CN118546240A - Recombinant human fibronectin and preparation method and application thereof - Google Patents

Abstract

The present invention relates to a recombinant human fibronectin and a preparation method and application thereof, belonging to the technical field of protein engineering and genetic engineering. The present invention screens and splices a recombinant human fibronectin with high activity, high stability and good water solubility having an integrin binding site and a collagen binding domain through simulation software, and the coding gene thereof is optimized by Pichia pastoris codon preference, and a recombinant strain construction method mediated by a multi-site integration plasmid is used to construct an engineering yeast strain with high yield of recombinant human fibronectin, and the concentration of the target protein in the fermentation supernatant of the strain is increased by 1.5 times compared with the single-site integration recombinant strain. Compared with the commercially available recombinant fibronectin products, the recombinant human fibronectin prepared by the present invention has a better skin barrier repair effect, and therefore has good practical application value.

Description

Recombinant human fibronectin and its preparation method and application

Technical Field

The invention belongs to the technical field of protein engineering and gene engineering, and specifically relates to a recombinant human fibronectin and a preparation method and application thereof.

Background Art

Fibronectin (FN) is a high molecular weight glycoprotein in the extracellular matrix. It is widely involved in various physiological activities of cells, including cell migration, adhesion, proliferation, hemostasis and tissue repair. Fibronectin can also mobilize the phagocyte system to remove harmful substances from damaged tissues, which is conducive to rapid wound repair and has the function of a growth factor. Fibronectin can also significantly increase the synthesis rate of DNA, RNA and protein, and has become a basic substance for the production of new drugs using large-scale cell culture technology.

Fibronectin is widely used, but the production of natural fibronectin is extremely limited and the cost is high, which limits its application. Traditional fibronectin is mainly obtained by step-by-step separation and purification of animal plasma. The production process is time-consuming, the extraction process is complicated, and the final yield is extremely low. At the same time, the safety of animal raw materials also limits the application of traditional extraction methods. With the continuous development of synthetic biology, genetic engineering technology is used to design recombinant fibronectin fragments with specific biological functions to achieve their efficient expression in microbial expression systems, solving the shortcomings of traditional extraction methods such as viral risks. At the same time, it also improves the hydrophilicity and immune rejection of fibronectin, which is of great significance to promote the application of fibronectin in industrial production. The expression systems of animals and plants are high in cost and long in cycle, and it is difficult to meet the needs of industrialization. In contrast, the production of recombinant fibronectin by microbial fermentation is low in cost, short in cycle, easier to cultivate, and easier to commercialize. However, the bacterial expression system is prone to residual pyrogens, resulting in impure expression products that are difficult to use in clinical practice. In addition, the target protein is usually expressed in the form of inclusion bodies, and the product purification process is complicated. In addition, the post-translational processing and modification system of the prokaryotic expression system is imperfect, and the biological activity of the expression product is low, which affects the quality of the product.

Pichia pastoris is one of the most common heterologous protein expression systems. It grows fast, has a clear genetic background, and is easy to operate. It is an ideal host for producing large amounts of protein in a short period of time. In addition, Pichia pastoris also has a systematic gene editing system, a complete protein secretion expression mechanism, and the ability to post-translationally modify heterologous proteins. It is now a widely used heterologous host in the field of food and medicine.

Summary of the invention

In view of the shortcomings of the prior art, the purpose of the present invention is to provide a recombinant human fibronectin and its preparation method and application. Based on the natural human fibronectin amino acid sequence, the present invention designs a recombinant human fibronectin with high activity, high stability and good water solubility having an integrin binding site and a collagen binding domain through simulation software analysis.

The technical solution provided by the present invention is as follows:

In a first aspect of the present invention, a recombinant human fibronectin is provided, wherein the recombinant human fibronectin:

a1) having the amino acid sequence shown in SEQ ID NO.1; or,

a2) A protein having the amino acid sequence as shown in SEQ ID NO.1 with one or more amino acid residues substituted and/or deleted and/or added and having the same function.

The second aspect of the present invention provides a nucleic acid molecule, which can encode the above-mentioned recombinant human fibronectin.

In a preferred technical solution of the present invention, the nucleotide sequence of the nucleic acid molecule is shown as SEQ ID NO.2.

The third aspect of the present invention provides a recombinant expression vector, which comprises the nucleic acid molecule described in the second aspect.

In a preferred technical solution of the present invention, the plasmid vector of the recombinant expression vector is pPIC9K or pGAPZαA.

The fourth aspect of the present invention provides an engineered strain, which contains the recombinant expression vector described in the third aspect of the present invention or contains the nucleic acid molecule described in the second aspect of the present invention or is capable of expressing the recombinant human fibronectin described in the first aspect of the present invention.

In a preferred technical solution of the present invention, the host bacteria of the engineering strain is Pichia pastoris; more preferably, it is Pichia pastoris GS115.

In a preferred technical solution of the present invention, the nucleic acid molecule encoding recombinant human fibronectin in the engineered strain is integrated into the host bacterial genome and expressed under the regulation of a single promoter or simultaneously expressed under the co-regulation of multiple promoters.

The fifth aspect of the present invention provides a method for preparing the recombinant human fibronectin described in the first aspect of the present invention, comprising the steps of: culturing the engineered strain described in the fourth aspect of the present invention to express the recombinant human fibronectin; and isolating and purifying the recombinant human fibronectin.

The sixth aspect of the present invention provides the use of the above-mentioned recombinant human fibronectin in the preparation of food, medicine or daily chemical products.

A seventh aspect of the present invention provides a cosmetic, wherein the cosmetic comprises at least the recombinant human fibronectin described in the first aspect.

Beneficial effects:

The present invention screened and spliced a recombinant human fibronectin with high activity, high stability and good water solubility having integrin binding sites and collagen binding domains through simulation software, and its coding gene was optimized by Pichia pastoris codon preference, and a recombinant strain construction method mediated by multi-site integration plasmid was used to construct an engineered yeast strain with high yield of recombinant human fibronectin, and the concentration of the target protein in the fermentation supernatant of the strain was increased by 1.5 times compared with the single-site integration recombinant strain. Compared with the commercially available recombinant fibronectin products, the recombinant human fibronectin prepared by the present invention has better skin barrier repair effect, so it has good practical application value.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is the SDS-PAGE protein electrophoresis and Western-Blot detection diagram of the recombinant Pichia pastoris fermentation supernatant in Example 3; wherein, Figure A is the SDS-PAGE protein electrophoresis diagram, lane M represents the standard protein with a molecular weight of 180 kDa; lane 1 represents the recombinant Pichia pastoris GS115/pPIC9K fermentation supernatant; lane 2 represents the recombinant Pichia pastoris GS115/pPIC9K-FN1 ^opt fermentation supernatant; Figure B is the Western-Blot detection diagram.

Figure 2 is the SDS-PAGE protein electrophoresis diagram of the recombinant Pichia pastoris fermentation supernatant in Example 5; wherein, lane M represents a standard protein with a molecular weight of 180 kDa; lane 1 represents the fermentation supernatant of recombinant Pichia pastoris GS115/pPIC9K-FN1 ^opt ; lane 2 represents the fermentation supernatant of recombinant Pichia pastoris GS115/pPIC9K-FN1 ^opt /pGAPZαA-FN1 ^opt using methanol as the carbon source; lane 3 represents the fermentation supernatant of recombinant Pichia pastoris GS115/pPIC9K-FN1 ^opt /pGAPZαA-FN1 ^opt using glycerol as the carbon source.

Figure 3 is a bar graph of the relative mRNA expression of FLG in HaCaT cells under the action of different fibronectins in Example 6; 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 human fibronectin FN1 group prepared by the present invention; in the figure, compared with control group 1, P<0.01 (**), P<0.0001 (****), which are considered to be significantly different.

DETAILED DESCRIPTION

The technical scheme of the present invention is further described below in conjunction with the embodiments. The reagents and materials involved in the embodiments are all common commercially available products unless otherwise specified. The experimental operations involved in the embodiments are all conventional operations in the art unless otherwise specified.

The culture medium involved in the embodiment:

YPD medium: yeast extract 10 g/L, peptone 20 g/L, glucose 20 g/L;

BMGY medium: yeast extract 10 g/L, peptone 20 g/L, K ₂ HPO _{4 3} g/L, KH ₂ PO ₄ 11.8 g/L, YNB 3.4 g/L, ammonium sulfate 10 g/L, biotin 4×10 ^-4 g/L, glycerol 10 g/L;

BMMY medium: yeast extract 10 g/L, peptone 20 g/L, K ₂ HPO _{4 3} g/L, KH ₂ PO ₄ 11.8 g/L, YNB 3.4 g/L, ammonium sulfate 10 g/L, biotin 4×10 ^-4 g/L, methanol 10 mL/L;

BMGYP medium: yeast extract 10 g/L, peptone 20 g/L, K ₂ HPO _{4 3} g/L, KH ₂ PO ₄ 11.8 g/L, YNB 3.4 g/L, ammonium sulfate 10 g/L, biotin 4×10 ^-4 g/L, glycerol 40 g/L.

Commercially available recombinant fibronectin was purchased from Shanghai Zeye Biotechnology Co., Ltd., product number: ZY637Ra01P.

Example 1: Sequence selection of recombinant human fibronectin

Based on the natural human fibronectin amino acid sequence (NCBI accession number: P02751.5), a recombinant human fibronectin FN1 with high activity, high stability and good water solubility with integrin binding sites and collagen binding domains was designed through simulation software analysis. The recombinant human fibronectin contains 254 amino acids, and a 6×HIS tag is added to the carboxyl end of the amino acid sequence to facilitate downstream purification. The final amino acid sequence of the recombinant human fibronectin FN1 is shown in SEQ ID NO.1, which is nearly 100% homologous to the natural human fibronectin sequence and has low immunogenicity.

Example 2: Construction of an expression system containing a recombinant human fibronectin encoding gene (FN1 ^opt )

The coding gene of recombinant human fibronectin FN1 was sequence optimized according to the codon preference of Pichia pastoris. The nucleotide sequence of the optimized recombinant human fibronectin coding gene FN1 ^opt is shown in SEQ ID NO.2, wherein gaattc at the 5' end of the sequence is an EcoRI restriction site, and gcggccgc at the 3' end is a NotI restriction site. The codon-optimized recombinant human fibronectin coding gene FN1 ^opt was commissioned to Nanjing GenScript Biotechnology Co., Ltd. for full gene synthesis and cloned into the EcoRI and NotI restriction sites of the Pichia pastoris expression vector pPIC9K to obtain the recombinant expression vector pPIC9K-FN1 ^opt . DNA sequencing and alignment showed that the recombinant sequence was correct. The recombinant expression plasmid pPIC9K-FN1 ^opt was linearized by SalI fast cutting enzyme and then electroporated into the Pichia pastoris GS115 host cell. The recombinant transformants were screened by Geneticin G418 to obtain high-copy recombinant Pichia pastoris GS115/pPIC9K-FN1 ^opt .

Example 3: Shake flask fermentation of recombinant human fibronectin FN1 engineered yeast strain

The recombinant Pichia pastoris GS115/pPIC9K-FN1 ^opt obtained in Example 2 was subjected to shake flask fermentation culture, and the recombinant Pichia pastoris GS115/pPIC9K without pPIC9K was used as a control.

The fermentation steps are as follows: Pick a single clone and inoculate it into 40mL of YPD medium, and culture it at 30℃ and 200rpm for 24h. Transfer it to 40mL of initial expression medium BMGY medium at a 10% inoculum volume, and culture it at 30℃ and 200rpm for 24h. Collect the bacteria by centrifugation, wash the bacteria with physiological saline, and then inoculate all of them into 40mL of induced expression medium BMMY medium, culture it at 30℃ and 200rpm, add pure methanol to the medium every 24h to a final concentration of 1.0% (v/v), and induce expression for 96h.

The fermentation supernatant of the recombinant Pichia was subjected to SDS-PAGE protein electrophoresis analysis, and the electrophoresis analysis results are shown in Figure 1 A. Near the theoretical protein molecular weight of 29.1 kDa (the apparent molecular weight shown by SDS-PAGE is larger than the theoretical molecular weight), the fermentation supernatant of the recombinant Pichia GS115/pPIC9K-FN1 ^opt (lane 2) had an additional protein band (indicated by the arrow position), and further Western-Blot detection, the detection results are shown in Figure 1 B, confirming that the band at this position is the recombinant human fibronectin FN1 expressed by the constructed recombinant Pichia GS115/pPIC9K-FN1 ^opt .

Example 4: Construction of an engineered yeast strain that produces high levels of recombinant human fibronectin FN1

The production of recombinant human fibronectin FN1 was increased by a multi-site integration plasmid-mediated approach, and a recombinant strain expressing dual promoters was constructed.

The recombinant vector pPIC9K-FN1 ^opt was double-digested with EcoRI and NotI, and the recombinant human fibronectin encoding gene FN1 ^opt was collected and connected with the linear expression vector pGAPZαA treated with the same enzymes. The ligation product was transformed into Escherichia coli TOP10, and the recombinant plasmid pGAPZαA-FN1 ^opt was obtained under the premise of ensuring that the reading frame was not shifted. The recombinant sequence was correct after DNA sequencing. The recombinant plasmid pGAPZαA-FN1 ^opt was linearized with restriction endonuclease AvrII and then electroporated into recombinant Pichia pastoris GS115/pPIC9K-FN1 ^opt cells. The recombinant transformants were screened with Zeocin to obtain recombinant Pichia pastoris GS115/pPIC9K-FN1 ^opt /pGAPZαA-FN1 ^opt with high copy of the recombinant human fibronectin encoding gene. In the recombinant Pichia pastoris, the recombinant human fibronectin encoding gene was simultaneously expressed under the co-regulation of dual promoters.

Example 5: Shake flask fermentation of an engineered yeast strain that produces high levels of recombinant human fibronectin FN1

The obtained recombinant Pichia pastoris GS115/pPIC9K-FN1 ^opt /pGAPZαA-FN1 ^opt was subjected to shake flask fermentation culture according to the method of Example 3. The fermentation supernatant of recombinant Pichia pastoris GS115/pPIC9K-FN1 ^opt /pGAPZαA-FN1 ^opt (lane 2) and the fermentation supernatant of recombinant Pichia pastoris GS115/pPIC9K-FN1 ^opt (lane 1) were subjected to SDS-PAGE protein electrophoresis analysis. The results are shown in FIG2 . The band brightness of lane 2 is significantly greater than that of lane 1. The BioAnalysis biological analysis software shows that the protein expression level of the recombinant Pichia pastoris GS115/pPIC9K-FN1 ^opt /pGAPZαA-FN1 ^opt regulated by the dual promoter is 1.5 times higher than that of the recombinant Pichia pastoris GS115/pPIC9K-FN1 ^opt regulated by the single promoter.

At the same time, the recombinant Pichia pastoris GS115/pPIC9K-FN1 ^opt /pGAPZαA-FN1 ^opt was fermented in a shake flask using glycerol as the carbon source. The fermentation steps were as follows: a single clone was picked and inoculated into 40 mL of YPD medium and cultured at 30°C and 200 rpm for 24 h. A 10% inoculation amount was transferred to 40 mL of expression medium BMGYP medium and cultured at 30°C and 200 rpm for 96 h.

The SDS-PAGE results of the fermentation supernatant with glycerol as the carbon source are shown in lane 3 of Figure 2. There is an obvious protein band at a theoretical molecular weight of about 29.1 kDa, indicating that the recombinant Pichia pastoris can produce recombinant human fibronectin FN1 using both methanol and glycerol as the carbon source.

Example 6: Barrier repair experiment of recombinant human fibronectin FN1

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.

Human immortalized keratinocytes (HaCaT cells) were collected, and a cell suspension was prepared with a high-glucose DMEM cell culture medium. 2 mL of the cell suspension was added to each well of a 6-well plate, and the number of cells was 2.6×10 ⁵ /well. A blank control group (control group 1), a commercially available recombinant fibronectin control group (control group 2), and a recombinant human fibronectin group prepared by the present invention (experimental group) were set up in the experiment, and 3 duplicate wells were set up in each group. The 6-well plate was placed in a cell culture incubator (5% CO ₂ , 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 the recombinant fibronectin in the high-glucose DMEM cell culture medium was 1 mg/mL. After the 6-well plate was placed in an incubator (5% CO ₂ , 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 then collected. 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, and the 2 ^-△△CT method was used for calculation.

The results of different fibronectins promoting the mRNA expression of FLG in HaCaT cells are shown in Figure 3. Compared with the blank control group (control group 1), the commercially available recombinant fibronectin (control group 2) and the recombinant human fibronectin FN1 prepared by the present invention (experimental group) can both increase the relative mRNA expression of FLG. The recombinant human fibronectin FN1 prepared by the present invention has a stronger ability to increase the relative mRNA expression of FLG than the commercially available recombinant fibronectin. Therefore, the recombinant human fibronectin FN1 prepared by the present invention has a more obvious skin barrier repair function than the commercially available recombinant fibronectin.

Claims (10)

1. A recombinant human fibronectin, characterized in that the recombinant human fibronectin: a1) having the amino acid sequence shown in SEQ ID NO.1; or, a2) A protein having the amino acid sequence as shown in SEQ ID NO.1 with one or more amino acid residues substituted and/or deleted and/or added and having the same function. 2. A nucleic acid molecule, characterized in that the nucleic acid molecule can encode the recombinant human fibronectin according to claim 1. 3. The nucleic acid molecule according to claim 2, characterized in that the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO.2. 4. A recombinant expression vector, characterized in that the recombinant expression vector comprises the nucleic acid molecule according to claim 2. 5 . The recombinant expression vector according to claim 4 , wherein the plasmid vector of the recombinant expression vector is pPIC9K or pGAPZαA. 6. An engineered strain, characterized in that the engineered strain contains the recombinant expression vector according to claim 4, or contains the nucleic acid molecule according to claim 2, or is capable of expressing the recombinant human fibronectin according to claim 1. 7. The engineered strain according to claim 6, characterized in that the host bacteria of the engineered strain is Pichia pastoris; more preferably Pichia pastoris GS115; Preferably, the nucleic acid molecule encoding the recombinant human fibronectin in the engineered strain is integrated into the host bacterial genome and expressed under the regulation of a single promoter or simultaneously expressed under the co-regulation of multiple promoters. 8. A method for preparing the recombinant human fibronectin according to claim 1, characterized in that the steps include: culturing the engineered strain according to claim 6 to express the recombinant human fibronectin; and isolating and purifying the recombinant human fibronectin. 9. Use of the recombinant human fibronectin according to claim 1 in the preparation of food, medicine or daily chemical products. 10 . A cosmetic, characterized in that the cosmetic contains at least the recombinant human fibronectin according to claim 1 .