WO2009124503A1 - Use of protein extract from plant seed embryo and composition thereof - Google Patents

Abstract

Disclosed is a use of a plant seed embryo protein extract for stabilizing the bioactivity of an active protein and a method for preparing said protein extract. The present invention also provides a composition comprising at least one kind of protein extract from plant seed embryo, an active protein, and an acceptable pharmaceutical carrier or zymological carrier.

Description

 Application of protein extract of plant seed embryo and composition thereof

 The invention belongs to the field of biotechnology, and particularly relates to the application of a protein extract of a plant seed embryo for stabilizing the active protein biological activity, a preparation method of the protein extract and a composition containing the protein extract. Background technique

 Seeds are a period in the life history of seed plants at the highest stage of plant kingdom evolution and are the beginning of the next generation of independent plants. Mature seeds are composed of three parts: embryo, endosperm and seed coat. The embryo is the most important part of the seed, it is the original body of the new plant, from the fertilized egg (zygotic). A fully developed embryo consists of a plumule, a radicle, a hypocotyl, and a cotyledon. The seeds of angiosperms are encased in the fruit developed by the ovary; the gymnosperms have no ovary and the seeds are bare. During the development of the seeds of angiosperms, the endosperm is absorbed as seeds without endosperm, such as soybean, cotton, and rapeseed; the endosperm is not absorbed, which is called endosperm seeds, such as seeds of wheat, rice, and corn. The seeds are prepared for transmission from the structure and physiology, and sufficient nutrients are stored for the seedlings to grow into autotrophs. The protein plays an extremely important role in seed formation, development and seedling formation. It provides nutrients for seed growth and development, and also regulates various physiological and biochemical reactions and metabolic processes of seeds.

 Protein is one of the most basic substances in life, the material basis of life activities. Life activities are almost always realized by proteins. Some proteins are structural substances in organisms, and some proteins are functional substances in organisms (such as living organisms). Catalyst enzyme). In addition, many proteins, such as insulin, interferons, immunoglobulins, etc., can be used as drugs for the treatment of diseases.

Due to the large molecular weight and complex structure of proteins, protein molecules are very unstable, susceptible to physical or chemical factors and denatured, losing their biological activity. The instability of proteins is due to the instability caused by covalent bonds, such as hydrolysis, oxidation and racemization, in addition to the unique reaction of proteins, namely the breaking and exchange of disulfide bonds. In addition, instability caused by non-covalent bonds, such as aggregation, macroscopic precipitation, surface adsorption and protein denaturation. Especially when the concentration of the biologically active protein is low, protein degradation or protein adsorption by the wall is prone to occur, thereby affecting the biological activity of the protein preparation. One way to address the stability of proteinaceous drugs is to add protein stabilizers to low concentrations of protein drugs to protect proteins from degradation. These protein stabilizers are typically proteins of animal origin, such as human serum albumin. However, the use of animal proteins presents a serious potential risk that animal proteins are contaminated by animal viruses that are not completely killed. For example, cases of public hazards in human serum albumin products due to HIV contamination have been reported. In addition, animal proteins generally only competitively prevent biologically active proteins from being physically adsorbed by the tube wall, or are damaged by physical, chemical and biological factors in the environment, so their protective properties are also limited. Therefore, there is an urgent need in the art to provide a novel protein stabilizer that is safe, inexpensive, and efficient. Summary of the invention The invention relates to the use of a protein extract of a plant seed embryo for stabilizing the biological activity of an active protein.

 In a preferred embodiment, the method for preparing the protein extract of the plant seed embryo comprises using a protein extract (preferably a salt solution, more preferably 0.5% to 2.5% (W/), which does not cause severe degradation or inactivation of the protein. V) NaCl solution, preferably 0.9% to 2% (w/v) NaCl solution, most preferably 0.9% (w/v) physiological saline) is a step of extracting protein from plant seed embryos.

 In a more preferred embodiment, the method for preparing a protein extract of the plant seed embryo comprises the steps of: 1) degreasing and drying the embryo of the plant seed; 2) using protein extraction that does not cause severe degradation or inactivation of the protein. Liquid (preferably a salt solution, more preferably 0.5% to 2.5% (WA NaCl solution, still more preferably 0.9% to 2% (WA NaCl solution, most preferred)

0.9% (W/V) saline was extracted to obtain a protein extract; 3) The protein extract obtained in the step 2) was sterilized.

 In a still further preferred embodiment, the method for preparing a protein extract of the plant seed embryo comprises the steps of: 1) degreasing and drying the embryo of the plant seed; 2) using deionized water or steaming water for step 1 Extracting the embryos of the degreased, dried plant seeds, removing the water-soluble protein extract to obtain a precipitate; 3) using a salt solution (preferably 0.5% to 2.5% (W/V) NaCl solution, more preferably 0.9) %~2%CWA NaCl solution) The precipitate obtained in step 2) is extracted to collect the salt-soluble protein extract; 4) The salt-soluble protein extract obtained in step 3) is sterilized.

 In a preferred embodiment, the plant seed is selected from the group consisting of corn, wheat, rice, sorghum, barley, rye, oats, soybeans, peas or soybeans or a combination thereof.

 In another preferred embodiment, the active protein is selected from the group consisting of an enzyme, an antibody, an antigen, an allergen, an interleukin, a tumor necrosis factor, a transforming growth factor, an interferon, an insulin or a glucagon-like peptide. Wherein, the enzyme is preferably amylase, phytase, lipase, xylanase, cellulase, alkaline mannanase, superoxide dismutase, nucleic acid restriction enzyme, exonuclease Or a nucleic acid ligase or the like.

 The invention also provides a composition comprising at least one protein extract of a plant seed embryo, an active protein, and a pharmaceutically or enzymatically acceptable carrier. In one embodiment, the composition is comprised of a protein extract of at least one plant seed embryo, an active protein, and a pharmaceutically or enzymatically acceptable carrier.

 In a preferred embodiment, the active protein is selected from the group consisting of an enzyme, an antibody, an antigen, an allergen, an interleukin, a tumor necrosis factor, a transforming growth factor, an interferon, an insulin or a glucagon-like peptide. Wherein, the enzyme is preferably amylase, phytase, lipase, xylanase, cellulase, alkaline mannanase, superoxide dismutase, nucleic acid restriction enzyme, exonuclease Or a nucleic acid ligase or the like.

 In another preferred embodiment, the plant seed is selected from the group consisting of corn, wheat, rice, sorghum, barley, rye, oats, soybeans, peas or soybeans or a combination thereof.

 In a preferred embodiment, the method for preparing the protein extract of the plant seed embryo comprises using a protein extract (preferably a salt solution, more preferably 0.5% to 2.5% (W/), which does not cause severe degradation or inactivation of the protein. V) NaCl solution, preferably 0.9% to 2% (w/v) NaCl solution, most preferably 0.9% (w/v) physiological saline) is a step of extracting protein from plant seed embryos.

In a more preferred embodiment, the method for preparing a protein extract of the plant seed embryo comprises the steps of: 1) degreasing and drying the embryo of the plant seed; 2) using protein extraction that does not cause severe degradation or inactivation of the protein. Liquid (preferably salt The solution is more preferably 0.5% to 2.5% (WA NaCl solution, more preferably 0.9% to 2% (WA NaCl solution, most preferred)

0.9% (W/V) saline was extracted to obtain a protein extract; 3) The protein extract obtained in the step 2) was sterilized.

 In a still further preferred embodiment, the method for preparing a protein extract of the plant seed embryo comprises the steps of: 1) degreasing and drying the embryo of the plant seed; 2) using deionized water or steaming water for step 1 Extracting the embryos of the degreased, dried plant seeds, removing the water-soluble protein extract to obtain a precipitate; 3) using a salt solution (preferably 0.5% to 2.5% (W/V) NaCl solution, more preferably 0.9) %~2%CWA NaCl solution) The precipitate obtained in step 2) is extracted to collect the salt-soluble protein extract; 4) The salt-soluble protein extract obtained in step 3) is sterilized.

 In the composition of the present invention, the ratio of the amount of the active protein to the protein amount of the protein extract of the plant seed embryo is preferably 1:0.1 to 1:500, preferably 1:1-1:400, more preferably 1 : 5-1: 200, most preferably 1: 10-1: 100.

 Still another aspect of the invention relates to a method of preparing a preparation for stabilizing the biological activity of an active protein, characterized in that the method comprises the steps of: plant seed embryos with a protein extract which does not cause severe degradation or inactivation of the protein The protein is extracted to obtain the preparation for stabilizing the biological activity of the active protein.

 In a preferred embodiment, the method comprises the steps of: 1) defatting and drying the embryos of the plant seeds; 2) using a protein extract (preferably a salt solution, more preferably 0.5%) that does not cause severe degradation or inactivation of the protein. ~2.5% (W / V) NaCl solution, preferably 0.9% ~ 2% (W / V) NaCl solution, most preferably 0.9% (W / V) saline) extraction, to obtain protein extract; 3) the steps 2) The obtained protein extract is sterilized.

 In a more preferred embodiment, the method comprises the steps of: 1) defatting and drying the embryos of the plant seeds; 2) treating the degreased, dried plants of step 1) with deionized water or distilled water. The embryo of the seed is extracted, the water-soluble protein extract is removed, and a precipitate is obtained; 3) a salt solution (preferably 0.5% to 2.5% (W/V) NaCl solution, more preferably 0.9% to 2% (W/V) NaCl is used. Solution) The precipitate obtained in the step 2) is extracted to collect the salt-soluble protein extract; 4) The salt-soluble protein extract obtained in the step 3) is sterilized.

 Another aspect of the invention relates to a method of increasing the stability of an active protein in a formulation, the method comprising adding to the formulation a protein extract of at least one plant seed embryo as described above.

 The data of the examples of the present invention confirmed that the protein extract of the plant seed embryo of the present invention was used as a stabilizer for the active protein to obtain an excellent effect. Protein extracts from plant seed embryos can be used as a safe, inexpensive, and efficient new protein stabilizer. DRAWINGS

 Figure 1 shows the SDS-PAGE electropherogram of the three wheat seed protein extracts of Example 2. In the figure, Lane 1, protein extract of wheat seed embryo (water salt step extraction method) (protein extract obtained in Example 1 (3)); Lane 2, protein extract of wheat seed embryo (physiological saline one-step method) (Example 1 (b) protein extract obtained); Lane 3, wheat seed whole wheat (embryo + endosperm) protein extract (physiological saline one-step method) (Example 1 (a) obtained protein extract).

Figure 2 shows a graph of the results of the digestion of Example 3 (3). In the figure, Lane 1, negative control (Example 3 (B)); Lane 2, positive control (Example 3 (-)); Lanes 3-18, corresponding sample dilution Table 1 Samples 3-18 as EcoRI Protectant , plasmid pET-28b EcoRI digestion electropherogram. Figure 3 shows the results of the digestion of Example 3 (3). In the figure, lane 1, negative control (Example 3 (B)); Lane 2, positive control (Example 3 (-)); Lane 3-18, corresponding sample dilution Table 2, Sample 3-18 as EcoRI Protectant , plasmid pET-28b EcoRI digestion electropherogram.

 Figure 4 shows the results of the digestion of Example 3 (4). In the figure, Lane 1, negative control (Example 3 (B)); Lane 2, positive control (Example 3 (-)); Lanes 3-9, corresponding sample dilution Table 3, Samples 3-9 as EcoRI protection U, plasmid pET-28b EcoRI digestion electrophoresis map; lanes 10-16, corresponding sample dilution Table 3 samples 10-16 as EcoRI protective agent, plasmid pET-28b EcoRI digestion electropherogram. Specific implementation

 In particular, the invention relates to the use of a protein extract of a plant seed embryo for stabilizing the biological activity of an active protein. The term "plant seed" has the meaning commonly recognized or accepted by those skilled in the art, and plant seeds of the present invention may include, but are not limited to, seeds derived from the following plants:

 a plant seed belonging to the Poaceae family, such as the genus Hordeum, the rye Secale, and the oats

(Avena) ^Sorghum), Andropogori, .Holcus, Panicum, Oryza, Zea (Zea), Tritico saecale, Triticum, such as barley

( Hordeum vulgare ), Hordeum jubatum, Hordeum murinum, Hordeum secalinum, Hordeum distichon, Hordeum aegiceras, Hordeumhexastichon, cultivated six-row barley ( Hordeum Hexastichum ), Hordeum irregulare, barley

(Hordeum sativum ) ^ Hordeum secalinum [barley, pearl barley, foxtail barley wall barley meadow barley], rye (Secale cereale) [rye], oatmeal (Avena sativa, Avena fatua, Avena byzantina Avena fatua var. sativa, hybrid oatmeal (Avena hybrida [oat], two-color sorghum bico r, Sorghum halepense) Sorghum saccharatum (.Sorghum vulgare) Andropogon drummondii, Holcus biolor, 蜀黍Holcus sorghum), Sorghum aethiopicum, Sorghum arundinaceum, Carver sorghum (.Sorghum caffrorum) Sorghum sorghum <i Sorghum cernuum ) Sorghum Dochna, Sorghum drummondii, Sorghum durra ^ Sorghum guineense, Sorghum lanceolatum, Sorghum nervosum Sorghum saccharatum, Sorghum subglabrescens^ Sorghum verticilliflorum S ( Sorghum vulgare ) Sago sorghum. Holcus Halepensis) ^ Sorghum mili Acium millet, wild scorpion (Panicum militaceum) [sorghum, millet], 3⁄4 (Oryza sativa), 3⁄4 (Oryza latifolia) [禾S], maize (.Zea mays) [corn (corn), corn (maize) ], Triticum aestivum, Triticum durum, Triticum turgidum, Triticum hybernum, Maca wheat

( Triticum macha), common wheat ( Triticum >rat ra ) or common wheat ( Triticum ra / gorre ) [wheat (wheat), bread wheat (bread wheat) common wheat (common wheat), Tritico saecale ["small rye ( Tricale) "] Plant seeds.

Plant seeds belonging to the family Fatoceae, such as Pisum, Acacia, Albizicd, Acacia cacic, Glycine, genus Dolichos, genus Phaseol, wild soybean Lsojcd, etc., such as pea ί Pi sum sativum pea pea { Pi sum arvense Pi sum humile [pea], Albizia julibrissin ^ Da Xian ( Glycine max), Glycine gracilis (Glycine hispida), soy iPhaseolus max), pressed x [soybean] and other plant seeds.

 Plant seeds belonging to the family Walnut ( /^/"/3⁄4foc^^ ), such as Juglas Jugl ms, Wallia, etc., such as walnut (regia), Japanese walnut (Jug lans ailanthifolia, pecan uglans sieboldiana), gray walnut uglans ci re O, Wallia cinerea, Juglans bixbvi, uglans calif omica) ^ Indian black walnut uglans hindsi, Juglans intermedia, Juglansjamaice is, large walnut (Juglans major small fruit walnut i Juglans microcarpa, {Juglans nigra) or Wallia nigral^ (walnut) ^ (black walnut) ^ common walnut ^ persian walnut {white walnut} ^ gray moon peach (butternut) and other plant seeds.

 Plant seeds belonging to the family Linaceae, such as Linum Linum, Adeno!inum, etc., such as Linum (Linum usitatissimum Linum humile, Linus austriacum Linum bienne, Linum angustifolium, ^ Write linen { Linum catharticum (Linum flavum), large flowered linen {Linum grandiflorum Adenolinum grandiflorum, Yis linen (Linum lewisii, 3⁄4|side linen (Linum narbonense) (Linum narbonense) {Linum perenne Linum perenne var. iewisii, Linum pratense or Linum r Plant seeds such as gy layer [flax, linseed].

Plant seeds belonging to the family Brassicaceae, such as Brassica iBrassiccd, Melanosinapis, iSinapis, iArabadopsis, Thelhmgiella, etc., such as Brassica napus and Brassica rapa ssp. [canola, cane (oilseed rape), cabbage (turnip rape)], wild mustard (Sinpis arvensis mustard (Brssica juncea mustard iBrassica juncea var. juncea, mustard mustard {Brassica juncea var. crispifolia), big leaf mustard ί Brassica juncea var. foliosa) Black mustard {Brassica nigra), salt mustard (Thellungiella salsuginea), small salt mustard (Thellungiella halophila), Brassica sinapioides, Melanosinapis

Cabbage (Brassica oleracea) [feeding canola] or plant seeds such as Arabidopsis thaliana.

 It belongs to the genus iAsteraceae, such as Calendula (Centhamus Centrorea, Cichorium, Cynara, Helianthus, and Mangosteen). Lactuca ), Locusta, marigold ( Tagetes Valeriana et al, ( Calendula officinalis έΙ 3⁄4 ( Carthamus tinctorius ) ^ Bluebonnet ( Centaurea cyanus Chicory ( Cichorium intybus ) [ Blue Daisy ] , Artichoke ( Cynara scolymus ) , Sunflower {Helianthus annus ) ^Manicola (Lactuca sativa borage (. ssp. sativa) ^ Valeriana locusta [Vancho], Tagetes lucida, Tagetes erecta or Tagetes tenuifolia [Marigold] Plant seeds.

It belongs to the lacquer W "Anacardiaceae (such as Pistacia, Mango genus iMangifera, Cashew (A acardium, etc.; belongs to the birch family ( ^ / / "< ^ ^), such as genus (Co / j / ); It belongs to the family Bromeliaceae ( /Ό Τ^/ "<^^ ), such as the genus Bromelia (A mcO, Bromelia, etc.; belongs to the papaya family (Caricaceae, such as papaya Carica, etc.; Convolvulaceae, such as Ipomea, Convolvulus, etc.; belonging to the family Chenopodiaceae, such as the genus Beta, etc.; belonging to the genus Cucurbitaceae, such as the genus iCucubita ); belongs to Euphorbiaceae, such as cassava (Manihot, ricin Rici s, etc.; belongs to the family Lauraceae, such as Avocado Persea, Laurel (aurus, etc.; belongs to the Malvaceae (. Malvaceae), such as Gossypium, etc.; belonging to the family Musaceae, such as Musa (M cf); belonging to the palm family iPalmae), such as the palm family Elacis; belonging to the Pedaliaceae, such as the genus Sesamum; It belongs to the longan mProteaceae), such as Macafo a, etc.; belongs to the family Leo (^o/awaceae), such as the genus Capsicum (Ca/A«'CM), Nicotiana genus (Solanum)^ Genus (Lycopersicon), etc.; belongs to the family tea (Theaceae), such as the camellia CamellicO and other plant seeds.

Also it includes plant seeds belonging to other families such as, but not limited to, Cocos Cod Saccharum S _aCC hamm, Arachis Arachi, Punica (Ptmica), pepper genus Piper, peperomia Peperomia), of the genus Capsicum (Capsicum), coffee Is a Coffea, Theobroma, Spamgus, Citrullus, Cucumis, Cyrtomium, Elaeis, Fragaria, Hyoseyamus, Apple Genus (Pythium CPyrus) ^ Prunus CPrunus) ^ radish CRaphanus) ^ Carrot aucus, Senecio), Fenugreek mgomlkd, Vicia, Vicia, Vitis and other plant seeds.

 In a preferred embodiment, the plant seed suitable for use in the present invention is preferably an edible plant seed. In a more preferred embodiment, the plant seed is selected from the group consisting of Hordeum, rye (Recede, Avena, Sorghum, Panicum), Oryza, Corn. (Zea) A genus of Triticum, a genus of the genus Pi, a genus of Glycine, a species of the genus Phylogenus, or a combination thereof. In a still further preferred embodiment, the plant seed is selected from the group consisting of corn, wheat, rice, sorghum, barley, rye, oat, soybean, pea, soybean, or the like, or a combination thereof, especially those plants mature Seed (fully developed seeds).

The inventors compared the protective effects of protein extracts of various parts of plant seeds on active proteins by experiments, and found that protein extracts of plant seed embryos have outstanding protective effects. Therefore, in the present invention, the inventors selected a protein extract of a plant seed embryo as a protective agent for the active protein. Mature plant seeds are composed of embryos (emb _r yo), endosperm and seed coat. The fully developed embryo is composed of four parts: plumule, radicle hypocotyl and cotyledon. Therefore, in the present invention, the embryo of the plant seed is composed of the above-mentioned germ, radicle, hypocotyl and cotyledon. In a preferred embodiment, when the plant seed is an endosperm-free seed, the embryo of the plant seed preferably does not comprise cotyledons.

The embryos of the plant seeds of the present invention can be obtained directly from the market or can be isolated from intact plant seeds using physical and mechanical methods well known to those skilled in the art. The protein extract of the plant seed embryo of the present invention is a mixture containing a plurality of protein components obtained by extracting a protein extract which does not cause severe degradation or inactivation of the protein without causing severe degradation or inactivation of the protein. Among them, protein extracts are well known to those skilled in the art, including but not limited to, distilled water, deionized water, salt solution (such as physiological saline, phosphate buffer, etc.), dilute alkali solution, organic solvent (such as alcohol, etc.). ). Of course, in order to remove unnecessary impurities (such as lipids), it may be treated as needed during the extraction process or before extraction. The embryos of the seeds are subjected to processing steps such as grinding, drying, and degreasing. It should be noted that in the preparation of protein extracts of plant seed embryos, it should be ensured that the proteins therein are not degraded, destroyed or inactivated.

 In a preferred embodiment, the method for preparing the protein extract of the plant seed embryo comprises the steps of: 1) degreasing and drying the embryo of the plant seed; 2) using protein extraction without causing severe degradation or inactivation of the protein. a liquid (preferably a salt solution, more preferably 0.5% to 2.5% (WA NaCl solution, further preferably 0.9% to 2% (WA NaCl solution, most preferably 0.9% (W/V) physiological saline) is extracted to obtain a protein extract; 3) The protein extract obtained in step 2) is sterilized.

 In a more preferred embodiment, the method for preparing a protein extract of the plant seed embryo comprises the steps of:

(1) Degreasing, drying: For easy extraction, the embryo of the plant seed can be ground into a powder. A preferred embodiment may employ wheat germ powder ground from the embryo of wheat seeds. Among them, the degreasing agent used for degreasing is preferably an organic solvent such as acetone or diethyl ether. Specific steps: for example, the embryo of the plant seed can be continuously immersed in acetone, degreased 2 to 4 times, 0.1 to 24 hours each time, until the acetone after degreasing is colorless, the degreased solid is naturally dried to no acetone Weighed afterwards. Laboratory operations should be performed in a fume hood, and a vacuum concentration extraction tank is used in the pilot test.

 (2) Extraction: The embryos of the degreased and dried plant seeds are sufficiently contacted with the protein extract for 0.1 to 72 hours (preferably for 1 to 24 hours), centrifuged or filtered, and the protein extract is collected. Wherein, the extraction operation is preferably carried out at a low temperature (preferably 2 to 8 ° C); the protein extract is preferably a salt solution (preferably a salt solution, more preferably 0.5% to 2.5% (W/V) NaCl solution, A 0.9% to 2% (w/v) NaCl solution is preferred, with 0.9% (w/v) physiological saline being most preferred. In order to prevent or inhibit the growth of pathogenic microorganisms, an appropriate amount of preservative may be added to the extract, and the preservative may be selected from, but not limited to, phenol, thimerosal, benzoic acid, parabens (ni Potting type), etc.; or / and sterilizing the extract in advance. Specific steps, for example, the embryos of the degreased dried plant seeds and the salt solution (such as physiological saline) are 1:2~1: 50 W/V (ie, the embryos of the plant seeds after 1 gram of defatting and drying) 2-50 ml of salt solution extraction) (preferably 1: 5 to 1: 15 W/V, more preferably 1: 10 W/V). Proportion extraction, extraction should be completed at a low temperature of 2 to 8 ° C. The mixture was stirred intermittently for 72 hours (each stirring time was 8 hours, and after standing overnight, magnetic stirring was again performed for 8 hours, and thus repeated), and filtered or centrifuged with ordinary filter paper to obtain a protein extract.

 (3) Sterilization: The protein extract obtained in the step (2) is subjected to sterilization treatment to obtain a protein extract of the plant seed embryo. In a preferred embodiment, a method of filter sterilization is used. For example, a sterile microporous membrane having a pore diameter of not more than Ιμιη (preferably 0.22 μm) can be used for filtration sterilization to obtain a protein extract of a plant seed embryo in a desired solution form, and stored at a low temperature of 4 °C.

Furthermore, the inventors have unexpectedly found that the protective active substance is mainly concentrated in the salt-soluble protein component of the protein extract of the plant seed embryo by analyzing specific experimental data of the examples of the present invention; It contains less protective active substances and may also contain some component (heteroprotein) that inhibits the activity of protective proteins. However, the protein extract of the plant seed embryo obtained by directly extracting the aforementioned salt solution (for example, 0.9% (w/v) physiological saline) contains both a salt-soluble protein and a water-soluble protein. Therefore, in a more preferred embodiment of the present invention, the method for preparing a protein extract of the plant seed embryo comprises the steps of: 1) degreasing and drying the embryo of the plant seed; 2) using deionized water or steaming Water extracting the embryos of the degreased, dried plant seeds of step 1), removing the water-soluble protein extract to obtain a precipitate; 3) using a salt solution (preferably 0.5% to 2.5% (W/V) NaCl The solution, more preferably 0.9%~2% (W/V) NaCl solution) extracts the precipitate obtained in step 2), collects the salt-soluble protein extract; 4) sterilizes the salt-soluble protein extract obtained in step 3) .

 In a still further preferred embodiment, the method of preparing a protein extract of the plant seed embryo comprises the steps of:

(1) Degreasing, drying: For easy extraction, the embryo of the plant seed can be ground into a powder. A preferred embodiment may employ a powder milled from wheat seed embryos. Among them, the degreasing agent used for degreasing is preferably an organic solvent such as acetone or diethyl ether. Specific steps: for example, the embryo of the plant seed can be continuously immersed in acetone, degreased 2 to 4 times, 0.1 to 24 hours each time, until the acetone after degreasing is colorless, the degreased solid is naturally dried to no acetone Weighed afterwards. Laboratory operations should be completed in a fume hood, and a vacuum concentration extraction tank should be used in the pilot test.

 (2) Removal of water-soluble protein: Embrying of degreased, dried plant seeds with distilled water or deionized water (pre-sterilized, or / and adding an appropriate amount of preservative) to 1: 2 to 1: 50 W/V (preferably 1: 5 to 1: 15 W/V, more preferably 1: 10 W/V) Fully immersed for 0.1 to 24 hours (preferably for 1 to 12 hours, more preferably 2 to 2) 6 hours), centrifuge or filter with ordinary filter paper to remove the water-soluble protein extract. Among them, the extraction operation is preferably carried out at a low temperature (preferably 2 to 8 ° C) and magnetic stirring. Repeat the above steps at least 3 times (preferably 10 to 15 times) to remove the water-soluble protein to the utmost extent and leave the precipitated material for later use.

 (3) Obtaining a salt-soluble protein: a precipitate obtained in the step (2) and a salt solution (preferably 0.5% to 2.5% (W/V) NaCl solution, more preferably 0.9% to 2% (W/V) NaCl solution) ( It should be pre-sterilized, or / and add an appropriate amount of preservative) to 1: 2~1: 50 W/V (preferably 1: 5~1: 15, more preferably 1: 10 W/V) The contact leaching is carried out for 0.1 to 24 hours (preferably for 1 to 12 hours, more preferably 2 to 6 hours), and the mixture is centrifuged or filtered with a common filter paper to collect a salt-soluble protein extract. Among them, the extraction operation is preferably carried out at a low temperature (preferably 2 to 8 ° C) and magnetic stirring. Repeat the above steps 1~10 times to combine all the salt-soluble protein extracts.

 (4) Sterilization: The protein extract obtained in the step (3) is subjected to sterilization treatment to obtain a protein extract of the plant seed embryo. In a preferred embodiment, a method of filter sterilization is used. For example, a sterile microporous membrane having a pore diameter of not more than Ιμιη (preferably 0.22 μm) can be used for filtration sterilization to obtain a protein extract of a plant seed embryo in a desired solution form, and stored at a low temperature of 4 °C.

 It is to be noted that, in describing the preparation method of the protein extract of the plant seed embryo, the present invention uses the sentence pattern of "the preparation method includes the following steps: ...". That is, those skilled in the art can also add other technical steps in preparing the protein extract of the plant seed embryo as needed, such as, but not limited to, using the existing conventional techniques to treat the plant seed embryo. The protein extract is concentrated and lyophilized; or the protein extract of the plant seed embryo is subjected to mass detection (for example, determination of protein content, etc.); in addition, when the plant seed used is an endosperm-free seed, preferably in the embryo of the plant seed The cotyledon fraction is removed prior to defatting and drying. Of course, the added technical steps must ensure that the protein extract of the plant seed embryo is not substantially affected as an active protein stabilizer.

The term "protein extract of plant seed embryos" may be in liquid form (for example in the form of an extract) or in solid form (for example in the form of a lyophilized powder). Furthermore, the "protein extract of plant seed embryo" of the present invention may be a protein extract of a plant seed embryo of the same source (such as a protein extract of wheat seed embryo); or may be a protein extract of a plant seed embryo of various origins. A composition of matter (such as a combination of a protein extract of wheat seed embryos and a protein extract of corn seed embryos). The "active protein" of the present invention can be extracted from human body, animal, plant, microorganism, etc. by any means in the prior art without degrading or inactivating the protein, and genetically engineered means can be used to obtain biologically active recombination. Protein, or obtained directly from the market.

 It will be understood that the protein extract of the plant seed embryo of the present invention is a mixture containing a plurality of protein components, and therefore, unless otherwise specifically described herein, and in the appended claims, "active protein" or "enzyme" is used. None of the substances (active proteins), such as proteolytic enzymes, which can substantially degrade, destroy or inactivate proteins.

 The biological functions of various "active proteins" are well known to those skilled in the art, such as various enzymes used as biocatalysts, including but not limited to, industrial enzymes (including chemical manufacturing, foods, detergents, textiles, Enzymes used in paper, feed, cosmetics, wastewater treatment, etc., such as amylase, phytase, lipase, xylanase, cellulase, alkaline mannanase, superoxide dismutase (SOD) ); non-industrial enzymes (including enzymes used in fields such as genetic engineering), such as nucleic acid restriction enzymes, exonucleases, nucleic acid ligases, and the like.

 Various active proteins used as drugs for the prevention, diagnosis or treatment of diseases, including but not limited to, interleukin IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7 , IL-8, IL-9, IL-10, IL-1K IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18 and all their subtypes ( Such as IL-la, Ιί-1β), tumor necrosis factor (such as TNF ou TNF β), transforming growth factor (such as TGF-p, TGF-a), interferon (such as IFN-a, IFN-IFN-y) insulin , glucagon-like peptides (eg, GLP-1), migration inhibitory factor (MIF), granulocyte macrophage colony stimulating factor (GM-CSF), monocyte macrophage colony stimulating factor (M-CSF) ), granulocyte colony stimulating factor (G-CSF), chemokine (eg IL-8, RANTES) adhesion factor (eg soluble ICAM-1), asialo-serum mucin (ASOR), transferrin , asialoglycoprotein, stem cell factor (SCF), erythropoietin (EPO), allergen (eg dust mite allergen, pollen allergen), antigen, antibody (eg monoclonal antibody, dok) Antibody) and so on.

 Among them, some active proteins can also be applied to the field of genetic engineering (laboratory), such as, but not limited to, genetic engineering tools, enzymes, antigens, antibodies, and the like.

 In a preferred embodiment, the active protein is selected from the group consisting of an enzyme, an antibody, an antigen, an allergen, an interleukin, a tumor necrosis factor, a transforming growth factor, an interferon, an insulin, a glucagon-like peptide, and the like.

 The protein extract of the plant seed embryo of the present invention exhibits an excellent effect in stabilizing the biological activity of the active protein, i.e., the "stability" (especially "thermal stability") of the active protein is greatly improved. Here, "stability" is defined as the ability of an active protein to retain biological activity under various conditions. The "stabilization effect" of the protein extract of the plant seed embryo on the active protein involves the entire life cycle of the active protein (especially protein preparations, such as enzyme preparations, protein pharmaceutical preparations, etc.), generally including a preparation process, such as preparation, dilution, and assignment. Shape, tableting, granulation, lyophilization, drying, coating, etc.; circulation process, such as transportation, storage, etc.; use process.

The invention also provides a composition comprising at least one protein extract of said plant seed embryo, said active protein, and a pharmaceutically or enzymatically acceptable carrier. "Containing" means that the composition may also contain any other components which may be present in any amount as long as the component present at that level functions for the other components of the composition of the present invention. Or the effect has no substantial impact. In still another embodiment of the present invention, the composition is a protein extract from at least one of the plant seed embryos, the active protein, and pharmaceutically or enzymatically Consisting of an acceptable carrier.

 In the composition of the present invention, the ratio of the amount of the active protein to the protein amount of the protein extract of the plant seed embryo is preferably 1:0.1 to 1:500, preferably 1:1-1:400, more preferably 1 : 5-1: 200, most preferably 1: 10-1: 100. The specific concentration of the active protein is not strictly limited as long as it meets the general requirements of the technical field, or can be determined by a person skilled in the art through a limited number of experiments. For example, when the active protein in the composition is used as a therapeutic disease drug, the active protein is preferably a therapeutically effective amount of the active protein.

 The "pharmaceutically or enzymatically acceptable carrier" in the compositions of the present invention should be capable of being mixed with the active protein without substantially reducing the biological activity (biological function) of the active protein in the composition, or The protein extract of the plant seed embryo is greatly affected as an active protein stabilizer. Conventional "pharmaceutically or enzymatically acceptable carriers" include, but are not limited to, sugars such as lactose, glucose, sucrose, etc.; starches such as corn starch, potato starch, etc.; cellulose or derivatives thereof, such as carboxymethyl Cellulose sodium, ethyl cellulose, methyl cellulose, etc.; tragacanth powder; gelatin; talc; solid lubricants, such as stearic acid, magnesium stearate, etc.; calcium sulfate; vegetable oils, such as peanut oil, cottonseed Oil, sesame oil, olive oil, corn oil, cocoa butter, etc.; polyols such as propylene glycol, glycerin, sorbitol, mannitol, polyethylene glycol, etc.; aminocaproic acid, alginic acid, amino acids; emulsifiers, such as spit Wetting agent, such as sodium lauryl sulfate; slow release agents, such as aluminum hydroxide; preservatives, such as phenol, thimerosal, benzoic acid, sorbic acid, parabens (parabens) ), benzyl alcohol, phenylethyl alcohol, etc.; antioxidants such as sulfite, bisulfite, thiourea, methionine, ethylenediaminetetraacetate (EDTA), dibutylhydroxytoluene (BHT) , t-butyl hydroxyanisole (BHA), dithiothreitol (DTT), etc.; coloring agent; flavoring agent; co-solvent; excipient; tablet; diluent; pyrogen-free water; Isotonic saline solution; buffer, etc., or a combination thereof.

 It will be appreciated that in formulating the compositions of the present invention, one skilled in the art will be able to determine the specific protein extract, active protein, and pharmaceutically or enzymatically acceptable carrier of a suitable plant seed embryo by a limited number of experiments. Kinds to avoid possible interactions between them.

The type, amount, and usage of the pharmaceutically or enzymatically acceptable carrier in the composition of the present invention are related to the type of active protein in the composition, and are related to the specific technical field to which the composition is to be applied, and also The physical form of the composition (e.g., the dosage form of the pharmaceutical composition, etc.) is relevant. Of course, which carrier, and amount thereof, are used in formulating the composition are well known to those skilled in the art. For example, in one embodiment of the present invention, the active protein in the composition is a house dust mite allergen, the composition is to be applied in the field of medicine, and the composition is a liquid preparation (sublingual agent), Preferred carriers in the composition include: a pharmaceutically acceptable preservative (0.01% ( _w /v) thiomersal), a polyol (50% ( _v /v) glycerin), a diluent (physiological saline), and the like.

 In order to better embody the protective effect of the protein extract of the plant seed embryo in the composition of the invention, and the biological activity of the active protein in the composition, one skilled in the art can determine the composition of the invention by a limited number of experiments. The appropriate salt concentration. For example, in one embodiment of the present invention, the salt (NaCl) concentration in the restriction enzyme EcoRI composition is preferably from 10 mol/L to 35 mmol/L.

The composition of the present invention may be in the form of a liquid or a solid or a paste (liquid crystal). When the active protein in the composition is used as a medicament for preventing or treating diseases, the administration route of the composition (ie, the pharmaceutical composition) is packaged. These include, but are not limited to, oral administration, injection (including intramuscular, subcutaneous, intravenous, intrathecal), sublingual and buccal mucosal administration, rectal and colonic administration, and transdermal (transdermal) administration. Inhalation administration, etc.; the dosage forms include, but are not limited to: oral liquid, capsules, tablets, injections, sublingual preparations, suppositories, patches, rubs, ointments, creams, pastes, sprays Wait.

 The invention will be described in further detail below with reference to examples. However, it is to be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the invention. Example 1 Preparation of Three Wheat Seed Protein Extracts

 (1) Preparation of wheat seed whole wheat (embryo + endosperm) protein extract (one-step method of physiological saline)

 1) Degreasing and drying: The mature wheat seeds are dried, ground into powder, and continuously degreased three times with acetone for 4 hours. After the degreased acetone is colorless, the degreased solids are dried and weighed. weight. The degreasing process should be done in a fume hood or in a vacuum concentrating extraction tank for safety.

 2) Extraction: In order to ensure that the active ingredient is not inactivated, the extraction should be completed at a low temperature of 2-8 °C. The detailed steps are as follows: The degreased, dried wheat flour obtained in step 1) is extracted with a sterile 0.9% (w/v) physiological saline at a ratio of 1:10 (W/V) (ie, 1 g of degreased wheat flour is used). Extracted by 10 ml of physiological saline, intermittent magnetic stirring at 4 ° C for 72 hours (each stirring time was 8 hours, and after standing overnight, magnetic stirring again for 8 hours, repeated), filtered through ordinary filter paper to obtain a crude filtrate.

 3) Sterilization: The crude filtrate was sterilized by filtration using a 0.22 μηη sterile microporous membrane, and the obtained filtrate was a wheat seed whole wheat (embryo + endosperm) protein extract (physiological saline one-step method).

 4) Determination of total protein concentration by BCA protein assay (using Pierce's BCA protein assay kit).

 (ii) Preparation of protein extracts from wheat seed embryos (physiological saline one-step method)

 1) Degreasing and drying: The embryos of mature wheat seeds are dried, ground into powder (wheat germ powder), and degreased three times in acetone for 4 hours each time. After the degreased acetone is colorless, it will be degreased. The solid matter after drying was weighed. The degreasing process should be done in a fume hood or vacuum concentration extraction tank for safety.

 2) Extraction: In order to ensure that the active ingredient is not inactivated, the extraction should be completed at a low temperature of 2-8 °C. The detailed steps are as follows: The degreased, dried wheat germ powder obtained in step 1) is extracted with a sterile 0.9% (w/v) physiological saline at a ratio of 1:10 (W/V) (ie, after 1 gram of degreased The wheat germ powder was extracted with 10 ml of physiological saline), and the magnetic stirring was intermittently carried out at 4 ° C for 72 hours (each stirring time was 8 hours, and after standing overnight, magnetic stirring was again performed for 8 hours, and thus repeated), and filtered with ordinary filter paper to obtain a crude filtrate. .

 3) Sterilization: The crude filtrate was sterilized by filtration using a 0.22 μηη sterile microporous membrane, and the obtained filtrate was a protein extract of wheat seed embryo (physiological saline one-step method).

 4) Determination of total protein concentration by BCA protein assay (using Pierce's BCA protein assay kit).

 (3) Preparation of protein extracts from wheat seed embryos (water-salt step extraction method)

1) Degreasing and drying: The embryos of mature wheat seeds are dried, ground into powder (wheat germ powder), and degreased three times in acetone for 4 hours each time. After the degreased acetone is colorless, it will be degreased. The solid matter after drying was weighed. Degreasing process should be Complete in a fume hood or vacuum concentrated extraction tank for safety.

 2) Extraction: In order to ensure that the active ingredient is not inactivated, the extraction should be completed at a low temperature of 2-8 °C. The detailed steps are:

 (a) The degreased, dried wheat germ powder obtained in step 1) and the sterilized distilled water are extracted at a ratio of 1:10 (W/V) (ie, 10 ml of steamed wheat germ powder per 1 g of degreased wheat germ powder) Water extraction), magnetic stirring at 4 ° C for 2 hours, centrifugation at 5000 rpm for 15 minutes, remove the supernatant to obtain a precipitate; (The removed supernatant can be additionally retained, and the protein extract of the wheat seed embryo is obtained after filtration and sterilization ( Water soluble protein)).

 (b) Continue to add the sterilized distilled water to the precipitate in the same proportion, and repeat step (a) 10 times or more to remove the water-soluble protein to the utmost extent.

 (c) extracting the precipitate obtained in step (b) with a sterile 2% (W/V) NaCl solution at a ratio of 1:10 (W/V) (ie, extracting each 1 gram of the precipitate with 10 ml of 2% NaCl solution) After magnetic stirring at 4 ° C for 2 hours, centrifugation was performed at 5000 rpm for 15 minutes, and the supernatant was collected.

 (d) Repeat step (c) twice, and combine 3 times of 2% (W/V) NaCl solution extract to obtain a salt-soluble protein extract.

3) Sterilization: The extract obtained in the step 2) is filtered and sterilized by a 0.22 μm sterile microporous membrane, and the obtained filtrate is a protein extract of wheat seed embryo (water salt stepwise extraction method) (salt) Soluble protein).

 4) Determination of total protein concentration by BCA protein assay (using Pierce's BCA protein assay kit). Example 2 Three wheat seed protein extracts by SDS-PAGE electrophoresis

 The three wheat seed protein extracts obtained in Example 1 were subjected to SDS-PAGE electrophoresis according to the method of protein SDS-PAGE electrophoresis in the Handbook of Protein Technology (Wang Jiazheng, edited by Fan Ming, 2000, Science Press). . The experimental results are shown in Fig. 1. From the experimental results, it is known that the protein composition of the three wheat seed protein extracts prepared by the three methods of Example 1 has a large difference. By SDS-PAGE electrophoresis qualitative analysis, the protein components and components in the three protein extracts were all different. Example 3 Protection experiments of three wheat seed protein extracts on restriction endonuclease EcoRI

 (1) EcoRI digestion system (positive control)

 Add 0.6 μl (9 U, ie 9 ng) of restriction enzyme EcoRI (Cat. No. D1040A, Takara, Japan) to a 500 μl centrifuge tube, 2 μl of lO XH Buffer (included in the enzyme preparation) ), 3 μl (4 μg) plasmid pET-28b (Novagen, Cat. No. 69865-3, Merck, Germany) (containing a single EcoRI cleavage site) and 14.4 μl sterilized distilled water; vortex mixing After homogenization, briefly centrifuge at 12,000 rpm and add 30 μl of sterilized paraffin oil to seal. After incubating for 1 hour at 37 ° C, add 2 μl of 10 X Loading Buffer (included in the enzyme preparation) and mix. After 8 μl of sample electrophoresis, electrophoresis was carried out on a 0.8% agarose gel at 5 V/cm for 40 minutes, and then the enzyme digestion was detected on a Tanon GIS-2008 gel imaging system.

 (ii) EcoRI inactivation system (negative control)

Add 0.6 μl (9 U, ie 9 ng) of restriction enzyme EcoRI (Cat. No. D1040A, Takara, Japan) and 5 μl of sterilized distilled water in a 500 μl centrifuge tube, vortex mixed After mixing, centrifuge briefly at 12000 rpm and add 30 μl of sterilization. Paraffin oil was sealed and incubated at 65 °C for 30 minutes to completely inactivate EcoRI. Allow to cool to room temperature, then add 2 μl of 10 XH Buffer (included in the enzyme preparation), 3 μl (4 μg) of plasmid pET-28b (Novagen, Cat. No. 69865-3, Merck, Germany) ( Containing a single EcoRI cleavage site) and 9.4 microliters of sterilized distilled water; after vortexing, centrifuge briefly at 12,000 rpm. After incubating for 1 hour at 37 ° C, add 2 μl of 10 X Loading Buffer (included in the enzyme preparation) and mix. After 8 μl of sample electrophoresis, electrophoresis was carried out on a 0.8% agarose gel at 5 V/cm for 40 minutes, and then the enzyme digestion was detected on a Tanon GIS-2008 gel imaging system.

 (iii) Protection of wheat seed whole wheat/embryo protein extract by restriction endonuclease EcoRI

 Add 0.6 μl (9 U, ie 9 ng) of restriction enzyme EcoRI (Cat. No. D1040A, Takara, Japan) and 5 μl of different dilutions in a 500 μl centrifuge tube (with sterile distillation) Water-diluted wheat seed protein extract (Example 1 (a), (b) two protein extracts) sample, vortexed, centrifuged at 12,000 rpm, and added 30 μl of sterilized paraffin oil seal, 65 Incubate for 30 minutes at °C. Allow to naturally cool to room temperature, then add 2 μl of 10 XH Buffer (included in the enzyme preparation), 3 μl (4 μg) of plasmid pET-28b (Novagen, Cat. No. 69865-3, Merck, Germany) ( Containing a single EcoRI cleavage site) and 9.4 microliters of sterile distilled water; after vortexing, briefly centrifuge at 12,000 rpm. After incubation at 37 ° C for 12 hours (select a longer digestion time to better compare the protective effect of the two protein extracts on EcoRI), add 2 μl of 10 X Loading Buffer (included in the enzyme preparation) uniform. After 8 μl of sample electrophoresis, electrophoresis was carried out on a 0.8% agarose gel at 5 V/cm for 40 minutes, and then the enzyme digestion was detected on a Tanon GIS-2008 gel imaging system.

 The protein extract of wheat seed embryo (one-step method of physiological saline) (the protein extract obtained in Example 1 (b)) is shown in the sample dilution table 1. The experimental results are shown in Fig. 2. The wheat seed whole wheat (embryonic + endosperm) protein extract (one-step method of physiological saline) (the protein extract obtained in Example 1 (a)) is diluted in the sample dilution table 2, and the experimental results are shown in Fig. 3.

 From the experimental results, the whole wheat (embryo + endosperm) protein extract (physiological saline one-step method) of wheat seeds could not effectively protect EcoRI under the condition of incubation at 65 °C for 30 minutes; The protein extract (one-step method of physiological saline) has a good protective effect on the activity of EcoRI in a certain protein concentration range, and the reason for the poor protection of the stock solution may be related to the excessive salt (NaCl) concentration.

 Sample dilution table 1

10 50,000 χ 31.7 ng/ml 2.9 μηιοΐ/ΐ

 11 250,000χ 6.34 ng/ml 580 nmol/1

 12 Ι, ΟΟΟ, ΟΟΟχ 1585 pg/ml 145 nmol/1

 13 5,000,000χ 317 pg/ml 29 nmol/1

 14 25,000,000χ 63.4 pg/ml 5.8 nmol/1

 15 ΙΟΟ, ΟΟΟ, ΟΟΟχ 15.85 pg/ml 1.45 nmol/1

 16 500,000,000 χ 290 pmol/1

 17 2,500,000,000χ 634 fg/ml 58 pmol/1

 18 ΙΟ,ΟΟΟ,ΟΟΟ,ΟΟΟχ 158.5 fg/ml 14.5 pmol/1

 Sample dilution table 2

m

 (iv) Protection experiments of protein extracts from two wheat seed embryos against restriction endonuclease EcoRI

 In order to further determine whether the protective active substance is mainly concentrated in the salt-soluble protein component, the inventors designed the following experiment:

Example 1 (3) of 5 μl of different dilutions (diluted with sterile distilled water) The resulting protein extract (salt soluble protein) of wheat seed embryos was respectively 0.6 μl (9 U, ie 9 n)克) restriction enzyme EcoRI (Cat. No. D1040A, Takara, Japan) Vortex and mix, briefly centrifuge at 12000 rpm, add 30 μl of sterilized paraffin oil seal, and incubate at 65 °C for 30 minutes. Allow to cool to room temperature, then add 2 μl of 10 XH Buffer (included in the enzyme preparation), 3 μl (4 μg) of plasmid pET-28b (Novagen, Cat. No. 69865-3, Merck, Germany) ( Containing a single EcoRI cleavage site) and 9.4 microliters of sterilized distilled water; after vortexing, briefly centrifuge at 12,000 rpm. After incubating for 1 hour at 37 ° C, add 2 μl of 10 X Loading Buffer (included in the enzyme preparation) and mix. After 8 μl of sample electrophoresis, electrophoresis was carried out on a 0.8% agarose gel at 5 V/cm for 40 minutes, and then the enzyme digestion was detected on a Tanon GIS-2008 gel imaging system.

 5 μl of different dilutions (diluted with sterile distilled water) Example 1 (3) 2) (a) The wheat seed embryo protein extract (water-soluble protein) samples were respectively 0.6 μl (9U) , ie 9 ng) restriction enzyme EcoRI

(Cat. No. D1040A, Takara, Japan) Vortex and mix, 12000 r.p.m., briefly centrifuge, add 30 μl of sterilized paraffin oil seal, and incubate at 65 °C for 30 minutes. Allow to cool to room temperature, then add 2 μl of 10 XH Buffer (included in the enzyme preparation), 3 μl (4 μg) of plasmid pET-28b (Novagen, Cat. No. 69865-3, Merck, Germany) ( Containing a single EcoRI cleavage site) and 9.4 microliters of sterilized distilled water; after vortexing, briefly centrifuge at 12,000 rpm. After incubating for 1 hour at 37 ° C, add 2 μl of 10 X Loading Buffer (included in the enzyme preparation) and mix. After 8 μl of sample electrophoresis, electrophoresis was carried out on a 0.8% agarose gel at 5 V/cm for 40 minutes, and then the enzyme digestion was detected on a Tanon GIS-2008 gel imaging system.

 The dilution of the protein extracts of the two wheat seed embryos is shown in Table 3 of the sample dilution. Among them, samples 3-9 are samples of different dilutions of protein extracts (salt-soluble proteins) of wheat seed embryos; samples 10-16 are protein extracts of wheat seed embryos.

(Water-soluble protein) Samples with different dilution factors.

 Sample dilution table 3

16 Ι, ΟΟΟ, ΟΟΟχ 1.25 ng/mL one

 The experimental results are shown in Fig. 4. Since the salt concentration of the protein extract (salt-soluble protein) of the wheat seed embryo is too high, the enzyme digestion and electrophoresis are affected (Fig. 4, lane 3), but the dilution is 10 times to 1000 times. The activity of EcoRI can be effectively protected (Fig. 4, lanes 4-6); while the protein extract (water-soluble protein) of wheat seed embryos is less effective in protecting EcoRI activity (Fig. 4, lanes 10-16), enzyme Not completely cut, indicating that it contains less protective active protein components, and may also contain substances that inhibit the activity of protective proteins (heteroproteins). Example 4 Preparation of household dust mite allergen leaching solution

 (1) Cleaning, grinding, degreasing, drying

 Dust mite was cultured in a medium (2 parts of laboratory animal feed, 2 parts of dry yeast, 1 part of dried fish meal, medium humidity 16%) to a density of 300-500/g. The mixture was suspended and saturated with a saturated NaCl solution to collect dust particles. The obtained household dust mites are suspended in physiological saline, dried, and stored at -20 ° C for later use. Weigh the worm body, grind the liquid body with liquid nitrogen, and immerse it in acetone for 3 times, 4 hours each time. After the degreased acetone is colorless, the solid material after degreasing is naturally dried to the smell of no acetone. weight.

 (2) Extraction

 Both D. sylvestris and physiological saline were soaked in 1: 25 (W/V) (ie, extracted with 25 ml of physiological saline solution after 1 gram of degreased house dust mites), and intermittently magnetically stirred at 4 ° C for 72 hours. (Each stirring time was 8 hours, and after standing overnight, magnetic stirring was again performed for 8 hours, and thus repeated).

 (3) to slag

 After the completion of the stirring, the physiological saline solution leaching solution was filtered through a common filter paper to obtain a crude filtrate.

 (4) Fine filtration

 The obtained crude filtrate was filtered and sterilized by a sterile microporous membrane having a pore size of 0.22 μm, and the obtained filtrate was a house dust mite allergen leaching solution, and stored at a low temperature of 4 °C. Protein content was determined using Pierce's BCA Protein Assay Kit. The protein concentration of the household dust extract was 1.0 mg/ml.

 Example 5 Protection experiment of three wheat seed protein extracts against house dust mite allergen

 (1) Test method for household dust mite allergen activity

 The serum of the household dust mite allergen to be tested is mixed with the serum of the standard dust bank of the household dust mites (Zhejiang Iwu Biotechnology Co., Ltd.), and the serum in the standard serum library is used as a control, and the temperature is maintained at 37 ° C. Incubate for 1 hour, then remove and place in a refrigerator at 4 °C overnight (9-12 hours). The sample after overnight at 4 ° C was transferred to a sterilized glass test tube, and the content of the household dust mite allergen slgE was measured using a UnicaplOO instrument (Swedish Pharmacia). (According to the Immun CAP diagnostic system from the Swedish Pharmacia, Uni CAP automatic in vitro detection of allergen system instructions.) The relative serum level of slgE measured in the standard serum pool was 85.73 KUA/L.

The serum in the standard serum pool of the house dust mite contains specific IgE ( _S IgE) for the house dust mite allergen. The allergen active ingredient in the test substance will combine with the slgE in the serum to form a complex, which will reduce the concentration of free slgE in the serum. The UniCAP system detects the amount of slgE in serum. The higher the allergen activity in the test article, the lower the slgE concentration will decrease after the serum and the test substance are applied. At this time, using the UniCAP system test, it is found that the free slgE in the serum will be correspondingly reduced, and the activity of the allergen in the test article can be obtained by comparing the changes of the serum slgE concentration before and after the test article is added. In the case where serum slgE is not completely bound by the allergen in the test article, the allergen activity in this process is positively correlated with the decrease in serum slgE concentration. According to the inhibition rate of slgE, the activity of the allergen in the test article can be obtained. The higher the inhibition rate of slgE, the higher the allergen activity in the test article.

 (II) Protection experiments of three wheat seed protein extracts on house dust mite allergen

 The household dust mite allergen extract obtained in Example 4 and the three wheat seed protein extracts obtained in Example 1 were formulated into different compositions according to the formulations of Table 1, Table 3, and Table 5 (under the tongue). Containing the agent). The formulation of the control in Table 2 is the same as the formulation of Table 1 in Table 5, the formulation of the control in Table 4 is the same as the formulation of Table 3 in Table 5, and the formulation in Table 6 is the same as the formulation in Table 5, but the wheat seed protein in all the control formulations. The extract was first hydrolyzed by pepsin and papain for 6 hours, and the enzyme was inactivated at 100 ° C for 2 hours, and the remaining ingredients were added. The "wheat seed whole wheat/embryo protein" in the formulations of Table 1, Table 3, and Table 5 refers to the wheat seed whole wheat/embryo protein extract obtained by the three methods of Example 1, respectively, and each subsequent Values represent the quality of the protein in the extract.

 The composition was filtered and sterilized, placed at 25 ° C for stability test, and samples were taken at 0, 6, 12, 18, 24, 30, and 36 months to determine the activity of the house dust mite allergen. The activity at 0 months was 100%, and the values measured at other times were compared with the values at 0 months to detect changes in activity at different time points. Three wheat seed protein extracts were determined for house dust mite allergens. The effect of activity.

 The experimental results are shown in Table 2, Table 4 and Table 6. From the experimental results, it is known that the wheat seed whole protein extract (physiological saline one-step method) has a better effect than the wheat seed whole wheat protein extract (physiological salt water one-step method) for the protection of the house dust mite allergen. This indicates that the protective active protein component is mainly present in the embryo of the plant seed. The water-salt step-by-step extraction method is more advantageous than the one-step method of physiological saline, especially in the low-concentration formula of wheat embryo protein extract (compared with the experimental results of Table 3 formula 1# and Table 5 formula 1#), water and salt step by step. The wheat embryo protein obtained by the extraction method has a better protective effect. Table 1 Formula of the mixture of house dust mite allergen and wheat seed whole wheat protein extract (one-step method of physiological saline)

Table 2 Effect of wheat seed whole wheat protein extract (one-step saline method) on the activity of house dust mite allergen

Table 3 Formula of mixed protein extract of house dust mite allergen and wheat seed embryo (one-step method of physiological saline)

table

25 °C, 36 months, not «72.16 80.78 82.00 82.71 83.01 not « Table 5 Protein extract of house dust mite allergen and wheat seed embryo (water salt step extraction method)

Table

 Example 6 Preparation of protein extract of soybean seed embryo (water salt stepwise extraction method)

 1) Degreasing and drying: Remove the cotyledons from the mature soybean seed embryos, then dry them, grind them into powder, and soak them in acetone for 3 times for 4 hours each time. After the degreased acetone is colorless, it will be degreased. The solid matter was dried and weighed. The degreasing process should be done in a fume hood or in a vacuum concentration tank to ensure safety.

 2) Extraction: In order to ensure that the active ingredient is not inactivated, the extraction should be completed at a low temperature of 2-8 °C. The detailed steps are:

 (a) The degreased, dried soybean germ powder obtained in step 1) and the sterilized distilled water are extracted at a ratio of 1:10 (W/V) (ie, 10 ml of steamed soybean powder per 1 g of degreased soybean meal) Water extraction), magnetic stirring at 4 ° C for 2 hours, centrifugation at 5000 rpm for 15 minutes, the supernatant was removed, and a precipitate was obtained.

 (b) Continue to add the sterilized distilled water to the precipitate in the same proportion, and repeat step (a) 10 times or more to remove the water-soluble protein to the utmost extent.

(c) extracting the precipitate obtained in step (b) with a sterilized 2% (w/v) NaCl solution at a ratio of 1:10 (W/V) (ie Each 1 gram of the precipitate was extracted with 10 ml of 2% NaCl solution), magnetically stirred at 4 ° C for 2 hours, centrifuged at 5000 rpm for 15 minutes, and the supernatant was collected.

 (d) Repeat step (c) twice, and combine 3 times of 2% (W/V) NaCl solution extract to obtain a salt-soluble protein extract.

 3) Sterilization: The extract obtained in the step 2) was sterilized by filtration using a 0.22 μηη sterile microporous membrane, and the obtained filtrate was a protein extract of soybean seed embryo (water salt stepwise extraction method).

 4) Determination of total protein concentration by BCA protein assay (using Pierce's BCA protein assay kit). Example 7 Protection of p53 monoclonal antibody by protein extract of soybean seed embryo

 (a) Preparation of p53 monoclonal antibody

 A hybridoma cell line capable of secreting p53 monoclonal antibody (provided by Zhejiang Wowu Biotechnology Co., Ltd.) was injected into the peritoneal cavity of Blab/c mice to produce ascites, and ascites was purified by saturated ammonium sulfate salting out method. The protein concentration was 2.0 mg/ml and was stored in a -70 ° C freezer.

 (B) Determination of p53 monoclonal antibody activity

In this experiment, the indirect ELISA method was used to determine the effective dilution of p53 monoclonal antibody to reflect the change of its activity. The specific steps are: the antigen (recombinantly expressed p53 protein) is diluted to 10 μ _§ /ιη1 with a coating solution (pH 9.6 0.05M carbonate buffer), coated onto a 96-well plate, 50 μΐ/well, 4° The C package was left overnight. Wash 5 times with washing solution (pH 7.4 PBS) for 3 minutes each time. Dissolve/dilut the p53 monoclonal antibody into different dilutions (1: 1000, 1: 2000, 1: 4000, ...) with a diluent (0.1 g of bovine serum albumin plus pH 7.4 PBS to 100 ml). The diluted monoclonal antibody was added to a well-coated 96-well plate (negative control without addition of monoclonal antibody, replaced with a diluent), and reacted at 37 ° C for 2 hours. Wash 5 times with washing solution (pH 7.4 PBS) for 3 minutes each time. ΙΟΟ μΙ goat anti-mouse Ig antibody-HRP (1:4000 diluted with dilution) was added to each well, and 37 was reacted for 1 hour. Wash 5 times with washing solution (pH 7.4 PBS) for 3 minutes each time. 100 μl of 3,3,5,5-tetramethylbenzidine TB substrate containing 3⁄40 ₂ was added and developed for 10 minutes. After termination of the reaction with ₂ Μ H ₂ S0 ₄ 100 μΐ, the OD value was measured at a wavelength of 450 nm, and the effective dilution of each monoclonal antibody sample was determined by the maximum dilution when the OD value was 2.5 times larger than the negative control.

 (III) Protection of p53 monoclonal antibody by protein extract of soybean seed embryo

 The p53 monoclonal antibody was mixed with the protein extract of different concentrations of soybean seed embryos (water salt stepwise extraction method) (Example 6) in equal volume, divided, lyophilized, and the effective dilution of p53 monoclonal antibody was detected before and after lyophilization. (Active); The sample of the monoclonal antibody after lyophilization (solid) was placed at 25 ° C, and the effective dilution (activity) of the p53 monoclonal antibody was detected at 1, 2, 3, and 4 weeks.

 The p53 monoclonal antibody was mixed with protein extracts of different concentrations of soybean seed embryos (water-salt step extraction method) (Example 6) in equal volume, and the monoclonal antibody sample (liquid) was placed at 4 ° C, 0. The effective dilution (activity) of p53 monoclonal antibody was detected at 1, 2, 3, and 4 weeks.

From the experimental results, Table 7 shows that although the effective dilution of all the monoclonal antibody samples before and after lyophilization did not change, in the OD value detection at 1: 128000 dilution, the monoclonal antibody samples of protein extracts of different concentrations of soybean seed embryos were added. The ratio of OD value to the negative control was significantly higher than the OD value of the monoclonal antibody sample and the negative control of the protein extract not added to the soybean seed embryo. Ratio. This result indicates that the protein extract of soybean seed embryo has a certain protective effect on the activity of p53 monoclonal antibody during lyophilization. In addition, the protein extract of soybean seed embryos protected the activity of p53 monoclonal antibody (solid) more significantly during the subsequent 25 °C placement.

 Similarly, from the experimental results, Table 8 shows that the protein extract of soybean seed embryos also has a significant protective effect on the activity of p53 monoclonal antibody (liquid).

Table 7 Effect of protein extracts from soybean seed embryos on p53 monoclonal antibody (solid)

Table 8 Effect of protein extracts from soybean seed embryos on p53 monoclonal antibody (liquid)

Example 8 Preparation of Three Corn Seed Protein Extracts

 (1) Preparation of corn seed endosperm protein extract (physiological saline one-step method)

 1) Degreasing and drying: The endosperm of mature corn seeds is air-dried, ground into powder, and continuously degreased three times with acetone for 4 hours. After the degreased acetone is colorless, the degreased solids are dried. Weighed afterwards. The degreasing process should be done in a fume hood or in a vacuum concentration tank to ensure safety.

2) Extraction: In order to ensure that the active ingredient is not inactivated, the extraction should be completed at a low temperature of 2-8 °C. The detailed steps are as follows: The degreased, dried corn endosperm powder obtained in step 1) is extracted with a sterile 0.9% (w/v) physiological saline at a ratio of 1:10 (W/V) (ie, after 1 gram of degreased Corn endosperm powder was extracted with 10 ml of normal saline), intermittent magnetic stirring at 4 ° C for 72 hours (per The stirring time was 8 hours, and after standing overnight, the magnetic stirring was again performed for 8 hours, and thus repeated, and filtered with ordinary filter paper to obtain a crude filtrate.

 3) Sterilization: The crude filtrate was sterilized by filtration using a 0.22 μηη sterile microporous membrane, and the obtained filtrate was a corn seed embryo protein extract (physiological saline one-step method).

 4) Determination of total protein concentration by BCA protein assay (using Pierce's BCA protein assay kit).

 (ii) Preparation of protein extracts from maize seed embryos (one-step method of physiological saline)

 1) Degreasing and drying: The embryos of mature corn seeds are dried, ground into powder, and continuously degreased three times with acetone for 4 hours. After the degreased acetone is colorless, the degreased solids are dried. Weighed afterwards. The degreasing process should be done in a fume hood or in a vacuum concentration tank to ensure safety.

 2) Extraction: In order to ensure that the active ingredient is not inactivated, the extraction should be completed at a low temperature of 2-8 °C. The detailed steps are as follows: The degreased, dried corn germ powder obtained in step 1) and the sterilized 0.9% (w/v) saline are extracted at a ratio of 1: 10 (W/V).

(ie, every 1 gram of degreased corn germ powder is extracted with 10 ml of physiological saline), intermittent magnetic stirring at 4 ° C for 72 hours (each stirring time is 8 hours, after standing overnight, magnetic stirring again for 8 hours, so repeated), Filtration with ordinary filter paper gave a crude filtrate.

 3) Sterilization: The crude filtrate was sterilized by filtration using a 0.22 μηη sterile microporous membrane, and the obtained filtrate was a protein extract of corn seed embryo (physiological saline one-step method).

 4) Determination of total protein concentration by BCA protein assay (using Pierce's BCA protein assay kit).

 (III) Preparation of protein extracts from maize seed embryos (water-salt step extraction method)

 1) Degreasing and drying: The embryos of mature corn seeds are dried, ground into powder, and continuously degreased three times with acetone for 4 hours. After the degreased acetone is colorless, the degreased solids are dried. Weighed afterwards. The degreasing process should be done in a fume hood or in a vacuum concentration tank to ensure safety.

 2) Extraction: In order to ensure that the active ingredient is not inactivated, the extraction should be completed at a low temperature of 2-8 °C. The detailed steps are:

 (a) The degreased, dried corn germ powder obtained in step 1) and the sterilized distilled water are extracted at a ratio of 1:10 (W/V) (ie, 10 ml of steamed corn germ powder per 1 g of degreased corn) Water extraction), magnetic stirring at 4 ° C for 2 hours, centrifugation at 5000 rpm for 15 minutes, the supernatant was removed, and a precipitate was obtained.

 (b) Continue to add the sterilized distilled water to the precipitate in the same proportion, and repeat step (a) 10 times or more to remove the water-soluble protein to the utmost extent.

 (c) extracting the precipitate obtained in step (b) with a sterilized 0.9% (w/v) NaCl solution at a ratio of 1:10 (W/V) (ie, extracting 10 ml of 0.9% NaCl solution per 1 gram of precipitate) After magnetic stirring at 4 ° C for 2 hours, centrifugation was performed at 5000 rpm for 15 minutes, and the supernatant was collected.

 (d) Repeat step (c) twice and combine the three 0.9% (w/v) NaCl solution extracts to obtain a salt-soluble protein extract.

3) Sterilization: The extract obtained in the step 2) is filtered and sterilized by a 0.22 μm sterile microfiltration membrane to obtain a filtration solution. The liquid is a protein extract of corn seed embryos (water-salt step extraction method).

 4) Determination of total protein concentration by BCA protein assay (using Pierce's BCA protein assay kit). Example 9 Protection of recombinant human interferon oc-2b by three kinds of corn seed protein extracts

 The recombinant human interferon a-2b (IFN a-2b) (provided by Zhejiang Wowu Biotechnology Co., Ltd.) and the three corn seed protein extracts obtained in Example 8 were prepared according to the formulations of Table 9, Table 11, and Table 13, respectively. , formulated into different compositions (spray). The formulation of the control in Table 10 is the same as the formulation of Table 9 in Table 9, the formulation of the control in Table 12 is the same as the formulation of Table 11 in Table 11, and the formulation in Table 14 is the same as that in Formulation #5 of Table 13, but the corn seed protein in all the control formulations. The extract was first hydrolyzed by pepsin and papain for 6 hours, and the enzyme was inactivated at 100 ° C for 2 hours, and the remaining ingredients were added. The "protein of corn seed endosperm/embryo" in the formulations of Table 9, Table 11, and Table 13 refers to the protein extract of corn seed endosperm/embryo prepared by the three methods of Example 8, respectively, and the respective numerical values thereafter represent The quality of the protein in the extract.

 The composition was filtered and sterilized, placed at 40 ° C for stability (acceleration) test, and samples were taken at 0, 1, 5, and 10 days for measurement of IFN a-2b activity (determination of interferon biological activity according to SFDA) The standard method "cell lesion inhibition method" is carried out, the People's Republic of China Pharmacopoeia 2005 edition three appendices 56 to 57 pages). The activity at 0 days was 100%. The values measured at other times were compared with the values at 0 days. The changes in activity at different time points were examined to determine the effects of three corn seed protein extracts on IFN a-2b activity.

 The experimental results are shown in Table 10, Table 12, and Table 14. From the experimental results, it was found that the activity of IFN a-2b decreased to 0 after one day in an accelerated experiment at 40 ° C without adding any plant seed protein protectant (0 # and control). The three corn seed protein extracts have different degrees of protection against IFN a-2b. Among them, the corn seed endosperm protein extract has only a weak protective effect, while the corn seed embryo protein extract has better protection effect. And the effect of the water-salt step extraction method is better than the one-step method of physiological saline.

Table 10 Effect of corn seed endosperm protein extract (one-step saline method) on IFN a-2b activity

Place at 40 ° C for 1 day 0 33.28 56.74 61.90 84.27 82.51 0

Placed at 40 ° C for 5 days Not tested 0 31.43 40.05 62.35 60.99 Not tested

40°C for 10 days Untested Untested 15.60 29.53 43.48 42.76 Not tested Table ll Formulation of IFNa-2b and corn seed embryo protein extract (physiological saline one-step method)

Table 12 Protein extract of maize seed embryos (one-step method of physiological saline) on the activity of IFNa-2b

Table 14 Jade's white extract (salt method) IFNa-2b ringing

Place at 40 ° C for 1 day 0 95.46 97.68 98.70 97.52 96.48 0

Placed at 40 ° C for 5 days Not tested 92.66 95.28 96.85 96.76 95.27 Not tested

40°C, 10 days, not tested, 81.93 89.36 90.74 94.83 93.95 Not possible, although the present invention has been described in detail, it will be apparent to those skilled in the art that, without departing from the spirit and scope of the invention Various changes and modifications can be made to the invention. Therefore, the appended claims cover all such variations within the scope of the invention.

Claims

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 1. Application of protein extracts of plant seed embryos to stabilize the biological activity of active proteins.

 2. The use according to claim 1, wherein the method for preparing a protein extract of the plant seed embryo comprises extracting a protein of a plant seed embryo with a protein extract that does not cause severe degradation or inactivation of the protein. step.

 3. The use according to claim 2, wherein the method for preparing the protein extract of the plant seed embryo comprises the following steps:

 1) Degreasing and drying the embryos of plant seeds;

 2) extracting the embryos of the degreased and dried plant seeds of step 1) with deionized water or distilled water to remove the water-soluble protein extract to obtain a precipitate;

 3) extracting the precipitate obtained in the step 2) with a salt solution, and collecting the salt-soluble protein extract;

 4) The salt-soluble protein extract obtained in the step 3) is sterilized.

 4. Use according to claim 1, characterized in that the plant seed is selected from the group consisting of corn, wheat, rice, sorghum, barley, rye, oats, soybeans, peas or soybeans or a combination thereof.

 5. The use according to claim 1, wherein the active protein is selected from the group consisting of an enzyme, an antibody, an antigen, an allergen, an interleukin, a tumor necrosis factor, a transforming growth factor, an interferon, an insulin or a pancreas. High blood peptide.

 6. A composition comprising at least one protein extract of a plant seed embryo, an active protein, and a pharmaceutically or enzymatically acceptable carrier.

 The composition according to claim 6, wherein the method for preparing the protein extract of the plant seed embryo comprises extracting the protein of the plant seed embryo with a protein extract which does not cause severe degradation or inactivation of the protein. A step of.

 The composition according to claim 7, wherein the method for preparing the protein extract of the plant seed embryo comprises the following steps:

 1) Degreasing and drying the embryos of plant seeds;

 2) extracting the embryos of the degreased and dried plant seeds of step 1) with deionized water or distilled water to remove the water-soluble protein extract to obtain a precipitate;

 3) extracting the precipitate obtained in the step 2) with a salt solution, and collecting the salt-soluble protein extract;

 4) The salt-soluble protein extract obtained in the step 3) is sterilized.

 The composition according to claim 6, wherein the ratio of the amount of the active protein to the protein amount of the protein extract of the plant seed embryo is 1: 0.1-1:500.

 10. A method of preparing a preparation for stabilizing the biological activity of an active protein, the method comprising the steps of: extracting a protein of a plant seed embryo with a protein extract that does not cause severe degradation or inactivation of the protein, The formulation for stabilizing the biological activity of the active protein is obtained.

 11. A method of increasing the stability of an active protein in a formulation, the method comprising adding to the formulation a protein extract of at least one plant seed embryo.