WO2017219952A1 - 一种大豆抗冻蛋白的制备方法、应用 - Google Patents

一种大豆抗冻蛋白的制备方法、应用 Download PDF

Info

Publication number
WO2017219952A1
WO2017219952A1 PCT/CN2017/089103 CN2017089103W WO2017219952A1 WO 2017219952 A1 WO2017219952 A1 WO 2017219952A1 CN 2017089103 W CN2017089103 W CN 2017089103W WO 2017219952 A1 WO2017219952 A1 WO 2017219952A1
Authority
WO
WIPO (PCT)
Prior art keywords
protein
antifreeze
short peptide
preparation
use according
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/CN2017/089103
Other languages
English (en)
French (fr)
Inventor
郑易之
刘国宝
陈丽伊
彭显明
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shenzhen University
Original Assignee
Shenzhen University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Shenzhen University filed Critical Shenzhen University
Publication of WO2017219952A1 publication Critical patent/WO2017219952A1/zh
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Images

Classifications

    • 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
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/10—Preservation of living parts
    • A01N1/12—Chemical aspects of preservation
    • A01N1/122—Preservation or perfusion media
    • A01N1/125—Freeze protecting agents, e.g. cryoprotectants or osmolarity regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
    • 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
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • 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
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8273—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for drought, cold, salt resistance

Definitions

  • the invention belongs to the field of biotechnology, and in particular relates to a preparation method and application of soybean antifreeze protein.
  • the organism When the organism is subjected to low temperature stress, it causes dehydration and crystallization of the intracellular environment, which in turn causes cell membrane structure to rupture or protein inactivation, and the cell or organelle is destroyed, causing the organism to be damaged. Therefore, the most important and basic elements for the organism to resist low temperature freezing and cold damage are as follows: 1. Avoid freezing in cells; 2. Improve the low temperature stability of cell membranes (structures) and biological macromolecules. Under low temperature conditions, some organisms produce proteins with enhanced anti-freezing ability. In the 1960s, a specific protein-antifreeze protein (AFP) was found in the marine fish serum of the polar region to prevent the growth of ice crystals. ). Most of the antifreeze proteins found so far are ordered proteins, which have certain selection characteristics for the protected objects. The discovery of antifreeze proteins has laid an important foundation for a wide range of potential applications in agricultural low temperature breeding, food industry, and pharmaceutical industries.
  • AFP protein-antifreeze protein
  • PM1 protein is a kind of late embroygenesis abundant protein (LEA), which belongs to intrinsic disordered protein. , showing an unstructured state in a natural solution. Compared with ordered proteins, disordered proteins have thermostable, highly soluble properties and also have non-specific binding properties to protected objects.
  • LAA late embroygenesis abundant protein
  • the existing extraction method of PM1 protein is cumbersome, and the extracted PM1 protein has low purity; for PM1 protein, the generally accepted viewpoint is that it has broad application prospects, but its specific use is still uncertain.
  • the invention adopts a preparation method and application of soybean antifreeze protein, and aims to expand the specific use of PM1 protein and PM1-N short peptide (N-terminal short peptide of PM1) as antifreeze protein.
  • the present invention is achieved by the method for preparing a soybean antifreeze protein, comprising the following steps:
  • the plasmid containing the antifreeze protein expression sequence is transformed into E. coli for protein expression;
  • the His tag is excised with thrombin to obtain the antifreeze protein; the antifreeze protein is a PM1 protein or a PM1-N short peptide.
  • column parameters used in the affinity chromatography were as follows: Filler: Chelating Sepharose Fast Flow 4BTM, flow rate of 3 mL/min.
  • the invention also provides the use of PM1 protein or PM1-N short peptide in the preparation of transgenic antifreeze or cold resistant plants.
  • the invention also provides the use of a PM1 protein or a PM1-N short peptide for the preparation of a cryoprotectant for a biological product.
  • the biological product comprises a protein and an enzyme preparation.
  • the biological article comprises a cell, an embryo, a tissue or an organ.
  • the biological product is rabbit red blood cells.
  • cryoprotectant further contains glycerin.
  • cryoprotectant further contains trehalose.
  • the PM1 protein or the PM1-N short peptide is a recombinant protein.
  • the invention has the beneficial effects that the preparation method of the PM1 protein or the PM1-N short peptide provided by the invention has a simple preparation process, and the obtained protein has high purity and is convenient for large-scale fermentation production.
  • the present invention provides the function of the PM1 protein and the PM1-N short peptide by using the PM1 protein or the PM1-N short peptide to prepare a transgenic antifreeze or cold resistant plant; and the PM1 protein is used for preparing a biological product.
  • a cryoprotectant comprising cells, embryos, tissues or organs, and protein and enzyme preparations.
  • the application of the PM1 protein or the PM1-N short peptide provided by the invention not only opens up a new space for the application of the PM1 protein, but also indicates the direction for the application of the PM1 protein, and accelerates the research of the PM1 protein related research. exhibition.
  • FIG. 1 is a sequence and an electropherogram of a PM1 protein and a short peptide PM1-N thereof according to an embodiment of the present invention
  • FIG. 2 is a schematic diagram showing the results of turbidity detection of the liposome after freeze-thaw treatment according to Example 1 of the present invention
  • FIG. 3 is a schematic diagram showing the results of measuring the particle size of the liposome after freeze-thaw treatment according to the first embodiment of the present invention
  • FIG. 4 is a microscopic view of a sample before and after freeze-thaw treatment according to Embodiment 1 of the present invention, wherein FIG. 4a is a group of PM1 without PM1, a group of PM1 is added, and FIG. 4b is a group of PM1, and FIG. 4c is a PM1- N short peptide group;
  • Fig. 5 is a graph showing the results of measuring the protective effect of PM1 protein on rabbit red blood cells according to Example 2 of the present invention.
  • PM1 amino acid sequence of a protein on the NCBI is: MQGGKKAGESIKETATNIGASAKAGMEKTKATVQEKAERMTARDPVQKELATQKKEAKMNQAELDKQAARQHNTAAKQSATTAGHMGHGHHTTGTGTGTATYSTTGEYGQPMGAHQTSAMPGHGTGQPTGHVTEGVVGSHPIGTNRGPGGTATAHNTRAGGKPNDYGYGTGGT (SEQ ID NO: 1), (available from website http://www.ncbi.nlm.nih.gov/protein/NP_001238562.1 Get in).
  • the PM1-N short peptide is an N-terminal short peptide of PM1, and its amino acid sequence is: MQGGKKAGESIKETATNIGASAKAGMEKTKATVQEKAERMTARDPVQKELATQKKEAKMNQAELDKQAARQHNTAAKQSATTAG (SEQ ID NO: 2).
  • the PM1 protein or the PM1-N short peptide is prepared according to the technical scheme of the present invention, and the process is as follows:
  • the pET28a/PM1 plasmid and the pET28a/PM1-N plasmid were transformed into E. coli BL21Star for protein expression;
  • the His tag was excised with thrombin to obtain PM1 protein and PM1-N short peptide, respectively.
  • column parameters used in the affinity chromatography were as follows: column length: 20 cm; diameter: 16 mm, packing: Chelating Sepharose Fast Flow 4BTM, packing volume: 10 mL; manufacturer: Amersham Biosciences, flow rate 3 mL/min .
  • the electropherogram of the prepared PM1 protein and PM1-N short peptide is shown in Fig. 1. It can be seen from the figure that the molecular weight of the PM1 protein is about 20-22 kDa, and the molecular weight of the PM1-N short peptide is about 11 kDa.
  • the PM1 protein and the PM1-N short peptide are intrinsically disordered proteins, and they are in a disordered structure state in a natural solution, and have no obvious selectivity to a protected object.
  • the functions of the prepared PM1 protein and PM1-N short peptide were tested by specific examples to explore their applications.
  • the chemical composition of the cell membrane is mainly composed of phospholipids, a small amount of protein and polysaccharides.
  • POPC oleoylphosphatidylcholine
  • the press was repeatedly extruded to form a liposome having a diameter of about 100 nm through a 100 nm polycarbonate film.
  • the prepared liposome was quantified by a phospholipid quantification kit and diluted to 20 mg/ml. Two groups of 100 ⁇ l of liposome were added to an equal volume of phosphate buffer (control group without PM1 protein), 0.8 mg/ml. PM1 protein, 0.8 mg/ml PM1-N short peptide (plus PM1 protein, PM1-N short peptide group). After incubation at 28 ° C for 30 min, it was frozen at -80 ° C for 30 min, dissolved at 28 ° C for 30 min, and repeated 3 times.
  • Turbidity is an optical effect that can be inversely mapped to the interaction of light with suspended particles in solution, characterizing the extent to which light is blocked as it passes through the water layer.
  • the measured turbidity reflects the amount of transmitted light or the amount of scattered light of the sample, that is, the smaller the transmitted light intensity of the solution or the greater the intensity of the scattered light, the greater the turbidity of the aqueous solution.
  • Liposomes without PM1 protein and PM1-N short peptide group had lower OD 400 values; after freeze-thaw treatment, the turbidity of the solution increased significantly, indicating that the liposome after freeze-thaw treatment changed, meaning The liposomes are destroyed. The turbidity of the liposome sample with PM1 protein and PM1-N short peptide was not changed or slightly increased. This result indicates that PM1 protein and PM1-N short peptide are used for freeze-thawed liposome. Very good stability.
  • the particle size analyzer uses light scattering technology to measure the size distribution of particles in a solution to determine the diameter of the particles in the solution. 80 ⁇ l of the freshly prepared liposome was diluted to 4 ml with a phosphate buffer, and its particle size was measured by a particle size analyzer as shown in FIG.
  • the prepared liposome has a particle size of 100-200 nm; after repeated freeze-thaw treatment, 73% of the liposome has no change in particle size, and 27% of the larger particle size liposomes (about 1000) -3000nm), indicating that the particle size of some liposomes after freeze-thaw treatment is significantly increased, that is, the freeze-thaw treatment causes liposome rupture and then fuses to form liposomes with larger particle size.
  • the sample of PM1 protein was added to the liposome solution. After freeze-thaw treatment, the 100% liposome particle size did not change significantly.
  • the liposome solution with PM1-N short peptide was freeze-thawed and 95% fat.
  • the plastid particle size ranges from 100 to 200 nm. The above results indicate that the PM1 protein and the PM1-N short peptide can maintain, or better maintain, the stability of the liposome after freeze-thaw treatment.
  • Microscopy can be used to visually reflect the morphological changes of liposomes after freeze-thaw treatment. 5 ⁇ l of the prepared liposome sample was observed under a phase contrast microscope, and the observed field of view was homogeneous (Fig. 4a left). After freeze-thaw treatment, a clear aggregated, massive liposome aggregate appeared in the visual field. ( Figure 4a right). A sample of PM1 protein, PM1-N short peptide was added to the liposome solution, and only a small amount of liposome aggregates appeared in the sample after freeze-thaw treatment (Fig. 4b, Fig. 4c).
  • the freeze-thaw treatment can change the volume of the liposome, that is, the freeze-thaw can destroy the liposome, and the damaged liposome can also undergo re-fusion, which is the freeze-thaw induced liposome.
  • freezing and thawing may result in an increase in the viscosity of the liposomes such that they aggregate to form liposome aggregates.
  • a mixture of 50 ⁇ l of 0.2% fresh rabbit red blood cells and an equal volume of citrate buffer was prepared. Further, PM1 (final concentration: 5 mg/ml), glycerin (final concentration: 5%), and PM1 + glycerol were added to the solution containing rabbit red blood cells. After the solution was incubated at 4 ° C for 30 min, the number of rabbit blood cells was counted using a cell counter. The solution was again frozen at -20 ° C for 30 min, and thawed at room temperature for 30 min. After repeated three treatments, cell counting was performed.
  • rabbit red blood cells 0.2% were mixed with PM1 (concentration 5 mg/mL), trehalose (final concentration 0.75 mg/mL), and PM1 + trehalose. Incubate for 30 min at 4 °C. The number of rabbit blood cells was counted using a cell counter. After adding the above mixture to PM1 protein, the sample was frozen at -20 ° C for 30 min, room Warm melt solution for 30min. After three treatments, the cells were counted. Calculate the ratio of the number of remaining cells after freezing and thawing to the number of unfrozen and fused red blood cells and compare them.
  • soybean PM1 protein and PM1-N short peptide can be used as plant antifreeze protein for the production of transgenic antifreeze plants; PM1 protein can also be used as a cryopreservation agent for biological products, such as protein. (Enzyme preparation), cells (such as egg cells, red blood cells), embryos, tissues, organs (such as donated organs); can also be used for food freezing, storage, transportation, thawing and the like.
  • the combination of soybean PM1 protein and PM1-N short peptide with glycerin or trehalose can enhance the protective effect.
  • the PM1 protein and the PM1-N short peptide are disordered proteins, they have a disordered structure state and have no obvious selectivity to the protected object. Therefore, it has a broader application prospect.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Molecular Biology (AREA)
  • Zoology (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Physics & Mathematics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Plant Pathology (AREA)
  • Medicinal Chemistry (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Botany (AREA)
  • Analytical Chemistry (AREA)
  • Dentistry (AREA)
  • Environmental Sciences (AREA)
  • Peptides Or Proteins (AREA)

Abstract

本发明提供了一种大豆抗冻蛋白的制备方法，包括以下步骤：将含有所述抗冻蛋白表达序列的质粒转化至大肠杆菌中，进行蛋白表达；用亲和层析法对蛋白进行纯化；纯化后用凝血酶切除His标签，获得抗冻蛋白。所述抗冻蛋白为PM1蛋白或PM1-N短肽。本发明还提供了PM1蛋白或PM1-N短肽在制备转基因抗冻或抗寒植物、生物类制品的冻存保护剂方面的应用。

Description

一种大豆抗冻蛋白的制备方法、应用 技术领域
本发明属于生物技术领域，尤其涉及一种大豆抗冻蛋白的制备方法、应用。
背景技术
生物在遭受低温胁迫时，造成细胞内环境脱水、形成结晶，继而造成细胞膜结构破裂或蛋白质失活，细胞或细胞器受到破坏，导致生物体受到损伤。因此，生物体要抵御低温冻害、寒害，最重要、最基本的要素有：一、避免细胞内结冰；二、提高细胞膜(结构)及生物大分子的低温稳定性。在低温条件下，有的生物体内会产生具有提高生物抗冻能力的蛋白质，如上世纪60年代在极区的海洋鱼类血清中发现一种可阻止冰晶生长的特异性蛋白质-抗冻蛋白(AFP)。目前发现的抗冻蛋白多为有序蛋白，对被保护对象有一定的选择特性。抗冻蛋白的发现，为在农业低温育种、食品工业、医药行业上的广泛的潜在应用奠定了重要基础。
大豆是我国的主要经济作物，科学工作者已从大豆种子中发现了一种新型抗冻蛋白一PM1蛋白，PM1蛋白是LEA蛋白(late embroygenesis abundant protein，LEA)的一种，属于固有无序蛋白，在天然溶液中呈现无结构状态。与有序蛋白相比较，无序蛋白具有对热稳定、高可溶性特性，也具有可与被保护对象无特异性结合的特性。现有的PM1蛋白的提取方法较为繁琐，且提取的PM1蛋白纯度较低；对于PM1蛋白，普遍认可的观点是其具有广泛的应用前景，但对于它的具体用途，目前尚无法确定。
发明内容
本发明采用一种大豆抗冻蛋白的制备方法、应用，旨在拓展PM1蛋白和PM1-N短肽(PM1的N端短肽)作为抗冻蛋白的具体用途。
本发明是这样实现的，一种大豆抗冻蛋白的制备方法，包括以下步骤：
将含有所述抗冻蛋白表达序列的质粒转化至大肠杆菌中，进行蛋白表达；
用亲和层析法对蛋白进行纯化；
纯化后用凝血酶切除His标签，获得所述抗冻蛋白；所述抗冻蛋白为PM1蛋白或PM1-N短肽。
进一步地，所述亲和层析法中所用层析柱参数如下：填料：Chelating Sepharose Fast Flow 4BTM，流速为3mL/min。
本发明还提供了PM1蛋白或PM1-N短肽在制备转基因抗冻或抗寒植物方面的应用。
本发明还提供了PM1蛋白或PM1-N短肽用于制备生物类制品的冻存保护剂的应用。
进一步地，所述生物类制品包括蛋白及酶制剂。
进一步地，所述生物类制品包括细胞、胚胎、组织或器官。
进一步地，所述生物类制品为兔红细胞。
进一步地，所述冻存保护剂中还含有甘油。
进一步地，所述冻存保护剂中还含有海藻糖。
进一步地，所述PM1蛋白或PM1-N短肽为重组蛋白。
本发明的有益效果在于：本发明提供的PM1蛋白或PM1-N短肽的制备方法，制备过程简单，获得的蛋白纯度高，便于大规模发酵生产。本发明通过研究提供了PM1蛋白和PM1-N短肽的功能，将所述PM1蛋白或PM1-N短肽用于制备转基因抗冻或抗寒植物；将所述PM1蛋白用于制备生物类制品的冻存保护剂，所述生物类制品包括细胞、胚胎、组织或器官，以及蛋白及酶制剂。本发明所提供的PM1蛋白或PM1-N短肽的应用，不仅为PM1蛋白的应用拓展了新的空间，为PM1蛋白的应用指明了方向，更加速了PM1蛋白相关研究的进 展。
附图说明
图1是本发明实施例提供的PM1蛋白及其短肽PM1-N的序列及电泳图；
图2是本发明实施例1提供的经冻融处理后的脂质体的浊度检测结果示意图；
图3是本发明实施例1提供的经冻融处理后的脂质体的粒径测定结果示意图；
图4是本发明实施例1提供的冻融处理前后的样品的显微镜显示图，其中图4a为未加PM1、PM1-N短肽组，图4b为加PM1蛋白组、图4c为加PM1-N短肽组；
图5是本发明实施例2提供的PM1蛋白对兔红细胞的保护作用的测定结果示意图。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白，以下结合附图及实施例，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。
PM1蛋白在NCBI(美国国立生物技术信息中心)上的氨基酸序列为：MQGGKKAGESIKETATNIGASAKAGMEKTKATVQEKAERMTARDPVQKELATQKKEAKMNQAELDKQAARQHNTAAKQSATTAGHMGHGHHTTGTGTGTATYSTTGEYGQPMGAHQTSAMPGHGTGQPTGHVTEGVVGSHPIGTNRGPGGTATAHNTRAGGKPNDYGYGTGGT(SEQ ID NO：1)，(可从http：//www.ncbi.nlm.nih.gov/protein/NP_001238562.1网页中获取)。
PM1-N短肽是PM1的N端短肽，其氨基酸序列为：MQGGKKAGESIKETATNIGASAKAGMEKTKATVQEKAERMTARDPVQKELATQKKEAKMNQAELDKQAARQHNTAAKQSATTAG(SEQ ID NO：2)。
按照本发明的技术方案制备PM1蛋白或PM1-N短肽，过程如下：
将pET28a/PM1质粒、pET28a/PM1-N质粒转化至大肠杆菌BL21Star中，进行蛋白表达；
用亲和层析法对蛋白进行纯化；
纯化后，用凝血酶切除His标签，分别获得PM1蛋白、PM1-N短肽。
具体地，所述亲和层析法中所用层析柱参数如下：柱长：20cm；直径：16mm，填料：Chelating Sepharose Fast Flow 4BTM，填料体积：10mL；厂家：Amersham Biosciences，流速为3mL/min。
所制得的PM1蛋白、PM1-N短肽的电泳图如图1中所示，从图中可以看出PM1蛋白的分子量约为20-22kDa，PM1-N短肽的分子量约为11kDa。
PM1蛋白、PM1-N短肽为固有无序蛋白，在天然溶液中它们呈无序结构状态，对被保护对象没有明显的选择性。以下通过具体的实施例对所制得的PM1蛋白、PM1-N短肽的功能进行测试，以探索其应用。
实施例1人工脂质体的制备及PM1蛋白、PM1-N短肽对经冻融处理后脂质体的稳定作用
细胞膜的化学成分主要是由磷脂、少量蛋白质和多糖组成。我们选用POPC(油酰磷脂酰胆碱)制备人工脂质体。称取40mg的POPC，用500μl的氯仿将POPC完全溶解，用氮气缓慢除去氯仿后将POPC置于真空环境中1h，随后用1ml的磷酸缓冲液(pH 7.4)重新溶解POPC，溶解的POPC用挤压机反复挤压，使其通过100nm的聚碳酸脂膜形成直径约为100nm的脂质体。制备好的脂质体用磷脂定量试剂盒定量后稀释至20mg/ml，取两组100μl的脂质体分别加入等体积的磷酸缓冲液(未加PM1蛋白的对照组)、0.8mg/ml的PM1蛋白、0.8mg/ml的PM1-N短肽(加PM1蛋白、PM1-N短肽组)。28℃孵育30min后，置于-80℃冻30min，28℃溶解30min，反复3次。
(一)经冻融处理后的脂质体的浊度检测：
浊度是一种光学效应，可以反映射入光线与溶液中悬浮颗粒的相互作用， 表征光线透过水层时受到阻碍的程度。用测量浊度反映样品透射光的量或散射光的量，即溶液的透射光强度越小或散射光强度越大，表征水溶液的浊度越大。取冻融处理前后的样品各20μl用缓冲液稀释至200μl，用紫外分光光度计测定其OD400值，如图2所示。未加PM1蛋白及PM1-N短肽组的脂质体的OD400值较低；经冻融处理后，其溶液浊度明显上升，表明经冻融处理后的脂质体发生了改变，意味着脂质体受到破坏。而添加PM1蛋白、PM1-N短肽的脂质体样品，其浊度未发生改变、或略有所上升，这一结果表明PM1蛋白和PM1-N短肽对冻融后的脂质体起很好的稳定作用。
(二)经冻融处理后脂质体的粒径测定：
当光线通过不均匀介质时，会发生光散射现象，散射光中包含有颗粒大小、形状、结构以及成分、组成和浓度等信息。粒度仪，就是利用光散射技术测量溶液中颗粒的尺寸分布，确定溶液中颗粒的直径大小。取新鲜制备的脂质体80μl用磷酸缓冲液稀释至4ml，用粒度仪测定其粒径，如图3所示。制备好的脂质体粒径大小为100-200nm；经反复冻融处理后，73％的脂质体粒径未发生改变，同时出现了27％的较大粒径脂质体(约为1000-3000nm)，表明经冻融处理后的部分脂质体粒径明显增大，即冻融处理会引起脂质体破裂继而发生融合，形成较大粒径的脂质体。在脂质体溶液中添加PM1蛋白的样品，经过冻融处理后，100％的脂质体粒径未发生明显改变；添加PM1-N短肽的脂质体溶液经冻融后，95％脂质体粒径范围为100-200nm。上述结果表明，PM1蛋白和PM1-N短肽可维持、或较好的维持脂质体在冻融处理后的稳定。
(三)经冻融处理后脂质体的显微镜下观察：
采用显微镜术可直观地反映经冻融处理后脂质体的形态学变化。取制备好脂质体样品5μl置于相差显微镜下观察，所观察视野中为均质状态(图4a左)；经冻融处理后，视野中出现了明显聚集、呈块状的脂质体聚集物(图4a右)。在脂质体溶液中添加PM1蛋白、PM1-N短肽的样品，在冻融处理后，样品中仅出现少量的脂质体聚集物(图4b，图4c)。
综合以上研究结果，可以看出冻融处理可使脂质体的体积发生改变，即冻融可破坏脂质体，而破坏的脂质体也可发生再融合，这是冻融导致脂质体粒径变大的主要原因。此外，也存在另一种可能性，即冻融可导致脂质体粘度增大，使得它们聚集形成脂质体聚集物。在脂质体溶液中加入PM1蛋白、PM1-N短肽后，可阻止冻融引起的脂质体破坏及再融合、也可阻止脂质体发生彼此间聚集。
实施例2PM1蛋白对兔红细胞的保护作用测定
制备50μl的0.2％的新鲜兔血红细胞与等体积的柠檬酸缓冲液的混合液。再在含兔血红细胞的溶液中分别加入PM1(终浓度为5mg/ml)、甘油(终浓度为5％)、PM1+甘油。溶液在4℃孵育30min后，用细胞计数器统计兔血细胞数量。再将上述溶液置于-20℃冻30min，室温融解30min，反复三次处理后，进行细胞计数。
结果如图5所示，经冻融处理后的兔红细胞数明显减少，细胞剩余量仅为54％；在兔红细胞中加入PM1蛋白后再经冻融处理，剩余细胞数为72％，显著高于经冻融处理、未添加PM1蛋白的兔红细胞剩余量。若在兔红细胞溶液中加入5％甘油，经冻融后的兔红细胞数剩余量约58％，结果表明5％甘油未对经冻融的兔红细胞有保护作用；若在兔红细胞溶液中同时加入PM1蛋白和5％甘油，剩余细胞量提高至87％，表明它们对经冻融处理的兔红细胞保护作用更加明显，可见PM1蛋白与甘油对兔红细胞的有显著的协同保护作用。
由以上结果可知，在兔红细胞中添加PM1蛋白和甘油，可显著提高细胞在冻融处理后的稳定性。这一结果与PM1对脂质体的冻融下的保护作用是一致的。
实施例3PM1蛋白+海藻糖对兔红细胞的保护作用
将0.2％的兔血红细胞分别与PM1(浓度5mg/mL)、海藻糖(终浓度0.75mg/mL)、PM1+海藻糖混合。在4℃孵育30min。用细胞计数器计算兔血细胞数量。再将上述混合液加入PM1蛋白后，将样品置于-20℃冻30min，室 温融解30min。反复三次处理后，在经细胞计数。计算冻融后剩余细胞数量与未冻融血红细胞数量的比例并进行比较。
结果如图5所示，在兔红细胞溶液中加0.75mg/ml的海藻糖，经冻融的剩余的兔红细胞量为61％，即海藻糖的添加未显示出对经冻融处理的兔红细胞的保护及稳定作用；若在兔红细胞溶液中加入PM1和0.75mg/mL海藻糖后，经冻融的兔血红细胞剩余量为89％，可见PM1和0.75mg/mL对兔血红细胞具有显著的、协同的保护性效果。由此可知，在兔红细胞中添加PM1蛋白和海藻糖，可显著提高细胞在冻融处理后的稳定性。这一结果与PM1对脂质体的冻融下的保护作用是一致的。
综合以上实施例可知，大豆PM1蛋白及PM1-N短肽可以作为植物抗冻蛋白，用于制备转基因抗冻植物的生产；也可以将PM1蛋白用于生物类制品的冻存保存剂，如蛋白(酶制剂)、细胞(如卵细胞、红细胞)、胚胎、组织、器官(如捐赠的器官)；还可以用于食品的冷冻、储藏、运输、解冻等方面。此外，大豆PM1蛋白及PM1-N短肽与甘油或海藻糖组合，可以起到增强保护作用的效果。
由于PM1蛋白及PM1-N短肽为无序蛋白，呈无序结构状态，对被保护对象没有明显的选择性。因此，有着更加广阔的应用前景。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明的保护范围之内。

Claims (10)

  1. 一种大豆抗冻蛋白的制备方法，其特征在于，包括以下步骤：
    将含有所述抗冻蛋白表达序列的质粒转化至大肠杆菌中，进行蛋白表达；
    用亲和层析法对蛋白进行纯化；
    纯化后用凝血酶切除His标签，获得所述抗冻蛋白；所述抗冻蛋白为PM1蛋白或PM1-N短肽。
  2. 如权利要求1所述的制备方法，其特征在于，所述亲和层析法中所用层析柱参数如下：填料：Chelating Sepharose Fast Flow 4BTM，流速为3mL/min。
  3. PM1蛋白或PM1-N短肽在制备转基因抗冻或抗寒植物方面的应用。
  4. PM1蛋白或PM1-N短肽用于制备生物类制品的冻存保护剂的应用。
  5. 如权利要求4所述的应用，其特征在于，所述生物类制品包括蛋白及酶制剂。
  6. 如权利要求4所述的应用，其特征在于，所述生物类制品包括细胞、胚胎、组织或器官。
  7. 如权利要求4所述的应用，其特征在于，所述生物类制品为兔红细胞。
  8. 如权利要求4所述的应用，其特征在于，所述冻存保护剂中还含有甘油。
  9. 如权利要求4所述的应用，其特征在于，所述冻存保护剂中还含有海藻糖。
  10. 如权利要求4所述的应用，其特征在于，所述PM1蛋白或PM1-N短肽为重组蛋白。
PCT/CN2017/089103 2016-06-21 2017-06-20 一种大豆抗冻蛋白的制备方法、应用 Ceased WO2017219952A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201610451059.1A CN105969788B (zh) 2016-06-21 2016-06-21 一种大豆抗冻蛋白的制备方法、应用
CN201610451059.1 2016-06-21

Publications (1)

Publication Number Publication Date
WO2017219952A1 true WO2017219952A1 (zh) 2017-12-28

Family

ID=57022113

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2017/089103 Ceased WO2017219952A1 (zh) 2016-06-21 2017-06-20 一种大豆抗冻蛋白的制备方法、应用

Country Status (2)

Country Link
CN (1) CN105969788B (zh)
WO (1) WO2017219952A1 (zh)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105969788B (zh) * 2016-06-21 2019-09-17 深圳大学 一种大豆抗冻蛋白的制备方法、应用
CN108094405B (zh) * 2017-12-21 2021-06-18 湖南丰晖生物科技有限公司 细胞冻存组合物及其应用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102010869A (zh) * 2010-04-26 2011-04-13 吉林大学 一种腊梅胚胎发育晚期丰富蛋白的基因及其抗低温应用
CN103415533A (zh) * 2011-03-04 2013-11-27 株式会社钟化 源自植物种子的冰结晶化抑制物质
CN105969788A (zh) * 2016-06-21 2016-09-28 深圳大学 一种大豆抗冻蛋白的制备方法、应用

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1117497A (zh) * 1994-08-24 1996-02-28 中国科学院发育生物学研究所 高活性植物抗冻蛋白及其分离技术
US20030233681A1 (en) * 2002-05-01 2003-12-18 Arizona Bd Of Regents/Behalf Of Univ. Of Arizona ICE1, a regulator of cold induced transcriptome and freezing tolerance in plants

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102010869A (zh) * 2010-04-26 2011-04-13 吉林大学 一种腊梅胚胎发育晚期丰富蛋白的基因及其抗低温应用
CN103415533A (zh) * 2011-03-04 2013-11-27 株式会社钟化 源自植物种子的冰结晶化抑制物质
CN105969788A (zh) * 2016-06-21 2016-09-28 深圳大学 一种大豆抗冻蛋白的制备方法、应用

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
CHEN LIYI ET AL.: "Protection effect of soybean PM1 protein on liposome and the rabbit red blood cells", JOURNAL OF SHENZHEN UNIVERSITY SCIENCE & ENGINEERING, vol. 5, no. 33, 30 September 2016 (2016-09-30) *
GAO YANG: "Expression of soybean GmPM1 and GmPM9 proteins enhances resistance to copper stress in plant", CHINA MASTER'S THESES, no. 12, 31 December 2015 (2015-12-31), pages 39 - 41 *
SHIH M: "Characterization of two soybean (Glycine max L.) LEA IV pro- teins by circular dichroism and fourier transform infrared spectrometry", PLANT AND CELL PHYSIOLOLGY, vol. 51, no. 3, March 2010 (2010-03-01) - 31 December 2010 (2010-12-31), pages 395 - 407, XP055601941, ISSN: 0032-0781, DOI: 10.1093/pcp/pcq005 *

Also Published As

Publication number Publication date
CN105969788B (zh) 2019-09-17
CN105969788A (zh) 2016-09-28

Similar Documents

Publication Publication Date Title
Lu et al. Mitochondria-targeted antioxidant MitoTEMPO improves the post-thaw sperm quality
Mazur Principles of cryobiology
Prathalingam et al. Impact of antifreeze proteins and antifreeze glycoproteins on bovine sperm during freeze-thaw
Grier Development of the follicle complex and oocyte staging in red drum, Sciaenops ocellatus Linnaeus, 1776 (Perciformes, Sciaenidae)
Bang et al. Antifreeze peptides and glycopeptides, and their derivatives: potential uses in biotechnology
Liu et al. Exosomes released by human umbilical cord mesenchymal stem cells protect against renal interstitial fibrosis through ROS-mediated P38MAPK/ERK signaling pathway
Chen et al. Luteolin enhanced antioxidant capability and induced pyroptosis through NF-κB/NLRP3/Caspase-1 in splenic lymphocytes exposure to ammonia
CN103467585B (zh) 一种鱼源胶原蛋白抗冻多肽及其制备方法
Najafi et al. Comparing the effect of rooster semen extender supplemented with gamma-oryzanol and its nano form on post-thaw sperm quality and fertility
Jing et al. Metabolic Modulation of Intracellular Ammonia via Intravesical Instillation of Nanoporter‐Encased Hydrogel Eradicates Bladder Carcinoma
CN103333225A (zh) 抗菌肽及其制备方法和应用
WO2017219952A1 (zh) 一种大豆抗冻蛋白的制备方法、应用
Yanuhar et al. The identification of plankton, water quality, blood cell, and histology in culture pond of tilapia Oreochromis niloticus which infected by viral nervous necrosis (VNN)
Muhammad et al. Co-delivery of azithromycin and ibuprofen by ROS-responsive polymer nanoparticles synergistically attenuates the acute lung injury
CN102986648B (zh) 松材线虫的玻璃化冷冻保存及解冻复苏方法
CN104604843A (zh) 一种精液冷冻保存液及其制备方法
CN105646647A (zh) 一种鸡胚多肽及其制备方法与应用
CN103467586A (zh) 一种保护细菌低温冻融损伤的抗冻多肽及其制备方法
Wang et al. The Trp-rich antimicrobial amphiphiles with intramolecular aromatic interactions for the treatment of bacterial infection
Kimura et al. Inhibition of cryoaggregation of phospholipid liposomes by an Arabidopsis intrinsically disordered dehydrin and its K-segment
Guan et al. Expression and characterization of a novel endolysin LysPFX32 as potential biological antimicrobial agent against Pseudomonas fluorescens for pork preservation
CN106939108A (zh) 丝素纳米银/pva抗菌薄膜的制备方法及其产品和应用
CN120775002A (zh) 一种鳗鲡抗菌肽Anguinin及其应用
Xie et al. PH-sensitive BSA-modified resveratrol micelles targeting macrophages alleviate symptoms of rheumatoid arthritis
Yan et al. Cold tolerance of postharvest fungal pathogens is regulated by the conserved high osmolarity glycerol (HOG) pathway

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 17814690

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC (EPO FORM 1205A DATED 16/04/2019)

122 Ep: pct application non-entry in european phase

Ref document number: 17814690

Country of ref document: EP

Kind code of ref document: A1