Mala tube protein UP5 of tangut moth and application thereof

Abstract

The invention discloses a yellow thorn moth Martens tube protein UP5 and application thereof, the protein can be combined with white energy and calcium ions, has a remarkable capability of inhibiting formation of calcium oxalate crystals, and can be used for preventing and treating the most common kidney stone-calcium oxalate stone, so that a new strategy is expected to be provided for treatment of kidney stone, and the application of the yellow thorn moth Martens tube protein UP5 is worthy of popularization and application. The invention provides a novel medicine application of the Mala tube protein UP5 of the Cannidosa hemsleyana.

Description

Technical Field

[0001] The invention relates to the field of biomedicine, in particular to a Malpighian tubule protein UP5 of the yellow spur moth, and also to an application of the Malpighian tubule protein UP5. Background Art

[0002] Biomineralization is a ubiquitous phenomenon in nature. Through biomineralization, animals, plants and microorganisms can precisely regulate the formation of inorganic minerals. Biomineralization is mostly beneficial to organisms. For example, the skeleton of vertebrates is composed of collagen, non-collagenous proteins (glycoproteins, proteoglycans and Gla proteins) and hydroxyapatite crystals, and the shells of molluscs are 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, bone support and sensory detection.

[0003] When the internal chemical environment of the human body fluctuates abnormally, pathological mineralization will occur, and harmful inorganic or organic salt crystals will precipitate, which is called pathological (or pathological) crystallization, which in turn induces diseases such as kidney stones, gallstones, malaria, and gout. In humans, calcium oxalate is the main component of urinary stones, and calcium oxalate stones account for about 80% of all stones. Therefore, most studies on urolithiasis focus on calcium oxalate stones. The formation of kidney stones involves a series of key events, including 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 may affect this process in a variety of ways.

[0004] The Malpighian tubules are urinary organs in insects, similar to the kidneys of mammals, and their function is to maintain the body's osmotic regulation, electrolyte balance, and waste elimination. There is increasing evidence that the kidney stones produced by insects have significant similarities to those of mammals. Oxalic acid in animals is mainly derived from plant-based diets, and the oxalates absorbed from the diet are absorbed by the intestines and produced through metabolism. In order to prevent the formation of calcium oxalate stones, proteins that inhibit the formation of calcium oxalate crystals must exist in the Malpighian tubules of insects.

[0005] The Malpighian tubules of the yellow spur moth have a very high concentration of calcium oxalate, but no stones are formed, indicating that there are very good calcium oxalate crystal inhibitory proteins in the Malpighian tubules of the yellow spur moth. A protein rich in acidic amino acids, UP5, was found in the Malpighian tubules of the yellow spur moth. This protein is specifically expressed in the Malpighian tubules and has a significant ability to inhibit the formation of calcium oxalate crystals. The specific details are as follows: UP5 was found to be specifically expressed in the Malpighian tubules by quantitative PCR, and the UP5 protein was obtained by prokaryotic expression. Microthermophoresis and circular dichroism spectroscopy revealed that UP5 has a strong interaction with calcium; in vitro simulated mineralization experiments revealed that UP5 can inhibit the growth of calcium oxalate crystals. Summary of the invention

[0006] In view of this, one of the objects of the present invention is to provide a yellow spur moth Malpighian tubule protein UP5; the second object of the present invention is to provide a gene encoding the yellow spur moth Malpighian tubule protein UP5; the third object of the present invention is to provide a recombinant vector containing the yellow spur moth Malpighian tubule protein UP5 gene; the fourth object of the present invention is to provide a transformant containing the yellow spur moth Malpighian tubule protein UP5 gene; the fifth object of the present invention is to provide the use of the yellow spur moth Malpighian tubule protein UP5 in the preparation of a calcium ion adsorbent; the sixth object of the present invention is to provide the use of the yellow spur moth Malpighian tubule protein UP5 in the preparation of a drug for reducing the formation of calcium oxalate crystals; the seventh object of the present invention is to provide the use of the yellow spur moth Malpighian tubule protein UP5 in the preparation of a drug for preventing or treating calcium oxalate stones.

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

[0008] 1. The Malpighian tubule protein UP5 of the yellow-spurred moth, wherein the amino acids of the protein UP5 are shown in SEQ ID NO.4.

[0009] 2. A gene encoding the Malpighian tubule protein UP5 of the yellow-spurred moth, the gene sequence of which is shown in SEQ ID NO.3.

[0010] 3. A recombinant vector containing the Malpighian tubule protein UP5 gene of the yellow spur moth.

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

[0012] 4. A transformant containing the Malpighian tubule protein UP5 gene of the yellow spur moth.

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

[0014] Preferably, the present invention uses the Malpighian tubule protein UP5 of the yellow spur moth in the preparation of a calcium ion adsorbent.

[0015] 5. Use of the Malpighian tubule protein UP5 of the yellow spur moth in the preparation of drugs for reducing the formation of calcium oxalate crystals.

[0016] 6. Use of the Malpighian tubule protein UP5 of the yellow spur moth in the preparation of a drug for preventing or treating calcium oxalate stones.

[0017] The beneficial effects of the present invention are as follows: the present invention provides a Malpighian tubule protein UP5 of the yellow spur moth, which can bind to calcium ions and has a significant ability to inhibit the formation of calcium oxalate crystals, and can be used to prevent and treat the most common kidney stones - calcium oxalate stones; the method is based on the mechanism that the Malpighian tubule UP5 protein of the yellow spur moth can bind to calcium ions and inhibit the growth of calcium oxalate crystals, and provides a new strategy for the treatment of kidney stones. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to make the purpose, technical solution and beneficial effects of the present invention clearer, the present invention provides the following drawings for illustration:

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

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

[0021] Figure 3 The calcium binding capacity analysis of UP5 protein (A: determination of the binding content of UP5 and soluble calcium; B: analysis of the binding capacity of UP5 and calcium oxalate);

[0022] Figure 4 The SEM images of UP5 co-incubated with calcium chloride and sodium oxalate;

[0023] Figure 5 This is a statistical chart of the area of ​​calcium oxalate crystals. DETAILED DESCRIPTION

[0024] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0025] Example 1: Expression profile analysis of UP5 protein of the yellow spur moth

[0026] Various tissues of the yellow spur moth larvae on the first day of clustering were collected for quantitative PCR analysis. On the first day of clustering, seven tissues including head, midgut, fat body, epidermis, fat body, Malpighian tubules and silk glands were collected. The silk gland was further divided into five parts. Total RNA was isolated using TRIzol reagent (Invitrogen, USA). Digestion with RNase-free DNase I (Promega, USA) at 37°C for 30 minutes to remove contaminating genomic DNA, and then total RNA was reverse transcribed into cDNA using M-MLV reverse transcriptase (Invitrogen). Quantitative real-time PCR (qRT-PCR) was performed on a qTOWER 2.2qRT-PCR instrument (Analytik Jena Biometra, Germany) using SYBR Premix Ex Taq (TaKaRa, Japan). The amplification reaction was performed under the following cycle conditions: initial denaturation at 95°C for 30 seconds, followed by 40 cycles of denaturation at 95°C for 10 seconds, annealing at 60°C for 30 seconds, and extension at 72°C for 35s. Translation elongation factor 2 (TEF2) was used as a housekeeping gene. The expression values ​​of the target gene 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: -Δ(Ct-Ch) The statistical results are as follows Figure 1 As shown in A. The results showed that UP5 was specifically expressed in the Malpighian tubules.

[0027] Example 2: Prokaryotic expression and purification of the UP5 protein of the yellow spur moth

[0028] The UP5 gene amplification primers (UP5-F: 5'-CGC GGATCC TCTTATATCCCGCAAAAAGATACATC-3'(SEQ ID NO.1); UP5-R: 5'-ATAAGAAT GCGGCCGC CTAGTTTTGGGAATTACATACATTTTCAT-3'(SEQ ID NO.2)). In the above primer sequences, the bold sequence is the protection base sequence, and the underlined sequence is the restriction site sequence. Using the cDNA of the Malpighian tubules of the yellow spur moth larvae as a template, a UP5 sequence with a length of 1527bp was cloned, and its nucleic acid sequence is shown in SEQ ID NO.3, and its 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 Escherichia coli BL21 (DE3) cells. IPTG was added to induce the expression of UP5 protein at 37°C.

[0029] Purify by nickel column affinity chromatography, and elute with protein buffer containing different concentrations of imidazole. The elution concentration and volume are 0mM 200mL, 20mM 200mL, 50mM 20mL, 100mM 10mL, 200mM 10mL, 300mM5mL, 400mM5mL, and collect the eluate on ice. The collected flow-through and eluate are subjected to SDS-PAGE electrophoresis and observed with Coomassie Brilliant Blue. The results are as follows: Figure 1 As shown in B. The results showed that relatively pure UP5 protein was eluted with 200-400 mM imidazole, but some impurities still existed after nickel column affinity chromatography, which might interfere with subsequent experiments, so the eluted protein was collected for further purification.

[0030] Ultrafiltration was performed on the imidazole solution with a single target protein band in the protein electrophoresis detection diagram. The ultrafiltration solution was then subjected to gel filtration chromatography. According to the peak diagram, protein electrophoresis was performed. The purified proteins detected as usable were combined into a tube and concentrated using a 10kDa ultrafiltration tube. The protein concentration was determined using a Bradford protein quantitative detection kit, and the corresponding labels were made, and the samples were quickly frozen in liquid nitrogen and stored at -80°C for later use. The results are shown in Figure 1. Figure 1 As shown in C. The results showed that UP5 protein with high purity was obtained by gel filtration chromatography separation.

[0031] Example 3: Circular dichroism analysis of UP5 protein of the yellow spur moth

[0032] Circular Dichroism (CD) is a technique commonly used to determine the secondary structure of proteins. UP5 protein was dissolved in 0.1M phosphate buffer with different pH values ​​(4, 5, 6, 7) and calcium nitrate (Ca(NO3)2) solutions with different concentrations (0, 2, 4, 8, 16, 32, 64 and 128 mM). The spectra were recorded using a MOS-500 CD spectrophotometer with a scan range set to 190-250 nm. The data were analyzed using Origin software. The results are shown in the figure. Figure 2 The results showed that in a neutral pH buffer, the UP5 protein was mainly composed of an a-helical structure ( Figure 2 , A). This is consistent with the results of the previous structural prediction. When the pH value of the buffer gradually changes from neutral to acidic, the trough blue-shifts and the wavelength becomes smaller and smaller, indicating that there are more and more irregular structures, indicating that the UP5 protein is more stable under neutral conditions and that acidity will destroy its helical structure. As the calcium ion concentration increases, the trough red-shifts and the helical structure becomes more and more ( Figure 2 , B), indicating that Ca 2+It promotes protein aggregation, which is important for protein stability, and indirectly indicates that UP5 may interact with calcium ions.

[0033] Example 4: Microthermophoresis analysis of the UP5 protein of the yellow spur moth

[0034] In order to explore the binding of UP5 protein to calcium ions, microthermophoresis analysis was performed using a Monolith NT.115 instrument. Proteins were labeled using the MonolithTM RED-NHS second-generation protein labeling kit. All experiments were performed at room temperature, and the binding buffer (Assay Buffer) contained 50mM Tris-HCl (pH 7.8), 150mM NaCl, 10mMMgCl2, and 0.05% Tween 20. Prepare 16 PCR tubes and mark them as 1-16. Add 5μL Assay Buffer to each of PCR tubes 2-16; add 10μL 10mM CaCl2 to tube 1, pipette 5μL to tube 2, mix by blowing repeatedly with a pipette, then pipette 5μL from tube 2 to tube 3, dilute tubes 4-16 in the same way, and finally discard the excess 5μL from tube 16; add 5μL of the labeled protein (Target) to each of tubes 1-16, mix by blowing thoroughly, centrifuge instantly, pipette the samples in tubes 1-16 with a capillary pipette, and then test on the machine. The obtained binding curve is analyzed by MO.AffinityAnalysis software, and each data point is the average of 3 independent MST measurements. The dissociation constant (KD) between UP5 and calcium ions is calculated to be 38.9μM through the data obtained from the MST experiment. The experimental results showed that the interaction between UP5 and calcium ions has a micromolar affinity, showing a high affinity binding of the complex ( Figure 2 , C).

[0035] Example 5: Determination of UP5 calcium binding capacity

[0036] It is known that UP5 can interact with calcium ions. In order to explore the calcium binding ability of UP5, the binding content of UP5 and soluble calcium was first determined. The specific method is as follows: the UP5 protein solution was diluted to 0.5 mg / mL with 20mM Tris-HCl (pH 8.0) and 150mM NaCl, and then incubated with 5mM CaCl2 solution at 37°C for 2h. Then 6 volumes of anhydrous ethanol were added, mixed and incubated at room temperature for 10min, and then centrifuged at 12000g for 10min. The precipitate was freeze-dried and then dissolved in 20mM Tris-HCl (pH 8.0) and 150mM NaCl buffer solution. The re-dissolved solution was used to determine the calcium content using a calcium content colorimetric detection kit (Beyotime, China). The results showed that 1mg of positive control casein could bind 35.34μg of calcium, and 1mg of UP5 could bind 20.988μg of calcium ( Figure 3 , A). Then, the ability of UP5 protein to bind to insoluble calcium - calcium oxalate was investigated ( Figure 3 , B). The specific steps are 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. 50 μL of the protein solution was mixed with 10 mg of calcium oxalate powder, incubated at 37 ° C for 12 h, and then centrifuged at 12000 g for 15 min at 4 ° C to collect the supernatant. The precipitate was washed 3 times with ddH2O and 0.5 M NaCl, and each wash was centrifuged at 12000 g for 15 min at 4 ° C to collect the supernatant. The precipitate was then washed with 50 μL of 0.5 M EDTA at 4 ° C overnight, and then centrifuged at 12000 g for 15 min at 4 ° C to collect the supernatant. Each supernatant was concentrated to 100 μL using a 10 kDa ultrafiltration tube for SDS-PAGE detection. The results showed that after incubation of UP5 protein with calcium oxalate, the supernatant protein decreased, indicating that UP5 protein can bind to calcium oxalate. The subsequent extraction of UP5 protein bound to calcium oxalate by EDTA further verified the fact that UP5 can bind to calcium oxalate.

[0037] Example 6: Determination of the binding ability of UP5 and calcium oxalate

[0038] In order to evaluate whether UP5 affects the formation and growth of calcium oxalate crystals, an in vitro simulated mineralization experiment was conducted, using bovine serum albumin and protein buffer as controls, and the shapes of calcium oxalate crystals induced at different time points were observed under an electron microscope. The silicon wafer was cut into 5mm×5mm sizes and placed in a 96-well plate, and then 1mM CaCl2, 1mM sodium oxalate solution, and protein solution (UP5 or BSA in 20mM Tris-HCl, 150mM NaCl, pH 7.5) were added to the 96-well plate in sequence.

[0039] 8.0, the final concentration was 0.5 mg / mL). The total volume was 200 μL, and the same buffer without protein was used as a negative control. Then, the 96-well plate was statically incubated at 37°C, and the reaction was terminated at different times. After the silicon wafer was removed, it was washed with distilled water 3-4 times, dried at 65°C, gold-coated, and observed using a SU3500 scanning electron microscope (Hitachi, Japan). The results showed that racetrack-shaped calcium oxalate crystals were formed in the absence of protein and with the addition of bovine serum albumin ( Figure 4 In sharp contrast, the calcium oxalate particles with the addition of the yellow spur moth UP5 protein are very small, about 1000 times smaller than the calcium oxalate crystals induced by bovine serum albumin and protein buffer ( Figure 5 ), indicating that UP5 protein in the Malpighian tubules of the yellow-spurred moth can inhibit the formation of calcium oxalate stones.

[0040] Therefore, an acidic silk protein UP5 was discovered from the Malpighian tubules of the yellow spur 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 the treatment of kidney stones can be divided into the following three points:

[0041] 1) Interaction with calcium ions: The high affinity binding of UP5 protein to calcium ions indicates that UP5 may reduce the formation of calcium oxalate crystals by binding to calcium ions in urine and reducing the participation of calcium ions in crystal formation.

[0042] 2) Inhibit the formation of calcium oxalate crystals: In vitro simulated mineralization experiments showed that UP5 protein can inhibit the growth and morphology of calcium oxalate crystals. This inhibitory effect may help prevent the formation of kidney stones.

[0043] 3) New ideas for biomineralization treatment: The study of UP5 protein provides new ideas for the biomineralization treatment 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 showing great potential in inhibiting the formation of calcium oxalate crystals.

[0044] UP5 protein can subsequently be used for preventive treatment: UP5 protein can be used as part of preventive treatment, through dietary supplements or in the form of medicines, to reduce the formation of calcium oxalate crystals. Drug treatment: UP5 protein or its derivatives may be targeted for drug development to treat already formed kidney stones. Combination therapy: UP5 protein may be used in combination with other kidney stone treatment drugs to enhance the therapeutic effect. Future research needs to further explore the clinical application of UP5 protein, including its safety, effectiveness, and optimal route of administration. At the same time, more research is needed to understand the specific mechanism of action of UP5 protein in kidney stone formation in order to better develop and utilize this natural resource.

[0045] The above-described embodiments are only preferred embodiments for fully illustrating the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or changes made by those skilled in the art based on the present invention are within the protection scope of the present invention. The protection scope of the present invention shall be subject to the claims.

Claims

1. A protein UP5 from the Malpighian tubules of the yellow-spurred moth, characterized in that: The amino acids of the protein UP5 are shown in SEQ ID NO.

4.

2. A gene encoding the Malpighian tubule protein UP5 of the yellow-spurred moth, characterized in that: The gene sequence of the protein UP5 is shown in SEQ ID NO.

3.

3. A recombinant vector containing the Malpighian tubule protein UP5 gene of the yellow spur moth as claimed in claim 2.

4. The recombinant vector according to claim 3, characterized in that: The recombinant vector is obtained by connecting the sequence shown in SEQ ID NO.3 into the pET-28a vector expression frame.

5. A transformant containing the Malpighian tubular protein UP5 gene of the yellow spur moth according to claim 2.

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

7. Use of the Malpighian tubule protein UP5 of the yellow spur moth as claimed in claim 1 in the preparation of a calcium ion adsorbent.

8. Use of the Malpighian tubule protein UP5 of the yellow spur moth as claimed in claim 1 in the preparation of a drug for reducing the formation of calcium oxalate crystals.

9. Use of the Malpighian tubule protein UP5 of the yellow spur moth as claimed in claim 1 in the preparation of a medicament for preventing or treating calcium oxalate calculi.

Images