ANGPTL-7 recombinant protein, gene, recombinant vector, recombinant strain, composition and application of ANGPTL-7 recombinant protein, gene, recombinant vector, recombinant strain and composition

Abstract

The invention relates to the field of wound tissue repair, and discloses an ANGPTL-7 recombinant protein, a gene, a recombinant vector, a recombinant strain, a composition and applications thereof. The recombinant protein has an amino acid sequence selected from any one of the following (a)-(c): (a) an amino acid sequence as shown in SEQ ID NO: 1; (b) a derivative protein obtained by substitution, deletion or addition of at least one amino acid to the amino acid sequence represented by SEQ ID NO: 1, wherein the protein activity of the derivative protein is not lower than 95% of the protein activity of (a); (c) an amino acid sequence of which the amino terminal and / or carboxyl terminal of the amino acid sequence (a) or (b) are / is connected with a label; and (d) an amino acid sequence formed by connecting a signal sequence to the amino terminal of the amino acid sequence (a) or (b). The recombinant protein can promote tissue repair and accelerate wound healing, and is expected to be widely applied to clinical treatment.

Description

Technical Field

[0001] The present invention relates to the field of wound tissue repair, and in particular, to an ANGPTL-7 recombinant protein, a gene, a recombinant vector, a recombinant strain, a composition and applications thereof. Background Art

[0002] As the aging of the population intensifies, refractory wounds have become a global health problem. Common types of wounds include diabetic ulcer wounds, vascular wounds, pressure injury wounds, etc. These diseases have poor prognosis and limited treatment options. Prolonged wound healing can easily lead to serious consequences such as decreased quality of life, amputation, and death, which imposes a huge burden on families and society. The Journal of the American Medical Association has published articles for two consecutive years pointing out that the development of new drugs is the key method to solve the problem of refractory wounds. The Chinese medicine five-grain worm is a classic Chinese medicine for external treatment, which has excellent efficacy for refractory wounds. The external treatment of five-grain worms is called maggot therapy or biological debridement technology in Europe and the United States. The Lancet magazine praised it as a promising wound treatment method. The US Food and Drug Administration (FDA) has approved medical maggots as Class III medical devices for clinical use. However, this therapy has certain shortcomings, such as limited sources, high prices, pain, allergies, and increased psychological burden.

[0003] The main reason why refractory wounds cannot heal on their own is that the blood supply to the wound base and wound edge is poor, the function of the remaining cells is limited, and the proliferation and repair of wound cells cannot be achieved. During the progression of the disease, the metabolism of nutrients in the wound lesion area is difficult, and the secretion of growth factor substances is insufficient, which ultimately leads to the prolonged healing of the wound. The key to solving this pathophysiological problem lies in the discovery of new drugs that promote the proliferation and repair of residual cells in the wound. The angiopoietin-like protein family is a new protein family with 8 members. Some member proteins are expressed in trace amounts in the human body. Angiopoietin-like protein-1 and angiopoietin-like protein-2 are secreted in the mouthparts of mosquitoes, which are also dipteran insects; although Science magazine has conducted in-depth discussions on the tissue regeneration function of members of this family, the efficacy of angiopoietin-like proteins in medical maggots in promoting the healing of refractory wounds has not yet been found.

[0004] In order to find the key substances that medical maggots can use to promote wound healing and solve the problem of treating refractory wounds, existing studies have conducted a combined analysis of the whole genome, transcriptome and proteome of Lucilia sericata, the only medical maggot species certified by the FDA, and identified the key substances that medical maggots can use to promote wound healing through differential analysis.

[0005] Therefore, we used multi-omics combined analysis to identify the differential expression of angiogenin-like proteins in medical maggots, and screened out protein drugs that promote the healing of refractory wounds. Then, we expressed them through transfection of microorganisms, and finally obtained recombinant worm-derived proteins through cell wall purification. Summary of the invention

[0006] The purpose of the present invention is to overcome the problems existing in the prior art and to provide an ANGPTL-7 recombinant protein, gene, recombinant vector, recombinant strain, composition and their applications. The recombinant protein can promote tissue repair and accelerate wound healing, and is expected to be widely used in clinical treatment.

[0007] In order to achieve the above object, the present invention provides, in a first aspect, an ANGPTL-7 recombinant protein having an amino acid sequence selected from any one of the following (a)-(c):

[0008] (a) the amino acid sequence shown in SEQ ID NO: 1;

[0009] (b) a derivative protein obtained by substituting, deleting or adding at least one amino acid to the amino acid sequence shown in SEQ ID NO: 1, wherein the protein activity is not less than 95% of the protein activity of (a);

[0010] (c) an amino acid sequence having a tag connected to the amino terminus and / or carboxyl terminus of the amino acid sequence described in (a) or (b);

[0011] (d) An amino acid sequence in which a signal sequence is linked to the amino terminus of the amino acid sequence described in (a) or (b).

[0012] The second aspect of the present invention provides a gene encoding an ANGPTL-7 recombinant protein, wherein the gene has a nucleotide sequence encoding the ANGPTL-7 recombinant protein as described above.

[0013] Preferably, the gene has a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:1.

[0014] Preferably, the gene has the nucleotide sequence shown in SEQ ID NO: 2, or has a nucleotide sequence that is at least 90% identical to SEQ ID NO: 2, preferably at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical.

[0015] The third aspect of the present invention provides a recombinant vector, which contains the gene as described above.

[0016] Preferably, the expression vector of the recombinant vector is pET28a-SUMO plasmid.

[0017] The fourth aspect of the present invention provides a recombinant strain, which contains the recombinant vector as described above.

[0018] Preferably, the host cell of the recombinant strain is a prokaryotic host cell, more preferably Escherichia coli.

[0019] The fifth aspect of the present invention provides a method for preparing ANGPTL-7 recombinant protein, comprising the following steps:

[0020] (1) culturing the recombinant strain as described above to induce the expression of the gene encoding the ANGPTL-7 recombinant protein;

[0021] (2) Isolate and purify the expressed ANGPTL-7 recombinant protein.

[0022] Preferably, the culture conditions at least include: a temperature of 15-25°C and a culture time of 2-5h.

[0023] Preferably, the separation and purification process comprises: resuspending, crushing I, and solid-liquid separation I of the cultured bacteria to obtain crushed cells, dissolving, crushing II, and solid-liquid separation II of the crushed cells to obtain a crude protein solution, and subjecting the crude protein solution to chromatography.

[0024] Preferably, the conditions for the crushing I and the crushing II independently include: ultrasonic power of 150-250 W, working for 2-4 s, pausing for 3-5 s, and a total time of 25-30 min.

[0025] The conditions of the solid-liquid separation I and the solid-liquid separation II independently include: a rotation speed of 14000-18000 rpm, a temperature of 1-10° C., and a time of 40-60 min.

[0026] Preferably, the dissolving solution used for the dissolution has a pH of 7.5-8.5 and contains 15-25 mM Tris-HCl and 0.3-0.8 M NaCl.

[0027] Preferably, the chromatography is selected from at least one of ion exchange chromatography, affinity chromatography and gel filtration chromatography, more preferably affinity chromatography.

[0028] A sixth aspect of the present invention provides a composition comprising the ANGPTL-7 recombinant protein as described above, or the ANGPTL-7 recombinant protein prepared according to the method as described above.

[0029] Preferably, the composition further comprises a solvent and a hydrogel.

[0030] Preferably, relative to 100 mL of the solvent, the content of the ANGPTL-7 recombinant protein is 0.2-0.6 mg, and the content of the hydrogel is 2-12 g.

[0031] Preferably, the hydrogel is selected from at least one of methacrylate gelatin, chitosan and sodium alginate, more preferably methacrylate gelatin.

[0032] In a seventh aspect, the present invention provides the use of at least one of the aforementioned ANGPTL-7 recombinant protein, the aforementioned gene, the aforementioned recombinant vector, the aforementioned recombinant strain, the ANGPTL-7 recombinant protein obtained by the aforementioned method, and the aforementioned composition in the preparation of a drug for promoting tissue repair and / or a drug for promoting wound healing.

[0033] Preferably, the promoting of tissue repair is promoting the proliferation of human skin fibroblasts and / or promoting the expression of cytokines in human skin fibroblasts.

[0034] Through the above technical scheme, the ANGPTL-7 recombinant protein provided by the present invention can stimulate human skin fibroblasts, has a significant effect of promoting cell proliferation, and can significantly increase the expression of cell growth factors; after being mixed with hydrogel and applied, it can accelerate the healing of diabetic rat wounds. The ANGPTL-7 recombinant protein provided by the present invention can be used as an external medicine to promote tissue repair and accelerate wound healing, which has great clinical significance and broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 The electrophoresis diagram of the insect-derived ANGPTL-7 recombinant protein before and after induction in Example 2, from left to right, lane 1: Marker: 14.4, 18.4, 25, 35, 45, 66.2, 116 (kDa), lane 2: negative control, lane 3: after induction (obvious expression bands can be seen) (theoretical target protein 61kD);

[0036] Figure 2 It is a gel electrophoresis diagram of the purification of the insect-derived ANGPTL-7 recombinant protein in Example 2; from left to right, lane 1: supernatant after induced lysis, lane 2: sample penetration, lane 3: 60mM imidazole eluent, lane 4: 200mM imidazole eluent, lane 5: 200mM imidazole eluent, lane 6: 500mM imidazole eluent-1, lane 7: Marker: 14.4, 18.4, 25, 35, 45, 66.2, 116 (kDa), lane 8: 500mM imidazole eluent-2;

[0037] Figure 3This is a graph showing the effect of different concentrations of ANGPTL-7 on the proliferation of human dermal fibroblasts in Example 3 (CCK-8 method);

[0038] Figure 4 This is a graph showing the effect of different concentrations of ANGPTL-7 on the expression of growth factors FGF-2 and EGF in human dermal fibroblasts in Example 4;

[0039] Figure 5 This is a diagram showing the therapeutic effects of wound healing in the control group and the experimental group in Example 5. DETAILED DESCRIPTION

[0040] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0041] The first aspect of the present invention provides an ANGPTL-7 recombinant protein having an amino acid sequence selected from any one of the following (a)-(c):

[0042] (a) the amino acid sequence shown in SEQ ID NO: 1;

[0043] (b) a derivative protein obtained by substituting, deleting or adding at least one amino acid to the amino acid sequence shown in SEQ ID NO: 1, wherein the protein activity is not less than 95% of the protein activity of (a);

[0044] (c) an amino acid sequence having a tag connected to the amino terminus and / or carboxyl terminus of the amino acid sequence described in (a) or (b);

[0045] (d) An amino acid sequence in which a signal sequence is linked to the amino terminus of the amino acid sequence described in (a) or (b).

[0046] In the present invention, the ANGPTL-7 recombinant protein refers to the recombinant protein of angiopoietin-like protein-7. Specifically, the amino acid sequence shown in SEQ ID NO: 1 is:

[0047] ENEPEYILACDEFPTLDFNRFESQLEGMKTGNDLLRLHLENIQFRMDKQEEILKSLEKKIDNIPHSVDILLQSLEEKLIKIPQNIESKIKTESHQLNDLIGKQY QLIQTQLQELPVNDKWQILSDIKEIVKGHNVSLESFEDKLKRMENVISKATLATRELGDSVFKNLTILEKAYQRNGNGTAEAIVKPEDQELVCNIYPSSCSDFQE NYCDNNKCRISNPIFGPKSFVVACDSEGGGWTLIQRRINGSVDFYRNWSEYKAGFGDIDGEFFIGLEKLHALTTALQPMELMIQLQDFDDIVKYAKYDGFVIGN ETDNYKLIKVGSFSGTAGDSFSYHQGFKFTTKDRDNDMADDRNCAITKMGAWWYQKCLRSNLNGKYYNNPLPPLNGQGICWGDFHGYEYSMKFVQMMIRPKRI*.

[0048] The present invention conducts a three-omics joint analysis of the whole genome, transcriptome and proteome of Lucilia sericata, and determines the key substances that medical maggots use to promote wound healing through differential analysis; further, with the help of multi-omics joint analysis, the differential expression of angiogenin-like proteins in medical maggots is clarified and optimized, thereby obtaining the ANGPTL-7 recombinant protein with the effect of promoting the healing of refractory wounds.

[0049] In some embodiments of the present invention, the activity of the ANGPTL-7 recombinant protein containing the amino acid sequence (b) may be 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.8%, 99.9%, 100% of the activity of the ANGPTL-7 recombinant protein containing the amino acid sequence (a), or any intermediate value between any two of the above values. In the present invention, the protein activity of the ANGPTL-7 recombinant protein can be measured and characterized by a cytotoxicity assay (CCK assay), for example, a CCK-8 cell proliferation assay.

[0050] The 20 amino acid residues that make up proteins can be divided into four categories according to the polarity of their side chains: 1. Non-polar amino acids: alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), methionine (Met), phenylalanine (Phe), tryptophan (Trp) and proline (Pro); 2. Polar uncharged amino acids: glycine (Gly), serine (Ser), threonine (Thr), cysteine ​​(Cys), aspartic acid (Asn), glutamine (Gln) and tyrosine (Tyr); 3. Positively charged amino acids: arginine (Arg), lysine (Lys) and histidine (His); 4. Negatively charged amino acids: aspartic acid (Asp) and glutamic acid (Glu) (see "Biochemistry" (Second Edition), Volume 1, Shen Tong, Wang Jingyan, pages 82-83, Higher Education Press, December 1990). If amino acid residues belonging to the same category are substituted in a protein, such as Arg replacing Lys or Leu replacing Ile, the role of the residue in the protein domain (such as providing positive charge or forming a hydrophobic pocket structure) does not change, so the three-dimensional structure of the protein is not affected, and the function of the protein can still be achieved. The amino acid residue substitution belonging to the same category can occur at any amino acid residue position of the above-mentioned ANGPTL-7 recombinant protein.

[0051] In addition to the amino acid residue substitutions described above, the ANGPTL-7 recombinant protein provided by the present invention also includes a protein in which one or more amino acid residues are deleted or added at any position of amino acid residues 1-332, or both, compared to the amino acid sequence shown in SEQ ID NO: 1.

[0052] As mentioned above, the ANGPTL-7 recombinant protein provided by the present invention can also be modified or mutated to obtain a derivative protein. A derivative protein refers to a protein having a difference in amino acid sequence from the ANGPTL-7 recombinant protein having the above amino acid sequence, or a difference in a modification form that does not affect the sequence, or both. These proteins include natural or induced genetic variants, and the induced variants can be obtained by various techniques, such as random mutations caused by radiation or mutagens, or by site-directed mutagenesis or other known molecular biological techniques. Modifications (usually without changing the primary structure, i.e., without changing the amino acid sequence) include: chemical derivatization forms of proteins in vivo or in vitro, such as acetylation or carboxylation; modifications also include glycosylation, such as those produced by glycosylation modification during protein synthesis and processing or in further processing steps. This modification can be achieved by exposing the protein to a glycosylation enzyme (such as a mammalian glycosylase or deglycosylase). Modified forms also include sequences with phosphorylated amino acid residues (such as phosphotyrosine, phosphoserine, phosphothreonine).

[0053] In order to facilitate purification, (a) or (b) can also be modified by adding tags commonly used in the art. The tags will not affect the activity of the ANGPTL-7 recombinant protein of the present invention. In actual application, whether to add tags can be selected according to needs. Preferably, in order to further improve the purification effect, the amino acid tag is a histidine tag or a tag with an amino acid sequence as shown in SEQ ID NO: 9.

[0054] MAHHHHHHMSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKI KKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDII EAHREQIGGGSEFRT (SEQ ID NO: 9).

[0055] In the present invention, the amino terminus of the ANGPTL-7 recombinant protein is connected to a signal sequence in the protozoan of Lucilia sericata, and its amino acid sequence is shown in SEQ ID NO: 11.

[0056] MILKALLLLYFVCNSLHCAKA (SEQ ID NO: 11).

[0057] The above-mentioned ANGPTL-7 recombinant protein can be obtained by artificial synthesis, or its encoding gene can be synthesized first and then obtained by biological expression.

[0058] The second aspect of the present invention provides a gene encoding an ANGPTL-7 recombinant protein, wherein the gene has a nucleotide sequence encoding the ANGPTL-7 recombinant protein as described above.

[0059] In the present invention, there is no particular limitation on the specific sequence of the coding gene, as long as it can encode the aforementioned ANGPTL-7 recombinant protein. Those skilled in the art can design and construct the relevant coding gene based on the amino acid sequence of the ANGPTL-7 recombinant protein and the codon table and gene construction technology known in the art.

[0060] It is known in the art that, among the 20 different amino acids constituting proteins, except for Met (ATG) or Trp (TGG) which are respectively encoded by a single codon, the other 18 amino acids are respectively encoded by 2-6 codons (Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory Press, New York, USA, second edition, 1989, see Appendix D on page 950). That is, due to the degeneracy of the genetic code, there are mostly more than one codons that determine an amino acid, and the substitution of the third nucleotide in a triplet codon often does not change the composition of the amino acids, so the nucleotide sequences of genes encoding the same protein may be different. According to the known codon table, those skilled in the art can deduce the nucleotide sequences of the genes that can encode them from the amino acid sequence shown in SEQ ID NO: 1 provided by the present invention, and obtain the nucleotide sequences by biological methods (such as PCR method) or chemical synthesis methods, so the partial nucleotide sequences should all be included in the scope of the present invention.

[0061] On the contrary, using the DNA sequence disclosed herein, it is also possible to modify the nucleic acid sequence provided by the present invention by methods known in the art, such as the method of Sambrook et al. (Molecular Cloning, Cold Spring Harbor Laboratory Press, New York, USA, second edition, 1989), to obtain an amino acid sequence consistent with the activity of the ANGPTL-7 recombinant protein of the present invention. In the present invention, the coding gene can also be codon-optimized according to the characteristics of the expression vector or the needs of in vitro expression to improve the expression efficiency or expression amount in the in vitro expression vector. The present invention has no particular limitation on the specific codon liberalization method, as long as the optimized coding gene can express a protein having the aforementioned amino acid sequence (a) or (b).

[0062] As described above, accordingly, the 5' end and / or 3' end of the nucleotide sequence may also be connected to a coding sequence of a tag.

[0063] According to the present invention, preferably, the gene has a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:1.

[0064] In some embodiments of the present invention, Escherichia coli is used as an expression system for ANGPTL-7 recombinant protein. In order to obtain higher expression efficiency and higher expression amount, codon optimization is performed when expressing exogenous protein, which is suitable for Escherichia coli expression and improves protein expression efficiency. The obtained gene has the nucleotide sequence shown in SEQ ID NO: 2; or has a nucleotide sequence that is at least 90% identical to SEQ ID NO: 2, preferably at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical.

[0065]

[0066] In the present invention, the percentage of "identity", such as 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5% identity, refers to the degree of similarity between amino acid sequences or nucleotide sequences determined by sequence alignment is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%. For example, the ratio of the number of positions with the same base or amino acid residue to the total number of positions after two sequences have the same residue at as many positions as possible by introducing spaces, etc. The percentage of "identity" can be determined using software programs known in the art, preferably using default parameters for comparison.

[0067] When the ANGPTL-7 recombinant protein is modified with a tag with an amino acid sequence as shown in SEQ ID NO: 9, the tag nucleotide sequence may be as shown in SEQ ID NO: 10.

[0068] ATGGCTCACCATCATCATCATCATATGTCGGACTCAGAAGTCAATCAAGAAGCTAAGCCAGAGGTCAAGCCAGAAGTCAAGCCTGAGACTCACATCAATTTAAAGGTGTCCGATGGATCTTCAGAGATCTTCTTCAAGATCAAAAAGACCACTCCTTTAAGAAGGCTGAT GGAAGCGTTCGCTAAAAGACAGGGTAAGGAAATGGACTCCTTAAGATTCTTGTACGACGGTATTAGAATTCAAGCTGATCAGACCCTGAAGATTTGGACATGGAGGATAACGATATTATTGAGGCTCACAGAGAACAGATTGGTGGTGGATCCGAATTCCGGACC(SEQ ID NO: 10).

[0069] When the amino acid sequence of the ANGPTL-7 recombinant protein is connected to the signal sequence as shown in SEQ ID NO: 11, the nucleotide sequence of the signal sequence connected to the amino acid sequence of the recombinant protein in the protozoa of Lucilia sericata can be shown as SEQ ID NO: 12.

[0070] ATGATTCTTAAAGCGTTGTTATTATTATATTTTGTTTGCAATTCTC TGCATTGTGCAAAAGCT (SEQ ID NO: 12).

[0071] The nucleotide sequence provided by the present invention can usually be obtained by polymerase chain reaction (PCR) amplification, recombination, or artificial synthesis. For example, those skilled in the art can easily obtain templates and primers based on the nucleotide sequence provided by the present invention, and amplify the relevant sequence using PCR. Once the relevant nucleotide sequence is obtained, the relevant amino acid sequence can be obtained in large quantities by recombination. The obtained nucleotide sequence is usually cloned into a vector, then transferred into a genetically engineered bacterium, and then the relevant nucleotide sequence is isolated from the host cell after proliferation by conventional methods.

[0072] In addition, the relevant nucleotide sequences can also be synthesized by known artificial chemical synthesis methods.

[0073] The third aspect of the present invention provides a recombinant vector, which contains the gene as described above.

[0074] In the present invention, the "vector" used in the recombinant vector can be selected from various vectors known in the art, such as various commercially available plasmids, cosmids, phages and retroviruses, etc., which can be selected according to specific circumstances. Preferably, in order to further increase the expression amount of the recombinant protein and the extracellular expression of the recombinant protein, the expression vector is a pET28a-SUMO plasmid. The recombinant vector construction can be obtained by enzyme cutting with various nucleases that can have a cleavage site in the vector multiple cloning site to obtain a linear plasmid, and connected with a gene fragment cut with the same nuclease to obtain a recombinant vector.

[0075] Illustratively, the gene sequence shown in SEQ ID NO: 2 has been codon-optimized, and the gene is inserted into the Escherichia coli expression vector pET28a-SUMO by genetic engineering methods to form a recombinant vector, which is kanamycin-resistant and has a histidine-small ubiquitin-like modifier (His-SUMO) double tag.

[0076] The fourth aspect of the present invention provides a recombinant strain, which contains the recombinant vector as described above.

[0077] In the present invention, any existing expression system in the art can be used to construct the recombinant strain, as long as it can express the aforementioned ANGPTL-7 recombinant protein. In some embodiments of the present invention, when the recombinant strain contains a recombinant vector as described above, the recombinant vector can be transformed, transduced or transfected into a host cell (strain) by conventional methods in the art, such as chemical transformation by calcium chloride method, high voltage electroporation transformation, preferably electroporation transformation. The host cell can be a prokaryotic host cell or a eukaryotic host cell, preferably a prokaryotic host cell; more preferably, at least one of Escherichia coli, Bacillus subtilis and yeast. Further preferably, the host cell is Escherichia coli, such as Escherichia coli Rosetta (DE3), which has the advantages of short cycle, low cost and high expression amount.

[0078] The fifth aspect of the present invention provides a method for preparing ANGPTL-7 recombinant protein, comprising the following steps:

[0079] (1) culturing the recombinant strain as described above to induce the expression of the gene encoding the ANGPTL-7 recombinant protein;

[0080] (2) Isolate and purify the expressed ANGPTL-7 recombinant protein.

[0081] The present invention has no particular restrictions on the specific culture methods and conditions of the recombinant strains, and those skilled in the art can adjust the culture conditions according to actual conditions and needs (such as the selected strain type and culture requirements, expression efficiency and expression amount requirements, etc.).

[0082] In some preferred embodiments of the present invention, when the recombinant strain is Escherichia coli, the culturing comprises: first culturing the recombinant strain in a culture medium containing kanamycin to obtain a culture solution, and mixing the culture solution with IPTG for a second culturing to induce the expression of the gene encoding the ANGPTL-7 recombinant protein. Preferably, the conditions of the first culturing include at least: a temperature of 30-45°C, culturing until OD 600 is 0.4-0.6; the conditions of the second culture include at least: a temperature of 15-25°C and a time of 2-5h; using this induction condition, the recombinant protein can be expressed more slowly, and there is sufficient time for the formation of a spatial conformation, which plays a very important role in the function of the recombinant protein.

[0083] In the present invention, the kanamycin-containing culture medium can be a conventional culture medium suitable for recombinant strains with kanamycin added thereto, for example, the formula of the conventional culture medium can be: 5-15 g / L tryptone, 1-10 g / L yeast extract, 5-15 g / L NaCl (specifically, LB culture medium), and 40-60 μg / mL kanamycin added. The final concentration of IPTG used in the second culture is preferably 0.5-1.5 mM.

[0084] In the present invention, any existing method and conditions for separating and purifying culture products can be used to separate and purify the ANGPTL-7 recombinant protein, as long as the separation and purification conditions have no effect or substantially no effect on the activity of the ANGPTL-7 recombinant protein. Preferably, the separation and purification process comprises: enzymatically hydrolyzing and crushing the cultured bacterial cells, performing solid-liquid separation to obtain a crude protein solution, and performing chromatography on the crude protein solution.

[0085] According to the present invention, preferably, the enzymatic hydrolysis uses lysozyme, and the conditions for the fragmentation at least include: an ultrasonic power of 150-250 W, working for 2-4 seconds, pausing for 3-5 seconds, and a total time of 25-30 minutes. In this preferred manner, excessive fragmentation can be avoided to avoid loss of recombinant protein.

[0086] According to the present invention, preferably, the conditions for solid-liquid separation include at least: a rotation speed of 14000-18000 rpm, a temperature of 1-10° C., and a time of 40-60 min;

[0087] In the present invention, the chromatography can be performed in a variety of ways, for example, the chromatography is selected from at least one of ion exchange chromatography, affinity chromatography and gel filtration chromatography. Preferably, the chromatography is affinity chromatography, and since the His-SUMO tag is added to the recombinant protein, one-step purification can achieve a higher purity. Exemplarily, the process of nucleophilic chromatography includes: passing the crude protein solution through a Ni-NTA column, then washing the column with a buffer containing 20mM Tris-HCl, 0.5M NaCl and pH 8.0 until the effluent does not contain protein; then eluting with 20mM, 60mM, 200mM and 500mM imidazole buffers, respectively, and collecting the eluate in sections to elute the ANGPTL-7 recombinant protein.

[0088] The sixth aspect of the present invention provides a composition, which contains the ANGPTL-7 recombinant protein as described above, or contains the ANGPTL-7 recombinant protein prepared according to the method as described above. The composition uses the ANGPTL-7 recombinant protein as an active ingredient and has a promoting effect on wound healing.

[0089] According to the present invention, preferably, the composition further contains a solvent and a hydrogel. The solvent may be water, glycerin or other agents that are safe for the skin or wound surface; the hydrogel may be any one selected from methacrylate gelatin, chitosan and sodium alginate, more preferably methacrylate gelatin.

[0090] According to the present invention, preferably, relative to 100 mL of the solvent, the content of the ANGPTL-7 recombinant protein is 0.2-0.6 mg, and the content of the hydrogel is 2-12 g.

[0091] The present invention also provides a method for wound healing, comprising applying the above-mentioned composition to the wound surface and performing photocuring in the presence of a photoinitiator. The photoinitiator can be added to the composition for use, or can be applied after the composition is applied.

[0092] Preferably, the photoinitiator can be any suitable photoinitiator, more preferably a water-soluble, cell-compatible type I photoinitiator, such as lithium phenyl-2,4,6-trimethylbenzoylphosphinate, etc.; the photocuring conditions can be selected according to the photoinitiator and the hydrogel. Preferably, the wavelength of the light source is 350-450nm, and the luminous power is 1000-2000mW / cm 2 , the cross-linking time is 15-25s.

[0093] In a seventh aspect, the present invention provides the use of at least one of the aforementioned ANGPTL-7 recombinant protein, the aforementioned gene, the aforementioned recombinant vector, the aforementioned recombinant strain, the ANGPTL-7 recombinant protein obtained by the aforementioned method, and the aforementioned composition in the preparation of a drug for promoting tissue repair and / or a drug for promoting wound healing.

[0094] In the present invention, the term "wound" refers to a type of wound in which the dermis of the skin is damaged, thereby forming a tear, cut, puncture, incision, tear, abrasion, split, knife mark, scratch, crack or rupture in the skin of an animal. In various embodiments, the wound is selected from ulcers, chronic slow healing wounds and open wounds. "Wound healing" refers to a series of pathological and physiological processes in which local tissues are repaired by regeneration, repair and reconstruction after tissue loss due to the action of wounding factors.

[0095] In the present invention, tissue repair refers to the process in which adjacent healthy cells regenerate and replace local tissues and cells after they are damaged and die due to pathogenic factors to restore tissue integrity. Preferably, the promotion of tissue repair is to promote the proliferation of human skin fibroblasts and / or promote the expression of cytokines in human skin fibroblasts.

[0096] The present invention will be described in detail below through examples.

[0097] Unless otherwise stated, implied from the context, or customary in the art, the contents of any patents, patent applications, or publications referred to in this application are incorporated herein by reference in their entirety, where applicable, and if the definition of a specific term disclosed in the prior art is inconsistent with any definition provided in this application, the definition of the term provided in this application shall prevail.

[0098] The molecular biology experimental methods not specifically described in the following examples are all carried out in accordance with the specific methods listed in the book Molecular Cloning Experimental Guide (4th Edition) (J. Sambrook, MR Green, 2017), or in accordance with the kits and product instructions; other experimental methods, unless otherwise specified, are conventional methods.

[0099] The instruments and equipment used in the following examples, unless otherwise specified, are all conventional laboratory instruments and equipment; the experimental materials used in the following examples, unless otherwise specified, are all purchased from conventional biochemical reagent stores.

[0100] In the following examples, LB liquid medium: yeast powder 5g / L, peptone 10g / L, sodium chloride 10g / L;

[0101] LB solid medium: yeast powder 5g / L, peptone 10g / L, sodium chloride 10g / L, agar powder 20g / L.

[0102] pET28a-SUMO was purchased from Shanghai Zeye Biotechnology Co., Ltd.

[0103] Example 1 Construction of insect-derived ANGPTL-7 recombinant protein gene expression vector

[0104] The gene sequence of the insect-derived ANGPTL-7 recombinant protein was codon-optimized and synthesized by Shanghai Haoyuan Biotechnology Co., Ltd. The synthesized gene was inserted into the Escherichia coli expression vector pET28a-SUMO through genetic engineering methods to form a recombinant expression vector with kanamycin resistance and a histidine-small ubiquitin-like modifier (His-SUMO) double tag.

[0105] Specifically, the ANGPTL-7 recombinant protein gene sequence synthesized by Shanghai Haoyuan Biotechnology Co., Ltd. and the pET28a-SUMO plasmid were double-digested with Cpo I and Not I and recovered by agarose electrophoresis, and then the two digestion products were ligated at 4°C for 16 hours. The ligation reaction system (200 μL) is shown in Table 1 to obtain the recombinant vector pET28a-SUMO-ANGPTL-7.

[0106] Table 1

[0107] Gene template 4μL Forward Primer (50μM) 2μL Reverse Primer (50μM) 2μL 10×PCR Buffer 20μL dNTP Mixture 16μL DNA Polymerase 2μL <![CDATA[dH2O]]> 154μL

[0108] The amino acid sequence of the insect-derived ANGPTL-7 recombinant protein is as follows (the amino acid sequence of the ANGPTL-7 recombinant protein is shown in SEQ ID NO: 1, and is modified with a tag with an amino acid sequence shown in SEQ ID NO: 9):

[0109] *;

[0110] The nucleotide sequence encoding the insect-derived ANGPTL-7 recombinant protein gene is shown in SEQ ID NO: 2, and is modified with a tag sequence shown in SEQ ID NO: 10.

[0111]

[0112] Example 2 Expression and purification of insect-derived ANGPTL-7 protein gene

[0113] The expression and purification experiment of insect-derived ANGPTL-7 protein was carried out as follows:

[0114] (1) Protein expression

[0115] The recombinant vector pET28a-SUMO-ANGPTL-7 of the gene corresponding to the ANGPTL-7 protein obtained in Example 1 was transformed into Escherichia coli Rosetta2 (DE3) competent cells and then spread on LB solid medium plates containing 50 μg / mL kanamycin (purchased from Sangon Biotech (Shanghai) Co., Ltd.), cultured at 37°C overnight, and a single clone colony was picked and cultured with 300 mL of LB medium containing 50 μg / mL kanamycin at 37°C until OD 600 When the p-value reached about 0.6, IPTG (purchased from Sangon Biotech (Shanghai) Co., Ltd., catalog number: IB0168) with a final concentration of 1 mM was used to induce expression. After shaking culture at 18°C ​​for 3 h, the culture solution was centrifuged at 12000×g for 2 min to collect the bacteria.

[0116] Proteins were extracted from the cells before and after IPTG induction, and the target protein was detected by PAGE gel electrophoresis. The results were as follows: Figure 1 As shown, the bacteria before IPTG induction were used as negative control.

[0117] Figure 1 Lane 1 shows Marker: 14.4, 18.4, 25, 35, 45, 66.2, 116 (kDa), Lane 2 is the negative control, and Lane 3 is the induced bacteria, where obvious expression bands can be seen (the theoretical size of the target protein ANGPTL-7 is 61 kD).

[0118] (2) Protein purification and renaturation

[0119] Buffer Binding Buffer (50 mL, containing 20 mM Tris-HCl, 0.5 M NaCl, pH 8.0) was mixed with the bacteria, and lysozyme was first used for enzymolysis to preliminarily lyse the cells and release some proteins, and then ultrasonic bacteria were used to further break the cells and release the remaining proteins. The specific conditions were: 200w, ultrasonic 3s, interval 4s, a total of 25-30min; the broken solution was centrifuged at 4°C and 16000rpm for 50min, and the supernatant protein solution and precipitate (broken cells) were collected after ultrasonic disruption, and the supernatant protein solution was filtered with a 0.22μm filter for later use;

[0120] Prepare a Ni-NTA column, use a buffer Binding Buffer (containing 20 mM Tris-HCl, 0.5 M NaCl, pH 8.0) to buffer and equilibrate the column; load the supernatant protein solution at a flow rate of 1 mL / min, wash the column with a buffer Binding Buffer (containing 20 mM Tris-HCl, 0.5 M NaCl, pH 8.0) until the effluent contains no protein; elute with 20 mM, 60 mM, 200 mM and 500 mM imidazole buffers, respectively, collect the eluate in sections until the G250 detection solution does not change color; wash the column material with 3 times the column volume of deionized water, and seal the column with 20% ethanol.

[0121] The collected eluate was subjected to SDS-PAGE electrophoresis. Figure 2 shown. Figure 2 The channels from left to right in the middle are: lane 1 is the supernatant protein solution after induced lysis, lane 2 is the load penetration solution (the solution after the supernatant protein solution is filtered through a 0.22μm filter), lane 3 is 60mM imidazole eluent, lane 4 is 200mM imidazole eluent, lane 5 is 200mM imidazole eluent, lane 6 is 500mM imidazole eluent-1, lane 7 is Marker: 14.4, 18.4, 25, 35, 45, 66.2, 116 (kDa), and lane 8 is 500mM imidazole eluent-2; finally, 500mM imidazole eluent-2 was collected, concentrated and sent for quality control, and then packaged and frozen as ANGPTL-7 recombinant protein samples.

[0122] Example 3 Insect-derived ANGPTL-7 promotes proliferation of human dermal fibroblasts

[0123] The ANGPTL-7 recombinant protein sample obtained in Example 2 was used to prepare 1640 culture medium containing different concentrations of ANGPTL-7 (purchased from Thermo Fisher Scientific (China) Co., Ltd.), and the concentrations of ANGPTL-7 were set to 0 μg / ml, 0.125 μg / ml, 0.25 μg / ml, 1 μg / ml, 2 μg / ml, and 4 μg / ml, respectively; human dermal fibroblasts were seeded in a 96-well plate at 5,000 cells per well, and after adherence, the 1640 culture medium containing different concentrations of ANGPTL-7 was replaced, and the serum content was 2% (South American fetal bovine serum, purchased from Thermo Fisher Scientific (China) Co., Ltd.); after culturing for three days, the culture medium was removed, the PBS buffer was rinsed once, and after aspiration, a culture medium containing 10% CCK-8 (purchased from Selleck Biotechnology Co., Ltd., USA) was added, and the cells were incubated in an incubator for 1 hour, and the absorbance was measured at 450 nm in an ELISA reader. The results are as follows: Figure 3 shown.

[0124] The results showed that when the concentration of ANGPTL-7 was greater than 1 μg / ml, the OD value at 450 nm gradually increased, indicating that insect-derived ANGPTL-7 had an effect of promoting cell proliferation.

[0125] Example 4: Insect-derived ANGPTL-7 promotes the expression of human dermal fibroblast growth factor

[0126] The ANGPTL-7 recombinant protein sample obtained in Example 2 was used to prepare 1640 culture medium containing different concentrations of ANGPTL-7 (purchased from Thermo Fisher Scientific (China) Co., Ltd.), and the concentrations of ANGPTL-7 were set to 0 μg / ml, 0.25 μg / ml, 1 μg / ml, and 4 μg / ml, respectively; human dermal fibroblasts were seeded in a 6-well plate at 80,000 cells per well, and after adherence, the 1640 culture medium containing different concentrations of ANGPTL-7 was replaced, and the serum content was 2% (South American fetal bovine serum, Thermo Fisher Scientific (China) Co., Ltd.); after three days of culture, the culture medium was removed, the plate was rinsed twice with PBS buffer, Trizol was added, RNA was lysed and extracted, the RNA absorption value was determined by ultraviolet absorption measurement, and the concentration was calculated to evaluate the RNA purity. Reverse transcriptase was added to reverse transcribe the sample RNA into cDNA. After relative quantification, the target genes (fibroblast growth factor FGF-2 and epidermal growth factor EGF) and the housekeeping gene (GAPDH) were amplified by PCR. The nucleotide sequences of the primer pairs required for the FGF-2, EGF and GAPDH gene sequences are shown in SEQ ID NO: 3-SEQ ID NO: 8, as shown in Table 2.

[0127] Table 2

[0128]

[0129] The target genes FGF-2 and EGF were diluted in series to determine the standard curve. The standard curve samples and the samples to be tested were added to the Real-time RT-PCR reaction solution containing SYBR Green dye for amplification and detection to obtain the relative content of the target genes (FGF-2 and EGF). The above extraction and detection reagents were purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd. Figure 4 shown.

[0130] The results showed that after three days of stimulation with insect-derived ANGPTL-7, the expression levels of growth factors FGF-2 and EGF in human dermal fibroblasts increased significantly.

[0131] Example 5 Preparation and Application of Gelma Hydrogel Containing ANGPTL-7

[0132] The ANGPTL-7 (4 μg / ml) obtained in Example 2, Gelma (methacrylated gelatin, purchased from Suzhou Yongqinquan Intelligent Equipment Co., Ltd.) (10% w / v), and lithium phenyl-2,4,6-trimethylbenzoylphosphinate (purchased from Huaxia Siyin Biotechnology Co., Ltd.) (0.3% w / v) were dissolved in ultrapure water and mixed evenly to form a precursor solution, which was stored in warm water at 40°C away from light.

[0133] Diabetic rats (purchased from Shanghai Shengchang Biotechnology Co., Ltd.) were selected, and the hair on the back was shaved, disinfected and draped, and full-thickness skin defect wounds with a diameter of 1 cm were made on both sides of the spine; after hemostasis, the wounds on both sides were covered and fixed with sterile gauze and treated with drug-containing hydrogel, and the dressings were changed every 3 days after surgery; the control group only replaced the sterile gauze, and the experimental group replaced the drug-containing hydrogel. The specific method of using the drug-containing hydrogel is as follows: apply the precursor fluid to the wound, and achieve light curing through a 405nm UVLED light source with a light output of 1500mW / cm 2 , cross-linking time is 20s, treatment results are shown in Figure 5 .

[0134] Observation of wound healing showed that the wound healing speed in the experimental group was significantly better than that in the control group.

[0135] The above results show that the use of the insect-derived ANGPTL-7 recombinant protein of the present invention to stimulate human skin fibroblasts has a significant effect on promoting cell proliferation and can significantly increase the expression of cell growth factors. Gelma hydrogel prepared using insect-derived ANGPTL-7 can accelerate wound healing in diabetic rats. The insect-derived ANGPTL-7 provided by the present invention can be used as an external medicine to promote tissue repair and accelerate wound healing, and is expected to be widely used in clinical treatment.

[0136] The preferred embodiments of the present invention are described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims

1. A recombinant ANGPTL-7 protein, characterized in that: The recombinant protein has an amino acid sequence selected from any one of the following (a)-(c): (a) the amino acid sequence shown in SEQ ID NO: 1; (b) a derivative protein obtained by substituting, deleting or adding at least one amino acid to the amino acid sequence shown in SEQ ID NO: 1, wherein the protein activity is not less than 95% of the protein activity of (a); (c) an amino acid sequence having a tag connected to the amino terminus and / or carboxyl terminus of the amino acid sequence described in (a) or (b); (d) An amino acid sequence in which a signal sequence is linked to the amino terminus of the amino acid sequence described in (a) or (b).

2. A gene encoding ANGPTL-7 recombinant protein, characterized in that: The gene has a nucleotide sequence encoding the ANGPTL-7 recombinant protein according to claim 1.

3. The gene according to claim 2, characterized in that The gene has a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 1; Preferably, the gene has the nucleotide sequence shown in SEQ ID NO: 2, or has a nucleotide sequence that is at least 90% identical to SEQ ID NO: 2, preferably at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical.

4. A recombinant vector, characterized in that: The recombinant vector contains the gene according to claim 2 or 3; Preferably, the expression vector of the recombinant vector is pET28a-SUMO plasmid.

5. A recombinant strain, characterized in that: The recombinant strain contains the recombinant vector according to claim 4; Preferably, the host cell of the recombinant strain is a prokaryotic host cell, more preferably Escherichia coli.

6. A method for preparing ANGPTL-7 recombinant protein, characterized in that: The following steps are involved: (1) culturing the recombinant strain according to claim 5 to induce the expression of the gene encoding the ANGPTL-7 recombinant protein; (2) Isolate and purify the expressed ANGPTL-7 recombinant protein.

7. The method according to claim 6, characterized in that The culturing comprises: performing a first culturing on the recombinant strain in a culture medium containing kanamycin to obtain a culture solution, and mixing the culture solution with IPTG to perform a second culturing; Preferably, the first culture conditions include at least: a temperature of 30-45°C, culture until OD 600 is 0.4-0.6; the conditions of the second culture at least include: temperature is 15-25℃, time is 2-5h.

8. The method according to claim 6 or 7, characterized in that: The separation and purification process comprises: enzymatically hydrolyzing and crushing the cultured bacteria, performing solid-liquid separation to obtain a crude protein solution, and performing chromatography on the crude protein solution; Preferably, the enzymatic hydrolysis uses lysozyme, and the conditions for the fragmentation at least include: ultrasonic power of 150-250W, working for 2-4s, pausing for 3-5s, and a total time of 25-30min; The solid-liquid separation conditions include at least: a rotation speed of 14000-18000 rpm, a temperature of 1-10° C., and a time of 40-60 min; Preferably, the chromatography is selected from at least one of ion exchange chromatography, affinity chromatography and gel filtration chromatography, more preferably affinity chromatography.

9. A composition, characterized in that The composition contains the ANGPTL-7 recombinant protein according to claim 1, or contains the ANGPTL-7 recombinant protein prepared according to the method of any one of claims 6 to 8; Preferably, the composition further comprises a solvent and a hydrogel; Preferably, relative to 100 mL of the solvent, the content of the ANGPTL-7 recombinant protein is 0.2-0.6 mg, and the content of the hydrogel is 2-12 g; Preferably, the hydrogel is selected from at least one of methacrylate gelatin, chitosan and sodium alginate, more preferably methacrylate gelatin.

10. Use of at least one of the ANGPTL-7 recombinant protein according to claim 1, the gene according to claim 2 or 3, the recombinant vector according to claim 4, the recombinant strain according to claim 5, the ANGPTL-7 recombinant protein obtained by the method of any one of claims 6 to 8, and the composition according to claim 9 in the preparation of a drug for promoting tissue repair and / or a drug for promoting wound healing; Preferably, the promoting of tissue repair is promoting the proliferation of human skin fibroblasts and / or promoting the expression of cytokines in human skin fibroblasts.

Images