Recombinant human chemical fibronectin as well as coding gene and application thereof

Abstract

The invention discloses recombinant human chemical fibronectin as well as a coding gene and application thereof. The method comprises the following steps: performing codon optimization on an amino acid sequence of recombinant human chemical fibronectin according to codon preference of pichia pastoris to obtain a coding gene nucleotide sequence SEQ ID NO.2, cloning the coding gene nucleotide sequence SEQ ID NO.2 between EcoRI and NotI restriction enzyme cutting sites of a pichia pastoris expression vector pPIC9K to obtain a recombinant expression vector pPIC9K-FNc, and transferring the recombinant expression vector pPIC9K-FNc into a P.pastoris GS115 expression host cell to obtain the recombinant human chemical fibronectin. Recombinant pichia pastoris P.pastoris GS115 / pPIC9K-FNc is obtained through genetic mycin G418 screening, then recombinant bacteria are subjected to fermentation culture and are subjected to induced expression by using methanol, and then the recombinant human chemical fibronectin is obtained after purification. The fibronectin produced by the method is large in molecular weight and has better biological activities such as wrinkle resistance and barrier repair.

Description

Technical Field

[0001] The invention belongs to the technical field of protein engineering and gene engineering, and specifically relates to a recombinant humanized fibronectin and a coding gene and application thereof. Background Art

[0002] Fibronectin (FN) is widely present in the animal kingdom. It is a high molecular weight glycoprotein in the extracellular matrix, with a sugar content of 4.5% to 9.5%. Its subunit molecular weight is 220 to 250 kDa, and it is composed of about 2500 amino acid residues. The subunit structure of FN from different tissues is not exactly the same, but very similar. The common feature of its peptide chain is that it is composed of several globular domains composed of some repeated amino acid sequences. Each globular domain can bind to different macromolecules or cell surface specific receptors, making it a multifunctional molecule. FN is widely distributed in the body, existing in soluble form in plasma and various body fluids, and in insoluble form in the extracellular matrix and cell surface.

[0003] As a matrix for cell culture, fibronectin can improve the adhesion rate and confluence rate of various cells, enhance the cell metabolism rate, and significantly increase the synthesis rate of DNA, RNA and protein. Applying fibronectin to microsphere carriers as a medium for mass production of cells can save space and raw materials, and become the basic material for the production of new drugs using large-scale cell culture technology. In the repair and healing of skin wounds, fibronectin can shorten the wound healing time and reduce wound scars. In skin care, the repair effect of fibronectin is manifested in promoting normal physiological metabolism of the skin, smoothing fine lines, delaying aging, and preventing abnormal secretion of melanocytes, that is, whitening and lightening. Therefore, fibronectin has broad application prospects in the fields of medicine, beauty, and skin care.

[0004] Fibronectin is widely used, but the yield of natural fibronectin extracted from tissues is extremely limited, and its application is limited by its high cost and low purity. The use of genetic engineering technology to design recombinant fibronectin fragments with specific biological functions and achieve their efficient expression in microbial expression systems has become a major trend in fibronectin production. At present, most recombinant fibronectin studies are on the expression of truncated fragments of fibronectin, with molecular weights ranging from 15-40kDa (such as CN110590939B, CN115785280A, CN117586379A, etc.). Fibronectin is a macromolecular functional protein with a molecular weight of 220 to 250kDa. Truncated fragments with too small a molecular weight cannot play a good role in the biological function of human fibronectin. Summary of the invention

[0005] In view of the deficiencies of the prior art, the present invention provides a recombinant humanized fibronectin and its encoding gene and application. The present invention first optimizes the amino acid sequence of the recombinant humanized fibronectin shown in SEQ ID NO.1 according to the codon preference of Pichia pastoris to obtain the encoding gene nucleotide sequence SEQ ID NO.2, and then clones it between the EcoRI and NotI restriction sites of the Pichia pastoris expression vector pPIC9K to obtain the recombinant expression vector pPIC9K-FNc, and then transfers it into the P. pastoris GS115 expression host cell, and obtains the high-copy recombinant Pichia pastoris P. pastoris GS115 / pPIC9K-FNc by screening with geneticin G418. Then, the recombinant bacteria P. pastoris GS115 / pPIC9K-FNc are fermented and cultured and induced to express using methanol, and then purified to obtain the recombinant humanized fibronectin FNc. Experiments have shown that the fibronectin produced by the present invention has a large molecular weight, can better exert the biological function of humanized fibronectin, and has better biological activities such as anti-wrinkle and barrier repair.

[0006] In order to achieve the above technical objectives, the technical solution of the present invention is as follows:

[0007] A recombinant humanized fibronectin, whose amino acid sequence is shown in SEQ ID NO.1.

[0008] A gene encoding recombinant humanized fibronectin, which encodes the above recombinant humanized fibronectin. Preferably, the nucleotide sequence of the encoding gene is as shown in SEQ ID NO.2, and is named FNc.

[0009] A recombinant expression vector containing the above-mentioned recombinant humanized fibronectin coding gene. Preferably, the plasmid vector of the recombinant expression vector is pPIC9K, and the recombinant expression vector is pPIC9K-FNc.

[0010] A recombinant expression strain containing the above-mentioned recombinant humanized fibronectin coding gene or the above-mentioned recombinant expression vector. Preferably, the host bacteria of the recombinant expression strain is Pichia pastoris. Further preferably, the Pichia pastoris is GS115, and the recombinant expression strain is GS115 / pPIC9K-FNc.

[0011] The invention also discloses the application of the recombinant expression strain in preparing recombinant humanized fibronectin.

[0012] The present invention also discloses a method for preparing recombinant humanized fibronectin by using the above-mentioned recombinant expression strain, comprising the steps of: fermenting and culturing the above-mentioned recombinant expression strain, and inducing the expression of recombinant humanized fibronectin by using methanol; specifically: picking a single clone and inoculating it in a YPD culture medium, and culturing it at 28-32°C for 12-36h; then transferring it to an initial expression culture medium BMGY at an inoculation rate of 5-10%, and culturing it at 28-32°C for 12-36h; collecting the bacterial cells by centrifugation, washing the bacterial cells with physiological saline, and then replacing them with an induced expression culture medium BMMY, culturing at 28-32°C, adding pure methanol to the culture medium every 24h to a final concentration of 1.0% (v / v), and inducing expression for 72-108h.

[0013] The initial expression medium BMGY was composed of: yeast extract 10 g / L, peptone 20 g / L, K2HPO4 3 g / L, KH2PO4 11.8 g / L, YNB 3.4 g / L, ammonium sulfate 10 g / L, biotin 4 × 10 -4 g / L, glycerol 10g / L.

[0014] The induction expression medium BMMY was composed of: yeast extract 10 g / L, peptone 20 g / L, K2HPO4 3 g / L, KH2PO4 11.8 g / L, YNB 3.4 g / L, ammonium sulfate 10 g / L, biotin 4 × 10 -4 g / L, methanol 10mL / L.

[0015] The above-mentioned method for purifying recombinant humanized fibronectin comprises the steps of: centrifuging the fermentation broth after the above-mentioned recombinant expression strain is induced and cultured to obtain a supernatant, and then clarifying the supernatant with a hollow fiber membrane, concentrating and replacing with an ultrafiltration membrane, anion exchange chromatography, ultrafiltration desalting and freeze-drying to obtain a purified recombinant humanized fibronectin. Preferably, the molecular weight cutoff of the hollow fiber membrane is 500kDa, and the molecular weight cutoff of the ultrafiltration membrane is 10kDa.

[0016] The use of the recombinant humanized fibronectin prepared by the above method in the preparation of food, medicine, cosmetics, health care products or device products.

[0017] The technical effects of the present invention are as follows: the fibronectin produced by the present invention has a large molecular weight and can better exert the biological function of human fibronectin; it does not contain any exogenous amino acids such as labels, is recombinant humanized fibronectin, and has good safety; it has better biological activities such as anti-wrinkle and barrier repair; and it has a high protein yield and low purification cost, and is more suitable for industrial large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1The results of SDS-PAGE protein electrophoresis and Western Blot of the fermentation supernatant of the recombinant humanized fibronectin FNc in Example 3 of the present invention are shown; wherein, Figure A is an SDS-PAGE protein electrophoresis diagram, in which lane M represents a standard protein with a molecular weight of 180 kDa; lane 1 represents the fermentation supernatant of the control bacteria P. pastoris GS115 / pPIC9K; lane 2 represents the fermentation supernatant of the recombinant bacteria P. pastoris GS115 / pPIC9K-FNc; and Figure B is a Western-Blot result diagram;

[0019] Figure 2 This is a comparison chart of the results of promoting the secretion of COL-I in HSF cells by different samples in Example 5 of the present invention; wherein, control group 1 represents a blank control group; control group 2 represents a commercially available recombinant fibronectin group; and the experimental group represents a recombinant humanized fibronectin FNc group prepared by the present invention; in the figure, compared with control group 1, P<0.01 (**), P<0.001 (***), which is considered to have significant differences;

[0020] Figure 3 It is a bar graph of the relative mRNA expression of FLG in HaCaT cells under the action of different samples in Example 6 of the present invention; wherein, control group 1 represents a blank control group; control group 2 represents a commercially available recombinant fibronectin group; and the experimental group represents a recombinant humanized fibronectin FNc group prepared by the present invention; in the figure, compared with control group 1, P<0.0001 (****), which is considered to be significantly different. DETAILED DESCRIPTION

[0021] The effects are described below in conjunction with the embodiments and drawings.

[0022] Example 1: Sequence design of recombinant humanized fibronectin

[0023] The natural human fibronectin amino acid sequence (Swiss-Prot: P02751.5) was obtained using the NCBI database, and the sequence information was deeply mined and analyzed through the AlphaFold protein structure database. The specific structural domains and corresponding functions of the sequence were mastered to perform sequence design, and the hydrophilicity and charge distribution of amino acids were analyzed using bioinformatics and other means, so that the recombinant humanized fibronectin can achieve higher expression efficiency and high stability while maintaining high biological activity. The recombinant collagen of the present invention has a total of 831 amino acids, and the specific amino acid sequence is shown in SEQ ID NO.1. The sequence contains the collagen binding domain, integrin binding site and type III FN functional fragments of human fibronectin, which ensures the realization of the biological function of recombinant fibronectin, and the sequence does not contain any exogenous amino acids such as labels, which is a recombinant humanized fibronectin with high biocompatibility and low risk of use.

[0024] Example 2: Construction of recombinant bacteria P. pastoris GS115 / pPIC9K-FNc

[0025] According to the codon preference of Pichia pastoris, the amino acid sequence of the recombinant humanized fibronectin shown in SEQ ID NO.1 in Example 1 was codon optimized, and the optimized nucleotide sequence of the coding gene was shown in SEQ ID NO.2, named FNc. The nucleotide sequence of FNc was commissioned to Nanjing GenScript Biotechnology Co., Ltd. for full gene synthesis, and cloned between the EcoRI and NotI restriction sites of the Pichia pastoris expression vector pPIC9K to obtain the recombinant expression vector pPIC9K-FNc. After DNA sequencing and comparison, the recombinant sequence was correct. The recombinant expression plasmid pPIC9K-FNc was linearized by SalI fast cutting enzyme and then electroporated into the Pichia pastoris P. pastoris GS115 expression host cell, and the recombinant transformant was screened by geneticin G418 to obtain a high copy recombinant Pichia pastoris P. pastoris GS115 / pPIC9K-FNc.

[0026] Example 3: Inducible expression of recombinant bacteria P. pastoris GS115 / pPIC9K-FNc

[0027] The obtained recombinant engineered bacteria P. pastoris GS115 / pPIC9K-FNc was fermented in a shake flask. The fermentation steps are as follows: Pick a single clone and inoculate it in 40mL of YPD medium (yeast extract 10g / L, peptone 20g / L, glucose 20g / L), and culture it at 30°C and 200rpm for 24h. Transfer the inoculum to 40mL of the initial expression medium BMGY (yeast extract 10g / L, peptone 20g / L, K2HPO4 3g / L, KH2PO4 11.8g / L, YNB 3.4g / L, ammonium sulfate 10g / L, biotin 4×10 -4 g / L, glycerol 10g / L), 30℃, 200rpm culture for 24h. The cells were collected by centrifugation, washed with physiological saline and replaced with 40mL induced expression medium BMMY (yeast extract 10g / L, peptone 20g / L, K2HPO4 3g / L, KH2PO411.8g / L, YNB 3.4g / L, ammonium sulfate 10g / L, biotin 4×10 -4 g / L, methanol 10mL / L), cultured at 30°C, 200rpm, pure methanol was added to the culture medium every 24h to a final concentration of 1.0% (v / v), and induced expression for 96h. Centrifugation was performed to obtain the fermentation supernatant of the recombinant engineered bacteria for testing.

[0028] A blank control strain (P. pastoris GS115 / pPIC9K) was constructed, and the above shake flask fermentation culture and methanol induction steps were repeated, and the obtained fermentation supernatant was used for comparative test.

[0029] The recombinant engineered bacteria fermentation supernatant and the control bacteria (P. pastoris GS115 / pPIC9K) fermentation supernatant were subjected to SDS-PAGE protein electrophoresis analysis. The theoretical size of the recombinant humanized fibronectin is 92.5 kDa. The SDS-PAGE results are as follows: Figure 1 As shown in A, the fermentation supernatant of the recombinant bacteria P. pastoris GS115 / pPIC9K-FNc (lane 2) compared with the blank control bacteria P. pastoris GS115 / pPIC9K (lane 1) has an obvious protein band (indicated by the arrow) above the theoretical size (the apparent molecular weight of this detection method is usually greater than the theoretical molecular weight). Further Western-Blot detection, the test results are as follows Figure 1 As shown in B, the band at this position is determined to be the recombinant humanized fibronectin FNc expressed by the constructed engineering bacteria. The above results show that the high molecular weight recombinant humanized fibronectin of the present invention can be successfully induced to express.

[0030] Example 4: Purification and preparation of recombinant humanized fibronectin FNc

[0031] After the recombinant bacteria P. pastoris GS115 / pPIC9K-FNc were subjected to large-scale shake flask fermentation according to the method of Example 3, the fermentation supernatant after the fermentation broth was centrifuged was subjected to the following purification steps to obtain a pure recombinant humanized fibronectin FNc product, and the specific steps are as follows:

[0032] ① Clarification: The fermentation supernatant is filtered through a 500kDa hollow fiber membrane to sterilize and remove large molecular impurities.

[0033] ②Ultrafiltration membrane concentration and replacement: Use a 10kDa ultrafiltration membrane to concentrate the feed solution and replace it with ion A solution (20mMTris, pH8.0) until the conductivity of the permeate end is equivalent to that of ion A solution;

[0034] ③ Ion exchange chromatography: Anion exchange chromatography (filler Q Sepharose Fast Flow) was used. The recombinant humanized fibronectin was negatively charged in a pH 8.0 buffer and eluted with 150 mM sodium chloride (pH 8.0), and the elution peak was collected.

[0035] ④Ultrafiltration membrane desalination: Use a 10kDa ultrafiltration membrane package to desalinate the feed solution until the conductivity at the permeate end drops to 70μS / cm;

[0036] ⑤ Freeze-drying: The ultrafiltration concentrate obtained above was filtered through a 0.45 μm microfiltration membrane and placed in a freeze-drying tray for freeze-drying.

[0037] The purity of the protein FNc purified through the above steps is greater than 95%, and the yield is greater than 60%.

[0038] Example 5: Anti-wrinkle experiment

[0039] Collagen is one of the important components of the dermis, providing support for the skin and giving it elasticity and texture. Type I collagen (COL-I) accounts for 80-85% of the collagen in the skin. Promoting the increase in COL-I content can achieve a certain effect in resisting the formation of wrinkles.

[0040] Human skin fibroblasts (HSF cells) with good growth status were collected and cell suspension was prepared with high-glucose DMEM cell culture medium. 2 mL of cell suspension was added to each well of a 6-well plate. The number of cells was 2.6×10 5 / well. The experiment set up a blank control group (control group 1), a commercially available recombinant fibronectin (Escherichia coli source, molecular weight 62.6kDa, 574 amino acids, purity greater than 95%) control group (control group 2), and a recombinant humanized fibronectin FNc group prepared by the present invention (experimental group), and each group was set up with 3 duplicate wells. The 6-well plate was placed in a cell culture incubator (5% CO2, 37°C) and incubated for 24 hours, and the cell fusion rate reached 60% to 70%. The culture medium was discarded, and 2mL of low-glucose DMEM cell culture medium was added to each well of the blank control group; 2mL of low-glucose DMEM cell culture medium containing the corresponding fibronectin was added to the remaining groups, wherein the concentration of recombinant fibronectin in the low-glucose DMEM cell culture medium was 1mg / mL. After the administration was completed, the 6-well plate was placed in an incubator (5% CO2, 37°C) and cultured for 24 hours, and then the HSF cell culture medium was collected, and the cell precipitate was collected after centrifugation at 12000rpm for 10 minutes, and the type I collagen content was detected according to the instructions of the ELISA kit.

[0041] The results of different collagens promoting the secretion of COL-I in HSF cells are as follows Figure 2 As shown, compared with the blank control group (control group 1), both the commercially available recombinant fibronectin (control group 2) and the recombinant humanized fibronectin FNc prepared by the present invention (experimental group) can increase the secretion of COL-I, among which the recombinant humanized fibronectin FNc prepared by the present invention has a stronger ability to promote the secretion of COL-1 than the commercially available recombinant fibronectin. Therefore, the recombinant humanized fibronectin FNc of the present invention has an obvious anti-wrinkle effect.

[0042] Example 6: Barrier repair experiment

[0043] Filaggrin (FLG) is one of the important components of the cornified envelope, ensuring the integrity of the skin barrier. FLG deficiency can lead to disordered lipid bilayer structure and delayed maturation, while also causing reduced compactness of stratum corneum cells, increased intercellular permeability and decreased photoprotection, ultimately leading to damage to the skin barrier.

[0044] Human immortalized keratinocytes (HaCaT cells) were collected and cell suspension was prepared with high-glucose DMEM cell culture medium. 2 mL of cell suspension was added to each well of a 6-well plate. The cell number was 2.6 × 10 5 / well. The experiment set up a blank control group (control group 1), a commercially available recombinant fibronectin (Escherichia coli source, molecular weight 62.6kDa, 574 amino acids, purity greater than 95%) control group (control group 2), and a recombinant humanized fibronectin FNc group prepared by the present invention (experimental group), and each group was set up with 3 duplicate wells. The 6-well plate was placed in a cell culture incubator (5% CO2, 37°C) and incubated for 24 hours. When the cell fusion rate reached 50% to 60%, the culture medium was discarded, and 2 mL of high-glucose DMEM cell culture medium was added to each well of the blank control group; 2 mL of high-glucose DMEM cell culture medium containing the corresponding recombinant fibronectin was added to the remaining groups, wherein the concentration of recombinant humanized fibronectin FNc in the high-glucose DMEM cell culture medium was 1 mg / mL. The 6-well plate was placed in an incubator (5% CO2, 37°C) and incubated for 24 h. The cells were washed twice with 2 mL of PBS buffer in each well. According to the Total RNA extraction kit, 1 mL of RNAiso Plus was added, and the cells were lysed by blowing and collecting samples. According to the instructions of the kit, RNA extraction, reverse transcription and fluorescence quantitative PCR detection experiments were carried out to detect the relative expression of mRNA of barrier-related protein FLG. -△△CT Method to calculate.

[0045] The ability of different samples to promote the mRNA expression of FLG in HaCaT cells Figure 2 As shown, compared with the blank control group (control group 1), the commercially available recombinant fibronectin (control group 2) and the recombinant humanized fibronectin FNc prepared by the present invention (experimental group) can increase the relative mRNA expression of FLG. The recombinant humanized fibronectin FNc prepared by the present invention has a higher ability to increase the relative mRNA expression of FLG than the commercially available recombinant fibronectin. Therefore, the recombinant humanized fibronectin FNc prepared by the present invention has a more obvious skin barrier repair function than the commercially available recombinant fibronectin.

Claims

1. A recombinant humanized fibronectin, the amino acid sequence of which is shown in SEQ ID NO.

1.

2. A gene encoding recombinant humanized fibronectin, which encodes the recombinant humanized fibronectin according to claim 1, which is FNc, and the nucleotide sequence is shown in SEQ ID NO.

2.

3. A recombinant expression vector comprising the gene encoding the recombinant humanized fibronectin according to claim 2, wherein the plasmid vector of the recombinant expression vector is pPIC9K, and the recombinant expression vector is pPIC9K-FNc.

4. A recombinant expression strain, comprising the gene encoding the recombinant humanized fibronectin according to claim 2 or the recombinant expression vector according to claim 3, wherein the host bacteria of the recombinant expression strain is Pichia pastoris.

5. The recombinant expression strain according to claim 4, characterized in that: The Pichia pastoris is GS115, and the recombinant expression strain is GS115 / pPIC9K-FNc.

6. Use of the recombinant expression strain according to claim 4 or 5 in preparing recombinant humanized fibronectin.

7. A method for preparing recombinant humanized fibronectin using the recombinant expression strain according to claim 5, characterized in that: A single clone was picked and inoculated into YPD medium and cultured at 28-32°C for 12-36h; then, it was transferred to the initial expression medium BMGY at an inoculation rate of 5-10% and cultured at 28-32°C for 12-36h; the bacteria were collected by centrifugation, washed with physiological saline and replaced with the induced expression medium BMMY, cultured at 28-32°C, pure methanol was added to the culture medium every 24h to a final concentration of 1.0%, the expression was induced for 72-108h, and the recombinant humanized fibronectin was obtained by further purification.

8. The method for preparing recombinant humanized fibronectin according to claim 7, characterized in that: The initial expression medium BMGY was composed of: yeast extract 10 g / L, peptone 20 g / L, K2HPO4 3 g / L, KH2PO4 11.8 g / L, YNB 3.4 g / L, ammonium sulfate 10 g / L, biotin 4×10 -4 g / L, glycerol 10g / L. The induction expression medium BMMY is composed of: yeast extract 10 g / L, peptone 20 g / L, K2HPO4 3 g / L, KH2PO4 11.8 g / L, YNB 3.4 g / L, ammonium sulfate 10 g / L, biotin 4×10 -4 g / L, methanol 10mL / L.

9. The method for preparing recombinant humanized fibronectin according to claim 7, characterized in that: The method for purifying recombinant humanized fibronectin comprises the following steps: the fermentation liquid after induction culture is centrifuged to obtain a supernatant, the supernatant is clarified by a hollow fiber membrane, concentrated and replaced by an ultrafiltration membrane, anion exchange chromatography, ultrafiltration desalination and freeze-dried to obtain a purified recombinant humanized fibronectin; the molecular weight cutoff of the hollow fiber membrane is 500kDa, and the molecular weight cutoff of the ultrafiltration membrane is 10kDa.

10. Use of the recombinant humanized fibronectin prepared by the method according to any one of claims 7 to 9 in the preparation of food, medicine, cosmetics, health care products or device products.

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