Use of rprp1 protein in the preparation of products against oxidative and high temperature stress

Abstract

This invention belongs to the field of bioengineering technology, specifically relating to the use of rPRP1 protein in the preparation of antioxidant and heat stress-resistant products. The rPRP1 protein can significantly enhance the tolerance of *C. elegans* to oxidative and heat stress, reduce the levels of oxidative damage markers in vivo, and upregulate the expression of mitochondrial antioxidant-related genes. This protein has broad application prospects in the development of functional products with antioxidant, anti-aging, and stress-enhancing properties.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to the use of rPRP1 protein in the preparation of antioxidant and high-temperature stress-resistant products. Background Technology

[0002] Environmental stress is a significant factor limiting the growth and development of organisms and affecting their survival rate. Among these, oxidative stress and high-temperature stress are the most common and extremely destructive. Under oxidative stress, reactive oxygen species (ROS) accumulate in large quantities within organisms, disrupting the redox balance and leading to lipid peroxidation, protein degradation, and DNA damage, which can even trigger apoptosis in severe cases. High-temperature stress not only directly causes protein denaturation and inactivation and damage to biomembrane structures but also induces secondary severe oxidative stress; that is, high temperatures are often accompanied by a surge in ROS. Therefore, oxidative stress and high-temperature stress are frequently intertwined.

[0003] To address the aforementioned issues, various antioxidants and heat protectants have been developed in the prior art. However, most existing protein protectants (such as conventional heat shock proteins or antioxidant enzymes) have limited functions and can only cope with single oxidative or high-temperature stresses, making it difficult to achieve synergistic resistance. On the other hand, chemically synthesized antioxidants (such as BHA and BHT) pose potential biotoxicity and safety risks.

[0004] Therefore, there is an urgent need in this field to develop a bioactive substance that is safe and natural in origin and has both antioxidant and high-temperature stress tolerance. Summary of the Invention

[0005] This invention identifies and isolates proteins with dual stress-resistance activities from natural agricultural products, and successfully clones a gene encoding a small nucleoprotein U1-C from red rice (named...). rPRP1 The study identified a gene and demonstrated that its encoded rPRP1 protein significantly promotes oxidative stress tolerance in the body, providing a new candidate gene and material basis for developing antioxidant functional factors based on rice.

[0006] One objective of this invention is to provide the use of a biomaterial in the preparation of products that enhance animal oxidative stress tolerance, heat tolerance, and / or anti-aging; The biomaterial is selected from any one of the following: (1) From red rice ( Oryza sativa cv. Dahonggu Proteins isolated from [organism name] that possess antioxidant stress activity, high temperature stress tolerance, and / or anti-aging properties; (2) A protein having at least 70%, 75%, 80%, 85%, 90%, 95% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 2; (3) Isolated nucleic acid molecules that encode proteins as described in (1) or (2); (4) A recombinant vector comprising the nucleic acid molecules described in (3); (5) Host cell; which contains nucleic acid molecules as described in (3) and / or recombinant vectors as described in (4).

[0007] In some preferred embodiments, the biomaterial is selected from any of the following: (1) Proteins with amino acids as shown in SEQ ID NO: 2; (2) The isolated nucleic acid molecules that encode the protein described in (1); (3) A recombinant vector comprising the nucleic acid molecules described in (2); (4) Host cell; which contains nucleic acid molecules as described in (2) and / or recombinant vectors as described in (3).

[0008] In some embodiments, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO: 1.

[0009] In some embodiments, the recombinant vector is pCold I.

[0010] In some embodiments, the host cell includes eukaryotes and prokaryotes.

[0011] In some preferred embodiments, the host cell is Escherichia coli.

[0012] In some embodiments, the product includes food, food additives, health food, health food additives, feed, feed additives, and drugs.

[0013] In some preferred embodiments, the product includes feed and feed additives.

[0014] In some embodiments, the product is used to scavenge reactive oxygen species (ROS), reduce lipofuscin accumulation, and / or regulate the expression levels of antioxidant-related genes.

[0015] In some preferred embodiments, the antioxidant-related genes include, but are not limited to: MEV-1 , ISP-1 , GST-4 , GSC-1 .

[0016] In some preferred embodiments, the animal is a nematode.

[0017] A second objective of this invention is to provide a composition for anti-stress and / or anti-aging, comprising an effective amount of amino acids such as the protein shown in SEQ ID NO: 2, and a physiologically acceptable carrier.

[0018] In some embodiments, the composition further includes other antioxidants, heat protectants, and / or anti-aging products.

[0019] A third objective of this invention is to provide a method for enhancing oxidative stress tolerance and / or heat tolerance in animals for non-disease treatment, the method comprising: The animal is given an effective amount of the amino acid protein as shown in SEQ ID NO: 2, or the anti-stress composition.

[0020] In some preferred embodiments, the animal is a nematode.

[0021] SEQ ID NO: 1: .

[0022] SEQ ID NO: 2: MPRYYCDYCDTYLTHDSPSVRKQHNAGYKHKANVRTYYQQFEEQQTQSLIDQRIKEHLGQAAAFQVGAPFNQHLLSFPGGVPRPRLPILPTPGMPLGVPQVPGAPLMPGVRPPIL PAPGIPGYPGAPNVPTMPQTGAPPGSMPPGSMPPGSMPMQMAPLPRPPTLPPPTSGAPGAPIPNSGAPPAMYQTNPPQPAGPTSGAPPPVSAPPPAAPPQAPFSYAQPPEGNH*.

[0023] This invention provides the use of rPRP1 protein derived from red rice in the preparation of products that enhance animal oxidative stress tolerance, heat tolerance, and / or anti-aging. rPRP1 protein significantly enhances the tolerance of *C. elegans* to oxidative and heat stress, reduces the levels of oxidative damage markers in vivo, and upregulates the expression of mitochondrial antioxidant-related genes. This protein has broad application prospects in the development of functional products (such as foods, health products, and feed additives) with antioxidant, anti-aging, and stress-enhancing properties. Attached Figure Description

[0024] Figure 1 The electrophoresis results of rPRP1 protein induction expression are shown. CK represents the control group; rPRP1 represents the rPRP1 proteome.

[0025] Figure 2 The survival rates of *Caenorhabditis elegans* after treatment with 8 mM H2O2 for different durations are shown in the figure (* indicates...). P <0.01 indicates P <0.05).

[0026] Figure 3 The results show the survival rate comparison of Caenorhabditis elegans after heat treatment at 37℃ for different times.

[0027] Figure 4 The effect of feeding rPRP1 on the relative content of lipofuscin in nematodes was shown.

[0028] Figure 5 The effect of feeding rPRP1 on ROS levels in nematodes is shown (fluorescence imaging and quantitative analysis). In the figures, A shows images of the nematode control group and the group fed the target protein under white light and fluorescence, respectively; B shows a comparison of the relative body length of nematodes; and C shows the results of the comparative analysis of the relative ROS content in nematodes.

[0029] Figure 6This shows antioxidant-related genes in nematodes after feeding with rPRP1. GST-4 (A), MEV-1 (B) ISP-1 (C), GSC-1 (D) relative expression level. Detailed Implementation

[0030] To enable those skilled in the art to better understand and implement the technical solutions of the present invention, the present invention will be further described below in conjunction with specific embodiments and accompanying drawings.

[0031] Unless otherwise specified, all reagents used in this invention are commercially available, and all methods used are conventional techniques in the art.

[0032] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly used in the field to which this invention pertains. For the purposes of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural forms, and vice versa.

[0033] Unless the context clearly indicates otherwise, the terms “a” and “an” as used herein include plural references. For example, reference to “a cell” includes multiple such cells and equivalents known to those skilled in the art, etc.

[0034] The numerical ranges used in this article should be understood as including all numbers within that range. For example, the range 1 to 20 should be understood to include any number, combination of numbers, or subrange from the following group: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0035] As used herein, the term "comprising" or "including" means "including, but not limited to." This term is intended to be open-ended to specify the presence of any of the stated features, elements, integers, steps, or components, but does not exclude the presence or addition of one or more other features, elements, integers, steps, components, or groups thereof. Therefore, the term "comprising" includes the more restrictive terms "consisting of" and "substantially consisting of." In one embodiment, the term "comprising" as used throughout the application, particularly in the claims, may be replaced by the term "consisting of."

[0036] As used herein, the terms “optional,” “any,” “arbitrary,” or “any one” mean that the event or situation described below may, but does not have to, occur, including the circumstances in which the event or situation occurs or does not occur. As used herein, “an” and “a” refer to one or more grammatical objects.

[0037] The term “and / or” as used herein should be understood to mean any one of the options or any combination of two or more of the options.

[0038] Example 1: Cloning and Sequence Analysis of the rPRP1 Gene in Red Rice Materials: Red rice variety "Da Hong Gu" cultivar.

[0039] Methods: The full-length CDS of the target gene was amplified from Dahonggu cDNA by RT-PCR using specific primers rPRP1-F (SEQ ID NO: 3): ATGCCTCGGTACTATTGCGA and rPRP1-R (SEQ ID NO: 4): TCAGTGGTTGCCCTCAGGAG.

[0040] Results: The 693 bp rPRP1 gene coding sequence (SEQ ID NO: 1) was obtained, encoding 230 amino acids (SEQ ID NO: 2). Sequence alignment showed that it encodes the small nucleoprotein U1-C, which is highly conserved in the Oryza genus.

[0041] Example 2 Prokaryotic expression of rPRP1 protein Construction of expression vector: The rPRP1 gene with BamHI and EcoRI restriction sites was ligated into the pColdI vector to construct the recombinant plasmid pCold1-rPRP1.

[0042] Induction of expression: The recombinant plasmid was transformed into E. coli BL21 and expression was induced at 16℃ with 1.0 mM IPTG. Transformation was performed by heat shock: 50 μL of competent E. coli cells were thawed on ice, and 5 μL to 10 μL of recombinant plasmid was added in a clean bench and gently mixed. After incubating on ice for 30 minutes, the cells were heat-shocked at 42℃ for 90 seconds, immediately followed by an ice bath for 2 minutes. 400 μL of antibiotic-free LB broth was added, and the cells were cultured at 37℃ with shaking for 1 hour. 100 μL of the bacterial culture was spread onto LB agar plates containing the corresponding antibiotics, dried in a clean bench, and then cultured at 37℃ for 12 to 16 hours.

[0043] The culture of single colonies after transformation was used as the seed culture. If the above culture was stored for too long, a portion of pCold I / BL21 and pCold I-rPRP1 / BL21 strains frozen at -80℃ were streaked on LB+Amp plates and incubated overnight at 37℃. Single colonies were then picked and placed in centrifuge tubes containing 5 mL of LB+Amp liquid medium and incubated overnight at 37℃ and 200 rpm. 300 µL of the seed culture was transferred to Erlenmeyer flasks containing 30 mL of LB liquid medium and cultured with shaking at 37℃ and 200 rpm until mid-log (OD600 value of 0.50). The culture was then cooled on ice, and a certain volume of 1 M IPTG was added in a clean bench to bring the final concentration to 1.0 mM. The culture was then incubated at 200 rpm at 16℃, 20℃, and 28℃ for 6 to 8 hours each.

[0044] The results are as follows Figure 1 As shown, SDS-PAGE analysis revealed a specific induced band at approximately 25 kDa, consistent with the predicted molecular weight of rPRP1, confirming successful expression.

[0045] Example 3: Effects of rPRP1 protein on oxidation and heat tolerance in *C. elegans* Nematode treatment: Rinse the plate surface with sterile M9 buffer. Transfer the rinse solution containing adult nematodes and eggs into a 1.5 mL EP tube. Centrifuge at 3000 rpm for 1 min at room temperature. Discard the supernatant and rinse once with sterile water. Add 1 mL of 2× nematode lysis buffer to the precipitate, gently invert to mix, and incubate at room temperature for 5-7 min. During incubation, shake intermittently to allow the adult nematode body walls to rupture and release the eggs. Add the same volume of M9 buffer to stop the lysis. The entire lysis process should not exceed 10 min. Centrifuge again at 3000 rpm for 1 min. Discard the supernatant and collect the egg precipitate. Rinse once with sterile water and repeat the washing process three times with M9 buffer to remove all lysis buffer residue. Finally, resuspend the cleaned eggs in M9 buffer and incubate in a shaker at 20°C in the dark. After the eggs hatch into L1 stage larvae, a synchronized nematode population with completely uniform developmental stages can be obtained. L1 stage nematodes were transferred to NGM medium containing OP50 and grew to L4 stage after about two days.

[0046] After IPTG induction, strain pCold1-rPRP1 / BL21 containing the target vector and OP50 cells were collected separately. The OD600 of the culture was adjusted to 0.5 with LB liquid medium. After mixing thoroughly, the cultures were centrifuged, and the supernatant was discarded, leaving 100 μL of concentrated bacterial solution. Control nematodes (Ck): pCold1 / BL21 strain and OP50 cells were collected separately after IPTG induction. The OD600 of the culture was adjusted to 0.5 with LB liquid medium. After mixing thoroughly, the cultures were centrifuged, and the supernatant was discarded, leaving 100 μL of concentrated bacterial solution. The concentrated bacterial solutions were spread onto plates containing 80 mM 5-FUDR medium and incubated overnight at 37°C. Then, synchronized nematodes that had reached the L4 stage (approximately 2 days) were inoculated.

[0047] The synchronized L4 stage Caenorhabditis elegans N2 was divided into two groups, with n=12 nematodes in each group: Experimental group (+rPRP1 group): BL21 / pCold1-rPRP1 induced cells with OD600=0.5 and OP50 cells (mixed in a 1:1 volume ratio) were fed to the whole group, with a total volume of 1 mL. Control group (Ck group): BL21 / pCold I empty vector induced bacterial cells with OD600=0.5 were fed with OP50 bacterial cells (mixed in a 1:1 volume ratio), with a total volume of 1 mL.

[0048] (1) Oxidative stress experiment: Nematodes were treated with 20 μL 8 mM H2O2 for 20 min, and the survival rate was counted at regular intervals.

[0049] Results: The survival rate of nematodes in the experimental group was significantly higher than that in the control group at all time points. P <0.05%. After 15 minutes of treatment, the survival rate in the experimental group was 63.9%, while that in the control group was 33.3%. Figure 2 ).

[0050] (2) Heat stress experiment: Nematodes were incubated at 37°C for 90 min, and the survival rate was counted at regular intervals.

[0051] Results: The experimental group of nematodes showed significantly enhanced heat tolerance. After 90 minutes of treatment, the survival rate of the experimental group reached 58.3%, while that of the control group was only 16.7%. Figure 3 ).

[0052] (3) Physiological index testing: L4 stage nematodes cultured for 8 days were rinsed with M9 buffer and transferred to 1.5 mL centrifuge tubes. Centrifugation was performed at 3000 rpm for 1 minute at room temperature. After centrifugation, the supernatant was discarded, and the nematodes were washed twice with M9 buffer to thoroughly remove any residual E. coli OP50 biofilm and metabolic products from their bodies. The nematode pellet was resuspended in an appropriate amount of M9 buffer. A small amount of the suspension was taken and added to a glass slide, glycerol was added, and a coverslip was placed on top to remove any air bubbles. Finally, the slide was placed under a fluorescence microscope, and blue fluorescence was selected for observation and imaging. Twenty nematodes were observed in each group, with three parallel experiments.

[0053] Lipofuscin detection: L4 nematodes cultured for 8 days were rinsed with M9 buffer and transferred to 1.5 mL centrifuge tubes. Centrifugation was performed at 3000 rpm for 1 minute at room temperature. After centrifugation, the supernatant was discarded, and the nematodes were washed twice with M9 buffer to thoroughly remove residual E. coli OP50 biofilm and nematode metabolic products. The nematode pellet was resuspended in an appropriate amount of M9 buffer. A small amount of liquid was taken from the nematode suspension and added to a glass slide. Glycerol was added, and a coverslip was placed on top to remove air bubbles. Finally, the pellet was placed under a fluorescence microscope, and blue fluorescence was selected for observation and imaging. Twenty nematodes were observed in each group, and three parallel experiments were set up. The results showed that the lipofuscin content in the experimental group nematodes was significantly lower than that in the control group. P <0.05)( Figure 4 ).

[0054] ROS level detection: Nematodes treated for three days were collected and rinsed twice with M9 solution. Then, 2',7'-dichlorofluorescein diacetate (2DCFDA) was added to a final concentration of 0.1 mM and incubated in a 37°C water bath for 30 min. The nematodes were then transferred to a glass slide, excess water was removed, a small amount of glycerol was added, and a coverslip was placed on top. Imaging was performed under a stereofluorescence microscope (objective ×1.6, zoom ×8, exposure time 1.5 s). Each group consisted of 20 nematodes, and the experiment was repeated three times. Images were processed using ImageJ software to analyze their relative fluorescence intensity.

[0055] Using the H2DCF-DA fluorescent probe, the results showed that the body length of the nematodes in the experimental group did not change significantly compared with that in the control group, but the relative ROS level of the nematodes in the experimental group was significantly lower than that in the control group. P <0.01), a decrease of approximately 50% ( Figure 5 ).

[0056] (4) Gene expression analysis: Antioxidant-related genes in nematodes were detected by qRT-PCR: MEV-1 , ISP-1 , GST-4 and GSC-1 According to TaKaRa SYBR® Premix Ex Taq TM (Tli RNaseH Plus), the reaction system was prepared according to the ROX plus kit instructions. CK group nematode cDNA was used as the control sample. CeAct As internal reference genes, their expression levels were detected using a StepOne Plus real-time PCR instrument. The reaction program was set as follows: 95℃, 2 min pre-denaturation; 95℃, 15 s denaturation; 58℃, 15 s annealing; 72℃, 30 s extension (signal collection); the melting program was set as follows: 95℃, 15 s; 60℃, 1 min; from 60℃ to 95℃: the temperature increment / step size was set to +0.5℃ (signal collection), 95℃, 15 s (signal collection), for 40 cycles. Each primer was repeated three times. The primers are shown in Table 1.

[0057] Table 1 Primers used for qRT-PCR The results showed that the nematodes in the experimental group MEV-1 and ISP-1 Gene expression levels were 2.4 times and 1.6 times higher than those of the control group, respectively. P <0.05), GST-4 and GSC-1 Genes were also significantly upregulated ( Figure 6 ).

[0058] It should be noted that, since the steps and methods used are the same as in the embodiments, preferred embodiments are described in this invention to avoid redundancy. Although preferred embodiments of the present invention have been described, those skilled in the art, once they understand the inventive concept of the present invention, can make other changes and modifications to these embodiments, and all such changes and modifications fall within the scope of the present invention.

[0059] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. If such modifications and variations fall within the scope of equivalents of this invention, then this invention also intends to include these modifications and variations.

Claims

1. Use of a biomaterial for the preparation of a product for enhancing the oxidative stress tolerance, heat tolerance and / or anti-aging of an animal, characterized in that, The biomaterial is selected from any one of the following: (1) Proteins with amino acids as shown in SEQ ID NO: 2; (2) The isolated nucleic acid molecules that encode the protein described in (1); (3) A recombinant vector comprising the nucleic acid molecules described in (2); (4) Host cell; which contains nucleic acid molecules as described in (2) and / or recombinant vectors as described in (3).

2. The use according to claim 1, characterized in that, The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO:

1.

3. The use according to claim 1 or 2, characterized in that, The products include feed and feed additives.

4. The use according to claim 3, characterized in that, The product is used to scavenge reactive oxygen species, reduce lipofuscin accumulation, and / or regulate the expression levels of antioxidant-related genes.

5. The use according to claim 4, characterized in that, The antioxidant-related genes include MEV-1 , ISP-1 , GST-4 , GSC-1 .

6. The use according to claim 5, characterized in that, The animal in question is a nematode.

7. A composition for stress resistance and / or anti-aging, characterized in that, It contains an effective amount of the protein as shown in SEQ ID NO: 2, and a physiologically acceptable carrier.

8. A method for enhancing oxidative stress tolerance and / or heat tolerance in animals for non-disease treatment, characterized in that, The method includes: The animal is given an effective amount of the amino acid protein as shown in SEQ ID NO: 2, or the composition of claim 7.

9. The method according to claim 8, characterized in that, The animal in question is a nematode.

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