A UP5 of mariner channel protein of yellow dragon moth and application of the UP5

Abstract

The application discloses a protein UP5 of the Malpighian tubule of Euproctis pseudoconspersa and application thereof, the protein can be combined with calcium ions, and has a significant ability of inhibiting formation of calcium oxalate crystals, and can be used for preventing and treating calcium oxalate stones which are the most common kidney stones, so that a new strategy for treating kidney stones is expected to be provided, and the protein UP5 of the Malpighian tubule of Euproctis pseudoconspersa provides a new drug use.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, in particular to a protein UP5 in the Malpighian tubule of Leucoma salicis, and the application of the protein UP5 in the Malpighian tubule of Leucoma salicis. BACKGROUND

[0002] Biomineralization is a ubiquitous phenomenon in nature. Through biomineralization, animals, plants and microorganisms can precisely control the formation of inorganic minerals. Most of the biomineralization is beneficial to the organism, such as the skeleton of vertebrates is composed of collagen, non-collagen proteins (glycoproteins, proteoglycans and Gla proteins) and hydroxyapatite crystals, the shell of mollusks is composed of about 95% calcium carbonate crystals and <5% organic matrix, mainly including acidic proteins, chitin and silk-like proteins. Biominerals have multiple functions such as protection, skeletal support and sensory detection.

[0003] When the internal chemical environment of the human body fluctuates abnormally, pathologic mineralization occurs, harmful inorganic or organic salt crystals are precipitated, which are called pathological (or pathologic) crystals, and then induce diseases such as kidney stones, gallstones, malaria, gout, etc. In humans, calcium oxalate is the main component of urinary tract stones, and calcium oxalate stones account for about 80% of all stones. Therefore, most of the research on urolithiasis has focused on calcium oxalate stones. The formation of kidney stones involves a series of key events, including the supersaturation of urine with insoluble salts, crystal nucleation, crystal growth and aggregation. Crystal nucleation is considered to be the first step in the formation of kidney stones, and macromolecules in urine can affect this process in various ways.

[0004] The Malpighian tubule is the excretory organ of insects, which is similar to the kidney of mammals, and its function is to maintain the osmotic regulation, electrolyte balance and waste elimination of the body. More and more evidence shows that the kidney stones produced by insects have significant similarities with the kidney stones of mammals. The oxalate in the animal body mainly comes from the plant diet, and the oxalate salt absorbed from the diet is absorbed by the intestinal tract and produced by metabolism. In order to prevent the formation of calcium oxalate stones, there must be proteins in the Malpighian tubule of insects that can inhibit the formation of calcium oxalate crystals.

[0005] The Malpighian tubule of Leucoma salicis has a very high concentration of calcium oxalate, but does not form stones, indicating that there is an excellent calcium oxalate crystal inhibiting protein in the Malpighian tubule of Leucoma salicis. A protein UP5 rich in acidic amino acids was found in the Malpighian tubule of Leucoma salicis, which is specifically expressed in the Malpighian tubule and has a significant ability to inhibit the formation of calcium oxalate crystals. The specific details are as follows: UP5 is specifically expressed in the Malpighian tubule by quantitative PCR, and UP5 protein is obtained by prokaryotic expression. It is found by micro-thermal mobility and circular dichroism that UP5 has a strong interaction with calcium; it is found by in vitro simulation mineralization experiment that UP5 can inhibit the growth of calcium oxalate crystals. SUMMARY

[0006] Therefore, one of the purposes of the present application is to provide a yellow stick moth Malpighian tubule protein UP5; the second purpose of the present application is to provide a yellow stick moth Malpighian tubule protein UP5 gene, the third purpose of the present application is to provide a recombinant vector containing the yellow stick moth Malpighian tubule protein UP5 gene; the fourth purpose of the present application is to provide a transformant containing the yellow stick moth Malpighian tubule protein UP5 gene; the fifth purpose of the present application is to provide the application of the yellow stick moth Malpighian tubule protein UP5 in preparing calcium ion adsorbent; the sixth purpose of the present application is to provide the application of the yellow stick moth Malpighian tubule protein UP5 in preparing a drug for reducing the formation of calcium oxalate crystals; the seventh purpose of the present application is to provide the application of the yellow stick moth Malpighian tubule protein UP5 in preparing a drug for preventing or treating calcium oxalate stones.

[0007] In order to achieve the above purposes, the present application provides the following technical solutions:

[0008] 1. A yellow stick moth Malpighian tubule protein UP5, wherein the amino acid sequence of the protein UP5 is shown as SEQ ID NO. 4.

[0009] 2. A yellow stick moth Malpighian tubule protein UP5 gene, wherein the gene sequence of the protein UP5 is shown as SEQ ID NO. 3.

[0010] A recombinant vector containing the yellow stick moth Malpighian tubule protein UP5 gene.

[0011] Preferably, the recombinant vector is obtained by connecting the sequence shown as SEQ ID NO. 3 into the expression frame of a pET-28a vector.

[0012] A transformant containing the yellow stick moth Malpighian tubule protein UP5 gene.

[0013] Preferably, the transformant is Escherichia coli BL21 (DE3).

[0014] Preferably, the application of the yellow stick moth Malpighian tubule protein UP5 in preparing a calcium ion adsorbent.

[0015] The application of the yellow stick moth Malpighian tubule protein UP5 in preparing a drug for reducing the formation of calcium oxalate crystals.

[0016] 6. The application of the yellow stick moth Malpighian tubule protein UP5 in preparing a drug for preventing or treating calcium oxalate stones.

[0017] The application has the advantages that the application provides a UP5 protein of the Malacosoma neustria tubule, which can bind with calcium ions and has a significant ability to inhibit the formation of calcium oxalate crystals, and can be used for preventing and treating the most common kidney stones, i.e. calcium oxalate stones; and the method provides a new strategy for treating kidney stones based on the mechanism that the UP5 protein of the Malacosoma neustria tubule can bind with calcium ions and inhibit the growth of calcium oxalate crystals. BRIEF DESCRIPTION OF DRAWINGS

[0018] In order to make the objectives, technical solutions and advantages of the application clearer, the application is described below with reference to the following drawings:

[0019] Figure 1 UP5 protein expression profile analysis and purification (A: UP5 protein expression profile; B: SDS-PAGE diagram of UP5 protein eluted by different concentrations of imidazole; C: gel filtration chromatography of UP5 protein);

[0020] Figure 2 UP5 protein and calcium ion interaction (A: secondary structure change of UP5 at different pH values; B: secondary structure change of UP5 at different Ca ions; C: MST binding curve of UP5 and Ca ions);

[0021] Figure 3 UP5 protein calcium binding ability analysis (A: UP5 and soluble calcium binding content determination; B: UP5 and calcium oxalate binding ability analysis diagram);

[0022] Figure 4 UP5 and calcium chloride and sodium oxalate co-incubation SEM diagram;

[0023] Figure 5 Calcium oxalate crystal area statistical diagram. DETAILED DESCRIPTION

[0024] The application will be further described below with reference to the drawings and specific examples, so that those skilled in the art can better understand the application and implement it, but the examples are not used as a limitation to the application.

[0025] Example 1: UP5 protein expression profile analysis of Malacosoma neustria

[0026] Quantitative PCR analysis was performed on the tissues of the 1st instar larvae of E. uniformis. Seven tissues, including head, midgut, fat body, epidermis, Malpighian tubules and silk glands, were collected on the first day of the 5th instar. The silk glands were further divided into five parts. Total RNA was isolated using TRIzol reagent (Invitrogen, USA). Contaminated genomic DNA was removed by DNase I (Promega, USA) digestion at 37°C for 30 min, and then the total RNA was reverse-transcribed into cDNA using M-MLV reverse transcriptase (Invitrogen). Quantitative real-time PCR (qRT-PCR) was performed using SYBR Premix Ex Taq (TaKaRa, Japan) on a qTOWER 2.2 qRT-PCR instrument (Analytik Jena Biometra, Germany). The amplification reaction was performed under the following cycling conditions: initial denaturation at 95°C for 30 s, followed by 40 cycles of denaturation at 95°C for 10 s, annealing at 60°C for 30 s, and extension at 72°C for 35 s. Translation elongation factor 2 (TEF2) was used as a housekeeping gene. The expression values of the target genes and the housekeeping gene were automatically obtained by the system software and recorded as Ct and Ch, respectively. The relative expression value of each gene was calculated using the following formula: 2 −Δ(Ct−Ch) Statistical results are shown in Table A of Figure 1 The results show that UP5 is specifically expressed in the Malpighian tubules.

[0027] Example 2, Prokaryotic expression and purification of E. uniformis UP5 protein

[0028] UP5 gene amplification primers with BamH I and Not I restriction sites were designed (UP5-F: 5'-CGCGGATCCTCTTATATCCCGCAAAAAGATACATC-3' (SEQ ID NO. 1); UP5-R: 5'-ATAAGAATGCGGCCGCCTAGTTTTGGGAATTACATACATTTTCAT-3' (SEQ ID NO. 2)). In the above primer sequences, the bold sequence is a protection base sequence, and the underlined sequence is a restriction site sequence. The UP5 sequence with a length of 1527 bp was cloned from the Malpighian tubule cDNA of E. uniformis larvae, and the nucleic acid sequence is shown in SEQ ID NO. 3, and the amino acid sequence is shown in SEQ ID NO. 4. The UP5 gene was integrated into the BamH I and Not I restriction sites in the pET-28a vector, and the recombinant plasmid was transformed into E. coli BL21 (DE3) cells. Add IPTG to induce the expression of UP5 protein at 37°C.

[0029] Purification was performed by nickel column affinity chromatography, followed by elution with protein buffers containing different concentrations of imidazole. Elution concentrations and volumes were 0 mM 200 mL, 20 mM 200 mL, 50 mM 20 mL, 100 mM 10 mL, 200 mM 10 mL, 300 mM 5 mL, and 400 mM 5 mL. The eluent was collected on ice. The collected flow-through and eluent were analyzed by SDS-PAGE electrophoresis and observed using Coomassie Brilliant Blue. Results are shown below. Figure 1 As shown in Figure B. The results show that relatively pure UP5 protein was eluted with 200-400 mM imidazole. However, some impurities remained after nickel column affinity chromatography, which may interfere with subsequent experiments. Therefore, the eluted proteins were collected for further purification.

[0030] The imidazole solution containing a single band of the target protein in the protein electrophoresis image was ultrafiltered. The ultrafiltered solution was then subjected to gel filtration chromatography, and protein electrophoresis was performed based on the peak chromatogram. The purified proteins that were detected as usable were combined into a single tube and concentrated using a 10 kDa ultrafiltration tube. Protein concentration was determined using a Bradford protein quantification kit, and appropriate labels were applied. The tubes were then flash-frozen in liquid nitrogen and stored at -80°C for later use. Results are as follows: Figure 1 As shown in Figure C. The results show that UP5 protein with high purity was obtained by gel filtration chromatography.

[0031] Example 3: Circular dichroism analysis of UP5 protein from the yellow-spotted moth.

[0032] Circular dichroism (CD) is a technique commonly used to determine the secondary structure of proteins. UP5 protein was dissolved in 0.1 M phosphate buffer at different pH values ​​(4, 5, 6, 7) and in calcium nitrate (Ca(NO3)2) solutions at different concentrations (0, 2, 4, 8, 16, 32, 64, and 128 mM). Spectra were recorded using a MOS-500 CD spectrophotometer with a scan range of 190-250 nm. Data were analyzed using Origin software. The results are shown below. Figure 2 As shown. The results indicate that in a neutral pH buffer, the UP5 protein predominantly exists in an α-helix structure. Figure 2 This is consistent with previous structural predictions. As the pH of the buffer gradually changes from neutral to acidic, the troughs exhibit a blue shift, with the wavelengths becoming smaller, indicating an increase in random structures. This suggests that the UP5 protein is more stable under neutral conditions, and acidity disrupts its helical structure. With increasing calcium ion concentration, the troughs exhibit a red shift, indicating an increase in helical structures. Figure 2 (B) Explain Ca 2＋The protein aggregation is promoted, which is important for protein stability, and it is side evidence that UP5 can interact with calcium ions.

[0033] Example 4, Microscale thermophoresis analysis of Euproctis similis UP5 protein

[0034] In order to explore the combination of UP5 protein and calcium ions, microscale thermophoresis analysis was performed using Monolith NT.115 instrument. The protein was labeled using MonolithTM RED - NHS second-generation protein labeling kit. All experiments were carried out at room temperature, and the binding buffer (Assay Buffer) contained 50 mM Tris-HCl (pH 7.8), 150 mM NaCl, 10 mM MgCl2, 0.05% Tween 20. 16 PCR tubes were prepared and labeled as No. 1-16, 5 μL Assay Buffer was added to No. 2-16 PCR tubes; 10 μL 10 mM CaCl2 was added to No. 1 tube, 5 μL was taken and added to No. 2 tube, and the pipette was repeatedly blown and mixed, 5 μL was taken from No. 2 tube and added to No. 3 tube and mixed, and the same method was used to complete No. 4-16 tubes. Finally, discard the excess 5 μL from No. 16 tube; 5 μL of labeled protein (Target) was added to No. 1-16 tubes and mixed thoroughly, and after centrifugation, the samples in No. 1-16 tubes were taken with a capillary pipette and detected on the instrument. The obtained binding curve was analyzed by MO.Affinity Analysis software, and each data point was the average of 3 independent MST measurements. The data obtained by MST experiment showed that the dissociation constant (KD) between UP5 and calcium ions was 38.9 μM. The experimental results showed that the interaction between UP5 and calcium ions had micromolar level affinity, which showed high affinity binding of the complex Figure 2 , C).

[0035] Example 5, Determination of calcium binding ability of UP5

[0036] UP5 is known to interact with calcium ions. To investigate the calcium-binding capacity of UP5, the binding content of UP5 with soluble calcium was first determined. The specific method is as follows: The UP5 protein solution was diluted to 0.5 mg / mL with 20 mM Tris-HCl (pH 8.0) and 150 mM NaCl, and then incubated with 5 mM CaCl2 solution at 37°C for 2 h. Then, 6 volumes of anhydrous ethanol were added, and the mixture was incubated at room temperature for 10 min, followed by centrifugation at 12000 g for 10 min. The precipitate was freeze-dried and then dissolved in a 20 mM Tris-HCl (pH 8.0) and 150 mM NaCl buffer solution. The calcium content of the redissolved solution was determined using a calcium content colorimetric assay kit (Beyotime, China). The results showed that 1 mg of the positive control casein could bind 35.34 μg of calcium, and 1 mg of UP5 could bind 20.988 μg of calcium. Figure 3 A). Subsequently, the binding affinity of UP5 protein to insoluble calcium oxalate was investigated. Figure 3 (B) The specific steps are as follows: Dilute the UP5 protein solution to 0.5 mg / mL with 20 mM Tris-HCl (pH 8.0) and 150 mM NaCl. Mix 50 μL of the protein solution with 10 mg of calcium oxalate powder and incubate at 37°C for 12 h. Then centrifuge at 12000 g for 15 min at 4°C to collect the supernatant. Wash the precipitate three times with ddH2O and 0.5 M NaCl, and centrifuge at 12000 g for 15 min at 4°C after each wash to collect the supernatant. Next, wash the precipitate overnight at 4°C with 50 μL of 0.5 M EDTA, and then centrifuge at 12000 g for 15 min at 4°C to collect the supernatant. Concentrate each supernatant to 100 μL using a 10 kDa ultrafiltration tube for SDS-PAGE detection. The results showed that the amount of protein in the supernatant decreased after incubation of UP5 protein with calcium oxalate, indicating that UP5 protein can bind to calcium oxalate. Furthermore, subsequent extraction of UP5 protein bound to calcium oxalate using EDTA further confirmed this binding ability.

[0037] Example 6: Determination of the binding capacity of UP5 with calcium oxalate

[0038] To evaluate whether UP5 would affect the formation and growth of calcium oxalate crystals, in vitro simulated mineralization experiments were performed with bovine serum albumin and protein buffer as controls, and the shapes of calcium oxalate crystals induced at different time nodes were observed under an electron microscope. Silicon chips were cut into 5 mm × 5 mm in size and placed in a 96-well plate, then 1 mM CaCl2, 1 mM sodium oxalate solution and protein solution (UP5 or BSA in 20 mM Tris-HCl, 150 mM NaCl, pH 8.0, final concentration 0.5 mg / mL) were added to the 96-well plate in turn. The total volume was 200 µL, and the same buffer without protein was used as a negative control. Then, the 96-well plate was incubated statically at 37°C, and the reaction was terminated at different times. After the silicon chips were removed, they were washed 3-4 times with distilled water, dried at 65°C, gold plated, and observed using a SU3500 scanning electron microscope (Hitachi, Japan), which showed that in the absence of protein and with the addition of bovine serum albumin, both formed track-type calcium oxalate crystals (Fig. 1). Figure 4 In sharp contrast, the addition of UP5 protein from the yellow stick moth (Monema flavescens) Malpighian tubules resulted in very small calcium oxalate particles, about 1000 times smaller than those induced by bovine serum albumin and protein buffer (Fig. 2). Figure 5 This indicates that the UP5 protein in the Malpighian tubules of the yellow stick moth can inhibit the formation of calcium oxalate stones.

[0039] Therefore, an acid silk protein UP5 was discovered in the Malpighian tubules of the yellow stick moth (Monema flavescens), and through a series of experiments it was found that UP5 has a high affinity for calcium ions and can inhibit the formation of calcium oxalate crystals. The potential of UP5 protein for kidney stone treatment can be summarized as follows:

[0040] 1)Interaction with calcium ions: The high affinity of UP5 protein for calcium ions indicates that UP5 may bind to calcium ions in urine, reducing the participation of calcium ions in crystal formation, thereby reducing the formation of calcium oxalate crystals.

[0041] 2)Inhibition of calcium oxalate crystal formation: In vitro simulated mineralization experiments showed that UP5 protein can inhibit the growth and morphology of calcium oxalate crystals, which may help prevent the formation of kidney stones.

[0042] 3)New ideas for biological mineralization therapy: The study of UP5 protein provides new ideas for the biological mineralization therapy of kidney stones, which may prevent or treat kidney stones by regulating the formation of calcium oxalate crystals in urine. The discovery of UP5 protein provides a new perspective for the treatment of kidney stones, especially in the inhibition of calcium oxalate crystal formation, which shows great potential.

[0043] UP5 protein can be used for preventive treatment: UP5 protein can be used as part of preventive treatment, through dietary supplements or drug forms, to reduce the formation of calcium oxalate crystals. Drug treatment: UP5 protein or its derivatives may be the target of drug development for the treatment of kidney stones that have already formed. Combination therapy: UP5 protein may be used in combination with other drugs for the treatment of kidney stones to enhance the therapeutic effect. Future research needs to further explore the clinical application of UP5 protein, including its safety, effectiveness and optimal administration route. At the same time, more research is needed to understand the specific mechanism of UP5 protein in the formation of kidney stones, so as to better develop and utilize this natural resource.

[0044] The above-described embodiments are only preferred embodiments of the present application, and the protection scope of the present application is not limited thereto. Any equivalent replacement or transformation made by those skilled in the art based on the present application is within the protection scope of the present application. The protection scope of the present application is subject to the claims.

Claims

1. A Malacosoma protein of the UP5 tubules of the Yellow-Tail Moth, characterized in that: The amino acid sequence of the protein UP5 is shown as SEQ ID NO.

4.

2. A gene encoding a Malacosoma protein of the tubules UP5, characterized in that: The gene sequence of the protein UP5 is shown as SEQ ID NO.

3.

3. A recombinant vector containing the gene of the protein UP5 of the Malacosoma neustria tubule of claim 2.

4. The recombinant vector of claim 3, wherein: The recombinant vector is obtained by connecting the sequence shown as SEQ ID NO. 3 into the expression frame of a pET-28a vector.

5. A transformant containing the gene of the protein UP5 of the Malacosoma neustria tubule of claim 2.

6. The transformant according to claim 5, characterized by: The transformant is Escherichia coli BL21 (DE3).

7. Use of the protein UP5 of the Malacosoma neustria tubule of claim 1 in the preparation of a drug for preventing or treating calcium oxalate stones.

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