Function-enhanced recombinant fibronectin and its preparation method and application

Abstract

The present invention discloses a functionally enhanced recombinant fibronectin, a preparation method and an application thereof. The amino acid sequence of the recombinant fibronectin is shown in SEQ ID No. 3, which is formed by fusion of the main structural domain FN III8-10 of human fibronectin with proline at position 171 and the main structural domain FN III12-14 of human fibronectin and cytokine binding. The recombinant fibronectin of the present invention has enhanced cell proliferation and adhesion performance of promoting human fibroblasts, exhibits excellent cell biological efficacy, can be efficiently expressed in Pichia pastoris engineering strains, and has broad application prospects in the fields of skin care, beauty and tissue engineering.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a recombinant fibronectin with enhanced function and a preparation method and application thereof. Background Art

[0002] Fibronectin (FN) is a macromolecular glycoprotein present in the extracellular matrix and basement membrane of many animal cells. It plays a key role in cell adhesion and is also involved in cell proliferation, differentiation, hemostasis and wound repair. It has broad application prospects in cancer diagnosis, premature birth prediction, animal cell culture and beauty and skin care.

[0003] Fibronectin is a modular protein, mainly composed of type I, type II and type III repeating units. Due to the existence of alternative splicing, the molecular weight of its monomers fluctuates between 230kD-270kD. Furthermore, two monomers are connected by a disulfide bond at the C-terminus to form a dimer with a molecular weight of about 500kD, which binds to collagen / gelatin, integrin, heparin, fibronectin and other extracellular molecules through different binding sites to exert corresponding functions.

[0004] With the rapid development of synthetic biology technology, technologies such as DNA recombination, protein de novo design and structure prediction have greatly promoted the rational design and efficient industrial production of functional proteins. For example, the applicant's previous research results (Chinese patent application CN 116574173A) disclose a recombinant human fibronectin mutant with enhanced protein stability and its expression strain, which, on the basis of the key functional domain FN III9-10 of human fibronectin, mutates the 28th or 78th amino acid in the recombinant human fibronectin to form a recombinant human fibronectin mutant with enhanced protein stability, and the recombinant human fibronectin mutant 1 and mutant 2 can further promote the cell proliferation, migration and adhesion performance of human fibroblasts compared to the original recombinant human fibronectin. The applicant further developed on the basis of the above research results and obtained the functionally enhanced recombinant fibronectin of the present invention. Summary of the invention

[0005] The invention provides a recombinant fibronectin with enhanced function, and a preparation method and application thereof.

[0006] The technical solution of the present invention is as follows:

[0007] The present invention places the main structural domain FN III8-10 of human fibronectin binding to integrin whose 171st position of the amino acid sequence as shown in SEQ ID No.1 mutated to proline at the C-terminus, and places the main structural domain FN III12-14 of human fibronectin binding to cytokines as shown in SEQ ID No.2 at the N-terminus, and performs fusion expression to obtain a recombinant fibronectin FN III12-14-8-10 with enhanced function.

[0008] The amino acid sequence of the functionally enhanced recombinant fibronectin FN III12-14-8-10 of the present invention is shown in SEQ ID No.3.

[0009] According to the codon preference of Pichia pastoris, the present invention performs codon optimization on recombinant fibronectin FN III12-14-8-10 to obtain a gene encoding the recombinant fibronectin FN III12-14-8-10, which is formed by fusion of a nucleotide sequence of a human fibronectin FN III8-10 functional region in which position 171 is mutated to proline as shown in SEQ ID No.4 and a nucleotide sequence of a human fibronectin FN III12-14 functional region as shown in SEQ ID No.5, and the nucleotide sequence is shown in SEQ ID No.6.

[0010] Furthermore, the present invention constructs a strain that efficiently expresses recombinant fibronectin FN III12-14-8-10. First, the nucleotide sequences of the functional region of human fibronectin FN III8-10 and the functional region of human fibronectin FN III12-14 in which the 171st position is mutated to proline are synthesized respectively, and then PCR amplified and seamlessly cloned into the pPIC9K empty vector respectively. The correctly sequenced recombinant plasmids are enriched and linearized by SalI enzyme digestion and then transformed into Pichia pastoris. The Pichia pastoris genetically engineered bacteria that efficiently secrete and express the recombinant fibronectin FN III12-14-8-10 are obtained through G418 resistance gradient screening.

[0011] Furthermore, the present invention provides a method for preparing the above-mentioned recombinant fibronectin FN III12-14-8-10, specifically: inoculating Pichia pastoris genetically engineered bacteria that efficiently express recombinant fibronectin FN III12-14-8-10 into BMMY culture medium, inducing expression with methanol at 30°C for 48-72 hours, collecting the supernatant, and purifying to obtain recombinant fibronectin FN III12-14-8-10.

[0012] The present invention also provides the use of the recombinant fibronectin FN III12-14-8-10 in the preparation of skin care products.

[0013] The present invention fuses the human fibronectin FN III8-10 and FN III12-14 regions that control the binding of integrins and growth factors on the basis of the recombinant human fibronectin mutants in Chinese patent application CN 116574173 A, and mutates the 171st position of FN III8-10 to proline, thereby obtaining a functionally enhanced recombinant fibronectin FN III12-14-8-10. The recombinant fibronectin FN III12-14-8-10 of the present invention exhibits a better effect of promoting cell proliferation and cell adhesion than the above-mentioned recombinant human fibronectin mutants, and has important application prospects in the fields of skin care products and the like. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 Schematic diagram of the recombinant plasmid containing recombinant fibronectin FN III12-14-8-10.

[0015] Figure 2 This is a nucleic acid electrophoresis diagram during the construction of the FN III12-14-8-10 recombinant plasmid, wherein lane 1: PCR amplified fragment of the human fibronectin FN III12-14 functional region; lane 2: 5kb DNA marker; lane 3: PCR amplified fragment of the human fibronectin FN III8-10 functional region with position 171 mutated to proline.

[0016] Figure 3 This is the nucleic acid electrophoresis diagram of Escherichia coli colony PCR during the construction of FN III12-14-8-10 recombinant plasmid, where lane 1: 5kb DNA marker; lane 2: FN III12-14-8-10 recombinant fragment.

[0017] Figure 4 This is an SDS-PAGE image of the culture supernatant of a high-copy Pichia pastoris genetically engineered strain expressing recombinant fibronectin FN III12-14-8-10 (induced for 72 hours).

[0018] Figure 5 This is a graph showing the effect of recombinant fibronectin FN III12-14-8-10 on the proliferation of human fibroblasts.

[0019] Figure 6 This is a graph showing the effect of recombinant fibronectin FN III12-14-8-10 on the relative adhesion rate of human fibroblasts. DETAILED DESCRIPTION

[0020] The present invention is further described below by specific examples and drawings, but the present invention is not limited to the scope of the examples. The experimental methods without specific conditions in the following examples are selected according to conventional methods and conditions, or according to the product specifications.

[0021] In the following examples, Pichia pastoris GS115 was used as the starting strain. Recombinant fibronectin FN III12-14-8-10 used pPIC9K as the integration vector, and the nucleotide sequences of SEQ ID No. 4 and SEQ ID No. 5 synthesized by Shanghai Qingke Biotechnology Co., Ltd. were PCR amplified and purified, and seamlessly cloned into the pPIC9K empty plasmid.

[0022] Example 1

[0023] Construction of high-copy Pichia pastoris genetic engineering strain:

[0024] (1) The sequences selected in the present invention are the human fibronectin FNIII8-10 and FN III12-14 regions that control the binding of integrins and growth factors, respectively, and the 171st position of FN III8-10 is mutated to proline, and the optimized nucleotide sequences SEQ ID No. 4 and SEQ ID No. 5 are obtained based on the codon preference of Pichia pastoris.

[0025] (2) The nucleotide sequences SEQ ID No. 4 and SEQ ID No. 5 were commissioned to be synthesized by Shanghai Qingke Biotechnology Co., Ltd., and the two gene fragments were amplified using primer pairs P1 (nucleotide sequence as shown in SEQ ID No. 7) and P2 (nucleotide sequence as shown in SEQ ID No. 8), P3 (nucleotide sequence as shown in SEQ ID No. 9) and P4 (nucleotide sequence as shown in SEQ ID No. 10), respectively, and seamlessly cloned into the pPIC9K empty vector (purchased from Thermo Fisher Scientific), and the recombinant plasmid containing the recombinant fibronectin FN III12-14-8-10 was obtained as shown in Figure 1 As shown. After successful sequencing verification, the correct recombinant plasmid was enriched, the recombinant plasmid was linearized using SalI single enzyme digestion, and electroporated into Pichia pastoris. Finally, a high-copy Pichia pastoris genetic engineering strain expressing recombinant fibronectin FN III12-14-8-10 was obtained through G418 resistance gradient screening. The nucleic acid electrophoresis diagram of the FNIII12-14-8-10 recombinant plasmid construction process is shown in Figure 2 As shown, lane 1: PCR amplification fragment of human fibronectin FNIII12-14 functional region; lane 2: 5kb DNA marker; lane 3: PCR amplification fragment of human fibronectin FN III8-10 functional region with position 171 mutated to proline. The E. coli colony PCR nucleic acid electrophoresis diagram during the construction of FN III12-14-8-10 recombinant plasmid is shown in Figure 3As shown, lane 1: 5kb DNA marker; lane 2: FN III12-14-8-10 recombinant fragment.

[0026] Example 2

[0027] Fermentation of high-copy Pichia pastoris genetic engineering strain and expression of recombinant fibronectin FN III12-14-8-10:

[0028] The high-copy Pichia pastoris genetic engineering strain expressing recombinant fibronectin FN III12-14-8-10 was inoculated into 30 mL BMGY medium (the medium consisted of 20 g / L peptone, 10 g / L yeast extract, 10×YNB 100 ml, 10× potassium phosphate buffer pH 6.0 100 ml, 10×glycerol 100 ml, and the balance was water, and the percentages were weight percentages), and cultured for 16 to 20 hours, and then the initial OD 600 The inoculum size was 1 and inoculated into 30 mL BMMY medium (the medium consisted of 20 g / L peptone, 10 g / L yeast extract, 10×YNB 100 ml, 10× potassium phosphate buffer pH 6.0 100 ml, and the balance was water, and the percentages were mass percentages) for cultivation. 300 μL of filtered and sterilized methanol was added every 24 hours, and 1 mL of fermentation broth was collected after 72 hours. The expression level of FN III12-14-8-10 was detected by HPLC to be about 0.2 g / L. 500 μL of supernatant was taken out by centrifugation at 12000 rpm for 10 min, and 500 μL of supernatant was sterilized through a 0.22 μm sterilization filter membrane. 24 μL of the treated supernatant and 6 μL of 5× loading buffer were mixed evenly and heated at 100°C for 10 min, and then 15 μL was spotted on SDS-PAGE for protein electrophoresis detection. The results of the detection are shown in Figure 4 .

[0029] Example 3

[0030] 1. Cell Proliferation Assay

[0031] The MTT method was used to detect the ability of recombinant fibronectin FN III12-14-8-10 to promote cell proliferation using human fibroblasts as experimental cells. Figure 5As shown, 12-14-8-10 refers to recombinant fibronectin FN III12-14-8-10, and 9-10 (1408) refers to the recombinant human fibronectin mutant 2 disclosed in Chinese patent application CN 116574173 A. It can be seen from the figure that 12-14-8-10 (fibronectin Fn02) has a significant proliferative effect on cells at a concentration of 25%, and compared with 9-10 (1408) (fibronectin Fn01), its proliferative effect is significantly enhanced.

[0032] 2. Cell Adhesion Assay

[0033] The centrifugation method was used to detect the ability of recombinant fibronectin FN III12-14-8-10 to promote cell adhesion using human fibroblasts as experimental cells. Figure 6 As shown, 12-14-8-10 refers to recombinant fibronectin FN III12-14-8-10, and 9-10 (1408) refers to the recombinant human fibronectin mutant 2 disclosed in Chinese patent application CN 116574173 A. It can be seen from the figure that 12-14-8-10 (fibronectin Fn02) has a significant adhesion-promoting effect on cells at a concentration of 25%-1%, and has the strongest adhesion-promoting effect at a concentration of 20%. Compared with 9-10 (1408) (fibronectin Fn01), its adhesion-promoting effect is significantly enhanced.

[0034] Based on the above cell proliferation and cell adhesion experiments, it can be determined that the recombinant fibronectin FN III12-14-8-10 has an enhanced effect of promoting cell proliferation and cell adhesion.

[0035] The above are only preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be regarded as limiting the present invention, and the protection scope of the present invention should be based on the scope defined by the claims. For ordinary technicians in this technical field, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A recombinant fibronectin with enhanced function, characterized in that: It is recombinant fibronectin FN III12-14-8-10, and its amino acid sequence is shown in SEQ ID No.

3.

2. The gene encoding the recombinant fibronectin according to claim 1, characterized in that: Its nucleotide sequence is shown in SEQ ID No.

6.

3. A strain that efficiently expresses the recombinant fibronectin as claimed in claim 1, characterized in that: The construct was constructed by the following method: first, the nucleotide sequences of the human fibronectin FN III 8-10 functional region with position 171 mutated to proline as shown in SEQ ID No.4 and the human fibronectin FN III 12-14 functional region as shown in SEQ ID No.5 were synthesized, and then PCR amplified and seamlessly cloned into the pPIC9K empty vector. The correctly sequenced recombinant plasmids were enriched and Sal After linearization by enzyme I, the strain was transformed into Pichia pastoris, and genetically engineered Pichia pastoris that efficiently expressed the recombinant fibronectin FN III12-14-8-10 was obtained through G418 resistance gradient screening.

4. The method for preparing recombinant fibronectin according to claim 1, characterized in that: Specifically, the genetically engineered Pichia pastoris bacteria efficiently expressing the recombinant fibronectin FN III12-14-8-10 according to claim 3 is inoculated into BMMY medium, induced to express with methanol at 30°C for 48-72 hours, the supernatant is collected, and the recombinant fibronectin FN III12-14-8-10 is purified.

5. Use of the recombinant fibronectin according to claim 1 in preparing skin care products.

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