2 represents the elution and collection solution of pH 6.5 and 50 mM sodium chloride in the non-reducing state; 3 represents the elution and collection solution of pH 7.0 and 50 mM sodium chloride in the reducing state 4 means pH 7.0, 25mM sodium chloride eluted collection solution in reducing state; 5 means pH 7.5, 50mM sodium chloride eluting collection solution in reducing state; 6 means pH 8.0, 50mM sodium chloride eluting solution in reducing state Collection fluid.
  • 1 represents the elution collection solution of pH 6.5 and 50 mM sodium chloride in the reducing state
  • 2 represents the elution and collection solution of pH 6.5 and 50 mM sodium chloride in the non-reducing state
  • 3 represents the elution and collection
  • Figure 14 The chromatograms of the three batches of the pilot process. Wherein A is the chromatogram of Unigel80SP, and B is the chromatogram of Unigel 80Q; 20200804, 20200805, and 20200806 represent different production batch numbers, respectively.
  • FIG. 15 Three batches of small-scale electrophoresis detection results (reduction), where M is the standard molecular weight Marker; 1 represents batch 20200804, 2 represents batch 20200805, and 3 represents batch 20200806; reduction means adding reducing agent ⁇ -mercaptoethanol during sample preparation.
  • the rice-specific promoter Gtl3a and its signal peptide were used to mediate the expression of recombinant human chymotrypsinogen gene in rice endosperm cells.
  • human chymotrypsin gene sequence Genbank accession number: NP_001897.4
  • Nanjing GenScript Biotechnology Co., Ltd. was entrusted to synthesize according to the genetic code preferred by rice, as shown in SEQID NO.
  • the nucleotide changes of 15%, the codon changes 35%, but the corresponding amino acid sequence does not change, the constructed plasmid is pOsPMP799 ( Figure 1).
  • a rice-specific promoter and its signal peptide were used to mediate the expression of human chymotrypsinogen gene in rice endosperm cells.
  • the synthetic codon-optimized human chymotrypsinogen gene (SEQ ID NO.
  • the pOsPMP801 plasmid was transformed into Agrobacterium tumefaciens EHA105 (Invitrogen, USA), and POsPMP801 was transformed into the callus regeneration tissue of rice variety TP309 through Agrobacterium tumefaciens-mediated transformation, and complete plants were formed after culture, selection and induction.
  • the specific method is as follows:
  • the Agrobacterium containing the expression vector pOsPMP801 was expanded. Spread the bacteria on karamycin-resistant plates and cultivate in a 28°C incubator for 2-3 days.
  • a single colony of Agrobacterium was picked with an inoculation loop and picked into a suspension medium (AAM liquid medium), and cultured at 28° C. with shaking (160 rpm). Generally, 100mL of medium can be scraped into 3 to 4 loops with an inoculation loop.
  • HPT-resistant calli were transferred to differentiation medium (N6 medium), and cultured at 26°C in light for 20-30 days.
  • the differentiated seedlings were selected from the differentiation medium and transferred to MS medium containing 1/2 for rooting. After culturing in the light at 28°C for 30 days, they were transferred to the field for growth.
  • Extract genomic DNA from leaves at the seedling stage using human chymotrypsinogen forward primer Gtl3a-F (SEQ ID NO.2:5'-CACATCCATCATTATCCATCCACC-3') and reverse primer CT-R (SEQ ID NO.3:5') -ACTTCGGGTTCTTGAACACCTT-3') was amplified by PCR, the theoretical size of the product was 547bp, and the genetically engineered plant containing the target gene was identified by PCR.
  • the PCR detection method was as follows:
  • the PCR amplification reaction system contains 2.5 ⁇ L of 10 ⁇ Buffer; 0.15 ⁇ L of 5U/ ⁇ L rTaq enzyme, 4 ⁇ L of 2.5 mM dNTP, and 0.5 ⁇ L of forward and reverse primers; add ddH 2 O to 25 ⁇ L.
  • the obtained recombinant human chymotrypsinogen-positive seedlings were transplanted into a greenhouse to grow to maturity, and a single plant that could normally bear fruit was harvested. Since there is currently no commercially available detection kit for quantitatively detecting the content of recombinant human chymotrypsinogen, high-expressing individual plants were preliminarily screened by SDS-PAGE, and negative individual plants were eliminated. The highly expressed single plant was continued to be planted as a candidate single plant, and the expression level comparison and homozygous screening were performed again until a homozygous line with stable and high expression of recombinant human chymotrypsinogen was obtained.
  • Figure 5 shows the results of SDS-PAGE detection of the expression levels of some recombinant human chymotrypsinogen in rice. According to preliminary estimation, the highest expression level of recombinant human chymotrypsin can reach more than 2g/kg brown rice.
  • the genetically engineered rice is hulled and processed into semi-finished rice, which is ground into 80-100 mesh rice flour.
  • the rice flour was mixed with disodium hydrogen phosphate-citric acid extraction buffers of different pH in a ratio of 1:5 (weight/volume, kg/L), and the extraction was carried out according to the extraction conditions designed in Table 1. After the extraction, the supernatant was taken for SDS-PAGE detection. The results showed that under each extraction condition, there was no significant difference in the amount of target protein extracted in the extract (see Figure 6).
  • the extraction conditions were further optimized.
  • the genetically engineered rice is hulled and processed into semi-finished rice, which is ground into 80-100 mesh rice flour.
  • the rice flour was mixed at a ratio of 1:5 (weight/volume, kg/L) with disodium hydrogen phosphate-citric acid extraction buffer at pH 2.5, pH 3.0 and pH 3.5, respectively, and then placed at 25 °C for extraction for 1 h. .
  • the extraction buffer with pH 3.0 was designed to be stirred for 1 h, 2 h and 4 h at 20°C, 25°C and 30°C, respectively. After the extraction, centrifugation was performed, and the supernatant was taken for SDS-PAGE detection.
  • the crude extracts containing recombinant human chymotrypsinogen were prepared according to the extraction conditions determined in Example 2, and then loaded onto chromatographic columns packed with Unigel 80SP, Unigel 80CM, and Unigel MMC 50S chromatographic media, followed by phosphoric acid with different pH. Gradient elution was performed with disodium hydrogen-citrate buffer. The results show that the above three chromatographic media, in the pH gradient elution, the elution peak tailing is more serious, and the resolution is poor.
  • the electrophoresis detection results are shown in Figure 8. Unigel 80CM penetrates significantly (Figure 8B), that is, the loading of the sample for 20 CV is overloaded.
  • the crude extract (ie the loading solution) of recombinant human chymotrypsinogen was prepared according to the extraction conditions determined in Example 2.
  • the crude extract of recombinant human chymotrypsinogen was loaded (0.5mL/min flow rate) into a 1mL Unigel80SP chromatography column.
  • a distribution collector was used to automatically collect the permeate at about 2mL/tube.
  • the crude extract of recombinant human chymotrypsinogen was prepared according to the extraction conditions determined in Example 2. 50 mL (50 CV) of crude recombinant human chymotrypsinogen extract was loaded into 1 mL of Unigel 80SP chromatography medium, followed by gradient elution with pH 4.0 containing 0-200 mM sodium chloride in disodium hydrogen phosphate-citric acid buffer . The results are shown in Figure 10. Under the conditions of pH4.0 and 0mM sodium chloride washing, a small amount of target protein was eluted. When the salt concentration was increased to 30mM sodium chloride, the target protein was obviously washed from the chromatography medium. take off.
  • the target protein is easily eluted, and the washing space is very small.
  • pH 4.0, 30mM sodium chloride washes impurities more loss of target protein, and there is a tailing phenomenon
  • pH 4.0, 0mM sodium chloride washes impurities although there is a trace loss of target protein, but more impurity proteins can be removed
  • the washing peak and the subsequent elution peak have a certain degree of separation, therefore, the washing conditions are determined to be pH4.0, 0mM sodium chloride.
  • pH4.0 100mM sodium chloride elution (collection volume 27CV) and pH 4.0, 150mM sodium chloride elution (collection volume 25CV) have no significant difference in the purity of the target protein between the two elution fractions, the elution conditions were determined. It is pH 4.0, 100-150 mM sodium chloride, preferably pH 4.0, 150 mM sodium chloride elution (the elution volume is relatively small).
  • the primary product of recombinant human chymotrypsinogen was prepared according to the extraction and cation chromatography conditions determined in Examples 2 and 3. Since the first step of primary purification used a cationic medium, the second step of refinement was to use an anionic medium.
  • the primary product is properly concentrated with a 5-10kDa ultrafiltration membrane package, and then dialyzed with a pH 7.9-8.1, conductance 1.5-2.0mS/cm disodium hydrogen phosphate-citric acid buffer or phosphate buffer solution until the conductance is 1.5- 2.5mS/cm, adjust the pH to 7.9-8.1, as the loading solution of the second step chromatography.
  • the pretreated sample solution in the first step was loaded into a chromatography column filled with 1 mL of anion chromatography media Unigel 80Q, NanoGel 50Q, MMA-50S, and Q Bestrose FF at a flow rate of 0.5 mL/min, and then used Sodium chloride gradient elution.
  • the results are shown in Figure 11.
  • the target protein is weakly bound to the Q series chromatography medium. Under the lower conductance (25-50mM sodium chloride), the target protein is gradually eluted, and the elution peak tailing phenomenon is serious.
  • Q BestroseFF 25mM sodium chloride can elute some impurities, but the target protein cannot be shed.
  • the results showed that the purity of the target protein in the final eluate was basically the same regardless of the anion chromatography medium ( Figure 11).
  • Unigel 80Q is preferred as the second-step chromatography medium.
  • Example 4 the chromatography loading solution of Example 4 was prepared, and then the loading volume of 40 mL (40 CV) was applied to a 1 mL Unigel 80Q chromatographic column, and then with pH 7.9 to 8.1, Elution was performed with 50 mM sodium chloride in disodium hydrogen phosphate-citric acid (or 10 mM phosphate buffer) buffer.
  • the results are shown in Figure 12, the eluate of Unigel 80Q showed three protein bands in the reducing state, but a single band in the non-reducing condition. After analysis, it may be that recombinant human chymotrypsinogen occurs self-activation in the process of protein expression, transport and storage.
  • the Unigel 80Q chromatography elution conditions of recombinant human chymotrypsin were as follows: 10 mM phosphate buffer, pH 6.5-8.0, 2-50 mM sodium chloride.
  • the optimal activation conditions for recombinant human chymotrypsin in combination with Example 6 are: recombinant human chymotrypsinogen concentration 1 mg/mL, pH 8.0.
  • Ungel 80Q The optimal elution conditions of Ungel 80Q are 10 mM phosphate buffer, pH 8.0, 50 mM sodium chloride (the protein content of the collected solution under this elution condition is about 1 mg/mL, and the collected solution does not need to adjust pH, and can be directly used for activation ).
  • Unigel 80SP chromatographic collection solution is properly concentrated with a 5 ⁇ 10kDa ultrafiltration membrane bag, and used pH 7.9-8.1, conductance 1.5 ⁇ 2.0 mS/cm disodium hydrogen phosphate-citric acid buffer (or 10mM phosphate buffer at the same pH) was dialyzed until the conductance decreased to 1.5-2.5mS/cm, and the pH was adjusted to 7.9-8.1, as the upper part of this step of chromatography.
  • Sample solution eluted with a buffer solution with pH of 7.9 ⁇ 8.1, containing 50mM sodium chloride, 20.41g/L sodium hydrogen phosphate dodecahydrate, 15.02g/L citric acid monohydrate at a flow rate of 382 ⁇ 840cm/h , and collect the eluate containing recombinant human chymotrypsinogen to obtain recombinant human chymotrypsinogen (purified product) with a purity of more than 95%.
  • the chromatograms are shown in Figure 14, and the chromatograms of the three batches are basically the same.
  • the purity and yield test results are shown in Table 3.
  • the purity of the three batches of small-scale recombinant human chymotrypsinogen is greater than 95%, and the average yield can reach 1.2g/kg genetically engineered rice flour. It is verified that the RSD value of the three batches is less than 10%. , indicating that the process has good consistency and controllable quality.
  • the purified recombinant human chymotrypsin prepro is inactive, and it needs to be cut off by trypsin to form a biologically active recombinant human chymotrypsin. Therefore, it is necessary to activate recombinant human chymotrypsin by enzymatic hydrolysis.
  • the activated samples were tested for activity according to the method described in the 2020 edition of the Chinese Pharmacopoeia. The results are shown in Table 4. After being activated by different concentrations of trypsin for 24-72 h at 2-8 °C, the enzymatic activity of recombinant human chymotrypsin all reached the requirement of 1000 U/mg in the 2020 edition of the Chinese Pharmacopoeia, and There was no significant difference in the enzyme activity between different concentrations of trypsin and activation time points. However, with the same concentration of trypsin, the activity of recombinant human chymotrypsin was extremely low, only 200-300U/mg after being activated at 25°C for 72h.
  • the concentration of recombinant human chymotrypsinogen (set at 1-5 mg/mL) and the pH of the solution (7.0-8.5) were optimized on the basis of the above.
  • the amount of trypsin added was 0.1% (w/w)
  • the pH of the solution was 8.0 during activation, and the activation was performed at 2-8 °C for 40 hours.
  • the optimal activation conditions are determined as follows: the concentration of recombinant human chymotrypsinogen is 1-5 mg/mL, preferably 1 mg/mL. The amount of trypsin added is 0.05% to 0.5%, preferably 0.1%; the activation pH is 7.0 to 8.5, preferably pH 8.0.
  • Chymotrypsin is relatively stable in solid state, but its solution is unstable, therefore, to facilitate storage, recombinant human chymotrypsin needs to be freeze-dried.
  • the activated recombinant human chymotrypsin in Example 7 was appropriately concentrated using a 5-10kDa ultrafiltration membrane bag, and dialyzed with ultrapure water until the conductance was below 0.5 mS/cm, and then filtered through a 0.22 ⁇ m membrane, and the filtrate was divided into two parts. Freeze-dried after packaging.
  • the freeze-dried product is a white crystalline powder, which can be dissolved quickly after adding water, and its titer is 1500U/mg after testing.
  • the electrophoresis detection result of the preparation is shown in Figure 18, wherein the commercially available drug is chymotrypsin for injection (freeze-dried powder injection) produced by Shanghai Shanghai Pharmaceutical First Biochemical Pharmaceutical Co., Ltd.; the USP reference substance is the USP chymotrypsin standard product .
  • the electrophoresis behavior of recombinant human chymotrypsin is consistent with that of the USP reference substance, while the commercially available drugs have partially degraded fragments; while in the reduced state, the recombinant human chymotrypsin exhibits 3 electrophoresis bands, of which the polymer band is suspected It is partially inactivated recombinant human chymotrypsinogen.
  • the production process of the recombinant human chymotrypsin established by Examples 1-8 is as follows: planting of genetically engineered rice—rice processing (hulling, milling)—extraction—preliminary purification by cation chromatography—fine purification by anion chromatography—activation—concentration, Dialysis - deployment - sterile filtration - filling - freeze drying.

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Abstract

提供了一种高效表达糜蛋白酶原的基因工程水稻稻谷的制备方法及从表达人糜蛋白酶原的基因工程稻谷制备重组人糜蛋白酶的方法。将高效表达经水稻密码子优化的人糜蛋白酶原基因的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人糜蛋白酶原的粗提取物；将含重组糜蛋白酶原的粗提取物经过阳离子交换层析和阴离子交换层析，获得重组人糜蛋白酶原目标物。该重组人糜蛋白酶原用胰蛋白酶激活，冷冻干燥后获得重组人糜蛋白酶。该方法操作简单，无动物源性材料，获得的糜蛋白酶纯度和活性高，成本低，易于工业放大。

Description

一种利用基因工程水稻表达人糜蛋白酶原和制备重组人糜蛋白酶的方法 技术领域
本发明涉及生物技术领域，具体涉及以基因工程水稻为生物反应器，表达人糜蛋白酶原和制备重组人糜蛋白酶的方法。
背景技术
糜蛋白酶(Chymotrypsin)也叫胰凝乳蛋白酶，属于丝氨酸蛋白酶家族，能专一性水解芳香族氨基酸羧基形成的肽键。糜蛋白酶原由胰腺分泌，随胰液进入小肠后，在Arg15和Ile16之间(按Bovine chymotrypsinA序列编号)被胰蛋白酶切为通过二硫键连接的两部分，随后糜蛋白酶在Leu13酶切去短肽Serl4-Argl5，在Tyrl46和Asn148酶切去短肽Tyr147-Asn148，形成由二硫键相连的三条多肽链并具有生物活性。
糜蛋白酶在临床上具有广泛的用途，药理作用是分解蛋白质，促进血凝块、脓性分泌物和坏死组织等清除，主要用于眼科手术以松弛睫状韧带，减轻创伤性虹膜睫状体炎；也可用于创口或局部炎症，以减少局部分泌和水肿。
由于糜乳蛋白酶能消化各种蛋白质，因而在其他表达体系表达时，会导致宿主细胞的维系生命的蛋白质降解，导致现有表达体系表达量不高。因此，现有糜蛋白酶制备依然从牛或者猪的胰脏中提取，经分级盐析后先制成糜蛋白酶原，经胰蛋白酶激活后，再用硫酸铵、乙醇重结晶精制而得。该工艺提取特异性差，操作时间长，收率低。从牛或者猪的胰脏中提取的糜蛋白酶，其氨基酸序列与人糜蛋白酶不同，其中牛源糜蛋白酶的氨基酸同人糜蛋白酶的同源性为82％，猪源糜蛋白酶的氨基酸序列与人糜蛋白酶的同源性仅为43％。现有动物源糜蛋白酶不仅存在引入动物源性病毒的风险，还会因异源蛋白进入人体后可能会引起免疫反应，这大大增加了用药的安全风险。
人糜蛋白酶原为一条含有263个氨基酸的肽链，在体内激活过程中首先通过信号肽到达胞外，信号肽经肽酶水解后蛋白分子所含氨基酸数变为245,即糜蛋白酶原。当糜蛋白酶原被运输到小肠后才会被胰蛋白酶激活形成活性形式。
无动物源性和人源化的生物制品是目前生物医药行业的发展趋势。从1982年第一个无动物源性的重组人胰岛素替代从动物胰脏中提取，到2008 年第一个无血浆制品重组凝血酶上市，再到2020年第一个无动物源性植物源重组人血清白蛋白在美国完成1期临床研究。研发无动物源性和人源化的糜蛋白酶符合生物药物的发展趋势。
国内外目前尚没有重组人糜蛋白酶的医药产品上市。专利CN104342423A公开了一种高活性重组人糜蛋白酶的制备方法和应用，该方法是采用大肠杆菌表达重组人糜蛋白酶的包涵体，经变性和复性处理后，通过离子交换色谱层析获得较高纯度的重组人糜蛋白酶。
发明内容
本发明利用水稻胚乳细胞生物反应器来高效表达重组人糜蛋白酶原，从基因工程水稻稻谷中提取和纯化重组人糜蛋白酶原，然后通过激活获得具有生物活性的重组人糜蛋白酶。
本发明的一个目的是在基因工程水稻稻谷中提供一种表达重组人糜蛋白酶原的方法，包括以下步骤：
(1)合成如SEQ ID NO.1所示的经水稻密码子优化的人糜蛋白酶原基因序列；
(2)构建水稻胚乳细胞特异性表达的重组人糜蛋白酶原表达载体；
(3)将步骤(2)所获得载体转化到水稻品种的愈伤再生组织中；
(4)培养上述愈伤再生组织，经筛选和诱导获得转重组人糜蛋白酶原基因工程水稻植株；
(5)培养重组人糜蛋白酶原基因工程水稻植株，获得重组人糜蛋白酶原基因工程水稻稻谷。
其中重组人糜蛋白酶原表达载体优选具有如图3所示的结构。
本发明的另一个目的是提供一种从重组人糜蛋白酶原的基因工程稻谷中制备重组人糜蛋白酶的方法，包括如下步骤：
提取、纯化重组人糜蛋白酶原的方法，包括以下步骤：
(1)从重组人糜蛋白原的基因工程水稻稻谷中提取含有重组人糜蛋白酶原的粗提取物；
(2)将含有重组人糜蛋白原的粗提取物经Unigel 80SP阳离子交换层析，得到初级产物；
(3)将初级产物经Unigel 80Q阴离子交换层析，得到纯化的重组人糜蛋白酶原；
将上述纯化后的重组人糜蛋白酶原经胰蛋白酶激活，冷冻干燥后，获得重组人糜蛋白酶。
进一步的，提取、纯化重组人糜蛋白酶原的方法包含以下步骤：
1)将重组人糜蛋白酶原的基因工程稻谷脱壳加工成半精米，研磨成80～100目的米粉。将米粉与提取缓冲液以1:5(重量/体积，kg/L)的比例混合，于4～30℃提取1～16h后加入2～5％的珍珠岩压滤，滤液再经0.22μm滤膜过滤后即为重组人糜蛋白酶原的粗提取物。所述提取缓冲液的成分为：4.44～69.66g/L十二水合磷酸氢二钠、0.58～19.71g/L一水合柠檬酸，pH2.5～8.0。
2)釆用阳离子层析介质进行初级分离纯化，阳离子层析介质包括SPBestrose FF(博格隆(上海)生物技术有限公司)、Unigel 80SP(苏州纳微科技股份有限公司)、Unigel 80CM(苏州纳微科技股份有限公司)、Unigel MMC 50S(苏州纳微科技股份有限公司)。优选使用Unigel 80SP层析介质。在一种实施方式中，采用4～6倍柱体积(CV)的pH为3.0～3.5的磷酸氢二钠-柠檬酸的缓冲液，以300～600cm/h的线性流速平衡层析柱；以步骤1)的粗提取物为上样液，其中上样液pH为3.0～3.5，上样体积不超过74CV；用5～10CV、pH为3.5～4.0的磷酸氢二钠-柠檬酸缓冲液，以300～600cm/h的线性流速进行杂质蛋白的洗脱；用pH3.9～4.1、含100～200mM氯化钠的磷酸氢二钠-柠檬酸缓冲液，以300～600cm/h的线性流速进行重组人糜蛋白酶原的洗脱，收集含有重组人糜蛋白酶原的洗脱液，获得含重组人糜蛋白酶原的初级产物；
3)釆用阴离子层析介质进行精度纯化，阴离离子层析介质包括Unigel80Q(苏州纳微科技股份有限公司)、NanoGel 50Q(苏州纳微科技股份有限公司)、MMA-50S(苏州纳微科技股份有限公司)、Q Bestrose FF(博格隆(上海)生物技术有限公司)、Unigel 30Q(苏州纳微科技股份有限公司)、Q Bestrose HP(博格隆(上海)生物技术有限公司)。优选使用Unigel80Q层析介质。采用10～15倍柱体积的pH 7.9～8.1、电导1.5～2.0mS/cm的磷酸氢二钠-柠檬酸的缓冲液，以382～840cm/h的流速平衡柱子；将步骤2)的含重组人糜蛋白酶原的洗脱液用5～10kDa超滤膜包适当浓缩，并用pH7.9～8.1,电导1.5～2.0mS/cm的磷酸氢二钠-柠檬酸缓冲液透析至电导降低至1.5～2.5mS/cm，调节pH至7.9～8.1，作为此步层析的上样液；用pH6.5～8.0，含25～75mM氯化钠的磷酸氢二钠-柠檬酸缓冲液以382～840cm/h的流速进行洗脱，收集含有重组人糜蛋白酶原的洗脱液，获得纯度95％以上的的重组人糜蛋白酶原。
进一步的，本申请提供的重组人糜蛋白酶原激活的方法，包括以下步骤：
(1)在0.75～4mg/mL的重组人糜蛋白酶原溶液中按照0.05％～0.5％(w/w)的比例加入激活剂胰蛋白酶，2～8℃，静置激活36～72h。
(2)激活结束后，调节pH至2.5～3.5，以终止激活。
(3)将步骤2的重组人糜蛋白酶用5～10kDa超滤膜包(聚醚砜材质)适当浓缩，并用超纯水透析至电导降低至0.5mS/cm以下，调节pH至5.5～6.5。装盘、冻干。
本发明也提供按照本发明方法用水稻胚乳细胞表达制备的重组人糜蛋白酶原。
与现有的技术相比，本发明的优点和积极效果是：
本发明依托两大优势平台—水稻胚乳细胞蛋白质表达平台(Oryz HiExp)和蛋白质纯化平台(Oryz Pur)，采用水稻胚乳细胞高效表达重组人糜蛋白酶原，经过提取及两步层析后可获得SEC-HPLC纯度高于99％的重组人糜蛋白酶原。重组人糜蛋白酶原经活化后得到重组人糜蛋白酶，其活性可达1500U/mg以上，高于2020版中国药典1000U/mg的标准。本发明所述的生产工艺相比传统生产工艺，无动物源性材料、操作更为简单、生产时间短、安全性更高、环境友好，具有重要的应用前景，符合未来生物制药的发展趋势。
附图说明
图1.pOsPMP799质粒结构示意图。
图2.pOsPMP800质粒结构示意图。
图3.pOsPMP801质粒结构示意图。
图4.基因工程水稻部分植株的目的基因的PCR检测。其中M为DNA标准分子量Marker；1-23分别为T1代基因工程材料的不同植株；P为阳性对照质粒；红色箭头所指为目的基因。
图5.基因工程水稻部分株系稻谷表达量检测。其中M为DNA标准分子量Marker；2、7、8、9、16、20分别表示基因工程水稻选育过程中不同株系；红色箭头所指为目标蛋白。
图6.不同提取条件下重组人糜蛋白酶原粗提取物SDS-PAGE检测结果。其中M为标准分子量Marker；A-C中的1-27分别表示表1中1-27不同提取条件。
图7.不同pH、温度及提取时间下重组人糜蛋白酶原粗提取物的电泳检测结果。其中M为标准分子量Marker。
图8.Unigel 80SP、Unigel 80CM及Unigel MMC 50S层析洗脱组分SDS-PAGE检测结果。其中A为Unigel80SP层析洗脱组分的SDS-PAGE检测结果；B为Unigel80CM层析洗脱组分的SDS-PAGE检测结果；C为Unigel50S层析洗脱组分的SDS-PAGE检测结果；M为标准分子量Marker；Wash表示洗杂；CIP1和CIP2分别表示再生1和再生2；FT2为再平衡时拖尾部 分的穿透液。
图9.Unigel 80SP穿透液电泳检测结果，其中M为标准分子量Marker，L为层析上样液，25～36分别代表收集管的编号。
图10.pH4.0不同盐浓度洗杂层析组分SDS-PAGE检测结果。其中M为标准分子量Marker；Wl-1，W1-2为洗杂峰分段收集；Elu为洗脱收集液；CIP为再生液。
图11.不同阴离子层析电泳检测结果。其中FT为穿透液；25、50、75、100、150分别表示不同含mM氯化钠的洗脱组分；红色箭头所示为目的蛋白。
图12.Unigel 80Q层析组分SDS-PAGE检测结果。其中M为标准分子量marker；L为上样液；FT为穿透液；W为洗杂收集液；Elu为洗脱收集液；CIP为再生液；还原即在电泳制样过程中加入β-巯基乙醇，非还原即为制样过程中不加β-巯基乙醇。
图13.Unigel 80Q层析不同洗脱条件层析收集液电泳检测。其中1表示还原状态下pH 6.5、50mM氯化钠洗脱收集液；2表示非还原下pH 6.5、50mM氯化钠洗脱收集液；3表示还原状态下pH 7.0、50mM氯化钠洗脱收集液；4表示还原状态下pH 7.0、25mM氯化钠洗脱收集液；5表示还原状态下pH 7.5、50mM氯化钠洗脱收集液；6表示还原状态下pH 8.0、50mM氯化钠洗脱收集液。
图14.三批小试工艺层析图谱。其中A为Unigel80SP层析图谱，B为Unigel 80Q层析图谱；20200804、20200805、20200806分别表示不同的生产批号。
图15.三批小试电泳检测结果(还原)，其中M为标准分子量Marker；1表示20200804批，2表20200805批，3表示20200806批；还原表示制样过程中加入还原剂β-巯基乙醇。
图16.激活前重组人糜蛋白酶SEC-HPLC检测图谱。
图17.激活后重组人糜蛋白酶SEC-HPLC检测图谱。
图18.重组人糜蛋白酶SDS-PAGE检测结果。
具体实施方式
下文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。
以下实施例中使用的试剂和仪器，除特别说明以外，均为普通市售。
【实施例1】高效表达重组人糜蛋白酶原基因工程水稻的制备
1.重组人糜蛋白酶原基因表达载体的构建
本实施例选用水稻特异性启动子Gtl3a及其信号肽来介导重组人糜蛋白酶原基因在水稻胚乳细胞中的表达。根据人糜蛋白酶基因序列(Genbank登记号：NP_001897.4)，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SEQID NO.1所示，经水稻偏好密码子优化后的核苷酸改变了15％，密码子改变了35％，但其相应的氨基酸序列没有变化，构建的质粒为pOsPMP799(图1)。本实施例选用水稻特异性启动子及其信号肽来介导人糜蛋白酶原基因在水稻胚乳细胞中的表达。将所述合成的经密码子优化的人糜蛋白酶原基因(SEQIDNO.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pOsPMP800(图2)；然后用HindIII和EcoRI酶切pOsPMP800，将长度为2263bp的含Gtl3a启动子/信号肽序列以及密码子优化的人糜蛋白酶原基因和Nos终止子的表达盒插入到经HindIII和EcoRI酶切双元表达载体pc1300，构建农杆介导菌质粒，命名为pOsPMP801(图3)。
2.基因工程水稻遗传转化
将pOsPMP801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转化将POsPMP801转化到水稻品种TP309的愈伤再生组织中，经培养、筛选和诱导后形成完整的植株。具体方法如下：
(1)愈伤诱导
1)将成熟水稻种稻谷脱壳后用70％酒精浸泡灭菌1min，20％次氯酸钠再处理30min；
2)用灭菌无菌水清洗5-7次；
3)将处理好的稻谷接种到诱导培养基(N6培养基)上，每皿接种6-8粒；
4)在32℃光照处理5-7天。
(2)农杆菌制备
将含有表达载体pOsPMP801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。
(3)用接种环取农杆菌单菌落挑取到悬浮培养基(AAM液体培养基)中，在28℃摇菌(160rpm)培养。一般100mL培养基可用接种环刮入3至4环。
(4)农杆菌侵染(共培养)
1)将愈伤组织转移到灭菌的三角瓶中；
2)调整农杆菌悬浮液OD600值于0.05～0.1之间；
3)将稻谷悬浮在AAM培养基中，侵染1.5min，其间持续摇晃；
4)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30～45min；
5)将无菌滤纸放在2N6-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，将侵染过的愈伤放在滤纸上，25℃黑暗共培养3天。
(5)水洗和筛选
1)将经过共培养的愈伤组织转移到灭菌的三角瓶中；
2)用灭菌水清洗愈伤组织5-7次；
3)用含有0.5g/L浓度的头抱霉素灭菌水浸泡感染的愈伤组织约30min,然后于28℃、180～200rpm摇20～30min；
4)倒去含有抗生素的灭菌水，将三角瓶倒置于含滤纸的灭菌培养皿中大约15min；
5)将愈伤组织在灭菌的滤纸上晾干；
6)转移愈伤组织转移到含有HPT抗生素的筛选培养基上培养20-30天。
(6)愈伤组织分化
将经过20～30天选择，将具有HPT抗性愈伤组织转移到分化培养基(N6培养基)上，在26℃光照培养20～30天。
(7)生根
从分化培养基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，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)基因组DNA的提取
取T 0代HPT阳性再生苗的叶片约2cm，分别放入离心管，加入600μL CTAB提取缓冲液(2％CTAB，1.38MNaCL 0.1M Tris-HCl，20mM EDTA,PH8.0)，在震荡破碎机破碎后，在65℃温浴60min，再加入等体积的氯仿/异戊醇，轻缓颠倒混合，12000rpm离心l0min；将上清液转入另一新1.5mL离心管中，加入等体积的异丙醇，缓慢倒置混合，在室温下放置60min；12000rpm离心l0min，去上清液，70％乙醇漂洗DNA沉淀，风干后加入80μL的TE缓冲液溶解DNA，-20℃保存备用。
(2)PCR扩增
以苗期取叶片提取基因组DNA，采用人糜蛋白酶原正向引物Gtl3a-F(SEQ ID NO.2:5’-CACATCCATCATTATCCATCCACC-3’)和反向引物CT-R(SEQ IDNO.3:5'-ACTTCGGGTTCTTGAACACCTT-3')进行PCR扩增，产物理论大小为547bp，PCR鉴定含有目的基因的基因工程植株，PCR检测方法如下：
1)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；
2)PCR扩增反应体系包含2.5μL的10×Buffer；0.15μL的5U/μL的rTaq酶，4μL的2.5mMdNTP，正向和反向引物各0.5μL；加ddH 2O至25μL。
3)在94℃预变性5min，94℃变性30s，58℃退火反应30s，72℃延伸30s，35个循环，最后72℃延伸10min。
4)PCR扩增产物经1％琼脂胶电泳，EB染色后在凝胶成像仪里观察结果。
鉴定结果表明，通过根癌农杆菌介导转化获得56株独立的重组人糜蛋白酶原基因工程水稻。部分基因工程水稻目的基因PCR鉴定结果如图4所示。
4.重组人糜蛋白酶原的表达量的鉴定
将获得的重组人糜蛋白酶原阳性苗移栽到温室中生长至成熟，收种能够正常结实的单株。由于目前无市售的可用来定量检测重组人糜蛋白酶原含量的检测试验盒，故通过SDS-PAGE法初步筛选高表达的单株，剔除阴性单株。将高表达的单株作为候选单株继续种植，再次进行表达量比较及纯合子筛选，直至获得稳定高效表达重组人糜蛋白酶原的纯合株系。部分重组人糜蛋白酶原稻谷表达量的SDS-PAGE检测结果如图5所示。经初步估算，重组人糜蛋白酶的最高表达量可达2g/kg糙米以上。
SEQ ID NO.1
Figure PCTCN2021125969-appb-000001
SEQ ID NO.2
Figure PCTCN2021125969-appb-000002
SEQ ID NO.3
Figure PCTCN2021125969-appb-000003
【实施例2】从基因工程水稻中提取重组人糜蛋白酶原
1.重组人糜蛋白酶原提取条件初步筛选
将基因工程稻谷脱壳加工成半精米，研磨成80～100目的米粉。将米粉与不同pH的磷酸氢二钠-柠檬酸提取缓冲液以1:5(重量/体积，kg/L)的比例混合，按照表1所设计的提取条件进行提取。提取结束后，取上清液进行SDS-PAGE检测。结果显示，各提取条件，提取液中目的蛋白提取量无显著差异(见图6)。
进一步分析发现，试验处理2、3、6、9、20、26的杂质蛋白相对较少，几乎无高分子杂质蛋白。其中以处理3(提取液pH3.0，0mMNaCl)，提取时间8.5h，提取温度17℃时的纯度相对较高。通过上述结果可以看出，pH对目的蛋白的提取率无显著影响，但是对提取液中目的蛋白的纯度影响显著，pH越低，杂质越少；氯化钠浓度、提取时间及提取温度等对目的蛋白的提取率无显著影响。
表1不同提取条件设计
处理NO. pH 氯化钠浓度(mM) 时间(h) 温度(℃)
1 5.5 0 16 17
2 3 250 8.5 30
3 3 0 8.5 17
4 5.5 500 8.5 4
5 5.5 250 16 30
6 3 250 8.5 4
7 5.5 500 1 17
8 5.5 250 16 4
9 3 250 16 17
10 8 250 1 17
11 5.5 500 8.5 30
12 5.5 0 8.5 4
13 8 500 8.5 17
14 5.5 500 16 17
15 5.5 250 1 4
16 8 250 16 17
17 5.5 0 1 17
18 8 0 8.5 17
19 5.5 0 8.5 30
20 3 500 8.5 17
21 5.5 250 8.5 17
22 5.5 250 8.5 17
23 8 250 8.5 4
24 5.5 250 1 30
25 5.5 250 8.5 17
26 3 250 1 17
27 8 250 8.5 30
2.重组人糜蛋白酶原提取条件确定
根据上述的初步筛选结果，进一步对提取条件进行优化。将基因工程稻谷脱壳加工成半精米，研磨成80-100目的米粉。将米粉以1:5(重量/体积，kg/L)分别采用pH2.5、pH3.0及pH3.5的磷酸氢二钠-柠檬酸提取缓冲液的比例混合,然后置于25℃提取1h。同时设计pH3.0的提取缓冲液分别于20℃.25℃及30℃搅拌提取lh、2h、4h。提取结束后，离心，取上清液进行SDS-PAGE检测。结果显示，各提取条件下，提取液中目的蛋白提取量无显著差异(图7)。从经济角度考虑，优选提取时间越短，温度20～30℃(室温)，成本最低。因此，确定重组人糜蛋白酶原的提取条件：缓冲液(磷酸氢二钠-柠檬酸)pH2.5～3.5；提取时间1～2h；提取温度20～30℃。
【实施例3】重组人糜蛋白酶原的阳离子层析介质初级纯化
1.初级纯化阳离子层析介质的选择
根据实施例2确定的提取条件制备含重组人糜蛋白酶原的粗提物，然后分别上样至装填有Unigel 80SP、Unigel 80CM、Unigel MMC 50S层析介质的层析柱，随后采用不同pH的磷酸氢二钠-柠檬酸缓冲液进行梯度洗脱。结 果显示上述3种层析介质，在pH梯度洗脱时，洗脱峰拖尾较为严重，分离度较差。电泳检测结果见图8，Unigel 80CM穿透明显(图8B)，即上样20CV即已过载。
Unigel MMC 50S在平衡过程中，拖尾严重，拖尾处可见有明显的目的蛋白脱落(图8C)，目的蛋白在各pH条件下均有洗脱，主要集中在pH6.0和pH7.0洗脫组分中。Unigel 80SP上样及再平衡过程未见目的蛋白脱落，目的蛋白主要集中在pH4.5和pH5.0的洗脱组分中，但这两个条件洗脱峰拖尾严重。考虑到Unigel 80CM载量低及Unigel MMC50S再平衡拖尾严重且有目的蛋白损失，因此最终优选Unigel 80SP作为初级纯化的层析介质。
2.阳离子Unigel 80SP层析载量测定
根据实施例2确定的提取条件制备重组人糜蛋白酶原的粗提物(即上样液)。将重组人糜蛋白酶原的粗提物上样(0.5mL/min流速)至1mL Unigel80SP层析柱中，当上样至20CV时，开始采用分布收集器按照约2mL/管自动收集穿透液。对收集的穿透液进行SDS-PAGE检测，结果如图9所示，收集液的27管即74CV(20+27×2＝74CV)可见目的蛋白穿透。因此，在此上样条件下，Unigel 80SP的最大载量为74CV。
3.阳离子Unigel 80SP层析优化
根据实施例2确定的提取条件制备重组人糜蛋白酶原的粗提物。将50mL(50CV)的重组人糜蛋白酶原粗提物上样至1mL Unigel 80SP层析介质，然后用pH 4.0的含0～200mM氯化钠的磷酸氢二钠-柠檬酸缓冲液进行梯度洗脱。结果如图10所示，在pH4.0、0mM氯化钠洗杂条件下有微量的目的蛋白洗脱，当盐浓度提高至30mM氯化钠时，目的蛋白明显的从层析介质上被洗脱。由上可知，目的蛋白极易被洗脱，洗杂空间极小。考虑到pH 4.0、30mM氯化钠洗杂目的蛋白损失较多，且存在拖尾现象，而pH 4.0、0mM氯化钠洗杂时虽然有微量目的蛋白损失，但是可去除较多的杂质蛋白，同时洗杂峰与后续的洗脱峰(pH 4.0,100mM氯化钠洗脱)有一定的分离度，因此，确定洗杂条件为pH4.0、0mM氯化钠。由于pH4.0、100mM氯化钠洗脱(收集体积27CV)和pH 4.0、150mM氯化钠洗脱(收集体积25CV)两种洗脱组分目的蛋白的纯度无显著差异，因此确定洗脱条件为pH 4.0、100～150mM氯化钠，优选pH 4.0、150mM氯化钠洗脱(洗脫体积相对较小)。
【实施例4】重组人糜蛋白酶原的阴离子层析介质精细纯化
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人糜蛋白酶原的初级产物，由于第一步初级纯化釆用阳离子介质，故第二步精细纯化拟釆用阴离子介质。
1.初级产物预处理
首先将初级产物用5～10kDa超滤膜包适当浓缩后，用pH 7.9～8.1，电导1.5～2.0mS/cm的磷酸氢二钠-柠檬酸缓冲液或者磷酸盐缓冲液透析至电导为1.5～2.5mS/cm，调节pH至7.9～8.1，作为第2步层析的上样液。
2.阴离子层析介质筛选
将第1步预处理的上样液，以0.5mL/min的流速分别上样至装填有1mL阴离子层析介质Unigel 80Q、NanoGel 50Q、MMA-50S、Q Bestrose FF的层析柱中，然后采用氯化钠梯度洗脱。结果如图11所示，目的蛋白与Q系列的层析介质结合较弱，在较低电导下(25-50mM氯化钠)，目的蛋白均逐渐洗脱，且洗脱峰拖尾现象严重，尤以Q BestroseFF，25mM氯化钠可洗脱部分杂质，但目的蛋白不能被脱落。结果显示不论何种阴离子层析介质，最终洗脱液中目的蛋白的纯度基本相同(图11)。从层析介质成本及耐用性方面考虑，优选Unigel 80Q作为第2步层析介质。
3.Unigel 80Q层析优化
根据实施例2～3确定的条件，制备实施例4的层析上样液，然后以40mL(40CV)的上样量，上样至1mL Unigel 80Q层析柱，然后用pH7.9～8.1,含50mM氯化钠的磷酸氢二钠-柠檬酸(或者10mM的磷酸盐缓冲液)缓冲液进行洗脱。结果如图12所示，Unigel 80Q的洗脱液在还原状态下呈三条蛋白带，而在非还原条件下呈现单一条带。经分析，可能为重组人糜蛋白酶原在蛋白质表达、转运及储存过程中发生自激活现象。
由于上述层析条件洗脱时收集体积大，说明洗脱强度低，阴离子层析可通过降低洗脱pH来提高洗脱强度，故拟通过降低洗脱液的pH对层析的洗脱条件进行优化。结果如表2所示，当釆用pH 6.5、50mM氯化钠，10mM磷酸盐缓冲液洗脱时，收集体积大大减小。经洗脱条件优化后，样品纯度无显著变化(见表2及图13)。
综上所述，重组人糜乳蛋白酶的Unigel 80Q层析洗脱条件为：10mM磷酸盐缓冲液，pH6.5～8.0、2～50mM氯化钠。结合实施例6重组人糜蛋白酶的最优激活条件为：重组人糜蛋白酶原浓度lmg/mL，pH8.0。
Ungel 80Q的最优洗脱条件为10mM磷酸盐缓冲液，pH 8.0、50mM氯化钠(此洗脱条件收集液的蛋白质含量约为1mg/mL，而且收集液无需调节pH，可直接用于激活)。
表2不同洗脱层析洗脱收集体积
Figure PCTCN2021125969-appb-000004
Figure PCTCN2021125969-appb-000005
【实施例5】重组人糜蛋白酶原三批小试工艺验证
根据实施例2～4确定的最优提取、初步纯化及精细纯化条件，进行三批小试工艺验证，验证过程及验证结果如下所述。
1.重组人糜蛋白酶原粗提取物制备(提取)
称取500g重组人糜蛋白酶原米粉，加入2500mL含14.72g/L十二水合磷酸氢二钠、16.69g/L一水合柠檬酸提取缓冲液，在20～30℃下搅拌提取1～2h。提取结束后，加入2％～5％(w/v)助滤剂，釆用正压过滤的方式进行压滤。初滤液经0.22μm滤膜过滤后，即为重组人糜蛋白酶原粗提取物(Unigel 80SP层析上样液)。
2.重组人糜蛋白酶原初步纯化
采用4～6CV的pH 3.3-3.5的20.4lg/L十二水合磷酸氢二钠、15.02g/L一水合柠檬酸缓冲液，以300～600cm/h的线性流速平衡填充有Unigel 80SP，装柱高度19～21cm的XK16/40层析柱。以上述重组人糜蛋白酶原粗提取物为上样液进行上样，上样体积50CV。用8～10倍柱体积、pH3.9～4.1的含7.61g/L十二水合磷酸氢二钠、12.91g/L一水合柠檬酸的缓冲液以300～600cm/h的线性流速进行杂质蛋白的洗脱；用pH3.9～4.1的含150mM氯化钠的上述磷酸氢二钠-柠檬酸缓冲液，以300～600cm/h的线性流速进行重组人糜蛋白酶原的洗脱，收集含有重组人糜蛋白酶原的洗脱液，获得含重组人糜蛋白酶原的初级产物。
3.重组人糜蛋白酶原精细纯化
釆用10～15倍柱体积的pH 7.9～8.1，电导1.5～2.0mS/cm的磷酸氢二钠-柠檬酸的缓冲液，以382～840cm/h的流速平衡填充有Unigel 80Q，装柱高度27～29cm的C10/40层析柱；将上述重组人糜蛋白酶原初级产物(即Unigel 80SP层析收集液)用5～10kDa超滤膜包适当浓缩，并用pH 7.9-8.1，电导1.5～2.0mS/cm的磷酸氢二钠-柠檬酸缓冲液(或者相同pH的10mM磷酸盐缓冲液)透析至电导降低至1.5～2.5mS/cm,调节pH至7.9～8.1,作为此步层析的上样液；用pH为7.9～8.1，含50mM氯化钠、20.41g/L十二水合磷酸氢二钠、15.02g/L一水合柠檬酸的缓冲液以382～840cm/h的流速进行洗脱，收集含有重组人糜蛋白酶原的洗脱液，获得纯度95％以上的的重组人糜蛋白酶 原(精纯产物)。层析图谱如图14所示，三批小试层析图谱基本一致。
纯度及收率检测结果如表3所示，三批小试重组人糜蛋白酶原的纯度均大于95％，平均收率可达1.2g/kg基因工程水稻米粉，验证3批RSD值小于10％，说明该工艺一致性良好，质量可控。
表3三批小试纯度及收率
Figure PCTCN2021125969-appb-000006
【实施例6】重组人糜蛋白酶原的激活
纯化后的重组人糜蛋白前原是没有活性的，需要经过胰蛋白酶切去部分肽段后才形成具有生物学活性的重组人糜蛋白酶。因此需要通过酶解方式激活重组人糜蛋白酶。
1.激活条件初步摸索
将实施例5制备的重组人糜蛋白酶原纯化样品(lmg/mL)按照10mL/管分为若干份，分别加入终浓度为0.5％、0.25％、0.125％、0.05％(w/w,胰蛋白酶/重组人糜蛋白酶原)的胰蛋白酶(活性＞2500U/mg)，然后分别置于2～8℃及25±2.5℃静置激活。分别在激活的24h、32h、48h、56h、72h各取样1mL,加入2～3μL 50％HCl终止激活(pH3～4)，激活后的样品按照《中国药典》2020版所述方法进行活性检测。结果如表4所示，在2～8℃条件下加入不同浓度的胰蛋白酶分别激活24～72h后，重组人糜蛋白酶的酶活性均达到《中国药典》2020版的1000U/mg的要求，且不同浓度的胰蛋白酶以及各激活时间点的酶活性无明显的差异。但相同浓度的胰蛋白酶，在25℃条件下激活72h，重组人糜蛋白酶的活性极低，仅有200～300U/mg。
表4不同激活条件重组人糜蛋白防活性检测结果
Figure PCTCN2021125969-appb-000007
Figure PCTCN2021125969-appb-000008
2.激活条件优化
为了优选重组人糜蛋白酶原的激活条件，在上述基础上对激活时重组人糜蛋白酶原的浓度(设置1～5mg/mL)、溶液的pH(7.0-8.5)进行优化。当胰蛋白酶加入量为0.1％(w/w)，激活时溶液pH为8.0，在2～8℃条件下激活40h，发现随着激活前重组人糜蛋白酶原浓度的升高，重组人糜蛋白酶的活性逐渐降低(表5)；当胰蛋白酶加入量为0.1％(w/w)，不同pH下2～8℃激活40h，发现激活溶液pH为8.0时，重组人糜蛋白酶的活性相对最高(表6)。激活前后重组人糜蛋白酶(原)的SEC-HPLC如图16及图17所示，激活后重组人糜蛋白酶纯度达到95％，相比激活前重组人糜蛋白酶原99％的纯度，略微降低。
因此确定最佳的激活条件为：重组人糜蛋白酶原浓度1～5mg/mL优选lmg/mL。胰蛋白酶加入量0.05％～0.5％，优选0.1％；激活pH为7.0～8.5，优选pH 8.0。
表5糜蛋白酶原浓度对激活后活性的影响
Figure PCTCN2021125969-appb-000009
表6不同pH激活后活性的影响测定结果
Figure PCTCN2021125969-appb-000010
【实施例8】重组人糜蛋白酶制剂研究
糜蛋白酶在固体状态时比较稳定，但其溶液不稳定，因此，为了便于储存，需要对重组人糜蛋白酶进行冷冻干燥。将实施例7激活后的重组人糜蛋 白酶，采用5～10kDa超滤膜包适当浓缩，并用超纯水超滤透析至电导为0.5mS/cm以下，然后用0.22μm的滤膜过滤，滤液分装后冷冻干燥。冻干成品为白色结晶性粉末，加水后可迅速溶解，经测定其效价为1500U/mg。
制剂的电泳检测结果如图18所示，其中市售药物为上海上药第一生化药业有限公司生产的注射用糜蛋白醱(冻干粉针剂)；USP对照品为美国药典糜蛋白酶标准品。
在非还原状态下，重组人糜蛋白与USP对照品电泳行为一致，而市售药物存在部分降解片段；而在还原状态下，重组人糜蛋白酶呈现3条电泳条带，其中高分子条带疑似为部分未激活的重组人糜蛋白酶原。
【实施例9】重组人糜蛋白酶的生产工艺
由实施例1-8建立重组人糜蛋白的生产工艺如下：基因工程水稻的种植—稻谷加工(脱壳、磨粉)—提取—阳离子层析初步纯化—阴离子层析精细纯化—激活—浓缩、透析—调配—除菌过滤—灌装—冷冻干燥。

Claims (15)

  1. 一种表达人糜蛋白酶原的基因工程水稻稻谷的制备方法，包括以下步骤：
    (1)合成具有如SEQ ID NO.1所示序列的经水稻密码子优化的人糜蛋白酶原基因；
    (2)将步骤(1)的人糜蛋白酶原基因，与水稻特异性启动子Gtl3a及其信号肽导入质粒载体构建水稻胚乳细胞特异性表达的人糜蛋白酶原表达载体；
    (3)将步骤(2)获得的人糜蛋白酶原表达载体转化到水稻的愈伤再生组织中；
    (4)培养所述愈伤再生组织，经筛选和诱导获得表达人糜蛋白酶原的基因工程水稻植株；
    (5)培育所述表达人糜蛋白酶原的基因工程水稻植株，获得表达人糜蛋白酶原的基因工程水稻稻谷。
  2. 根据权利要求1所述的表达人糜蛋白酶原的基因工程水稻稻谷的制备方法，其特征在于，步骤(2)所述质粒载体为pOsPMP801。
  3. 一种利用表达人糜蛋白酶原的基因工程水稻稻谷制备重组人糜蛋白酶的方法，包括如下步骤：
    (1)从表达人糜蛋白酶原的基因工程水稻稻谷中提取含有重组人糜蛋白酶原的粗提取物；其中，所述表达人糜蛋白酶原的基因工程水稻稻谷按照权利要求1所述的方法制备；
    (2)将所述含有重组人糜蛋白酶原的粗提取物经阳离子交换层析，得到初级产物；
    (3)将所述初级产物经阴离子交换层析，得到纯化的重组人糜蛋白酶原；
    (4)将所述纯化的重组人糜蛋白酶原经胰蛋白酶激活，冷冻干燥后，获得重组人糜蛋白酶。
  4. 根据权利要求3所述的利用表达人糜蛋白酶原的基因工程水稻稻谷制备重组人糜蛋白酶的方法，其特征在于，步骤(1)所述含有重组人糜蛋白酶原的粗提取物是通过下述方法制备：
    1)将表达重组人糜蛋白酶原的基因工程水稻稻谷脱壳加工成半精米，研磨成米粉；
    2)将上述米粉与提取缓冲液混合，所述提取缓冲液为pH 2.5～8.0的磷酸氢二钠-柠檬酸缓冲液，于4～30℃下提取，获得混合物；
    3)将步骤2)的提取混合物入2％～5％的珍珠岩压滤，滤液再经滤膜过滤后即为重组人糜蛋白酶原的粗提取物。
  5. 根据权利要求4所述的利用表达人糜蛋白酶原的基因工程水稻稻谷制备重组人糜蛋白酶的方法，其特征在于，所述重组人糜蛋白酶原的提取缓冲液为pH 2.5～3.5的磷酸氢二钠-柠檬酸缓冲液，提取时间为1～2h，提取温度为20～30℃。
  6. 根据权利要求3所述的利用表达人糜蛋白酶原的基因工程水稻稻谷制备重组人糜蛋白酶的方法，其特征在于，步骤(2)所述阳离子交换层析介质为SPBestroseFF、Unigel 80SP、Unigel 80CM或UnigelMMC 50S。
  7. 根据权利要求6所述的利用表达人糜蛋白酶原的基因工程水稻稻谷制备重组人糜蛋白酶的方法，其特征在于，所述阳离子交换层析介质为Unigel 80SP。
  8. 根据权利要求7所述的利用表达人糜蛋白酶原的基因工程水稻稻谷制备重组人糜蛋白酶的方法，其特征在于，步骤(2)所述重组人糜蛋白酶原初级产物是通过下述方法制备：
    1)采用4～6倍柱体积(CV)的pH为3.0～3.5的磷酸氢二钠-柠檬酸的缓冲液，以300～600cm/h的线性流速平衡Unigel 80SP层析柱；
    2)以所述重组人糜蛋白酶原的粗提取物为上样液，其中上样液pH为3.0～3.5，上样体积不超过75CV；
    3)采用5～10CV、pH3.5～4.0，含0～30mM氯化钠的磷酸氢二钠-柠檬酸缓冲液，以300～600cm/h的流速进行杂质蛋白的洗脱；洗杂缓冲液为pH4.0，不含氯化钠的磷酸氢二钠-柠檬酸缓冲液；
    4)用pH 3.9-4.1，含100～200mM氯化钠的磷酸氢二钠-柠檬酸缓冲液，以300～600cm/h的线性流速进行重组人糜蛋白酶原的洗脱，收集富含重组人糜蛋白酶原的洗脱液，获得含有重组人糜蛋白酶原的初级产物。
  9. 根据权利要求3所述的利用表达人糜蛋白酶原的基因工程水稻稻谷制备重组人糜蛋白酶的方法，其特征在于，步骤(3)所述的阴离子交换层析介质为Unigel 80Q、NanoGel 50Q、MMA-50S、Q Bestrose FF、Unigel 30Q或Q Bestrose HP。
  10. 根据权利要求9所述的利用表达人糜蛋白酶原的基因工程水稻稻谷制备重组人糜蛋白酶的方法，所述阳离子层析介质为Unigel 80Q。
  11. 根据权利要求10所述的利用表达人糜蛋白酶原的基因工程水稻稻谷制备重组人糜蛋白酶的方法，其特征在于，步骤(3)所述纯化的重组人糜蛋白酶原是通过下述方法制备：
    1)釆用10～15CV的pH 7.9～8.1，电导1.5～2.0mS/cm的磷酸氢二钠-柠檬酸的缓冲液或者磷酸盐缓冲，以382～840cm/h的流速平衡Unigel 80Q层析柱；
    2)将所述初级产物用5～10kDa超滤膜包适当浓缩，并用pH 7.9～8.1， 电导1.5～2.0mS/cm的磷酸氢二钠-柠檬酸缓冲液或者磷酸盐缓冲液透析至上样液电导降低至1.5～2.5mS/cm,调节pH至7.9～8.1，上样至阴离子交换层析；
    3)用pH 6.5～8.0，含25～75mM氯化钠的磷酸氢二钠-柠檬酸缓冲液或者磷酸盐缓冲液以382～840cm/h的流速进行洗脱，收集含有重组人糜蛋白酶原的洗脱液，获得纯度99％以上纯化的重组人糜蛋白酶原。
  12. 根据权利要求3所述的利用表达人糜蛋白酶原的基因工程水稻稻谷制备重组人糜蛋白酶的方法，其特征在于，步骤(4)所述重组人糜蛋白酶是通过下述方法制备：
    1)在0.75～4mg/mL的所述纯化的重组人糜蛋白酶原中按照0.05％～0.5％(w/w)的比例加入激活剂胰蛋白酶，2～8℃，静置激活36～72h；
    2)激活结束后，调节pH至2.5～3.5，终止激活；
    3)将激活后的重组人糜蛋白酶用聚醚砜材质的5～10kDa超滤膜包适当浓缩，并用超纯水透析至电导降低至0.5mS/cm以下，调节pH至5.5～6.5；装盘、冷冻干燥，获得重组人糜蛋白酶冻干粉。
  13. 根据权利要求3～12任一项所述方法制备的重组人糜蛋白酶。
  14. 一种人糜蛋白酶原基因，其序列如SEQ ID NO.1所示。
  15. 一种人糜蛋白酶原表达载体，其特征在于，将权利要求14所述的人糜蛋白酶原基因与水稻特异性启动子Gtl3a及其信号肽导入质粒载体构建而成。
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DATABASE Necleotide GenBank; ANONYMOUS : "Homo sapiens chymotrypsinogen B1 (CTRB1), transcript variant 1, mRNA ", XP055927265, retrieved from NCBI *
DATABASE Nucleotide GenBank; ANONYMOUS : "Homo sapiens chymotrypsinogen B1 (CTRB1), transcript variant 1, mRNA ", XP055927264, retrieved from NCBI *
LIU, XIAO, LI SU-XIA: "Study On the Expression, Purification and Properties of Recombinant Human Chymotrypsin", CHINESE JOURNAL OF BIOCHEMICAL PHARMACEUTICS, QUANGUO SHENGHUA ZHIYAO QINGBAO ZHONGXINZHAN, NANJING, CN, vol. 34, no. 2, 28 April 2014 (2014-04-28), CN , pages 71 - 74, XP055927267, ISSN: 1005-1678 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115521962A (zh) * 2022-09-26 2022-12-27 福瑞施生物医药科技(深圳)有限公司 水稻多肽组合物及其制备方法和应用
CN115521962B (zh) * 2022-09-26 2024-03-15 福瑞施生物医药科技(深圳)有限公司 水稻多肽组合物及其制备方法和应用

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