CN118255862B - Recombinant humanized silk fibroin and preparation method and application thereof - Google Patents
Recombinant humanized silk fibroin and preparation method and application thereof Download PDFInfo
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- CN118255862B CN118255862B CN202410686199.1A CN202410686199A CN118255862B CN 118255862 B CN118255862 B CN 118255862B CN 202410686199 A CN202410686199 A CN 202410686199A CN 118255862 B CN118255862 B CN 118255862B
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- silk fibroin
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Abstract
The invention discloses a recombinant humanized silk fibroin and a preparation method and application thereof. In the recombinant humanized filaggrin, a humanized FLG protein fragment with a KIF polymerization function is obtained by mutating a proFLG protein fragment by at least one amino acid, wherein the mutation is that arginine is point mutated into lysine, the proFLG protein fragment comprises a plurality of basic tetrapeptides which can form a beta-turn structure and have the KIF polymerization function, and at least one of the basic tetrapeptides contains one or more arginines. The recombinant humanized silk fibroin has long-acting property, can be applied to the purposes of skin barrier damage repair, allergy relief of dry sensitive skin, repair and moisturizing, and symptom depth relief of atopic dermatitis and the like caused by silk fibroin deficiency, and can also be used for preparing dressing, skin external medicines, cosmetics, tissue engineering materials and silk fibroin sponge.
Description
Technical Field
The invention belongs to the biomedical field, and in particular relates to a recombinant humanized silk fibroin and a preparation method and application thereof.
Background
Atopic dermatitis (Atopic Dermatitis, AD) is a common recurrent chronic inflammatory skin disease, and has various clinical phenotypes and internal phenotypes, such as stinging, burning, erythema, itching, erosion, exudation, desquamation and other symptoms, and inflammation caused by overactivation of TH2 type immune response is one of the basic characteristics and key factors of AD, another key factor is abnormal epidermal cell differentiation, and the most common factor causing abnormal epidermal cell differentiation is related to filaggrin. It is currently accepted in the academy that Filaggrin (FLG) mutations are the most common factor in triggering AD. Filaggrin (FLG) mutations can cause filaggrin deficiency, thus causing problems with keratinocyte junctions, compromising skin barrier function, allowing the penetration of strain sources and microorganisms into the epidermis, and initiating Th 2-type inflammation; the Th2 inflammatory factor can further inhibit the expression of skin barrier related proteins including silk fibroin, so that the problem of damaged skin barrier is aggravated, AD is caused to become a common and recurrent chronic inflammatory skin disease, and great trouble is brought to patients. Current treatments or relief of atopic dermatitis symptoms are mainly directed to AD inflammation, and it is actually necessary to consider how to repair skin barriers to prevent penetration of strain sources and microorganisms into the epidermis, while it is necessary to solve the problem of filaggrin deficiency after filaggrin mutation in view of repairing skin barriers.
Filaggrin (FLG) is an important molecule in keratinocytes for connecting keratin intermediate filaments (KERATIN INTERMEDIATE FILAMENTS, KIF), and keratin fibers are regularly arranged and aggregated with the help of FLG monomers to participate in forming a stable desmosome structure, and form a physical barrier (skin barrier) on the outer layer of epidermis, so that water loss and invasion of strain sources and microorganisms are prevented. Filaggrin is not only an important protein involved in the corneocytes, but also maintains the intercellular connection of corneocytes, and its degradation products are essential components for maintaining the integrity of skin barrier and the hydration function of epidermis. In the granulosa layer cell, the expression product of the FLG gene is a non-active profilaggrin (proFilaggrin, proFLG), each individual proFLG contains 2 incomplete FLG monomer fragments and 10 complete FLG monomer fragments, the monomers are connected by a linker with an amino acid sequence of FLYQVST (or a homologous sequence), and in the process of converting the granulosa layer into the stratum corneum, functional FLG monomers (hereinafter referred to as FLGs) are generated after the linker sequence is subjected to dephosphorylation and protease cleavage. FLG interacts with charged residues of KIF through alternately arranged charges in a beta-turn structure and promotes the regular arrangement of the charged residues, and further research finds that FLG and KIF mainly form ionic salt bridges through positive and negative charge interactions and assist KIF to be regularly gathered into bundles, and meanwhile participate in the formation of desmosome structures, so that horny layer cells are firmly connected to form a 'brick-wall structure' of skin, the integrity and strength of horny layer are maintained, normal skin barrier is maintained, and invasion of strain sources and microorganisms is reduced. During the transformation of keratinocytes, arginine in the amino acid sequence of FLG is converted to citrulline to lose its positive charge under the catalysis of the peptide acyl arginine deiminase-1/3 (PEPTIDYLARGININE DEIMINASE-1/3, PAD-1/3), thereby allowing dissociation of FLG from KIF at the upper layer of the stratum corneum. The dissociated FLG is degraded with the participation of various enzymes, and the degradation products participate in the formation of natural moisturizing factors (Natural Moisturizing Factor, NMF). Therefore, when the skin barrier damage caused by FLG deficiency and skin problems such as AD caused by FLG gene mutation and the like are alleviated, the problem of long-term action under limited dosage needs to be considered for direct supplementation of FLG.
At present, the treatment of Atopic Dermatitis (AD) takes anti-inflammatory treatment as a main means, and long-term external or oral antihistamine, anti-inflammatory, glucocorticoid and other medicines for controlling inflammation and itch are needed, and skin cleaning products and moisturizers are used for assisting, so that the symptoms are reduced or lightened, the aim of improving the life quality of patients is fulfilled, but the treatment effect is large in difference, easy to repeat and often accompanied with different types of side effects. The existing direct supplementing technology aiming at the deficiency of FLG does not consider subcellular localization of FLG, only supplements the FLG by external use of skin, only plays the role of moisturizing the FLG, and cannot play the role of regular arrangement of the FLG after aggregation of KIF; some exogenously-supplemented FLG proteins have too large molecular weight, and the special structure of keratinocyte parts is not considered when the Cell-PENETRATING PEPTIDES, CPPS is designed, so that the membrane-penetrating efficiency is low, and the requirement of a large number of KIF regular arrangement cannot be met.
Disclosure of Invention
Aiming at the fundamental demand problem in the existing atopic dermatitis treatment, the inventors have conducted intensive studies. The invention innovatively proposes:
1. Selecting proFLG protein fragments which can form a beta-turn structure and have a KIF polymerization function, and improving the membrane penetration efficiency by moderately reducing the molecular weight of the protein under the condition of maintaining the functionality; the distribution and the duty ratio of the amino acid types of the selected proFLG protein fragment are basically consistent with those of the FLG complete monomer, so that the selected proFLG protein fragment can be degraded, enough amino acids are reserved for generating NMF, and the near natural proportion of each component of NMF is maintained;
2. The proFLG protein fragments are fragments among the FLG natural enzyme cleavage sites, and the sites participating in subsequent enzymatic degradation reaction of the FLG are reserved;
3. Because lysine is not catalyzed by PAD-1/3 enzyme to lose charge, under the premise of retaining possible Calpain-1 enzyme cleavage site, the selected proFLG protein fragment is modified, partial arginine is point mutated into lysine, so that the recombinant humanized silk fibroin retains positive charges and charge arrangement sequence on FLG natural site, positive and negative charge interaction between FLG and KIF is not influenced, and simultaneously positive charges provided by lysine can be retained for a longer time after PAD-1/3 enzyme reaction, thereby prolonging the action process with KIF and strengthening the stability of the epidermal desmosome structure of AD patients;
4. The Cell-PENETRATING PEPTIDES, CPPS, which is redesigned by taking the special structure of the keratinocyte part into full consideration, is taken as a Cell-penetrating carrier, and forms fusion protein with the proFLG protein fragment after modification, so as to guide the recombinant humanized silk fibroin to penetrate the Cell membrane, exert the KIF polymerization function and improve the skin barrier integrity and the epidermis hydration function of AD patients.
The invention solves the problem of the root demand in the treatment of atopic dermatitis, namely, the deficiency or lack of FLG is effectively supplemented, and the action progress of FLG and KIF is prolonged by modifying the key amino acid providing positive charge in FLG protein, thus providing more choices for improving the damage of skin barrier function of AD patients and various symptoms caused by the damage.
In order to achieve the purpose, the invention adopts the following technical scheme:
A recombinant humanized filaggrin, wherein a humanized FLG protein fragment with a KIF polymerization function in the recombinant humanized filaggrin is obtained by mutating at least one amino acid of a basic tetrapeptide which can form a beta-turn structure in a proFLG protein fragment and has the KIF polymerization function, wherein the amino acid is mutated from arginine point mutation to lysine;
The proFLG protein fragment comprises a plurality of basic tetrapeptides which can form a beta-turn structure and have a KIF polymerization function, and at least one of the basic tetrapeptides contains one or more arginines.
Those skilled in the art know that for the basic tetrapeptides of the present invention that do not contain arginine, which can form a beta-turn structure and have KIF polymerization function, the amino acid mutation from arginine point mutation to lysine is not generated.
In one embodiment of the invention, proFLG protein fragments are protein fragments between FLG monomer-related linker cleavage sites and Caspase-14 cleavage sites at the N-terminus or between Caspase-14 cleavage sites and FLG monomer-related linker cleavage sites at the C-terminus in human proFLG protein, or between two Caspase-14 cleavage sites, wherein the protein fragments comprise one or more basic tetrapeptides which can form a beta-turn structure and have a KIF polymerization function.
Preferably, a protein fragment with a high net positive charge at pH7.0 is selected, and more preferably a protein fragment with an amino acid type distribution and a ratio close to that of the FLG complete monomer.
In some preferred embodiments of the invention, the humanized FLG protein fragment in the recombinant humanized silk fibroin is obtained by modifying the p796 to p949 fragment (sequence shown in SEQ ID No. 2) of human proFLG protein. Human proFLG protein has a plurality of fragments with similar characteristics to the sequence shown in SEQ ID No. 2.
In one embodiment of the present invention, the humanized FLG protein fragment having KIF polymerization function in the recombinant humanized silk fibroin is obtained by mutating at least 1 amino acid in proFLG protein fragment having KIF polymerization function, which contains a β -turn structure, and the amino acid is mutated from arginine to lysine.
In one embodiment of the invention, the mutated amino acid position may be selected based on the amino acid sequence of the proFLG protein fragment.
In an embodiment of the present invention, the humanized FLG protein fragment is obtained by mutating a sequence shown in SEQ ID No.2, and the mutated amino acid positions may be at least 1 position selected from the group consisting of R22, R31, R35, R47, R54, R55, R57, R68, R84, R89, R95, R99, R102, R108, R112, R117, R129, R142, R145, and R146 in SEQ ID No. 2.
In a preferred embodiment of the invention, the arginine at the possible Calpain-1 cleavage site is not mutated.
In a preferred embodiment of the invention, arginine is avoided in consecutive mutations in the amino acid sequence at closely located positions.
In one embodiment of the present invention, the recombinant humanized silk fibroin comprises an amino acid sequence obtained by mutating a part of amino acids from the amino acid sequence represented by SEQ ID No.2, wherein the mutation site is selected from at least 1 site of R22, R31, R35, R47, R54, R55, R57, R68, R84, R89, R95, R99, R102, R108, R112, R117, R129, R142, R145, R146 in SEQ ID No.2, and the amino acid is mutated from arginine to lysine.
In some preferred embodiments of the invention, the sequence of the humanized FLG protein fragment in the recombinant humanized silk fibroin is shown in SEQ ID No. 3. The method can also be applied to a plurality of proFLG protein fragments, and good effects are obtained.
In one embodiment of the invention, the recombinant humanized silk fibroin carries a protein tag that is easy to purify, such as a His tag, an HA tag, a Flag tag, a GST tag, a Myc tag, or the like, or a combination of protein tags. In some embodiments, the N-terminus thereof carries a His tag.
In one embodiment of the invention, the recombinant humanized silk fibroin carries a transmembrane peptide having a function of penetrating a cell membrane. The penetrating peptide may be a known penetrating peptide, for example, considering that the epidermal cells include keratinocytes, it is preferable to be a penetrating peptide having an amino acid sequence shown in SEQ ID No. 1.
In one embodiment of the present invention, the transmembrane peptide may be located at the C-terminus of the recombinant humanized silk fibroin.
In one embodiment of the invention, a protein linker is used to link the transmembrane peptide to the remaining protein segments of the recombinant humanized silk fibroin, preferably the protein linker is a natural FLG inter-monomer linker segment.
In one embodiment of the invention, the amino acid sequence of the recombinant humanized silk fibroin is shown as SEQ ID No. 4.
The invention also provides a method for modifying proFLG protein fragments, wherein the proFLG protein fragments comprise a plurality of basic tetrapeptides which can form a beta-turn structure and have a KIF polymerization function, at least one of the basic tetrapeptides contains one or more arginines, and the method comprises the following steps:
One or more arginine points in the base tetrapeptides comprising arginine are mutated to lysine.
In an embodiment of the invention, the FLG protein fragment or proFLG protein fragment is a protein fragment with an N-terminal being a linker cleavage site between FLG monomers and a C-terminal being a Caspase-14 cleavage site, or is a protein fragment with an N-terminal being a Caspase-14 cleavage site and a C-terminal being a linker cleavage site between FLG monomers, or is a protein fragment between two Caspase-14 cleavage sites, and the protein fragment contains one or more basic tetrapeptides capable of forming a β -turn structure and having a KIF polymerization function.
Among them, a protein fragment having a high positive net charge at pH7.0 is more preferable, and a protein fragment having an amino acid type distribution and a ratio close to those of the FLG intact monomer is more preferable.
In some preferred embodiments of the invention, the p796 to p949 fragment of human proFLG protein (sequence shown as SEQ ID No. 2) is engineered. Human proFLG protein has a plurality of fragments with similar characteristics to the sequence shown in SEQ ID No. 2.
In one embodiment of the present invention, the mutated amino acid site may be selected according to the composition of the proFLG protein fragment, preferably, arginine is mutated to lysine based on the retention of positive charges and charge arrangement order on the natural site of FLG.
In one embodiment of the present invention, the amino acid position of the proFLG fragment (shown in SEQ ID No. 2) may be mutated at least 1 position selected from the group consisting of R22, R31, R35, R47, R54, R55, R57, R68, R84, R89, R95, R99, R102, R108, R112, R117, R129, R142, R145, R146 in SEQ ID No. 2.
In a preferred embodiment of the invention, the arginine at the possible Calpain-1 cleavage site is not mutated in the FLG protein or FLG protein fragment or proFLG protein or proFLG protein fragment.
In a preferred embodiment of the invention, arginine is avoided in consecutive mutations in the amino acid sequence at closely located positions.
In one embodiment of the present invention, a part of the amino acids in the proFLG fragment (shown in SEQ ID No. 2) is mutated at least 1 site selected from the group consisting of R22, R31, R35, R47, R54, R55, R57, R68, R84, R89, R95, R99, R102, R108, R112, R117, R129, R142, R145, R146 in SEQ ID No.2, and the amino acid is mutated from an arginine point to a lysine.
The method can also be applied to a plurality of proFLG protein fragments, and good effects can be obtained.
The invention also provides an application of the recombinant humanized silk fibroin in preparing a silk fibroin product.
In one embodiment of the invention, the silk fibroin product is selected from the group consisting of dressing, skin external drug, cosmetic, tissue engineering material, and silk fibroin sponge.
The invention also provides a composition containing the recombinant humanized silk fibroin, which contains the recombinant humanized silk fibroin.
In one embodiment of the invention, the composition comprising the recombinant humanized silk fibroin comprises at least about 0.01% recombinant humanized silk fibroin by weight to volume ratio (W/V, meaning the mass of solute per 100ml solution, the mass units of solute being g, the same applies below).
In example 4, 0.1-0.15% (W/V) of recombinant humanized silk fibroin was contained.
The invention also provides application of the recombinant humanized silk fibroin and a composition containing the recombinant humanized silk fibroin.
The reagents and materials used in the present invention are commercially available.
The invention has the positive progress effects that:
1. The recombinant humanized silk fibroin comprises CPPs designed aiming at the special structure of a keratinocyte part, the CPPs can guide the humanized FLG protein fragments which are fused and expressed with the CPPs to penetrate through cell membranes, so that the accurate subcellular localization of the recombinant humanized silk fibroin is realized, the function of FLG polymerization KIF is exerted, and the defect that FLG which is simply exogenously supplemented cannot penetrate through the cell membranes to reach an action site is avoided.
2. The humanized FLG protein fragment in the recombinant humanized silk fibroin is from proFLG, the fragment between the natural restriction sites of the FLG is selected, and the site involved in the subsequent enzymatic degradation reaction of the FLG is reserved. The selected proFLG protein fragment not only maintains the capability of combining with KIF, but also reduces the molecular weight of the protein and improves the membrane penetrating efficiency, so that a large number of recombinant humanized silk fibroin penetrate cell membranes to promote KIF polymerization, and further a stable bridged particle structure is formed to improve the damaged skin barrier function. The amino acid type distribution and proportion of the proFLG protein fragment are close to the proportion of FLG monomer, so that the type and proportion of NMF generated by degradation are close to natural state.
3. In the proFLG fragment selected in the description 2, part of arginine which is possibly not involved in Calpain-1 enzyme digestion is mutated into lysine which cannot be acted by PAD-1/3, and when the recombinant humanized silk fibroin is acted by PAD-1/3, more positive charges can be reserved for the lysine because the lysine is not acted by the PAD-1/3, so that the acting time of the recombinant humanized silk fibroin and KIF is prolonged, the long-time stability of the desmosome structure of keratinocytes of AD patients is promoted, and the skin barrier function is improved.
4. The humanized FLG protein fragment and CPPs in the recombinant humanized silk fibroin are connected by the FLG natural linker fragment, the FLG natural linker fragment can be identified and cut by intracellular protease to release the humanized FLG protein fragment with KIF polymerization function, and the influence of CPPs in the fusion protein on polymerization can be reduced.
Drawings
FIG. 1 is a diagram of agarose gel electrophoresis for double restriction identification of recombinant plasmids pET30a-Genemae-hFLG-1, pET30a-hFLG-w and pET30 a-hFLG-m; wherein, each lane is explained as follows:
FIG. 1-A: m: DNA electrophoresis Marker DL5000, 1-5: pET30a-Genemae-hFLG-1 recombinant plasmid sample No. 1-5.
Fig. 1-B: m: DNA electrophoresis Marker DL5000, 1-5: pET30a-hFLG-w recombinant plasmid samples No. 1-5.
Fig. 1-C: m: DNA electrophoresis Marker DL5000, 1-5: pET30a-hFLG-m recombinant plasmid samples No. 1-5.
FIG. 2 is a graph showing the results of partial sequencing of recombinant plasmids pET30a-Genemae-hFLG-1, pET30a-hFLG-w and pET30 a-hFLG-m.
FIG. 3 is a chart showing coomassie brilliant blue staining and Western Blot detection results for recombinant protein expression; wherein, each lane is explained as follows:
Fig. 3-a: the bacterium is BL21 (DE 3) escherichia coli transformed with pET30a-Genemae-hFLG-1 recombinant plasmid; c1: BSA (1 μg); c2: BSA (2 μg); m: protein electrophoresis markers; NC1: no bacterial lysate was induced; 1: IPTG induction for 16h bacterial lysate at 15 ℃;2: IPTG induction at 37℃for 4h of bacterial lysate; NC2: uninduced bacterial lysate supernatant; 3: IPTG induction of 16h bacterial lysate supernatant at 15 ℃;4: IPTG induction at 37℃for 4h of bacterial lysate supernatant; NC3, not inducing bacterial lysate to precipitate; 5: IPTG induced 16h bacterial lysate precipitation at 15 ℃;6: IPTG induction at 37℃for 4h of bacterial lysate precipitation.
Fig. 3-B: m: protein electrophoresis markers; 1: genemae-hFLG-1 nickel column to purify the sample.
Fig. 3-C: the bacterium is BL21 (DE 3) escherichia coli transformed with pET30a-hFLG-w recombinant plasmid; c1: BSA (1 μg); c2: BSA (2 μg); m: protein electrophoresis markers; NC1: no bacterial lysate was induced; 1: IPTG induction for 16h bacterial lysate at 15 ℃;2: IPTG induction at 37℃for 4h of bacterial lysate; NC2: uninduced bacterial lysate supernatant; 3: IPTG induction of 16h bacterial lysate supernatant at 15 ℃;4: IPTG induction at 37℃for 4h of bacterial lysate supernatant; NC3, not inducing bacterial lysate to precipitate; 5: IPTG induced 16h bacterial lysate precipitation at 15 ℃;6: IPTG induction at 37℃for 4h of bacterial lysate precipitation.
Fig. 3-D: m: protein electrophoresis markers; 1: phFLG-w Nickel column purification of samples.
Fig. 3-E: the bacterium is BL21 (DE 3) escherichia coli transformed with pET30a-hFLG-m recombinant plasmid; c1: BSA (1 μg); c2: BSA (2 μg); m: protein electrophoresis markers; NC1: no bacterial lysate was induced; 1: IPTG induction for 16h bacterial lysate at 15 ℃;2: IPTG induction at 37℃for 4h of bacterial lysate; NC2: uninduced bacterial lysate supernatant; 3: IPTG induction of 16h bacterial lysate supernatant at 15 ℃;4: IPTG induction at 37℃for 4h of bacterial lysate supernatant; NC3, not inducing bacterial lysate to precipitate; 5: IPTG induced 16h bacterial lysate precipitation at 15 ℃;6: IPTG induction at 37℃for 4h of bacterial lysate precipitation.
Fig. 3-F: m: protein electrophoresis markers; 1: phFLG-m Nickel column the samples were purified.
FIG. 4 is a graph showing the results of recombinant protein Co-IP experiments.
FIG. 5 is a graph showing the comparison of the effects of applying recombinant humanized silk fibroin and hyaluronic acid after laser spot removal.
Figure 6 is a graph of the results of measurements of the moisture content of the stratum corneum in the cheek test and control areas after use of the composition.
FIG. 7 is a graph showing the results of a percutaneous moisture loss assay in the cheek test and control areas after use of the composition.
Detailed Description
In order to make the contents of the present invention more easily understood, the technical scheme of the present invention will be further described with reference to the accompanying drawings and the detailed description, but the present invention is not limited thereto.
Example 1 design of recombinant proteins, construction of recombinant vectors, expression and identification of recombinant proteins
1. Design of recombinant proteins
In this example, based on the inter-monomer linker site and Caspase-14 cleavage site in human proFLG protein, the binding fragment was selected under conditions of pH7.0 for net charge number, possible cleavage site of Calpain-1, amino acid class classification and duty ratio, etc., and p796 to p949 protein fragment was designated hFLG-w and amino acid sequence was SEQ ID No.2. Under the premise of retaining possible Calpain-1 cleavage sites in SEQ ID No.2, replacing part of arginine in SEQ ID No.2 with lysine to obtain an improved humanized FLG protein fragment, which is named hFLG-m, wherein the amino acid sequence is SEQ ID No.3, and the specific mutation sites are: R22K, R35K, R54K, R68K, R84K, R108K, R129K, R146K in the sequence of SEQ ID No.2.
Recombinant humanized silk fibroin design:
His protein tag is added at N end of hFLG-m, FLG natural linker (SGSFLYQVSTHKQSES) fragment is used for connecting hFLG-m protein fragment and membrane penetrating peptide (SEQ ID No. 1) at C end to obtain recombinant humanized silk fibroin, which is named Genemae-hFLG-1 and has amino acid sequence of SEQ ID No.4.
The purpose of the experimental design of this example 1 was to verify:
1. Whether the penetrating peptide (the sequence is shown as SEQ ID No. 1) has the function of guiding penetrating;
2. whether the recombinant humanized silk fibroin has the ability to bind to KIF;
3. whether a partial arginine point mutation to lysine would result in an altered interaction of the recombinant humanized silk fibroin with KIF.
Based on this, two recombinant proteins, phFLG-w (comprising the human proFLG protein fragment) and phFLG-m (comprising the engineered humanized FLG protein fragment), were designed in this example 1:
his and Myc protein label combinations connected by a (G4S) 1 protein linker are added at the N end of hFLG-w, a hFLG-w protein fragment and a membrane penetrating peptide (the sequence is shown as SEQ ID No. 1) are connected at the C end by an FLG natural linker sequence (SGSFLYQVSTHKQSES), and a recombinant protein containing a human proFLG protein fragment is obtained, and is named phFLG-w, and the amino acid sequence is shown as SEQ ID No.5.
His and HA protein label combinations connected by (G4S) 1 protein linker are added at the N end of hFLG-m, a hFLG-m protein fragment and a transmembrane peptide (the sequence is shown as SEQ ID No. 1) are connected at the C end by an FLG natural linker (SGSFLYQVSTHKQSES) fragment, and a recombinant protein containing the modified humanized FLG protein fragment is obtained, and is named phFLG-m, and the amino acid sequence is SEQ ID No.6.
2. Construction of recombinant vectors
The E.coli expression vector was selected as pET30a, and recombinant vectors were constructed by selecting Nde I and Hind III restriction enzyme sites. After reverse translation of the amino acid sequences SEQ ID No.4, SEQ ID No.5 and SEQ ID No.6 into DNA sequences and substitution of partial codons into E.coli preference, adding a base CAT upstream of the sequences, forming Nde I cleavage site sequences with the codon ATG of the first amino acid methionine, adding termination codons TAA and Hind III cleavage site sequences downstream, obtaining corresponding DNA fragments named nGenemae-hFLG-1, nhFLG-w and nhFLG-m respectively, and synthesizing gene fragments artificially.
The empty vector pET30a and nGenemae-hFLG-1 fragments were double digested with Nde I and HindIII restriction enzymes, respectively, and the double digestion reaction system is shown in Table 1 below:
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After the double digestion system is incubated at 37 ℃ for 30 minutes, the restriction enzyme is inactivated after the double digestion system is incubated at 65 ℃ for 20 minutes, nGenemae-hFLG-1 fragment (system 1) and pET30a empty vector fragment (system 2) are respectively recovered through a Cycle Pure Kit, and then the two fragments are connected by T4 DNA LIGASE, and the reaction system is shown in the following table 2:
；
After the ligation system was incubated at 16℃for 12 hours, the ligation products were all transformed into E.coli DH 5. Alpha. Competent cells by heat shock, and the transformed competent cells were plated on kanamycin-resistant LB plates for overnight culture. After 16 hours of culture, 5 positive clones were picked and inoculated into liquid LB medium resistant to kanamycin, respectively, for overnight shake culture.
1Ml of plasmid was extracted from each of 5 overnight cultured bacterial cells in a small amount. The extracted 5 recombinant vector samples were subjected to double digestion identification, and the reaction conditions are shown in the following table 3:
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And carrying out agarose gel electrophoresis on 5 double enzyme digestion identification products, wherein the electrophoresis result is shown in a figure 1-A, an M lane is DNA MARKER DL < 5000 >, 1-5 lanes are respectively pET30a-Genemae-hFLG-1 recombinant plasmid samples No. 1-5, and the result shows that all 5 recombinant plasmids contain insertion fragments. The recombinant vector containing the insert was sequenced, and the sequencing results were aligned to show that the insert sequence was correct, and the partial sequencing results are shown in FIG. 2-A. The recombinant plasmid sequenced correctly was maintained and designated pET30a-Genemae-hFLG-1.
NhFLG-w and nhFLG-m are respectively used for constructing recombinant vectors with pET30a, the experimental scheme and the construction steps are the same as those of pET30a-Genemae-hFLG-1, and the recombinant plasmids are named pET30a-hFLG-w and pET30a-hFLG-m. The result of the double enzyme digestion identification agarose gel electrophoresis of pET30a-hFLG-w is shown as figure 1-B, the M lane is DNA MARKER DL5000, the 1-5 lanes are respectively pET30a-hFLG-w recombinant plasmid samples 1-5, the result shows that 5 recombinant plasmids all contain insertion fragments, the sequencing result is compared, the insertion fragment sequence is correct, and the partial sequencing result is shown as figure 2-B. The result of the agarose electrophoresis for the double enzyme digestion identification of pET30a-hFLG-M is shown as figure 1-C, the M lane is DNA MARKER DL5000, the 1-5 lanes are respectively pET30a-hFLG-M recombinant plasmid samples 1-5, the result shows that 5 recombinant plasmids all contain insertion fragments, the sequencing result is compared, the insertion fragment sequence is correct, and the partial sequencing result is shown as figure 2-C.
3. Expression and identification of recombinant proteins
Expression of recombinant humanized silk fibroin:
The pET30a-Genemae-hFLG-1 recombinant plasmid is transformed into BL21 (DE 3) competent cells by a heat shock method, and a monoclonal colony is obtained after kanamycin resistance plate screening.
BL21 (DE 3) monoclonal colonies were inoculated into 5ml of kanamycin-resistant liquid LB medium and cultured overnight at 37℃under shaking at 250 RPM. The overnight culture broth was inoculated at a ratio of 1:50 into 2 shake flasks containing 50ml of kanamycin-resistant fresh medium, shake-cultured at 37℃until OD 600 was about 0.6, and 1ml of broth was aspirated and stored as a control group for uninduced expression, and then the inducer IPTG was added to the shake flasks to a final concentration of 0.5mM. The 2 shake flasks were induced to express at 15℃for 16 hours and 37℃for 4 hours, respectively, at 200RPM.
Identification of recombinant humanized silk fibroin:
After the induction expression was completed, 450. Mu.l of the non-induced control group, the induced group bacterial liquid at 15℃and 37℃were aspirated, and 300. Mu.l of the lysate was added and subjected to ultrasonic lysis for 10 minutes. 100 μl of the lysate was aspirated and stored as whole cell lysate. 200 μl of the cell lysate was taken and centrifuged at 15000RPM for 10 minutes, the supernatant was the cell lysate supernatant, and the pellet was the cell lysate pellet. And respectively adding a proper amount of 5 Xloading buffer solution into the whole cell lysate, the cell lysate supernatant and the cell lysate sediment, uniformly mixing, heating at 100 ℃ for 5 minutes, and centrifuging at 15000RPM for 5 minutes to obtain an SDS-PAGE detection sample. Mu.l of whole cell lysate sample, cell lysate supernatant sample and cell lysate pellet sample were separately aspirated and added to SDS-PAGE gel wells for electrophoresis, as shown in FIG. 3-A, and the gel was stained with Coomassie Brilliant blue to show that E.coli can express recombinant humanized silk fibroin (indicated by the arrow in the figure). After recovering the recombinant humanized silk fibroin by using a nickel column, the primary antibody is a mouse anti-His tag antibody by Western Blot detection, and clear bands can be seen as shown in the figure 3-B, which shows that the recombinant humanized silk fibroin Genemae-hFLG-1 is successfully purified.
The protocols for phFLG-w and phFLG-m protein expression and identification were identical to those of recombinant humanized silk fibroin Genemae-hFLG-1. phFLG-w is expressed by Escherichia coli and the result of Western Blot detection of the recovered sample purified by the nickel column is shown in FIG. 3-C (the position indicated by the arrow in the figure). phFLG-m is expressed by Escherichia coli and the result of Western Blot detection of the recovered sample purified by the nickel column is shown in FIG. 3-F.
Example 2
FLG is known to bind to KIF assembled from keratin 1/10 (KRT 1/10) in keratinocytes, and thus target proteins phFLG-w and phFLG-m that bind to KIF were detected using KRT1 as a decoy protein.
Keratinocytes were cultured in 6-well plates, phFLG-w (set as control group) and phFLG-m (set as experimental group) proteins purified in example 1 were taken in the same number of moles, and after Co-culturing with keratinocytes in 6-well plates at 37℃for 1 hour, the cells were lysed on ice with a non-denatured lysate containing a protease inhibitor, and the supernatant was centrifuged and the total protein concentration of the supernatant was measured for Co-IP experiments.
The specific experimental procedure for Co-IP was as follows:
Supernatant with the same total protein amount of the control group and the experimental group is taken as an Input positive control group, and the experimental process is as follows: and adding a proper amount of 5X loading buffer solution into protein supernatant of the control group and the experimental group, uniformly mixing, heating at 100 ℃ for 5 minutes, adding the samples into SDS-PAGE gel for electrophoresis, and performing transfer after the electrophoresis is finished. After the transfer, myc tag (i.e., phFLG-w protein), HA tag (i.e., phFLG-m protein), KRT1 and beta-actin protein levels were detected with Myc tag, HA tag, KRT1 and beta-actin antibodies, respectively, as shown in FIG. 4: in the Input positive control, the brightness of the beta-actin strips of the internal reference proteins of the experimental group and the control group are consistent, the result shows that the two groups have the same sample loading quantity, the KRT1 strip is visible in the experimental group and the control group, the anti-Myc strip (phFLG-w protein) is visible in the control group, the anti-HA strip (phFLG-m protein) is visible in the experimental group, and the brightness of the two strips is consistent, and the result shows that: 1. the membrane penetrating peptide can effectively guide the recombinant protein to penetrate through cell membranes; 2. arginine point mutation and protein tag are different, so that the membrane penetrating efficiency of two recombinant proteins is not affected.
KRT1 is taken as bait protein, target proteins phFLG-w and phFLG-m are detected, and the experimental process is as follows: supernatants of the same total protein mass as the control and experimental groups were taken separately, and equal amounts of KRT1 antibody-conjugated beads were added to each and the mixture was incubated with slow shaking at 4℃for 2 hours. After incubation, the beads-antigen-antibody complex was collected by centrifugation and washed. After washing, adding a proper amount of 2X loading buffer solution into the complex solution, uniformly mixing, heating at 100 ℃ for 5 minutes, centrifuging the sample, taking supernatant, adding the supernatant into SDS-PAGE gel for electrophoresis, and carrying out transfer after the electrophoresis is finished. After the transfer, protein levels of KRT1, myc tag (i.e., phFLG-w protein) and HA tag (i.e., phFLG-m protein) were detected with KRT1, myc tag and HA tag antibodies, respectively. As can be seen from the KRT1-IP results in FIG. 4, the anti-HA band was brighter than the anti-Myc band, which indicated that phFLG-m, which had some arginine point mutations to lysine, bound more to KIF than phFLG-w.
Combining the above experimental results with the property that lysine cannot be catalyzed by PAD-1/3 to lose positive charge, the Co-IP experimental results show that: compared with the human proFLG protein fragment, the binding time of the humanized FLG protein fragment after partial arginine point mutation to lysine and KIF can be prolonged.
EXAMPLE 3 recombinant humanized silk fibroin alone application
The recombinant humanized silk fibroin is found in a single application test, and has obvious effects on reducing scab formation and scab falling and wound healing time after laser speckle removal operation.
In this example, the test subjects rubbed 0.5% (W/V) aqueous hyaluronic acid with the left cheek as the control group and 0.25% (W/V) aqueous recombinant humanized silk fibroin with the right cheek as the test group after performing the pulse laser spot removal operation. The skin burning pain of the test subjects was lost 2 days after the operation, and the control subjects were lost 4 days after the operation. Results fig. 5 shows that the test group had significantly shorter scab, scab shedding and wound healing times than the control group. After the cheek on the two sides is scabbed and falls off, the skin is free from pigmentation, hyperplasia and the like.
EXAMPLE 4 use of recombinant humanized silk fibroin in compositions
The present example provides the use of a composition having a profound relief of AD symptoms and an anti-allergic, soothing dry sensitive skin effect.
1. Raw materials
The recombinant humanized silk fibroin prepared in example 1; deionized water is prepared; sodium hyaluronate is commercially available; glycerol is commercially available; butanediol is commercially available; carbomers are commercially available; triethanolamine is commercially available; 1, 2-pentanediol is commercially available; 1,2 hexanediol is commercially available.
2. Preparation method
Adding the raw materials according to the proportion of 0.02-0.1% of recombinant humanized silk fibroin, 0.5-2.5% of sodium hyaluronate, 10-20% of glycerin, 3-8% of butanediol, 0.2-1.0% of carbomer, 0.5-2.0% of 1, 2-pentanediol and 0.8-2.5% of 1, 2-hexanediol into deionized water, uniformly stirring, regulating the pH value of the solution to 6.2 by using triethanolamine, and continuously stirring the solution at proper temperature and rotating speed to obtain the required water-in-oil composition solution I; mixing the sodium hyaluronate solution with the recombinant humanized silk fibroin solution, and stirring at a proper temperature and rotating speed to ensure that the sodium hyaluronate wraps the recombinant humanized silk fibroin to prepare the required water-in-oil composition solution II. Finally, mixing the water-in-oil composition solution I and the solution II in proportion, and continuing to stir uniformly to ensure that the final concentration of the recombinant human silk fibroin is 0.1-0.15% (W/V), thus obtaining the final composition.
3. Test method
In order to determine the efficacy of the resulting composition in alleviating AD symptoms and anti-allergic soothing dry sensitive skin, the most annoying dry skin and sensitive itching symptoms of AD patients were considered, and dry skin skinned subjects were selected in this example using the lactic acid stinging test as the determination method. The lactic acid stinging test method comprises the following steps: at room temperature, 50 μl of 10% aqueous lactic acid solution was applied to one cheek of the face after cleaning and drying, and subjective symptoms of the subjects were asked at 30s, 2.5min, and 5min, respectively, and scored by a score 4 method (0 for no tingling sensation, 1 for mild tingling, 2 for moderate tingling, and 3 for heavy tingling). Then the fractions of 2.5min and 5min are added, and the total fraction is more than or equal to 3 and the lactic acid stinging reaction is positive. Skin moisture content and transcutaneous moisture loss (TEWL) measurements were performed simultaneously and recorded.
1. Subject inclusion criteria: (1) 37 healthy Chinese females 20-40 years old with dry skin and sensitive itching; (2) The non-pregnancy, non-lactation, no major underlying diseases and immune system diseases, no allergic history; (3) Hormonal drugs and immunosuppressive drugs have not been used for nearly 3 months; (4) disabling any cosmetic product 3 days before the start of the test; (5) Willing to participate in the test, the test scheme can be strictly adhered to and follow-up can be completed.
2. Test composition: the composition obtained in example 4.
3. Test procedure: (1) In the test, the left cheek of the subject was numbered as region a, and the right cheek of the subject was numbered as region B. According to the random number table, the test area of different subjects can be an A area or a B area, and after the test area is selected, the other side is a control area. (2) After the subjects were instructed to clean the face, 50 μl of 10% aqueous lactic acid was applied to the test area, and the subjects were asked to feel consciously about the test area at 30s,2.5min,5min, respectively, and scored by a score 4 method (score 0 for no tingling, score 1 for light tingling, score 2 for moderate tingling, score 3 for heavy tingling), and then the scores at 2.5min,5min were added, with the total score being 3 or more positive for the lactic acid tingling test. (3) The subject was instructed to wipe the composition from example 4 above on the test area and 0.5mL deionized water on the control area; (5) The subjects rubbed the aforementioned composition once a day at night in each designated test area and 0.5mL deionized water in the control area over the following 28 days; (6) Subjects performed skin stratum corneum moisture content measurements, transdermal moisture loss (TEWL) and lactic acid stinging tests before, after, 7, 14, 21 and 28 days, respectively, and recorded the results; (7) collating test data and performing statistical analysis.
4. Test results
The test was performed for 28d, during which 2 persons were released from the group, and 35 persons completed the test. The data obtained were statistically analyzed using SPSS11.0 software and data were averaged ± standard deviationThe comparison of the two sets of data indicated that P <0.05 was a significant level of difference using the t-test.
As shown in Table 4, after the cheek application composition, the lactic acid stinging test score of the test group was significantly reduced compared with that of the control group, the moisture content of the skin horny layer of the test group was significantly higher than that of the control group as shown in FIG. 6, the percutaneous water loss of the test group was significantly lower than that of the control group as shown in FIG. 7, and the difference was statistically significant. The result shows that the skin dryness sensitivity is reduced, the skin barrier is enhanced, and the composition has the effects of deeply relieving the dryness sensitivity symptoms and the allergy skin resistance and the relaxation of AD patients, and can effectively enhance the skin barrier:
；
Note that: * P <0.05 is indicated with statistical differences.
The above is merely an example of the present application, and the technology disclosed in the present application is applied to a plurality of proFLG protein fragments to find an excellent effect, so the present application is not limited to the field of the present application, and specific structures and characteristics of common knowledge known in the art are not described in any way herein, so a person of ordinary skill in the art knows all the prior art in the field before the date of application or priority, and has the ability to apply the conventional experimental means before the date, and a person of ordinary skill in the art can complete and implement the present application in combination with its own ability, and some typical known structures or known methods should not be an obstacle for a person of ordinary skill in the art to practice the present application, given the present application. It should be noted that modifications and improvements can be made by those skilled in the art without departing from the structure of the present application, and these should also be considered as the scope of the present application, which does not affect the effect of the implementation of the present application and the practical applicability of the present application. The protection scope of the present application is subject to the content of the claims, and the description of the specific embodiments and the like in the specification can be used for explaining the content of the claims.
Claims (7)
1. A method of engineering proFLG protein fragments, wherein said proFLG protein fragments comprise a plurality of base tetrapeptides having a β -turn structure and a KIF polymerization function, at least one of said base tetrapeptides comprising one or more arginines, said method comprising:
mutating one or more arginine points in the base tetrapeptide comprising arginine to lysine;
The amino acid sequence of the proFLG protein fragment is shown as SEQ ID No. 2;
The amino acid sequence of the reconstructed proFLG protein fragment is shown as SEQ ID No. 4.
2. A recombinant humanized filaggrin, wherein a humanized FLG protein fragment with KIF polymerization in the recombinant humanized filaggrin is obtained by mutating a proFLG protein fragment to lysine using the method of claim 1, wherein the amino acid is mutated from arginine to lysine;
the amino acid sequence of the recombinant humanized silk fibroin is shown as SEQ ID No. 4.
3. The recombinant humanized silk fibroin of claim 2, wherein the amino acid sequence of the proFLG protein fragment is set forth in SEQ ID No. 2.
4. The recombinant humanized silk fibroin of claim 3, wherein the amino acid sequence of the humanized FLG protein fragment is set forth in SEQ ID No. 3.
5. The recombinant humanized silk fibroin of any one of claims 2-4, wherein the recombinant humanized silk fibroin carries a transmembrane peptide, the transmembrane peptide is linked to the remaining protein fragments of the recombinant humanized silk fibroin by a protein linker, and the amino acid sequence of the transmembrane peptide is shown in SEQ ID No. 1.
6. Use of a recombinant humanized silk fibroin according to any one of claims 2-5 for the preparation of a silk fibroin product.
7. The use according to claim 6, wherein the silk fibroin product is selected from the group consisting of dressing, skin external medicine, cosmetics, tissue engineering materials, and silk fibroin sponge.
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