A recombinant collagen-like protein for biological defense and wound repair

Abstract

The present invention provides a protein, characterized in that the protein comprises a sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 1. The present invention also provides a nucleic acid encoding the protein, a vector, a cell comprising the same, a composition, and the use of the protein in the preparation of an antiviral drug or a wound repair drug.

Description

Technical Field

[0001] The present invention relates to the fields of medicine and cosmetics, and in particular to a recombinant collagen and application thereof. Background Art

[0002] Collagen is the main structural protein in the extracellular matrix of mammals. It is widely present in skin, bones, muscles and other tissues. It is mainly involved in physiological and biochemical behaviors such as cell proliferation, differentiation, migration and signal transmission, and plays a role in supporting, repairing and protecting tissue cells. Due to its good biological properties, collagen has a wide range of applications in tissue engineering, clinical medicine, food industry, packaging materials, cosmetics, medical cosmetology, biomaterials and medical devices.

[0003] Human papillomavirus (HPV) is a non-enveloped DNA virus belonging to the genus Papillomavirus of the family Papillomaviridae. This type of virus infects the epidermis and mucous membranes of the human body. Sometimes HPV can cause warts or even cancer after invading the human body. HPV can be divided into five genera based on the differences in their L1 gene DNA sequences. It is reported that 70% of cervical cancer cases are caused by two high-risk types, HPV-16 and HPV-18.

[0004] Currently, cervical cancer is the only cancer caused by HPV that can be screened for. Screening can detect patients who have no symptoms. The goal of cervical cancer screening is to detect precancerous cell changes before the cells become cancerous and when they can be prevented from developing into cancer through treatment. Cervical cancer screening is an important part of routine health care for patients with a cervix.

[0005] Female genital trauma is clinically divided into uterine prolapse, anterior vaginal wall prolapse and posterior vaginal wall prolapse. When the female reproductive organs are connected to the adjacent urinary tract or intestine due to injury, a urinary fistula or fecal fistula is formed. Childbirth is the main cause of vulvar and vaginal trauma, and it can also be caused by trauma, such as accidental falls, vulva touching sharp objects, etc. Trauma can injure the vagina or pass through the vagina to injure the urethra, bladder or rectum. Previous cervical medical procedures or surgeries may cause cervical trauma, leading to cervical insufficiency. Curettage may also be associated with cervical insufficiency. In a few cases, cervical tears that occurred during previous childbirth may also be a risk factor for cervical insufficiency. Summary of the invention

[0006] In a first aspect of the present invention, a protein is provided, characterized in that the protein comprises a sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:1.

[0007] In some embodiments of the invention, the protein comprises a sequence having at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or 100% sequence identity to the amino acid sequence shown in SEQ ID NO: 1. In some embodiments of the invention, the protein comprises a sequence having 100% sequence identity to the amino acid sequence shown in SEQ ID NO: 1.

[0008] In some embodiments of the present invention, the protein is recombinant collagen.

[0009] In some embodiments of the present invention, the protein has antiviral activity. Optionally, the virus includes but is not limited to HPV virus. Further optionally, the virus is HPV-18 virus.

[0010] In some embodiments of the present invention, the protein has wound repair activity.

[0011] In some embodiments of the present invention, the protein comprises the amino acid sequence shown in SEQ ID NO:1.

[0012] In some embodiments of the present invention, the protein is further fused with a homologous or heterologous domain, such as a purification tag, a reporter domain, and the like.

[0013] In some embodiments of the present invention, the amino acid sequence of the protein is shown in SEQ ID NO:1.

[0014] In the second aspect of the present invention, a nucleic acid molecule is provided, characterized in that the nucleic acid molecule encodes the protein as described in the first aspect of the present invention.

[0015] In some embodiments of the present invention, the nucleic acid molecule is a DNA sequence. In some embodiments of the present invention, the nucleic acid molecule is an RNA sequence. In some embodiments of the present invention, the DNA sequence is double-stranded DNA or single-stranded DNA.

[0016] In some embodiments of the invention, the nucleic acid molecule is codon optimized. In some embodiments of the invention, the codon optimization is codon optimization for expression in prokaryotes. In some embodiments, the prokaryotes are bacteria, such as Escherichia coli. In some embodiments of the invention, the codon optimization is codon optimization for expression in eukaryotes. In some embodiments, the eukaryotes are mammals such as humans.

[0017] In some embodiments of the present invention, the nucleic acid molecule comprises modified nucleotides. In some embodiments of the present invention, the modification includes but is not limited to 2'-oxymethylation modification, 2'-ethyloxymethylation modification, and phosphorothioate modification.

[0018] In the third aspect of the present invention, a vector is provided, characterized in that the vector comprises the nucleic acid molecule as described in the second aspect of the present invention.

[0019] In some embodiments of the present invention, the vector comprises a nucleic acid sequence encoding a protein whose amino acid sequence is shown in SEQ ID NO:1.

[0020] In some embodiments of the invention, the vector comprises a promoter and / or an enhancer sequence.

[0021] In some embodiments of the invention, the vector comprises a promoter sequence.

[0022] In the fourth aspect of the present invention, a cell is provided, characterized in that the cell comprises the protein as described in the first aspect of the present invention, the nucleic acid molecule as described in the second aspect of the present invention or the vector as described in the third aspect of the present invention.

[0023] In some embodiments of the invention, the cell is a prokaryotic cell. In some embodiments of the invention, the cell is a bacterium. In some embodiments of the invention, the cell is Escherichia coli.

[0024] In some embodiments of the invention, the cells are cultured cells.

[0025] In some embodiments of the invention, the cell is a mammalian cell.

[0026] In some embodiments of the present invention, the cell is selected from: CHO cells, BHK cells, NS0 cells, SP2 / 0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells, hybridoma cells, yeast cells, insect cells and plant cells.

[0027] In the fifth aspect of the present invention, a composition is provided, characterized in that the composition comprises the protein as described in the first aspect of the present invention.

[0028] In some embodiments of the invention, the composition is a pharmaceutical composition.

[0029] In some embodiments of the present invention, the composition further comprises a pharmaceutically acceptable excipient.

[0030] In the sixth aspect of the present invention, there is provided use of the protein as described in the first aspect of the present invention in the preparation of antiviral drugs or wound repair drugs.

[0031] In some embodiments of the present invention, there is provided a use of the protein as described in the first aspect of the present invention in the preparation of an antiviral drug. In some embodiments of the present invention, the virus is an HPV virus. In some embodiments of the present invention, the virus is an HPV-18 virus.

[0032] In some embodiments of the present invention, there is provided use of the protein as described in the first aspect of the present invention in the preparation of a wound repair drug.

[0033] The recombinant collagen of the present invention has strong antiviral activity and damage repair function, and also has certain antibacterial activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The structure of the recombinant collagen of the present invention is shown: PEP1-L1-PEP2-L2-PEP3-L3-PEP4-L4-PEP5-PEP6, wherein PEP1 is a SUMO tag, PEP2 is a carrier functional peptide, PEP3 is a human type I collagen domain variant, PEP4 is a human type III collagen domain variant, PEP5 is a biological defense functional peptide high activity variant, and PEP6 is a His tag. L1-L4 are linker sequences.

[0035] Figure 2 The SDS-PAGE electrophoresis diagrams of the intracellular supernatant (A) and inclusion bodies (B) after bacterial cell disruption are shown. To improve the purity, the intracellular supernatant was subsequently used for purification. DETAILED DESCRIPTION

[0036] Definition section

[0037] As used herein, the terms "protein" and "polypeptide" are used interchangeably. It refers to a molecule consisting of a plurality of amino acid residues linearly linked by peptide bonds (amide bonds). The term protein generally refers to any chain having a plurality of amino acid residues, and is not limited to a specific length. The term protein also includes products modified after the expression of the polypeptide, including but not limited to phosphorylation, glycosylation, acylation, derivatization by connecting known protective or blocking groups, products cleaved by protease hydrolysis, or modifications by non-naturally occurring amino acids. Proteins can be generated by recombinant technology or obtained by chemical synthesis. The protein of the present invention can contain about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500 or 2000 amino acid residues. The polypeptide may have a defined three-dimensional structure or may not have a defined three-dimensional structure. A polypeptide having a defined three-dimensional structure is referred to as a folded polypeptide, while a polypeptide that does not have a defined three-dimensional structure and can adopt a large number of different conformations is referred to as an unfolded polypeptide.

[0038] As used herein, the terms "amino acid" and "amino acid residue" can be used interchangeably. Amino acid refers to an amino acid having a D stereo configuration or an L stereo configuration, and can also refer to a synthetic, non-natural amino acid whose side chain is different from 20 common amino acids. Non-natural amino acids can be obtained from commercial channels. Amino acids can also be optionally further substituted, for example, including modifications to their -NH2 groups, -COOH groups or side chain groups, which modifications include some protective groups commonly used in peptide synthesis. As used herein, with respect to a protein having an amino acid sequence with at least, for example, 95% identity with a reference amino acid sequence of the present invention, it is meant that the rest of the amino acid sequence of the protein is consistent with the reference sequence except that the amino acid sequence may include up to five point mutations per 100 amino acids of the reference amino acid sequence. That is, in order to obtain a protein having an amino acid sequence with at least 95% identity with a reference amino acid sequence, up to 5% of the amino acids in the reference sequence may be deleted or replaced by another amino acid, or up to 5% of the number of amino acids of the total number of amino acids in the reference sequence may be inserted into the reference sequence. These changes to the reference sequence may occur at the N- or C-terminal position of the reference nucleotide sequence or anywhere between those terminal positions, or may be interspersed individually among the amino acid residues of the reference sequence, or may be interspersed in one or more consecutive groups within the reference sequence. In some embodiments of the invention, known free or commercially available computer programs may be used to determine whether any particular amino acid sequence has at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity with an amino acid sequence of the invention.

[0039] As used herein, the terms "nucleic acid", "nucleic acid molecule", "nucleic acid sequence", "polynucleotide" and "nucleotide sequence" are used interchangeably herein, specifically, refer to a series of consecutive nucleotides in DNA and RNA, and are any chains comprising two or more nucleotides. Nucleotide sequences, nucleic acids, polynucleotides, etc. can be single-stranded, double-stranded, chimeric mixtures of single-stranded or double-stranded, sense and antisense polynucleotides, or derivatives or modified forms thereof. For example, by way of non-limiting example, they can be modified at the phosphate backbone at any position, or at the base or sugar portion of any 1, 2, 3, 4 or more nucleotides, for example, to improve the stability of the molecule, hybridization parameters, etc. Nucleotide sequences generally carry genetic information, including but not limited to information used by cells to make proteins and enzymes.

[0040] As used herein, the term "nucleotide" is intended to mean a glycoside comprising a sugar moiety, a base moiety, and a covalently linked group such as a phosphate or phosphorothioate internucleotide linking group, and the term encompasses naturally occurring nucleotides such as DNA nucleotides or RNA nucleotides, as well as non-naturally occurring nucleotides comprising modified sugar and / or base moieties, also referred to as "nucleotide analogs". One of ordinary skill in the art will appreciate that the 5' terminal nucleotide of an oligonucleotide does not comprise a 5' internucleotide linking group, although a 5' terminal group may or may not be included. Non-naturally occurring nucleotides include nucleotides having modified sugar moieties, such as bicyclic nucleotides or 2' modified nucleotides, such as 2' substituted nucleotides.

[0041] As used herein, the term "vector" or "expression vector" has the same meaning as "expression construct" and can be used interchangeably. The term includes vectors that are self-replicating nucleic acid structures, as well as vectors that are integrated into the genome of a host cell. The expression vector disclosed herein comprises an expression cassette. The expression vector allows for a large amount of stable mRNA transcription. Once the expression vector is inside the host cell, the mRNA molecule or protein encoded by the gene is produced by the cell transcription and / or translation machinery.

[0042] Example

[0043] Example 1: Protein Design

[0044] Based on years of in-depth research in the field of collagen, the applicant designed highly active variants of human type I collagen domain and human type III collagen domain, as well as highly active variants of biological defense functional peptides, and then added tag sequences to fuse them to obtain Figure 1The recombinant collagen-like protein PHPV1.X shown has an amino acid sequence as shown in SEQ ID NO: 1. The highly active variant fragment of the biological defense functional peptide segment contains a highly active mutant of human α-defensin 5 (HD-5). During the design, the three pairs of cysteines of HD-5 were not changed, the important arginines at positions 28 and 9 were not changed, and the threonine at position 12 was mutated to the most hydrophobic tryptophan to enhance the amphiphilicity; the original valine at position 19 in the middle position is an aliphatic amino acid with a hydrophobic side chain, which mutated to a threonine with a neutral side chain to adjust the overall hydrophobicity of the fragment. The recombinant collagen-like protein has strong antiviral, antibacterial and wound repair activities.

[0045] Example 2: Preparation of recombinant collagen

[0046] The nucleic acid fragment encoding the recombinant collagen-like protein obtained by outsourcing synthesis, as shown in SEQ ID NO: 1, was cloned into the BlpI and XbaI restriction sites of the pET28a (+) vector. The reaction solution was transformed into the stbl3 competent medium, cultured for 14 hours, and then the positive clones were selected for sequencing for identification. After culturing the positive clones with correct sequencing, the expression vector plasmid of the recombinant collagen-like protein (SEQ ID NO: 2) was extracted.

[0047] The obtained vector plasmid was transformed into BL21 (DE3) E. coli competent cells (NEB, C2527I). Cultured in LB culture medium (containing kanamycin) at 37°C for 15 hours. IPTG was added to a concentration of 0.5 mM and induced at 16°C for 21 hours. Centrifuged and collected the cells, added lysis buffer and ultrasonically disrupted them, and centrifuged again to obtain the supernatant containing recombinant collagen protein ( Figure 2 ). After filtering with a 0.45 μm filter membrane, the mixture was purified by Ni-NTA affinity chromatography and then ion chromatography. The eluate was freeze-dried to obtain a recombinant collagen-like protein (SEQ ID NO: 1) with a purity of 96.7%. This sample showed only a single band in SDS-PAGE.

[0048] Example 3: Antiviral activity detection

[0049] The antiviral activity of the recombinant collagen prepared by the method of the above embodiment was determined according to a method substantially the same as that in the literature Int J Mol Sci. 2019 Nov 1; 20(21): 5455. The details are as follows.

[0050] HaCaT cells (Yuanjing Bio, YC-A018) transduced with retrovirus (the virus contains an expression frame that drives the expression of HPV-18E6 and HPV-18E7 with HPV-18URR) were prepared, cultured and exposed to aqueous solutions of recombinant collagen-like protein (SEQ ID NO: 1) and human α-defensin 5 (SEQ ID NO: 3, MCE Cat. No. HY-P5407) at different concentrations (2 μg / mL, 20 μg / mL) from day 6 to day 12; the control group was treated with DMSO. The percentage of HPV-18 DNA copies per cell in the culture compared to the DMSO group was detected by RT-qPCR on day 13. The experiment was repeated 5 times.

[0051] The results are shown in Table 1, expressed as mean ± standard deviation. The activity of recombinant collagen in inhibiting HPV viral DNA was better than that of human α-defensin 5, with a statistically significant difference (P<0.01).

[0052] Table 1. Antiviral activity test results

[0053]

[0054] Example 4: Antimicrobial Activity Detection

[0055] Escherichia coli (ATCC 8739) was inoculated on a nutrient agar plate to activate it, and a single colony was selected and then inoculated into MH agar medium and cultured at 37°C for 15 hours. 7 CFU / mL of bacterial suspension.

[0056] The minimum inhibitory concentration (MIC) was determined by agar plate dilution method: the recombinant collagen (SEQ ID NO: 1) was dissolved in sterile water to obtain the required test solutions of various concentrations; sterile water was set as a negative control group. After heating the sterilized MH agar medium, it was cooled to about 50°C, 9 mL was added to the culture dish, and then 1 mL of the protein test solution was quickly added and mixed, so that the protein concentration was 1024, 512, 256, 128, 64, 32, 16 or 8 μg / mL. Cool to room temperature until the culture medium solidifies, add 1 μL of the above bacterial suspension to the culture medium and spread it evenly, culture at 37°C for 24 hours, and the lowest concentration that can inhibit bacterial growth visible to the naked eye is taken as MIC (μg / mL). The results are shown in Table 2. It shows that the recombinant collagen has certain antibacterial activity.

[0057] Table 2. MIC test results of Escherichia coli

[0058]

[0059] Example 5. Testing of wound repair

[0060] Take 10-week-old BALB / c male mice, weighing 21-24g, and randomly divide them into groups, with 10 mice in each group. Feed them normally for 5 days, remove the hair on the back the day before the experiment, and anesthetize them by intraperitoneal injection of 2% sodium pentobarbital on the day of the experiment. After deep anesthesia takes effect, place the animals under an argon ion laser, and irradiate the mouse back skin with a wavelength of 458 nanometers to 514 nanometers vertically, with a power of about 240 milliwatts, a spot diameter of 4 mm, and irradiate each point for 3 minutes, with an energy density of about 345 joules per square centimeter. After laser irradiation-induced injury, take pictures to record the wound conditions and calculate the wound area (original surface area). For each animal in the recombinant collagen group, apply 1 mL (6 mg / mL) of the recombinant collagen aqueous solution prepared as in Example 2 to the left side of the back thoracic spine. For each animal in the collagen group, apply an equal amount of collagen (Sigma, C7774) aqueous solution 1 mL (6 mg / mL) to the left side of the back thoracic spine. For each animal in the negative control group, an equal amount of normal saline was applied externally on the left side of the dorsal thoracic spine. Apply twice a day, 12 hours apart. After 7 days of administration, take photos to record the wound healing and calculate the wound area (current wound area). The wound healing rate was calculated according to the following formula: Wound healing rate (%) = (original wound area - current wound area) / original wound area × 100%, which was used to evaluate the wound healing rate. The results are shown in Table 3, and the data of each group are expressed as mean ± standard deviation. The wound healing rate of mice in the recombinant collagen group was higher than that in the collagen group, with a statistically significant difference (P<0.001).

[0061] Table 3. Mouse wound repair test results

[0062] Grouping Wound healing rate Negative control group 49％±3％ Recombinant collagen group 96％±2％ Collagen Group 58％±5％

[0063] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the embodiments here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A protein, characterized in that The amino acid sequence of the protein is shown in SEQ ID NO:

1.

2. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the protein according to claim 1.

3. A carrier, characterized in that The vector comprises a nucleic acid sequence encoding a protein whose amino acid sequence is shown in SEQ ID NO:

1.

4. A cell, characterized in that The cell comprises the protein of claim 1, the nucleic acid molecule of claim 2 or the vector of claim 3.

5. A composition, characterized in that The composition comprises the protein of claim 1.

6. Use of the protein according to claim 1 in the preparation of antiviral drugs or wound repair drugs, wherein the virus is HPV-18 virus.

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