Recombinant humanized collagen and application thereof

Abstract

The present invention discloses a recombinant humanized collagen and uses thereof, the recombinant humanized collagen contains a humanized LEKTI protein fragment and a type III human collagen fragment, and the two protein fragments are connected through a linker containing a furin enzyme cutting site. After the recombinant humanized collagen is cut by the furin protease, two protein fragments are generated and exert respective functions. The recombinant humanized collagen has the prospect of being developed into medicines, cosmetics or medical instruments for treating diseases which are treated or relieved by supplementing III-type human collagen fragments and / or inhibiting kallikrein KLKs; diseases treated or relieved by supplementing the III-type human collagen fragment and / or inhibiting the KLKs include atopic dermatitis, internal sephton syndrome and tumors with high expression of the KLKs.

Description

Technical Field

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

[0002] Collagen is the most abundant and widely distributed protein in the human body. Currently, 28 types have been discovered, accounting for up to 30 - 40% of the proteins in the human body. Among them, type III collagen is an important component widely present in the dermis and constitutes the extracellular matrix (ECM). Collagen has a repetitive G-X-Y triplet structure, where G is glycine, and X and Y represent other amino acids, most commonly proline and hydroxyproline. Collagen has characteristics such as good biocompatibility and degradability. Glycine and many other amino acids can keep the skin surface moist and soft, improving the dry skin condition. Therefore, it is widely used in fields such as cosmetics and medical devices. The traditional method of extracting collagen from animal tissues such as pigs and cows has problems such as low protein purity, easy transmission of pathogenic microorganisms, and large batch-to-batch differences, which limit the use of collagen. The method of genetic engineering can well solve the above problems, and by selecting fragments with reasonable distribution of moisturizing amino acids, high water solubility, and good stability in wild collagen for expression, the use effect of collagen is further improved.

[0003] In the physiological process, serine protease inhibitor Kazal type 5 (SPINK5, also known as LEKTI, hereinafter referred to as LEKTI) acts as an inhibitor of kallikrein-related peptidases (KLKs), strictly regulating the activities of various KLKs. The stratum corneum cells orderly shed under the synergistic action of KLKs and LEKTI, maintaining the balance with the proliferation and differentiation rates of keratinocytes, and thus maintaining the normal thickness of the stratum corneum and the skin barrier function. LEKTI is a Kazal type serine protease inhibitor, containing 15 highly similar domains. There are furin protease cleavage sites between multiple domains. After the LEKTI protein is cleaved multiple times by furin protease, several active fragments composed of individual or multiple domains in series are generated, inhibiting the activities of KLKs. As a negative regulator of KLKs, when its expression level is dysregulated or it loses its activity due to gene mutation, patients may present atopic dermatitis (AD) or Netherton syndrome (NS).

[0004] In the epidermis, the KLKs involved in stratum corneum desquamation are mainly KLK5, KLK7, and KLK14, etc. Among them, KLK5 is a key molecule for the activation of multiple KLKs. KLK5 can autoactivate, and the activated KLK5 generates active KLK7 and KLK14 by cleaving proKLK7 (the precursor of KLK7) and proKLK14 (the precursor of KLK14). The activated KLK14 can in turn activate KLK5, overall increasing the activity of KLKs. KLK5 and KLK14 have trypsin activity, and KLK7 has chymotrypsin activity. They act together to cleave the desmosomes between keratinocytes, separating and shedding the surface stratum corneum cells. KLKs also regulate the expression of various inflammatory factors. KLK5 can significantly promote the expression of Thymic Stromal Lymphopoietin (TSLP), and TSLP is a key molecule initiating Th2-type inflammation. Inhibiting the activity of KLK5 can significantly reduce the expression of TSLP and Th2-type inflammation. Th2-type inflammation is a basic feature of Atopic Dermatitis (AD). When LEKTI cannot effectively inhibit the high activity of KLKs, there will be excessive dissociation of the stratum corneum and damage to the skin barrier. The invasion of microorganisms and allergens in the environment further promotes the increased expression of TSLP, and TSLP induces CD4 + T cells to differentiate into T helper 2 cells (Th2 cells) expressing pro-inflammatory factors such as IL-4, IL-5, and IL-13. IL-4 and IL-13 can stimulate sensory neurons to produce itching. IL-4 and IL-13 can also increase the expression of KLK5 and KLK7 that damage the skin barrier, while decreasing the expression of filaggrin (FLG), desmoglein 1 (DSG1), LEKTI, etc. that maintain the skin barrier, further aggravating the scaling symptoms. Due to itching and scaling, patients will scratch the affected area, and this physical stimulus of scratching can also upregulate the level of TSLP. Considering the above multiple factors, the symptoms of scaling, itching, and skin barrier damage become increasingly severe, causing serious damage to the quality of life and mental health of AD patients.

[0005] When LEKTI loses its inhibitory activity due to gene mutation, LEKTI completely loses its inhibition of KLKs, leading to Netherton syndrome (NS), a rare but severe autosomal recessive genetic disorder. Its clinical manifestations are: congenital ichthyosiform erythroderma, circumflex linear ichthyosis, bamboo-like hair, and atopic diathesis with higher IgE. NS can occur in the neonatal period, accompanied by symptoms such as dry skin, itching, and desquamation. Due to severe impairment of the skin barrier function, patients are prone to severe life-threatening symptoms such as repeated infections, dehydration, and hypernatremia. At the same time, due to the lack of inhibition of KLKs activity, the expression of various inflammatory factors and IgE is excessively elevated, and patients are prone to diseases such as allergies and asthma. In treatment, exogenous supplementation of LEKTI to inhibit KLKs has very positive significance for controlling the symptoms of AD and NS from the source.

[0006] In the processes of the occurrence, development, invasion, and metastasis of various tumors such as cervical cancer, ovarian cancer, endometrial cancer, esophageal cancer, gastric cancer, breast cancer, and pancreatic cancer, abnormally highly expressed KLKs also play important roles. KLKs can degrade the ECM and release various growth factors bound to the ECM, such as bound epidermal growth factor (bEGF), bound fibroblast growth factor (bFGF), bound vascular endothelial growth factor (bVEGF), etc., making them soluble growth factors such as sEGF, sFGF, sVEGF, etc., to promote tumor growth. KLKs promote tumor invasion and metastasis by hydrolyzing adhesion proteins such as desmocollin (DSC), desmoglein (DSG), and epithelial cadherin (E-cadherin, CDH1) between tumor cells, reducing cell adhesion. KLKs also indirectly regulate cell migration by regulating the expression of other genes. Highly expressed KLK14 can upregulate matrix metalloproteinases (MMPs) to promote tumor invasion and metastasis. Effectively reducing the activity of KLKs has a positive effect on inhibiting the occurrence, development, invasion, and metastasis of tumors.

[0007] Currently, the treatment for AD and NS mainly involves topical application of emollients, glucocorticoids, calcineurin inhibitors, etc. to control symptoms such as inflammation, itching, and desquamation, but the treatment effects vary from person to person, with large individual differences. 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 KLKs inhibition scheme with high specificity and clear effects.

[0008] In order to overcome the existing deficiencies, the present invention is specifically proposed. Summary of the Invention

[0009] The object of the present invention is to overcome the deficiencies of the prior art and provide a recombinant humanized collagen and its uses.

[0010] The above object of the present invention is achieved by the following technical solutions: A recombinant humanized collagen contains a humanized LEKTI protein fragment. The sequence of the humanized LEKTI protein fragment is shown in SEQ ID No.2 and is obtained by mutating some amino acids in the human LEKTI protein fragment with the sequence shown in SEQ ID No.1. By mutating some amino acids in the human LEKTI protein fragment, the stability and inhibitory activity against KLKs of the humanized LEKTI protein fragment are greatly improved. The recombinant humanized collagen also contains a human type III collagen fragment, which can keep the skin moist and improve the dry skin state.

[0011] Preferably, the recombinant humanized collagen also contains a protein linker (linker, hereinafter referred to as linker) for connecting the humanized LEKTI protein fragment and the human type III collagen fragment. The protein linker contains a furin protease cleavage site, enabling the recombinant humanized collagen to be cleaved by the protease furin to release the LEKTI protein fragment with inhibitory effect on KLKs and the human type III collagen fragment for improving the dry skin state.

[0012] More preferably, the amino acid sequence of the linker is shown in SEQ ID No.7.

[0013] More preferably, the sequence of the human type III collagen fragment is shown in SEQ ID No.3, and the sequence of the recombinant humanized collagen is shown in SEQ ID No.8.

[0014] More preferably, the sequence of the human type III collagen fragment is shown in SEQ ID No.4, and the sequence of the recombinant humanized collagen is shown in SEQ ID No.9.

[0015] More preferably, the sequence of the human type III collagen fragment is shown in SEQ ID No.5, and the sequence of the recombinant humanized collagen is shown in SEQ ID No.10.

[0016] More preferably, the sequence of the human type III collagen fragment is shown in SEQ ID No.6, and the sequence of the recombinant humanized collagen is shown in SEQ ID No.11.

[0017] Use of any of the above recombinant humanized collagens for preparing drugs, cosmetics or medical devices for treating diseases that can be treated or alleviated by supplementing human type III collagen and / or inhibiting kallikrein KLKs.

[0018] Preferably, the diseases treated or alleviated by supplementing human type III collagen and / or inhibiting kallikrein KLKs include atopic dermatitis, Netherton syndrome, and tumors with high expression of KLKs.

[0019] Beneficial effects: Those skilled in the art know that reducing the enzymatic activity of KLKs plays an important role in controlling the symptoms of atopic dermatitis and Netherton syndrome, as well as inhibiting the occurrence, development, invasion, and metastasis of tumors with high expression of KLKs. LEKTI can treat and alleviate related diseases by inhibiting KLKs including KLK5. The recombinant humanized collagen provided by the present invention has an inhibitory effect on multiple KLKs including KLK5 through the humanized LEKTI protein fragment contained therein, and its inhibitory intensity on KLK5 is nearly 3 times that of the human wild-type LEKTI protein fragment with the sequence shown in SEQ ID No. 12 (see Figures 17 - 21 the IC50 values shown).

[0020] The recombinant humanized collagen also contains a human type III collagen fragment, which is connected to the humanized LEKTI protein fragment through a linker containing a furin cleavage site. After cleavage by furin protease in the intercellular space, the human type III collagen fragment is released to improve skin dryness and keep the skin moist.

[0021] Therefore, the recombinant humanized collagen provided by the present invention has the prospect of being developed into cosmetics, drugs, or medical devices for treating diseases that can be treated or alleviated by supplementing type III human collagen fragments and / or inhibiting KLKs. The diseases that can be treated or alleviated by inhibiting KLKs include atopic dermatitis, Netherton syndrome, and tumors with high expression of KLKs. Description of the drawings

[0022] Figure 1 is a partial sequencing result of the recombinant vector pET30a-nrhCOL_1#; Figure 2 is a partial sequencing result of the recombinant vector pET30a-nrhCOL_2#; Figure 3 is a partial sequencing result of the recombinant vector pET30a-nrhCOL_3#; Figure 4 is a partial sequencing result of the recombinant vector pET30a-nrhCOL_4#; Figure 5 is a partial sequencing result of the recombinant vector pET30a-nrhLEKTI_wt; Figure 6SDS-PAGE Coomassie brilliant blue staining map of the expression of recombinant humanized collagen rhCOL_1#; among them: M1: Protein electrophoresis Marker; PC1: BSA (1 μg); PC2: BSA (2 μg); NC: Uninduced bacterial lysate; 1: Bacterial lysate induced for expression overnight at 20 °C; NC1: Supernatant of uninduced bacterial lysate; 2: Supernatant of bacterial lysate induced for expression overnight at 20 °C; NC2: Precipitate of uninduced bacterial lysate; 5: Precipitate of bacterial lysate induced for expression overnight at 20 °C; Figure 7 SDS-PAGE Coomassie brilliant blue staining map of the expression of recombinant humanized collagen rhCOL_2#; among them: PC1: BSA (1 μg); PC2: BSA (2 μg); M1: Protein electrophoresis Marker; NC: Uninduced bacterial lysate; 1: Bacterial lysate induced for expression overnight at 20 °C; NC1: Supernatant of uninduced bacterial lysate; 2: Supernatant of bacterial lysate induced for expression overnight at 20 °C; NC2: Precipitate of uninduced bacterial lysate; 5: Precipitate of bacterial lysate induced for expression overnight at 20 °C; Figure 8 SDS-PAGE Coomassie brilliant blue staining map of the expression of recombinant humanized collagen rhCOL_3#; among them: PC1: BSA (1 μg); PC2: BSA (2 μg); M1: Protein electrophoresis Marker; NC: Uninduced bacterial lysate; 1: Bacterial lysate induced for expression overnight at 20 °C; NC1: Supernatant of uninduced bacterial lysate; 2: Supernatant of bacterial lysate induced for expression overnight at 20 °C; NC2: Precipitate of uninduced bacterial lysate; 5: Precipitate of bacterial lysate induced for expression overnight at 20 °C; Figure 9 SDS-PAGE Coomassie brilliant blue staining map of the expression of recombinant humanized collagen rhCOL_4#; among them: M1: Protein electrophoresis Marker; PC1: BSA (1 μg); PC2: BSA (2 μg); NC: Uninduced bacterial lysate; 1: Bacterial lysate induced for expression overnight at 20 °C; NC1: Supernatant of uninduced bacterial lysate; 2: Supernatant of bacterial lysate induced for expression overnight at 20 °C; NC2: Precipitate of uninduced bacterial lysate; 5: Precipitate of bacterial lysate induced for expression overnight at 20 °C; Figure 10 SDS-PAGE Coomassie brilliant blue staining map of the expression of human wild-type LEKTI protein rhLEKTI_wt; M1: Protein electrophoresis Marker; PC1: BSA (1 μg); PC2: BSA (2 μg); NC: Uninduced bacterial lysate; 1: Bacterial lysate induced for expression overnight at 20 °C; NC1: Supernatant of uninduced bacterial lysate; 2: Supernatant of bacterial lysate induced for expression overnight at 20 °C; NC2: Precipitate of uninduced bacterial lysate; 5: Precipitate of bacterial lysate induced for expression overnight at 20 °C; Figure 11 The experimental system for detecting the inhibitory activity of rhLEKTI_wt, rhCOL_1#, rhCOL_2#, rhCOL_3# and rhCOL_4# against KLK5; wherein: (0.1×) (0.4×)……: indicates that the molar ratios of rhLEKTI_wt, rhCOL_1#, rhCOL_2#, rhCOL_3# and rhCOL_4# to KLK5 in the experimental system are 0.1, 0.4……; Figure 12 The experimental results of the inhibitory activity of rhLEKTI_wt; Figure 13 The experimental results of the inhibitory activity of rhCOL_1#; Figure 14 The experimental results of the inhibitory activity of rhCOL_2#; Figure 15 The experimental results of the inhibitory activity of rhCOL_3#; Figure 16 The experimental results of the inhibitory activity of rhCOL_4#; Figure 17 The IC50 results of rhLEKTI_wt; Figure 18 The IC50 results of rhCOL_1#; Figure 19 The IC50 results of rhCOL_2#; Figure 20 The IC50 results of rhCOL_3#; Figure 21 The IC50 results of rhCOL_4#; Figure 22 The results of establishing the mouse AD model. A is the right ear of the mice in the modeling Vehicle group; B is the right ear of the mice in the AD group; Figure 23 The treatment effect of the mouse AD model. A is the right ear of the mice in the treatment Vehicle group; B is the right ear of the mice in the positive control group; C is the right ear of the mice in the treatment group. Specific implementation mode

[0023] The following specifically introduces the substantial content of the present invention in combination with embodiments, but does not limit the protection scope of the present invention thereto.

[0024] Example 1: Design of recombinant protein, construction of recombinant vector, expression and identification of recombinant protein I. Design of recombinant protein 1. Design of recombinant humanized collagen In this embodiment, partial amino acid sites in the human wild-type LEKTI protein fragment with the mutant sequence as shown in SEQ ID No.1 were mutated to obtain a humanized LEKTI protein fragment with the sequence as shown in SEQ ID No.2, named mhLEKTI.

[0025] Protein fragments in human type III collagen were screened according to factors such as solubility, stability, and amino acid sequence distribution. The sequences are as shown in SEQ ID No.3, SEQ ID No.4, SEQ ID No.5, and SEQ ID No.6, named mhCOL_1#, mhCOL_2#, mhCOL_3#, and mhCOL_4#.

[0026] A protein fragment containing a furin protease cleavage site with the sequence as shown in SEQ ID No.7 was used as a linker to connect mhCOL_1#, mhCOL_2#, mhCOL_3#, and mhCOL_4# with mhLEKTI 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 multiple recombinant humanized collagens, named: rhCOL_1# (sequence as shown in SEQ ID No.8), rhCOL_2# (sequence as shown in SEQ ID No.9), rhCOL_3# (sequence as shown in SEQ ID No.10), and rhCOL_4# (sequence as shown in SEQ ID No.11).

[0027] 2. Design of Recombinant Human Wild-Type LEKTI Protein To detect the inhibitory activity of recombinant humanized collagen on KLKs, methionine (M) was added to the N-terminus of the human wild-type LEKTI protein fragment with the sequence as shown in SEQ ID No.1, and a furin protease cleavage site and a His protein tag (sequence: HHHHHH) were added to the N-terminus to obtain a recombinant human wild-type LEKTI protein with the sequence as shown in SEQ ID No.12, named rhLEKTI_wt.

[0028] II. Construction of Recombinant Vectors The amino acid sequence of rhCOL_1# (sequence as shown in SEQ ID No.8) was reverse translated into a DNA sequence, codon-optimized for the E. coli expression system, with the base CAT added to the 5' end of the optimized DNA sequence, and a stop codon TAA and Hin a d III cleavage site (sequence: AAGCTT) added to the 3' end of the DNA sequence to obtain a DNA sequence as shown in SEQ ID No.13, named nrhCOL_1#.

[0029] After synthesizing the artificial nrhCOL_1# DNA fragment, the Escherichia coli expression vector pET30a and the nrhCOL_1# fragment were respectively double-digested with Nde I and Hin d III restriction endonucleases. After the double-digestion system was incubated 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: ; The double-digested nrhCOL_1# fragment and the pET30a empty vector fragment were respectively recovered using the Cycle Pure Kit. Then, after the two fragments were mixed in a suitable molar ratio, they were ligated with T4 DNA Ligase. The ligation system was incubated at 16 °C overnight. The recombinant vector was named pET30a-nrhCOL_1#. The ligation reaction system is as follows: ; The ligation product was transformed into DH5α Escherichia coli competent cells by heat shock method, and spread on a kanamycin-resistant LB plate and cultured overnight. After monoclonal colonies grew on the plate, monoclonal colonies were picked and amplified in a kanamycin-resistant LB liquid medium, plasmids were extracted and sequenced. Part of the sequencing results are as Figure 1 shown. The sequencing results were compared with nrhCOL_1# (the sequence is shown in SEQ ID No. 13), and the results showed that the inserted fragment sequence was correct.

[0030] The operations of rhCOL_2#, rhCOL_3#, rhCOL_4# and rhLEKTI_wt were the same as above, and the DNA sequences were respectively obtained and named nrhCOL_2# (its sequence is shown in SEQ ID No. 14), nrhCOL_3# (its sequence is shown in SEQ ID No. 15), nrhCOL_4# (its sequence is shown in SEQ ID No. 16) and nrhLEKTI_wt (its sequence is shown in SEQ ID No. 17). The operations for constructing the recombinant vectors were the same as those for rhCOL_1#. The recombinant vectors were respectively named pET30a-nrhCOL_2#, pET30a-nrhCOL_3#, pET30a-nrhCOL_4# and pET30a-nrhLEKTI_wt. Part of the sequencing results of the recombinant vector pET30a-nrhCOL_2# are as Figure 2 shown; part of the sequencing results of the recombinant vector pET30a-nrhCOL_3# are as Figure 3 shown; part of the sequencing results of the recombinant vector pET30a-nrhCOL_4# are as Figure 4 shown; part of the sequencing results of the recombinant vector pET30a-nrhLEKTI_wt are as Figure 5As shown, the sequencing result comparison shows that the sequences of the inserted fragments in the recombinant vectors pET30a-nrhCOL_2#, pET30a-nrhCOL_3#, pET30a-nrhCOL_4# and pET30a-nrhLEKTI_wt are correct.

[0031] III. Expression and Identification of Recombinant Proteins 1. Expression of Recombinant Humanized Collagen SHuffle T7 Escherichia coli, which is beneficial to the correct folding of proteins containing disulfide bonds, was selected as the expression strain. After transforming the recombinant vector pET30a-nrhCOL_1# into Escherichia coli competent cells by heat shock method, positive clones were obtained by screening on kanamycin-resistant LB plates. The SHuffle T7 Escherichia coli monoclonal was inoculated into 5 ml of liquid LB medium with kanamycin resistance and cultured overnight at 37°C with shaking at 250 RPM. The overnight cultured bacterial solution was inoculated into 2 fresh media with kanamycin resistance 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, and then the inducer IPTG (final concentration 0.3 mM) was added to the two flasks respectively, and the two flasks were induced to express overnight at 20°C with shaking at 200 RPM.

[0032] 2. Identification of Recombinant Humanized Collagen After the overnight induced expression was completed, 450 μl of the bacterial solutions of the non-induced control group and the 20°C induction group were taken, 300 μl of lysis buffer was added, and ultrasonic lysis was performed for 10 minutes to obtain bacterial lysates. 100 μl was taken and stored; 200 μl of the bacterial lysate was centrifuged at 15000 RPM for 10 minutes, and the supernatant and precipitate of the bacterial lysate were stored separately. Appropriate amounts of the bacterial lysate, the supernatant of the bacterial lysate, and the precipitate of the bacterial lysate were taken, and appropriate amounts of 5× loading buffer were added respectively. After mixing, they were heated at 100°C for 5 minutes to obtain test samples. The test samples of the bacterial lysate, the supernatant of the bacterial lysate, and the precipitate of the bacterial lysate were respectively aspirated for SDS-PAGE electrophoresis. After the electrophoresis was completed, the gel was stained with Coomassie Brilliant Blue, and the results were as Figure 6 shown. The arrow indicates the recombinant humanized collagen rhCOL_1# expressed by Escherichia coli, which was recovered by nickel column for subsequent experiments.

[0033] The operations of rhCOL_2#, rhCOL_3#, rhCOL_4# and rhLEKTI_wt were the same as above. The SDS-PAGE electrophoresis results of the test samples of the bacterial lysate, the supernatant of the bacterial lysate, and the precipitate of the bacterial lysate of rhCOL_2# were as Figure 7 shown. The SDS-PAGE electrophoresis results of the test samples of the bacterial lysate, the supernatant of the bacterial lysate, and the precipitate of the bacterial lysate of rhCOL_3# were asFigure 8 As shown in the figure, the SDS-PAGE electrophoresis results of the detection samples of rhCOL_4# bacterial lysate, bacterial lysate supernatant and bacterial lysate precipitate are as follows Figure 9 As shown in the figure, the SDS-PAGE electrophoresis results of the detection samples of rhLEKTI_wt bacterial lysate, bacterial lysate supernatant and bacterial lysate precipitate are as follows Figure 10 As shown in the figure. The arrows in the figure indicate the corresponding recombinant proteins, which were used in subsequent experiments after nickel column recovery.

[0034] Example 2: Detection of the inhibitory activity of recombinant humanized collagen on KLK5 rhLEKTI_wt, rhCOL_1#, rhCOL_2#, rhCOL_3# and rhCOL_4# are all inhibitors of KLK5. Using rhLEKTI_wt as the positive control group and rhCOL_1#, rhCOL_2#, rhCOL_3# and rhCOL_4# as the experimental groups, the inhibitory activities of the inhibitors on the enzyme activity of KLK5 at different concentrations were detected. Boc-Val-Pro-Arg-AMC was selected as the catalytic substrate of KLK5. Active KLK5 catalyzed the substrate to release free AMC fluorescent groups. The luminescence intensity of the free fluorescent groups was detected by a microplate reader (excitation at 380 nm and detection at 450 nm). The difference in luminescence intensity corresponded to the difference in the inhibitory activity of different inhibitors on the enzyme. After the enzyme and the inhibitor were mixed, they were first incubated at 37 °C for 15 minutes, and then the substrate was added and placed in the microplate reader for enzyme kinetics detection 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 is as follows Figure 11 .

[0035] The experimental results are as follows Figures 12 - 16 As shown in the figure, when the inhibitors (rhLEKTI_wt, rhCOL_1#, rhCOL_2#, rhCOL_3# and rhCOL_4#) were in different molar ratios with KLK5, the fluorescence intensities of the rhCOL_1#, rhCOL_2#, rhCOL_3# and rhCOL_4# groups were significantly lower than those of the corresponding rhLEKTI_wt groups, and the inhibition rates of rhCOL_1#, rhCOL_2#, rhCOL_3# and rhCOL_4# on the enzyme activity of KLK5 were significantly higher than that of rhLEKTI_wt; as Figures 17 - 21 shown in the figure, calculated from the fluorescence value at the 20th minute of the experiment, the IC50 value of rhCOL_1# was 1.34 nM, the IC50 value of rhCOL_2# was 1.41 nM, the IC50 value of rhCOL_3# was 1.31 nM, and the IC50 value of rhCOL_4# was 1.47 nM, which were much lower than the IC50 value of rhLEKTI_wt, 3.67 nM.

[0036] The above results suggest that the inhibitory activities of recombinant humanized collagens rhCOL_1#, rhCOL_2#, rhCOL_3# and rhCOL_4# against KLK5 enzyme activity are greatly improved compared to the human wild-type LEKTI protein rhLEKTI_wt. By inhibiting the activity of KLK5 enzyme, the activities of KLK7 and KLK14 are indirectly inhibited, as well as the reverse activation of proKLK5 by KLK14, reducing the overall enzyme activity of KLKs, which is beneficial to controlling the abnormal desquamation of the stratum corneum and the invasion and metastasis of tumors. The improvement of the inhibitory activity of recombinant humanized collagens against KLKs enhances their application potential in cosmetics, medical devices and drugs.

[0037] Example 3: Therapeutic experiment of recombinant humanized collagen on mouse AD model An AD model of BALB / c mice was established using the inducer calcipotriol (MC903) for 14 days. The specific operation was as follows: 34 mice were divided into a modeling Vehicle group (6 + 1 mice) and an AD group (24 + 3 mice); in the modeling Vehicle group, 20 μl of absolute ethanol was applied to the right ear of the mice once a day, and in the AD group, 20 μl of 1.5 nM MC903 ethanol solution was applied to the right ear of the mice once a day; on the 14th day, the right ears of the mice were photographed, the ear thickness was measured and recorded, and the right ear specimens of 6 mice from each group were preserved. As Figure 22 shown, there were no obvious changes in the right ears of the mice in the modeling Vehicle group, while the right ears of the mice in the AD group showed obvious swelling, thickening and desquamation, indicating that the modeling was successful and could be used for subsequent therapeutic experiments.

[0038] The AD mice were divided into a treatment Vehicle group (6 + 1 mice), a positive control group (6 + 1 mice) and a treatment group (6 + 1 mice) to detect the therapeutic function of recombinant humanized collagen on AD. The experiment lasted for 14 days. The specific operation was as follows: in the treatment Vehicle group, 20 μl of PBS solution was applied to the right ear of the mice once a day, in the positive control group, 20 μl of 0.5‰ (W / V, calculated as betamethasone) betamethasone sodium phosphate PBS solution was applied to the right ear of the mice once a day, and in the treatment group, 20 μl of 2‰ (W / V) rhCOL_3# protein PBS solution was applied to the right ear of the mice once a day. At the end of the experiment, the right ears of the mice in each group were photographed, the ear thickness was measured and recorded, and the right ear specimens of 6 mice from each group were preserved. As Figure 23 shown, the degree of desquamation and dryness of the right ears of the mice in the treatment group were significantly reduced compared to the treatment Vehicle group and the positive control group, indicating that the humanized LEKTI protein fragment in recombinant humanized collagen can effectively inhibit the desquamation symptoms of AD, and the type III collagen fragment can effectively improve skin dryness and increase skin moisture.

[0039] In summary: Those skilled in the art know that KLKs play an important role in diseases such as atopic dermatitis, Netherton syndrome, and tumors with high KLK expression. By regulating KLKs, LEKTI can treat or relieve related diseases. The recombinant humanized collagen provided by the present invention contains a humanized LEKTI protein fragment, which is obtained by mutating some amino acid sites of the human wild-type LEKTI protein fragment, and its inhibitory activity against KLK5 is nearly 3 times that of the recombinant human wild-type LEKTI protein (see Figures 17 - 21 IC50 value).

[0040] The recombinant humanized collagen also contains a human type III collagen fragment, which 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 human type III collagen fragment is released to improve skin dryness and keep the skin moist.

[0041] Therefore, the recombinant humanized collagen provided by 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 relieved by inhibiting kallikrein KLKs. Diseases that can be treated or relieved by inhibiting kallikrein KLKs include atopic dermatitis, Netherton syndrome, and tumors with high KLK expression.

[0042] 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 this specific embodiment.

Claims

1. A recombinant humanized collagen, characterized in that: The recombinant humanized collagen contains a humanized LEKTI protein fragment, and the sequence of the humanized LEKTI protein fragment is shown in SEQ ID No.2; the recombinant humanized collagen also contains a type III human collagen fragment.

2. The recombinant humanized collagen according to claim 1, wherein: The recombinant humanized collagen also contains a linker that connects the humanized LEKTI protein fragment and the type III human collagen fragment, and the linker contains a furin cleavage site.

3. The recombinant humanized collagen according to claim 2, characterized in that: The amino acid sequence of the linker is shown in SEQ ID No.

7.

4. The recombinant humanized collagen according to claim 3, wherein: The sequence of the type III human collagen fragment is shown in SEQ ID No.3, forming a recombinant humanized collagen with the amino acid sequence shown in SEQ ID No.

8.

5. The recombinant humanized collagen according to claim 3, wherein: The sequence of the type III human collagen fragment is shown in SEQ ID No.4, forming a recombinant humanized collagen with the amino acid sequence shown in SEQ ID No.

9.

6. The recombinant humanized collagen according to claim 3, wherein: The sequence of the type III human collagen fragment is shown in SEQ ID No.5, forming a recombinant humanized collagen with the amino acid sequence shown in SEQ ID No.

10.

7. The recombinant humanized collagen according to claim 3, wherein: The sequence of the type III human collagen fragment is shown in SEQ ID No.6, forming a recombinant humanized collagen with the amino acid sequence shown in SEQ ID No.

11.

8. Use of the recombinant humanized collagen according to any one of claims 1 to 7 for the preparation of a drug, a cosmetic or a medical device for treating a disease that can be treated or alleviated by supplementing a type III human collagen fragment and / or inhibiting kallikrein KLKs.

9. The use according to claim 8, characterized in that: The diseases that can be treated or alleviated by supplementing a type III human collagen fragment and / or inhibiting kallikrein KLKs include atopic dermatitis, Netherton syndrome and tumors with high expression of KLKs.

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