A recombinant humanized filaggrin and its uses

Abstract

The present invention discloses a recombinant humanized filaggrin and its uses. The recombinant humanized filaggrin contains a humanized LEKTI protein fragment, and the humanized LEKTI protein fragment contains the humanized Kazal motif protein sequence shown in SEQ ID No.2; it also contains a humanized FLG protein fragment, and the humanized FLG protein fragment is linked to the humanized LEKTI protein fragment through a linker containing a furin cleavage site. After cleavage by interstitial furin protease, the two protein fragments perform their respective functions. The recombinant humanized filaggrin of the present invention has the prospect of being developed into a drug, a cosmetic or a medical device for treating diseases that can be treated or alleviated by supplementing FLG and / or inhibiting kallikrein KLK5. Diseases that can be treated or alleviated by supplementing FLG and / or inhibiting KLK5 include atopic dermatitis, Netherton syndrome and tumors with high expression of KLKs.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, relates to recombinant proteins, and specifically relates to a recombinant humanized filaggrin and its uses. Background Art

[0002] Atopic Dermatitis (AD) is a common chronic inflammatory skin disease characterized by repeated attacks, often accompanied by symptoms such as itching, dry skin, and desquamation. There are many causes of AD, and among them, the mutation of filaggrin (FLG) 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 isopeptide bonds with loricrin under the catalysis of transglutaminase (TGM) and participates in the formation of CE; FLG is a key molecule for polymerizing keratin intermediate filaments (KIF) in keratinocytes. FLG helps the regular arrangement of KIF through salt bridges and aggregates them into bundles. With the participation of FLG, a stable and firm CE and regularly bundled KIF are formed, which constitute the basis of the desmosome structure between keratinocytes, enabling the tight connection between keratinocytes and thus forming a skin barrier to prevent the entry of allergens, microorganisms, environmental pollutants, etc. and the loss of moisture; when FLG dissociates from KIF, it is cleaved and degraded by various enzymes such as Caspase14, PAD-1, and BLMH, and finally forms approximately 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, and further damage the skin barrier function, resulting in the entry of allergens, microorganisms, environmental pollutants, etc. and the loss of moisture.

[0003] Under physiological conditions, keratinocytes are continuously shed from the surface of the stratum corneum, and this process proceeds orderly under the regulation of kallikreins (KLKs) and their inhibitors. KLK5 is a key factor for the activation of multiple KLKs. ProKLK5 (the precursor of KLK5) is converted into active KLK5 through autoactivation. Subsequently, the activated KLK5 activates proKLK7 (the precursor of KLK7) and proKLK14 (the precursor of KLK14). The activated KLK14 can further activate proKLK5 to increase the enzymatic activity of KLK5. KLK5 is the initiating molecule for the activation of multiple KLKs. KLK5 and KLK14, through their trypsin activities, and KLK7, through its chymotrypsin activity, hydrolyze desmosomes to separate keratinocytes and cause them to 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 (Serine Protease Inhibitor Kazal-type 5, SPINK5, also known as LEKTI, hereinafter referred to as LEKTI), so that the rate of stratum corneum shedding is balanced with the rate of keratinocyte proliferation and differentiation to maintain the normal thickness of the stratum corneum and skin barrier function. The LEKTI protein has 15 Kazal-type protease inhibitor domains. Except for the 2nd and 5th domains that form 3 pairs of disulfide bonds, the remaining domains all form 2 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 activities 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-type inflammation. Inhibiting the activity of KLK5 can downregulate the expression of TSLP and alleviate Th2-type inflammation. Th2-type inflammation is a basic feature of AD. Due to reasons such as FLG and LEKTI mutations, the skin barrier function is damaged. After allergens, microorganisms, environmental pollutants, etc. invade, keratinocytes are promoted to express TSLP 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 forming the barrier function. The reduction of FLG and Caspase14 not only further damages the barrier function but also reduces the FLG metabolites that maintain the weak acidity of the epidermis. The increase in the epidermal pH value further enhances the activity of KLKs. Along with the upregulation of KLK expression and the increase in activity, TSLP is upregulated by activating PAR2, which promotes the differentiation of Th2 cells through positive feedback. 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 upregulation of KLK5 and KLK7 expression and activity, as well as the downregulation of LEKTI expression, desmosomes are excessively cleaved, resulting in obvious scaling. Itching and scaling cause AD patients to scratch the affected area, and this physical stimulus of scratching can also promote TSLP expression. In summary, the interaction between skin barrier damage, inflammation, and scratching accelerates the progression of AD and may lead to its chronicity through a positive feedback loop. By inhibiting KLKs and thus inhibiting the expression of the AD initiation molecule TSLP, scaling is reduced, skin barrier repair is promoted, and ultimately, AD symptoms are alleviated.

[0005] Netherton Syndrome (NS) is another disease related to LEKTI. Its pathogenesis is a rare and severe autosomal recessive genetic disease caused by mutations in the SPINK5 gene. The clinical features of NS are congenital ichthyosiform erythroderma, circumflex linear ichthyosis, bamboo-like hair, and an atopic diathesis with a high IgE level. 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. Infants develop generalized congenital ichthyosiform erythroderma with fine scales within a few weeks after birth. As the disease progresses, the erythroderma gradually develops into circumflex linear ichthyosis, accompanied by skin dryness, itching, and scaling. Due to severe impairment of the skin barrier function, patients are prone to recurrent skin infections, severe dehydration, hypernatremia, hypothermia, and even septicemia, which can endanger their lives. Due to the high activity of KLKs and skin barrier damage, the levels of various inflammatory factors and IgE in patients are significantly increased, making them prone to allergic diseases, 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 various tumors such as pancreatic cancer, cervical cancer, endometrial cancer, esophageal adenocarcinoma, gastric cancer, ovarian cancer, breast cancer, prostate cancer, and lung cancer. KLKs regulate the aggregation of tumor cells by degrading intercellular junction proteins (such as DSG, DSC), and increase the invasion and metastasis of tumor cells by degrading adhesion proteins (such as integrins, cadherins, etc.). KLKs can also remodel the extracellular matrix (ECM). KLKs change the composition of the ECM by degrading ECM protein substrates and release ECM-bound factors such as bound epidermal growth factor (bEGF), bound fibroblast growth factor (bFGF), bound vascular endothelial growth factor (bVEGF), etc. to become soluble growth factors sEGF, sFGF, sVEGF, etc., which 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 multiple KLKs including KLK7. In various cancers, KLKs are key molecules. Therefore, the development of inhibitors against KLKs has very positive significance for controlling tumor occurrence, development, invasion and metastasis.

[0007] Currently, the treatment regimens for AD and NS are different. However, due to the type of inflammation and the characteristics of skin lesions, traditional treatments mostly use topical emollients, 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. Existing treatment regimens have not proposed effective solutions for damaged CE structure and excessive KLKs activity. In tumor treatment, although it is known that KLKs are involved in the occurrence, development, invasion and metastasis of various tumors, there is still no efficient regimen for inhibiting KLKs. Summary of the Invention

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

[0009] The above object of the present invention is achieved by the following technical solutions:

[0010] A recombinant humanized filaggrin, which contains a humanized LEKTI protein fragment, and the humanized LEKTI protein fragment contains a humanized Kazal motif protein sequence of formula I.

[0011] Formula I:

[0012] C-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-Xaa7-Xaa8-Xaa9-Xaa 10 -Xaa 11 -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。

[0013] In formula I, Xaa n represents the amino acid numbered n, and Xaa n has the following meanings:

[0014] Xaa2 is: H, D, S, K, A, R, L, M, I or N;

[0015] Xaa3 is: E, D, A, Q, H, P, Y, S, R or N;

[0016] Xaa4 is: F or Y;

[0017] Xaa5 is: R, Q, K or E;

[0018] Xaa6 is: A, K, P, N, D, H, R, E, S, V, T, Q, L, Y, F, M, I or W;

[0019] Xaa7 is: F, G, Q, L, S, Y or M;

[0020] Xaa8 is: M, E, V, A, R, L, Q, I, P, T, W, D, Y, N, F or H;

[0021] Xaa9 is: K, R, Q, E, P, I, A, V, L or T;

[0022] Xaa 10 is: N, D or I;

[0023] Xaa 11 is: G or N;

[0024] Xaa 12 is: K, D, R, I, T, E, Y or V;

[0025] Xaa 13 is: L, F or Y;

[0026] Xaa 14 is: F, I, G, A, P, S, N, L, H, W, Y, V, T, or absent;

[0027] Xaa 16 is: T, P, D, N, E, A, V, S or Q;

[0028] Xaa 18 is: E, D, Y, R, S, G, P, N, T, M, W, H, L, V, F or I;

[0029] Xaa 19 is: N, K, Y, S, H, L, T, G, R, M, V, I, F, W, E, Q, A or D;

[0030] Xaa 21 is: P, F or A;

[0031] Xaa 22 is: V, I or F;

[0032] Xaa 23 is: Q, C, L, R, E, H, T, F, V, W, Y, I or N;

[0033] Xaa 24 is: G, S, D, W or F;

[0034] Xaa 25 is: P, L, T, A or D;

[0035] Xaa 26 is: D or Y;

[0036] Xaa 27 is: G;

[0037] Xaa 28 is: K, I, R, Q, V or M;

[0038] Xaa 29 is: M, T, V, I, T, S or F;

[0039] Xaa 30 is: H, F, Y or W;

[0040] Xaa 31 is: G, I, D, N, T, E, F, R, S, L, Y, H, V, P, W, A, K or Q;

[0041] Xaa 32 is: N;

[0042] Xaa 33 is: K, R, L, T, Q, P, W, I, M, E, V or F;

[0043] Xaa 35 is: S, A, T, Y, M, N, D, K, L, R, Q or E;

[0044] Xaa 36 is: M, T or L.

[0045] Preferably, the amino acid sequence of the humanized Kazal motif protein fragment is as shown in SEQ ID No.2.

[0046] Preferably, the amino acid sequence of the humanized LEKTI protein fragment is as 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 recombinant humanized filaggrin, but also enhances its inhibitory effect on KLKs. By mutating the TGM1 action sites in the humanized Kazal motif protein fragment and / or the protein fragments on both sides thereof, the free property of the humanized LEKTI protein fragment is increased without reducing its inhibitory effect on KLKs.

[0047] Preferably, the recombinant humanized filaggrin further contains a humanized FLG protein fragment, which is formed by connecting multiple human proFLG protein fragments with the amino acid sequence as shown in SEQ ID No.1. The functional fragment of human proFLG that binds to loricrin is selected, and the humanized FLG protein fragment is obtained by appropriately repeating this functional fragment, supplementing the FLG functional fragment participating in CE assembly, improving the insufficient CE components caused by FLG mutations and other reasons, and ultimately enhancing the skin barrier function.

[0048] Preferably, the recombinant humanized filaggrin further contains a linker (hereinafter referred to as linker) that connects the humanized LEKTI protein fragment and the humanized FLG protein fragment, and the linker contains a furin cleavage site. A fragment containing the natural furin cleavage site of the human wild-type LEKTI protein is selected as the linker between 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 inhibits KLKs and the humanized filaggrin fragment participating in CE assembly.

[0049] More preferably, the amino acid sequence of the linker is as shown in SEQ ID No.6.

[0050] More preferably, the humanized FLG protein fragment is formed by connecting 3 human proFLG protein fragments shown in SEQ ID No.1, forming a recombinant humanized filaggrin with the amino acid sequence as shown in SEQ ID No.7.

[0051] More preferably, the humanized FLG protein fragment is formed by connecting 5 human proFLG protein fragments shown in SEQ ID No.1, forming a recombinant humanized filaggrin with the amino acid sequence as shown in SEQ ID No.8.

[0052] More preferably, the humanized FLG protein fragment is formed by connecting 7 human proFLG protein fragments shown in SEQ ID No.1, forming a recombinant humanized filaggrin with the amino acid sequence as shown in SEQ ID No.9.

[0053] Use of any of the above recombinant humanized filaggrins for the preparation of drugs, cosmetics or medical devices for treating diseases that can be treated or alleviated by supplementing FLG and / or inhibiting kallikrein KLK5.

[0054] Preferably, the diseases that can be treated or alleviated by supplementing FLG and / or inhibiting kallikrein KLK5 include atopic dermatitis, Netherton syndrome and tumors with high expression of KLKs.

[0055] Beneficial effects:

[0056] Those skilled in the art know that KLKs, including KLK5, play important roles in diseases such as atopic dermatitis, Netherton syndrome, and tumors with high KLK expression. LEKTI can treat or relieve related diseases by regulating KLK5. The recombinant humanized filaggrin provided by the present invention contains a humanized LEKTI protein fragment, and the humanized LEKTI protein fragment contains the humanized Kazal motif protein sequence shown in SEQ ID No.2, and its inhibitory intensity on 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).

[0057] The recombinant humanized filaggrin also contains a humanized FLG protein fragment. The humanized FLG protein fragment is linked to the humanized LEKTI protein fragment through a linker containing a furin cleavage site. After cleavage by furin protease in the intercellular space, the humanized FLG protein fragment is released, and it plays the function of FLG as introduced in the background technology. For example, it binds to CE under the action of TGM enzyme in the intercellular space to strengthen the CE intensity, and finally enhances the skin barrier function, reduces water loss and allergen invasion.

[0058] Therefore, the recombinant humanized filaggrin provided by the present invention has the prospect of being developed into drugs, cosmetics or medical devices for treating diseases that can be treated or relieved by supplementing FLG and / or inhibiting kallikrein KLK5. Diseases that can be treated or relieved by inhibiting kallikrein KLK5 include atopic dermatitis, Netherton syndrome, and tumors with high KLK expression. Description of the Drawings

[0059] Figure 1 Partial sequencing result of recombinant vector pET30a-n_rhFLG_1#;

[0060] Figure 2 Partial sequencing result of recombinant vector pET30a-n_rhFLG_2#;

[0061] Figure 3 Partial sequencing result of recombinant vector pET30a-n_rhFLG_3#;

[0062] Figure 4 Partial sequencing result of recombinant vector pET30a-n_hLEKTI_wt;

[0063] Figure 5SDS-PAGE Coomassie brilliant blue staining map of the expression of recombinant humanized filaggrin rhFLG_1#; among them: M1: Protein electrophoresis Marker; PC1: BSA (1 μg); PC2: BSA (2 μg); NC: Uninduced bacterial lysate; 1: Bacterial lysate induced overnight at 20 °C; 2: Bacterial lysate induced for 4 h at 37 °C; NC1: Supernatant of uninduced bacterial lysate; 3: Supernatant of bacterial lysate induced overnight at 20 °C; 4: Supernatant of bacterial lysate induced for 4 h at 37 °C; NC2: Precipitate of uninduced bacterial lysate; 5: Precipitate of bacterial lysate induced overnight at 20 °C; 6: Precipitate of bacterial lysate induced for 4 h at 37 °C;

[0064] Figure 6 SDS-PAGE Coomassie brilliant blue staining map of the expression of recombinant humanized filaggrin rhFLG_2#; among them: PC1: BSA (1 μg); PC2: BSA (2 μg); M1: Protein electrophoresis Marker; NC: Uninduced bacterial lysate; 1: Bacterial lysate induced overnight at 20 °C; 2: Bacterial lysate induced for 4 h at 37 °C; NC1: Supernatant of uninduced bacterial lysate; 3: Supernatant of bacterial lysate induced overnight at 20 °C; 4: Supernatant of bacterial lysate induced for 4 h at 37 °C; NC2: Precipitate of uninduced bacterial lysate; 5: Precipitate of bacterial lysate induced overnight at 20 °C; 6: Precipitate of bacterial lysate induced for 4 h at 37 °C;

[0065] Figure 7 SDS-PAGE Coomassie brilliant blue staining map of the expression of recombinant humanized filaggrin rhFLG_3#; among them: M1: Protein electrophoresis Marker; PC1: BSA (1 μg); PC2: BSA (2 μg); NC: Uninduced bacterial lysate; 1: Bacterial lysate induced overnight at 20 °C; 2: Bacterial lysate induced for 4 h at 37 °C; NC1: Supernatant of uninduced bacterial lysate; 3: Supernatant of bacterial lysate induced overnight at 20 °C; 4: Supernatant of bacterial lysate induced for 4 h at 37 °C; NC2: Precipitate of uninduced bacterial lysate; 5: Precipitate of bacterial lysate induced overnight at 20 °C; 6: Precipitate of bacterial lysate induced for 4 h at 37 °C;

[0066] Figure 8SDS-PAGE Coomassie brilliant blue staining map of the expressed wild-type human LEKTI protein fragment hLEKTI_wt; where: M1: Protein electrophoresis Marker; PC1: BSA (1 μg); PC2: BSA (2 μg); NC: Uninduced bacterial lysate; 1: Bacterial lysate induced overnight at 20 °C; 2: Bacterial lysate induced for 4 h at 37 °C; NC1: Supernatant of uninduced bacterial lysate; 3: Supernatant of bacterial lysate induced overnight at 20 °C; 4: Supernatant of bacterial lysate induced for 4 h at 37 °C; NC2: Precipitate of uninduced bacterial lysate; 5: Precipitate of bacterial lysate induced overnight at 20 °C; 6: Precipitate of bacterial lysate induced for 4 h at 37 °C;

[0067] Figure 9 Experimental system for detecting the inhibitory 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…;

[0068] Figure 10 Experimental results of hLEKTI_wt inhibitory activity detection;

[0069] Figure 11 Experimental results of rhFLG_1# inhibitory activity detection;

[0070] Figure 12 Experimental results of rhFLG_2# inhibitory activity detection;

[0071] Figure 13 Experimental results of rhFLG_3# inhibitory activity detection;

[0072] Figure 14 hLEKTI_wt IC50 results;

[0073] Figure 15 rhFLG_1# IC50 results;

[0074] Figure 16 rhFLG_2# IC50 results;

[0075] Figure 17 rhFLG_3# IC50 results;

[0076] Figure 18 Results of establishing a mouse AD model. A is the right ear of a mouse in the Vehicle group for model establishment; B is the right ear of a mouse in the model group;

[0077] Figure 19For the treatment effect of 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 implementation mode

[0078] The following combines the embodiments to specifically introduce the substantial content of the present invention, but does not limit the protection scope of the present invention thereby.

[0079] Example 1: Design of recombinant protein, construction of recombinant vector, expression and identification of recombinant protein

[0080] I. Design of recombinant protein

[0081] In this example, the protein fragment in proFLG (the sequence is shown in SEQ ID No.1) is repeated 3 times, 5 times, and 7 times, and this humanized FLG protein fragment is named mhFLG_1#, mhFLG_2#, and mhFLG_3# respectively.

[0082] The sequence of the humanized Kazal motif protein fragment is shown in SEQ ID No.2, the protein fragment sequence at the N-terminal of the humanized Kazal motif protein fragment is shown in SEQ ID No.3, and the protein fragment sequence at the C-terminal of the humanized Kazal motif protein fragment is shown in SEQ ID No.4. The above three sequences are ligated to obtain the humanized LEKTI protein fragment named mhLEKTI (the sequence is shown in SEQ ID No.5). There is no TGM1 action site in mhLEKTI.

[0083] Select the fragment containing the furin protease cleavage site in the human wild-type LEKTI protein (the sequence is shown in SEQ IDNo.6) as the linker, ligate mhLEKTI with mhFLG_1#, mhFLG_2#, and mhFLG_3# respectively, and add methionine (M) at the N-terminal of the recombinant protein and His protein tag (the sequence is: HHHHHH) at the C-terminal to obtain recombinant humanized filaggrin, named rhFLG_1# (the sequence is shown in SEQ ID No.7), rhFLG_2# (the sequence is shown in SEQ ID No.8), and rhFLG_3# (the sequence is shown in SEQ ID No.9) respectively. For the functional verification of the recombinant humanized filaggrin, design the 6th domain of the recombinant human wild-type LEKTI protein, that is: add methionine at the N-terminal of the amino acid sequence of the 6th domain of the human wild-type LEKTI protein and His protein tag (the sequence is: HHHHHH) at the C-terminal, named hLEKTI_wt (the sequence is shown in SEQ ID No.10).

[0084] II. Construction of recombinant vector

[0085] The DNA sequence was reverse-translated from rhFLG_1# (the sequence is shown in SEQ ID No. 7) and codon-optimized for Escherichia coli. The CAT base was added to the 5' end of the DNA sequence to form an Nde I restriction enzyme site with the start codon ATG, and the stop codon TGA and Hin the d III restriction enzyme site sequence were added to the 3' end of the DNA sequence. The resulting DNA sequence was named n_rhFLG_1# (its sequence is shown in SEQ ID No. 11).

[0086] The selected Escherichia coli expression vector was pET30a. The pET30a empty vector and the synthesized n_rhFLG_1# fragment were separately digested with Nde I and Hin d III restriction endonucleases. After incubating the double digestion system at 37 °C for 2 hours, it was incubated at 65 °C for 20 minutes to inactivate the restriction endonucleases. The double digestion reaction system is as follows:

[0087] Table 1 Double digestion reaction system of restriction endonucleases

[0088] ,

[0089] The n_rhFLG_1# fragment (System 1) and the pET30a empty vector fragment (System 2) were separately recovered using Cycle Pure Kit, and then the two fragments were ligated with T4 DNA Ligase. The ligation system was incubated at 16 °C for 12 hours. The recombinant vector was named pET30a-n_rhFLG_1#. The ligation reaction system is as follows:

[0090] Table 2 Ligation reaction system of DNA ligase

[0091] ,

[0092] The ligation product was transformed into DH5α Escherichia coli competent cells by heat shock and spread on a kanamycin-resistant LB plate for overnight culture. After overnight culture, positive clones were picked for amplification culture, and then plasmids were extracted and the inserted fragments were sequenced. Part of the sequencing results is as Figure 1 shown. Comparing the sequencing results with n_rhFLG_1# (its sequence is shown in SEQ ID No. 11) showed that the sequence of the inserted fragment was correct.

[0093] The operations for rhFLG_2#, rhFLG_3# and hLEKTI_wt were the same as above. The DNA sequences were obtained respectively 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 for constructing the recombinant vectors was the same as that for 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 as Figure 2 shown. The partial sequencing results of the recombinant vector pET30a-n_rhFLG_3# are as Figure 3 shown. The partial sequencing results of the recombinant vector pET30a-n_hLEKTI_wt are as Figure 4 shown. The sequencing result alignment 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.

[0094] III. Expression and Identification of Recombinant Proteins

[0095] 1. Expression of Recombinant Humanized Filaggrin

[0096] Since the recombinant protein contains disulfide bonds, SHuffle T7 Escherichia coli, which is beneficial to the correct folding of proteins containing disulfide bonds, was selected as the expression strain. After the recombinant plasmid pET30a-n_rhFLG_1# was transformed into Escherichia coli by heat shock method, positive clones were obtained through screening on kanamycin-resistant plates.

[0097] The monoclonal of SHuffle T7 Escherichia coli was inoculated into 5 ml of kanamycin-resistant LB medium and cultured overnight with shaking at 250 RPM at 37 °C. The overnight culture was inoculated into 2 fresh 50 ml kanamycin-resistant media at a ratio of 1:50 respectively and cultured with shaking at 37 °C until the OD 600 was approximately 0.6. 1 ml of the bacterial solution was aspirated as the non-induced expression control group. Then, the inducer IPTG (final concentration 0.3 mM) was added to the two flasks respectively, and the two flasks were induced for expression at 20 °C / overnight and 37 °C / 4 hours respectively, with the rotation speed of 200 RPM.

[0098] 2. Identification of Recombinant Humanized Filaggrin

[0099] After the induction expression ended, 450 μl of the bacterial solutions from the uninduced control group, the 20 °C / overnight group, and the 37 °C / 4-hour group were taken, 300 μl of lysis buffer was added, and ultrasonic lysis was performed for 10 minutes. At this time, the lysis buffer was the whole cell lysate, and 100 μl was taken for preservation; 200 μl of the whole cell lysate was centrifuged at 15,000 RPM for 10 minutes. The obtained supernatant was the cell lysate supernatant, and the precipitate was the cell lysate precipitate. Appropriate amounts of 5× loading buffer were added to the whole cell lysate, the cell lysate supernatant, and the cell lysate precipitate respectively. After mixing, they were heated at 100 °C for 5 minutes to obtain the detection samples. The detection samples of the whole cell lysate, the cell lysate supernatant, and the cell lysate precipitate were respectively aspirated for SDS-PAGE electrophoresis. After the electrophoresis ended, Coomassie brilliant blue staining was performed, and the results were as Figure 5 shown. The arrow indicates the recombinant humanized filaggrin rhFLG_1# expressed by Escherichia coli, which was recovered by nickel column for subsequent experiments.

[0100] The same operations were performed for rhFLG_2#, rhFLG_3#, and hLEKTI_wt. The SDS-PAGE electrophoresis results of the detection samples of the whole cell lysate, the cell lysate supernatant, and the cell lysate precipitate of rhFLG_2# were as Figure 6 shown. The SDS-PAGE electrophoresis results of the detection samples of the whole cell lysate, the cell lysate supernatant, and the cell lysate precipitate of rhFLG_3# were as Figure 7 shown. The SDS-PAGE electrophoresis results of the detection samples of the whole cell lysate, the cell lysate supernatant, and the cell lysate precipitate of hLEKTI_wt were as Figure 8 shown. The arrow in the figure indicates the corresponding recombinant protein, which was recovered by nickel column for subsequent experiments.

[0101] Example 2: Detection of the inhibitory activity of recombinant humanized filaggrin on KLK5

[0102] Taking hLEKTI_wt as the positive control group and rhFLG_1#, rhFLG_2#, and rhFLG_3# as the experimental groups, the inhibitory activities of the inhibitors on the enzyme activity of KLK5 at different concentrations were detected. The substrate of KLK5 was Boc-Val-Pro-Arg-AMC. KLK5 catalyzed the hydrolysis of the substrate to release the free AMC fluorescent group, and its luminescence intensity was detected by a microplate reader (excitation at 380 nm, detection at 450 nm). After the enzyme and the inhibitor were mixed, they were first incubated at 37 °C for 15 minutes. After adding the substrate, they were placed in the microplate reader, and enzyme kinetics detection was performed at 25 °C (the initial fluorescence intensity was detected when the 96-well plate was placed in the microplate reader, and then detected once every 1 minute for a total of 20 times). The experimental system was as Figure 9 .

[0103] The experimental results were as Figures 10 to 13As shown, for the inhibitors (hLEKTI_wt, rhFLG_1#, rhFLG_2# and rhFLG_3#) at different molar ratios with KLK5, the fluorescence intensities of the rhFLG_1#, rhFLG_2# and rhFLG_3# groups were significantly lower than those of the corresponding hLEKTI_wt groups, and the inhibition rates of rhFLG_1#, rhFLG_2# and rhFLG_3# on the enzyme activity of KLK5 were significantly higher than that of hLEKTI_wt; as Figures 14 to 17 shown, calculated from the fluorescence values at the 20th minute of the experiment, the IC50 value of rhFLG_1# was 6.10 nM, the IC50 value of rhFLG_2# was 6.50 nM, and the IC50 value of rhFLG_3# was 7.03 nM, which were significantly lower than the IC50 value of 22.34 nM of hLEKTI_wt.

[0104] Collectively, the above results suggest that the inhibitory activities of recombinant humanized filaggrin rhFLG_1#, rhFLG_2# and rhFLG_3# on the enzyme activity of KLK5 are greatly improved compared to the human wild-type LEKTI protein hLEKTI_wt. Effectively inhibiting the enzyme activity of KLK5 can further inhibit the activation of proKLK7 and proKLK14, and the reverse activation of proKLK5 by KLK14, ultimately reducing the overall enzyme activity of KLKs and inhibiting the abnormal desquamation of the stratum corneum and the invasion and metastasis of tumors. Considering that the experiment was carried out in a weakly alkaline system, combined with the situation of skin barrier disruption and increased pH in AD and NS patients due to FLG mutations, LEKTI mutations, etc., it is suggested that recombinant humanized filaggrin still has good inhibitory effects on the enzyme activity of KLK5 in a high-pH environment, which improves its application potential in cosmetics, medical devices and drugs.

[0105] Example 3: Therapeutic experiment of recombinant humanized filaggrin on a mouse AD model

[0106] An AD mouse model was established using the inducer calcipotriol (MC903), 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 vehicle control group for modeling (6 + 1 mice) and a model group (24 + 3 mice). In the vehicle control group for modeling, 20 μl of absolute ethanol was applied to the right ear of each mouse every day; in the model group, 20 μl of 1.5 nM MC903 ethanol solution was applied to the right ear of each mouse 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 from each of the vehicle control group for modeling and the model group were taken for preservation. According to Figure 18 shown, there were no obvious changes in the right ears of the mice in the vehicle control group for modeling, while the right ears of the mice in the model group showed swelling, thickening and desquamation, indicating successful modeling.

[0107] After successful modeling, the remaining mice in the model group were regrouped into: treatment Vehicle group (6 + 1 mice), positive control group (6 + 1 mice), and treatment group (6 + 1 mice) for the experiment of recombinant human filaggrin in the treatment of AD. The treatment lasted for 14 days, and the specific operations were as follows: the treatment Vehicle group was applied with 20 μl of PBS solution to the right ear every day; the positive control group was applied with 20 μl of 0.5‰ (W / V, calculated as betamethasone) betamethasone sodium phosphate PBS solution to the right ear every day; the treatment group was applied with 20 μl of 2‰ (W / V) rhFLG_1# protein PBS solution to the right ear every day. After the treatment, the right ears of the mice in each group were photographed, 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 Figure 19 As shown, the degree of desquamation and dryness of the right ears of the mice in the treatment group was significantly reduced compared with those in the treatment Vehicle group and the positive control group, suggesting that the humanized LEKTI protein fragment in the recombinant human filaggrin rhFLG_1# can effectively inhibit the desquamation symptoms of AD, and the humanized FLG protein fragment can strengthen the CE to repair the skin barrier and increase the epidermal water content.

[0108] In summary, it can be seen that:

[0109] Those skilled in the art know that KLKs, including KLK5, play important roles in diseases such as atopic dermatitis, Netherton syndrome, and tumors with high expression of KLKs. LEKTI can treat or relieve related diseases by regulating KLK5. The recombinant human filaggrin provided by the present invention contains a humanized LEKTI protein fragment, and the humanized LEKTI protein fragment contains the humanized Kazal motif protein sequence shown in SEQ ID No.2, and its inhibitory intensity on KLK5 is more than 3 times that of the recombinant human wild-type LEKTI protein on KLK5 (see Figures 14 to 17 IC50 value).

[0110] The recombinant human filaggrin also contains a humanized FLG protein fragment, and the humanized FLG protein fragment is linked to the humanized LEKTI protein fragment through a linker containing a furin cleavage site. After cleavage by furin protease in the intercellular space, the humanized FLG protein fragment is released to exert the functions of FLG introduced in the background technology, such as binding to CE under the action of intercellular TGM enzyme to strengthen the CE intensity, and ultimately enhancing the skin barrier function, reducing water loss and allergen invasion.

[0111] Therefore, the recombinant human filaggrin provided by the present invention has the prospect of being developed into a drug, cosmetic, or medical device for treating diseases that can be treated or relieved by inhibiting kallikrein KLK5. Diseases that can be treated or relieved by inhibiting kallikrein KLK5 include atopic dermatitis, Netherton syndrome, and tumors with high expression of KLKs.

[0112] The function of the above embodiments is to specifically introduce the substantial content of the present invention. However, 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: Its amino acid sequence is shown in SEQ ID No.

7.

2. Use of the recombinant humanized filaggrin according to claim 1 for preparing medicines, cosmetics or medical devices for treating atopic dermatitis.

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