Recombinant humanized fibronectin and its encoding gene and application

Abstract

The application discloses a kind of recombinant human fibronectin and its coding gene and application.The amino acid sequence of the recombinant human fibronectin is codon-optimized according to the codon preference of Pichia pastoris first, and the nucleotide sequence of the coding gene is obtained as SEQ ID NO.2, then it is cloned into the EcoRI and NotI enzyme cutting sites between pPIC9K of Pichia pastoris expression vector, and the recombinant expression vector pPIC9K-FNc is obtained, and then it is transferred into P.pastoris GS115 expression host cell, and recombinant P.pastoris GS115 / pPIC9K-FNc is obtained by geneticin G418 screening, then the recombinant bacteria are fermented and expressed by methanol induction, and the recombinant human fibronectin is obtained after purification.The fibronectin produced by the application has large molecular weight and better biological activities such as wrinkle resistance and barrier repair.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of protein engineering and genetic engineering, and particularly relates to a recombinant humanized fibronectin, a coding gene thereof and an application thereof. BACKGROUND

[0002] Fibronectin (FN) is widely present in the animal kingdom and is a high relative molecular mass glycoprotein in the extracellular matrix, containing 4.5% to 9.5% of sugar, with a subunit molecular weight of 220 to 250 kDa, and being composed of about 2500 amino acid residues. The FN subunits from different tissues are not exactly the same, but are very similar. The common feature of the peptide chains is that they are composed of several spherical domains of repeated amino acid sequences, and each spherical domain can be combined with different macromolecules or cell surface specific receptors, thereby making it a multifunctional molecule. FN is widely distributed in the body, and exists in a soluble form in plasma and various body fluids, and in an insoluble form in the extracellular matrix and cell surface.

[0003] As a substrate for cell culture, fibronectin can improve the adhesion rate, confluence rate and metabolic rate of various cells, and significantly increase the synthesis rate of DNA, RNA and protein. Coating fibronectin on microsphere carriers as a medium for large-scale cell production can save space and raw materials, and become a basic material for the production of new drugs by applying scale cell culture technology. In skin wound repair and healing, fibronectin can shorten the wound healing time and reduce the scar of the wound. In terms of skin care, the repair effect of fibronectin is to promote normal physiological metabolism of the skin, smooth fine lines, delay aging, and prevent abnormal secretion of melanocytes, that is, to whiten and fade spots. Therefore, fibronectin has a wide application prospect 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 the high cost and low purity limit its application. Using genetic engineering technology to design a recombinant fibronectin fragment with specific biological functions and realize its efficient expression in a microbial expression system has become a trend in fibronectin production. At present, most of the research on recombinant fibronectin is the expression of truncated fragments of fibronectin, with a molecular weight of 15-40 kDa (such as CN110590939B, CN115785280A, CN117586379A, etc.). Fibronectin is a large functional protein with a molecular weight of 220-250 kDa, and the truncated fragment with too small a molecular weight cannot well exert the biological function of human fibronectin. SUMMARY

[0005] In view of the deficiencies of the prior art, the present application provides a recombinant humanized fibronectin, a coding gene thereof and an application thereof. The present application first optimizes the amino acid sequence of the recombinant humanized fibronectin shown in SEQ ID NO. 1 according to the codon bias of Pichia pastoris to obtain a coding gene nucleotide sequence SEQ ID NO. 2, then clones it between the EcoRI and NotI enzyme cutting sites of the Pichia pastoris expression vector pPIC9K to obtain a recombinant expression vector pPIC9K-FNc, and then transfers it into a P.pastoris GS115 expression host cell to obtain a high-copy recombinant P.pastoris GS115 / pPIC9K-FNc through geneticin G418 screening. Then the recombinant bacteria P.pastoris GS115 / pPIC9K-FNc is subjected to fermentation culture and methanol induction expression, and the recombinant humanized fibronectin FNc is obtained after purification. It has been proved through tests that the fibronectin produced by the present application has a large molecular weight and can better exert the biological functions of human fibronectin, and has better biological activities such as anti-wrinkle and barrier repair.

[0006] In order to achieve the above technical purposes, the technical scheme of the present application is as follows:

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

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

[0009] A recombinant expression vector containing the coding gene of the above-mentioned recombinant humanized fibronectin. 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 coding gene of the above-mentioned recombinant humanized fibronectin 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 present application also discloses the application of the above-mentioned recombinant expression strain in the preparation of a recombinant humanized fibronectin.

[0012] The application further discloses a method for preparing the recombinant humanized fibronectin by using the recombinant expression strain, which comprises the following steps: fermenting and culturing the recombinant expression strain, and inducing the expression of the recombinant humanized fibronectin by using methanol; specifically, a single colony is inoculated in a YPD culture medium and cultured at 28-32 DEG C for 12-36 hours; then, the single colony is inoculated in an initial expression culture medium BMGY at a inoculation amount of 5-10%, and cultured at 28-32 DEG C for 12-36 hours; the bacterial bodies are collected by centrifugation, washed with normal saline, and then replaced into an induction expression culture medium BMMY, and cultured at 28-32 DEG C; pure methanol is added into the culture medium every 24 hours to reach a final concentration of 1.0% (v / v), and the induction expression is performed for 72-108 hours.

[0013] The initial expression culture medium BMGY is 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, and glycerol 10 g / L.

[0014] The induction expression culture medium BMMY is 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, and methanol 10 mL / L.

[0015] The purification method of the recombinant humanized fibronectin comprises the following steps: the fermentation liquor of the recombinant expression strain after induction culture is centrifuged to obtain supernatant, the supernatant is clarified by a hollow fiber membrane, concentrated by an ultrafiltration membrane, subjected to anion exchange chromatography, desalted by ultrafiltration, and then freeze-dried to obtain the purified product of the recombinant humanized fibronectin. Preferably, the molecular weight cut-off of the hollow fiber membrane is 500 kDa, and the molecular weight cut-off of the ultrafiltration membrane is 10 kDa.

[0016] The recombinant humanized fibronectin prepared by the method is applied to the preparation of food, medicine, cosmetics, health products or instrument products.

[0017] The fibronectin produced by the application has a large molecular weight, can better exert the biological function of human fibronectin, is a recombinant humanized fibronectin without any label or other exogenous amino acids, has good safety, has better biological activities such as wrinkle resistance and barrier repair, has high protein yield, has low purification cost, and is more suitable for industrial large-scale production. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1SDS-PAGE protein electrophoresis and Western Blot results of the fermentation supernatant of the recombinant humanized fibronectin FNc in Example 3 of the present application; wherein, Figure A is the SDS-PAGE protein electrophoresis chart, and in the chart, lane M represents standard proteins 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 the Western-Blot results chart;

[0019] Figure 2 Comparison chart of the COL-I secretion promoting abilities of different samples in Example 5 of the present application; wherein, control group 1 represents the blank control group; control group 2 represents the commercially available recombinant fibronectin group; and the experimental group represents the recombinant humanized fibronectin FNc group prepared by the present application; in the chart, P<0.01(**), P<0.001(***) compared with the control group 1, and it is considered to have a significant difference;

[0020] Figure 3 Column chart of the mRNA relative expression amount of FLG in HaCaT cells under the action of different samples in Example 6 of the present application; wherein, control group 1 represents the blank control group; control group 2 represents the commercially available recombinant fibronectin group; and the experimental group represents the recombinant humanized fibronectin FNc group prepared by the present application; in the chart, P<0.0001(****) compared with the control group 1, and it is considered to have a significant difference. DETAILED DESCRIPTION

[0021] The effects will be described below in combination with the examples and the accompanying drawings.

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

[0023] The natural human fibronectin amino acid sequence (Swiss-Prot: P02751.5) is obtained by using the NCBI database, the sequence information is analyzed in depth by using the AlphaFold protein structure database, the specific domain and the corresponding function of the sequence are mastered after the sequence design, and the hydrophobicity, charge distribution and the like of the amino acid are analyzed by using bioinformatics and the like, so that the recombinant humanized fibronectin realizes higher expression efficiency and higher stability on the premise of maintaining high biological activity. The recombinant collagen protein has 831 amino acids, and the specific amino acid sequence is shown as SEQ ID NO. 1. The sequence includes the collagen binding domain, the integrin binding site and the functional fragment of type III FN of human fibronectin, which ensures the realization of the biological function of the recombinant fibronectin, and the sequence does not contain any tag and other exogenous amino acids. The recombinant humanized fibronectin has high biocompatibility and low use risk.

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

[0025] The amino acid sequence of the recombinant humanized fibronectin shown in SEQ ID NO. 1 was codon-optimized according to the codon bias of Pichia pastoris. The optimized nucleotide sequence of the coding gene is shown in SEQ ID NO. 2, named FNc. The nucleotide sequence of FNc was synthesized by Nanjing Kingsriver Biotech Co., Ltd. and cloned into the EcoRI and NotI enzyme cutting sites of the Pichia pastoris expression vector pPIC9K to obtain the recombinant expression vector pPIC9K-FNc. The recombinant sequence was correct after DNA sequencing comparison. The recombinant expression plasmid pPIC9K-FNc was linearized by SalI fast enzyme and electroporated into Pichia pastoris GS115 expression host cells. The recombinant transformants were screened by geneticin G418 to obtain high-copy recombinant Pichia pastoris GS115 / pPIC9K-FNc.

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

[0027] The obtained recombinant engineering bacteria P. pastoris GS115 / pPIC9K-FNc were subjected to shake flask fermentation culture. The fermentation steps were as follows: a single colony was inoculated in 40 mL of YPD culture medium (yeast extract 10 g / L, peptone 20 g / L, glucose 20 g / L) and cultured at 30°C, 200 rpm for 24 h. The inoculum was transferred to 40 mL of initial expression medium BMGY (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 x 10 -4 g / L, glycerol 10 g / L) at a 10% inoculum, and cultured at 30°C, 200 rpm for 24 h. The bacterial cells were collected by centrifugation and washed with physiological saline, and then replaced into 40 mL of induction expression medium BMMY (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 x 10 -4 g / L, methanol 10 mL / L) and cultured at 30°C, 200 rpm. Pure methanol was added to the culture medium every 24 h to a final concentration of 1.0% (v / v) for induction expression for 96 h. The fermentation supernatant of the recombinant engineering bacteria was obtained by centrifugation and subjected to tests.

[0028] The blank control bacteria (P. pastoris GS115 / pPIC9K) was constructed, and the shake flask fermentation culture and methanol induction steps were repeated, and the obtained fermentation supernatant was subjected to comparative test.

[0029] The fermentation supernatant of the recombinant engineering bacteria was subjected to SDS-PAGE protein electrophoresis analysis together with the fermentation supernatant of the control bacteria (P. pastoris GS115 / pPIC9K). The theoretical size of the recombinant humanized fibronectin is 92.5 kDa, and the SDS-PAGE result is as shown in Figure 1 As shown in FIG. 2A, the fermentation supernatant (lane 2) of the recombinant bacteria P. pastoris GS115 / pPIC9K-FNc has an obvious protein band (indicated by an arrow) above the theoretical size (the apparent molecular weight of the detection method is usually greater than the theoretical molecular weight) compared with the blank control bacteria P. pastoris GS115 / pPIC9K (lane 1). Further Western-Blot detection was performed, and the detection result is as shown in Figure 1 As shown in FIG. 2B, it is determined that the position band is 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 application can be successfully induced and expressed.

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

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

[0032] ① Clarification: The fermentation supernatant was filtered through a 500 kDa hollow fiber membrane to remove bacteria and remove macromolecular impurities.

[0033] ② Ultrafiltration membrane concentration replacement: The feed liquid was concentrated by a 10 kDa ultrafiltration membrane, and at the same time, ion A liquid (20 mM Tris, pH 8.0) was used for replacement until the conductivity of the permeate end was equivalent to that of the ion A liquid;

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

[0035] ④ Ultrafiltration membrane desalination: The feed liquid was desalted by a 10 kDa ultrafiltration membrane until the conductivity of the permeate end was reduced to 70 μS / cm;

[0036] ⑤ Freeze-drying: The ultrafiltration concentrated solution obtained above is filtered by a 0.45 μm microfiltration membrane and then placed in a freeze-drying tray for freeze-drying.

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

[0038] Example 5: Anti-wrinkle experiment

[0039] Collagen is one of the important components of the dermis layer, which provides support for the skin and makes the skin elastic and textured. Among them, type I collagen (COL-I) accounts for 80-85% of the collagen component of the skin, and increasing the content of COL-I can achieve the effect of resisting the formation of wrinkles.

[0040] Human skin fibroblasts (HSF cells) in a good growth state are collected, and a cell suspension is prepared with high-glucose DMEM cell culture solution. 2 mL of the cell suspension is added to each well of a 6-well plate, and the number of cells is 2.6 x 10 5 / well. A blank control group (control group 1), a commercially available recombinant fibronectin (E. coli source, molecular weight 62.6 kDa, 574 amino acids, purity greater than 95%) control group (control group 2), and a recombinant humanized fibronectin FNc group (experimental group) prepared by the present application are set up, with 3 replicate wells in each group. The 6-well plate is incubated in a cell culture incubator (5% CO2, 37°C) for 24 h, and the cell fusion rate reaches 60%-70%. The culture solution is discarded, 2 mL of low-glucose DMEM cell culture solution is added to each well of the blank control group, and 2 mL of low-glucose DMEM cell culture solution containing the corresponding fibronectin is added to each well of the other groups. The concentration of the recombinant fibronectin in the low-glucose DMEM cell culture solution is 1 mg / mL. After the administration is completed, the 6-well plate is placed in the incubator (5% CO2, 37°C) for 24 h, and then the HSF cell culture solution is collected. The cells are collected by centrifugation at 12,000 rpm for 10 min, and the content of type I collagen is detected according to the ELISA kit instructions.

[0041] The results of the ability of different collagens to promote the secretion of COL-I by HSF cells are shown in Table 1. Figure 2 As shown in Table 1, 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 application (experimental group) can all increase the secretion of COL-I. The recombinant humanized fibronectin FNc prepared by the present application has a stronger ability to promote the secretion of COL-I than the commercially available recombinant fibronectin, so the recombinant humanized fibronectin FNc of the present application has a significant effect of resisting wrinkles.

[0042] Example 6: Barrier repair experiment

[0043] Filaggrin (FLG) is one of the important components of the keratin envelope, which guarantees the integrity of the skin barrier. The lack of FLG will cause the disorder of the lipid bilayer structure, the delay of maturation, and at the same time, cause the decrease of the tightness of the keratin layer cells, the increase of the permeability between cells and the decrease of the light protection, finally leading to the damage of the skin barrier.

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

[0045] The mRNA expression promoting ability of different samples in HaCaT cells is shown in Figure 2 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 application (experimental group) can all increase the relative expression of the mRNA of FLG. The recombinant humanized fibronectin FNc prepared by the present application has a higher ability to increase the relative expression of the mRNA of FLG than the commercially available recombinant fibronectin, so the recombinant humanized fibronectin FNc prepared by the present application 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 as SEQ ID NO.

1.

2. A gene encoding the recombinant humanized fibronectin of claim 1, which is FNc, the nucleotide sequence of which is shown as SEQ ID NO.

2.

3. A recombinant expression vector containing the gene encoding the recombinant humanized fibronectin of claim 2, the plasmid vector of the recombinant expression vector being pPIC9K, and the recombinant expression vector being pPIC9K-FNc.

4. A recombinant expression strain containing the gene encoding the recombinant humanized fibronectin of claim 2 or the recombinant expression vector of claim 3, the host strain of the recombinant expression strain being Pichia pastoris.

5. The recombinant expression strain of claim 4, wherein, The Pichia pastoris is GS115, and the recombinant expression strain is GS115 / pPIC9K-FNc.

6. Use of the recombinant expression strain of claim 4 or 5 in the preparation of recombinant humanized fibronectin.

7. A method for producing recombinant humanized fibronectin using the recombinant expression strain of claim 5, characterized in that, The single clone is inoculated in YPD medium and cultured at 28-32℃ for 12-36 h, then inoculated in the initial expression medium BMGY at a ratio of 5-10%, and cultured at 28-32℃ for 12-36 h, then centrifuged to collect the bacterial cells, washed with physiological saline, and then transferred to the induction expression medium BMMY, and cultured at 28-32℃, and pure methanol is added to the medium every 24 h to a final concentration of 1.0%, and the expression is induced for 72-108 h, and the recombinant humanized fibronectin is further purified.

8. The method for preparing the recombinant humanized fibronectin of claim 7, wherein the purification method of the recombinant humanized fibronectin comprises the following steps: centrifuging the fermentation broth after induction culture to obtain supernatant, then clarifying the supernatant by hollow fiber membrane, concentrating and replacing the supernatant by ultrafiltration membrane, performing anion exchange chromatography, desalting by ultrafiltration, and freeze-drying to obtain the purified product of the recombinant humanized fibronectin; the molecular weight cut-off of the hollow fiber membrane is 500 kDa, and the molecular weight cut-off of the ultrafiltration membrane is 10 kDa. The initial expression medium BMGY is: 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 4x10 -4 g / L, glycerol 10 g / L; The induction expression medium BMMY is: yeast extract 10 g / L, proteose peptone 20 g / L, K2HPO4 3 g / L, KH2PO4 11.8 g / L, YNB 3.4 g / L, ammonium sulfate 10 g / L, biotin 4x10 -4 g / L, methanol 10 mL / L.

9. The method for preparing recombinant human fibronectin as described in claim 7, characterized in that, 10. Use of the recombinant humanized fibronectin prepared by the method of any one of claims 7-9 in the preparation of cosmetic or instrument products. ​

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