A snail antifreeze protein and its preparation method and application

Abstract

The application discloses a snail anti-freezing protein and a preparation method and application thereof, and relates to the technical field of anti-freezing. The amino acid sequence of the anti-freezing protein is shown as SEQ ID NO:1. The protein has a high proportion of hydrophilic and hydrophobic amino acids, and shows obvious thermal hysteresis activity and ice crystal recrystallization inhibition capacity. The application further provides a preparation method of the protein, which comprises extraction from a snail body or recombinant expression through genetic engineering.

Description

Technical Field

[0001] This invention relates to the field of antifreeze technology, and more specifically to a snail antifreeze protein, its preparation method, and its application. Background Technology

[0002] In the natural environment, low temperatures, especially sub-zero temperatures, pose a severe survival challenge to organisms. When body temperature drops below freezing, internal fluids gradually freeze to form ice crystals. These ice crystals can not only pierce cell membranes and organelles, causing mechanical damage, but also disrupt the concentration balance of solutions inside and outside the cell, causing a sharp change in osmotic pressure, leading to cell death and tissue necrosis. However, over the long course of evolution, some organisms living in cold regions—such as polar fish, insects, plants, and certain bacteria—have gradually evolved a highly efficient life protection mechanism, enabling them to survive in icy environments. Among these mechanisms, the production of antifreeze proteins is considered to be at the core of this adaptive strategy.

[0003] Antifreeze proteins are a class of proteins with special functions. They can significantly lower the freezing point of body fluids in a non-colligeral manner, with minimal effect on the melting point. This characteristic is known as "thermal hysteresis activity." Unlike mechanisms that rely on small molecule solutes (such as salts or glycerol) to lower the freezing point through colligative principles, antifreeze proteins can selectively adsorb onto the surface of ice crystals, thereby inhibiting further growth and recrystallization of ice crystals and creating a significant temperature difference between the freezing and melting points (i.e., thermal hysteresis). Specifically, antifreeze proteins can bind to specific crystal faces of ice crystals (such as primary or secondary facets), inhibiting growth in that direction and thus altering the normal morphology of ice crystals, causing them to exhibit abnormal shapes such as needle-like, bipyramidal, hexagonal plate-like, or dendritic forms. At the same time, through their adsorption and inhibition mechanisms, antifreeze proteins can also effectively prevent small ice crystals from fusing into large, destructive ice crystals at low temperatures, i.e., inhibiting ice crystal recrystallization. These effects work together to ensure that biological fluids do not form sharp, large ice crystals at low temperatures, thereby reducing the damage of freezing to cells and tissues. Given the thermal hysteresis activity, ice crystal morphology regulation ability, and recrystallization inhibition functions of antifreeze proteins, they have shown broad application potential in the field of cryopreservation of sperm, egg cells, embryos, and even various foods and meat products.

[0004] Located in the high-latitude, high-altitude southern mountainous region of Shihezi, Xinjiang Uygur Autonomous Region, the winter climate is extremely cold. A certain type of snail discovered in this region exhibits a strong overwintering ability and a relatively abundant population. Microscopic observation of its tissue homogenate using a nano-osmometer revealed that the ice crystals exhibit a sharply edged hexagonal structure, suggesting the possible presence of antifreeze proteins within the snail, making it an ideal material for studying biological antifreeze mechanisms. Furthermore, the known types of insect antifreeze proteins are currently limited, mainly concentrated in Coleoptera, Lepidoptera, and Collembola, with no reports of their presence in mollusks.

[0005] Therefore, discovering and elucidating a novel antifreeze protein derived from snails and its potential applications has become an urgent task for researchers in this field. Summary of the Invention

[0006] In view of this, the present invention provides a snail antifreeze protein, its preparation method and application.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] A snail antifreeze protein, the antifreeze protein being numbered Gene.43641, has an amino acid sequence as shown in SEQ ID NO:1.

[0009] Furthermore, protein molecules with antifreeze function can be formed by replacing, deleting, or inserting one or more amino acid residues in the amino acid sequence shown in SEQ ID NO: 1.

[0010] A DNA molecule encoding the snail antifreeze protein, said DNA molecule being (a) or (b): (a) The nucleotide sequence is shown in SEQ ID NO: 2; (b) A DNA molecule encoding the snail antifreeze protein formed by replacing, deleting or inserting one or more nucleotides of the nucleotide sequence shown in SEQ ID NO: 2.

[0011] A recombinant expression vector comprising the DNA molecule and a regulatory sequence for expression operatively linked to the DNA molecule.

[0012] The host cell includes the DNA molecule or the recombinant vector.

[0013] The preparation method of snail antifreeze protein includes the following steps: (1) Construct a recombinant expression vector containing a nucleic acid sequence encoding the snail antifreeze protein; (2) The recombinant expression vector was transformed into Escherichia coli host cells to obtain recombinant engineered bacteria; (3) The recombinant engineered bacteria were cultured under induction conditions to express soluble His-SnailAFP fusion snail antifreeze protein; (4) Lyse the bacterial cells to obtain a crude extract containing the fusion protein; (5) The crude extract was purified by Ni Agarose Resin column affinity chromatography to obtain high-purity snail antifreeze protein.

[0014] Furthermore, the induction conditions in step (3) are as follows: induction is performed using isopropyl-β-D-thiogalactoside (IPTG) at a concentration of 0.8 mM, an induction temperature of 20°C, and an induction time of 5-6 hours.

[0015] Furthermore, the elution conditions for the Ni Agarose Resin column affinity chromatography in step (5) are to use buffers containing 20 mM and 500 mM imidazole to wash away impurities and elute the target protein, respectively.

[0016] Application of snail antifreeze protein in the field of antifreeze.

[0017] As can be seen from the above technical solution, compared with the prior art, the beneficial effects of the present invention are as follows: This invention incorporates antifreeze proteins against Escherichia coli. E. coli (BL21) exhibits significant cryoprotective effects, effectively maintaining survival in low-temperature environments. Today, thanks to its unique functional properties, antifreeze proteins have found important applications in various fields such as food, agriculture, and medicine: in the food industry, they can improve the texture and preservation of frozen foods; in agriculture, they provide new biotechnological means for cold-resistant crop breeding; and in the medical field, they facilitate the long-term low-temperature storage of biological samples and provide auxiliary support for clinical practices such as organ transplantation. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0019] Figure 1 A three-dimensional structural model of snail antifreeze protein; Figure 2SDS-PAGE images of different BL21 bacterial cells before and after induction expression: Lane A: E. coli BL21 (pET32a-AFP) cells without IPTG induction; Lane B: E. coli BL21 (pET32a-AFP) cells after IPTG induction to express the antifreeze protein Trx-AFP; Lane C: Supernatant after sonication of E. coli BL21 (pET32a-AFP) cells after IPTG induction to express the antifreeze protein Trx-AFP; Lane D: Precipitate after sonication of E. coli BL21 (pET32a-AFP) cells after IPTG induction to express the antifreeze protein Trx-AFP; Lane E: E. coli BL21 (pET32a empty vector) cells without IPTG induction; Lane F: E. coli BL21 (pET32a empty vector) cells endogenously expressing the tag protein Trx of the empty vector plasmid in IPTG induction. Figure 3 SDS-PAGE image of IPTG-induced expression in E. coli; Lane A shows the fusion antifreeze protein Trx-AFP expressed by IPTG-induced E. coli BL21 (pET32a-AFP); Lane B shows the SDS-PAGE image of the target protein Trx solution after purification by nickel column chromatography of the tag protein Trx (thioreductase protein) of the empty vector plasmid expressed by IPTG-induced E. coli BL21 (pET32a empty vector). Figure 4 The middle figure shows the growth process of a single ice crystal without antifreeze proteins. Figure 4 A is simple phosphate-buffered saline (PBS). Figure 4 B is a PBS solution of E. coli BL21 (pET32a empty vector) after endogenous expression of the tag protein Trx induced by IPTG, followed by sonication and disruption. Figure 4 C represents the growth and change of a single ice crystal in the supernatant of PBS solution after disruption of E. coli BL21 (pET32a-AFP) without IPTG induction, with a scale bar of 10 μm; Figure 5 The growth and changes of a single ice crystal in the supernatant of PBS solution after IPTG-induced disruption of Escherichia coli BL21 (pET32a-AFP) are shown. The scale bar is 10 μm. Figure 6 This is a diagram illustrating the growth and changes of ice crystals after IPTG-induced expression in E. coli. Figure 6 A represents the growth and changes of individual ice crystals in the Trx-AFP fusion solution of *E. coli* (pET32a-AFP) after IPTG-induced expression of the antifreeze protein Trx-AFP, followed by nickel column purification. Figure 6B is a graph showing the growth process of a single ice crystal in the target protein Trx solution after purification by nickel column chromatography. The tag protein Trx (thioreductoid) of the empty vector plasmid expressed by IPTG-induced Escherichia coli BL21 (pET32a empty vector) is 10 μm. Figure 7 The thermal hysteresis activity of BSA, purified tag protein Trx, and Trx-AFP fusion antifreeze protein solution as a function of concentration is shown in the graph. Figure 8 The growth curves of BL21 bacteria from different treatment groups after treatment at -80℃ for 24 h and addition to new culture medium. Detailed Implementation

[0020] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0021] Example 1 In January 2024, this invention discovered a snail with significant overwintering ability in the high-latitude, high-altitude Nanshan region of Shihezi City, Xinjiang Uygur Autonomous Region. Observation of its tissue homogenate using nanoliter osmotic pressure technology revealed that the ice crystals exhibited a typical hexagonal growth morphology, suggesting that the snail may contain antifreeze active substances.

[0022] Subsequently, based on existing snail transcriptome data, researchers identified several uncharacterized gene sequences suspected of encoding antifreeze proteins. By combining existing research experience and analyzing their structural patterns, a candidate gene, Gene.120703, was selected and analyzed using E. coli. E. coli Heterologous expression was performed using the (BL21) expression system. Recombinant protein was obtained after IPTG-induced expression and nickel column purification. Activity assays showed that the protein could still modify ice crystal growth morphology. At a protein concentration of approximately 1.08 mg / mL, as determined by UV-280, it exhibited thermal hysteresis activity at approximately 0.7 °C, further confirming its antifreeze function.

[0023] The amino acid sequence of snail antifreeze protein has a certain regularity. The sequence is composed of 41 amino acids in a unit, which is repeated 13 times. It has no homology with the sequences of known antifreeze proteins.

[0024] Nanshan Snail Bradybaena The amino acid sequence (521 residues) of the antifreeze protein Gene.120703 of sp. is shown in SEQ ID NO: 1: MTGFGAARHIADTRVEDITPDSHAVIGLQSGSNKFASQKGM ASFGAVRHVSDIRVDNQLQEGQGVLSLQSGTNKYESQKGM TSFGAVRHIADKRIDDMDQISQSIVRLQAGTNKLDSQKGM TSFGAVRHVSDIRAADINPEAHGVIGLQAGSNKYASQKGM TSFGAARHVSDIRADDINPEAHGVIGLQAGSNKYASQKGM TSFGSVRHVSDIRADDMNPEGQTVLSLQAGTNKFESQKGM TSFGSVRHVSDIRADDINPQGHGVIGLQAGSNKHASQKGM TSFGAARHVSDIRADDINPEAHGVIGLQSGSNKFASQKGM TSFGAVRHVSDIRSEDINPEAHGVIGLQSGSNKFASQKGM TSFGAARHVSDIHTEEMTPASEGIVRLQAGTNKMDSQKGM TAFGAVRHVSDIHSADMTPEGQSVIGLQAGSNKFASQKGM TSFGAVRHISDIRADAVDQKGQGVLSLQTGTNRFDSQKGM TAFGAVRHISDIRADQIVPDSHATIGLQAGSNKFASQKGM; The three-dimensional structural model of the snail antifreeze protein is shown below. Figure 1 .

[0025] Nanshan Snail Bradybaena The antifreeze protein gene sequence Gene.120703 (1206 bp) of sp. is shown in SEQ ID NO: 2:

[0026] (1) Preparation of recombinant engineered bacteria: A recombinant expression vector containing a nucleic acid sequence encoding the snail antifreeze protein was constructed; the recombinant expression vector was transformed into Escherichia coli host cells to obtain recombinant engineered bacteria; (2) Induced expression: Select a single colony that has been verified to be positive and inoculate it into LB liquid medium containing ampicillin (100 μg / mL). Incubate overnight at 37°C with shaking at 160 rpm / min. Add IPTG to the medium to a final concentration of 0.8 mM and lower the incubation temperature to 20°C. Continue to induce expression for 5-6 hours.

[0027] (3) Collection and disruption of bacterial cells: After induction, bacterial cells were collected by centrifugation at 8000 rpm / min for 5 minutes at 4°C. The cells were resuspended in 20 mL of PBS. Cell disruption was performed using an ultrasonic cell disruptor in an ice-water bath (125 W, 5 seconds sonication, 5-second interval, total duration 1 h). The disrupted bacterial solution was centrifuged at 10000 rpm / min for 30 minutes at 4°C, and the supernatant (the crude extract containing the soluble His-SnailAFP fusion protein) was collected. SDS-PAGE electrophoresis showed successful expression of the recombinant protein containing snail Gene.120703 in *E. coli*. The SDS-PAGE electrophoresis results are shown in [link to SDS-PAGE electrophoresis results]. Figure 2 .

[0028] (4) Ni Agarose Resin column affinity chromatography purification: Add 5 column volumes of double-distilled water to the packed column to flush out the ethanol in the tubing, then equilibrate the column with 10 column volumes of binding buffer. Load the supernatant through a 0.22 μm filter into the equilibrated nickel column. Wash with 15 column volumes of PBS containing 20 mM imidazole to remove unbound contaminating proteins. Then elute with 15 column volumes of elution buffer containing 250 mM imidazole and collect the eluted target protein.

[0029] (5) Dialysis: The eluted target protein was placed in a 3 kDa dialysis bag and dialyzed overnight with PBS at 4°C. The protein was then concentrated with sucrose powder to remove high concentrations of imidazole. After dialysis, high-purity recombinant snail antifreeze protein was obtained and stored at -20°C. SDS-PAGE electrophoresis showed a single protein band at the expected molecular weight, with a purity greater than 95%. The SDS-PAGE electrophoresis results are shown below. Figure 3 .

[0030] (6) Protein activity identification: The ice crystal modification activity of antifreeze proteins was observed using a Nanolitre Osmometer (Otago Osmometers Ltd, New Zealand) and a Zeiss AX10 microscope (Zeiss, German). 2 μL of sample was pipetteted into the sample well of the Nanolitre Osmometer's temperature control stage. The stage was placed on the microscope stage, and the temperature was rapidly decreased until the entire field of view was frozen. The temperature was then slowly increased, and the temperature at which melting into individual ice crystals began was recorded. This process was repeated until only a single ice crystal remained in the field of view. The temperature was then slowly decreased again, and the growth of individual ice crystals was observed, while the temperature at which individual ice crystal growth began was recorded. The ice crystal modification activities of the simple PBS solution, empty protein solution, un-IPTG-induced recombinant protein solution, and IPTG-induced recombinant protein solution are described in [reference needed]. Figure 4 , Figure 5 and Figure 6 The thermal hysteresis curves of PBS solution, empty protein solution, and IPTG-induced recombinant protein solution as a function of concentration are shown in the figure. Figure 7 .

[0031] (7) The cryoprotective effect of snail recombinant antifreeze protein on Escherichia coli: The experiment consisted of four groups: Group 1: pET32a-TrxA-AFP recombinant bacteria (induced by 0.8 mM IPTG for 6 h); Group 2: pET32a-TrxA empty vector bacteria (induced by 0.8 mM IPTG for 6 h) with exogenously added AFP; Group 3: pET32a-TrxA empty vector bacteria (induced by 0.8 mM IPTG for 6 h); and Group 4: pET32a-TrxA empty vector bacteria (without IPTG induction). Two mL of bacterial culture from Groups 1, 3, and 4 were each mixed with an equal volume (2 mL) of PBS and transferred to 5 mL sterile EP tubes. Another 2 mL of bacterial culture from Group 2 was mixed with an equal volume (2 mL) of AFP solution, resulting in a protein concentration of 200 μg / mL, and then transferred to 5 mL sterile EP tubes. All samples were thoroughly mixed and then frozen at -20 ℃ for 48 h. After thawing, the culture was transferred to 20 mL of LB medium and incubated at 37 °C and 160 rpm for growth recovery. OD600 absorbance was measured at 500 μL of bacterial culture every 2 h, and then every 1 h thereafter. Each group was divided into three replicates, and growth curves of *E. coli* were plotted. The *E. coli* growth curves are shown below. Figure 8 .

[0032] This invention retrieved a similar protein sequence with high homology to the Nanshan snail in other species, containing 41 amino acid repeats, suggesting that it may have a certain antifreeze effect.

[0033] Similar sequence of Taoyuan snail (944 residues), shown as SEQ ID NO: 3: MANREKPMGMDRALHSKVSAKYDVYAEREARDWILALTGEDIGSGAAEVERRLKDGQILVKLINAVYAKTPNLPEAARHVNLKGNTSELPFKQMENIEIFLKAAQLYGVPENSLFPTGDLFEGRNMAMVLATITQLGTEAQRNGFTGPTCGPKPTEKHVMEFSREQLKAGETIIGLQAGTNKLASQAGMKIGTPRHVADIRADDFVKEGQGVLGLQAGTNKFATQAGMSFGAVRHVSDIRADEFCREGQGVITLQSGTNKFASQKGMTGFGAARHIADTRVEDITPDSHAVIGLQSGSNKFASQKGMASFGAVRHVSDIRADNQLQEGQGVLSLQSGTNKYESQKGMTSFGAVRHIADKRIDDMDQIGQSIVRLQAGTNKLDSQKGMTSFGAVRHVSDIRAADINPEAHGVIGLQAGSNKFASQKGMTSFGAARHVSDIRADDINPEAHGVIGLQAGSNKYASQKGMTSFGSVRHVSDIRADEMNPEGQAVLSLQAGTNKFESQKGMTSFGSVRHVSDIRADDINPQGHGVIGLQAGSNKHASQKGMTSFGAARHVSDIRADDINPEAHGVIGLQSGSNKFASQKGMTSFGAVRHVSDIRSEDINPEAHGVIGLQSGSNKFASQKGMTSFGAARHVSDIHTEEMTPASEGIVRLQAGTNKMDSQKGMTAFGAVRHVSDIHSADMTPEGQSVIGLQAGSNKFASQKGMTSFGAVRHISDIRADELDQKGQGVLSLQTGTNQFDSQKGMTAFGAVRHISDIRADQIVPDSHATIGLQAGTNKYASQKGMSSFGAARHVSDIRADDMSQEGQGVIGLQAGSNKFASQKGMSSFGAARHVSDIRADDMSQEGQGVIGLQAGSNKFASQSGMTGFGAVRHIADSRIDEMNQEAQGVIGLQAGSNKFASQAGMTGFGAVRHIADLRIEDMNQEAQGVIGSQAGSNKFASQ。

[0034] Similar sequence of Gobi snail (587 residues), as shown in SEQ ID NO: 4: MANREKPMGMDRALHSKVSAKYDVYAEREARDWILALTGEDIGSGAAEVERRLKDGQILVKLINAVYAGTPNLPPAAREANLKGNTSELPFKQMENIEIFLKAAQLYGVPENSLFPTGDLFEGRNMAMVLATITQLGTEAQRNGFTGPTCGPKPTEKHVMEFSREQLKAGETIIGLQAGTNKLASQAGMKFGTARHVADIRADDFVKEGQGVLGLQAGTNKFATQAGMSFGSVRHVSDIRADELCREGQGVITLQSGTNKFASQKGMTGFGAARHIADTRVEDITPDSHAVIGLQSGSNKFASQKGMASFGAVRHVSDIRVDNQLQEGQGVLSLQSGTNRYESQKGMTSFGAVRHIADKRIDEMDQIGQSIVRLQAGTNKLDSQKGMTSFGAVRHVSDIRAADINPEAHGVIGLQAGSNKYASQKGMTSFGAARHVSDIRADDINPEAHGVIGLQAGSNKYASQKGMTSFGSVRHVSDIRADDMNPEGQSVLSLQAGTNKFESQKGMTSFGSVRHVSDIRADDINPQGHGVIGLQAGSNKHASQKGMTSFGSVRHVSDIRADDINPEAHGVIGLQSGSNKFASQKGM。

[0035] Similar sequence of Planorbidae snail (142 residues), as shown in SEQ ID NO: 5: FEGRNMAMVISTILQLGTEAQRHGFNGPVCGPKPSEKHVVEFTEAQLRAGHSIIGLQAGTNKCASQSGMSFGAVRHIADIRADDASREGQGVIGLQAGSNKGASQAGMSFGSVRHIADIRADDASREGQGHIGLQAGSNKRA。

[0036] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A snail antifreeze protein, characterized in that, The antifreeze protein has the amino acid sequence shown in SEQ ID NO:

1.

2. The snail antifreeze protein according to claim 1, characterized in that, Protein molecules with antifreeze function formed by replacing, deleting or inserting one or more amino acid residues of the amino acid sequence shown in SEQ ID NO:

1.

3. A DNA molecule encoding the snail antifreeze protein of claim 1, characterized in that, The DNA molecule is (a) or (b): (a) Its nucleotide sequence is shown in SEQ ID NO: 2; (b) A DNA molecule encoding the snail antifreeze protein as described in claim 1, formed by replacing, deleting or inserting one or more nucleotides of the nucleotide sequence shown in SEQ ID NO:

2.

4. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the DNA molecule of claim 3 and a regulatory sequence for expression operatively linked to the DNA molecule.

5. A host cell, characterized in that, The host cell comprises the DNA molecule of claim 3 or the recombinant vector of claim 4.

6. The method for preparing the snail antifreeze protein according to claim 1, characterized in that, Includes the following steps: (1) Construct a recombinant expression vector containing a nucleic acid sequence encoding the snail antifreeze protein; (2) The recombinant expression vector was transformed into Escherichia coli host cells to obtain recombinant engineered bacteria; (3) The recombinant engineered bacteria were cultured under induction conditions to express soluble His-SnailAFP fusion snail antifreeze protein; (4) Lyse the bacterial cells to obtain a crude extract containing the fusion protein; (5) The crude extract was purified by Ni Agarose Resin column affinity chromatography to obtain high-purity snail antifreeze protein.

7. The method according to claim 6, characterized in that, The induction conditions in step (3) are as follows: induction is performed using isopropyl-β-D-thiogalactoside (IPTG) at a concentration of 0.8 mM, at a temperature of 20°C, and for a duration of 5-6 hours.

8. The method according to claim 6, characterized in that, The elution conditions for Ni Agarose Resin column affinity chromatography in step (5) are to use buffers containing 20 mM and 500 mM imidazole to wash away impurities and elute the target protein, respectively.

9. The application of the snail antifreeze protein of claim 1 in the field of antifreeze.

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