WO2014071681A1 - 一种从水稻种子生产和分离纯化重组人抗胰蛋白酶（OsrAAT）的方法 - Google Patents

一种从水稻种子生产和分离纯化重组人抗胰蛋白酶（OsrAAT）的方法 Download PDF

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WO2014071681A1
WO2014071681A1 PCT/CN2013/000482 CN2013000482W WO2014071681A1 WO 2014071681 A1 WO2014071681 A1 WO 2014071681A1 CN 2013000482 W CN2013000482 W CN 2013000482W WO 2014071681 A1 WO2014071681 A1 WO 2014071681A1
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osraat
rice
sample
column
eluate
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PCT/CN2013/000482
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French (fr)
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杨代常
施倩妮
张莉平
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武汉禾元生物科技有限公司
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Priority to BR112015010455-0A priority Critical patent/BR112015010455B1/pt
Priority to EP13853496.1A priority patent/EP2918680B1/en
Priority to PL13853496T priority patent/PL2918680T3/pl
Priority to CA2890659A priority patent/CA2890659C/en
Publication of WO2014071681A1 publication Critical patent/WO2014071681A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
    • C07K14/8121—Serpins
    • C07K14/8125—Alpha-1-antitrypsin
    • 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)
    • C12N15/8241—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
    • C12N15/8243—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 involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/8251—Amino acid content, e.g. synthetic storage proteins, altering amino acid biosynthesis
    • 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)
    • C12N15/8241—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

Definitions

  • the invention belongs to the technical field of genetic engineering, and particularly relates to a rice genetic code optimized rA gene, a related vector and a method for producing, separating and purifying OsrAAT from the transgenic rice seed. Background technique
  • Human ⁇ ⁇ -antitrypsin also known as human ⁇ -protein, preparation ( ⁇ 1- ⁇ ), is the most serine-rich protease inhibitor in human peripheral blood. It is mainly synthesized in the liver and inhibits neutrophil elastin in the lungs
  • AAT deficiency is a hereditary disease associated with emphysema and liver disease (Eriksson, 1996).
  • Intravenous injection of plasma-derived human plasma-derived AAT (AAT) is the only viable clinical treatment for patients with AAT deficiency (Insisi and Stoller, 2008).
  • AAT has many therapeutic uses, such as prevention of type 1 diabetes in mice, treatment of skin diseases (Lewis, Shapiro et al., 2005; Brown, 2006), and plays an important anti-inflammatory role in the innate immune system. Dai et al., 2001).
  • the expressed rAAT can only be used in laboratory studies.
  • Yeast as a eukaryotic expression system can perform glycan modification of a high mannose type (Cregg, Cereghino et al., 2000), but this modification differs from that of a human glycan.
  • Deletion and abnormal glycosylation are major problems that prevent yeast expression systems from expressing recombinant polysaccharide proteins.
  • the yield of large-scale production of rAAT by batch fed yeast is as high as 1.23 g/L (Tamer and Chisti, 2001)
  • pharmacokinetic studies indicate that yeast-produced rAAT is 3 ⁇ 4 ⁇ 4 from the blood.
  • mice Such as mice, rabbits, goats and sheep, the expression systems also successfully expressed rAAT (Carlson, Ro g ers and the like,
  • rAAT plant expression systems
  • transgenic tomatoes Agarwal, Singh et al, 2008
  • chloroplasts Nadai, Bally et al, 2009.
  • the expression levels of rAAT in transgenic tomato and chloroplast reached 1.55% to 2% of total protein, respectively.
  • the yield of rAAT in rice cells reached 200 m3 ⁇ 4 / liter. It has recently been discovered that endosperm cells of cereal crops are highly promising expression systems for the production of recombinant proteins.
  • Rice endosperm has been used to express various recombinant drug proteins such as human lactoferrin (Suzuki, Kelleher et al., 2003), human lysogens
  • the present invention also provides a vector comprising the above ⁇ gene, preferably a rice endosperm cell-specific expression vector, and a vector having the structure shown in Fig. 3.
  • Another object of the present invention is to provide the use of the above vector for the preparation of OsrAAT transgenic rice seeds.
  • Another object of the present invention is to provide a method of preparing rA transgenic rice seeds, comprising the following steps:
  • the OsrAAT expression vector preferably has a structure as shown in FIG.
  • the selectable marker gene vector preferably has a structure as shown in FIG.
  • the above method includes the following steps:
  • the rice is shelled into semi-prepared rice and ground into 80-100 mesh rice flour, and the rice flour and extraction buffer are 1:5 by weight (kg) / volume (L).
  • the 6.8-7.1 100-1 lOmMPB buffer was eluted at a flow rate of 100-180 cm/h, and the eluate containing OsrAAT was collected to obtain a primary OsrAAT eluate;
  • the above method for separating and purifying OsrAAT includes the following steps:
  • the rice is shelled into semi-prepared rice and ground into 80-100 mesh rice flour, and the rice flour is mixed with the extraction buffer at a weight/volume ratio of 1 kg: 10 L, at room temperature. After 1 hour of extraction, the obtained mixture was subjected to pressure filtration through a filter-type plate and frame filter press to obtain a clear OsrAAT extract; wherein the composition of the extraction buffer was: 20 mM phosphate buffer, 1 mM mercaptoethanol, pH 7.0. ;
  • Figure 1 is a schematic view showing the structure of plasmid pOsPMP02.
  • Figure 2 is a schematic view showing the structure of plasmid pOsPMP131.
  • Figure 3 is a schematic view showing the structure of plasmid pOsPMP132.
  • Figure 4 is a schematic view showing the structure of plasmid pOsPMP135.
  • Figure 5 is the result of Western hybridization.
  • OsrAAT which was expressed in 9 different transgenic rice, was aggregated in its endosperm cells.
  • Figure 6 is a graph of Southern hybridization results.
  • Figure 7 shows the expression levels of OsrAAT in different plants.
  • Figure 8 is an electrophoresis pattern of chromatographic separation with different fillers by anion exchange chromatography as a primary purification; wherein, Figure 8A is a DEAE sepharose FF filler, Figure 8B is a Macroprep DEAE filler, Figure 8C is a Capto Q filler; M: molecular marker , S: sample loading, FT: breakthrough peak, Elu: OsrAAT elution peak,
  • Figure 9 is an electrophoresis pattern of a composite filler as a primary purification, chromatographed with Macroprep CHT-I; wherein, M: molecular marker, S: sample loading, FT: breakthrough peak, Elu: OsrAAT elution peak, CIP: Clean in place.
  • Figure 10 is an electrophoresis pattern of hydrophobic chromatography as intermediate purification and chromatography with different fillers
  • Figure 10A shows Phenyl sepharose HP filler
  • Figure 10B shows Phenyl sepharose FF HS filler
  • Figure 10C shows Octyl sepharose FF filler
  • M molecular marker
  • S sample loading
  • FT breakthrough peak
  • Elu Elu:
  • Figure 11 is an electrophoresis pattern of a composite filler as a medium-grade purification with different fillers
  • Figure 11 A is Macroprep CHT-I filler
  • Figure 1 IB is Capto MMC filler
  • Figure 11C is Capto
  • Figure 12 is an electrophoresis pattern of a composite filler as a final stage and chromatographed with Capto Adhere;
  • M molecular marker
  • S sample loading
  • FT breakthrough peak
  • Elu OsrAAT elution peak
  • CIP in-situ cleaning.
  • Figure 13 is an electrophoresis pattern of the affinity chromatography as the final purification, chromatography with different fillers; 13A is AAT-select filler, FIG. 13B is ConA sepharose 6B filler; M: molecular marker, S: sample loading, FT: breakthrough peak, Elu: OsrAAT elution peak.
  • Figure 14 is an electrophoresis pattern of crude liquid containing rAAT, which is purified by anion exchange chromatography, cation-bonded metal chelation, and anion-bound hydrophobic interaction chromatography;
  • the fillers used are DEAE sepharose FF, Macroprep CHT-I and Capto Adhere from left to right; M: molecular marker, S: sample loading, FT: breakthrough peak, Elu: OsrAAT elution peak.
  • Figure 15 is an HPLC purity map (HPLC-SEC) of OsrAAT obtained after purification.
  • Figure 16 is the results of analysis of the biological activity of OsrAAT; wherein, the left panel is the result of SDS-PAGE analysis of the band transfer, the right panel is the result of Western hybridization; M: the molecular marker, 1: 132-17 OsrAAT of the T1 plant, 2 : Human plasma sputum, 3: OsrAAT supplemented with porcine elastase, 4: Human plasma AAT supplemented with porcine elastase, 5: 7j extract of Daozhonghua 11.
  • Figure 17 shows the results of measurement of porcine elastase inhibitory activity of OsrAAT.
  • the Macroprep CHT-I filler used in the following examples was produced by BIO-RAD; DEAE Fast Flow Macroprep-DEAE Capto Q Phenyl sepharose HP Phenyl sepahrose FF HS Octyl sepharose FF, Capto MMC, Capto Adhere, AAT- Select, ConA sepharose 6B packing, manufacturer is GE Healthcare; Econo-column 15/20 column, purchased from BIO-RAD; XK16/20 column, purchased from General Electric ( GE Healthcare); Other implementation materials and reagents are commercially available unless otherwise stated.
  • Example 1 Construction of recombinant human antitrypsin vector specific for 7j rice and acquisition of transgenic rice plants
  • the rice-specific expression recombinant human anti-trypsin vector of the present invention and the transgenic rice plants are screened for the method of CN100540667, and the recombinant human serum albumin is replaced with the recombinant human antitrypsin of the invention.
  • a rice endosperm-specific expression cassette was constructed using the plasmid P OsPMP02 as shown in FIG.
  • the synthetic codon-optimized human ⁇ gene (SEQ ID N0.1) was digested with Myll and Xhol and cloned into pOsPMP02 to construct plasmid pOsPMP131, as shown in Figure 2; then ptsPMP131 was digested with ndm and EcoRI.
  • the promoter and its signal peptide sequence also have a codon-optimized gene and the entire expression cassette of the Nw terminator (shown as SEQ ID NO. 2) inserted into the binary expression vector JH2600 to construct an agro-mediated plasmid, named For pOsPMP132, as shown in Figure 3.
  • the pOsPMP132 plasmid and the pOsPMP135 plasmid shown in Figure 4 were transformed into Agrobacterium tumefaciens EHA105 (Invitrogen, USA), respectively, and pOsPMP132 and pOsPMP135 were transformed into the rice variety Zhonghua 11 by Agrobacterium tumefaciens-mediated co-transformation.
  • Southern hybridization was used to identify the T1 plants of the two transgenic rice lines 132-17 and 132-10 obtained above, and about 100 mg of young leaves were taken separately, and ground with liquid nitrogen and a rapid plant genomic DNA extraction system (China Tiangen) Biotechnology Co., Ltd.) extracted genomic DNA, and digested the genomic DNA with EcoRK ndm and EcoRI and ndin (New England Bio Company) at 37 ° C for 8 hours, respectively, and then separated by 0.8% agarose gel; After the gel was transferred to the MILLIPORE NY + membrane, hybridization was carried out according to the instructions of the Roche DIG High Prime DNA Labeling and Detection Starter Kit I; wherein, the following primers (5'-GCATCCATAAATCGCCCCATAG-3' and 5,-GCCCTTGAAGAAGATGTAGTTC-3) were used for amplification.
  • a 645 bp probe was obtained from the coding region of AAT; the results showed that two fragments were detected by digestion with EcoRI or Hndin digestive enzymes, consistent with the results of genetic analysis; single bands obtained by EcoRI and Hndm double digestion Comparing the entire expression cassette, the results show that the Agrobacterium bidirectional storage is in the entire expression cassette.
  • the expression level of OsrAAT in the above nine transgenic rice was also determined by the porcine elastase inhibitory activity assay, and the results showed that the expression level was 0.4- 2.24 mg OsrAAT per gram of brown rice, as shown in Fig. 7. Based on the above experimental results, the transgenic plants 132-17 with the highest expression level were expanded and cultured for further study.
  • the transgenic rice plants were finally screened.
  • Example 2 Chromatographic separation and purification of various media and elution conditions of OsrAAT
  • the present invention uses chromatography to separate and purify OsrAAT from rice seeds, and screens chromatographic media and elution conditions for each level as follows:
  • the inventors selected the fillers to be screened as anion exchange resins having a higher working flow rate, including Macro-prep DEAE manufactured by Burle and DEAE sepahrose FF and Capto Q produced by GE.
  • the target protein can be more effectively separated from impurities, while Macro-prep DEAE loses the target protein at a salt concentration higher than lOmMPB;
  • DEAE sepharose FF has an average particle size of 75 ⁇ m, while Macro The -prep DEAE has a mean diameter of 50 m, and the DEAE sepharose FF has a higher flow rate of use when the purification capacity is comparable.
  • the OsrAAT-containing extract was loaded onto the column containing DEAE sepharose FF at a lower conductivity, pH 7.0 to ensure that OsrAAT was completely adsorbed on the column due to the high activity loss of the target protein at low pH. It is not possible to elute with a pH gradient, so only the PB gradient method is used for elution. It was found that the target protein was eluted at 10-20% 500mMPB, but there was no eluted band before 10% 500mMPB, which means that the process of adding impurities before the elution of the target protein could not be performed. Accordingly, it is considered that at this pH, it is difficult to increase the purity of the target protein by merely increasing the elution salt concentration. Finally, considering the purification effect and recovery rate, 108 mMPB, pH 7.0 was used as the preferred elution condition.
  • the purification process can only be carried out under pH neutral conditions.
  • the selection of a general cation exchange packing inevitably causes the target protein to be unable to hang the column, but the cation exchange serves as a kind of agent to remove impurities such as pigments.
  • the medium has a high use value.
  • the inventors chose Macroprep CHT-I (cation-bound metal chelation) for primary purification.
  • Macroprep CHT-I The sample was activated by the negatively charged phosphate and positively charged calcium ions of Macroprep CHT-I. It was found that Macroprep CHT-I has a good target protein enrichment effect, but its flow rate and load are relatively The DEAE sepharose FF is slightly insufficient, and the extract is only once applied, causing dead adsorption of the pigment on the chromatographic medium, and the medium having about 5% column volume cannot be regenerated. Considering that Macroprep CHT-I has a better purification effect, it cannot be used as a primary purification filler due to the problem of media contamination.
  • the hydrophobic medium has a good removal effect on non-specific impurities in the transgenic rice.
  • the present invention separately uses a plurality of hydrophobic fillers having similar properties to perform the purification steps, including Phenyl sepharose HP, Phenyl sepharose FF (HS) and Octyl. Sepharose FF.
  • Phenyl sepharose FF (HS) has the same ligand and matrix as Phenyl sepharose HP, except for the diameter of the globular matrix and the density of the ligand.
  • the average particle size of the former is about 3 times larger than that of the latter, so it has a higher working flow rate. However, the latter has a finer particle size, which has inconvenience to the application, but has a higher resolution and can be obtained.
  • Phenyl sepharose FF The hydrophobicity of Phenyl sepharose FF is significantly higher than that of Phenyl sepharose HP. Under the condition of 0.8M ammonium sulfate, 80% of the target protein is suspended, the permeate, 50% 7j lotion and pure water eluate are collected and electrophoresed. Detection. As a result, it was found that the purity of the target protein was significantly improved, and the purity of the 40% 7j lotion was up to 80%, but the biological activity was still seriously lost.
  • the hydrophobicity of Octyl sepharose FF has the same matrix as the above filler, and the hydrophobicity is weak.
  • the degree of glycosidation modification of rAAT in transgenic rice is different, and the difference in hydrophobicity of each rAAT can be selectively separated on Octyl filler.
  • the sample treated with Octyl sepharose FF was adjusted to a concentration of 1M ammonium sulfate, loaded onto a column containing Octyl sepharose FF, and the permeate, 40% 7 wash solution and pure water eluate were collected and electrophoresed. Detection. The results showed that the purity of the target protein was significantly improved, and the loss of biological activity was significantly lower than that of Phenyl sepharose FF (HS) and Phenyl sepharose HP, but the activity recovery was still not high.
  • the three hydrophobic fillers have good purification ability for rAAT, but because this medium affects the activity of the target protein, it is not suitable for the purification of rAAT.
  • the hydrophobic environment may cause changes in the spatial structure of the target protein, resulting in protein inactivation.
  • the inventors defined the fillers to be screened as complex chromatography media with higher working flow rates, including macroprep CHT-L Capto MMC and Capto Adhere.
  • Capto MMC is a chromatographic medium with cation exchange and hydrophobic properties.
  • the experiment uses 100mMPB low salt loading and 1.5M ammonium sulfate high salt loading. It is found that the sample is more hydrophobic and is loaded on low salt. In the case where the protein is still suspended, the purity of the penetrating and eluting samples is low; when the salt is loaded, the purity of the penetrating protein is high, up to 85%, but the activity recovery is low. It was found through experiments that Macroprep CHT-I has the best purification effect and high activity recovery.
  • Capto MMC has better purification performance under high salt penetration conditions, but Capto Adhere has the best purification effect but high activity recovery, among which Macroprep Both CHT-I and Capto Adhere can be used for the purification of recombinant human antitrypsin.
  • Macroprep CHT-I can remove many miscellaneous bands, but Capto Adhere can specifically remove some of the bands that Macroprep CHT-I can't remove.
  • Macroprep CHT-I media matrix is rigid and easy to pack, and at high concentration of hydrogen. Excellent stability in sodium oxide, cleaning process and price are better than Capto Adhere.
  • Combining Macroprep CHT-I as a preferred composite chromatography medium, Capto Adhere will be used for the screening of final purification chromatographic media.
  • Capto Adhere in the hydrophobic medium and the composite medium with hydrophobic interaction, except for the anionic and hydrophobic interaction of Capto Adhere, although there are obvious de-doping effects, there are different degrees of loss of activity; Capto Adhere as a medium-grade purified chromatographic medium It does not affect the activity of the sample, and has excellent removal effect on some of the bands, but the removal ability of most of the bands is slightly insufficient.
  • the Macroprep CHT-I filler has obvious advantages in the removal of most impurities and the recovery of activity. Therefore, Macroprep CHT-I was identified as the preferred intermediate purification chromatographic medium; 108mMPB, pH7.0 as the preferred elution condition.
  • Capto adhere is a preferred composite chromatography medium due to the special removal capacity of high molecular weight hybrid bands that cannot be removed by Macroprep CHT-I fillers during intermediate purification.
  • the extract containing OsrAAT was applied to a Capto adhere-loaded column at pH 7.0, conductance at 3.0 ms/cm, and eluted with a mixed gradient of sodium chloride and pH to understand the basic elution conditions. It was found that OsrAAT was gradually eluted during the increase in pH-lowering conductance. When the elution pH is around 6.8 and the conductance reaches about 40 ms/cm, about 80% of the protein of interest is eluted. When the salt concentration is raised again or the pH is lowered, the heteroprotein starts to elute. Taking into account the purification effect and recovery rate, 0.4M sodium chloride containing 46mMPB, pH 6.8 as the elution conditions.
  • the inventors have defined the fillers to be screened as affinity fillers at higher working flow rates, including fillers such as ConA sepharose 6B and ⁇ -select. It was found through experiments that ConA sepharose 6B and AAT-select have good protein purification effects, and both can be used for the purification of recombinant human antitrypsin.
  • the recombinant protein of interest is a collection of proteins with different degrees of glycosidation, and ConA sepharose 6B can bind glycosylated rAAT with unglycosylated rAAT was isolated, and the activity assay showed that the rAAT activity was not affected by the degree of glycosidation.
  • ⁇ -select has more advantages as affinity chromatography for purine purification, it can not distinguish different glycosidation modified ⁇ , so different degrees of glycosylated rAAT are captured and obtained with impurities Effective separation, and the cleaning process and service life of the filler are better than ConAsepharose 6B. Finally, ⁇ -select is used as the preferred affinity chromatography medium.
  • Capto adhere and ⁇ -select can be used for the purification of recombinant human antitrypsin.
  • Capto adhere can remove the ⁇ polymer band that AAT-select can not remove, the HPLC purity can reach up to 97%, which is much higher than 85% of ⁇ -select; the elution condition of target protein in ⁇ -select is 2MMgcl 2 , The elution cost increases, and the high-salt sample is difficult to follow-up; Capto adhere is easier to operate than ⁇ -select chromatography, and the cleaning process and price are better than ⁇ -select.
  • Capto adhere was selected as the preferred filler for the final purification, with 0.4 M sodium chloride containing 46 mMPB and pH 6.8 as the elution conditions.
  • This example is a screening process for chromatographic separation and purification of various chromatographic media and elution conditions of OsrAAT according to Example 2, specifically for separating and purifying OsrAAT.
  • the rice of 132-17 transgenic rice is shelled into semi-prepared rice and ground into 80-100 mesh rice flour.
  • the rice flour and the extraction buffer were mixed at a ratio of 1 : 10 (weight/volume, kg/1), and extracted at room temperature for 1 hour.
  • the fraction of the extraction buffer was: 20 mM phosphate buffer, 1 mM mercaptoethanol, pH 7.0.
  • the mixture obtained above was subjected to pressure filtration through a filter cloth frame filter press to obtain a clear OsrAAT extract as a chromatographic sample.
  • Capto Q packing was placed on an XK16/20 column, and the column was equilibrated with 200 ml of buffer (20 mM phosphate buffer, pH 7.0) at a flow rate of 150 cm/h until the pH and conductance values were unchanged. The sample conductance was 5.3 ms/cm and the pH was 6.95. After the completion of the sample, the sample was eluted with a buffer (108 mMPB, pH 7.0) at a flow rate of 150 cm/h, and the eluate was collected to obtain a rAAT-containing fraction, and ⁇ -mercaptoethanol was added to a final concentration of 4 ⁇ . The chromatographic results are shown in Figure 8C.
  • Phenyl sepharose HP packing was placed on an XK16/20 column with 200 ml of buffer (108 mM phosphate buffer, ammonium sulfate 0.75 M, 1.2 M, 1.5 M, pH 7.0) at 150 cm/h. The flow rate balances the column until its pH and conductance values are unchanged.
  • the above-mentioned 2 ⁇ 1 (DEAE sepharose Fast Flow)-containing elution fraction containing OsrAAT was added to ammonium sulfate (0.75 M, 1 M, 1.5 M) to have an conductance of 95, 135, 165.0 ms/cm, and the pH was adjusted to 6.9.
  • the sample was loaded at a flow rate of 150 cm/h, and the penetrating solution was collected and ⁇ -mercaptoethanol was added to a final concentration of 4 mM for use. The result is shown in Fig. 10A.
  • Phenyl sepahrose FF HS packing was placed on an Econo-column 15/20 column, and the column was equilibrated with 200 ml of buffer (108 mM phosphate buffer, 1.0 M ammonium sulfate, pH 7.0) at a flow rate of 150 cm/h. Until the pH value and conductance value did not change.
  • buffer 108 mM phosphate buffer, 1.0 M ammonium sulfate, pH 7.0
  • the above-mentioned 2.1.1 (DEAE sepharose Fast Flow)-containing elution fraction containing OsrAAT was added to ammonium sulfate (1 M) to conduct electric conductivity of 135.0 ms/cm, adjusted to pH 6.9, and loaded at a flow rate of 150 cm/h.
  • the permeate was collected and ⁇ -mercaptoethanol was added to a final concentration of 4 mM for use. The result is shown in Fig. 10B.
  • the eluted fractions containing OsrAAT obtained in the above 2.1.1 were each 20 ml, diluted four times with water and used as a sample for loading.
  • Capto MMC packing was placed on an XK16/20 column, and the column was equilibrated with 200 ml of buffer (20 mM phosphate buffer, H7.0) at a flow rate of 150 cm/h until the pH and conductance values were unchanged.
  • the sample has a conductivity of 3.0 ms/cm and a pH of 6.9.
  • the sample was eluted with a buffer (108 mMPB, pH 7.0) at a flow rate of 150 cm/h, and the permeate and the eluate were collected for use. The result is shown in Fig. 11B.
  • Capto Adhere was placed on an Econo-column 15/20 column, and the column was equilibrated with 200 ml of buffer (10 mM phosphate buffer, H8.0) at a flow rate of 150 cm/h until the pH and conductance were absent. Variety.
  • the sample conductance was 3.0 ms/cm and the pH was 6.9.
  • the sample was eluted with a buffer (46 mMPB, 400 mM NaCl, pH 6.8) at a flow rate of 150 cm/h, and the eluate was collected to obtain a component containing OsrAAT, and ⁇ -mercaptoethanol was added to a final concentration of 4 mM. .
  • the result is shown in 11C.
  • the OsrAAT-containing eluate obtained by the above-mentioned macroprep CHT-I was used as the final purified sample.
  • Capto Adhere was placed on an Econo-column 15/20 column, and the column was equilibrated with 200 ml of buffer (10 mM phosphate buffer, H8.0) at a flow rate of 150 cm/h until the pH and conductance were absent. Variety.
  • the sample conductance was 3.0 ms/cm and the pH was 6.9.
  • the sample was eluted with a buffer (46 mMPB, 400 mM NaCl, pH 6.8) at a flow rate of 150 cm/h, and the eluate was collected to obtain a component containing OsrAAT.
  • Electrophoresis maps such as 12 Approximately 20 ml of AAT Select was placed on an XK16/20 column, and the column was equilibrated with 200 ml of buffer (20 mM Tris, 150 mM NaCI, pH 7.4) at a flow rate of 150 cm/h until the pH and conductance values were unchanged. The sample conductance was 10.9 ms/cm and the pH was 6.9. After the end of the loading, the sample was eluted with a buffer (20 mM Tris, 2M MgCI 2 , pH 7.4) at a flow rate of 150 cm/h, and the eluate was collected to obtain a component containing OsrAAT. The electropherogram is shown in Figure 13A.
  • the electrophoresis pattern of the preferred triterpene purification of the above primary, intermediate and final stages is shown in Fig. 14.
  • the final separation and purification of OsrAAT by HPLC indicates that the HPLC purity of OsrAAT is 97%, as shown in Fig. 15; and the OsrAAT is collected.
  • the rate is up to 18.89 ⁇ 3.19%, which is equivalent to 0.366g of OsrAAT per kilogram of coarse rice, as shown in Table 1 below.
  • the biological activity of OsrAAT expressed in rice endosperm was evaluated by band shift and porcine elastase inhibitory activity (Huang).
  • the band transfer assay showed that the band transfer of the complex covalently linked to porcine elastase in the crude protein extract was consistent with the plasma-derived AAT (plasma-derived ⁇ , ⁇ ) Western blot, indicating that OsrAAT is effective.
  • the ground is combined with a specific substrate, as shown in Fig.

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Abstract

本发明提供了一种水稻遗传密码子优化的OsrAAT基因，相关载体及其制备OsrAAT转基因水稻种子的方法和OsrAAT的分离纯化方法。通过水稻胚乳细胞特异性表达OsrAAT的表达载体来制备OsrAAT转基因水稻种子，并从中分离纯化OsrAAT，所获得OsrAAT的HPLC纯度为97%，收率可达18.89 ± 3.19%，相当于每公斤的粗米可生产0.366g的OsrAAT。

Description

'种^ 稻种子生产和分离纯化重组人抗胰蛋白酶（OsrAAT)的方法 技术领域
本发明属于基因工程技术领域，具体涉及一种水稻遗传密码子优化的 rA 基因、 相关载体及其从转基因水稻种子生产、 分离和纯化 OsrAAT的方法。 背景技术
人 α ΐ -抗胰蛋白酶（ΑΑΤ), 也称为人 αΐ-蛋白,制剂（α1- ΡΙ)， 是人外周血中最 富含丝氨酸的蛋白酶抑制剂。 其主要是在肝脏中合成， 并在肺中抑制中性粒细胞弹性蛋 白 ||(Blank、 Brantly, 1994)。 AAT缺乏症是一种与肺气肿和肝脏疾病相关的遗传性疾 病 (Eriksson, 1996)。静脉注射增加源自血浆的人 α 1 -抗胰蛋白酶（plasma-derived AAT, AAT) 是治疗 AAT缺乏症病人唯一可行的临床治疗方法 ( Heresi和 Stoller, 2008)。 此外， AAT还有许多的治疗用途，如在预防小鼠 I型糖尿病、治疗皮肤病 (Lewis, Shapiro 等， 2005; Brown, 2006)，并在先天免疫系统中发挥着重要的抗炎作用 (许、戴等， 2001)。
目前， 商业化生产的 pAAT主要是来自人血浆， 其产量受到血液供应的限制， 而且 人源 pAAT 具有传播新的或未知病原体的风险， 因此确保其安全性显得非常复杂 (Kamaukhova, Ophir等， 2006)。除此之外， 为了满足市场需求， 生产重组 αΐ-抗胰蛋白酶 (rAAT )并具有成本效益的替代方法也不断被开发出来。 1983年， 大肠杆菌首先被用 于生产无活性的 rAAT(Bollen等， 1983)。之后用大肠杆菌表达系统表达 rAAT的最高水 平可达到 38mg / L(Kamaukhova等， 2004)。 但是， 由于大肠杆菌表达系统缺乏转录后 的修饰作用， 其表达的 rAAT也只能用于实验室研究。 作为真核表达系统的酵母可以进 行高甘露糖类型的聚糖修饰作用 (Cregg, Cereghino等， 2000)，但这种修饰与人聚糖结构 不同。 缺失和异常的糖基修饰是妨碍酵母表达系统表达重组多糖蛋白的主要问题。 尽管 通过分批补料培养酵母来大规模生产 rAAT 的收率高达到 1.23g/ L(Tamer和 Chisti, 2001)，然而，药代动力学研究表明，酵母生产的 rAAT会被¾¾地从血液中清除 (Casolaro, Fells等， 1987)。 也有研究用黑曲霉表达 rAAT, 因为其糖基化模式是更类似于哺乳动物 (Maras, van Die等， 1999; Gerngross 2004; Ward, Lin等， 2004; Nevalainen, Te'o等， 2005)。 然而， 并没有研究这个系统中的聚糖修饰作用， 而且 50mg / L的表达水平是仍然很低。 因此这个系统表达的 rAAT也仅限于实验室研究。
如小鼠、 兔、 山羊和绵羊等动物表达系统也成功地表达了 rAAT(Carlson, Rogers等，
1989; Archibald, McClenaghan等， 1990; Massoud, Bischoff等， 1991； Wright, Carver等， 1991; Carver, Wright等， 1992; Ziomek, 1998)。也从绵羊奶 palrymple和 Garner, 1998) 和山羊奶 (Ziomek, 1998)中获得大规模生产的 rAAT。 尽管从转基因绵羊奶中分离的 rAAT纯度达 99.9% , 然而在人类体内， 痕量的天然绵羊 AAT和 αΐ-抗糜蛋白酶便可以 诱导一种全身性抗体应答 (Spencer, Humphries 等， 2005)。 也有用植物表达系统生产 rAAT,包括水稻细胞 (Terashima, Murai等， 1999)、转基因番茄 (Agarwal, Singh等， 2008) 和叶绿体 (Nadai, Bally等， 2009)。 其中， 转基因番茄和叶绿体中 rAAT的表达水平分别 达到总蛋白质的 1.55 %至 2 %。在水稻细胞中 rAAT的产量达到 200毫¾ /升。最近发现， 谷物作物的胚乳细胞是很有潜力的可用于生产重组蛋白质的表达系统。 水稻胚乳已被用 来表达各种重组药物蛋白，如人乳铁蛋白 (Suzuki, Kelleher等， 2003)、人溶菌 ||(Yang, Guo 等， 2003)、 rhIGF-1融合和人粒细胞-巨噬细胞群激活因子 (Ning, Xie等， 2008; Xie, Qiu 等， 2008)。 最近， 水稻种子成功地应用于大规模生产的重组人血清白蛋白 (He, Ning等， 2011 这些研究表明， 7j稻胚乳是具有成本效益和安全性的药物蛋白质表达平台。
尽管使用不同的表达系统来表达 rAAT已经取得了进歩， 然而大规模生产植物源重 组人抗胰蛋白酶仍受到表达水平的限制。 而且， 至今未见有关使用水稻胚乳来大规模生 产重组人抗胰蛋白酶的报道， 也未见有关从水稻种子中分离纯化重组人抗胰蛋白酶方法 的报道。 发明内容
本发明的一个目的是提供一种水稻遗传密码子优化的重组人抗胰蛋白酶基因， 以提 高人抗胰蛋白酶基因在水稻种子的表达水平；本发明称 OsrAAT COryza sativaAAT)基因， 具有如 SEQ ID N0.1所示的序列。
进一歩地， 本发明还提供了含有上述 Ο 基因的载体， 优选水稻胚乳细胞特异 性表达载体， 更 具有如图 3所示结构的载体。
本发明的另一个目的是提供上述载体在制备 OsrAAT转基因水稻种子中的应用。 本发明的另一个目的是提供一种制备 rA 转基因水稻种子的方法， 包括以下歩 骤：
( 1 )制备具有如 SEQ ID NO.1所示序列的 OsrAAT基因；
(2)构建水稻胚乳细胞特异性表达的 0^ 表达载体和选择性标记基因载体；
(3 )将歩骤 2所获得载体共转化到水稻品种的愈伤再生组织中；
(4)培养所述愈伤再生组织， 经筛选和诱导获得 (¾ΓΑ 转基因水稻植株；
(5)培养 (¾ΓΑ 转基因水稻植株， 获得 (¾ΓΑ 转因水稻种子。
其中， 所述 OsrAAT表达载体优选具有如图 3所示的结构。
其中， 所述选择性标记基因载体优选具有如图 4所示的结构。
本发明的再一个目的是提供一种从 rA 转基因水稻种子中分离纯化 OsrAAT的 方法， 包括以下歩骤：
( 1 ) 以 Os'rAAT转基因水稻种子为原料制备 OsrAAT提取液；
(2)以阴离子交换层析对 OsrAAT提取液进行初级纯化，获得初级 OsrAAT洗脱液， 其中所述阴离子交换层析的介质为 DEAE sepharose FF;
(3 ) 以阳离子交换结合金属螯合的复合层析对初级 OsrAAT洗脱液进行中级纯化， 获得中级 OsrAAT洗脱液， 其中所述复合层析的介质为 Macroprep CHT-I;
(4) 以阴离子交换结合疏水作用的复合层析对中级 OsrAAT洗脱液进行末级纯化， 获得纯化的 OsrAAT, 其中所述复合层析的介质为 Capto Adhere。
进一歩地， 上述方法包括以下歩骤：
(1 ) 以 (¾ΓΑ 转基因水稻种子为原料， 将稻谷脱壳成半精米并研磨成 80-100目 的米粉， 将所述米粉与提取缓冲液以重量 (kg) /体积 (L)为 1:5-1:10的比例混合， 常温下 提取 1小时后将得到的混合物经滤布式板框压滤机压滤， 得到澄清的 OsrAAT提取液； 其中所述提取缓冲液的成分为： 20-25mM磷酸盐缓冲液、 l-4mM巯基乙醇， pH6.9-7.1;
(2)采用 DEAE sepharose FF填料的层析柱进行初级分离纯化，采用 8-12个柱体积 的 pH为 6.9-7.1的 20-25mM磷酸盐缓冲液， 以 100-180cm/h的流速平衡柱子； 以歩骤 1 的 OsrAAT提取液作为上样样品， 其中样品电导为 2-3.5ms/cm， pH为 6.8-7.0; 用 pH为
6.8- 7.1的 100-1 lOmMPB缓冲液以 100-180cm/h的流速洗脱样品， 收集含有 OsrAAT的 洗脱液， 获得初级 OsrAAT洗脱液；
(3)采用 Macroprep CHT-I层析柱进行次级纯化分离， 采用 8-12个柱体积的 pH为
6.9- 7.2的 5-12mM磷酸盐缓冲液以 100-150cm/h的流速平衡柱子， 以稀释四倍的歩骤 2 中的初级 OsrAAT洗脱液为上样样品， 其中样品电导为 2-3.5ms/cm， pH为 6.8-7.0; 用 pH为 6.8-7.1的 100-llOmMPB缓冲液以 100-180cm/h的流速洗脱样品，收集含有 OsrAAT 的洗脱液， 获得中级 OsrAAT洗脱液；
(4)采用 Capto Adhere层析进行精纯，用 8-12倍柱体积的 pH为 7.5-8.2的 8-12mM 磷酸盐缓冲液以 100-180cm/h的流速平衡柱子， 以中级 OsrAAT洗脱液为上样样品， 其 中样品电导为 2-3.5ms/cm， pH为 6.8-7.1; 用 pH为 6.6-7.0的 46mMPB,400mMNaCl缓 冲液以 100-180cm/h的流速洗脱样品，收集含有 OsrAAT的洗脱液，获得纯化的 OsrAAT。
进一歩地， 上述分离纯化 OsrAAT的方法包括以下歩骤：
(1 ) 以 OsrAAT转基因水稻种子为原料， 将稻谷脱壳成半精米并研磨成 80-100目 的米粉， 将所述米粉与提取缓冲液以重量 /体积为 1 kg: 10L的比例混合， 常温下提取 1 小时后将得到的混合物经滤布式板框压滤机压滤， 得到澄清的 OsrAAT提取液； 其中所 述提取缓冲液的成分为： 20mM磷酸盐缓冲液、 ImM巯基乙醇， pH7.0;
(2)将 20ml的 DEAE sepharose FF填料装于 Econo-column 15/20层析柱上，用 200ml pH为 7.0的 20mM磷酸盐缓冲液， 以 150cm/h的流速平衡柱子； 以 OsrAAT提取液作为 上样样品， 其中样品电导为 2.6ms/cm， pH为 6.95; 用 pH为 7.0的 108mMPB缓冲液以 150cm/h的流速洗脱样品， 收集含有 OsrAAT的洗脱液， 获得初级 OsrAAT洗脱液；
(3 )将 20ml 的 Macroprep CHT-I填料装于 Econo-column 15/20层析柱上，用 200ml pH为 7.0的 ΙΟπ 磷酸盐缓冲液以 150cm/h的流速平衡柱子，以稀释四倍的歩骤 2中初 级 OsrAAT洗脱液为上样样品， 其中样品电导为 3.0ms/cm， pH为 6.9; 用 pH为 7.0的 108mMPB缓冲液以 150cm/h的流速洗脱样品， 收集含有 OsrAAT的洗脱液， 获得中级 OsrAAT洗脱液；
(4)将 10ml的 Capto Adhere装于 Econo-column 15/20层析柱上， 用 200ml pH为 8.0的 10mM磷酸盐缓冲液以 150cm/h的流速平衡柱子， 以中级 OsrAAT洗脱液为上样 样品， 其中样品电导为 3.0ms/cm， pH为 6.9; 用 pH为 6.8的 46mMPB,400mMNaCl缓 冲液以 150cm/h的流速洗脱样品， 收集含有 OsrAAT的洗脱液， 获得纯化的 OsrAAT。 附图说明
图 1是质粒 pOsPMP02的结构示意图。
图 2是质粒 pOsPMP131的结构示意图。
图 3是质粒 pOsPMP132的结构示意图。
图 4是质粒 pOsPMP135的结构示意图。
图 5是 Western杂交结果。
显示 9株不同的转基因水稻表达的 OsrAAT均聚集在其胚乳细胞中。
图 6是 Southern杂交结果图。
其中， 分别用 EcoRK ndm以及同时用 EcoRI和 ndm进行酶切。
图 7显示了不同植株中 OsrAAT的表达水平。
图 8是以阴离子交换层析作为初级纯化， 用不同填料进行层析的电泳图谱； 其中， 图 8A为 DEAE sepharose FF填料， 图 8B为 Macroprep DEAE填料， 图 8C 为 Capto Q填料； M: 分子标记， S: 上样样品， FT: 穿透峰， Elu: OsrAAT洗脱峰，
Elul: 杂质洗脱峰， Elu2: OsrAAT洗脱峰， CIP: 在位清洗。
图 9是以复合填料作为初级纯化， 用 Macroprep CHT-I层析时的电泳图谱； 其中， M: 分子标记， S: 上样样品， FT: 穿透峰， Elu: OsrAAT洗脱峰， CIP: 在 位清洗。
图 10是以疏水层析作为中级纯化， 用不同填料进行层析时的电泳图谱；
其中图 10A为 Phenyl sepharose HP填料， 图 10B为 Phenyl sepharose FF HS填料， 图 10C为 Octyl sepharose FF填料； M: 分子标记， S: 上样样品， FT: 穿透峰， Elu:
OsrAAT洗脱峰， CIP: 在位清洗。
图 11是以复合填料作为中级纯化， 用不同填料进行层析时的电泳图谱；
其中，图 11 A为 Macroprep CHT-I填料，图 1 IB为 Capto MMC填料，图 11C为 Capto
Adhere; M: 分子标记， S: 上样样品， FT: 穿透峰， Elu: OsrAAT洗脱峰， Elul : 杂 质洗脱峰， Elu2: OsrAAT洗脱峰， CIP: 在位清洗。
图 12是以复合填料作为末级纯化， 用 Capto Adhere层析时的电泳图谱；
其中， M: 分子标记， S: 上样样品， FT: 穿透峰， Elu: OsrAAT洗脱峰， CIP: 在 位清洗。
图 13是以亲和填料作为末级纯化， 用不同填料进行层析是的电泳图谱； 其中， 图 13A为 AAT-select填料， 图 13B为 ConA sepharose 6B填料； M: 分子标 记， S: 上样样品， FT: 穿透峰， Elu: OsrAAT洗脱峰。
图 14是含有 rAAT的粗体液依次经过阴离子交换层析、阳离子结合金属螯合作用层 析、 阴离子结合疏水作用层析进行纯化的电泳图谱；
其中使用的填料从左到右依次为 DEAE sepharose FF、 Macroprep CHT-I和 Capto Adhere; M: 分子标记， S: 上样样品， FT: 穿透峰， Elu: OsrAAT洗脱峰。
图 15是纯化后获得的 OsrAAT的 HPLC纯度图谱（HPLC-SEC) 。
图 16是对 OsrAAT生物活性的分析结果； 其中， 左图是条带转移的 SDS-PAGE分 析结果，右图是 Western杂交结果； M:分子标记， 1： 132-17 T1代植株的 OsrAAT, 2： 人血浆 ΑΑΤ, 3： 添加了猪弹性蛋白酶的 OsrAAT, 4： 添加了猪弹性蛋白酶的人血浆 AAT, 5： 7j稻中华 11的提取物。
图 17是 OsrAAT的猪弹性蛋白酶抑制活性的测定结果。 具体实施方式
以下通过结合附图详细说明本发明的特点和优点。 所提供的实施例仅是对本发明方 法的举例说明， 而不以任何方式限制本发明揭示的其余内容。
以下实施例中使用的 Macroprep CHT-I填料，生产商是伯乐（BIO-RAD)公司； DEAE Fast Flow Macroprep-DEAE Capto Q Phenyl sepharose HP Phenyl sepahrose FF HS Octyl sepharose FF、 Capto MMC、 Capto Adhere、 AAT-select、 ConA sepharose 6B填料， 生产商是通用电气 （GE Healthcare ) 公司； Econo-column 15/20 层析柱， 购自伯乐 (BIO-RAD)公司； XK16/20层析柱， 购自通用电气 (GE Healthcare)公司； 其他实 施材料和试剂除特别说明之处均为普通市售。
【实施例 1】 7j稻特异性表达重组人抗胰蛋白酶载体的构建及转基因水稻植株的获 得
人 基因（GenBank登记号： M001002235), 由 Heron Blue生物技术公司根据
7j稻偏好的遗传密码子合成， 将 46.5%的人 α ΐ -抗胰蛋白酶（ΑΑΤ)遗传密码子进行优 化并使 18.1%的人 α ΐ -抗胰蛋白酶核苷酸发生改变， 具体如 SEQ ID N0.1所示， 但是其 相应的氨基酸序列没有变化； 本实施例选用水稻特异性启动子 GtUa及其信号肽来介导 重组人抗胰蛋白酶基因在水稻胚乳细胞中的表达，具体参考公开号为 CN100540667中的 方法来构建本发明的水稻特异性表达重组人抗胰蛋白酶载体以及筛选转基因水稻植株， 将其中所述的重组人血清白蛋白换成本发明的重组人抗胰蛋白酶。 用如图 1所示的质粒 POsPMP02来构建水稻胚乳特异性表达盒。 将所述合成的经密码子优化的人 ΑΛΓ基因 (SEQ ID N0.1 )用 Myll和 Xhol酶切后克隆到 pOsPMP02中构建成质粒 pOsPMP131， 如图 2所示； 然后用 ndm和 EcoRI酶切 pOsPMP131 , 将长度为 2832bp的含 Gtl3a 启动子及其信号肽序列还有经密码子优化的 基因以及 Nw终止子的整个表达盒（如 SEQ ID N0.2 所示） 插入到双元表达载体 JH2600 , 构建农杆介导菌质粒， 命名为 pOsPMP132, 具体如图 3所示。 将所述 pOsPMP132质粒和如图 4所示的 pOsPMP135 质粒分别转化根癌农杆菌 EHA105 (美国 Invitrogen公司）， 通过根癌农杆菌介导共转化 将 pOsPMP132和 pOsPMP135转化到水稻品种中华 11的愈伤再生组织中， 经培养、 筛 选和诱导后形成完整的植株;然后，通过 PCR扩增来鉴别阳性转化植株，用起始于 (^7^7 信号肽的正向引物为 (5 , -GAGGGTGTGGAGGCTCTTGT-3 ' ：)，起始于 基因的反向 引物序列为 (5 ' GCCCTTGAAGAAGATGTAGTTC 3 ' )。 鉴别结果表明， 通过两株根癌 农杆菌介导转化获得 23株独立的重组人 α 1 -抗胰蛋白酶转基因水稻和 12株独立的高产 重组人 α 1 -抗胰蛋白酶转基因水稻。
进一歩地， 用 Western杂交来检测所获得的转基因水稻表达的 OsrAAT是否聚集在 胚乳细胞中， 取约 200mg 7j稻种子用 600μ1 PBS在 4°C条件下磨碎， 然后在 10620 xg 下离心 5分钟， 获得 40ml的粗蛋白提取物和 200ng的源于人体血液的 AAT, 并用 12% SDS-PAGE凝胶进行蛋白分离， 最后用 0.1 %的考马斯亮蓝 R-250进行染色； 结果显示 有 9株转基因水稻表达的 OsrAAT积累在水稻胚乳中，如图 5所示。另外，还采用 Southern 杂交鉴定以上获得的其中两株转基因水稻 132-17和 132-10的 T1植株，分别取约 lOOmg 的嫩叶， 用液氮研磨并用快捷型植物基因组 DNA提取系统（中国天根生物科技有限公 司）提取基因组 DNA, 分别用 EcoRK ndm以及同时用 EcoRI和 ndin (新英格兰 生物公司）在 37°C酶切所述基因组 DNA 8小时， 然后 0.8 %琼脂糖凝胶进行分离； 将 凝胶转移到 MILLIPORE NY +膜后， 按照罗氏 DIG High Prime DNA Labeling and Detection Starter Kit I 的 指 示 进 行 杂 交 ； 其 中 ， 用 以 下 引 物 (5'-GCATCCATAAATCGCCCCATAG-3' 以及 5，-GCCCTTGAAGAAGATGTAGTTC-3，) 扩增 AAT的编码区获得长度为 645bp的探针； 结果显示： 用 EcoRI或 Hndin消化酶酶 切均可检测到两个片段， 与遗传分析的结果一致； EcoRI和 Hndm双酶切获得的单个条 带与整个表达盒相比较， 结果显示所述农杆菌双向载储在于整个表达盒中， 如图 6所
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本实施例还通过猪弹性蛋白酶抑制活性法测定上述 9株转基因水稻中 OsrAAT的表 达水平， 结果显示其表达水平为每克糙米生产 0.4- 2.24mg OsrAAT, 如图 7所示。 综合 上述实验结果，将其中表达水平最高的转基因植株 132-17进行扩大培养用于进一歩的研 究， 为了了解转基因株系 132-17的特征， 分析其 T1种子的遗传分离， 结果表明， 其中 51粒种子表达 OsrAAT, 5粒五种子未表达 OsrAAT, 符合双基因座模型 (15:1, CfflTEST p = 0.408),表明了农杆菌双向载体或插入的基因存在于植株 132-17中。通过以上多种检 测方法， 最终筛选获得转基因水稻植株。
【实施例 2】层析法分离纯化 OsrAAT的各级介质和洗脱条件 本发明使用层析法从水稻种子中分离纯化 OsrAAT, 对各级层析介质和洗脱条件的 筛选如下：
1、 OsrAAT的初级纯化
1.1作为初级纯化的阴离子交换层析中层析介质和洗脱条件的筛选
本发明人将所需要筛选的填料选定为具有较高工作流速的阴离子交换树脂， 包括伯 乐公司生产的 Macro-prep DEAE以及 GE公司生产的 DEAE sepahrose FF和 Capto Q等 填料。
通过实验发现，在低盐上样的情况下， DEAE sepharose FF以及 Macro-prep DEAE蛋 白纯化效果较优， Capto Q纯化效果次之， 三者均可用于重组人抗胰蛋白酶的纯化。 但 是， 在相同的上样条件下， 两种弱阴离子交换介质表现出了较 Capto Q更高的保留时间 和分离效果； 由于水稻种子中具有较高含量的色素和多糖类物质， 使用阴离子填料必然 造成色素等在层析介质上的沉积， 导致载量和纯度的下降， Capto Q受此影响最为明显; 在提高平衡液盐浓度除杂的过程中， DEAE sepharose FF表现出了明显的高盐耐受性，使 得目的蛋白能够更有效的与杂质得到分离，而 Macro-prep DEAE在盐浓度高于 lOmMPB 的情况下， 目的蛋白穿出损失； DEAE sepharose FF平均粒径为 75 μ m， 而 Macro-prep DEAE均径为 50 m，在纯化能力相当的情况下， DEAE sepharose FF具有更高的使用流 速。
将含有 OsrAAT的提取液以较低电导， pH7.0上样于装有 DEAE sepharose FF 的层 析柱上， 以确保 OsrAAT能够完全被吸附在柱子上， 由于目的蛋白在低 pH下活性损失 严重， 不能采用 pH梯度洗脱， 故只采用 PB梯度方法进行洗脱。结果发现， 目的蛋白集 中在 10-20% 500mMPB下被洗脱下来， 但在 10%500mMPB前并无被洗脱下来的杂带， 意味着不能在目的蛋白洗脱前增加一歩除杂的过程， 据此认为， 在该 pH值下， 仅依靠 增加洗脱盐浓度， 很难再提高目的蛋白的纯度。 最后综合考虑纯化效果和回收率， 以 108mMPB,pH7.0作为优选的洗脱条件。
1.2作为初级纯化的复合介质中层析介质的筛选
考虑到目的蛋白的稳定性， 纯化过程只能在 pH中性的条件下进行， 选择一般的阳 离子交换填料必然造成目的蛋白的不能挂柱， 但是阳离子交换作为一种能辦子的去除色 素等杂质的介质又具有较高的使用价值。本发明人选择了 Macroprep CHT-I (阳离子结合 金属螯合作用）用于初歩纯化。
样品是通过 Macroprep CHT-I带负电荷的磷酸根和带正电荷的钙离子与介质发生作 用的， 结果发现， Macroprep CHT-I有较好的目的蛋白富集效果， 但其流速， 载量相对于 DEAE sepharose FF略显不足， 且提取液仅上样一次即造成色素等在层析介质上的死吸 附， 有约 5%柱体积的介质无法再生。 综合考虑， Macroprep CHT-I虽纯化作用较佳， 但 由于介质污染的问题无法作为初级纯化填料使用。
1.3初级纯化层析介质和洗脱条件的确定 综合各种因素， 将 DEAE sepharose FF 作为优选的初级纯化层析介质； 以 108mMPB,pH7.0作为优选的洗脱条件。
2.0srAAT的中级纯化
2.1作为中级纯化的疏水层析中层析介质和洗脱条件的筛选
疏水介质对转基因水稻中非特异性杂质有着较好的去除作用， 本发明分别用多种具 有相近性质的疏水性填料 ¾£行该纯化歩骤，包括 Phenyl sepharose HP、 Phenyl sepharose FF(HS)以及 Octyl sepharose FF。 Phenyl sepharose FF(HS)与 Phenyl sepharose HP相比， 具 有相同的配基和基质， 不同之处在于球状基质的直径和配基的密度。 前者的平均粒径比 后者大 3倍左右， 因此具备较高的工作流速； 但后者由于填料颗粒较细， 虽然给应用上 带来了不便， 但是具有较高的分辨率， 能取得较好的纯化效果。 将经过 Phenyl sepharose HP处理后的样品调整硫酸铵浓度至 0.75M、 1.2M、 1.5M, 上样于装有 Phenyl sepharose HP的层析柱上， 收集穿透液， 50%7j洗液和纯水洗脱液， 并进行电泳检测。 结果发现， 各浓度硫酸铵下均具有较好的杂质去除效果， 得到穿透样品的纯度约为 70%， 但是随着 硫酸铵浓度的升高， 挂柱的目的蛋白增多， 严重影响了目的蛋白的回收， 且穿透样品中 rAAT的生物活性损失严重。
Phenyl sepharose FF的疏水性明显高于 Phenyl sepharose HP, 在 0.8M硫酸铵的条件 下， 80%的目的蛋白挂柱， 收集穿透液， 50%7j洗液和纯水洗脱液， 并进行电泳检测。 结果发现， 目的蛋白纯度有明显改善， 40%7j洗液纯度最高可达到 80%， 但生物活性依 然损失严重。
Octyl sepharose FF 的疏水性与上述填料具有相同的基质不同的配基， 疏水性较弱。 rAAT在转基因水稻中表达的糖苷化修饰程度不同，各 rAAT的疏水性差异能在 Octyl填 料上得到选择性的分离。将经过 Octyl sepharose FF处理后的样品调整硫酸铵浓度至 1M， 上样于装有 Octyl sepharose FF的层析柱上， 收集穿透液， 40%7洗液和纯水洗脱液， 并 进行电泳检测。 结果发现， 目的蛋白纯度有明显改善， 生物活性损失较 Phenyl sepharose FF(HS)和 Phenyl sepharose HP明显减少， 但活性回收仍然不高。
综上所述， 三种疏水性填料对 rAAT具有较好的纯化能力， 但是由于此种介质影响 目的蛋白活性， 不适合用于 rAAT的纯化。 在样品经过疏水性填料的过程中， 疏水环境 可能造成目的蛋白空间结构的变化， 导致蛋白失活。
2.2作为中级纯化的复合层析中层析介质和洗脱条件的筛选
本发明人将所需要筛选的填料限定为且具有较高工作流速的复合性层析介质， 包括 Macroprep CHT-L Capto MMC和 Capto Adhere等填料。
Capto MMC为阳离子交换结合疏水性能的层析介质，实验采用了 lOOmMPB低盐上 样和 1.5M硫酸铵高盐上样两种方式， 结果发现， 样品的疏水性较强， 在低盐上样的情 况下仍有蛋白挂柱， 但穿透和洗脱样品纯度均较低； 高盐上样时， 穿透蛋白纯度较高， 最高达 85%, 但活性回收较低。 通过实验发现 Macroprep CHT-I的纯化效果最好且活性回收较高， Capto MMC在高 盐穿透的条件下纯化效果较好但活性不佳， Capto Adhere纯化效果次之但活性回收高， 其中 Macroprep CHT-I和 Capto Adhere均可用于重组人抗胰蛋白酶的纯化。 Macroprep CHT-I能去除的杂带白较多， 但 Capto Adhere可以针对性的去除部分 Macroprep CHT-I 不能去除的杂带； Macroprep CHT-I介质基质刚性强便于装柱，且在高浓度的氢氧化钠中 具有极佳的稳定性， 清洗工艺和价格均优于 Capto Adhere。综合各种因素， 将 Macroprep CHT-I作为优选的复合层析介质， Capto Adhere将用于末级纯化层析介质的筛选。
由于 CHT填料本身 pH不低于 7.0以及对磷酸盐敏感的特性， 采用磷酸盐梯度洗脱 的方法， 摸索出了 108mMPB， pH7.0的基本洗脱条件。继续对洗脱条件进行优化， 采用 氯化钠梯度洗脱的方法摸索出 Macroprep CHT-I去杂的条件并与原洗脱条件进行比较， 结果发现： 增加洗杂后， 纯度得到明显改善， HPLC的纯度最高可升高至 85%， 但回收 降低了近 10%, 该部分杂质有望在后续纯化歩骤中得到去除。 综合考虑纯化效果和回收 率， 确定了 Macroprep CHT-I不洗杂， 以 108mMPB,pH7.0作为优选的洗脱条件。
2.3中级纯化层析介质和洗脱条件的确定
疏水介质和具有疏水作用的复合介质中， 除了阴离子结合疏水作用的 Capto Adhere 夕卜， 虽均有较明显的去杂效果， 但是都存在不同程度活性丢失问题； Capto Adhere作为 中级纯化的层析介质并不影响样品的活性， 且对部分条带具有极好的去除作用， 但是对 大部分杂带的去除能力稍显不足。 而 Macroprep CHT-I填料在对大部分杂质的去除能力 和活性回收上均有着明显的优势， 因此确定 Macroprep CHT-I作为优选的中级纯化层析 介质； 以 108mMPB,pH7.0作为优选的洗脱条件。
3.0srAAT的末级纯化
3.1作为末级纯化的复合层析中层析介质和洗脱条件的筛选
由于 Capto adhere在中级纯化过程中所显示出的对 Macroprep CHT-I填料所不能去 除的高分子量杂带的特殊去除能力， 将其作为优选的复合层析介质。
将含有 OsrAAT的提取液以 pH7.0， 电导 3.0ms/cm上样与装有 Capto adhere的层析 柱上， 采用氯化钠和 pH混合梯度洗脱的方法， 了解基本的洗脱条件。 结果发现， 在 pH 降低电导升高的过程中， OsrAAT逐渐被洗脱下来。 当洗脱 pH在 6.8左右， 电导达到约 40ms/cm时， 约 80%目的蛋白被洗脱下来。 再升高盐浓度或者将低 pH值时， 杂蛋白开 始被洗脱下来。 综合考虑纯化效果和回收率， 以 0.4M氯化钠含 46mMPB, pH6.8作为 的洗脱条件。
3.2作为末级纯化的亲和层析中层析介质和洗脱条件的筛选
本发明人将所需要筛选的填料限定为较高工作流速的亲和填料， 包括 ConA sepharose 6B和 ΑΛΓ-select等填料。 通过实验发现， ConA sepharose 6B和 AAT-select均 具有较好的蛋白纯化效果， 两者均可用于重组人抗胰蛋白酶的纯化。 重组的目的蛋白为 糖苷化程度不同的蛋白集合体， ConA sepharose 6B可将糖苷化的 rAAT与未糖苷化的 rAAT分离开来， 活性测定结果表明 rAAT活性并不受糖苷化程度的影响， 挂柱洗脱的 rAAT纯度较高， 穿透出来的有活性无糖苷化的目的蛋白将被损失， 导致该方法蛋白与 活性回收均不高； ΑΛΓ-select作为专用于 ΑΛΓ纯化的亲和层析具有更多优势，它无法分 辨不同糖苷化修饰的 ΑΛΓ, 因而不同程度糖苷化的 rAAT均被捕获， 并与杂质得到有效 分离， 且填料的清洗工艺、使用寿命均优于 ConAsepharose 6B。最后将 ΑΛΓ-select作为 优选的亲和层析介质。
3.3末级纯化层析介质和洗脱条件的确定
Capto adhere和 ΑΛΓ-select均可用于重组人抗胰蛋白酶的纯化。但是 Capto adhere可 去除 AAT-select不能去除的 ΑΛΓ 多聚体条带， HPLC纯度最高可达到 97%, 远高于 ΑΛΓ-select的 85%; ΑΛΓ-select中目的蛋白的洗脱条件为 2MMgcl2,洗脱成本增加， 高盐 样品后续处理困难； Capto adhere较 ΑΛΓ-select层析操作容易，且清洗工艺和价格都优于 ΑΛΓ-select。
综合考虑，确定 Capto adhere作为末级纯化优选的填料，以 0.4M氯化钠含 46mMPB, pH6.8作为^ t的洗脱条件。
【实施例 3】 OsrAAT的分离纯化
本实施例是根据实施例 2中对层析法分离纯化 OsrAAT的各级层析介质和洗脱条件 的筛选过程， 具体对 OsrAAT进行分离纯化。
1.OsrAAT层析样品的制备
将 132-17转基因水稻的稻谷脱壳成半精米， 研磨成 80-100目的米粉。 将米粉与提 取缓冲液以 1 : 10 (重量 /体积， kg/1)的比例混合， 于常温提取 1小时。提取缓冲液的成 分为： 20mM磷酸盐缓冲液、 ImM巯基乙醇， pH7.0。 将上述得到的混合物经滤布式板 框压滤机压滤， 得到澄清的 OsrAAT提取液作为层析样品。
2.初级纯化
2.1通过阴离子交换层析进行初级纯化
2.1.1利用 DEAE sepharoseFast Flow进行阴离子交换层析
将 DEAE sepharoseFast Flow填料约 20ml装于 Econo-column 15/20层析柱上， 用 200ml缓冲液（20mM磷酸盐缓冲液， pH7.0) 以 150cm/h的流速平衡柱子， 直到其 pH 值、 电导值无变化。 样品电导为 2.6mS/cm， pH 为 6.95。 上样结束后， 用缓冲液 (108mMPB,pH7.0) 以 150cm/h的流速洗脱样品， 收集洗脱液， 得到含有 OsrAAT的组 分,添加 β -巯基乙醇至终浓度为 4mM备用。 层析结果如图 8A所示。
2.1.2利用 Macroprep-DEAE进行阴离子交换层析
将 Macroprep-DEAE填料约 16ml装于 XK16/20层析柱上，用 200ml缓冲液（20mM 磷酸盐缓冲液， H7.0) 以 150cm/h的流速平衡柱子， 直到其 pH值、 电导值无变化。 样 品电导为 5.3ms/cm， pH为 6.95。上样结束后， 用缓冲液（108mMPB,pH7.0)以 150cm/h 的流速洗脱样品， 收集穿透液和洗脱液备用。 层析结果如图 8B所示。 2.1.3利用 Capto Q进行阴离子交换层析
将 Capto Q填料约 15ml装于 XK16/20层析柱上， 用 200ml缓冲液（20mM磷酸盐 缓冲液， pH7.0) 以 150cm/h的流速平衡柱子， 直到其 pH值、 电导值无变化。样品电导 为 5.3ms/cm， pH为 6.95。 上样结束后， 用缓冲液 (108mMPB,pH7.0) 以 150cm/h的流 速洗脱样品， 收集洗脱液， 得到含有 rAAT的组分，添加 β -巯基乙醇至终浓度为 4π 备 用。 层析结果如图 8C所示。
2.2通过复合层析进行初级纯化
利用 Macroprep CHT-I进行阳离子交换结合金属螯合层析
将 Macroprep CHT-I填料约 20ml装于 Econo-column 15/20层析柱上，用 200ml缓冲 液（lOmM磷酸盐缓冲液， pH7.0) 以 150cm/h的流速平衡柱子， 直到其 pH值、 电导值 无变化。 样品电导为 1.5ms/cm， pH为 6.9。 上样结束后， 用缓冲液（108mMPB,pH7.0) 以 150cm/h的流速洗脱样品， 收集洗脱液， 得到含有 rAAT的组分，添加 β -巯基乙醇至终 浓度为 4mM备用。 层析结果如图 9所示。
以上四种不同层析介质的交换层析效果图如图 8和图 9所示， 将其中洗脱效果最佳 的 DEAE sepharosse Fast Flow中洗脱得到的含有 OsrAAT 的组分的洗脱液进行以下中级 纯化。
3.中级纯化
3.1通过疏水层析进行中级纯化
3.1.1 利用 Phenyl sepharose HP进行疏水层析
将 Phenyl sepharose HP填料约 20ml装于 XK16/20层析柱上， 用 200ml缓冲液 (108mM磷酸盐缓冲液， 硫酸铵分别为 0.75M, 1.2M, 1.5M, 调 pH7.0) 以 150cm/h的 流速平衡柱子， 直到其 pH值、 电导值无变化。将上述 2丄 1 (DEAE sepharoseFast Flow) 获得的含有 OsrAAT的洗脱流分中加入硫酸铵（0.75M, 1M， 1.5M), 使其电导为 95， 135， 165.0ms/cm， 调 pH为 6.9， 以 150cm/h的流速上样， 收集穿透液并添加 β -巯基乙 醇至终浓度为 4mM备用。 结果如图 10A所示。
3.1.2利用 Phenyl sepahrose FF HS进行疏水层析
将 Phenyl sepahrose FF HS填料约 20ml装于 Econo-column 15/20层析柱上，用 200ml 缓冲液（108mM磷酸盐缓冲液， 1.0M硫酸铵， pH7.0) 以 150cm/h的流速平衡柱子， 直 到其 pH值、电导值无变化。将上述 2.1.1 (DEAE sepharose Fast Flow)获得的含有 OsrAAT 的洗脱流分中加入硫酸铵（1M)， 使其电导为 135.0ms/cm， 调 pH为 6.9， 以 150cm/h的 流速上样， 收集穿透液并添加 β -巯基乙醇至终浓度为 4mM备用。 结果如图 10B所示。
3.1.3 利用 Octyl sepharose FF进行疏水层析
将 Octyl sepharose FF填料约 20ml装于 XK16/20层析柱上，用 200ml缓冲液 (20mM 磷酸盐缓冲液， 1M硫酸铵， pH7.0) 以 150cm/h的流速平衡柱子， 直到其 pH值、 电导 值无变化。将上述 2丄 1 (DEAE sepharose Fast Flow)获得的含有 OsrAAT的洗脱流分中 加入硫酸铵（1M)， 使其电导为 120.0ms/cm， 调 pH为 6.9， 以 150cm/h的流速上样， 收 集穿透液并添加 β -巯基乙醇至终浓度为 4mM备用。 结果如图 10C所示。
3.2通过复合层析进行中级纯化
3.2.1通过 Macroprep CHT-I进行阳离子交换结合金属螯合层析
取上述 2.1.1 (DEAE sepharose Fast Flow)获得的含有 OsrAAT的洗脱流分各 20ml,加 水稀释四倍后作为上样样品。
将 Macroprep CHT-I填料约 20ml装于 XK16/20层析柱上，用 200ml缓冲液（ ΙΟπ 磷酸盐缓冲液， H7.0) 以 150cm/h的流速平衡柱子， 直到其 pH值、 电导值无变化。 样 品电导为 3.0ms/cm， pH为 6.9。 上样结束后， 用缓冲液（108mMPB,pH7.0) 以 150cm/h 的流速洗脱样品， 收集洗脱液， 得到含有 OsrAAT 的组分，添加 β -巯基乙醇至终浓度为 4mM备用。 结果如图 11A所示。
3.2.2通过 Capto MMC进行阳离子结合疏水层析
将 Capto MMC填料约 20ml装于 XK16/20层析柱上， 用 200ml缓冲液（20mM磷 酸盐缓冲液， H7.0) 以 150cm/h的流速平衡柱子， 直到其 pH值、 电导值无变化。 样品 电导为 3.0ms/cm， pH为 6.9。上样结束后， 用缓冲液（108mMPB,pH7.0) 以 150cm/h的 流速洗脱样品， 收集穿透液和洗脱液备用。 结果如图 11B所示。
3.2.3通过 Capto Adhere进行阴离子结合疏水层析
将 Capto Adhere约 10ml装于 Econo-column 15/20层析柱上，用 200ml缓冲液（10mM 磷酸盐缓冲液， H8.0) 以 150cm/h的流速平衡柱子， 直到其 pH值、 电导值无变化。 样 品电导为 3.0ms/cm， pH为 6.9。 上样结束后， 用缓冲液（46mMPB,400mMNaCl,pH6.8) 以 150cm/h的流速洗脱样品， 收集洗脱液， 得到含有 OsrAAT的组分，添加 β -巯基乙醇 至终浓度为 4mM备用。 结果如 11C所示。
将上述洗脱效果最好的 Macroprep CHT-I所获得的含 OsrAAT的洗脱液作为以下末 级纯化的上样样品。
4.末级纯化
4.1通过复合层析进行末级纯化
4.1.1通过 Capto Adhere进行阴离子结合疏水层析
将 Capto Adhere约 10ml装于 Econo-column 15/20层析柱上，用 200ml缓冲液（10mM 磷酸盐缓冲液， H8.0) 以 150cm/h的流速平衡柱子， 直到其 pH值、 电导值无变化。 样 品电导为 3.0ms/cm， pH为 6.9。 上样结束后， 用缓冲液（46mMPB,400mMNaCl,pH6.8) 以 150cm/h的流速洗脱样品， 收集洗脱液， 得到含有 OsrAAT的组分。 电泳图谱如 12所 将 AAT Select约 20ml装于 XK16/20层析柱上，用 200ml缓冲液 (20mM Tris ,150mM NaCI,PH7.4) 以 150cm/h的流速平衡柱子， 直到其 pH值、 电导值无变化。 样品电导为 10.9ms/cm， pH为 6.9。上样结束后，用缓冲液（20mM Tris, 2M MgCI2 ,PH 7.4)以 150cm/h 的流速洗脱样品， 收集洗脱液， 得到含有 OsrAAT的组分。 电泳图谱如图 13A所示。
4.2.2利用 ConA Sepharose FF 6B进行亲和层析
将 ConA Sepharose FF 6B约 10ml装于 XK16/20层析柱上， 用 200ml缓冲液 (20mMTris-Hcl PH7.4含 0.5M Nacl ,lmM Mn2+.lmM Ca2+)以 150cm/h的流速平衡柱子， 直到其 pH值、 电导值无变化。样品电导为 10.9mS/cm， pH为 6.9。上样结束后， 用缓冲 液（0.1M glucose)以 150cm/h的流速洗脱样品，收集洗脱液，得到含有 OsrAAT的组分。 电泳图谱如 13B所示。
综合上述初级、中级和末级的优选三歩纯化的电泳图谱如图 14所示，最终分离纯化 获得的 OsrAAT经 HPLC检测， 表明 OsrAAT的 HPLC纯度为 97%, 如图 15所示； 且 OsrAAT的收率可达 18.89 士 3.19%， 相当于每公斤的粗米可产 0.366g的 OsrAAT, 具 体如下表 1所示。
Figure imgf000014_0001
【实施例 4】 OsrAAT的生物活性分析
采用条带转移 （band shift) 以及猪弹性蛋白酶抑制活性法（Huang)评估水稻胚乳 所表达的 OsrAAT的生物活性。 条带转移测定结果发现在粗蛋白提取物中与猪弹性蛋白 酶共价连接的复合物的条带转移与源自血浆的 AAT (plasma-derived ΑΑΓ, ρΑΛΓ) Western 印迹结果相一致，表明 OsrAAT可有效地与特定底物结合，具体如图 16所示； 为了进一 歩确定 OsrAAT是否具有与 ρΑΛΓ相同的猪弹性蛋白酶抑制活性， 猪弹性蛋白酶抑制活 性法检测结果发现： OsrAAT的猪弹性蛋白酶抑制活性与 ρΑΑΤ相一致， 具体如图 17所
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Claims

权利要求书
1.一种水稻遗传密码子优化的 Ο 基因， 具有如 SEQ ID N0.1所示的序列。
2.—种含有权利要求 1所述 C^rA 基因的载体。
3.权利要求 2所述的载体是水稻胚乳细胞特异性表达载体。
4.权禾腰求 3所述载体， 具有如图 3所示的结构。
5.权利要求 2-4中任一项所述的载体在制备 Ο 转基因水稻种子中的应用。
6.如权利要求 1所述的水稻遗传密码子优化的 0^ 基因在制备 rA 转基因 水稻种子中的应用。
7.一种制备 rA 转基因水稻种子的方法， 包括以下歩骤：
( 1 )制备具有如 SEQ ID NO.1所示序列的 OsrAAT基因；
(2)构建水稻胚乳细胞特异性表达的 0^ 表达载体和选择性标记基因载体；
(3 )将歩骤 2所获得载体共转化到水稻品种的愈伤再生组织中；
(4)培养所述愈伤再生组织， 经筛选和诱导获得 (¾ΓΑ 转基因水稻植株；
(5)培养 (¾ΓΑ 转基因水稻植株， 获得 (¾ΓΑ 转因水稻种子。
8.权利要求 7所述的方法，其特征在于，所述 0^ 表达载体具有如图 3所示的结
9.权利要求 7所述的方法， 其特征在于， 所 ¾t择性标记基因载体具有如图 4所示 的结构。
10.一种从 C^rA 转基因水稻种子中分离纯化 OsrAAT的方法， 包括以下歩骤：
( 1 ) 以 Os'rAAT转基因水稻种子为原料制备 OsrAAT提取液；
(2)以阴离子交换层析对 OsrAAT提取液进行初级纯化，获得初级 OsrAAT洗脱液， 其中所述阴离子交换层析的介质为 DEAE sepharose FF;
(3 ) 以阳离子交换结合金属螯合的复合层析对初级 OsrAAT洗脱液进行中级纯化， 获得中级 OsrAAT洗脱液， 其中所述复合层析的介质为 Macroprep CHT-I;
(4) 以阴离子交换结合疏水作用的复合层析对中级 OsrAAT洗脱液进行末级纯化， 获得纯化的 OsrAAT, 其中所述复合层析的介质为 Capto Adhere。
11.权利要求 10所述的方法， 其特征在于： 包括以下歩骤：
( 1 ) 以 (¾ΓΑ 转基因水稻种子为原料， 将稻谷脱壳成半精米并研磨成 80-100目 的米粉， 将所述米粉与提取缓冲液以重量 /体积为 1 :5-1 :10的比例混合， 常温下提取 1小 时后将得到的混合物经滤布式板框压滤机压滤， 得到澄清的 OsrAAT提取液； 其中所述 提取缓冲液的成分为： 20-25mM磷酸盐缓冲液、 l-4mM巯基乙醇， pH6.9-7.1 ;
(2)采用 DEAE sepharose FF填料的层析柱进行初级分离纯化，采用 8-12个柱体积 的 pH为 6.9-7.1的 20-25mM磷酸盐缓冲液， 以 100-180cm/h的流速平衡柱子； 以歩骤 1 的 OsrAAT提取液作为上样样品， 其中样品电导为 2-3.5ms/cm， pH为 6.8-7.0; 用 pH为 6.8-7.1的 100-1 lOmMPB缓冲液以 100-180cm/h的流速洗脱样品， 收集含有 OsrAAT的 洗脱液， 获得初级 OsrAAT洗脱液；
(3)采用 Macroprep CHT-I层析柱进行次级纯化分离， 采用 8-12个柱体积的 pH为 6.9-7.2的 5-12mM磷酸盐缓冲液以 100-150cm/h的流速平衡柱子， 以稀释四倍的歩骤 2 中的初级 OsrAAT洗脱液为上样样品， 其中样品电导为 2-3.5ms/cm， pH为 6.8-7.0; 用 pH为 6.8-7.1的 100-llOmMPB缓冲液以 100-180cm/h的流速洗脱样品，收集含有 OsrAAT 的洗脱液， 获得中级 OsrAAT洗脱液；
(4)采用 Capto Adhere层析进行精纯，用 8-12倍柱体积的 pH为 7.5-8.2的 8-12mM 磷酸盐缓冲液以 100-180cm/h的流速平衡柱子， 以中级 OsrAAT洗脱液为上样样品， 其 中样品电导为 2-3.5ms/cm， pH为 6.8-7.1; 用 pH为 6.6-7.0的 46mMPB,400mMNaCl缓 冲液以 100-180cm/h的流速洗脱样品，收集含有 OsrAAT的洗脱液，获得纯化的 OsrAAT。
12.权利要求 11所述的方法， 其特征在于： 包括以下歩骤：
(1 ) 以 (¾ΓΑ 转基因水稻种子为原料， 将稻谷脱壳成半精米并研磨成 80-100目 的米粉，将所述米粉与提取缓冲液以重量 /体积为 1:10的比例混合，常温下提取 1小时后 将得到的混合物经滤布式板框压滤机压滤， 得到澄清的 OsrAAT提取液； 其中所述提取 缓冲液的成分为： 20mM磷酸盐缓冲液、 ImM巯基乙醇， pH7.0;
(2)采用 DEAE sepharose FF填料的层析柱进行初级分离纯化， 采用 10个柱体积 的 pH为 7.0的 20mM磷酸盐缓冲液， 以 150cm/h的流速平衡柱子； 以歩骤 1的 OsrAAT 提取液作为上样样品，其中样品电导为 2.6ms/cm， pH为 6.95;用 pH为 7.0的 108mMPB 缓冲液以 150cm/h的流速洗脱样品， 收集含有 OsrAAT的洗脱液， 获得初级 OsrAAT洗 脱液；
(3)采用 Macroprep CHT-I层析柱进行次级纯化分离， 采用 10个柱体积的 pH为 7.0的 10mM磷酸盐缓冲液以 150cm/h的流速平衡柱子， 以稀释四倍的歩骤 2中初级 OsrAAT洗脱液为上样样品， 其中样品电导为 3.0ms/cm， pH为 6.9; 用 pH为 7.0 的 108mMPB缓冲液以 150cm/h的流速洗脱样品， 收集含有 OsrAAT的洗脱液， 获得中级 OsrAAT洗脱液；
(4)采用 Capto Adhere层析进行精纯， 用 10倍柱体积的 pH为 8.0的 10mM磷酸 盐缓冲液以 150cm/h的流速平衡柱子， 以中级 OsrAAT洗脱液为上样样品， 其中样品电 导为 3.0ms/cm， pH为 6.9; 用 pH为 6.8的 46mMPB,400mMNaCl缓冲液以 150cm/h的 流速洗脱样品， 收集含有 OsrAAT的洗脱液， 获得纯化的 OsrAAT。
13. 一种含有如权利要求 1所述水稻遗传密码子优化的 rA 基因的转基因水稻。
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CN111285929B (zh) * 2018-12-10 2023-09-19 武汉禾元生物科技股份有限公司 一种从基因工程水稻种子中分离纯化重组人表皮细胞生长因子的方法

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