Recombinant humanized silk protein and application thereof

Abstract

The invention discloses a recombinant humanized sericin and application thereof. The recombinant humanized sericin contains a humanized LEKTI protein fragment, and the humanized LEKTI protein fragment contains a humanized Kazal motif protein sequence as shown in SEQ ID No.2; the humanized LEKTI protein fragment is characterized by further containing a humanized FLG protein fragment, and the humanized FLG protein fragment is connected with the humanized LEKTI protein fragment through a linker containing a furin restriction enzyme cutting site. After the recombinant humanized silk protein is cut by intercellular gap furin protease, the two protein fragments perform respective functions. The recombinant humanized silk protein has the prospect of being developed into medicines, cosmetics or medical instruments for treating diseases treated or relieved by supplementing FLG and / or inhibiting kallikrein KLK5, and the diseases treated or relieved by supplementing FLG and / or inhibiting KLK5 comprise atopic dermatitis, internal sephton syndrome and tumors with high KLKs expression.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and relates to recombinant proteins, and in particular to a recombinant humanized filaggrin protein and a use thereof. Background Art

[0002] Atopic dermatitis (AD) is a common chronic inflammatory skin disease characterized by recurrent attacks, often accompanied by symptoms such as itching, dry skin, and desquamation. There are many causes of AD, among which filaggrin (FLG) mutation is one of the important factors. The precursor of FLG is profilaggrin (proFLG) expressed in the granular layer. After dephosphorylation and enzymatic cleavage, multiple functional FLG monomers are obtained. During the differentiation of keratinocytes into keratinocytes, the cell membrane is gradually replaced by the cornified envelope (CE). FLG forms an isopeptide bond with loricrin under the catalysis of transglutaminase (TGM) and participates in the formation of CE. FLG is a key molecule for the polymerization of keratin intermediate filaments (KIF) in keratinocytes. FLG assists KIF in regular arrangement and aggregation into bundles through salt bridges. With the participation of FLG, stable and firm CE and regularly bundled KIF are formed, forming the basis of the desmosome structure between corneocytes, making the corneocytes tightly connected to form a skin barrier, preventing the entry of allergens, microorganisms, environmental pollutants, etc. and the loss of water; when FLG dissociates from KIF, it is cut and degraded by various enzymes such as Caspase14, PAD-1, BLMH, and finally forms about 50% of the natural moisturizing factor (NMF). When FLG loses its biological function due to mutation, it will affect the structure of CE and desmosomes, thereby destroying the skin barrier function, leading to the entry of allergens, microorganisms, environmental pollutants, etc. and the loss of water.

[0003] Under physiological conditions, keratinocytes continuously shed from the surface of the stratum corneum, a process that proceeds in an orderly manner under the regulation of kallikreins (KLKs) and their inhibitors. KLK5 is a key factor in the activation of multiple KLKs. ProKLK5 (KLK5 precursor) is converted into active KLK5 through self-activation, and then activated KLK5 activates proKLK7 (KLK7 precursor) and proKLK14 (KLK14 precursor). Activated KLK14 can activate proKLK5 to further increase the enzyme activity of KLK5. KLK5 is the initiator molecule for the activation of multiple KLKs. KLK5 and KLK14 hydrolyze desmosomes through their trypsin activity, and KLK7 hydrolyzes desmosomes through its chymotrypsin activity, causing keratinocytes to separate and shed from the surface of the stratum corneum. The enzymatic activities of KLK5, KLK7, and KLK14 are strictly regulated by the serine protease inhibitor Kazal-type 5 (SPINK5, also known as LEKTI, hereinafter referred to as LEKTI), which balances the rate of stratum corneum shedding with the rate of keratinocyte proliferation and differentiation to maintain normal stratum corneum thickness and skin barrier function. The LEKTI protein has 15 Kazal-type protease inhibitor domains. Except for the second and fifth domains that form three pairs of disulfide bonds, the remaining domains form two pairs of disulfide bonds. The sequences of Kazal motifs between different domains are highly conserved. There are furin protease cleavage sites between multiple domains of LEKTI. Under the action of furin protease, multiple fragments containing single or multiple domains are generated, which inhibit the enzymatic activity of KLKs and ultimately regulate the rate of stratum corneum shedding. There are also TGM1 action sites on multiple domains, which bind to CE under the action of TGM1.

[0004] Thymic stromal lymphopoietin (TSLP) is a key molecule that initiates Th2 inflammation. Inhibiting KLK5 activity can downregulate TSLP expression and reduce Th2 inflammation. Th2 inflammation is a basic feature of AD. Due to FLG and LEKTI mutations, the skin barrier function is destroyed. After the invasion of allergens, microorganisms, and environmental pollutants, TSLP is promoted in keratinocytes through multiple signaling pathways. TSLP induces CD4 +T cells differentiate into T helper 2 cells (Th2 cells) that express pro-inflammatory factors such as IL-4, IL-5, and IL-13. IL-4 and IL-13 can promote the expression of KLK5 and KLK7, while inhibiting the expression of proteins such as FLG, Caspase14, LEKTI, and DSG1 that are involved in the formation of barrier function. The reduction of FLG and Caspase14 not only further destroys the barrier function, but also reduces the FLG metabolites that maintain the weak acidity of the epidermis. The increase in epidermal pH increases the activity of KLKs. With the upregulation of KLKs expression and activity, TSLP is upregulated by activating PAR2, and positive feedback promotes the differentiation of Th2 cells. IL-4 secreted by Th2 cells can not only promote the proliferation of Th2 cells themselves, but also induce B cells to produce IgE, and promote Th2 cells to express IL-5 and IL-13 through DC cells. IL-4 and IL-13 can directly stimulate sensory neurons to produce itching. Due to the up-regulation and increased activity of KLK5 and KLK7, and the down-regulation of LEKTI, desmosomes are over-cut and produce obvious desquamation. Itching and desquamation cause AD patients to scratch the affected area, and scratching as a physical stimulus can also promote TSLP expression. In summary, skin barrier damage, inflammation and scratching interact with each other in a positive feedback loop, accelerating the progression of AD and may lead to chronic AD. By inhibiting KLKs and then inhibiting the expression of TSLP, the AD initiating molecule, desquamation can be reduced, skin barrier repair can be promoted, and ultimately the symptoms of AD can be alleviated.

[0005] Netherton Syndrome (NS) is another LEKTI-related disease, which is caused by a rare and severe autosomal recessive genetic disease caused by a mutation in the SPINK5 gene. The clinical features of NS are congenital ichthyosis-like erythroderma, convoluted linear ichthyosis, bamboo-like hair, and atopic constitution (Atopic Diathesis) with high IgE. Most patients are accompanied by Th2-type inflammation. In NS, due to gene mutations, LEKTI completely loses its inhibitory effect on KLKs, and the disease can occur in the neonatal period. Children develop systemic congenital ichthyosis-like erythroderma within a few weeks after birth, accompanied by fine scales. As the onset time prolongs, the erythroderma gradually develops into convoluted linear ichthyosis, accompanied by dry skin, itching and desquamation. Due to severe damage to the skin barrier function, patients are prone to repeated skin infections, severe dehydration, hypernatremia, hypothermia, and even sepsis, which is life-threatening. Due to the high activity of KLKs and damage to the skin barrier, the levels of various inflammatory factors and IgE in patients are significantly increased, making patients susceptible to allergies, asthma and other diseases.

[0006] KLKs are also involved in the occurrence, development, invasion and metastasis of various tumors. High expression of one or more KLKs has been found in pancreatic cancer, cervical cancer, endometrial cancer, esophageal adenocarcinoma, gastric cancer, ovarian cancer, breast cancer, prostate cancer, lung cancer and other tumors. KLKs regulate the cohesion of tumor cells by degrading intercellular junction proteins (such as DSG and DSC) and increase the invasion and metastasis of tumor cells by degrading adhesion proteins (such as integrins and cadherins). KLKs can also reconstruct the extracellular matrix (ECM). KLKs change the composition of ECM by degrading ECM protein substrates and release ECM-bound proteins such as bound epidermal growth factor (bEGF), bound fibroblast growth factor (bFGF), bound vascular endothelial growth factor (bVEGF) to become soluble growth factors such as sEGF, sFGF, sVEGF, etc. to promote tumor growth. KLKs can also affect the tumor microenvironment and promote tumor development by activating the protease hydrolysis system. Typical proteases such as matrix metalloproteinases (MMPs) and urokinase plasminogen activator (uPA) are activated by a variety of KLKs including KLK7. KLKs are key molecules in many cancers, so the development of inhibitors against KLKs has a very positive significance for controlling tumor occurrence, development, invasion and metastasis.

[0007] Currently, the treatment plans for AD and NS are different, but due to the type of inflammation and the characteristics of skin lesions, traditional treatments mostly use topical moisturizers, glucocorticoids, calcineurin inhibitors, etc., in order to control symptoms such as inflammation, itching, and desquamation. However, there are problems such as large individual differences in treatment effects, large side effects, and easy recurrence after drug withdrawal. The existing treatment plans do not provide effective solutions for damaged CE structure and excessive KLKs activity. In tumor treatment, although KLKs are known to be involved in the occurrence, development, invasion, and metastasis of various tumors, there is still no efficient solution to inhibit KLKs. Summary of the invention

[0008] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a recombinant humanized filaggrin and its use.

[0009] The above-mentioned purpose of the present invention is achieved through the following technical solutions: A recombinant humanized filaggrin protein, wherein the recombinant humanized filaggrin protein contains a humanized LEKTI protein fragment, and the humanized LEKTI protein fragment contains a humanized Kazal motif protein sequence of formula I.

[0010] Formula I: C-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-Xaa7-Xaa8-Xaa9-Xaa 10 -Xaa11 -Xaa 12 -Xaa 13 -Xaa 14 -C-Xaa 16 -R / K-Xaa 18 -Xaa 19 -D / E-Xaa 21 -Xaa 22 -Xaa 23 -Xaa 24 -Xaa 25 -Xaa 26 -Xaa 27 -Xaa 28 -Xaa 29 -Xaa 30 -Xaa 31 -Xaa 32 -Xaa 33 -C-Xaa 35 -Xaa 36 -C.

[0011] In Formula I, Xaa n Indicates amino acid numbered n, Xaa n Has the following meanings: Xaa2 is: H, D, S, K, A, R, L, M, I, or N; Xaa3 is: E, D, A, Q, H, P, Y, S, R, or N; Xaa4 is: F or Y; Xaa5 is: R, Q, K or E; Xaa6 is: A, K, P, N, D, H, R, E, S, V, T, Q, L, Y, F, M, I or W; Xaa7 is: F, G, Q, L, S, Y, or M; Xaa8 is: M, E, V, A, R, L, Q, I, P, T, W, D, Y, N, F or H; Xaa9 is: K, R, Q, E, P, I, A, V, L, or T; XA 10 is: N, D or I; XA 11 Yes: G or N; XA 12 is: K, D, R, I, T, E, Y, or V; XA 13 is: L, F or Y; XA 14 is: F, I, G, A, P, S, N, L, H, W, Y, V, T, or missing; XA 16 is: T, P, D, N, E, A, V, S or Q; XA 18 is: E, D, Y, R, S, G, P, N, T, M, W, H, L, V, F, or I; XA 19 is: N, K, Y, S, H, L, T, G, R, M, V, I, F, W, E, Q, A, or D; XA 21 is: P, F or A; XA 22 is: V, I or F; XA 23 is: Q, C, L, R, E, H, T, F, V, W, Y, I, or N; XA 24 is: G, S, D, W or F; XA 25 is: P, L, T, A or D; XA 26 Yes: D or Y; XA 27 Yes: G; XA 28 is: K, I, R, Q, V, or M; XA 29 is: M, T, V, I, T, S, or F; XA 30 is: H, F, Y or W; XA 31 is: G, I, D, N, T, E, F, R, S, L, Y, H, V, P, W, A, K, or Q; XA 32 Yes: N; XA 33 is: K, R, L, T, Q, P, W, I, M, E, V, or F; XA 35 is: S, A, T, Y, M, N, D, K, L, R, Q, or E; XA 36 Yes: M, T or L.

[0012] Preferably, the sequence of the humanized Kazal motif protein fragment is shown as SEQ ID No.2.

[0013] Preferably, the amino acid sequence of the humanized LEKTI protein fragment is shown in SEQ ID No. 5. By combining the humanized Kazal motif protein fragment and the protein fragments on both sides thereof, the humanized LEKTI protein fragment is obtained, which not only improves the stability of the recombinant humanized filaggrin, but also improves its inhibitory effect on KLKs. By mutating the TGM1 action site in the humanized Kazal motif protein fragment and / or the protein fragments on both sides thereof, the freeness of the humanized LEKTI protein fragment is increased without reducing the inhibitory effect on KLKs.

[0014] Preferably, the recombinant humanized filaggrin also contains a humanized FLG protein fragment, which is formed by connecting multiple human proFLG protein fragments with amino acid sequences such as those shown in SEQ ID No. 1. A functional fragment that binds to Loricrin in human proFLG is selected, and the humanized FLG protein fragment is obtained by appropriately repeating the functional fragment to supplement the FLG functional fragment involved in CE assembly, improve the insufficient CE components caused by FLG mutation and the resulting unstable CE structure, and ultimately improve the skin barrier function.

[0015] Preferably, the recombinant humanized filaggrin further comprises a linker (hereinafter referred to as linker) for connecting the humanized LEKTI protein fragment and the humanized FLG protein fragment, and the linker comprises a furin cleavage site. A fragment comprising the natural furin cleavage site of the wild-type human LEKTI protein is selected as a linker of the humanized LEKTI protein fragment and the humanized filaggrin fragment, so that the recombinant humanized filaggrin can be cleaved by the protease furin in the epidermis, releasing the LEKTI protein fragment that has an inhibitory effect on KLKs and the humanized filaggrin fragment that participates in CE assembly.

[0016] More preferably, the amino acid sequence of the linker is as shown in SEQ ID No.6.

[0017] More preferably, the humanized FLG protein fragment is formed by connecting three human proFLG protein fragments shown in SEQ ID No.1 to form a recombinant humanized filaggrin having an amino acid sequence as shown in SEQ ID No.7.

[0018] More preferably, the humanized FLG protein fragment is formed by connecting 5 human proFLG protein fragments shown in SEQ ID No.1 to form a recombinant humanized filaggrin with an amino acid sequence as shown in SEQ ID No.8.

[0019] More preferably, the humanized FLG protein fragment is formed by connecting 7 human proFLG protein fragments shown in SEQ ID No.1 to form a recombinant humanized filaggrin with an amino acid sequence as shown in SEQ ID No.9.

[0020] Use of any of the above-mentioned recombinant humanized filaggrin for preparing drugs, cosmetics or medical devices for treating diseases that are treated or alleviated by supplementing FLG and / or inhibiting kallikrein KLK5.

[0021] Preferably, the diseases treated or alleviated by supplementing FLG and / or inhibiting kallikrein KLK5 include atopic dermatitis, Netherton syndrome and tumors with high expression of KLKs.

[0022] Beneficial effects: Those skilled in the art know that KLKs, including KLK5, play an important role in diseases such as atopic dermatitis, Netherton syndrome, and tumors with high expression of KLKs. LEKTI can treat or alleviate related diseases by regulating KLK5. The recombinant humanized filaggrin provided by the present invention contains a humanized LEKTI protein fragment, which contains a humanized Kazal motif protein sequence shown in SEQ ID No. 2. Its inhibitory strength against KLK5 is more than 3 times that of the recombinant human wild-type LEKTI protein shown in SEQ ID No. 5 (see Figures 14 to 17 IC50 value).

[0023] The recombinant humanized filaggrin also contains a humanized FLG protein fragment, which is connected to the humanized LEKTI protein fragment through a linker containing a furin cleavage site. The humanized FLG protein fragment is released after being cleaved by the intercellular furin protease, and exerts the function of FLG as introduced in the background technology, such as combining with CE under the action of the intercellular TGM enzyme to enhance the strength of CE, and ultimately enhancing the skin barrier function and reducing water loss and allergen invasion.

[0024] Therefore, the recombinant humanized filaggrin provided by the present invention has the prospect of being developed into a drug, cosmetic or medical device for treating diseases that are treated or alleviated by supplementing FLG and / or inhibiting kallikrein KLK5. Diseases that are treated or alleviated by inhibiting kallikrein KLK5 include atopic dermatitis, Netherton syndrome and tumors with high expression of KLKs. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is the partial sequencing result of the recombinant vector pET30a-n_rhFLG_1#; Figure 2This is the partial sequencing result of the recombinant vector pET30a-n_rhFLG_2#; Figure 3 This is the partial sequencing result of the recombinant vector pET30a-n_rhFLG_3#; Figure 4 This is the partial sequencing result of the recombinant vector pET30a-n_hLEKTI_wt; Figure 5 This is the SDS-PAGE Coomassie Brilliant Blue staining of the recombinant humanized filaggrin rhFLG_1# expression; where: M1: protein electrophoresis marker; PC1: BSA (1μg); PC2: BSA (2μg); NC: uninduced bacterial lysate; 1: bacterial lysate induced overnight at 20℃; 2: bacterial lysate induced at 37℃ for 4h; NC1: supernatant of uninduced bacterial lysate; 3: supernatant of bacterial lysate induced overnight at 20℃; 4: supernatant of bacterial lysate induced at 37℃ for 4h; NC2: precipitate of uninduced bacterial lysate; 5: precipitate of bacterial lysate induced overnight at 20℃; 6: precipitate of bacterial lysate induced at 37℃ for 4h; Figure 6 This is the SDS-PAGE Coomassie Brilliant Blue staining of the recombinant humanized filaggrin rhFLG_2# expression; where: PC1: BSA (1μg); PC2: BSA (2μg); M1: protein electrophoresis marker; NC: uninduced bacterial lysate; 1: bacterial lysate induced overnight at 20℃; 2: bacterial lysate induced at 37℃ for 4h; NC1: supernatant of uninduced bacterial lysate; 3: supernatant of bacterial lysate induced overnight at 20℃; 4: supernatant of bacterial lysate induced at 37℃ for 4h; NC2: precipitate of uninduced bacterial lysate; 5: precipitate of bacterial lysate induced overnight at 20℃; 6: precipitate of bacterial lysate induced at 37℃ for 4h; Figure 7 This is the SDS-PAGE Coomassie Brilliant Blue staining of the recombinant humanized filaggrin rhFLG_3# expression; where: M1: protein electrophoresis marker; PC1: BSA (1μg); PC2: BSA (2μg); NC: uninduced bacterial lysate; 1: bacterial lysate induced overnight at 20℃; 2: bacterial lysate induced at 37℃ for 4h; NC1: supernatant of uninduced bacterial lysate; 3: supernatant of bacterial lysate induced overnight at 20℃; 4: supernatant of bacterial lysate induced at 37℃ for 4h; NC2: precipitate of uninduced bacterial lysate; 5: precipitate of bacterial lysate induced overnight at 20℃; 6: precipitate of bacterial lysate induced at 37℃ for 4h; Figure 8This is the SDS-PAGE Coomassie Brilliant Blue staining of the expression of the human wild-type LEKTI protein fragment hLEKTI_wt; where: M1: protein electrophoresis marker; PC1: BSA (1μg); PC2: BSA (2μg); NC: uninduced bacterial lysate; 1: bacterial lysate induced overnight at 20℃; 2: bacterial lysate induced at 37℃ for 4h; NC1: supernatant of uninduced bacterial lysate; 3: supernatant of bacterial lysate induced overnight at 20℃; 4: supernatant of bacterial lysate induced at 37℃ for 4h; NC2: precipitate of uninduced bacterial lysate; 5: precipitate of bacterial lysate induced overnight at 20℃; 6: precipitate of bacterial lysate induced at 37℃ for 4h; Fig. 9 This is the experimental system for detecting the inhibition rate of hLEKTI_wt, rhFLG_1#, rhFLG_2# and rhFLG_3# on KLK5 enzyme activity; where: *: (1×) (2×)…: indicates that the molar ratio of hLEKTI_wt, rhFLG_1#, rhFLG_2# and rhFLG_3# to KLK5 in the experimental system is 1, 2…; Fig.10 The results of the hLEKTI_wt inhibitory activity test; Fig.11 The results of the rhFLG_1# inhibitory activity detection experiment; Fig.12 The results of the rhFLG_2# inhibitory activity detection experiment; Fig.13 The results of the rhFLG_3# inhibitory activity detection experiment; Fig.14 IC50 results for hLEKTI_wt; Fig.15 IC50 result for rhFLG_1#; Fig.16 IC50 result for rhFLG_2#; Fig.17 IC50 results for rhFLG_3#; Fig.18 The modeling results of the mouse AD model, A is the right ear of the mouse in the vehicle group; B is the right ear of the mouse in the model group; Fig.19 The results of the treatment on the mouse AD model. A is the right ear of the mouse in the treatment Vehicle group; B is the right ear of the mouse in the positive control group; C is the right ear of the mouse in the treatment group. DETAILED DESCRIPTION

[0026] The essential contents of the present invention are described in detail below in conjunction with the embodiments, but the protection scope of the present invention is not limited thereto.

[0027] Example 1: Design of recombinant protein, construction of recombinant vector, expression and identification of recombinant protein 1. Design of recombinant protein In this example, the protein fragment in proFLG (sequence shown in SEQ ID No. 1) was repeated 3 times, 5 times, and 7 times, and the humanized FLG protein fragments were named mhFLG_1#, mhFLG_2#, and mhFLG_3#, respectively.

[0028] The sequence of the humanized Kazal motif protein fragment is shown in SEQ ID No. 2, the sequence of the protein fragment at the N-terminus of the humanized Kazal motif protein fragment is shown in SEQ ID No. 3, and the sequence of the protein fragment at the C-terminus of the humanized Kazal motif protein fragment is shown in SEQ ID No. 4. The humanized LEKTI protein fragment obtained by connecting the above three sequences is named mhLEKTI (the sequence is shown in SEQ ID No. 5). mhLEKTI does not contain the TGM1 action site.

[0029] The fragment containing the furin protease cleavage site in the human wild-type LEKTI protein (sequence as shown in SEQ ID No.6) was selected as the linker, and mhLEKTI was connected to mhFLG_1#, mhFLG_2#, and mhFLG_3#, respectively. Methionine (M) was added to the N-terminus of the recombinant protein, and a His protein tag (sequence: HHHHHH) was added to the C-terminus to obtain recombinant humanized filaggrin, which were named rhFLG_1# (sequence as shown in SEQ ID No.7), rhFLG_2# (sequence as shown in SEQ ID No.8), and rhFLG_3# (sequence as shown in SEQ ID No.9). In order to verify the function of recombinant humanized filaggrin, the recombinant human wild-type LEKTI protein domain 6 was designed, that is, methionine was added to the N-terminus of the amino acid sequence of the human wild-type LEKTI protein domain 6, and a His protein tag (sequence: HHHHHH) was added to the C-terminus, named hLEKTI_wt (sequence as shown in SEQ ID No.10).

[0030] 2. Construction of recombinant vector

[0031] According to rhFLG_1# (sequence as shown in SEQ ID No.7), reverse translation was performed into DNA sequence, and codon optimization for E. coli was performed. CAT base was added to the 5' end of the DNA sequence to form a start codon ATG. N I restriction site, adding the stop codon TGA and Hind III restriction site sequence, and the obtained DNA sequence was named n_rhFLG_1# (its sequence is shown in SEQ ID No.11).

[0032] The selected E. coli expression vector was pET30a. The pET30a empty vector and the synthesized n_rhFLG_1# fragment were respectively N I and Hin d III restriction endonuclease double digestion. After the double digestion system was incubated at 37°C for 2 hours, the restriction endonuclease was inactivated at 65°C for 20 minutes. The double digestion reaction system is as follows: Table 1 Restriction endonuclease double digestion reaction system , The Cycle Pure Kit recovered the n_rhFLG_1# fragment (system 1) and the pET30a empty vector fragment (system 2), and then connected the two fragments with T4 DNA Ligase. The connection system was incubated at 16°C for 12 hours. The recombinant vector was named pET30a-n_rhFLG_1#. The connection reaction system is as follows: Table 2 DNA ligase ligation reaction system , The ligation product was transformed into DH5α E. coli competent cells by heat stimulation, and then spread on kanamycin-resistant LB plates and cultured overnight. After overnight culture, the positive clones were amplified and cultured, and the plasmids were extracted and the insert fragments were sequenced. Some of the sequencing results are shown in Figure 2. Figure 1 As shown, the sequencing result was compared with n_rhFLG_1# (whose sequence is shown in SEQ ID No.11) and it was shown that the sequence of the inserted fragment was correct.

[0033] The operations of rhFLG_2#, rhFLG_3# and hLEKTI_wt were the same as above, and the DNA sequences were obtained and named n_rhFLG_2# (its sequence is shown in SEQ ID No.12), n_rhFLG_3# (its sequence is shown in SEQ ID No.13) and n_hLEKTI_wt (its sequence is shown in SEQ ID No.14). The operation of recombinant vector construction was the same as rhFLG_1#. The recombinant vectors were named pET30a-n_rhFLG_2#, pET30a-n_rhFLG_3# and pET30a-n_hLEKTI_wt, respectively. The partial sequencing results of the recombinant vector pET30a-n_rhFLG_2# are shown in Figure 2 The partial sequencing results of the recombinant vector pET30a-n_rhFLG_3# are shown in Figure 3The partial sequencing results of the recombinant vector pET30a-n_hLEKTI_wt are shown in Figure 4 The sequencing results showed that the sequences of the inserted fragments of the recombinant vectors pET30a-n_rhFLG_2#, pET30a-n_rhFLG_3# and pET30a-n_hLEKTI_wt were correct.

[0034] 3. Expression and identification of recombinant proteins 1. Expression of recombinant humanized filaggrin Since the recombinant protein contains disulfide bonds, SHuffle T7 Escherichia coli, which is conducive to the correct folding of disulfide-bonded proteins, was selected as the expression strain. The recombinant plasmid pET30a-n_rhFLG_1# was transformed into Escherichia coli by heat shock method, and positive clones were obtained by kanamycin resistance plate screening.

[0035] Inoculate the SHuffle T7 E. coli monoclone into 5 ml of kanamycin-resistant LB medium and shake at 250 RPM at 37°C overnight. Inoculate the overnight cultured E. coli into two 50 ml fresh kanamycin-resistant medium at a ratio of 1:50 and shake at 37°C until OD 600 When the p-value was about 0.6, 1 ml of bacterial solution was aspirated as the uninduced expression control group, and then the inducer IPTG (final concentration 0.3 mM) was added to the two shake flasks respectively, and the two shake flasks were induced to express at 20℃ / overnight and 37℃ / 4 hours respectively, with the rotation speed of 200RPM.

[0036] 2. Identification of recombinant humanized filaggrin After the induction of expression is completed, take 450μl of the bacterial solution from the uninduced control group, 20℃ / overnight group and 37℃ / 4 hours group, add 300μl of lysis solution, and perform ultrasonic lysis for 10 minutes. At this time, the lysis solution is the whole cell lysis solution, and 100μl is taken for storage; take 200μl of whole cell lysate, centrifuge at 15000RPM for 10 minutes, the supernatant obtained is the cell lysis supernatant, and the precipitate is the cell lysis precipitate. Add appropriate amount of 5× loading buffer to the whole cell lysate, cell lysis supernatant and cell lysis precipitate, respectively, mix well, and heat at 100℃ for 5 minutes to obtain the test sample. The whole cell lysate, cell lysis supernatant and cell lysis precipitate test samples were respectively aspirated for SDS-PAGE electrophoresis. After the electrophoresis, they were stained with Coomassie Brilliant Blue. The results are as shown below. Figure 5 As shown, the arrow indicates the recombinant humanized filaggrin rhFLG_1# expressed by Escherichia coli, which was recovered by nickel column for subsequent experiments.

[0037] The operation of rhFLG_2#, rhFLG_3# and hLEKTI_wt was the same as above. The SDS-PAGE electrophoresis results of rhFLG_2# whole cell lysate, cell lysate supernatant and cell lysate precipitate test samples are shown in Figure 6 The SDS-PAGE electrophoresis results of rhFLG_3# whole cell lysate, cell lysate supernatant and cell lysate precipitate test samples are shown in Figure 7 The SDS-PAGE electrophoresis results of hLEKTI_wt whole cell lysate, cell lysate supernatant and cell lysate precipitate test samples are shown in Figure 8 The arrow in the figure indicates the corresponding recombinant protein, which was recovered by nickel column for subsequent experiments.

[0038] Example 2: Detection of the inhibitory activity of recombinant humanized filaggrin on KLK5 With hLEKTI_wt as the positive control group, and rhFLG_1#, rhFLG_2# and rhFLG_3# as the experimental groups, the inhibitory activity of the inhibitor on KLK5 enzyme activity at different concentrations was detected. The substrate of KLK5 is Boc-Val-Pro-Arg-AMC. KLK5 catalyzes the hydrolysis of the substrate to release the free AMC fluorescent group, and its luminescence intensity is detected by an enzyme reader (380nm excitation, 450nm detection). After the enzyme and inhibitor are mixed, they are first incubated at 37°C for 15 minutes, and then placed in the enzyme reader after adding the substrate, and the enzyme kinetics are detected at 25°C (the initial fluorescence intensity is detected when the 96-well plate is placed in the enzyme reader, and then it is detected every 1 minute, for a total of 20 times). The experimental system is as follows Fig. 9 .

[0039] The experimental results are as follows Figures 10 to 13 As shown, at different molar ratios of inhibitors (hLEKTI_wt, rhFLG_1#, rhFLG_2# and rhFLG_3#) to KLK5, the fluorescence intensity of the rhFLG_1#, rhFLG_2# and rhFLG_3# groups were significantly lower than that of the hLEKTI_wt corresponding group, and the inhibition rate of rhFLG_1#, rhFLG_2# and rhFLG_3# on KLK5 enzyme activity was significantly higher than that of hLEKTI_wt; Figures 14 to 17 As shown, based on the fluorescence value at the 20th minute of the experiment, the IC50 value of rhFLG_1# was 6.10nM, the IC50 value of rhFLG_2# was 6.50nM, and the IC50 value of rhFLG_3# was 7.03nM, which were significantly lower than the IC50 value of hLEKTI_wt of 22.34nM.

[0040] The above results suggest that the inhibitory activity of recombinant humanized filaggrin rhFLG_1#, rhFLG_2# and rhFLG_3# on KLK5 enzyme activity is greatly improved compared with the human wild-type LEKTI protein hLEKTI_wt. Effective inhibition of KLK5 enzyme activity can further inhibit the activation of proKLK7 and proKLK14, and the reverse activation of proKLK5 by KLK14, ultimately reducing the enzyme activity of KLKs as a whole, inhibiting the abnormal dissociation of the stratum corneum and the invasion and metastasis of tumors. Considering that the experiment was conducted in a weakly alkaline system, combined with the fact that AD and NS patients have skin barrier damage and increased pH due to FLG mutations, LEKTI mutations, etc., it is suggested that recombinant humanized filaggrin still has a good inhibitory effect on KLK5 enzyme activity in a high pH environment, which increases its application potential in cosmetics, medical devices and drugs.

[0041] Example 3: Recombinant humanized filaggrin for treatment of mouse AD model The inducer calcipotriol (MC903) was used to establish a mouse AD model, and the mice used were Balb / c mice. The modeling lasted for 14 days, and the specific operations were as follows: the AD model was divided into a modeling vehicle group (6+1 mice) and a model group (24+3 mice). The modeling vehicle group applied 20μl of anhydrous ethanol to the right ear every day; the model group applied 20μl of 1.5nM MC903 ethanol solution to the right ear every day. On the 14th day, the right ears of the mice were photographed, the thickness of the right ears was measured, and the right ear samples of 6 mice each in the modeling vehicle group and the model group were taken and preserved. According to Fig.18 As shown, there was no obvious change in the right ears of the mice in the Vehicle group, while redness, swelling, thickening and desquamation appeared in the right ears of the mice in the Model group, indicating that the model was successful.

[0042] After the model was successfully established, the remaining model group mice were regrouped into: treatment vehicle group (6+1 mice), positive control group (6+1 mice) and treatment group (6+1 mice) for recombinant humanized filaggrin for AD treatment experiments. The treatment lasted for 14 days, and the specific operations were as follows: the treatment vehicle group applied 20μl PBS solution to the right ear every day; the positive control group applied 20μl 0.5‰‌ (W / V, in terms of betamethasone) betamethasone sodium phosphate PBS solution to the right ear every day; the treatment group applied 20μl 2‰‌ (W / V) rhFLG_1# protein PBS solution to the right ear every day. After the treatment, the right ears of mice in each group were photographed and the thickness of the right ears was measured, and the right ear samples of 6 mice in each group were taken and preserved. According to Fig.19As shown, the desquamation and dryness of the right ear of the mice in the treatment group were significantly reduced compared with those in the treatment Vehicle group and the positive control group, indicating that the humanized LEKTI protein fragment in the recombinant humanized filaggrin rhFLG_1# can effectively inhibit the desquamation symptoms of AD, and the humanized FLG protein fragment can repair the skin barrier by strengthening CE and increase the moisture content of the epidermis.

[0043] In summary: Those skilled in the art know that KLKs, including KLK5, play an important role in diseases such as atopic dermatitis, Netherton syndrome, and tumors with high expression of KLKs. LEKTI can treat or alleviate related diseases by regulating KLK5. The recombinant humanized filaggrin provided by the present invention contains a humanized LEKTI protein fragment, which contains a humanized Kazal motif protein sequence shown in SEQ ID No. 2, and its inhibitory strength against KLK5 is more than 3 times that of the recombinant human wild-type LEKTI protein against KLK5 (see Figures 14 to 17 IC50 value).

[0044] The recombinant humanized filaggrin also contains a humanized FLG protein fragment, which is connected to the humanized LEKTI protein fragment through a linker containing a furin cleavage site. The humanized FLG protein fragment is released after being cleaved by the intercellular furin protease, and exerts the function of FLG as introduced in the background technology, such as combining with CE under the action of the intercellular TGM enzyme to enhance the strength of CE, and ultimately enhancing the skin barrier function and reducing water loss and allergen invasion.

[0045] Therefore, the recombinant humanized filaggrin provided by the present invention has the prospect of being developed into a drug, cosmetic or medical device for treating diseases that are treated or alleviated by inhibiting kallikrein KLK5, and the diseases that are treated or alleviated by inhibiting kallikrein KLK5 include atopic dermatitis, Netherton syndrome and tumors with high expression of KLKs.

[0046] The purpose of the above-mentioned embodiments is to specifically introduce the essential content of the present invention, but those skilled in the art should know that the protection scope of the present invention should not be limited to the specific embodiments.

Claims

1. A recombinant humanized filaggrin, characterized in that: The recombinant humanized filaggrin contains a humanized LEKTI protein fragment, and the humanized LEKTI protein fragment contains a humanized Kazal motif protein sequence shown in SEQ ID No.

2.

2. The recombinant humanized filaggrin according to claim 1, characterized in that: The amino acid sequence of the humanized LEKTI protein fragment is shown in SEQ ID No.

5.

3. The recombinant humanized filaggrin according to claim 1 or 2, characterized in that: The recombinant humanized filaggrin also contains a humanized FLG protein fragment, which is formed by connecting a plurality of human proFLG protein fragments having amino acid sequences as shown in SEQ ID No.

1.

4. The recombinant humanized filaggrin according to claim 3, characterized in that: The recombinant humanized filaggrin also contains a connector for connecting the humanized LEKTI protein fragment and the humanized FLG protein fragment, and the connector contains a furin enzyme cleavage site.

5. The recombinant humanized filaggrin according to claim 4, characterized in that: The amino acid sequence of the linker is shown in SEQ ID No.

6.

6. The recombinant humanized filaggrin according to claim 5, characterized in that: The humanized FLG protein fragment is formed by connecting three human proFLG protein fragments shown in SEQ ID No. 1 to form a recombinant humanized filaggrin with an amino acid sequence shown in SEQ ID No.

7.

7. The recombinant humanized filaggrin according to claim 5, characterized in that: The humanized FLG protein fragment is formed by connecting 5 human proFLG protein fragments shown in SEQ ID No.1 to form a recombinant humanized filaggrin with an amino acid sequence shown in SEQ ID No.

8.

8. The recombinant humanized filaggrin according to claim 5, characterized in that: The humanized FLG protein fragment is formed by connecting 7 human proFLG protein fragments shown in SEQ ID No.1 to form a recombinant humanized filaggrin with an amino acid sequence shown in SEQ ID No.

9.

9. Use of the recombinant humanized filaggrin according to any one of claims 1 to 8 for preparing drugs, cosmetics or medical devices for treating diseases that are treated or alleviated by inhibiting kallikrein KLK5, or use of the recombinant humanized filaggrin according to any one of claims 3 to 8 for preparing drugs, cosmetics or medical devices for treating diseases that are treated or alleviated by supplementing FLG and / or inhibiting kallikrein KLK5.

10. The use according to claim 9, characterized in that: The diseases that are treated or alleviated by supplementing FLG and / or inhibiting kallikrein KLK5 include atopic dermatitis, Netherton syndrome, and tumors with high expression of KLKs.

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