A gene encoding matrix metalloproteinase of ocnus cumlaude and application thereof

Abstract

The present application belongs to the field of genetic engineering, protein engineering and light industry technology, and particularly relates to a gene encoding a matrix metalloproteinase of Apostichopus japonicus and application thereof. The present application clones a matrix metalloproteinase (Smon_18113) from the body wall tissue of Apostichopus japonicus for the first time, the nucleotide sequence of which is shown as SEQ ID NO. 1, and the amino acid sequence of the matrix metalloproteinase encoded thereby is shown as SEQ ID NO. 3. The present application finds that a catalytic domain containing the 154th to 393rd amino acid residues is selected, a recombinant expression is carried out by using an E. coli prokaryotic expression system, a recombinant protein of Smon_18113 is obtained, and the recombinant protein shows a depolymerization effect on collagen fibers of Apostichopus japonicus, and has a wide application prospect in the fields of collagen extraction process, food, health care product, cosmetic, biological medicine and leather making industry.

Description

Technical Field

[0001] This invention belongs to the fields of genetic engineering, protein engineering, and light industry technology, specifically relating to a gene encoded by a matrix metalloproteinase in sea cucumber (Stichopus japonicus) and its applications. This invention is particularly applicable to collagen extraction processes, collagen fiber depolymerization treatment, and the efficient preparation of collagen in the food, health product, cosmetic, biopharmaceutical, and leather industries. Background Technology

[0002] Small warty sea cucumber ( Stichopus monotuberculatus The small-wart sea cucumber (Stichopus japonicus), commonly known as the yellow-fleshed sea cucumber and formerly called the "flower sea cucumber," is a tropical sea cucumber widely distributed, abundant, and economically valuable in the South my country Sea. Taxonomically, it belongs to the phylum Echinodermata, class Holothuroidea, order Sinatraceti, family Stichopidae, and genus Stichopus. Naturally distributed in the coral reefs of the Indo-West Pacific, it is mainly found in the South China Sea waters belonging to Guangdong, Guangxi, and Hainan provinces. The small-wart sea cucumber is a representative and valuable species of tropical sea cucumber. Its body wall is rich in collagen and various bioactive substances, making it suitable for direct use as a high-end food ingredient or for processing into health products and cosmetic raw materials. Therefore, the small-wart sea cucumber is the most promising species for the development of my country's tropical sea cucumber aquaculture industry. However, the small-wart sea cucumber exhibits a unique and abnormally rapid "autolysis" phenomenon. Under external stimuli such as mechanical damage, sudden temperature changes, and water quality fluctuations, the sea cucumber will trigger a violent autolysis phenomenon, manifested as the expulsion of internal organs, softening of the epidermis, tissue rupture, and liquefaction, ultimately forming a mucus-like substance. This phenomenon is essentially a structural breakdown of the collagen fiber network in the body wall, causing the originally regularly arranged collagen fiber bundles to break and reorganize in a disordered manner, ultimately leading to the loss of the integrity of the extracellular matrix. Based on this phenomenon, we have discovered a matrix metalloproteinase capable of hydrolyzing collagen fibers in the body wall tissue of *Staphylococcus aureus*.

[0003] Matrix metalloprotease (MMP) is a Zn-dependent protein. 2+ / Ca 2+The matrix metalloproteinase (MMP) family plays a central role in the degradation and remodeling of the extracellular matrix. MMPs participate in the synergistic degradation of collagen fibers and proteoglycan chains, causing fiber bundle depolymerization and triggering systemic tissue disintegration. Based on substrate specificity and structural similarity, MMPs can be classified into six subtypes, including collagenases, gelatinases, matrix hydrolases, matrix degradants, membrane matrix metalloproteinases, and other matrix metalloproteinases. Protein domain prediction shows that it contains a signal peptide, N-terminal propeptide, N-terminal peptide, catalytic domain, and heme-binding protein-like repeat region, exhibiting typical structural characteristics of metalloproteinases. Researching the catalytic activity and degradation function of sea cucumber MMPs can reveal their effects on specific substrates and explore their application potential in collagen extraction processes. Given the wide application of collagen in the food, health product, cosmetic, biomedical materials, and leather industries, the development of efficient and mild collagen fiber depolymerizing enzymes has significant industrial value. Summary of the Invention

[0004] The purpose of this invention is to address existing problems by providing a gene encoded by matrix metalloproteinase in sea cucumber and its application.

[0005] This invention is achieved through the following technical solution: The first objective of this invention is to provide a gene encoding matrix metalloproteinases in sea cucumbers with small warts. Smon_ 18113 The encoding gene Smon_18113 The nucleotide sequence is shown in SEQ ID NO.1.

[0006] The second objective of this invention is to provide an optimized encoding gene for matrix metalloproteinases in sea cucumbers with small warts. r- Smon_18113 The optimized coding gene r-Smon_18113 The nucleotide sequence is shown in SEQ ID NO.2.

[0007] The third objective of this invention is to provide a matrix metalloproteinase Smon_18113 for sea cucumbers, the amino acid sequence of which is shown in SEQ ID NO.3.

[0008] A fourth object of the present invention is to provide a method for amplifying the coding gene of claim 1. Smon_ 18113 The primer pair, wherein the upstream primer sequence is shown in SEQ ID NO.4 and the downstream primer sequence is shown in SEQ ID NO.5.

[0009] A fifth object of the present invention is to provide a product containing the said coding gene. Smon_18113The recombinant expression vector is characterized in that the recombinant expression vector is the prokaryotic expression vector pET-30a(+).

[0010] A sixth object of the present invention is to provide a host bacterium containing the recombinant expression vector, characterized in that the host bacterium is Escherichia coli BL21.

[0011] A seventh object of the present invention is to provide the coding gene. Smon_18113 Application in depolymerized collagen fibers.

[0012] An eighth object of the present invention is to provide the application of the matrix metalloproteinase Smon_18113 in depolymerized collagen fibers.

[0013] Furthermore, the matrix metalloproteinase Smon_18113 is its catalytic domain; The catalytic domain is located at positions 154 to 393 of the Smon_18113 amino acid sequence.

[0014] The present invention has the following advantages over the prior art: This invention marks the first cloning of a matrix metalloproteinase (Smon_18113) from the body wall tissue of the sea cucumber *Stichopus japonicus*. Its nucleotide sequence is shown in SEQ ID NO.1, and the amino acid sequence encoding the matrix metalloproteinase is shown in SEQ ID NO.3. This invention also reveals that by selecting the catalytic domain containing amino acid residues 154 to 393 and recombinantly expressing it using an *E. coli* prokaryotic expression system, a recombinant protein of Smon_18113 was obtained, exhibiting a depolymerization effect on collagen fibers in the body wall of *Stichopus japonicus*. This depolymerization effect can not only be used in the mild extraction process of sea cucumber collagen but can also be extended to the processing of collagen fibers from other animal sources, showing broad application prospects in the food industry (collagen peptide preparation), cosmetics industry (active collagen raw materials), biomedicine (medical collagen scaffolds), and light industry (leather processing). Attached Figure Description

[0015] Figure 1 The nucleotide sequence of the matrix metalloproteinase Smon_18113 of sea cucumber with small warts and its encoded protein; Figure 2 The domains and three-dimensional protein structure of the matrix metalloproteinase Smon_18113 from sea cucumber S. var. septemlobus; Figure 3 Recombinant expression and purification of the Smon_18113 matrix metalloproteinase (amino acid residues 154–393) of sea cucumber in Escherichia coli; Figure 4RFU standard curve of enzyme activity and specific activity of enzyme incubation substrate for recombinant matrix metalloproteinase Smon_18113 of sea cucumber; Figure 5 This study investigated the catalytic depolymerization of collagen fibers in the body wall of *Stichopus japonicus* by the recombinant matrix metalloproteinase Smon_18113. Detailed Implementation

[0016] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0017] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0018] Example 1: cDNA cloning and sequencing of Smon_18113, a matrix metalloproteinase from sea cucumber *Stichopus japonicus*. The transcript sequence of Smon_18113 was screened from the constructed body wall transcriptome library of *Sophora alopecuroides*. Based on the sequence, upstream primer SEQ ID NO.4 (5'-AAGCCAATGAATTTGCGACCT-3') and downstream primer SEQ ID NO.5 (5'-TTGGGGAATTGCTGTTTTGG-3') were designed to amplify Smon_18113.

[0019] Total RNA was extracted from the body wall tissue of *Stichopus japonicus* using the Trizol method (Invitrogen). Using the extracted RNA as a template, the first-strand cDNA was reverse transcribed into first-strand cDNA using the M-MuLV first-strand cDNA synthesis kit (Sangon Biotech Co., Ltd.). Using the cDNA as a template, the *Smon_18113* cDNA fragment was amplified by PCR using the aforementioned upstream and downstream primers. The PCR reaction system consisted of: 1 mL KOD One™ PCR Master Mix, 1 mL cDNA template, 0.75 mL upstream primer, 0.75 mL downstream primer, and 7.5 mL sterile water. The PCR program was: 94°C for 2 min pre-denaturation; 35 cycles: 98°C for 10 s denaturation, 55°C for 5 s annealing, 68°C for 11 s extension; and 72°C for 10 min final extension. The target band was recovered from agarose gel electrophoresis using an agarose gel recovery kit (Tiangen Biotech). TMThe Smon_18113 amplified fragment was cloned using the 18-T Vector Cloning Kit (TaKaRa). The ligation product was transformed into E. coli DH5α competent cells using a heat shock method. Positive clones were obtained through blue-white screening, expanded cultured, and plasmids were extracted for sequencing.

[0020] The results are as follows Figure 1 As shown, the obtained nucleotide sequence of Smon_18113 is shown in SEQ ID NO.1, and the corresponding amino acid sequence is shown in SEQ ID NO.3. The domain prediction and three-dimensional protein structure prediction of the matrix metalloproteinase Smon_18113 from the sea cucumber *Solanum tuberosum* are as follows: Figure 2 As shown.

[0021] Example 2: Prokaryotic recombinant expression of the Smon_18113 mature peptide catalytic domain protein The DNA fragment encoding the mature peptide catalytic domain (amino acid residues 154-393) of the Smon_18113 gene was obtained by PCR amplification. After restriction endonuclease digestion, it was subcloned into the prokaryotic expression vector pET-30a. Positive clones were obtained by kanapenem selection, transformed into BL21(DE3) Escherichia coli, and cultured on a large scale. Recombinant protein of the Smon_18113 mature peptide catalytic domain was obtained by induction with 0.5 mM IPTG at 37°C for 4 h. Purified Smon_18113 protein was obtained by inclusion body washing, denaturation, and renaturation. The induced and purified recombinant protein of the Smon_18113 mature peptide catalytic domain is shown below. Figure 3 As shown, the size of the Smon_18113 mature peptide catalytic domain recombinant protein (with the C-terminal histidine tag retained) is approximately 45 kDa, which is consistent with the expected size, indicating that the Smon_18113 mature peptide catalytic domain recombinant protein was successfully obtained.

[0022] Example 3: Enzyme activity assay of Smon_18113 recombinant protein Using Ca 2+ and Zn 2+ In the Tris-HCl buffer system, matrix metalloproteinases and MMP collagenase (as a control) were co-incubated with TF2-labeled fluorescent substrates. Fluorescence intensities were read using a microplate reader, and a standard curve was plotted by subtracting the blank from the fluorescence intensity values ​​of the TF2 gradient solutions. The formula was: Δfluorescence intensity (RFU) = (2907.10 (RFU) + 1101.77 (RFU / µM)) × product concentration (R0). 2=0.884), the enzyme fluorescence intensity of n=5 replicates was converted into product concentration using a standard curve, and then converted into enzyme activity (activity (U) = n / t (µmol / min)) and specific activity (specific activity (U / mg = activity (U) / enzyme protein mass (mg)). The catalytic ability of Smon_18113 recombinant protein for universal MMP substrates is as follows: Figure 4 As shown in the figure. The results showed that under the same conditions and enzyme amount (0.01 mg / mL, 30 min), the specific activity of the blank control group was 0.135 mU / mg, while the specific activity of matrix metalloproteinase was 1.014 mU / mg, which was significantly higher than that of the control group, showing stable and reproducible hydrolytic activity, indicating that the enzyme has catalytic ability for universal MMP substrates.

[0023] Example 4: Microscopic observation of the depolymerization effect of Smon_18113 recombinant protein on collagen fibers in the body wall of sea cucumber with small warts The body wall of *Stichopus japonicus* was sliced ​​into approximately 20 mg pieces on ice and immersed in two separate culture dishes containing 5 mL of buffer (50 mM Tris-HCl, 150 mM NaCl, 10 mM CaCl2, 20 µM ZnCl2, 0.05% Brij-35, pH 7.5) for 30 min on ice to inhibit the release and activation of endogenous enzymes. Recombinant Smon_18113 protein was added to one culture dish to a final concentration of 0.29 mg / mL, while PBS was added to the other dish as a control. After incubation at 30°C for 6 h and 12 h, the slices from each dish were transferred to 2.5% glutaraldehyde for fixation and then processed by scanning electron microscopy (SEM). The depolymerization effect of recombinant Smon_18113 protein on collagen fibers in the body wall of *Stichopus japonicus* was as follows: Figure 5 As shown in the figure. The results showed that most of the collagen fibers in the body wall tissue of the control group remained intact. In contrast, under the catalysis of matrix metalloproteinases, the collagen fibers in the body wall tissue of the experimental group underwent depolymerization, and the collagen fibers extensively depolymerized into collagen fiber bundles and single fibers.

[0024] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A gene encoding a matrix metalloproteinase in sea cucumber *Stichopus japonicus*. Smon_18113 Its characteristics are, The encoding gene Smon_18113 The nucleotide sequence is shown in SEQ ID NO.

1.

2. An optimized encoding gene for a matrix metalloproteinase in sea cucumber *Stichopus japonicus*. r-Smon_18113 Its characteristics are, The optimized coding gene r-Smon_18113 The nucleotide sequence is shown in SEQ ID NO.

2.

3. A matrix metalloproteinase Smon_18113 from sea cucumber *Stichopus japonicus*, characterized in that... The amino acid sequence of the matrix metalloproteinase Smon_18113 is shown in SEQ ID NO.

3.

4. A device for amplifying the encoding gene of claim 1 Smon_18113 The primer pair is characterized in that, The upstream primer sequence of the primer pair is shown in SEQ ID NO.4, and the downstream primer sequence is shown in SEQ ID NO.

5.

5. Containing the encoding gene of claim 1 Smon_18113 The recombinant expression vector, characterized in that, The recombinant expression vector is the prokaryotic expression vector pET-30a(+).

6. A host bacterium containing the recombinant expression vector of claim 5, characterized in that, The host bacterium is Escherichia coli BL21.

7. The encoding gene of claim 1 Smon_18113 Application in depolymerized collagen fibers.

8. The application of the matrix metalloproteinase Smon_18113 of claim 3 in depolymerizing collagen fibers.

9. The application according to claim 8, characterized in that, The matrix metalloproteinase Smon_18113 is its catalytic domain; The catalytic domain is located at positions 154 to 393 of the Smon_18113 amino acid sequence.

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