Recombinant human fibronectin with soothing compound function and preparation method and application thereof

Abstract

The present invention discloses a recombinant human fibronectin with soothing composite function and its preparation method and application. The recombinant human fibronectin with soothing composite function has an amino acid sequence as shown in SEQ ID NO.1. The present invention adds the hyaluronic acid interaction segment of human fibronectin while retaining the main segment of human fibronectin cell adhesion and cell integrin receptor interaction, and connects the two domains with a flexible "linker" amino acid sequence GGS to form a recombinant human FN protein with composite function. In addition to the core characteristics of traditional recombinant fibronectin, the recombinant human fibronectin also has the function of soothing cell stimulation. The recombinant human fibronectin prepared by the present invention will have a good effect in soothing skin irritation in the field of functional active skin care products.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and more specifically, to a recombinant human fibronectin with soothing compounding function and a preparation method and application thereof. Background Art

[0002] Fibronectin (FN) is widely present in the animal kingdom (from freshwater sponges to humans). It is a macromolecular extracellular membrane protein on the cell surface and the main non-collagenous glycoprotein in the extracellular matrix and basement membrane. Its subunit molecular weight is 220~250kDa and is composed of about 2500 amino acid residues. It plays a central role in cell adhesion and can regulate cell polarity, differentiation and growth. It is widely distributed in the body: it exists in plasma and various body fluids in a soluble form; it exists in the extracellular matrix (including certain basement membranes) and cell surfaces in an insoluble form. The former is called plasma FN and the latter is called cell FN. It can be cut into several domains by limited proteolysis and can bind to plasma fibrin, heparin, collagen and cell surface receptors. In the N-terminal domain of the FN protein, there is a binding region that can interact with hyaluronic acid HA, which also plays an important role in the binding of fibrin and heparin.

[0003] The molecular weight of natural fibronectin is too large, and it is composed of more than 2,000 amino acids, making it difficult to be absorbed by the skin. Therefore, genetic engineering technology is used to select domains of FN with different functions for expression to obtain recombinant fibronectin with different functions. At present, recombinant fibronectin has excellent skin care effects and has broad application prospects in the fields of medicine, beauty, and cosmetics. However, the current sequences of recombinant fibronectin are basically concentrated on the recombinant expression of the cell adhesion-related domain at the C-terminus of the protein, and there is no recombinant fibronectin that has been successfully fused, expressed, and purified with the N-terminal functional domain of recombinant fibronectin. Therefore, the functions of the currently synthesized recombinant proteins are limited, and there are few reports of cases in which the soothing effect of skin cell irritation of recombinant fibronectin is enhanced by the N-terminal domain function. Summary of the invention

[0004] In view of the problems existing in the above-mentioned prior art, the present invention utilizes technologies such as codon optimization and macromolecular protein solubility optimization to overcome the shortcomings of prokaryotic expression of macromolecular proteins and poor solubility of recombinant protein molecules containing the N-terminal functional domain of human fibronectin, and provides a macromolecular recombinant human fibronectin with soothing complex function and its preparation method and application.

[0005] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0006] In the first aspect, a recombinant human fibronectin with soothing complex function, the amino acid sequence of which is shown in SEQ ID NO.1.

[0007] The above-mentioned recombinant human fibronectin has a molecular weight of approximately 95 kDa, and comprises amino acids corresponding to the promoter, a human fibronectin fibrin and hyaluronic acid interaction region as shown in SEQ ID NO.2, and a human fibronectin cell adhesion-related and integrin receptor binding region as shown in SEQ ID NO.3, wherein the protein domains at both ends of SEQ ID NO.2 and SEQ ID NO.3 are connected by a flexible protein "linker" amino acid motif GGS, and the C-terminus of the recombinant protein carries a 6×His protein tag for protein purification.

[0008] The recombinant fibronectin of the present invention has a hyaluronic acid binding region and a segment related to the interaction between the extracellular matrix and the cell integrin receptor. The recombinant human fibronectin finally purified and harvested contains 780 amino acids, a molecular size of about 95 kDa, and a purity of more than 95%. This recombinant fibronectin has a significant promoting effect on the adhesion and proliferation of human keratinocyte cell line HaCat cells, and has a significant soothing effect on SDS stimulation of keratinocytes.

[0009] In the second aspect, a coding sequence of a recombinant human fibronectin with soothing complex function, the nucleotide sequence of which after codon optimization is shown in SEQ ID NO.4.

[0010] The coding sequence is a nucleotide sequence optimized through the codon of the prokaryotic expression system, including the promoter ATG. Both ends of the sequence are inserted into the pET28a prokaryotic expression vector through the restriction enzyme cutting sites "NdeI" (CATATG) and "XhoI" (CTCGAG). The nucleotide coding sequence "CACCACCACCACCACCAC" corresponding to the 6×His protein tag is included at the C-terminus of the recombinant protein, and the corresponding termination codon is TGA.

[0011] In a third aspect, a method for preparing a recombinant human fibronectin having a soothing compounding function comprises the following steps:

[0012] S1. According to the amino acid sequence SEQ ID NO.1, the codon of the E. coli expression system is optimized to generate the corresponding nucleotide sequence and construct a protein expression vector;

[0013] S2. Transform the expression vector into the Escherichia coli expression strain, screen the positive monoclonal colonies with higher expression levels, amplify and build a library; select the positive monoclonal colonies and inoculate them into liquid culture medium for culture; after amplification overnight, amplify the culture and induce protein expression the next day, and then collect the cells by centrifugation;

[0014] S3. Lyse the cells with lysis buffer, centrifuge and purify the protein;

[0015] S4. The purified harvested protein is concentrated by filtration to obtain the recombinant human fibronectin stock solution with soothing complex function.

[0016] Furthermore, in step S2, 1-2% (W / V, mass / volume) sorbitol is added to the liquid culture medium.

[0017] Furthermore, in step S2, the peptone of the liquid culture medium is selected from Angel Yeast FP210, FP220 and / or FP108 peptone.

[0018] Furthermore, in step S2, positive monoclonal colonies are picked, inoculated into an adapted liquid culture medium, cultured at 37°C for 10 hours, inoculated into a new culture medium containing kanamycin at an inoculum size of 1:100, and after culture at 37°C for 3-4 small tests, an inducer is added, and the OD600 value during induction is 0.8-2.0; the induction temperature is set to 20-24°C, and the induction is performed overnight.

[0019] Furthermore, the lysis buffer in step S3 is 10-40 mM phosphate buffer, and the sodium chloride concentration is 0-100 mM. 1%-2% arginine is also added to the lysis buffer and the chromatography buffer, and the pH value is adjusted to 7.2-7.6.

[0020] Furthermore, the resuspension ratio of the bacteria to the lysis buffer is 1:10-1:40 W / V, more preferably 1:20 W / V (ie, 1 g of bacteria is resuspended in 20 mL of lysis buffer).

[0021] Further, in step S3, the purified protein refers to the purified protein by affinity chromatography and anion exchange chromatography; the affinity chromatography filler uses Ni-NTA filler with polyacrylate microspheres as the matrix material, and the elution conditions are discontinuous gradient, 20mM phosphate buffer after equilibration, 50mM-100mM imidazole buffer washing, and 300mM imidazole buffer elution. A single purification can obtain a higher purity rhHA-FN. Further, the ion exchange chromatography uses DEAE Sepharose FF to elute with a sodium chloride concentration gradient. Using anion exchange chromatography, preferably DEAE Sepharose FF chromatography filler for secondary chromatography, a purity greater than 95% can be harvested, and the rhHA-FN recombinant protein with a high concentration of imidazole in the first chromatography is almost removed.

[0022] Among them, Ni-NTA: agarose is a nickel-containing affinity resin that can be used to purify recombinant proteins containing polyhistidine (6xHis) sequences; DEAE Sepharose FF: anion exchange chromatography filler is a weak anion exchange chromatography filler used for rapid protein purification.

[0023] Furthermore, the step S4 uses a 30KD ultrafiltration membrane package to concentrate and replace the solution, which can further wash away small molecule impurities. Compared with single chromatography, the volume of the liquid used for diafiltration is greatly reduced after the two-step chromatography, thereby reducing the protein loss during the diafiltration process.

[0024] In a fourth aspect, a recombinant human fibronectin with soothing complex function as described above or a recombinant human fibronectin with soothing complex function obtained by any of the above preparation methods is used in skin care products.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] By adding the hyaluronic acid interaction segment of human fibronectin, while retaining the main segment of human fibronectin cell adhesion and cell integrin receptor interaction, the two domains are connected by a flexible "linker" amino acid sequence GGS to form a recombinant human FN protein with complex functions. In addition to the core characteristics of traditional recombinant fibronectin, the recombinant human fibronectin also has the effect of soothing cell stimulation. The recombinant human fibronectin prepared by the present invention will have a good effect in soothing skin irritation in the field of functional active skin care products.

[0027] The molecular weight of the composite functional recombinant human fibronectin obtained by the preparation method of the present invention is about 95kDa, and the purity is higher than 95%. The recombinant human fibronectin with composite functions provided by the present invention has good activity through cell biology detection, and has the effect of promoting cell adhesion and cell proliferation. At the same time, it has a good protective effect in the keratinocyte anti-SDS stimulation test.

[0028] The present invention selects a culture medium formula of recombinant human fibronectin with complex functions and a bacterial lysis solution formula, so that the solubility of recombinant human fibronectin, which is mostly expressed as polymers, is greatly improved, thereby avoiding the problem of low renaturation efficiency of large molecular recombinant protein inclusion bodies in industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the solutions in the present application or the prior art, a brief introduction is given below to the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0030] Figure 1 This is a schematic diagram of the gene structure and vector construction of recombinant human fibronectin (rhHA-FN) with soothing complex function;

[0031] Figure 2The results of SDS-PAGE gel electrophoresis of the peptone in the culture medium for the expression of recombinant human fibronectin with soothing complex function were screened for soluble proteins;

[0032] Figure 3 The results of SDS-PAGE gel electrophoresis of soluble proteins were screened for the timing of inducing expression of recombinant human fibronectin with soothing complex function;

[0033] Figure 4 The results of SDS-PAGE gel electrophoresis were obtained for screening the concentration of sorbitol added to the culture medium for the expression of recombinant human fibronectin with soothing compounding function and soluble protein;

[0034] Figure 5 The results of SDS-PAGE gel electrophoresis were used to screen the soluble proteins in the bacterial lysate of recombinant human fibronectin with soothing compound function;

[0035] Figure 6 The results of SDS-PAGE gel electrophoresis were used to screen the soluble proteins of recombinant human fibronectin bacterial lysate with soothing compound function at pH value;

[0036] Figure 7 It is the chromatographic condition peak of recombinant human fibronectin with soothing complex function under different chromatographic conditions;

[0037] Figure 8 The results of SDS-PAGE gel electrophoresis are the results of chromatography of recombinant human fibronectin with soothing complex function;

[0038] Fig. 9 It is the result of HPLC test on the purity of recombinant human fibronectin with soothing compound function;

[0039] Fig.10 To test the cell adhesion promoting efficacy of three batches of recombinant human fibronectin samples with soothing compound function;

[0040] Fig.11 To test the cell proliferation efficacy of three batches of recombinant human fibronectin samples with soothing complex function;

[0041] Fig.12 To test the efficacy of recombinant human fibronectin with soothing compound function in soothing SDS-induced keratinocyte stimulation;

[0042] Fig.13 To test the efficacy of different concentrations of recombinant human fibronectin with soothing compound function in soothing SDS-induced keratinocyte stimulation;

[0043] Fig.14 To test the efficacy of different concentrations of rhFN without HA binding region in alleviating SDS stimulation on keratinocytes;

[0044] Fig.15 To test the efficacy of different concentrations of recombinant human fibronectin (prepared by China Invention Patent Publication No.: CN117586379A, invention name "A preparation method and application of recombinant fibronectin") in alleviating SDS stimulation on keratinocytes;

[0045] Fig.16 To test the efficacy of different concentrations of commercial rhFN-1 in alleviating SDS stimulation on keratinocytes;

[0046] Fig.17 This study was to test the efficacy of different concentrations of commercial rhFN-2 in alleviating SDS stimulation on keratinocytes. DETAILED DESCRIPTION

[0047] The present invention will be further described in detail below in conjunction with specific examples. The following examples are not intended to limit the present invention, but are only intended to illustrate the present invention. The experimental methods used in the following examples are generally conventional, unless otherwise specified, and the materials, reagents, etc. used in the following examples are commercially available, unless otherwise specified.

[0048] The term "about" used in the present invention means a range of ±20% of the value thereafter. In some embodiments, the term "about" means a range of ±10% of the value thereafter. In some embodiments, the term "about" means a range of ±5% of the value thereafter.

[0049] The term "comprises" or "comprising" as used in the present invention means "including but not limited to". The term is intended to be open-ended to specify the presence of any stated features, elements, integers, steps or components, but does not exclude the presence or addition of one or more other features, elements, integers, steps, components or groups thereof. Therefore, the term "comprising" includes the more restrictive terms "consisting of" and "consisting essentially of". In one embodiment, the term "comprising" used throughout the application, especially in the claims, may be replaced by the term "consisting of".

[0050] The terms "optionally", "any", "arbitrarily" or "any" used in the present invention mean that the event or environment described later may but need not occur, and the description includes occasions where the event or environment occurs or does not occur. For example, "optionally comprising an antibody heavy chain variable region" means that an antibody heavy chain variable region of a specific sequence may but does not have to be present.

[0051] The term "about" used in the present invention means a range of ±20% of the value thereafter. In some embodiments, the term "about" means a range of ±10% of the value thereafter. In some embodiments, the term "about" means a range of ±5% of the value thereafter.

[0052] The term "and / or" used in the present invention should be understood to mean any one of the optional items or a combination of any two or more of the optional items.

[0053] Example 1 Design of recombinant human fibronectin sequence with soothing complex function

[0054] A recombinant human fibronectin amino acid sequence comprising a human FN protein hyaluronic acid interaction domain and a human FN protein cell adhesion and integrin receptor interaction domain was designed and named rhHA-FN. The nucleotide sequence thereof is shown in SEQ ID NO.1, and is specifically as follows:

[0055] MPGCYDNGKHYQINQQWERTYLGNALVCTCYGGSRGFNCESKPEAEETCFDKYTGNTYRVGDTYERPKDSMIWDCTCIGAGRGRISCTIANRCHEGGQSYKIGDTWRRPHETGGYmLECVCLGNGKGEWTCKPIAEKCFDHAAGTSYVVGETWEKPYQGWMMVDCTCLGEGSGRITCTSRNRCNDQDTRTSYRIGDTWSKKDNRGNLLQCICTGNGRGEWKCERHGGSTDLSFVDITDSSIGLRWTPLNSSTIIGYRITVVAAGEGIPIFEDFVDSSVGYYTVTGLEPGIDYDISVITLINGGESAPTTLTQQTAVPPPTDLRFTNIGPDTMRVTWAPPPSIDLTNFLVRYSPVKNEEDVAELSISPSDNAVVLTNLLPGTEYVVSVSSVYEQHESTPLRGRQKTGLDSPTGIDFSDITANSFTVHWIAPRATITGYRIRHHPEHFSGRPREDRVPHSRNSITLTNLTPGTEYVVSIVALNGREESPLLIGQQSTVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRTEIDKPSQMQVTDVQDNSISVKWLPSSSPVTGYRVTTTPKNGPGPTKTKTAGPDQTEMTIEGLQPTVEYVVSVYAQNPSGESQPLVQTAVTNIDRPKGLAFTDVDVDSIKIAWESPQGQVSRYRVTYSSPEDGIHELFPAPDGEEDTAELQGLRPGSEYTVSVVALHDDMESQPLIGTQSTAIPLEHHHHHH。

[0056] The sequence of the interaction region of human fibronectin, fibrin and hyaluronic acid is shown in SEQ ID NO.2, as follows:

[0057] PGCYDNGKHYQINQQWERTYLGNALVCTCYGGSRGFNCESKPEAEETCFDKYTGNTYRVGDTYERPKDSMIWDCTCGARGRISCTIANRCHEGGQSYKIGDTWRRPHETG GYmLECVCLGNGKGEWTCKPIAEKCFDHAAGTSYVVGETWEKPYQGWMMVDCTCLGEGSGRITCTSRNRCNDQDTRTSYRIGDTWSKKDNRGNLLQCICTGNGRGEWKCERH.

[0058] The sequence of the cell adhesion-related and integrin receptor-binding region of human fibronectin is shown in SEQ ID NO.3, and is as follows:

[0059] .

[0060] Example 2 Gene design and preparation method of recombinant human fibronectin with soothing complex function

[0061] The method for preparing the recombinant human fibronectin with soothing complex function of this embodiment comprises the following steps:

[0062] S1. According to the amino acid sequence of Example 1, a codon-optimized gene sequence design was performed. The optimized recombinant rhHA-FN protein coding sequence was synthesized by gene synthesis, and an NdeI restriction site was added to the front of the sequence and an XhoI restriction site was added to the end of the sequence. The sequence was cloned into a pET28a vector and sequenced. The vector structure is shown in FIG. Figure 1 shown.

[0063] The gene coding sequence of the recombinant human fibronectin with soothing complex function is shown in SEQ ID NO.4, which is as follows:

[0064]

[0065] S2. Introduce the correctly sequenced cloning vector into the E. coli expression strain BL21-DE3, pick the positive monoclonal colony, inoculate it into the adapted liquid culture medium, culture it at 37℃ for 10 hours, inoculate it into a new culture medium containing kanamycin at an inoculum of 1:100, and culture it for 3-4 hours. When the OD600 value of E. coli reaches about 1.0, add 0.5mM IPTG (isopropyl-β-D-thiogalactoside) for induction. At this time, lower the temperature of the shaker or fermenter to 20℃, induce overnight (about 8-10 hours), and collect the bacteria. Take the preparation of 1L adapted liquid culture medium as an example, the formula is: 10g FP-220 peptone, 5g FM-888 yeast powder, 10g NaCl, 10g sorbitol. The culture medium is placed in a 3L shake flask with a baffle, sterilized at high temperature and high pressure, and placed at room temperature for use.

[0066] S3, bacterial lysis and protein chromatography purification: The lysis buffer is 20mM phosphate buffer PB, containing 100mM sodium chloride, 1% arginine, and pH 7.4. After the bacteria are broken and homogenized with lysis buffer at a ratio of 1:20 (W / V), the supernatant is centrifuged and purified by chromatography. The clarified bacterial lysate is affinity chromatographed with Ni-NTA filler using polyacrylate microspheres as the matrix material. The elution conditions are discontinuous gradient, 20mM phosphate buffer for balance, 5mM-10mM imidazole buffer for washing, and 300mM imidazole buffer for elution, and a relatively pure rhHA-FN protein peak can be harvested. For the rhHA-FN protein mixture with low chromatographic elution purity, anion exchange chromatography is performed, DEAE Sepharose FF is selected for elution with a sodium chloride concentration gradient, and high-purity rhHA-FN protein is harvested by gel chromatography purification using ion exchange filler DEAE Sepharose FF.

[0067] S4. After the protein is purified by chromatography, it is filtered, concentrated and the solution is replaced using a 30KD ultrafiltration membrane package, and finally the recombinant human fibronectin rhHA-FN protein stock solution with soothing complex function is obtained.

[0068] Example 3 Effect of peptone in bacterial liquid culture medium on expression level

[0069] Different expression media were prepared according to the LB medium formula using peptones of different types and sources (FP400, FP402, FP408, FP410, FP418, FP108, FP210, FP220, FP222, FP523 and FM885). After the transformation of E. coli carrying the rhHA-FN plasmid, they were inoculated into different types of culture media in the same proportion and cultured at 37°C, 200rpm for 3-4 hours. At this time, the OD value of the bacterial solution was about 0.8-2.0. 0.5mM IPTG was added to induce overnight at 20°C and the bacteria were harvested the next day. Resuspend the bacterial sludge at a ratio of 1:10 (W / V) (1g bacteria is resuspended in 10mL PB solution), add lysozyme at a final concentration of 2 mg / mL and lyse at 4℃ for 2 hours, vortex from time to time, add nuclease and treat at room temperature for 20 minutes, centrifuge at 12000rpm for 5 minutes, take the supernatant, add appropriate amount of SDS-PAGE (polyacrylamide gel electrophoresis) loading buffer, boil in water bath for 5-10 minutes, and detect the target protein in the supernatant by SDS-PAGE gel electrophoresis. Figure 2 As shown, after induction expression in FP220 medium, the amount of soluble rhHA-FN protein is the highest, and the amount of soluble protein in FP210, 222 and FM885 is relatively high. Therefore, the preferred culture medium peptone is FP220, and peptone FP210, FP222, and yeast powder FM885 are optional.

[0070] Example 4 Effect of induction timing on protein solubility

[0071] Prepare LB culture medium, revive Escherichia coli BL-21 (DE3) containing rhHA-FN plasmid, culture overnight at 37℃, 200rpm, and the next day, inoculate 1:100 in 100mL of fresh culture medium containing kanamycin, culture at 37℃, 200rpm for 2.5h, start collecting samples to detect the OD600 value of the bacterial solution and take samples for induction, record the OD600 value of the bacterial solution at the time of collection, and take the corresponding bacterial solution for induction, and collect the bacteria the next day. Resuspend the bacterial slurry at a ratio of 1:10 (W / V) (1g of bacteria is resuspended in 10mL PB solution), add lysozyme with a final concentration of 2mg / mL and lyse at 4℃ for 2 hours, vortex and oscillate from time to time, add nuclease and treat at room temperature for 20 minutes, centrifuge at 12000rpm for 5 minutes, take the supernatant, add an appropriate amount of SDS-PAGE loading buffer, boil in water bath for 5-10 minutes, and detect the target protein in the supernatant by SDS-PAGE gel electrophoresis. Figure 3 , the protein content of soluble rhHA-FN after induction was detected. It can be clearly seen that when the OD600 value of the bacterial sample was around 1.372 during induction, the expression level of soluble protein (in the supernatant) was significantly higher than that at other induction times.

[0072] Example 5 Effect of sorbitol in liquid culture medium on expression level

[0073] The inoculation and bacterial lysis methods of rhHA-FN in this example are basically the same as those in Example 3, except that FP-220 is used as the peptone in the LB culture medium, and 1% or 2% (W / V) sorbitol is added to the culture medium for comparison with no sorbitol. After lysis, the supernatant is taken and tested by SDS-PAGE gel electrophoresis. Figure 4 The addition of sorbitol to the culture medium can effectively increase the expression of the target protein and the concentration of the target protein in the soluble supernatant, among which the culture medium containing 2% sorbitol has the best effect.

[0074] Example 6 Effect of Sodium Chloride Concentration in Lysis Buffer on Protein Purification

[0075] The inoculation and bacterial lysis methods of rhHA-FN in this example are basically the same as those in Example 5, except that when the bacterial cells are lysed, the concentrations of sodium chloride (NaCl) in the sodium chloride-containing phosphate buffer PBS are 0, 50, 100, 250, and 500 mM, respectively. After bacterial lysis, the supernatant is taken for SDS-PAGE gel electrophoresis detection. Figure 5 As shown, when the NaCl concentration is 0, the soluble protein concentration is the highest, and the soluble protein content is ideal when the NaCl concentration is between 0-100 mM. Considering that NaCl can reduce non-specific binding in chromatography to a certain extent, the concentration of NaCl in the chromatography buffer is 0-100 mM.

[0076] Example 7 Effect of lysis buffer pH on protein purification

[0077] The inoculation and bacterial lysis methods of rhHA-FN in this example are basically the same as those in Example 5, except that when the bacterial cells are lysed, the buffer solution does not contain sodium chloride, and the pH value of the buffer solution is adjusted to 7.0, 7.4, 7.8, and 8.2, respectively. After bacterial lysis, the supernatant is taken for SDS-PAGE gel electrophoresis detection. Figure 6 As shown, it can be seen that when the pH value is about 7.4, the rhHA-FN content in the supernatant is the highest, and when the pH value is 7.8, it is the second highest. Therefore, the pH of the chromatography buffer is about 7.2-7.6.

[0078] Example 8 Effect of chromatography buffer on protein purification

[0079] The preparation method of rhHA-FN inoculation culture protein in this embodiment is basically the same as the steps of Example 2, except that 1% arginine is added to the lysis buffer and the chromatography buffer solution, or arginine is not added to the lysis buffer and the chromatography buffer solution. Ni-NTA affinity chromatography was performed twice in succession, the chromatography sample volume was 300 mL, and the chromatography buffer containing imidazole was used for discontinuous gradient elution. The imidazole concentrations in the phosphate buffer at the peak were 100mM, 200mM, 500mM, and 2M sodium chloride salt wash. Figure 7 As shown, under the chromatography conditions containing arginine, the chromatographic peak is higher than that under the chromatography conditions not containing arginine, and the collected liquid containing the target protein has a larger volume and a higher concentration.

[0080] The above Examples 3-8 are further verified by experimental data: the culture medium formula and bacterial lysis buffer solution formula that are most suitable for the recombinant human fibronectin with multiple functions in the present invention are selected to greatly improve the solubility of the recombinant protein that is originally mostly expressed as inclusion bodies.

[0081] Comparative Example 1

[0082] A vector was constructed to express and purify a recombinant FN protein without the HA binding region, which was named rhFN-without the HA binding region.

[0083] Comparative Example 2

[0084] According to the patent "A preparation method and application of recombinant fibronectin" (China invention patent publication number: CN117586379A), recombinant fibronectin containing fibronectin type III domain 8-10 was prepared.

[0085] The following effect examples are mainly used to verify the cell biological efficacy of rhHA-FN harvested by the method of Examples 2-8 and the recombinant fibronectin in Comparative Examples 1-2.

[0086] Effect Example 1 Expression and purity detection of recombinant human fibronectin with soothing compound function

[0087] SDS-PAGE gel electrophoresis was used to detect protein expression and chromatography purification. The specific steps were as follows: 5×SDS loading buffer was added to the supernatant of bacterial sample lysis or protein sample after chromatography, mixed well, heated the sample at 95-98℃ for 10 minutes, and centrifuged briefly. The protein was separated by electrophoresis using the prepared 10% SDS-PAGE gel. The results were as follows: Figure 8 As shown, the sample has a higher purity after chromatography.

[0088] The purity of the harvested recombinant protein stock solution was detected by high performance liquid chromatography (HPLC). The specific steps are as follows: the concentrated protein sample was filtered through a 0.22 μm filter membrane, added to the loading bottle, and automatically loaded. Fig. 9 As shown, a significant single main peak can be seen in the spectrum, and the peak map analysis results show that the single peak area accounts for about 97.616%, indicating that the purity of the recombinant protein rhHA-FN is greater than 97%.

[0089] Effect Example 2 Detection of the biological activity of recombinant human fibronectin with soothing compound function

[0090] Test 1: Test for promoting the adhesion of human keratinocytes HaCat

[0091] The recombinant human fibronectin rhHA-FN with soothing complex function harvested in Example 1 was treated with a cell culture plate at a concentration of 20 μg / mL, and PBS treatment was used as a control. Six replicate wells were set up in each group to observe the effect of FN on cell adhesion properties. The specific steps are as follows: Coat a 96-well plate with 20 μg / mL of recombinant protein rhHA-FN and treat at room temperature for 1 hour. After absorbing the protein solution, block it with 1% sterile bovine serum albumin BSA at room temperature for half an hour. The HaCat cells were resuspended in serum-free culture medium, and 20,000 cells were plated in each well of the 96 plates. After culturing at 37°C for 2 hours, each well was washed 3 times with PBS, and complete culture medium was added to observe and photograph the cell adhesion state. After overnight culture, the cell proliferation kit CCK8 was added and incubated for 2-4 hours, and the cell activity state was detected using an enzyme marker. The results are as follows Fig.10 , rhHA-FN has a significant effect in promoting cell adhesion.

[0092] Test 2: Detection of the proliferation of human keratinocytes HaCat

[0093] HaCat cells were plated in a 96-well plate, with 4,000 cells per well. The recombinant human fibronectin rhHA-FN with soothing complex function harvested in Example 1 was prepared at a final concentration of 200 μg / mL and added to the 96-well plate. The control group was an equal amount of PBS mixed with cell culture medium, with 6 replicates per group. After 48 hours of culture, CCK8 was added and incubated for 2-4 hours, and cell activity was detected using an ELISA instrument. Fig.11 As shown, rhHA-FN has a significant promoting effect on the proliferation of HaCat cells.

[0094] Test 3: Detection of the alleviating effect of recombinant fibronectin rhHA-FN on the SDS stimulation test of human keratinocytes

[0095] HaCat cells were prepared into a cell suspension and plated in a 96-well plate with about 10,000 cells per well for 18-24 hours. The culture medium in the original well was discarded, and MEM culture medium containing 0.01% SDS and 1% fetal bovine serum FBS was added to each well of the cells. A group of untreated blank controls was left, and the cells were placed in a 37°C incubator for incubation for 30 minutes. The culture medium in the well was discarded, washed once with PBS, and MEM culture medium containing 1% FBS was added to the model group, 1% FBS MEM culture medium containing dexamethasone was added to the positive control group, and 1% FBS MEM culture medium with a final concentration of 200μg / mL or 100μg / mL of rhHA-FN was added to the treatment group. The cells were placed in a 37°C incubator and continued to be cultured for about 24 hours, and then a certain amount of MTT maintenance culture medium was added to each well, incubated for 3-4 hours, the liquid was removed, 100μL DMSO was added to each well, and the mixture was shaken for 10-15 minutes. The absorbance was measured at a wavelength of 570nm using an enzyme reader. The results are as follows Fig.12 As shown, the cell viability of the rhHA-FN protein experimental group was significantly improved compared with the model group, and basically reached the level of the positive group treated with dexamethasone, indicating that the recombinant rhHA-FN protein helps keratinocytes resist SDS stimulation.

[0096] Test 4: Various recombinant fibronectins relieve SDS-stimulated human keratinocytes

[0097] The recombinant fibronectin rhHA-FN prepared by the method described in Example 2 was subjected to a human keratinocyte SDS stimulation experiment simultaneously with Comparative Example 1, Comparative Example 2, and two commercial recombinant human fibronectins, commercial rhFN-1 (full-length FN expressed in rice cells), and commercial rhFN-2 (GMP grade 65 kDa). MEM culture medium containing 0.01% SDS and 1% FBS was added to each well of cells, leaving a group of untreated blank controls, and incubated in a 37°C incubator for 30 minutes. The culture medium in the wells was discarded, and the wells were washed once with PBS. The model group was added with MEM culture medium containing 1% FBS, and the treatment group was added with 1% FBS MEM culture medium containing the above-mentioned recombinant proteins. The concentration gradient of each recombinant fibronectin was: 10, 25, 50, 100, 200 ug / mL, with 5 replicate wells in each group. The cells were placed in a 37°C incubator and continued to be cultured for about 24 hours. CCK8 was added and incubated for 2 hours. The absorbance values ​​of different wells at 450nm were detected by an enzyme reader, and the curve was calibrated according to the concentration gradient. Figure 13-17, respectively, recombinant human fibronectin with composite soothing function, recombinant human fibronectin without HA binding region, recombinant human fibronectin prepared by the patent "A preparation method and application of recombinant fibronectin" (China Invention Patent Publication No.: CN117586379A), commercial rhFN-1 protein, and commercial rhFN-2 protein. In the experiment of soothing SDS-induced keratinocyte stimulation, it can be seen that at different concentrations, the effect of the protein prepared by the present invention is the best.

[0098] Obviously, the embodiments described above are only some embodiments of the present application, rather than all embodiments. The preferred embodiments of the present application are given in the accompanying drawings, but they do not limit the patent scope of the present application. The present application can be implemented in many different forms. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present application more thorough and comprehensive. Although the present application is described in detail with reference to the aforementioned embodiments, for those skilled in the art, it is still possible to modify the technical solutions recorded in the aforementioned specific implementation methods, or to perform equivalent replacement of some of the technical features therein. Any equivalent structure made using the contents of the specification and drawings of this application, directly or indirectly used in other related technical fields, is similarly within the scope of patent protection of this application.

Claims

1. A recombinant human fibronectin with soothing complex function, characterized in that: Its amino acid sequence is shown in SEQ ID NO.

1.

2. The recombinant human fibronectin with soothing complex function according to claim 1, characterized in that: The molecular weight of the recombinant human fibronectin is 95 kDa.

3. A nucleotide encoding the recombinant human fibronectin with soothing complex function as claimed in claim 1, characterized in that: The nucleotide sequence is shown in SEQ ID NO.

4.

4. A method for preparing the recombinant human fibronectin with soothing complex function according to claim 1, characterized in that: It includes the following steps: S1. According to the amino acid sequence SEQ ID NO.1, the codon of the E. coli expression system is optimized to generate the corresponding nucleotide sequence and construct a protein expression vector; S2. The expression vector is transferred into the Escherichia coli expression strain, and the strain is cultured in liquid culture medium; then the bacteria are collected after induction; S3. Lyse the cells with lysis buffer, centrifuge and purify the protein; S4. The purified harvested protein is concentrated by filtration to obtain the recombinant human fibronectin stock solution with soothing complex function.

5. The method for preparing a recombinant human fibronectin with soothing complex function according to claim 4, characterized in that: In step S2, 1-2% W / V sorbitol is added to the liquid culture medium.

6. The method for preparing a recombinant human fibronectin with soothing complex function according to claim 4 or 5, characterized in that: In step S2, the peptone of the liquid culture medium is Angel Yeast FP210, FP220 and / or FP108 peptone, and the inoculation amount of the strain is 1:100; the OD600 value during induction is 0.8-2.0; the induction temperature is set to 20-24°C, and the induction is performed overnight.

7. The method for preparing a recombinant human fibronectin with soothing complex function according to claim 4, characterized in that: The lysis buffer in step S3 is 10-40 mM phosphate buffer, and the sodium chloride concentration is 0-100 mM.

8. The method for preparing a recombinant human fibronectin with soothing complex function according to claim 7, characterized in that: In step S3, 1%-2% arginine is added to the lysis buffer and the chromatography buffer for protein purification, and the pH value is adjusted to 7.2-7.

6.

9. The method for preparing a recombinant human fibronectin with soothing complex function according to claim 4, characterized in that: In the step S3, the purified protein refers to the purification of the protein by affinity chromatography and anion exchange chromatography; the affinity chromatography filler is Ni-NTA filler with polyacrylate microspheres as the matrix material, and the elution conditions are discontinuous gradient, 20mM phosphate buffer for equilibration, 5mM-10mM imidazole buffer for washing, and 300mM imidazole buffer for elution; the anion exchange chromatography uses DEAE Sepharose FF for elution with a sodium chloride concentration gradient.

10. Use of the recombinant human fibronectin with soothing complex function as claimed in claim 1 or the recombinant human fibronectin with soothing complex function obtained by any preparation method of claims 4 to 9 in preparing skin care products.

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