LRRC15 protein for promoting expression of hyaluronic acid as well as preparation method and application of LRRC15 protein

Abstract

The invention discloses a preparation method and application of an LRRC15 protein for promoting hyaluronic acid expression, and belongs to the technical field of molecular biotechnology and genetic engineering. The key points of the technical scheme are as follows: the LRRC15 protein for promoting hyaluronic acid expression is located on a human chromosome 3 q29, and the amino acid sequence of the LRRC15 protein is shown as SEQ ID NO: 2 in a sequence table. The invention further discloses a specific preparation method of the LRRC15 protein and application of the LRRC15 protein in preparation of drugs for promoting expression of low-molecular-weight hyaluronic acid and hyaluronic acid oligomers in fibroblasts. The LRRC15 protein provided by the invention has a remarkable effect in promoting the expression of low-molecular hyaluronic acid and hyaluronic acid oligomer, and has a very high application value.

Description

Technical Field

[0001] The present invention belongs to the fields of molecular biotechnology and genetic engineering technology, and particularly relates to an LRRC15 protein for promoting hyaluronic acid expression, a preparation method thereof, and an application thereof. Background Art

[0002] LRRC family proteins are widely distributed, expressed in many species, and LRRC family members play many important functions. LRRC15 is located on human chromosome 3 at 3q29 and is involved in the interaction between cells and the extracellular matrix. Studies have shown that in single-cell RNA sequencing data analysis, LRRC15 is expressed in both lung fibroblasts and lymphatic endothelial cells. Pro-inflammatory factors such as tumor necrosis factor-α, interleukin 1-β, and interferon-γ upregulate the expression of LRRC15 in C6 astrocytoma cells, while TGF-β upregulates the expression of LRRC15 in fibroblasts, and the expression of LRRC15 at the wound healing site is consistent with the role of TGF-β-activated fibroblasts in the wound healing process.

[0003] Hyaluronic acid is one of the main components of the extracellular matrix and is composed of repeating disaccharides of D-glucuronic acid and N-acetyl-D-glucosamine. It is synthesized into a high molecular weight (HMW) form (MW, >1000 kDa) by hyaluronic acid synthases (HAS1, HAS2, and HAS3). During tissue injury and inflammation, high molecular weight hyaluronic acid degrades into lower molecular weight hyaluronic acid and promotes the production of reactive oxygen species through hyaluronidase. The accumulated low molecular weight hyaluronic acid induces tissue injury and inflammation through the hyaluronic acid receptors CD44 and Toll-like receptor 4 (TLR4). The low molecular weight hyaluronic acid is further degraded into hyaluronic acid oligomers (<100 kDa) and then excreted from the body. In recent years, studies have shown that there is a close relationship between hyaluronic acid and fibrosis. Fibroblasts are the main cell type that releases hyaluronic acid. Hyaluronic acid fragments can stimulate fibroblasts to release cytokines and regulate the inflammatory response, thereby promoting TGF-β-mediated fibroblast proliferation.

[0004] At present, there are no relevant literatures and technical reports on the preparation method of LRRC15 protein and its relationship with hyaluronic acid. In view of this, the present invention is specifically proposed. Summary of the Invention

[0005] The object of the present invention is to provide an LRRC15 protein that promotes the expression of hyaluronic acid, its preparation method and application, to provide the amino acid sequence of the LRRC15 protein and the nucleotide sequence of the gene encoding the LRRC15 protein, and also to provide an expression vector containing the human LRRC15 protein and a host cell containing the expression vector. The LRRC15 protein, the expression vector or / and the host cell containing the expression vector can all promote the expression of low-molecular-weight hyaluronic acid in human fibroblasts.

[0006] The present invention adopts the following technical solutions to achieve the above object. An LRRC15 protein that promotes the expression of hyaluronic acid, wherein the LRRC15 protein is located on human chromosome 3 at 3q29, and the amino acid sequence of the LRRC15 protein is as shown in SEQ ID NO:2 in the sequence listing.

[0007] Preferably, the nucleotide sequence of the gene encoding the LRRC15 protein is as shown in SEQ ID NO:1 in the sequence listing.

[0008] Preferably, the expression vector containing the gene encoding the LRRC15 protein is pET-28a, and the host cell containing the gene encoding the LRRC15 protein is Escherichia coli Rosetta TM 。

[0009] A preparation method of an LRRC15 protein that promotes the expression of hyaluronic acid, the specific process is as follows: connecting the nucleotide sequence of the gene encoding the LRRC15 protein with an expression vector to obtain a recombinant expression vector; then introducing the recombinant expression vector into Escherichia coli competent cells; screening recombinant bacteria that stably and highly express the target protein; culturing the cells and inducing the expression of the fusion protein, collecting the bacterial cells, disrupting, centrifuging, denaturing, renaturing, and purifying to obtain the LRRC15 protein.

[0010] Preferably, the recombinant expression vector is transformed into the expression strain Rosetta TM , and the transformed single colony is inoculated into 15 mL of LB medium containing 50 μg / mL Kan at a volume ratio of 1:50, cultured at 37 °C, and when OD600 is between 0.6 and 1.0, induced for 24 h under the conditions of a temperature of 16 °C, a final concentration of IPTG of 0.1 mmol / L, and a rotation speed of 160 r / min. When expressing the LRRC15 protein under this induction condition, the expression level of the LRRC15 protein can be significantly improved.

[0011] Preferably, the recombinant expression vector is transformed into a host cell, after screening stable and highly expressing positive clones, culturing and inducing the expression of the target protein, centrifuging to collect the bacterial cells, ultrasonically disrupting, centrifuging, and performing ion exchange chromatography and gel filtration chromatography on the clarified supernatant to separate and purify the target fusion protein, and finally obtaining a high-purity LRRC15 protein.

[0012] The fibroblast cells were treated with the purified LRRC15 protein of the present invention, and hyaluronic acids with different molecular weights in the culture medium supernatant were detected. The results showed that it could promote the expression of low-molecular-weight hyaluronic acid and hyaluronic acid oligomers. The LRRC15 protein can be used to prepare drugs that promote the expression of low-molecular-weight hyaluronic acid or / and hyaluronic acid oligomers.

[0013] Compared with the prior art, the present invention has the following advantages and beneficial effects: The LRRC15 protein provided by the present invention has a significant effect in promoting the expression of low-molecular-weight hyaluronic acid and hyaluronic acid oligomers, and has high application value. Description of the Drawings

[0014] Figure 1 It is an SDS-PAGE analysis diagram of the expression of LRRC15 protein in Escherichia coli;

[0015] Figure 2 It is an SDS-PAGE analysis diagram of the purified LRRC15 protein;

[0016] Figure 3 It shows that the LRRC15 protein promotes the expression of low-molecular-weight hyaluronic acid. Detailed Embodiments

[0017] The following examples facilitate a better understanding of the present invention, but do not limit the present invention. The experimental methods in the following examples are all conventional methods unless otherwise specified. The test materials used in the following examples are all purchased from conventional biochemical reagent stores unless otherwise specified. In the following examples, quantitative tests are all set up with three repeated experiments, and the results are averaged.

[0018] Example 1

[0019] Preparation of LRRC15 Protein:

[0020] (1) Expression of LRRC15 Protein

[0021] The recombinant plasmid pET-28a-LRRC15 containing the correct sequence was transformed into the expression strain Rosetta TM。The transformed single colonies were inoculated into 1 mL of LB medium containing Kan (50 μg / mL) at a volume ratio of 1:50 and cultured at 37 °C for 10 h; inoculated into another 15 mL of LB medium containing Kan (50 μg / mL) at a volume ratio of 1:50 and cultured at 37 °C. When the OD600 was between 0.6 and 1.0, IPTG was added to a final concentration of 0.1 mmol / L for induction. The induction temperature was 16 °C, the rotation speed was 160 r / min. After 24 h, the cells were harvested, resuspended with 1×PBS, the cells were lysed and then centrifuged. The supernatant and precipitate were taken for 10 wt% SDS-PAGE electrophoresis analysis. The results showed that most of the LRRC15 protein was expressed in an insoluble form in the precipitate after lysis, as Figure 1 shown, M is the protein standard molecular weight Marker, A is the blank control (uninduced); B is the total cell protein, C is the supernatant after lysis; D is the precipitate after lysis.

[0022] (2) Purification of LRRC15 protein

[0023] Add 1 mL of denaturing lysis buffer (50 mmol / L Tris, 500 mmol / L, 8 mol / L urea and 6 mol / L guanidine hydrochloride) to the cells, and ultrasonically lyse the bacteria on ice (power 200 W, working for 2 s, interval 2 s, for a total of 15 min). After thorough lysis, centrifuge at 4 °C, 10000 g for 20 min, and take the supernatant. Take an appropriate amount of well-mixed 50 wt% BeyoGold TM His-tag PurificationResin (denaturation-resistant type), centrifuge at 4 °C (1000 g×10 s) to discard the storage solution, add one column volume of denaturing lysis buffer to the gel and mix well to equilibrate the gel, centrifuge at 4 °C (1000 g×10 s) to discard the liquid, and repeat the equilibration 1 - 2 times, discard the liquid. According to adding 4 mL of the supernatant of the bacterial lysate to every 0.5 mL of the gel solution (equivalent to 1 mL of 50 wt% gel), mix BeyoGold TM His-tag PurificationResin (denaturation-resistant type) and the supernatant of the bacterial lysate. Shake on a shaker at 4 °C for 60 min. Mix the lysate and BeyoGold TMA mixture of His-tag Purification Resin (denaturation-resistant formulation) was loaded into an empty column tube of an affinity chromatography column. The lid at the bottom of the purification column was opened, and the liquid in the column was allowed to flow out under the action of gravity. Approximately 20 μL of the flow-through was collected for subsequent analysis. The column was washed 5 times, each time adding 1 - 2 column volumes of denaturing lysis buffer, and approximately 20 μL of the lysate passing through the column was collected each time for subsequent analysis and detection. The column was washed again 5 times, each time adding 1 - 2 column volumes of denaturing wash buffer, and approximately 20 μL of the wash passing through the column was collected each time for subsequent analysis and detection. The target protein was eluted 5 - 10 times, each time using 1 column volume of denaturing elution buffer. The eluate from each time was collected into different centrifuge tubes and subjected to 10 wt% SDS-PAGE electrophoresis analysis. As Figure 2 shown, a purified His-tagged LRRC15 protein sample was obtained in the eluate. M is the protein standard molecular weight Marker; Con is the non-induced blank control; CL is the bacterial cell lysate; W1 is wash 1 of the lysate; FT is the flow-through; W2 - W4 are wash 2 - 4; E1 - E4 are elution 1 - 4.

[0024] Example 2

[0025] Cell model treatment:

[0026] The human MRC-5 cell line was purchased from ATCC. There were three groups in both the control group and the experimental group. The control group was treated with PBS for 48 h, and the experimental group was treated with LRRC15 protein for 48 h. At 48 h, the expression changes of hyaluronic acid with different molecular weights in the culture medium supernatant were detected using a hyaluronic acid kit, and the obtained experimental data were subjected to statistical analysis.

[0027] Example 3

[0028] LRRC15 protein promotes the expression of low molecular weight hyaluronic acid:

[0029] The results are as Figure 3 shown. The expression level of low molecular weight hyaluronic acid increased significantly compared with the control group, the medium molecular weight hyaluronic acid showed no significant change compared with the control group, and the expression level of high molecular weight hyaluronic acid decreased compared with the control group. Expression results: <100KD: hyaluronic acid with a molecular weight <100KD; 100 - 300KD: hyaluronic acid with a molecular weight of 100 - 300KD; 300 - 1000KD, hyaluronic acid with a molecular weight of 300 - 1000KD; >1000KD: hyaluronic acid with a molecular weight >1000KD. The above results indicate that LRRC15 protein can promote the expression of low molecular weight hyaluronic acid and hyaluronic acid oligomers in fibroblasts.

[0030] The basic principles, main features, and advantages of the present invention have been shown and described above. Without departing from the spirit and scope of the present invention, various changes and improvements are possible, and all such changes and improvements are intended to fall within the scope of protection of the present invention.

[0031] SEQUENCE LISTING

[0032] <110>Henan Normal University

[0033] <120>LRRC15 Protein for Promoting Hyaluronic Acid Expression, Preparation Method and Application Thereof

[0034] <160>2

[0035] <170>PatentIn version 3.3

[0036] <210>SEQ ID NO:1

[0037] <211>1764

[0038] <212>CDS

[0039] <213>artificial sequence

[0040] <400>SEQ ID NO:1

[0041]

[0042] <210>SEQ ID NO:2

[0043] <211>581

[0044] <212>PRT

[0045] <213>Artificial Sequence

[0046] <400>SEQ ID NO:2

[0047] MPLKHYLLLLVGCQAWGAGLAYHGCPSECTCSRASQVECTGARIVAVPTPLPWNAMSLQILNTHITELNESPFLNISALIALRIEKNELSRITPGAFRNLGSLRYLSLANNKLQVLPIGLFQGLDSLESLLLSSNQLLQIQPAHFSQCSNLKELQLHGNHLEYIPDGAFDHLVGLTKLNLGKNSLTHISPRVFQHLGNLQVLRLYENRLTDIPMGTFDGLVNLQELALQQNQIGLLSPGLFHNNHNLQRLYLSNNHISQLPPSVFMQLPQLNRLTLFGNSLKELSPGIFGPMPNLRELWLYDNHISSLPDNVFSNLRQLQVLILSRNQISFISPGAFNGLTELRELSLHTNALQDLDGNVFRMLANLQNISLQNNRLRQLPGNIFANVNGLMAIQLQNNQLENLPLGIFDHLGKLCELRLYDNPWRCDSDILPLRNWLLLNQPRLGTDTVPVCFSPANVRGQSLIIINVNVAVPSVHVPEVPSYPETPWYPDTPSYPDTTSVSSTTELTSPVEDYTDLTTIQVTDDRSVWGMTQAQSGLAIAAIVIGIVALACSLAACVGCCCCKKRSQAVLMQMKAPNEC 581。

Claims

1. A LRRC15 protein that promotes the expression of hyaluronic acid, characterized in that: The LRRC15 protein is located on human chromosome 3 3q29, and the amino acid sequence of the LRRC15 protein is shown in SEQ ID NO: 2 in the sequence listing.

2. The LRRC15 protein for promoting hyaluronic acid expression according to claim 1, characterized in that: The nucleotide sequence of the LRRC15 protein encoding gene is shown in SEQ ID NO: 1 in the sequence listing.

3. The LRRC15 protein for promoting hyaluronic acid expression according to claim 1, characterized in that: The expression vector containing the LRRC15 protein encoding gene is pET-28a, and the host cell containing the LRRC15 protein encoding gene is Escherichia coli Rosetta TM .

4. A method for preparing the LRRC15 protein for promoting hyaluronic acid expression according to any one of claims 1 to 3, characterized in that The specific process is: connect the nucleotide sequence of the LRRC15 protein encoding gene with the expression vector to obtain a recombinant expression vector; then introduce the recombinant expression vector into Escherichia coli competent cells; screen recombinant bacteria that stably and efficiently express the target protein; culture the cells and induce the expression of fusion protein, collect the bacteria, break, centrifuge, denature, renature, and purify to obtain the LRRC15 protein.

5. The method for preparing the LRRC15 protein that promotes hyaluronic acid expression according to claim 4, characterized in that: Transform the recombinant expression vector into the expression strain Rosetta TM The transformed single colony was inoculated into 15 mL of LB medium containing 50 μg / mL Kan at a volume ratio of 1:50 and cultured at 37°C. When OD600 was between 0.6 and 1.0, it was induced for 24 hours at a temperature of 16°C, a final IPTG concentration of 0.1 mmol / L, and a rotation speed of 160 r / min. When the LRRC15 protein was expressed under this induction condition, the expression level of the LRRC15 protein could be significantly improved.

6. The method for preparing the LRRC15 protein for promoting hyaluronic acid expression according to claim 4, characterized in that: The recombinant expression vector is transformed into host cells, and stable high-expression positive clones are screened and cultured to induce the expression of the target protein. The bacteria are collected by centrifugation, ultrasonically broken, and centrifuged. The clarified supernatant is subjected to ion exchange chromatography and gel filtration chromatography to separate and purify the target fusion protein, and finally a high-purity LRRC15 protein is obtained.

7. Use of the LRRC15 protein for promoting hyaluronic acid expression according to any one of claims 1 to 3 in the preparation of a drug for promoting the expression of low molecular weight hyaluronic acid or / and hyaluronic acid oligomers.

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