Recombinant monascus purpureus immunomodulatory protein and preparation and application thereof

Abstract

The application provides a recombinant Monascus purpureus immunomodulatory protein and a preparation and application thereof, and belongs to the technical field of microbial genetic engineering, wherein the amino acid sequence of the recombinant Monascus purpureus immunomodulatory protein is shown as SEQ ID No. 2, and the preparation method comprises the following steps: constructing a prokaryotic expression vector for expressing the recombinant Monascus purpureus immunomodulatory protein, transforming the prokaryotic expression vector into an E. coli competent cell, obtaining a recombinant genetic engineering strain, inducing expression, and purifying to obtain the recombinant Monascus purpureus immunomodulatory protein. The application discovers a kind of fungal immunomodulatory protein from Monascus purpureus for the first time through bioinformatics analysis, the recombinant protein has significant anti-aging effect, and provides a reference for the efficacy research and medicinal development of the fungal immunomodulatory protein.

Description

Technical Field

[0001] This invention relates to the field of microbial genetic engineering technology, specifically to a recombinant purple Monascus immunomodulatory protein and its preparation and application. Background Technology

[0002] As the global population ages increasingly, the proportion of people aged 65 and above is gradually rising, and the number of patients with age-related diseases is also increasing. For example, the incidence of neurodegenerative diseases such as Alzheimer's and Parkinson's, as well as chronic diseases such as cancer, is significantly increasing among the elderly. This phenomenon has drawn significant attention to aging-related issues, and effectively delaying aging is considered an important strategy for addressing these problems.

[0003] Recent studies have revealed that fungal immunomodulatory proteins (FIPs) possess a variety of biological activities, including anti-aging, antioxidant, anti-allergic, anti-tumor, hemagglutinating activity, stimulation of human peripheral blood lymphocyte proliferation, regulation of cytokine secretion, and reduction of transplant rejection. These properties make fungal immunomodulatory proteins of significant value in the development and utilization of pharmaceutical proteins.

[0004] Monascus purpureus, a traditional food fermentation starter, has been used for thousands of years. It is not only widely used in the food industry, but also shows broad application prospects in cosmetics and medicine due to its various physiological activities, including anti-cancer, anti-mutagenic, anti-obesity, hypoglycemic, and hypolipidemic effects. However, our understanding of its bioactive proteins, especially immunomodulatory proteins, is still insufficient. Summary of the Invention

[0005] In view of the technical problems existing in the background art, the present invention, through bioinformatics analysis of Monascus purpureus, has discovered an immunomodulatory protein of Monascus purpureus var. purpleis (…). Monascus purpureus The immunomodulatory protein (MPIMP) was further modified and a recombinant Monascus purpureus immunomodulatory protein was prepared using the Escherichia coli heterologous expression method. Monascus purpureus This recombinant protein (rMPIMP) is highly water-soluble and has significant anti-aging effects.

[0006] The specific technical solution of the present invention is as follows: A method for preparing recombinant Monascus purpureus immunomodulatory protein includes the following steps: S1. Construct a prokaryotic expression vector for recombinant Monascus purpureus immunomodulatory protein, wherein the amino acid sequence of the recombinant Monascus purpureus immunomodulatory protein is shown in SEQ ID No. 2; S2. Transform the prokaryotic expression vector into competent Escherichia coli cells to obtain a recombinant genetically engineered strain; S3. The recombinant genetically engineered strain was induced to express the protein, and after purification, recombinant Monascus purpureus immunomodulatory protein was obtained.

[0007] Preferably, in the above preparation method, step S1 specifically involves: using purple Monascus cDNA as a template, obtaining the MPIMP gene target fragment by PCR amplification, and then constructing the prokaryotic recombinant expression vector pMAL-C5X-HIS-MPIMP using the vector pMAL-C5X-HIS, wherein the sequences of the primers used for PCR amplification are shown in SEQ ID No. 3-4.

[0008] Preferably, in the above preparation method, the competent cells are Escherichia coli Rosetta (DE3) competent cells.

[0009] Preferably, in the above preparation method, step S3 uses isopropyl-β-D-thiogalactopyranoside (IPTG) for induction; more preferably, the induction conditions are: cell density (OD) 600 When the bacterial cell density (OD) is 0.4-0.8, add 0.2-1.0 mM IPTG and induce at 20-33℃ for 6-12 h. As in some embodiments of the present invention, when the bacterial cell density (OD) is... 600 When the concentration of rMPIMP was 0.6, the expression level of rMPIMP protein reached its peak of 41.31 mg / L after adding 0.6 mM IPTG and inducing at 30℃ for 10 h.

[0010] Preferably, in the above preparation method, the purification method is as follows: after expression, collect the bacterial cells, add PMSF and break the bacterial cells, centrifuge to collect the supernatant, and elute on a nickel column with imidazole elution buffer with a concentration of 250 mM.

[0011] The recombinant Monascus purpureus immunomodulatory protein prepared according to the method of the present invention is also within the scope of protection of the present invention.

[0012] Furthermore, this invention investigated the anti-aging effects of recombinant Monascus purpureus immunomodulatory protein using *C. purpureus*. Experimental results showed that the recombinant Monascus purpureus immunomodulatory protein provided by this invention does not affect the reproductive capacity of *C. purpureus*, and has significant anti-aging effects on *C. purpureus*, at least in the following aspects: a) Extends the lifespan of *C. elegans*; b) Enhance the locomotion function of Caenorhabditis elegans; c) Alleviate ultraviolet, high temperature and / or oxidative stress; d) Reduce the accumulation of lipofuscin in Caenorhabditis elegans; e) Increase the body length of Caenorhabditis elegans.

[0013] It is evident that the recombinant Monascus purpureus immunomodulatory protein provided by this invention can be used to prepare anti-aging products, such as anti-aging drugs and anti-aging cosmetics.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention, through bioinformatics analysis, has for the first time discovered a purple Monascus immunomodulatory protein from Monascus purpureus. Furthermore, considering the poor water solubility of the purple Monascus immunomodulatory protein and the time-consuming and expensive nature of traditional extraction methods, this invention further constructs the prokaryotic recombinant expression vector pMAL-C5X-HIS-MPIMP to obtain a genetically stable recombinant Escherichia coli engineered strain. This prokaryotic expression system enables the rapid and large-scale preparation of the soluble target protein (i.e., the recombinant purple Monascus immunomodulatory protein). Compared with the alkali-soluble acid precipitation method for extracting immunomodulatory proteins from Monascus purpureus, this method offers advantages such as higher efficiency, lower cost, environmental friendliness, safety, and no pollution.

[0015] The recombinant protein provided by this invention does not affect the reproductive capacity of *C. elegans*, and has a significant anti-aging effect on *C. elegans*, providing a reference for the efficacy research and pharmaceutical development of fungal immunomodulatory proteins. Attached Figure Description

[0016] Figure 1 This is an amino acid sequence comparison diagram of the Antrodia camphorata immunomodulatory protein and Monascus purpureus protein in Example 1 of the present invention; Figure 2 This is a sequence comparison diagram of the immunomodulatory protein of Monascus purpureus in Example 1 of the present invention and the amino acid sequence of the FIP containing the cerato-platanin domain; Figure 3 This is a phylogenetic tree analysis result of the immunomodulatory proteins of Monascus purpureus in Example 1 of the present invention; Figure 4 This is a comparison diagram of IPTG expression before and after induction in Example 2 of the present invention; Figure 5This is a comparison of the expression of the target protein rMPIMP under different conditions in Example 3 of the present invention. In the figure, M is the marker. Figure a shows the bacterial density screening, with the densities of lanes 1-5 being 0.2, 0.4, 0.6, 0.8, and 1.0, respectively. Figure b shows the IPTG concentration screening, with the IPTG concentrations of lanes 1-6 being 0, 0.2, 0.4, 0.6, 0.8, and 1.0 mmol / L, respectively. Figure c shows the temperature screening, with lanes 1-6 being 20, 23, 26, 30, 33, and 37 ℃, respectively. Figure d shows the time screening, with lanes 1-7 being 2, 4, 6, 8, 10, 12, and 24 h, respectively. Figure 6 This is a graph showing the effect of rMPIMP on the lifespan of nematodes in Example 4 of the present invention; Figure 7 This is a graph showing the effect of rMPIMP on the reproductive capacity of nematodes in Example 4 of the present invention; Figure 8 This is a graph showing the effect of rMPIMP on the motility of nematodes in Example 4 of the present invention. Figure 9 This is a graph showing the effect of rMPIMP on the body length and lipofuscin accumulation of nematodes in Example 4 of the present invention. Figure 10 The figure shows the effect of rMPIMP on the stress response of nematodes in Example 4 of the present invention. Detailed Implementation

[0017] The embodiments of the technical solution of the present invention will now be described in detail with reference to the accompanying drawings. These embodiments are merely illustrative of the technical solution of the present invention and are therefore intended to limit the scope of protection of the present invention.

[0018] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs; the terminology used herein is for the purpose of describing particular implementations only and is not intended to limit the invention; the terms “comprising” and “having” and any variations thereof are intended to cover non-exclusive inclusion.

[0019] Although Monascus purpureus has a long history of use and has been shown to possess various physiological activities, current research has very little understanding of its bioactive proteins, especially immunomodulatory proteins, which limits its application in this field. This invention utilizes Monascus purpureus (…). M. purpureus An immunomodulatory protein was successfully discovered in the study, and its in vitro recombinant preparation was further achieved. Furthermore, the anti-aging activity of this immunomodulatory protein was demonstrated using Caenorhabditis elegans as a model organism.

[0020] In a first aspect, embodiments of the present invention provide a purple Monascus immunomodulatory protein (MPIMP), which is composed of 147 amino acids, the amino acid sequence of which is shown in SEQ ID No. 1, the molecular weight is 15.16 KD (theoretical value 15161.84 Da), and the isoelectric point (PI) is 4.57; the protein molecular formula is C 658 H 1035 N 173 O 225 S6 has a total of 2097 atoms and a theoretical half-life of 30 h; its instability index is 22.27 (<40); its aliphatic coefficient is 76.39; it contains 10 negatively charged residues (Asp+Glu) and 7 positively charged residues (Arg+Lys), with an average hydrophobicity (GRAVY) of 0.031. MPIMP is rich in serine (Ser), glycine (Gly), alanine (Ala), leucine (Leu), cysteine ​​(Cys), and threonine (Thr), while its histidine (His), methionine (Met), and tryptophan (Trp) content is extremely low (<1.5%).

[0021] Considering that traditional extraction methods for proteins are time-consuming and expensive, and that MPIMP itself is poorly soluble in water, severely limiting its application, this invention modifies MPIMP to provide a recombinant Monascus purpureus immunomodulatory protein (rMPIMP) that is easily soluble in water while retaining its biological activity. It consists of 560 amino acids, with the amino acid sequence shown in SEQ ID No. 2, a molecular weight of 60.62 KD (theoretical value 60614.02 Da), and an isoelectric point (PI) of 5.24. The protein's molecular formula is C0. 2707 H 4174 N 712 O 842 S 14 It contains 8449 atoms and has a predicted in vitro half-life of approximately 30 hours; its instability index is 21.50, classifying it as a stable protein; its lipid coefficient is 77.79; rMPIMP contains 64 acidic amino acid residues (Asp+Glu) and 49 basic amino acid residues (Arg+Lys), with an average hydrophobicity (GRAVY) of -0.325. rMPIMP is rich in alanine (Ala), glycine (Gly), leucine (Leu), and aspartic acid (Asn), while its cysteine ​​(Cys) content is extremely low (<1.5%).

[0022] Secondly, embodiments of the present invention provide a method for preparing recombinant Monascus purpureus immunomodulatory protein, specifically including the following steps: (1) Using Monascus purpureus cDNA as a template, the target fragment of the MPIMP gene was obtained by PCR amplification, and then the prokaryotic recombinant expression vector pMAL-C5X-HIS-MPIMP was constructed using the vector pMAL-C5X-HIS. The sequences of the primers used for PCR amplification are shown in SEQ ID No. 3-4, and the amino acid sequence of the recombinant Monascus purpureus immunomodulatory protein is shown in SEQ ID No. 2. (2) The prokaryotic recombinant expression vector pMAL-C5X-HIS-MPIMP was transformed into Escherichia coli Rosetta (DE3) competent cells. Genetically stable recombinant genetically engineered strains were obtained by screening for resistance to ampicillin and chloramphenicol and by colony PCR verification. (3) Recombinant genetically engineered strains were induced to express by IPTG and purified to obtain recombinant purple Monascus immunomodulatory protein.

[0023] Furthermore, in some embodiments, the conversion is performed via thermal shock conversion.

[0024] Thirdly, the embodiments of the present invention, using *C. elegans* as an example, have demonstrated the anti-aging effects of MPIMP and rMPIMP, at least in the following aspects: a) It can prolong the lifespan of Caenorhabditis elegans; b) It can enhance the locomotion function of Caenorhabditis elegans; c) It can alleviate ultraviolet, high temperature and / or oxidative stress; d) It can reduce the accumulation of lipofuscin in Caenorhabditis elegans; e) It can increase the body length of Caenorhabditis elegans.

[0025] The following are some specific embodiments. It should be noted that the embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, they shall be performed in accordance with the techniques or conditions described in the literature in this field or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all conventional products that can be obtained commercially.

[0026] The specific composition of the culture medium used in the following examples is as follows: LB liquid medium: Weigh 5 g tryptone, 5 g sodium chloride and 2.5 g yeast extract powder, add deionized water to 0.5 L, dispense, stopper and wrap, and autoclave at 121℃ for 20 min; LB solid medium: Weigh 5 g tryptone, 5 g sodium chloride and 2.5 g yeast extract powder, add deionized water to 0.5 L, dispense, add 1.5%~2% agar powder, and autoclave at 121℃ for 20 min; Potato glucose broth (PDB): Wash and peel potatoes, weigh 100 g, cut into small pieces, add distilled water and boil for about 30 min, then filter with gauze, weigh 10 g of glucose and add, stir well, cool and then add distilled water to 0.5 L, dispense into test tubes or Erlenmeyer flasks, stopper and wrap, and autoclave at 121℃ for 20 min. Potato glucose agar (PDA): Wash and peel potatoes, weigh 100 g, cut into small pieces, add distilled water and boil for about 30 minutes, then filter with gauze, weigh 10 g of glucose and add, stir well, cool and add distilled water to 0.5 L, dispense, add 1.5%~2% agar powder, plug and wrap, and autoclave at 121℃ for 20 minutes.

[0027] In the following examples, the analytical method for SDS-PAGE gel electrophoresis is as follows: The test solution is mixed with 5×Loading Buffer at a volume ratio of 4:1, boiled in boiling water for 10 min, cooled, and centrifuged at 10000 r / min for 10 min. The supernatant is used for subsequent electrophoresis analysis. Electrophoresis is first performed at a constant voltage of 80 V for about 30 min, then the voltage is adjusted to 120 V and electrophoresis is continued for 90 min. After electrophoresis, the glass plate is removed for the next step of staining and destaining. Sufficient staining solution is added to cover the gel, and the gel is stained by shaking on a shaker for 2 h. After staining, the staining solution is discarded, and sufficient destaining solution is added to cover the gel. The gel is destained by shaking for 1 h, and then the destaining solution is replaced. The destaining process can be repeated multiple times until the gel background is clear.

[0028] In the following examples, the culture method of uracil-deficient Escherichia coli (E. coli OP50) was as follows: A sterile inoculating loop was used to pick up the bacterial culture and streak it onto the surface of LB solid medium, then inverted and cultured at 37°C for 12-16 hours. A single colony was picked and inoculated into 5 mL of LB liquid medium, and cultured at 37°C with shaking at 200 rpm until the logarithmic growth phase. An appropriate amount of the logarithmic growth phase bacterial culture was evenly spread onto the surface of NGM medium plates, inverted and cultured at 37°C for 12 hours, and then transferred to 4°C for short-term storage (1-2 months). The bacterial culture was mixed with an equal volume of 50% (v / v) glycerol, dispensed into cryovials, and stored at -80°C.

[0029] In the following examples, wild-type Caenorhabditis elegans (N2) was used. The nematode synchronization method was as follows: Nematode bodies containing a large number of pregnant adults were collected from the surface of NGM plates, gently rinsed with sterile water into 1.5 mL centrifuge tubes, centrifuged at 1000 rpm for 1 min, and the supernatant was discarded; approximately 700 μL of precipitate was retained, and 200 μL of 5 w / v% sodium hypochlorite and 100 μL of 5 M NaOH were added, vortexed until the nematode bodies were completely lysed, centrifuged at 2000 rpm for 1 min, and the supernatant was discarded; 1 mL of sterile water was added to the precipitate to resuspend the eggs, centrifuged at 2000 rpm for 1 min, and the supernatant was discarded, repeating the rinsing process twice; finally, 100 μL of the egg suspension was retained, evenly spread onto fresh NGM plates, and incubated in a 20°C incubator. The preservation method for nematodes is as follows: Wild-type N2 nematodes are starved to the Dauer stage. During synchronization, it is essential to ensure that the NGM medium is free from microbial contamination and residual E. coli. An appropriate amount of pre-chilled M9 buffer is added to NGM plates containing Dauer-stage nematodes. The nematode suspension is scraped and transferred to 1.5 mL centrifuge tubes, mixed with cryopreservation solution at a 1:1 (v / v) ratio, and aliquoted into pre-chilled sterile cryovials. The cryovials are placed in a cryopreservation foam box and kept at 4°C for 20 min, then transferred to -20°C for 20 min, and finally to -80°C for storage for several months. For long-term storage, transfer to liquid nitrogen. Simultaneously, 1-2 samples from the same batch should be taken out every month to test the recovery rate. When resuscitating nematodes, the cryovials are rapidly thawed in a 37°C water bath (<1 min). The suspension is evenly spread onto NGM plates covering OP50, incubated at 20°C for 24 h, and the larval hatching is observed. The nematode resuscitation method is as follows: Thaw cryovials rapidly in a 37°C water bath (<1 min), spread the suspension evenly onto an NGM plate covered with OP50, and incubate at 20°C for 24 h. Observe the larval hatching status afterward. Removal of contaminating microorganisms from nematodes: During nematode culture, due to limitations in the operating environment or experimental conditions, NGM medium may be contaminated by exogenous microorganisms (bacteria / fungi). To eliminate contamination interference, use a platinum inoculation needle to pick 3-5 healthy L4-stage adults from an uncontaminated area, wash three times with M9 buffer, and then transfer to an NGM plate containing fresh OP50 bacterial culture. Incubate at 20°C. If contamination persists, perform nematode synchronization treatment on the contaminated plates.

[0030] Example 1 This example, based on previously reported fungal immunomodulatory proteins, identified a purple Monascus immunomodulatory protein through bioinformatics analysis of Monascus purpureus. The specific procedures included: The amino acid sequences of typical members of reported fungal immunomodulatory proteins (such as Ganoderma lucidum and Antrodia camphorata) were collected, and multiple sequence alignment templates were constructed. Then, the homology search of Monascus purpureus proteome was performed using the BLASTP algorithm. The functional domains of candidate proteins were annotated using databases such as InterPro and Pfam. The primary and secondary structures of FIP feature structures were identified. A phylogenetic tree was constructed based on the Neighbor-Joining method to analyze the phylogenetic relationship between Monascus purpureus FIP homologous proteins and known families.

[0031] Sequence alignment analysis revealed that the hypothetical protein MPDQ_002695 from Monascus purpureus (GenBank TQB68824) is similar to the immunomodulatory protein ACA from Antrodia camphorata (… Antrodia camphorata The agent has a 59% sequence similarity. Figure 1 This suggests that it is a candidate for MPIMP. Amino acid composition analysis shows that MPIMP is rich in serine (Ser), glycine (Gly), alanine (Ala), leucine (Leu), and threonine (Thr), while its histidine (His), methionine (Met), and tryptophan (Trp) content is extremely low (<1.5%). This characteristic is highly similar to that of FIP family members such as Antrodia camphorata ACA and Trametes versicolor CMIMP. Although MPIMP has high sequence similarity to immunomodulatory proteins from other fungi, in terms of genetic evolution, it belongs to a different evolutionary branch than other immunomodulatory proteins from edible and medicinal fungi (such as Ganoderma lucidum LZ-8 and FIP-fve), and is genetically distant, but belongs to the same branch as ACA and CMIMP. Figure 2 and Figure 3 ).

[0032] Example 2 Based on the Monascus purpureus immunomodulatory protein in Example 1, this example provides a method for expressing a recombinant Monascus purpureus immunomodulatory protein, the preparation method of which includes the following steps: (1) Extraction of total RNA from Monascus purpureus.

[0033] Purple Monascus strain was aseptically inoculated into PDA under a clean bench and activated by static incubation at 30℃ for 5-7 days. The activated strain was then preserved as a secondary culture, and single colonies were picked and inoculated into PDA, then cultured in a shaker (30℃, 200 r / min) for 5-7 days. After fermentation, the fermentation mixture was removed and filtered through filter paper. The filter paper was pressed dry and placed in a 1.5 mL centrifuge tube (containing three 3 mm and 30 1 mm steel beads), and immediately placed in liquid nitrogen for freezing. Under liquid nitrogen conditions, the tissue was homogenized at 60 Hz for 90 s, repeated 5 times. Then, 1 mL of Trizol reagent was added to the clean bench. The mixture was rapidly and thoroughly shaken for 30 s and incubated at room temperature for 10-15 min. 200 μL of chloroform (CHCl3) was added, and the mixture was vigorously mixed for 30 s and incubated for 5 min. Centrifuge at 10,000 r / min for 10–15 min at 4 °C. Transfer the supernatant to a new 1.5 mL enzyme-free centrifuge tube, add an equal volume of pre-chilled isopropanol, gently invert to mix, and incubate on ice for 15 min. Centrifuge at 10,000 r / min for 10–15 min at 4 °C. Discard the supernatant, add 1 mL of pre-chilled 75% ethanol prepared with DEPC water, and mix by pipetting to wash the precipitate. Centrifuge at 8,000 r / min for 5 min at 4 °C. Discard the supernatant and allow the precipitate to air dry. Dissolve the RNA in an appropriate amount of DEPC water, determine the concentration, and store at -80 °C.

[0034] (2) Synthesis of Monascus cDNA.

[0035] cDNA was synthesized using the HiScript® II Q RT SuperMix for qPCR (+gDNA wiper) kit. The specific steps were as follows: 2 μg RNA diluent was added to a 1.5 mL enzyme-free PCR tube, followed by 4 μL of 4×gDNAwiper Mix. DEPC water was added to bring the volume to 16 μL, and the mixture was thoroughly mixed using a pipette. The reaction mixture was then placed in a PCR instrument and reacted at 42℃ for 2 min. 4 μL of 5×HiScript II Select qRTSuperMix II was added to the PCR tube, and the mixture was thoroughly mixed again. The reaction mixture was then placed in a PCR instrument and reacted at 50℃ for 15 min, followed by 85℃ for 5 s to complete cDNA synthesis.

[0036] (3) PCR amplification and purification of the target fragment.

[0037] Using the amplification primers MPIMP-F / R (sequence shown in SEQ ID No. 3-4) and the cDNA prepared in step (2) as materials, the PCR reaction was carried out according to the following system: 33 μL deionized water, 8 μL 5 × EVO Buffer, 3 μL template DNA, 2 μL MPIMP-F, 2 μL MPIMP-R, 1 μL dNTP Mix (10 mM each), and 1 μL DNA Polymerase (1 U / μL). All operations were performed on ice. After thoroughly mixing all components of the PCR system in the PCR tube, the PCR amplification program was set (pre-denaturation, 95℃, 3 min, 1 cycle; denaturation, 95℃, 15 s; annealing, 60℃, 15 s, 30 cycles; extension, 72℃, 30 s; extension, 72℃, 30 s, 1 cycle). After cooling to 4℃ for 1 min, the sample was collected for subsequent reactions or stored at -20℃.

[0038] The target DNA fragment was purified and recovered using the FastPure® Gel DNA Extraction Mini Kit. Specific operating procedures were performed according to the kit's instruction manual.

[0039] (4) Construction of expression carrier.

[0040] The target fragment and expression vector were double-digested with BamHI and EcoRI to give the target fragment the same sticky ends as the expression vector, linearizing the expression vector and providing an insertion site for the target fragment. The double digestion system was 50 μL (total volume, 2×Tango buffer, BamHI used was twice the amount of EcoRI). The operation was performed on ice at 37°C for 12 h. The digestion products were recovered using the FastPure® Gel DNA Extraction Mini Kit for subsequent experiments or storage at -20°C.

[0041] The double-digested target fragment and the linear expression vector were ligated using ligase. The ligation system was prepared according to the kit instructions (vector, volume approximately 50-100 ng; insert fragment volume approximately 3 times the molar volume of the vector; 1 μL 10× buffer; 0.5 μL T4 DNA ligase; add deionized water to a final volume of 10 μL). The ligation system was incubated at 16°C for 12 h.

[0042] (5) The ligation product was transformed into competent Escherichia coli.

[0043] Remove frozen E. coli competent cells from a -80°C freezer and allow them to thaw naturally on ice. Add an appropriate amount of the target plasmid. Gently tap the bottom of the centrifuge tube to mix, then incubate on ice for 30 min. Heat shock the centrifuge tube in a 42°C water bath for 90 s, then quickly immerse it in ice and incubate for 2 min. Add 500 μL of liquid LB medium, mix by pipetting, and incubate at 37°C for 1 h at 200 rpm on a shaker. Centrifuge at 6000 rpm for 1 min at room temperature to collect the cells, retaining approximately 100 μL of supernatant. Resuspend the cells by pipetting and spread evenly onto LB agar plates containing the appropriate antibiotic. Incubate the plates upside down in a 37°C incubator for 12–16 h. Identify positive transformants, and preserve the correctly sequenced strains in 50% glycerol (-20°C).

[0044] (6) Induced expression of recombinant proteins.

[0045] The successfully constructed recombinant expression strain Rosetta (DE3) was streaked onto LB agar plates containing Amp (100 μg / mL) and Chl (50 μg / mL) and cultured overnight at 37°C with the plates inverted for 12–16 h. Single colonies were picked and inoculated into 5 mL of LB liquid medium containing Amp (100 μg / mL) and Chl (50 μg / mL) and cultured with shaking at 200 r / min at 37°C for 12–16 h. 0.5 mL of the seed culture was inoculated into 50 mL of LB liquid medium containing Amp (100 μg / mL) and Chl (50 μg / mL) and cultured with shaking at 200 r / min at 37°C until the OD600 reached 0.6. IPTG was added to a final concentration of 0.6 mM and cultured with shaking at 200 r / min at 30°C for 10 h to induce protein expression.

[0046] After culture, bacterial cells were collected by centrifugation at 10000 r / min for 5 min (4℃), washed twice with pre-cooled PBS, and resuspended in 10 mL PBS. The cells were then disrupted using an ultrasonic cell disruptor. Before disruption, 100 μL of PMSF could be added to inhibit thiol proteases and serine proteases. Sonication was performed for 10 min at 50% power, with a 5 s cycle and a 5 s pause, maintaining the cell in an ice bath throughout the process. After centrifugation at 10000 r / min for 10 min (4℃), the supernatant and precipitate were collected, and SDS-PAGE gel electrophoresis was used to detect the expression of the target protein. The cells were eluted from the nickel column with 250 mM imidazole elution buffer, which removed most of the contaminating proteins, successfully separating rMPIMP protein.

[0047] Figure 4The expression of IPTG before (lane 1) and after (lane 1) induction was compared.

[0048] Example 3 Unlike Example 2, this example changes the conditions for recombinant protein expression in step (6), including bacterial density, IPTG induction concentration, induction temperature, and induction time.

[0049] The results are as follows Figure 5 As shown: with increasing cell density (OD) 600 With the increase of OD, the expression level of the fusion protein showed a trend of first increasing and then decreasing. 600 The peak value is reached at 0.6. Figure 5 a); As the concentration of IPTG inducer increases, the band of the target protein first increases and then decreases, reaching its maximum at 0.6 mM ( Figure 5 b); As the induction temperature increases, the target protein band reaches its maximum at 30℃ ( Figure 5 c); As the induction time increased, the target protein band first increased and then plateaued, reaching its maximum at 10 h ( Figure 5 d).

[0050] Example 4 Using *C. elegans*, this example tested the anti-aging activity of the recombinant *Monascus purpureus* immunomodulatory protein obtained in Example 2, including the following: (1) Nematode lifespan test.

[0051] Experimental grouping and administration: Wild-type N2 nematodes synchronized to the L4 stage were randomly divided into an administration group and a control group. The administration group received OP50 bacterial suspension supplemented with 1 mg / mL protein (rMPIMP or MBP) and NGM medium containing a final concentration of 50 μM FUDR to inhibit embryonic development. The control group received only NGM medium containing an equal volume of OP50 bacterial suspension and FUDR. Each group had three biological replicates, with 35 ± 5 nematodes (n ≥ 100) inoculated per plate.

[0052] Culture and monitoring: The plates were placed in a 20°C incubator and the culture medium was changed daily with fresh drug / control medium. Dead individuals (unresponsive to touch) were removed using a platinum needle, and the time of death and missing individuals (who died due to migration to the plate wall or mechanical damage) were recorded.

[0053] Survival curves were plotted using GraphPad Prism 9.0 software, and the results are as follows: Figure 6As shown, compared with the blank control and protein tag (MBP) groups, rMPIMP significantly prolonged the lifespan of *C. elegans* (p<0.05), and its lifespan curve shifted significantly to the right. The average lifespan of nematodes fed with rMPIMP was approximately 21.35 days, which was 19.47% longer than that of the blank control group; the median lifespan of nematodes increased significantly from 18.00 days to 22.33 days; the longest lifespan increased to 25 days, which was 17.21% longer than that of the blank control group; compared with the MBP group, the average lifespan and median lifespan of nematodes were increased by 17.89% and 19.6%, respectively, and the longest lifespan was increased by 15.36% compared with the MBP group (Table 1).

[0054] Table 1. Effects of rMPIMP on mean lifespan, median lifespan, and longest lifespan of nematodes.

[0055] (2) Test on the number of eggs laid by nematodes.

[0056] After the synchronized nematodes were cultured to the L4 stage, they were transferred to NGM medium in the drug treatment group and the blank control group respectively (the grouping method is the same as step (1)), with 1 nematode per plate; in order to facilitate the counting of the number of eggs laid, a 35 mm diameter culture dish was used, and after culturing at 20℃ for 3 days, the number of eggs laid was calculated.

[0057] The results are as follows Figure 7 As shown, compared with the blank control group and the MBP group, rMPIMP did not affect the oviposition rate of nematodes. Therefore, rMPIMP does not impair the reproductive capacity of nematodes.

[0058] (3) Nematode locomotion test.

[0059] After synchronization, the nematodes were transferred to the drug treatment group and the blank control group respectively (the grouping method is the same as in step (1)) and placed in liquid culture medium with FUDR. They were then placed in a constant temperature incubator at 20℃. The nematode movement of each group was observed and recorded on the 5th, 7th and 9th days after drug treatment. 100 μL of M9 solution was added to a glass slide. One nematode was picked up and placed in the M9 solution each time. The nematode was allowed to adapt to the M9 solution for 20 s. The nematode movement image was recorded under a microscope for 1 min. Within this 1 min, 20 s were randomly selected to count the number of nematode movements.

[0060] The results are as follows Figure 8As shown, compared with the blank control group, the frequency of body bending of nematodes increased from 29.17 times / 20s on day 5 to 34.8 times / 20s, from 16.89 times / 20s on day 7 to 33.2 times / 20s, and from 13.81 times / 20s on day 9 to 29.8 times / 20s, all of which significantly improved motility. Compared with the MBP group, the motility of nematodes in the rMPIMP intervention group was also significantly improved at all time points, indicating that rMPIMP activity originates from the MPIMP domain rather than the MBP-tagged protein.

[0061] (4) Determination of nematode body length and lipofuscin accumulation.

[0062] After synchronization, the nematodes were transferred to the FUDR-containing drug group and the blank control group (grouping method is the same as step (1)) liquid culture medium and cultured at a constant temperature of 20℃ for 7 days. After anesthetizing with NaN3, the nematodes were observed and images were collected using a fluorescence microscope. The body length and relative content of lipofuscin in the nematodes were analyzed using ImageJ software.

[0063] The results are as follows Figure 9 As shown: Compared with the blank control group, the body length of nematodes in the rMPIMP group increased by 15.67%; compared with the MBP group, the body length increased by 16.22%; compared with the blank control group, the lipofuscin level of nematodes in the rMPIMP treatment group was significantly reduced by 16.94%; compared with the MBP group, there was no significant difference in lipofuscin level in the rMPIMP treatment group.

[0064] (5) Nematode stress test.

[0065] UV stress test: After the synchronized nematodes were cultured to the L4 stage, they were transferred to NGM medium containing FUDR and blank control group (grouping method is the same as step (1)). They were cultured at 20℃ for 3 days. The two groups of nematodes were transferred to NGM medium without Escherichia coli OP50 and placed in a clean bench with the lid off for 1 h of UV light irradiation. Then the nematodes were transferred to the culture medium containing Escherichia coli OP50 and blank control group and placed in a 20℃ incubator for continued culture. All dead nematodes were removed every day to count the number of dead nematodes and the number of lost nematodes until all nematodes died.

[0066] High temperature stress test: After the synchronized nematodes were cultured to the L4 stage, they were transferred to NGM medium containing FUDR and blank control group (grouping method is the same as step (1)). After being cultured at a constant temperature of 20℃ for 3 days, they were placed in an incubator at 37℃ for high temperature treatment. Every hour, the nematode's head was touched with a platinum wire to check the nematode's survival ability and the number of surviving nematodes was recorded until all nematodes died.

[0067] Oxidative stress test: After the synchronized nematodes were cultured to the L4 stage, they were transferred to NGM medium containing FUDR and blank control group (grouping method is the same as step (1)). They were cultured at a constant temperature of 20℃ for 3 days. The nematodes in each group were then transferred to NGM containing 0.1% H2O2. The survival status of the nematodes was counted every 10 min until all the nematodes died.

[0068] The results are as follows Figure 10 As shown: the survival curve of nematodes in the rMPIMP group shifted significantly to the right, and their average lifespan under UV stress was extended by 40.98% compared with the blank control group. Figure 10 a) The average lifespan of those resistant to high-temperature stress was 75.52% longer than that of the control group. Figure 10 (b) The average lifespan resisting oxidative stress was 17.02% longer than that of the blank control group. Figure 10 c) The average lifespan resisting ultraviolet stress was 39.41% longer than that of the MBP group, the average lifespan resisting high temperature stress was 61.87% longer than that of the MBP group, and the average lifespan resisting oxidative stress was 10.03% longer than that of the MBP group.

[0069] In summary, this invention constructs the prokaryotic recombinant expression vector pMAL-C5X-HIS-MPIMP to obtain a genetically stable recombinant *E. coli* engineered strain. After IPTG induction and SDS-PAGE identification, rMPIMP was solublely expressed, and the rMPIMP protein was obtained through nickel column purification. The preparation method provided by this invention has the advantages of high efficiency, short processing time, simple operation, and low cost. The rMPIMP protein provided by this invention does not affect the reproductive capacity of *C. elegans* and has significant anti-aging effects, possessing extremely high value for promotion and application.

[0070] It should be noted that the present invention is not limited to the above-described embodiments. The above embodiments are merely examples, and any embodiments that have the same structure and perform the same effects as the technical concept within the scope of the present invention are included within the scope of the present invention. Furthermore, various modifications that can be conceived by those skilled in the art to the embodiments, and other ways of constructing by combining some of the constituent elements of the embodiments, without departing from the spirit of the present invention, are also included within the scope of the present invention.

Claims

1. A method for preparing recombinant Monascus purpureus immunomodulatory protein, characterized in that, Includes the following steps: S1. Construct a prokaryotic expression vector expressing recombinant Monascus purpureus immunomodulatory protein, wherein the amino acid sequence of the recombinant Monascus purpureus immunomodulatory protein is shown in SEQ ID No. 2; S2. Transform the prokaryotic expression vector into competent Escherichia coli cells to obtain a recombinant genetically engineered strain; S3. The recombinant genetically engineered strain was induced to express the protein, and after purification, recombinant Monascus purpureus immunomodulatory protein was obtained.

2. The preparation method according to claim 1, characterized in that, Step S1 is as follows: using purple Monascus cDNA as a template, the target fragment of the MPIMP gene is obtained by PCR amplification, and then the prokaryotic recombinant expression vector pMAL-C5X-HIS-MPIMP is constructed using the vector pMAL-C5X-HIS. The sequences of the primers used for PCR amplification are shown in SEQ ID No. 3-4.

3. The preparation method according to claim 1, characterized in that, The competent cells were Escherichia coli Rosetta (DE3) competent cells.

4. The preparation method according to claim 1, characterized in that, Step S3 uses IPTG for induction.

5. The preparation method according to claim 4, characterized in that, The induction conditions described in step S3 are as follows: when the cell density is 0.4-0.8, add 0.2-1.0 mM IPTG and induce at 20-33℃ for 6-12 h.

6. The preparation method according to claim 1, characterized in that, The purification method is as follows: after expression, the bacterial cells are collected, PMSF is added and the bacterial cells are broken, the supernatant is collected by centrifugation, and the supernatant is eluted on a nickel column with 250 mM imidazole elution buffer.

7. The recombinant Monascus purpureus immunomodulatory protein obtained by the preparation method according to any one of claims 1-6.

8. The application of the recombinant Monascus purpureus immunomodulatory protein as described in claim 7 in regulating the growth of Caenorhabditis elegans, characterized in that, At least one of the following: a) Extends the lifespan of *C. elegans*; b) Enhance the locomotion function of Caenorhabditis elegans; c) Alleviate ultraviolet, high temperature and / or oxidative stress; d) Reduce the accumulation of lipofuscin in Caenorhabditis elegans; e) Increase the body length of Caenorhabditis elegans.

9. The application of the recombinant Monascus purpleis immunomodulatory protein as described in claim 7 in the preparation of anti-aging products.

10. The application according to claim 9, characterized in that, The product is a medicine.

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