阅读说明：本技术 Fgf21温敏缓释载体和基因修饰方法以及其制备方法 (FGF21 temperature-sensitive slow-release carrier, gene modification method and preparation method thereof ) 是由 张银河 厉丁玮 李佳新 朱秀珍 罗雅雯 金剑锋 于 2021-06-17 设计创作，主要内容包括：本发明涉及的一种FGF21温敏缓释载体,所述FGF21温敏缓释载体包括一FGF21蛋白和一水凝胶,所述FGF21的基因序列的偏嗜密码子替换稀有密码子,以达到在Cys75-Cys93间构建工程二硫键,所述水凝胶为肝素一泊洛沙姆；同时涉及FGF21基因修饰方法以及水凝胶制备方法。(The invention relates to an FGF21 temperature-sensitive slow-release carrier, wherein the FGF21 temperature-sensitive slow-release carrier comprises an FGF21 protein and a hydrogel, a bias codon of a gene sequence of FGF21 replaces a rare codon so as to construct an engineering disulfide bond between Cys75 and Cys93, and the hydrogel is heparin-poloxamer; also relates to an FGF21 gene modification method and a hydrogel preparation method.)

1. An FGF21 temperature-sensitive slow-release carrier is characterized in that the FGF21 temperature-sensitive slow-release carrier comprises an FGF21 protein and hydrogel.

2. The FGF21 temperature-sensitive slow-release carrier of claim 1, wherein a bias codon of a gene sequence of the FGF21 recombinant protein replaces a rare codon so as to construct an engineered disulfide bond between Cys75 and Cys 93.

3. The FGF21 temperature-sensitive slow-release carrier of claim 2, wherein the hydrogel is a heparin-poloxamer mixture.

4. A method of genetic modification, the method comprising:

(a) modifying the FGF21 gene sequence by site-specific mutagenesis to introduce additional disulfide bonds based on distance and orientation constraints assessed by structural modeling;

(b) constructing an engineering disulfide bond between Cys75 and Cys93, changing G and C in a triplet codon into A and T on the premise of not changing Cys, replacing a rare codon with an optimal preference codon, and constructing a disulfide bond between Cys75 and Cys 93;

(c) constructing the full length of FGF21 recombinant gene on an expression vector of a CMV promoter, wherein the vector carries a His label, and pcDNF3.1 is taken as a vector;

(d) purifying the protein; and

(e) and (3) detecting the concentration and purity of the FGF21 protein.

5. The method of genetic modification of claim 4, wherein said step (c) further comprises: (c.1) prokaryotic escherichia coli expression and protein purification; or said step (c) further comprises: (c.2) transient expression and protein purification of eukaryotic HEK293t cells.

6. The method of genetic modification of claim 4, wherein said step (b) further comprises designing PCR primers: the 5' end primer comprises a restriction enzyme cutting site required by cloning and an upstream matching sequence containing an initiation codon ATG, and the length is about 26-30 bp; the 3' primer contains downstream sequence but does not contain stop codon, and HRV 3C Protease cleavage sequence is added to the end of the primer.

7. The method of genetic modification of claim 6, wherein step (f) further comprises the establishment of a stably transfected cell line with FGF 21.

8. The method of genetic modification of claim 7, wherein step (g) comprises lyophilization and storage of the FGF21 protein.

9. The gene modification method of claim 4, wherein the step (b) further comprises the steps of (b.1) finding three amino acid sequences that have a large influence on the level of 0-linked glycosylation, introducing an EcoRI cleavage site and an initiation codon ATG into a gene, excising the corresponding three gene sequences and a signal peptide gene with endonuclease, and ligating them with ligase.

10. A method of making a hydrogel, comprising:

(A) preparing a heparin-poloxamer powder;

(B) dissolving the lyophilized heparin-poloxamer powder and the lyophilized FGF21 powder in fresh saline (4C) to obtain 170mg/mI original hydrogel solution and the FGF21 solution, respectively; and

(C) and mixing the heparin-poloxamer powder and the FGF21 freeze-dried powder, and storing the mixture in a refrigerator at 4 ℃ overnight to prepare a solution, namely the FGF21 temperature-sensitive slow-release carrier.

11. The method for preparing a temperature-sensitive slow release carrier of FGF21 according to claim 10, wherein the step (a) further comprises:

(A.1) first reacting poloxamer 407 with 1.3mM 4-nitrophenyl chloroformate and diaminoethylene to give a monoamine-terminated poloxamer;

(A.2) coupling the intermediate with heparin salt in 2- (N-morpholine) sulfonic acid buffer at 25 ℃ for 1 day; and

and (A.3) dialyzing the reaction temperature-sensitive slow-release carrier for 3 days and freeze-drying to obtain the heparin-poloxamer freeze-dried powder.

Technical Field

The invention belongs to the field of biomedical materials, and particularly relates to an FGF21 temperature-sensitive slow-release carrier, a carrier construction method and a preparation method thereof.

Background

Diabetes has changed from rare pathological changes to epidemic diseases in China, the prevalence rate of diabetes of Chinese people is dramatically increased from 0.67% to 11.6% in average in recent 30 years, and the number of the people with diabetes is more than one hundred million people and seriously harms the health and safety of people in China, which is the country with the highest number of diabetes-related patients all over the world. One clinical complication that is quite common in diabetes is the presence of persistent chronic skin ulcers, such as diabetic feet. If the ulcers are not effectively controlled, progression will be further aggravated, and the risk of gangrene, amputation and even death will occur, while 40% of patients will relapse within 12 months after the foot ulcers heal. It is not exaggeration to say that diabetic foot is the most common and serious complication with the highest treatment cost for the diabetic, is one of the main causes of disability and death, and is also a major public health problem causing heavy burden to the society. Currently, there are various treatments for diabetic foot, including traditional methods of controlling blood glucose levels in diabetic patients, using anti-infective drugs, lowering blood lipid levels in diabetic patients, and skin grafting. However, these conventional treatments have many disadvantages such as labor and time for patient care, high treatment cost, and severe social and economic burden on patients, and thus do not achieve satisfactory therapeutic effects. Therefore, it is of great medical importance to find a safe, convenient and inexpensive method for diabetic wound healing.

Fibroblast growth factor-21 (fibroblast growth factor-21, FGF21), a new member of the FGF superfamily, is a pleiotropic endocrine factor. According to the reference of the literature, FGF21 plays an important role in the treatment of diabetic wounds, and can effectively promote the repair of the wounds, reduce blood sugar and the like. Although FGF21 does not directly promote proliferation, it has the ability to increase wound repair by regulating metabolism, such as promoting neovascularization, granulation tissue proliferation, and collagen deposition, to indirectly promote proliferation of cells and cell matrices. Meanwhile, FGF21 has the function of insulin, can enhance the sensitivity of the body to insulin, reduce blood sugar and protect the function of pancreatic beta cells, and has no obvious side effect. In addition, FGF21 is the only member of the FGF family that does not have mitogenic activity and does not cause carcinogenic events, and can be safely used in the clinic.

However, FGF21 has the defects of short half-life, poor stability, low protein expression quality and the like in vivo and in vitro, and the clinical application of FGF21 is limited to a great extent. On the other hand, conventional FGF21 self-assembled polymer micelles by a single polymer generally suffer from low drug loading, poor stability, and low bioavailability.

Disclosure of Invention

The invention aims to provide an FGF21 temperature-sensitive slow-release carrier, a gene modification method and a preparation method thereof, wherein the FGF21 temperature-sensitive slow-release carrier can improve the wound repair capability of diabetic feet by regulating metabolism.

Another object of the present invention is to provide an FGF21 temperature-sensitive slow-release vector and a gene modification method and a preparation method thereof, wherein the gene sequence of FGF21 is modified by specific mutagenesis to introduce additional disulfide into the amino acid sequence of the partial code to improve stability.

The invention also aims to provide an FGF21 temperature-sensitive slow-release carrier, a gene modification method and a preparation method thereof, wherein a preference codon of a gene sequence of FGF21 replaces a rare codon so as to construct an engineering disulfide bond between Cys75 and Cys 93.

Another object of the present invention is to provide a FGF21 temperature-sensitive slow-release vector, a gene modification method, and a preparation method thereof, wherein the substitution of the amino acid sequence of FGF21 reduces the level of O-linked glycosylation to improve protein quality.

Another object of the present invention is to provide an FGF21 temperature-sensitive slow-release vector and a gene modification method and a preparation method thereof, wherein a hydrogel in the FGF21 temperature-sensitive slow-release vector is a hydrophilic polymer having a three-dimensional structure, and the hydrogel is used for loading and delivering a growth factor to a damaged area.

The invention also aims to provide an FGF21 temperature-sensitive slow-release carrier, a gene modification method and a preparation method thereof, wherein the hydrogel is heparin-poloxamer, and the FGF21 protein and the heparin-poloxamer are combined to form the temperature-sensitive slow-release carrier so as to achieve the slow-release effect.

In order to achieve the purpose, the FGF21 temperature-sensitive slow-release carrier disclosed by the invention comprises FGF21 protein and hydrogel.

In a preferred embodiment, the bias codon of the gene sequence of FGF21 replaces the rare codon to achieve the engineered disulfide bond between Cys75-Cys 93.

In a preferred embodiment, the hydrogel is heparin-poloxamer.

A method of genetic modification comprising:

(a) based on the distance and orientation constraints assessed by structural modeling, we wanted to modify the FGF21 gene sequence by site-specific mutagenesis to introduce additional disulfide bonds;

(b) constructing an engineering disulfide bond between Cys75 and Cys93, changing G and C in a triplet codon into A and T on the premise of not changing Cys, replacing a rare codon with an optimal preference codon, and constructing a disulfide bond between Cys75 and Cys 93;

(b) constructing the full length of FGF21 recombinant gene on an expression vector of a CMV promoter, wherein the vector carries a His label, and pcDNF3.1 is taken as a vector;

(c) purifying the protein; and

(d) and (4) detecting the concentration and purity of the FGF21 protein.

In a preferred embodiment, the step (c) further comprises: (c.1) prokaryotic escherichia coli expression and protein purification; the step (c) further comprises: (c.2) transient expression and protein purification of eukaryotic HEK293t cells.

In a preferred embodiment, said step (b) further comprises designing PCR primers: the 5' end primer comprises a restriction enzyme cutting site required by cloning and an upstream matching sequence containing an initiation codon ATG, and the length is about 26-30 bp; the 3' primer contains downstream sequence but does not contain stop codon, and HRV 3C Protease cleavage sequence is added to the end of the primer.

In a preferred embodiment, the steps may further comprise (e) the establishment of a stably transfected cell line with FGF 21.

In a preferred embodiment, three amino acid sequences that have a greater effect on the level of 0-linked glycosylation are found, an EcoRI cleavage site and the initiation codon ATG are introduced into the gene, the corresponding three gene sequences and signal peptide gene are excised with endonuclease, and the step (f) of lyophilization storage of the FGF21 protein is ligated with ligase.

Finding three amino acid sequences which have great influence on the level of 0-linked glycosylation, introducing EcoRI enzyme cutting sites and initiation codon ATG into the gene, cutting out the corresponding three gene sequences and signal peptide gene by endonuclease, and connecting by ligase

A method for preparing a hydrogel comprises:

(A) preparing HP powder;

(B) dissolving the lyophilized HP powder and the lyophilized powder of novel FGF21 in fresh saline (4C) respectively to obtain a 170mg/mI original hydrogel solution and a required solution of novel FGF 21; and

(C) and mixing the HP powder and the novel FGF21 freeze-dried powder, and storing the mixture in a refrigerator at 4 ℃ overnight to prepare a solution, namely the FGF21 temperature-sensitive slow-release carrier.

In a preferred embodiment, the step (a) further comprises:

(A.1) first reacting poloxamer 407 with 1.3mM 4-nitrophenyl chloroformate and diaminoethylene to give a monoamine-terminated poloxamer;

(A.2) coupling the intermediate with heparin salt in 2- (N-morpholine) sulfonic acid buffer at 25 ℃ for 1 day; and

and (A.3) dialyzing the reaction temperature-sensitive slow-release carrier for 3 days and freeze-drying to obtain the heparin-poloxamer freeze-dried powder.

Drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed for the embodiments or the prior art descriptions will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.

FIG. 1 is a schematic diagram of the interaction of FGF21 and Heparin Poloxamer (HP) according to a preferred embodiment of the present invention.

FIG. 2 is a flow chart of hydrogel production by the sandwich cold method according to a preferred embodiment of the present invention.

Detailed Description

In order to make the technical problems, technical solutions and advantageous effects to be solved by the present invention more clearly apparent, the present invention is further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

It will be understood that when an element is referred to as being "secured to" or "disposed on" another element, it can be directly or indirectly secured to the other element. When an element is referred to as being "connected to" another element, it can be directly or indirectly connected to the other element. The terms "upper", "lower", "left", "right", "front", "rear", "vertical", "horizontal", "top", "bottom", "inner", "outer", and the like indicate orientations or positions based on the orientations or positions shown in the drawings, and are for convenience of description only and not to be construed as limiting the technical solution. The terms "first", "second" and "first" are used merely for descriptive purposes and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features. The meaning of "plurality" is two or more unless specifically limited otherwise.

At present, FGF21 plays a significant role in the repair of diabetic foot wounds. Therefore, FGF21 was selected for further study in this project group. Cell proliferation and cell matrix formation are essential to the skin wound healing process, and although FGF21 does not directly promote proliferation, it has the ability to increase wound repair by modulating metabolism, such as promoting neovascularization, granulation tissue proliferation, and collagen deposition, to indirectly promote proliferation of cells and cell matrices. Meanwhile, FGF21 has the function of insulin-like, can enhance the sensitivity of the body to insulin, reduce blood sugar, protect pancreatic beta cells, and has no obvious side effect, such as no edema and hypoglycemia caused by the blood sugar reduction by FGF 21. In addition, FGF21 is the only member of the FGF family that has no mitogenic activity, and it does not cause carcinogenic events and can be safely used in the clinic.

However, FGF21 has the disadvantages of short half-life, poor stability, low protein expression quality and the like in vivo and in vitro, and thus clinical application of FGF21 is limited to a great extent.

As shown in fig. 1 to 2, the present invention relates to an FGF21 temperature-sensitive slow-release vector, wherein the FGF2110 gene is optimally modified, and the FGF2110 gene sequence is modified by site-specific mutagenesis to introduce an additional engineered disulfide bond, i.e., disulfide stabilization engineering strategy, in the encoded amino acid sequence to improve stability, while reducing the level of 0-linked glycosylation by removing specific amino acid sequence substitutions to improve protein quality.

The invention designs a method for modifying the FGF2110 gene, which conveniently comprises the following steps:

(a) design of the FGF2110 recombinant protein. Based on the distance and orientation constraints assessed by structural modeling, we wanted to modify the FGF2110 gene sequence by site-specific mutagenesis to introduce additional disulfide bonds. The stability of FGF2110 can be significantly improved by literature review of the stability of FGF2110 by stabilizing the terminal domain of FGF2110C with an engineered disulfide bond at Cys75-Cys93 (cysteine at position 75 and cysteine at position 93). The FGF2110 protein expression quality is closely related to the level of 0-linked glycosylation. Therefore, to solve the problem of protein quality, we wanted to further modify the FGF2110 by deleting the corresponding amino acid sequence by deleting the relevant gene sequence for the purpose of reducing the level of 0-linked glycosylation.

(b) Modifying the sequence of the protein recombinant gene. The amino acid sequence and the base sequence of the FGF2110 protein are downloaded from GeneBank, and the original FGF2110 gene is optimized and modified by applying a genetic engineering technology. In order to construct an engineered disulfide bond between Cys75 and Cys93, G and C in a triplet codon are changed into A and T on the premise of not changing Cys, a rare codon is replaced by an optimal preference codon, and a disulfide bond is constructed between Cys75 and Cys 93. In order to improve the expression quality of the FGF2110 protein, the FGF2110 gene is optimized and modified again, three amino acid sequences which have a large influence on the level of 0-linked glycosylation are found through peptide diagram identification and by combining related literature research results, EcoRI enzyme cutting sites and an initiation codon ATG are directly introduced into the gene, corresponding three gene sequences and a signal peptide gene are cut by using related endonucleases and are connected by using related ligases, and the optimized FGF2110 gene is obtained.

(b) And (3) constructing an FGF2110 expression vector. The full length of the FGF2110 recombinant gene is constructed on an expression vector of a CMV promoter, the His tag is carried on the vector, and pcDNF3.1 is taken as a vector. Designing a PCR primer: the 5' end primer comprises a restriction enzyme cutting site required by cloning and an upstream matching sequence containing an initiation codon ATG, and the length is about 26-30 bp; the 3' primer contains downstream sequence but does not contain stop codon, and HRV 3C Protease cleavage sequence is added to the end of the primer. The FGF21 recombinant gene is amplified by a PCR amplification method and purified and recovered. The concentration of the product recovered in the previous step was identified, and 1. mu.g of the product was digested. After cleavage, the DNA was ligated to pcDNA 3.

(c) And (4) purifying the protein. The step (c) further comprises: (c.1) prokaryotic escherichia coli expression and protein purification; the step (c) further comprises: (c.2) transient expression and protein purification of eukaryotic HEK293t cells.

(d) And (4) detecting the concentration and purity of the FGF21 protein.

Further described, the above steps may further comprise (e) the establishment of stable transfected cell lines with FGF 2110; the step (f) of lyophilization and preservation of the FGF2110 proteins. And (e) and (f) are selected according to the preparation and the use of the FGF2110 temperature-sensitive slow-release carrier.

Further described, the step (b) further comprises the steps of (b.1) finding three amino acid sequences which have a large influence on the level of 0-linked glycosylation, introducing an EcoRI cleavage site and an initiation codon ATG into the gene, excising the corresponding three gene sequences and signal peptide gene with endonuclease, and ligating them with ligase.

Another technical feature of the present invention is that the FGF2110 temperature-sensitive slow-release carrier may be modified by the use of an ideal carrier in addition to the FGF2110 protein. A hydrogel is a hydrophilic polymer with a three-dimensional structure that is characterized by liquid fluidity and solid stability to load and deliver Growth Factors (GFs) to the lesion area. With conventional dressings, the aim is to cover skin defects to protect them from secondary damage. In contrast to conventional dressings, hydrogels have many advantages such as pain relief, wound healing promotion, wound microenvironment improvement, and bacterial growth inhibition. Thus, the hydrogels are useful for treating various skin wound related conditions such as abrasions, scratches, and pressure sores.

The FGF2110 temperature-sensitive slow-release carrier comprises heparin-poloxamer 20, wherein the heparin-poloxamer 20 is a temperature-sensitive hydrogel micelle material, and the heparin-poloxamer 20 is often used for modifying GFs to realize a bridge of the combined activity of the heparin-poloxamer 20. In short, the heparin-poloxamer 20 is a negatively charged linear polysaccharide that can readily bind to the GFs domain, which contains lysine and arginine residues that are rich in positive charges. Such heparin-GFs binding not only contributes to GFs stabilization, but also enhances GFs binding affinity to cell surface receptors, thereby initiating more intracellular signaling pathways. Thus, in the heparin-poloxamer 20 based hydrogel, it is the most efficient way to bind different growth factors and cytokines through the heparin-poloxamer 20 binding domain to form a (multi-) functional complex. And the heparin-poloxamer 20 can improve the solubility and stability through a proper formula. The heparin-poloxamer 20 is in a liquid state at 4 ℃ due to the temperature sensitivity, and is applied to an affected part at this time, so that the wound surface can be perfectly covered due to the good fluidity; after being applied, the heparin-poloxamer 20 quickly reaches the body temperature of 37 ℃, and at the moment, the heparin-poloxamer 20 becomes solid and tightly covers the surface of the wound, so that the possibility of bacterial infection can be isolated, and the loss of the medicine can be reduced. While the heparin-poloxamer 20 enhances peripheral nerve regeneration, the heparin-poloxamer 20 conjugated basic fibroblast growth factor (bFGF) promotes regeneration and functional recovery after spinal cord injury. Therefore, the FGF2110 can achieve the purpose of slowly releasing the drug, so that the half-life of the drug is prolonged, the bioavailability of the drug is improved, and the stability of the drug can be improved.

As shown in figure 2, the invention also relates to a preparation method of the FGF21 temperature-sensitive slow-release carrier, which comprises the following steps:

(A) preparing the heparin-poloxamer 20 powder;

(B) dissolving the lyophilized heparin-poloxamer 20 and the lyophilized FGF2110 powder in fresh saline (4C) to obtain 170mg/mI original hydrogel solution and the FGF2110 solution; and

(C) and mixing the heparin-poloxamer 20 powder and the novel FGF21 freeze-dried powder, and storing the mixture in a refrigerator at 4 ℃ overnight to obtain a clear solution, namely the FGF21 temperature-sensitive slow-release carrier.

Further described, the step (a) further comprises:

(A.1) first reacting poloxamer 40730 with 1.3mM 4-nitrophenyl chloroformate and diaminoethylene 40 to obtain monoamine-terminated poloxamer;

(A.2) coupling the intermediate with heparin salt in 2- (N-morpholine) sulfonic acid buffer 50 at 25 ℃ for 1 day; and

(A.3) dialyzing the reaction temperature-sensitive slow-release carrier for 3 days and freeze-drying to obtain the heparin-poloxamer 20 freeze-dried powder.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.