KR20220036820A - Silk fibroin protein composition and application method thereof - Google Patents

Abstract

The present invention relates to a composition including silk fibroin protein, and more particularly, to a method for using silk fibroin protein as a stable medium for a bodily fluid to perform telemedicine diagnosis or the like. In detail, according to the present invention, the composition includes silk fibroin protein extracted from a cocoon of a specific Bombyx mori species. The protein includes a heavy chain and a light chain having a molecular weight of approximately 35 to 400 kDa. The protein extracted from Bombyx mori is partially changed from an α-helix random coil into a β-sheet in terms of a three-dimensional structure due to interaction between the heavy chain and the light chain. Therefore, a specimen lacks stabilization ability, so that the protein is not suitable for purpose of use in the present invention. Thus, according to the present invention, a distribution content of Ala(α), Gly(α), Ala(β), and Gly(β) among amino acid repeating units of fibroin is changed, so that a biological sample is stabilized. In other words, according to the present invention, problems of conventional technologies based on cold chain storage, lyophilization, covalent immobilization, and cellulose are compensated, so that a wider selection range in on-demand or point-of-care applications is provided.

Description

Silk fibroin protein composition and application method thereof

The present invention relates to a composition containing silk fibroin protein, and more particularly, to a method of using silk fibroin protein as a stable medium for bodily fluid for telemedicine diagnosis, etc.

A common method for storing and handling blood or blood fractions (plasma, serum, etc.) currently used clinically and pathologically is by freezing (-20°C). In particular, in the case of very unstable RNA, there are great limitations such as storage and transportation at cryogenic temperatures (-80° C.) in order to preserve the safety of the sample. In other words, since the sample is transported using a Styrofoam box or dry ice, an expensive transport fee may be additionally borne during the transport process.

In some developing countries or remote field conditions, the minimum infrastructure to support continuous refrigeration is often not in place, limiting the range of precision diagnostics that can be performed.

Therefore, there is a pressing need for an inexpensive and stable storage format for monitoring therapeutic efficacy and guiding appropriate transition to second-line therapy for pharmacokinetic (PK) screening.

In particular, when a global pandemic occurs, secondary infection of the disease can be caused by face-to-face contact between medical staff and patients.

In order to prevent such secondary infection, the future direction of medical diagnosis is expected to minimize face-to-face contact, and the application of a new screening test through remote diagnosis is expected to become common.

Cellulose-based formats, which are currently most widely used, are not suitable to cover all analytes as potential analytes, and we found that some markers chemically react with the cellulose matrix, making it difficult to obtain sufficient recovery for analysis.

Several shortcomings of these cellulosic matrix-based blood and plasma stabilization systems have also been revealed through an extensive set of clinical evaluations lasting many years.

These deficiencies act as a limit to the lower limit of detection (LLD) achievable with dry blood specimens and the dynamic range for adequate quantitative identification, making it impossible to ensure the integrity of dry blood specimens, and to protect against RNA viruses. It may act as a limitation that it cannot be used as a transportation means for molecular testing.

Therefore, there is an urgent need to develop improved stabilization technology and products capable of sufficiently stabilizing biological samples and components, and recovering a sufficient amount of samples to perform various analyzes from the stabilized samples for detection and analysis.

The present invention provides a recombinant fibroin protein, characterized in that whole blood and specific blood components can be stabilized over a certain period of time and a storage temperature of a certain range, and sufficient recovery of the active ingredient for analysis is possible.

The composition of the present invention absorbs a small amount of body fluid in a sheet state, and then enters a stabilization phase. And it is air-dried as a mixed complex in a thin film. In addition, it is expected that the coagulation technology of the reconstituted mixed solution containing a small amount of unstable nucleic acid can be actively utilized for more stable pharmacokinetic screening by combining the freeze-drying technology.

Currently, the only technology used for dry storage of blood is cellulose-based technology, but this technology has not been widely adopted because it cannot match the cold chain performance matrix. The protein composition of the present invention can be used to replace the on-paper device, and is expected to present a new paradigm that does not depend on the existing method that relies on cold chain storage.

It is also expected that the present invention may find application in the diagnostic utility of another form of sample collection from urine, saliva and other bodily fluids or cells.

Republic of Korea Patent Registration No. 10-2071733

An object of the present invention for solving the above problems is to provide a composition of a specimen for the stable preservation of a biological sample.

Specifically, the present invention is a more stable protein by controlling the content of a unit polypeptide that has the potential to form a β structure among proteins extracted from the cocoon of Bombyx mori , thereby delaying the transfer to the Silk II structure as much as possible. The purpose is to prepare a composition.

The present invention reduces the spontaneous transition to the β structure by controlling the content of the unit polypeptide as described above. At the same time, even in a monomolecular state upon contact with a biological sample, it promotes the formation of a β structure with a biological sample and affects the interaction between molecular chains to transform it into a more stable and robust β structure, enabling more stable preservation of biological samples Do.

The present invention relates to a silk fibroin protein composition having excellent re-solubility, wherein the content of glycine is 40 to 50% by weight and the content of alanine is 25 to 35% by weight relative to the total amino acid content of the silk fibroin protein composition, and the silk fibroin protein composition It provides a silk fibroin protein composition that has an average molecular weight of 32 kDa to 340 kDa.

Specifically, the glycine is composed of glycine (β) and glycine (α), and the ratio of glycine (β) to glycine (α) may be 0.1: 99.9 to 10: 90, but is not limited thereto.

Specifically, the alanine is composed of alanine (β) and alanine (α), and the ratio of the alanine (β) to alanine (α) may be 0.1: 99.9 to 10: 90, but is not limited thereto.

In addition, specifically, the composition includes a heavy chain and a light chain, and the weight ratio of the heavy chain and the light chain is 0.1: 99.9 to 10: 90, It is not limited thereto.

The composition may be prepared within the range of pH 1 to 13, may be prepared within the range of 0.1 atm to 2.0 atm, may be prepared within the G-Force range of 3000RCF to 20000RCF, 60 minutes It may be prepared within a reaction time range of from to 240 minutes, but is not limited thereto.

Specifically, the composition may be in the form of a solid or liquid, or the composition may be in the form of a frozen and freeze-dried specimen applied to a cellulose base or a plastic stick.

In addition, the composition itself may be in the form of a frozen and freeze-dried specimen or in the form of dried powder particles, but is not limited thereto.

The composition of the present invention is characterized in that the fibroin protein is stably maintained for 3 to 14 days, and the composition of the present invention is characterized in that the storage stability is maintained at 22 °C to 38 °C for 30 days.

On the other hand, the composition of the present invention may add 20 to 90% by weight of glycerol as an additional additive based on the total weight, but is not limited thereto.

The composition of the present invention is characterized in that it is re-solubilized in water to form a silk fibroin solution without fibroin aggregates. In particular, it is possible to recover a detectable amount of a biological sample and an active ingredient contained in the sample after re-solubilization. do.

Conditions under which a detectable amount of a biological sample can be recovered after re-solubilization of the composition may be a time of 24 hours to 14 days and a temperature condition of 43° C. or less, and the biological sample is cells, tissues, blood ( For example, whole blood, plasma, serum, platelets, etc.), lactation products, amniotic fluid, sputum, urine, saliva, mucus, bronchial aspirate, or a fraction thereof or a combination thereof, or a combination thereof, may be one containing these, the active ingredient silver peptides, proteins, antibodies, enzymes, amino acids, hexanes, DNA and fractions thereof, RNA and fractions thereof, antisense oligonucleotides, metabolites, lipids, sugars, glycoproteins, peptidoglycans, microorganisms, cells and their It may be one or more selected from the group consisting of any combination, but is not limited thereto.

Specifically, the ratio of the fibroin protein composition to the biological sample may be about 1: 100 to 100: 1, but is not limited thereto.

Another aspect of the present invention provides a kit comprising a biological sample collection device comprising the fibroin protein composition.

Specifically, the kit may further include a container containing a solubilizing agent. The solubilizing agent may include water, a buffer solution, or a combination thereof.

In addition, specifically, the kit may include a container containing a stabilizer, and the stabilizer may be to stabilize the biological sample sample.

Specifically, the stabilizer may be selected from the group consisting of antioxidants, gelatin and derivatives thereof, albumin (human and bovine serum), amino acids, cationic and anionic surfactants, sugar alcohols, or any combination thereof. More specifically, the stabilizing agent may be a nuclease or proteinase inhibitor, and most specifically, the stabilizing agent may be an RNase inhibitor, but is not limited thereto.

In addition, specifically, the kit may be configured to include one or more selected from the group consisting of a nucleic acid amplifying agent, a component purification agent, an immune detection agent, or any combination thereof.

By using the silk fibroin protein composition of the present invention, it is possible to overcome the limitations of being affected by specific temperature and humidity, which are disadvantages of existing cellulose-based products, so that a more accurate diagnosis is possible by securing protein integrity in the blood sample during the transfer process. possible.

It should be understood that the effects of the present invention are not limited to the above-described effects, and include all effects that can be inferred from the configuration of the invention described in the detailed description or claims of the present invention.

1 shows an SEM photograph of fibroin dried through a refining process in the range presented in the present invention during the fibroin extraction process from Bombyx mori silkworm (A: range presented in the present invention, B: temperature, pressure, and time respectively 10% When increasing, C: when temperature, pressure, and time are increased by 20% each).
2 shows the molecular weight and chromatogram measurement data of the dry body fluid stabilization composition of the present invention (A: molecular weight distribution diagram of DHS, B: chromatogram in pH 2.5 aqueous solution, C: chromatogram in pH 3.0 aqueous solution, D : Chromatogram at pH 3.5 aqueous solution).
Figure 3 shows the data of measuring the stability according to the pH of the composition of the present invention.
FIG. 4 shows the data measured through Raman analysis of the change process according to the pH of FIG. 3 .
5 shows the SEM measurement data of the composition of the present invention (A: SEM data of the composition of the present invention, B: SEM data of applying plasma to the composition of the present invention).
6 shows the XRD measurement data of the composition of the present invention (A: immediately after the experiment, B: after 14 days).
7 is data obtained by measuring each biomarker after storing the sample at 38°C or 22°C for 30 days using the Luminex immunoassay system. White blocks are DHS (plasma), black blocks are DHS (blood), and gray blocks are represents liquid plasma (A: serum amyloid P component (SAP), B: soluble cell adhesion molecule (sVCAM-1), C: C-reactive protein (CRP)).
Figure 8 shows the total PSA measurement values of LP (Liquid Plasma), DBS (Dried Blood Spot (cellulose sheet)), DHS (Dried Humoral Spot (silk sheet)) immediately after and 2 weeks after the experiment (A: immediately after the experiment, B: After 2 weeks at 22 degrees and 43 degrees).
9 shows the leptin measurement values of LP (Liquid Plasma), DBS (Dried Blood Spot (cellulose sheet)), and DHS (Dried Humoral Spot (silk sheet)) immediately after and 2 weeks after the experiment (A: immediately after the experiment, B : after 2 weeks at 22°C and 43°C).
10 shows prolactin measurements of LP (Liquid Plasma), DBS (Dried Blood Spot (cellulose sheet)), and DHS (Dried Humoral Spot (silk sheet)) immediately after and 2 weeks after the experiment (A: immediately after the experiment, B : after 2 weeks at 22°C and 43°C).
11 shows data measured by chromatographic analysis of the amino acid content of different silk fibroin varieties (7 varieties) (A (Baekok jam), B (Geumgwang ju), C (Hansang Ⅱ), D (Hong bak) ), E (Imbakgal won), F (N74), G (SK), H (Won jam 125), I (Won won 126)).

The above objects, other objects, features and advantages of the present invention will be easily understood through the following preferred embodiments in conjunction with the accompanying drawings. However, the present invention is not limited to the embodiments described herein and may be embodied in other forms. Rather, the embodiments introduced herein are provided so that the contents of the present invention may be thorough and complete, and the intention of the technically implemented of the present invention may be sufficiently conveyed to those skilled in the art.

In describing each figure, like reference numerals have been used for like elements. In the accompanying drawings, numerical values are enlarged than actual figures for clarity of the present invention. Terms such as first, second, etc. may be used to describe various elements, but the elements should not be limited by the terms. The above terms are used only for the purpose of distinguishing one component from another. For example, without departing from the scope of the present invention, a first component may be referred to as a second component, and similarly, a second component may also be referred to as a first component. The singular expression includes the plural expression unless the context clearly dictates otherwise.

In the present invention, the terms used are intended to designate that there is a feature, number, step, element, or a combination thereof described in the specification, and one or more other features or number, step, element or a combination thereof It should be understood that it does not preclude the possibility of the existence or addition of one. In addition, when it is said that it is "on" another part, this includes not only the case where it is "immediately on" another part, but also the case where there is another part in the middle. Conversely, when another part is said to be "under", this includes not only the case where the other part is "directly under", but also the case where there is another part in the middle.

The refining method during the process of extracting fibroin from the Bombyx mori used in the present invention may be prepared by several methods, including known methods known to those skilled in the art.

In the present invention, the "fibroin (Fibroin)" refers to an insoluble protein contained in silk produced by other moth genus, such as house silkworm larvae.

In the present invention, the term “silk” may refer to fibroin and silk itself extracted from silkworms collectively called Bombyx mori silkworm ( Bombyx mori silkworm, silkworm) or Tussah, Giant silkworm, and, in the present invention, Although the composition of the present invention was prepared using Gajamgyeon, also called, it is not limited thereto.

In general, silkworms used in the present invention are not limited to Gajamgyeon or Nojamgyeon. However, in the case of the dog, the ratio of glycine (Glycin) 42 to 47 weight percentage, alanine (Alanine) 27 to 32 weight percentage, and serine (Serine) 10 to 14 weight percentage is kept constant, so the ratio of each component is predicted and adjustable, which is advantageous for use.

Silk fibroin, a protein extracted from silkworms, is mostly composed of a heavy chain having repeated hydrophobic and hydrophilic regions and a light chain composed of hydrophilic amino acids.

As described above, the scouring process of silkworms can be performed using several methods. However, in the case of performing the initial refining process in the conventional method, the heavy chain portion having a hydrophobic region is increased as shown in FIG. 1 . This increased heavy chain forms a beta-sheet linkage covalent bond, which can affect stabilization, the object of the present invention.

The present invention provides a process for refining and extracting Bombyx mori for preparing a fibroin composition for stabilizing body fluids.

Specifically, using Na ₂ CO ₃ , the refining was performed under conditions of 60 minutes to 120 minutes, a temperature of 100 to 120 degrees, and a pressure of 0.1 to 2.0 mbar.

When the refining is carried out in excess of the above conditions, since Bombyx mori is boiled and hydrolyzed fibroin is partially produced, the object of the present invention is not met.

The silk fibroin extracted from the Bombyx mori may be dissolved in an aqueous salt solution.

The aqueous solution for achieving the above object may use lithium bromide, lithium thiocyanate, calcium nitrate, calcium chloride mixture or other chemicals capable of dissolving the extract.

In general, when a lithium bromide solution is used, a decrease in molecular weight does not occur, but, for example, when a mixed solution containing CaCl ₂ is used, a decrease in molecular weight occurs.

In the present invention, silk fibroin was dissolved using a conventional method of 8 to 12 moles of lithium bromide, but the present invention is not limited thereto.

Thereafter, in the process of removing the salt solution, a membrane may be used as a conventional method, but is not limited thereto.

In one embodiment, the addition ratio of fibroin to the aqueous solution may be 1:5 to 1:100. For example at least 1:5, at least 1:10, at least 1:15, at least 1:20, at least 1:25, at least 1:30, at least 1:40, at least 1:45, at least 1:50, at least 1 :55, at least 1:60, at least 1:65, at least 1:70, at least 1:75, at least 1:80, at least 1:85, at least 1:90, at least 1:95, at least 1:100.

In some embodiments, the temperature may be between 50 degrees and 160 degrees C, but is not limited thereto.

In some embodiments, the time may be, for example, between at least 60 minutes and 240 minutes. For example, at least 60 minutes, at least 70 minutes, at least 80 minutes, at least 90 minutes, at least 100 minutes, at least 110 minutes, at least 120 minutes, at least 130 minutes, at least 140 minutes, at least 150 minutes, at least 160 minutes, at least 170 minutes. minutes, at least 180 minutes, at least 190 minutes, at least 200 minutes, at least 210 minutes, at least 220 minutes, at least 230 minutes, at least 240 minutes.

In some embodiments, the pressure may be between 0.1 and 2.0 atm. for example at least 0.1 atm, at least 0.2 atm, at least 0.3 atm, at least 0.4 atm, at least 0.5 atm, at least 0.6 atm, at least 0.7 atm, at least 0.8 atm, at least 0.9 atm, at least 1.0 atm, at least 1.1 atm, at least 1.2 atm , at least 1.3 atm, at least 1.4 atm, at least 1.5 atm, at least 1.6 atm, at least 1.7 atm, at least 1.8 atm, at least 1.9 atm, at least 2.0 atm.

In some embodiments, gravity can be between 3000 and 20000 RCF. For example at least 3000 RCF, at least 4000 RCF, at least 5000 RCF, at least 6000 RCF, at least 7000 RCF, at least 8000 RCF, at least 9000 RCF, at least 10000 RCF, at least 11000 RCF, at least 12000 RCF, at least 13000 RCF, at least 14000 RCF , at least 15000 RCF, at least 16000 RCF, at least 17000 RCF, at least 18000 RCF, at least 19000 RCF, at least 20000 RCF.

In some embodiments, double fibroin, which is composed of heavy chain, light chain and p25 protein, has 11 domains forming a repeating crystalline region of glycine, alanine and serine, a middle portion and a C-terminal N-terminal amorphous made up of areas

The weight ratio of the heavy chain and the light chain within the temperature, time, pressure, and gravity ranges of the ranges suggested in the above conditions is 0.1: 99.9 to 10.0: 90.0.

For example 0.1:99.9, at least 0.2:99.8, at least 0.3:99.7, at least 0.4:99.6, at least 0.5:99.5, at least 0.6:99.4, at least 0.7:99.3, at least 0.8:99.2, at least 0.9:99.1, at least 1.0: 99.0, at least 1.1:98.9, at least 1.2:98.8, at least 1.3:98.7, at least 1.4:98.6, at least 1.5:98.5, at least 1.6:98.4, at least 1.7:98.3, at least 1.8:98.2, at least 1.9:98.1, at least 2.0: 98.0, at least 2.1:97.9, at least 2.2:97.8, at least 2.3:97.7, at least 2.4:97.6, at least 2.5:97.5, at least 2.6:97.4, at least 2.7:97.3, at least 2.8:97.2, at least 2.9:97.1, at least 3.0: 97.0, at least 3.1:96.9, at least 3.2:96.8, at least 3.3:96.7, at least 3.4:96.6, at least 3.5:96.5, at least 3.6:96.4, at least 3.7:96.3, at least 3.8:96.2, at least 3.9:96.1, at least 4.0: 96.0, at least 4.1:95.9, at least 4.2:95.8, at least 4.3:95.7, at least 4.4:95.6, at least 4.5:95.5, at least 4.6:95.4, at least 4.7:95.3, at least 4.8:95.2, at least 4.9:95.1, at least 5.0: 95.0, at least, at least 5.1:94.9, at least 5.2:94.8, at least 5.3:94.7, at least 5.4:94.6, at least 5.5:94.5, at least 5.6:94.4, at least 5.7:94.3, at least 5.8:94.2, at least 5.9:94.1, at least 6.0:94.0, at least 6.1:93.9, at least 6.2:93.8, at least 6.3:93.7, at least 6.4:93.6, at least 6.5:93.5, at least 6.6:93.4, at least 6.7:93.3, at least 6.8:93.2, at least 6.9:93.1, at least 7.0:93.0, at least 7.1:92.9, at least 7.2:92.8, at least 7.3:92.7, at least 7.4:92.6, at least 7.5:92.5, at least 7.6:92.4, at least 7.7:92.3, at least 7.8:92.2, at least 7.9:92.1, at least 8.0:92.0, at least 8.1:91.9, at least 8.2:91.8, at least 8.3:91.7, at least 8.4:91.6, at least 8.5:91.5, at least 8.6:91.4, at least 8.7:91.3, at least 8.8:91.2, at least 8.9:91.1, at least 9.0:91.0, at least 9.1:90.9, at least 9.2:90.8, at least 9.3:90.7, at least 9.4:90.6, at least 9.5:90.5, at least 9.6:90.4, at least 9.7:90.3, at least 9.8:90.2, at least 9.9:90.1, at least 10.0:90.0.

In some embodiments, when the weight ratio of Gly is 40% within the range of temperature, time, pressure, and gravity in the ranges indicated in the above conditions, the ratio of Gly(β) to Gly(α) is 0.1 : 99.9 to 10.0 : can be set to 90.0.

For example, the ratio of Gly(β) to Gly(α) is 0.1:99.9, at least 0.2:99.8, at least 0.3:99.7, at least 0.4:99.6, at least 0.5:99.5, at least 0.6:99.4, at least 0.7:99.3, at least 0.8:99.2, at least 0.9:99.1, at least 1.0:99.0, at least 1.1:98.9, at least 1.2:98.8, at least 1.3:98.7, at least 1.4:98.6, at least 1.5:98.5, at least 1.6:98.4, at least 1.7:98.3, at least 1.8:98.2, at least 1.9:98.1, at least 2.0:98.0, at least 2.1:97.9, at least 2.2:97.8, at least 2.3:97.7, at least 2.4:97.6, at least 2.5:97.5, at least 2.6:97.4, at least 2.7:97.3, at least 2.8:97.2, at least 2.9:97.1, at least 3.0:97.0, at least 3.1:96.9, at least 3.2:96.8, at least 3.3:96.7, at least 3.4:96.6, at least 3.5:96.5, at least 3.6:96.4, at least 3.7:96.3, at least 3.8:96.2, at least 3.9:96.1, at least 4.0:96.0, at least 4.1:95.9, at least 4.2:95.8, at least 4.3:95.7, at least 4.4:95.6, at least 4.5:95.5, at least 4.6:95.4, at least 4.7:95.3, at least 4.8:95.2, at least 4.9:95.1, at least 5.0:95.0, at least, at least 5.1:94.9, at least 5.2:94.8, at least 5.3:94.7, at least 5.4:94.6, at least 5.5:94.5, at least 5.6:94.4, at least 5.7: 94.3, at least 5.8:94.2, at least 5.9:94.1, at least 6.0:94.0, at least 6.1:9 3.9, at least 6.2:93.8, at least 6.3:93.7, at least 6.4:93.6, at least 6.5:93.5, at least 6.6:93.4, at least 6.7:93.3, at least 6.8:93.2, at least 6.9:93.1, at least 7.0:93.0, at least 7.1: 92.9, at least 7.2:92.8, at least 7.3:92.7, at least 7.4:92.6, at least 7.5:92.5, at least 7.6:92.4, at least 7.7:92.3, at least 7.8:92.2, at least 7.9:92.1, at least 8.0:92.0, at least 8.1: 91.9, at least 8.2:91.8, at least 8.3:91.7, at least 8.4:91.6, at least 8.5:91.5, at least 8.6:91.4, at least 8.7:91.3, at least 8.8:91.2, at least 8.9:91.1, at least 9.0:91.0, at least 9.1: 90.9, at least 9.2:90.8, at least 9.3:90.7, at least 9.4:90.6, at least 9.5:90.5, at least 9.6:90.4, at least 9.7:90.3, at least 9.8:90.2, at least 9.9:90.1, at least 10.0:90.0.

In some embodiments, when the weight ratio of Gly is 45% within the ranges of temperature, time, pressure, and gravity in the ranges indicated in the above conditions, the ratio of Gly(β) to Gly(α) is at least 0.1 : 99.9 to You can make it 10.0 : 90.0.

For example, the ratio of Gly(β) to Gly(α) is 0.1:99.9, at least 0.2:99.8, at least 0.3:99.7, at least 0.4:99.6, at least 0.5:99.5, at least 0.6:99.4, at least 0.7:99.3, at least 0.8:99.2, at least 0.9:99.1, at least 1.0:99.0, at least 1.1:98.9, at least 1.2:98.8, at least 1.3:98.7, at least 1.4:98.6, at least 1.5:98.5, at least 1.6:98.4, at least 1.7:98.3, at least 1.8:98.2, at least 1.9:98.1, at least 2.0:98.0, at least 2.1:97.9, at least 2.2:97.8, at least 2.3:97.7, at least 2.4:97.6, at least 2.5:97.5, at least 2.6:97.4, at least 2.7:97.3, at least 2.8:97.2, at least 2.9:97.1, at least 3.0:97.0, at least 3.1:96.9, at least 3.2:96.8, at least 3.3:96.7, at least 3.4:96.6, at least 3.5:96.5, at least 3.6:96.4, at least 3.7:96.3, at least 3.8:96.2, at least 3.9:96.1, at least 4.0:96.0, at least 4.1:95.9, at least 4.2:95.8, at least 4.3:95.7, at least 4.4:95.6, at least 4.5:95.5, at least 4.6:95.4, at least 4.7:95.3, at least 4.8:95.2, at least 4.9:95.1, at least 5.0:95.0, at least, at least 5.1:94.9, at least 5.2:94.8, at least 5.3:94.7, at least 5.4:94.6, at least 5.5:94.5, at least 5.6:94.4, at least 5.7: 94.3, at least 5.8:94.2, at least 5.9:94.1, at least 6.0:94.0, at least 6.1:9 3.9, at least 6.2:93.8, at least 6.3:93.7, at least 6.4:93.6, at least 6.5:93.5, at least 6.6:93.4, at least 6.7:93.3, at least 6.8:93.2, at least 6.9:93.1, at least 7.0:93.0, at least 7.1: 92.9, at least 7.2:92.8, at least 7.3:92.7, at least 7.4:92.6, at least 7.5:92.5, at least 7.6:92.4, at least 7.7:92.3, at least 7.8:92.2, at least 7.9:92.1, at least 8.0:92.0, at least 8.1: 91.9, at least 8.2:91.8, at least 8.3:91.7, at least 8.4:91.6, at least 8.5:91.5, at least 8.6:91.4, at least 8.7:91.3, at least 8.8:91.2, at least 8.9:91.1, at least 9.0:91.0, at least 9.1: 90.9, at least 9.2:90.8, at least 9.3:90.7, at least 9.4:90.6, at least 9.5:90.5, at least 9.6:90.4, at least 9.7:90.3, at least 9.8:90.2, at least 9.9:90.1, at least 10.0:90.0.

In some embodiments, when the weight ratio of Gly is 50% within the temperature, time, pressure, and gravity ranges indicated in the above conditions, at least the ratio of Gly(β) to Gly(α) is 0.1 : 99.9 to You can make it 10.0 : 90.0.

For example, the ratio of Gly(β) to Gly(α) is 0.1:99.9, at least 0.2:99.8, at least 0.3:99.7, at least 0.4:99.6, at least 0.5:99.5, at least 0.6:99.4, at least 0.7:99.3, at least 0.8:99.2, at least 0.9:99.1, at least 1.0:99.0, at least 1.1:98.9, at least 1.2:98.8, at least 1.3:98.7, at least 1.4:98.6, at least 1.5:98.5, at least 1.6:98.4, at least 1.7:98.3, at least 1.8:98.2, at least 1.9:98.1, at least 2.0:98.0, at least 2.1:97.9, at least 2.2:97.8, at least 2.3:97.7, at least 2.4:97.6, at least 2.5:97.5, at least 2.6:97.4, at least 2.7:97.3, at least 2.8:97.2, at least 2.9:97.1, at least 3.0:97.0, at least 3.1:96.9, at least 3.2:96.8, at least 3.3:96.7, at least 3.4:96.6, at least 3.5:96.5, at least 3.6:96.4, at least 3.7:96.3, at least 3.8:96.2, at least 3.9:96.1, at least 4.0:96.0, at least 4.1:95.9, at least 4.2:95.8, at least 4.3:95.7, at least 4.4:95.6, at least 4.5:95.5, at least 4.6:95.4, at least 4.7:95.3, at least 4.8:95.2, at least 4.9:95.1, at least 5.0:95.0, at least, at least 5.1:94.9, at least 5.2:94.8, at least 5.3:94.7, at least 5.4:94.6, at least 5.5:94.5, at least 5.6:94.4, at least 5.7: 94.3, at least 5.8:94.2, at least 5.9:94.1, at least 6.0:94.0, at least 6.1:9 3.9, at least 6.2:93.8, at least 6.3:93.7, at least 6.4:93.6, at least 6.5:93.5, at least 6.6:93.4, at least 6.7:93.3, at least 6.8:93.2, at least 6.9:93.1, at least 7.0:93.0, at least 7.1: 92.9, at least 7.2:92.8, at least 7.3:92.7, at least 7.4:92.6, at least 7.5:92.5, at least 7.6:92.4, at least 7.7:92.3, at least 7.8:92.2, at least 7.9:92.1, at least 8.0:92.0, at least 8.1: 91.9, at least 8.2:91.8, at least 8.3:91.7, at least 8.4:91.6, at least 8.5:91.5, at least 8.6:91.4, at least 8.7:91.3, at least 8.8:91.2, at least 8.9:91.1, at least 9.0:91.0, at least 9.1: 90.9, at least 9.2:90.8, at least 9.3:90.7, at least 9.4:90.6, at least 9.5:90.5, at least 9.6:90.4, at least 9.7:90.3, at least 9.8:90.2, at least 9.9:90.1, at least 10.0:90.0.

In some embodiments, when the weight ratio of Ala is 25% within the temperature, time, pressure, and gravity ranges indicated in the above conditions, at least the ratio of Ala(β) to Ala(α) is 0.1 : 99.9 to You can make it 10.0 : 90.0.

For example, the ratio of Ala(β) to Ala(α) is 0.1 : 99.9, at least 0.2 : 99.8, at least 0.3 : 99.7, at least 0.4 : 99.6, at least 0.5 : 99.5, at least 0.6 : 99.4, at least 0.7 : 99.3, at least 0.8:99.2, at least 0.9:99.1, at least 1.0:99.0, at least 1.1:98.9, at least 1.2:98.8, at least 1.3:98.7, at least 1.4:98.6, at least 1.5:98.5, at least 1.6:98.4, at least 1.7:98.3, at least 1.8:98.2, at least 1.9:98.1, at least 2.0:98.0, at least 2.1:97.9, at least 2.2:97.8, at least 2.3:97.7, at least 2.4:97.6, at least 2.5:97.5, at least 2.6:97.4, at least 2.7:97.3, at least 2.8:97.2, at least 2.9:97.1, at least 3.0:97.0, at least 3.1:96.9, at least 3.2:96.8, at least 3.3:96.7, at least 3.4:96.6, at least 3.5:96.5, at least 3.6:96.4, at least 3.7:96.3, at least 3.8:96.2, at least 3.9:96.1, at least 4.0:96.0, at least 4.1:95.9, at least 4.2:95.8, at least 4.3:95.7, at least 4.4:95.6, at least 4.5:95.5, at least 4.6:95.4, at least 4.7:95.3, at least 4.8:95.2, at least 4.9:95.1, at least 5.0:95.0, at least, at least 5.1:94.9, at least 5.2:94.8, at least 5.3:94.7, at least 5.4:94.6, at least 5.5:94.5, at least 5.6:94.4, at least 5.7: 94.3, at least 5.8:94.2, at least 5.9:94.1, at least 6.0:94.0, at least 6.1: 93.9, at least 6.2:93.8, at least 6.3:93.7, at least 6.4:93.6, at least 6.5:93.5, at least 6.6:93.4, at least 6.7:93.3, at least 6.8:93.2, at least 6.9:93.1, at least 7.0:93.0, at least 7.1: 92.9, at least 7.2:92.8, at least 7.3:92.7, at least 7.4:92.6, at least 7.5:92.5, at least 7.6:92.4, at least 7.7:92.3, at least 7.8:92.2, at least 7.9:92.1, at least 8.0:92.0, at least 8.1: 91.9, at least 8.2:91.8, at least 8.3:91.7, at least 8.4:91.6, at least 8.5:91.5, at least 8.6:91.4, at least 8.7:91.3, at least 8.8:91.2, at least 8.9:91.1, at least 9.0:91.0, at least 9.1: 90.9, at least 9.2:90.8, at least 9.3:90.7, at least 9.4:90.6, at least 9.5:90.5, at least 9.6:90.4, at least 9.7:90.3, at least 9.8:90.2, at least 9.9:90.1, at least 10.0:90.0.

In some embodiments, when the weight ratio of Ala is 30% within the temperature, time, pressure, and gravity ranges indicated in the above conditions, at least the ratio of Ala(β) to Ala(α) is 0.1 : 99.9 to You can make it 10.0 : 90.0.

For example, the ratio of Ala(β) to Ala(α) is 0.1 : 99.9, at least 0.2 : 99.8, at least 0.3 : 99.7, at least 0.4 : 99.6, at least 0.5 : 99.5, at least 0.6 : 99.4, at least 0.7 : 99.3, at least 0.8:99.2, at least 0.9:99.1, at least 1.0:99.0, at least 1.1:98.9, at least 1.2:98.8, at least 1.3:98.7, at least 1.4:98.6, at least 1.5:98.5, at least 1.6:98.4, at least 1.7:98.3, at least 1.8:98.2, at least 1.9:98.1, at least 2.0:98.0, at least 2.1:97.9, at least 2.2:97.8, at least 2.3:97.7, at least 2.4:97.6, at least 2.5:97.5, at least 2.6:97.4, at least 2.7:97.3, at least 2.8:97.2, at least 2.9:97.1, at least 3.0:97.0, at least 3.1:96.9, at least 3.2:96.8, at least 3.3:96.7, at least 3.4:96.6, at least 3.5:96.5, at least 3.6:96.4, at least 3.7:96.3, at least 3.8:96.2, at least 3.9:96.1, at least 4.0:96.0, at least 4.1:95.9, at least 4.2:95.8, at least 4.3:95.7, at least 4.4:95.6, at least 4.5:95.5, at least 4.6:95.4, at least 4.7:95.3, at least 4.8:95.2, at least 4.9:95.1, at least 5.0:95.0, at least, at least 5.1:94.9, at least 5.2:94.8, at least 5.3:94.7, at least 5.4:94.6, at least 5.5:94.5, at least 5.6:94.4, at least 5.7: 94.3, at least 5.8:94.2, at least 5.9:94.1, at least 6.0:94.0, at least 6.1:9 3.9, at least 6.2:93.8, at least 6.3:93.7, at least 6.4:93.6, at least 6.5:93.5, at least 6.6:93.4, at least 6.7:93.3, at least 6.8:93.2, at least 6.9:93.1, at least 7.0:93.0, at least 7.1: 92.9, at least 7.2:92.8, at least 7.3:92.7, at least 7.4:92.6, at least 7.5:92.5, at least 7.6:92.4, at least 7.7:92.3, at least 7.8:92.2, at least 7.9:92.1, at least 8.0:92.0, at least 8.1: 91.9, at least 8.2:91.8, at least 8.3:91.7, at least 8.4:91.6, at least 8.5:91.5, at least 8.6:91.4, at least 8.7:91.3, at least 8.8:91.2, at least 8.9:91.1, at least 9.0:91.0, at least 9.1: 90.9, at least 9.2:90.8, at least 9.3:90.7, at least 9.4:90.6, at least 9.5:90.5, at least 9.6:90.4, at least 9.7:90.3, at least 9.8:90.2, at least 9.9:90.1, at least 10.0:90.0.

In some embodiments, when the weight ratio of Ala is 35% within the temperature, time, pressure, and gravity ranges indicated in the above conditions, at least the ratio of Ala(β) to Ala(α) is 0.1 : 99.9 to You can make it 10.0 : 90.0.

For example, 0.1:99.9, at least 0.2:99.8, at least 0.3:99.7, at least 0.4:99.6, at least 0.5:99.5, at least 0.6:99.4, at least 0.7:99.3, at least 0.8:99.2, at least 0.9:99.1, at least 1.0 : 99.0, at least 1.1:98.9, at least 1.2:98.8, at least 1.3:98.7, at least 1.4:98.6, at least 1.5:98.5, at least 1.6:98.4, at least 1.7:98.3, at least 1.8:98.2, at least 1.9:98.1, at least 2.0 : 98.0, at least 2.1:97.9, at least 2.2:97.8, at least 2.3:97.7, at least 2.4:97.6, at least 2.5:97.5, at least 2.6:97.4, at least 2.7:97.3, at least 2.8:97.2, at least 2.9:97.1, at least 3.0 : 97.0, at least 3.1:96.9, at least 3.2:96.8, at least 3.3:96.7, at least 3.4:96.6, at least 3.5:96.5, at least 3.6:96.4, at least 3.7:96.3, at least 3.8:96.2, at least 3.9:96.1, at least 4.0 : 96.0, at least 4.1:95.9, at least 4.2:95.8, at least 4.3:95.7, at least 4.4:95.6, at least 4.5:95.5, at least 4.6:95.4, at least 4.7:95.3, at least 4.8:95.2, at least 4.9:95.1, at least 5.0 : 95.0, at least, at least 5.1:94.9, at least 5.2:94.8, at least 5.3:94.7, at least 5.4:94.6, at least 5.5:94.5, at least 5.6:94.4, at least 5.7:94.3, at least 5.8:94.2, at least 5.9:94.1, at least 6.0:94.0, at least 6.1:93.9, at least 6.2:93.8, at least 6.3:93.7, at least 6.4:93.6, at least 6.5:93.5, at least 6.6:93.4, at least 6.7:93.3, at least 6.8:93.2, at least 6.9:93.1, at least 7.0:93.0, at least 7.1:92.9, at least 7.2:92.8, at least 7.3:92.7, at least 7.4:92.6, at least 7.5:92.5, at least 7.6:92.4, at least 7.7:92.3, at least 7.8:92.2, at least 7.9:92.1, at least 8.0:92.0, at least 8.1:91.9, at least 8.2:91.8, at least 8.3:91.7, at least 8.4:91.6, at least 8.5:91.5, at least 8.6:91.4, at least 8.7:91.3, at least 8.8:91.2, at least 8.9:91.1, at least 9.0:91.0, at least 9.1:90.9, at least 9.2:90.8, at least 9.3:90.7, at least 9.4:90.6, at least 9.5:90.5, at least 9.6:90.4, at least 9.7:90.3, at least 9.8:90.2, at least 9.9:90.1, at least 10.0:90.0.

In some embodiments, the mutual binding time between molecular chains may vary according to a change in the pH of the aqueous solution of the present composition.

That is, theoretically, in a protein including fibroin, a heavy chain, a light chain, and a random coil molecular chain form a zwitter ion near the isoelectronic point, so that the Ion repulsion is minimized. Accordingly, the β-sheet structure is formed by the increase or decrease of hydrophobic bonds, hydrogen bonds, ionic bonds, etc. between macromolecular chains and develops into a network structure, which affects the formation of gels.

In the composition produced in the present invention, when the pH is at least 1 or less, the mutual repulsive force between molecular chains acts and no gel is formed. When the pH was 1 to 13, the mutual repulsion between molecular chains decreased and the gel formation time was reduced. At pH 3, the mutual repulsion between molecular chains was minimal, and as the pH increased, the mutual repulsion between molecular chains increased and the gel formation time increased.

At least at pH 13 or higher, negative or positive charges between molecular chains dominate, and it was confirmed through Examples that aggregation did not occur between molecular chains due to strong ionic repulsion between molecules.

In some embodiments, the mutual binding time between molecular chains according to the pH change may vary depending on the concentration of the composition, and the concentration may be 0.1 to 60 weight fractions.

For example, the concentration can be at least when the concentration of the composition is 0.1 weight fraction, at least when the concentration of the composition is 1 weight fraction, at least when the concentration of the composition is 2 weight fractions, at least when the concentration of the composition is 3 weight fractions, at least when the concentration of the composition is 3 weight fractions, at least the concentration of the composition is 4 weight fractions, at least when the concentration of the composition is 5 weight fractions, at least when the concentration of the composition is 6 weight fractions, at least when the concentration of the composition is 7 weight fractions, at least when the concentration of the composition is 8 weight fractions, at least when the concentration of the composition is 8 weight fractions. is 9 weight fraction, at least when the concentration of the composition is 10 weight fractions, at least when the concentration of the composition is 11 weight fractions, at least when the concentration of the composition is 12 weight fractions, at least when the concentration of the composition is 13 weight fractions, at least when the concentration of the composition is 13 weight fractions. is 14 weight fractions, at least when the concentration of the composition is 15 weight fractions, at least when the concentration of the composition is 16 weight fractions, at least when the concentration of the composition is 17 weight fractions, at least when the concentration of the composition is 18 weight fractions, at least when the concentration of the composition is 18 weight fractions. is 19 weight fraction, at least when the concentration of the composition is 20 weight fractions, at least when the concentration of the composition is 21 weight fractions, at least when the concentration of the composition is 22 weight fractions, at least when the concentration of the composition is 23 weight fractions, at least when the concentration of the composition is 23 weight fractions. When is 24 weight fractions, at least when the concentration of the composition is 25 weight fractions, at least when the concentration of the composition is 26 weight fractions, at least when the concentration of the composition is 27 weight fractions, at least when the concentration of the composition is 28 weight fractions, at least when the concentration of the composition is 28 weight fractions. is 29 weight fractions, at least when the concentration of the composition is 30 weight fractions, at least when the concentration of the composition is 31 weight fractions, at least when the concentration of the composition is 32 weight fractions, at least when the concentration of the composition is 33 weight fractions, at least when the concentration of the composition is 33 weight fractions. is 34 weight fraction, at least when the concentration of the composition is 35 weight fraction, at least when the concentration of the composition is 36 weight fraction, at least when the concentration of the composition is 37 weight fraction, at least when the concentration is 38 weight fractions, at least when the concentration of the composition is 39 weight fractions, at least when the concentration of the composition is 40 weight fractions, at least when the concentration of the composition is 41 weight fractions, at least when the concentration of the composition is 42 weight fractions, at least when the concentration of the composition is 42 weight fractions. when the concentration is 43 weight fractions, at least when the concentration of the composition is 44 weight fractions, at least when the concentration of the composition is 45 weight fractions, at least when the concentration of the composition is 46 weight fractions, at least when the concentration of the composition is 47 weight fractions, at least when the concentration of the composition is 47 weight fractions. when the concentration is 48 weight fractions, at least when the concentration of the composition is 49 weight fractions, at least when the concentration of the composition is 50 weight fractions, at least when the concentration of the composition is 51 weight fractions, at least when the concentration of the composition is 52 weight fractions, at least when the concentration of the composition is 52 weight fractions. when the concentration is 53 weight fractions, at least when the concentration of the composition is 54 weight fractions, at least when the concentration of the composition is 55 weight fractions, at least when the concentration of the composition is 56 weight fractions, at least when the concentration of the composition is 57 weight fractions, at least when the concentration of the composition is 57 weight fractions. When the concentration is 58 weight fraction, at least when the concentration of the composition is 59 weight fraction, and at least when the concentration of the composition is 60 weight fraction, the above properties for pH are expressed.

In some embodiments, glycerol may be added as an additional additive in order to add stability to the composition of the present invention prepared in the whole process above.

The amount of glycerol added is at least 20 weight percent, at least 25 weight percent, at least 30 weight percent, at least 35 weight percent, at least 40 weight percent, at least 45 weight percent, at least 50 weight percent, at least 55 weight percent, relative to the aqueous composition of the present invention. percentage, at least 60 weight percent, at least 65 weight percent, at least 70 weight percent, at least 75 weight percent, at least 80 weight percent, at least 85 weight percent, at least 90 weight percent.

If it is more than the above range, the gelation time is gradually decreased as the glycerol content increases. This is because the strong hydrophilic action of glycerol increases the hydrophobicity of the protein by excluding water from the protein domain, thereby forming a structure affecting hydrophobic bonding and stabilizing it.

In some embodiments, the composition of the present invention prepared in the above process may be in the form of a frozen and freeze-dried specimen by applying it to a cellulose base.

In some embodiments, the composition of the present invention prepared in the above process may itself be in the form of a frozen and freeze-dried specimen.

In some embodiments, the composition of the present invention prepared in the above process may be in the form of dried powder particles.

In some embodiments, the composition of the present invention prepared in the above whole process may be in the form of a specimen frozen and freeze-dried by applying it to a plastic or similar stick.

In some embodiments, the composition of the present invention prepared in the above whole process may be a specimen in an aqueous solution state.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Although preferred embodiments have been illustrated and described in detail in the present invention, those skilled in the relevant art will appreciate that various modifications, additions, substitutions, etc. may be made without departing from the spirit of the invention and are considered within the scope of the invention as defined in the claims. make it clear that it can be It will be understood by those skilled in the art that any one of the various embodiments described and described herein may be further modified by introducing features shown in any other embodiment disclosed herein.

Hereinafter, the present invention will be described in detail by way of Examples. However, the following examples only illustrate the present invention, and the present invention is not limited by the following examples.

Example 1. Analysis of Ser, Gly, Ala Contents of Solid Specimens

In order to confirm the amino acid composition of the solid specimen of the present invention, the contents of serine (Ser), glycine (Gly) and alanine (Ala) were analyzed using an amino acid analyzer (L-8900, hitachi).

Specifically, after adding 10 mL of 6N hydrochloric acid to the composition of the present invention, the inside was substituted and sealed with nitrogen gas, heated at 110° C. for 24 hours to hydrolyze, and then amino acids were measured.

At this time, the ratio of fibroin and soluble salt was fixed at 1 to 40, G-Force was fixed at 3000 RCF, and the difference in the contents of Ser, Gly, and Ala among the primary amino acids was confirmed by changing the temperature, time, pressure and pH. .

Experiment 1 was prepared at a temperature of 50°C, Experiment 2 was prepared at a temperature of 140°C, the time was 60 minutes, the pressure was 0.1 atm, and the measurement was performed at pH 2.5.

Experiment 3 was prepared at time 60 minutes, Experiment 4 was prepared at time 240 minutes, and the temperature was 50 degrees, the pressure was 0.1 atm, pH 2.5 was prepared and measured.

Experiment 5 was prepared at a pressure of 0.1 atm, Experiment 6 was prepared at a pressure of 2.0 atm, and the temperature was 50 degrees, the time was 60 minutes, and pH 2.5 was prepared and measured.

Experiment 7 was prepared at pH 2.5, Experiment 8 was pH 3.0, and Experiment 9 was prepared at pH 3.5, and the temperature was 50 degrees, the time was 60 minutes, and the pressure was measured at 0.1 atm.

Experiment 1 Experiment 2 Experiment 3 Experiment 4 Experiment 5 Experiment 6 Experiment 7 Experiment 8 Experiment 9 Ser 2.5 4.0 1.8 1.3 3.6 3.2 3.9 3.2 3.5 Gly 40.4 40.6 41.0 46.5 40.8 40.2 40.6 40.4 46.4 Ala 30.4 28.3 28.3 30.7 28.4 29.8 27.9 28.2 27.1

As shown in Table 1 above, glycine and alanine content in the primary amino acid sequence of the silk fibroin protein composition prepared within the range presented in the present invention through this experiment was increased, while the content of serine was relatively decreased. It was confirmed that the composition can ensure stability in aqueous solution.

Example 2. Measurement of molecular weight distribution of dry body fluid spots (DHS)

To measure the molecular weight distribution, a freeze-dried specimen was added to urea to prepare a 4M silk aqueous solution, and then measured by Fast Protein Liquid Chromatography (FPLC) (AKTA Purifier, Amersham Biosciences). The column was superdex 200 10/300GL, and the flow rate was 0.5 ml/min.

As a result, as shown in FIG. 2 , the fibroin sample in the aqueous solution state had a sharp peak at 10 min corresponding to the upper fractionation limit of molecular weight in the chromatogram and a broad peak in a wide range from 15 min to 30 min. peak) was shown.

That is, it was confirmed that fibroin in an aqueous solution has a wide range of molecular weight distribution from a heavy chain peptide of about 350 kDa to 30 kDa or less, which is the fractionation limit of the column.

The peak appearing at 10 min partially confirmed that the heavy chain, light chain, and random coil form a gel by forming a β-sheet between molecular chains over time.

Since the molecular weight of the heavy chain calculated based on the gene sequence is 340 kDa, the fibroin composition of the present invention seems to inhibit the molecular chains from aggregating with each other to form a protein colloid.

In addition, it was confirmed that a more stabilized composition could be prepared by inhibiting the formation of a semi-solid gel in the dispersion system that forms a three-dimensional network structure connected to each other at the same time.

Example 3. 13C NMR structural analysis of dry body fluid spots (DHS)

In order to confirm the structural similarity to silk fibroin, 13C NMR analysis was performed.

In the sample obtained by crystallizing the silk protein composition prepared according to the present invention with water, as a result of spectroscopic analysis, Gly(C _α ) 44.6 ppm, Gly(C=O) 169.5 ppm, Ala(C _α ) 49.8 ppm, Ala(C _β ) 21.4 ppm , Ala (C=O) peak was observed at 173.0 ppm.

In the composition crystallized using methanol, Gly(C _α ) 45.6 ppm, Gly (C=O) 170.4 ppm, Ala(C _α ) 50.7 ppm, Ala(C _β ) 22.1 ppm, Ala(C=O) 173.9 ppm, respectively A peak was observed at

And the composition crystallized by a mixed solution of methanol and water (80:20) was Gly(C _α ) 44.5 ppm, Gly(C=O) 169.4 ppm, Ala(C _α ) 49.7 ppm, Ala(C _β ) 20.7 ppm, The peak was observed at 172.0 ppm of Ala(C=O).

Ala β carbon Ala(C _β ), which is characteristically shown in the structure of silk Ⅱ, was observed to appear near 20 ppm, and it was confirmed that all compositions prepared in the present invention exhibit structural characteristics similar to those of fibroin.

Example 4. Blood Recovery from Biological Specimens

In order to calculate the plasma and blood recovery rates of the biological specimens used in the present invention, the recovery efficiency was measured by accurately converting the levels of proteins recovered from the blood and plasma specimens into concentrations found in plasma consistent with the donor.

By drying whole blood or whole plasma for 24 hours in a fume hood for physical measurement of blood and plasma and measuring the residue, the weight/volume fraction of blood or plasma (wt%; blood per unit volume (ml)) or dried plasma (g)) was measured. In addition, the weight fraction of plasma solids per blood volume was estimated by assuming that the plasma volume in the blood was 55% according to the existing literature, and it is shown in Equations 1 and 2 below.

[Formula 1]

Solid plasma weight fraction in blood (g/mL) = plasma weight fraction (g/mL) X 0.55 (mL plasma volume/mL total blood volume)

[Formula 2]

Solid plasma fraction per weight of biological specimen (g/g) = Dry plasma weight fraction in blood (g/mL) X 0.02 mL blood

The mass normalization of solid plasma was multiplied by the plasma concentration (mg/mL) in the 40 mg specimen and normalized to the mass of the total solid plasma in the volume (about 1.02±0.05 mL) suspended in 960ul of 1X PBS, as shown in Calculations 3 and 4.

[Formula 3]

Theoretical plasma fraction (mg/mL) in a 40 mg living specimen = solid plasma per weight of plasma specimen (mg/mg) X specimen 40 mg X (plasma concentration (mg/mL)/(solid plasma weight fraction (mg/mL)) X PBS Suspension volume (mL))

[Formula 4]

Theoretical blood fraction (mg/mL) in a 40 mg biological specimen = solid plasma per weight of blood specimen (mg/mg) X specimen 40 mg X (plasma concentration (mg/mL)/(solid plasma weight fraction (mg/mL)) X PBS Suspension volume (mL))

Using the above method and calculation formula, 1 ml of blood was extracted from 5 people, and the average values thereof are shown in Table 2 below.

blood weight fraction plasma weight fraction solid plasma weight fraction per blood weight
solid plasma per plasma weight
solid plasma 0.22 0.10 0.06 0.04 0.07

From the above results, it was confirmed that the recovery rate of blood and plasma could reach a statistically significant level using the composition of the present invention. Therefore, using the composition of the present invention, regardless of the type of blood and plasma , it was confirmed that the analyte can be effectively recovered from the biological specimen.

Example 5. Recovery rate of C-reactive protein

Analyte recovery rate when using the composition of the present invention was measured. C-reactive protein was used as an analyte used as a marker, and it was confirmed that a statistically significant level was reached as a result of the measurement.

Specifically, in this experimental method, the content of C-reactive protein was measured using an immunoassay in a sample obtained by simple blood sampling.

As a commonly used immunoassay, particle immunoassay, enzyme immunoassay (EIA), radioimmunoassay, fluorescence immunoassay, chemiluminescence immunoassay, etc. may be used. In this example, the C-reactive protein was quantified using an enzyme immunoassay (EIA), but the present invention is not limited thereto.

An enzyme was pre-attached to the antibody binding to the two samples that had undergone the pre-treatment process, and an antigen-antibody reaction was induced. Thereafter, an enzyme reaction was induced by adding a substrate reacting to the bound enzyme.

The enzyme used in this case can be measured using any one of alkaline phosphatase, horseradish peroxidase, and metagalactosidase (β-galactosidase). And after the enzymatic reaction, the concentration of C-reactive protein was derived using a spectrophotometer.

As a result of measuring 10 healthy adult males, the average value of the plasma sample was 0.7±0.2 (mg/dL), and it was confirmed that the value measured in the present composition was 0.7±0.23 (mg/dL).

Example 6. Stabilization verification experiment of matrix versus liquid plasma

In this example, an experiment was conducted to compare the stabilization of the matrix compared to liquid plasma using the Luminex Assays protocol.

Specifically, three markers were measured in separate donor blood samples after samples were stored for 30 days in constant temperature incubators at 22° C. and 38° C. to simulate the storage state and sample transport time frame of the present composition.

As the three markers, SAP (serum amyloid P component), sVCAM-1 (soluble cell adhesion molecule), and CRP (C-reactive protein) were used.

Storage stability at 22°C is shown in Table 3, and storage stability at 38°C is shown in Table 4. At this time, the dry bodily fluid specimen (DHS) treated with plasma was denoted as DHS (plasma), and that treated with blood was denoted as DHS (blood). In addition, plasma was detected after freezing and thawing at -20°C.

DHS (plasma) DHS (Blood) plasma CRP% recovery 85 75 95 SAP % recovery 95 110 95 sVCAM-1% recovery 97 105 80

DHS (plasma) DHS (Blood) plasma CRP% recovery 80 75 95 SAP % recovery 110 110 110 sVCAM-1% recovery 95 105 40

As shown in the above results, plasma and blood treated with the present composition showed a similar level of recovery to plasma frozen at -20°C in sVCAM-1, CRP, and SAP, confirming that it had excellent stability.

Example 7. Recovery and stability experiments of biomarkers according to storage temperature and time

In addition to the three biomarkers identified in Example 6, prolactin, fetal cancer antigen (CEA), leptin, TNF-related apoptosis inducing ligand (tumor necrosis factor related apoptosis inducing ligand, TRAIL) and The recovery and stability tests according to temperature and time were performed for total prostate specific antigen (total PSA), and the results are shown in Table 5 below.

At this time, in Table 5 below, LP denotes liquid plasma, DHS denotes dry bodily fluid spots based on the composition sheet of the present invention, and DBS denotes dry bodily fluid spots based on cellulose sheet.

0 DAY After two weeks LP DHS DBS LP DHS DBS Storage temperature (℃) -80℃ 22℃ 22℃ 22℃ 22℃ 43℃ 22℃ 43℃ 22℃ 43℃
mind
big PROLACTIN ● ● ● ○ ○ ○ ● ● ○ ○ CEA ● ● ● ○ ● ○ ● ● ● ○ LEPTIN ● ● ● ○ ● ○ ◎ ● ○ ○ TRAIL ● ● ● ● ○ ● ● ● ● ○ TOTAL PSA ● ● ● ○ ○ ○ ● ● ○ ○ - ● : No significant difference
- ◎ : Significantly low level
- ○ : Stability problem occurred

As shown in the experimental results, it was confirmed that the composition of the present invention, DHS, exhibits excellent storage stability at room temperature and high temperature, and exhibits a significantly improved effect compared to the existing technology, cellulose sheet-based technology. Description of the present invention is for illustration, and those of ordinary skill in the art to which the present invention pertains will understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. For example, each component described as a single type may be implemented in a dispersed form, and likewise components described as distributed may be implemented in a combined form. The scope of the present invention is defined in the claims to be described later. All changes or modifications derived from the meaning and scope of the claims and their equivalents should be construed as being included in the scope of the present invention.

Claims (1)

In the silk fibroin protein composition with excellent re-solubility,
Compared to the total amino acid content of the silk fibroin protein composition, the content of glycine is 40.2 to 46.5% by weight, the content of alanine is 27.1 to 30.7% by weight, and the content of serine is 1.3 to 4.0% by weight,
The silk fibroin protein composition has an average molecular weight of 32 kDa to 340 kDa,
The composition is prepared within the range of pH 2.5 to 3.5, atmospheric pressure 0.1atm to 2.0atm, gravity acceleration 3000RCF, and reaction time 60 minutes to 240 minutes,
The composition is in any one of a frozen and freeze-dried form as it is, or a frozen and freeze-dried specimen applied to a cellulose base or a plastic stick,
The composition maintains storage and recovery stability of at least one biomarker selected from the group consisting of SAP, sVCAM-1, CRP, prolactin, CEA, leptin, TRAIL and PSA for at least 14 days at 22°C to 38°C and
As an additive, 20 to 90% by weight of glycerol is added based on the total weight, silk fibroin protein composition.

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