阅读说明：本技术 一种利用基因工程获得重组人纤连蛋白的方法 (Method for obtaining recombinant human fibronectin by using genetic engineering ) 是由 项琪 黄亚东 曹苗苗 于 2019-09-20 设计创作，主要内容包括：本发明属于基因工程领域,更具体地公开了一种密码子优化的重组纤连蛋白基因,通过基因工程的方法在体外进行表达纯化。本发明提供的利用毕赤酵母作为表达宿主细胞,可以解决人工提取纤连蛋白量少并且生物活性低的技术缺陷。(The invention belongs to the field of genetic engineering, and particularly discloses a recombinant fibronectin gene optimized by codons, which is expressed and purified in vitro by a genetic engineering method. The invention provides a method for using pichia pastoris as an expression host cell, which can overcome the technical defects of low quantity of artificially extracted fibronectin and low biological activity.)

1. A recombinant fibronectin protein comprising the amino acid sequence:

MHHHHHHNSDSECPLSHDGYCLHDGVCMYIEALDKYACNCVVGYIGERCQYRDLKWWELRGSEGSEGEGGSEGSEGEGGSEGSEGEGGSEGSEGEGGSEGSEGEGGSEGSEGEGITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRTEIDKP(SEQ ID NO:1)。

2. the recombinant fibronectin of claim 1, further comprising a Kex2 protease sequence, a secretion signal peptide sequence, a His tag for protein purification, or a combination thereof; and/or the recombinant fibronectin protein has a biological activity selected from the group consisting of: promoting cell adhesion, promoting cell proliferation, and promoting wound healing.

3. Nucleic acid encoding a recombinant fibronectin according to claim 1 or 2 having a yeast-preferred codon-optimized nucleotide sequence, preferably as set forth in SEQ ID No. 2 or SEQ ID No. 3, more preferably as set forth in SEQ ID No. 2.

4. The nucleic acid of claim 3, further comprising a nucleotide sequence encoding a Kex2 protease sequence, a secretion signal peptide sequence, a His tag for protein purification, or a combination thereof.

5. The nucleic acid according to claim 3 or 4, further comprising a promoter, preferably an AOX1 promoter, operably linked to the nucleotide sequence as set forth in SEQ ID NO 2 or 3.

6. An expression vector comprising the nucleic acid according to any one of claims 3 to 5, preferably the expression vector is pPICZ α A-rhFN.

7. A host cell comprising the expression vector according to claim 6, preferably the host cell is an E.coli or yeast cell, more preferably a Pichia pastoris, preferably a Pichia pastoris strain selected from: GS115, X33 and KM71, most preferably the GS115 strain.

8. A method of making the recombinant fibronectin of claim 1 or 2, comprising: (1) expressing a nucleic acid according to any one of claims 3-5 in a host cell; and (2) collecting and/or purifying the recombinant fibronectin.

9. The method according to claim 8, further comprising introducing the nucleic acid according to any one of claims 3-5 or the expression vector according to claim 6 into the host cell, preferably by electrical transformation.

10. Use of recombinant fibronectin according to claim 1 or 2 for the preparation of a cosmetic composition or a medicament for promoting cell adhesion, promoting cell proliferation and/or promoting wound healing.

Technical Field

The invention belongs to the field of genetic engineering, and particularly discloses a recombinant fibronectin gene optimized by codons, which is expressed and purified in vitro by a genetic engineering method.

Background

Fibronectin (Fibronectin), abbreviated as FN, is a glycoprotein of the extracellular matrix with a high molecular weight (220-250kD) and is usually formed by cross-linking two subunits via C-terminal disulfide bonds. Three major classes are involved, of which type I and II are stabilized primarily by disulfide bonds, and type III fibronectin lacks disulfide bonds, thereby allowing the development of a partially inverted beta-sheet under the influence of an external force. The different subunits of fibronectin are derived from the expression product of the same gene, and the mRNA formed after transcription of the gene can be cleaved by different enzymes, resulting in the formation of different fibronectin polypeptides. Each subunit of fibronectin contains several domains that bind to affinity sites of cell surface receptors, collagen, fibrin, and sulfated proteoglycans. The RGD (Arg-Gly-Asp) tripeptide sequence is the smallest subunit recognized by cells.

Fibronectin is widely involved in the processes of cell migration, adhesion, proliferation, hemostasis, tissue repair and the like, mobilizes a mononuclear phagocyte system to remove harmful substances at the damaged tissue, and has a growth factor-like effect. Fibronectin, as a matrix for cell culture, can improve the anchorage rate and the confluence rate of various cells, shorten the cell confluence time, ensure good morphological structure of the cells, enhance the metabolic rate and obviously improve the synthesis speed of DNA, RNA and protein. The fibronectin coated on the microsphere carrier can be used as a medium for mass production of cells, so that the space and raw materials can be saved, and the fibronectin becomes a basic substance for producing a new medicine by applying a large-scale cell culture technology.

FN currently used in domestic studies is mostly purchased abroad or natural fibronectin extracted from human or animal blood and tissues, but these methods have a limited amount of fibronectin and are expensive. On the other hand, wild-type FN is too large in molecular weight to be expressed recombinantly. Expression of foreign proteins is a very complex task, and it is unpredictable whether a particular protein can be expressed as desired or not and whether it has activity in E.coli, yeast, insect, mammalian cell, plant expression systems (Zhang Xiaoxia et al, research progress on the type of foreign protein expression system, division of medical and health sciences abroad, 2004, Vol. 31, No. 4, pp 203-208).

Disclosure of Invention

In order to solve the technical defects of low quantity and low biological activity of artificially extracted fibronectin in the prior art, the inventor selects a specific functional fragment of human fibronectin and utilizes a pichia pastoris expression system to express recombinant fibronectin with optimized codons, thereby solving the problem of mass production, reducing the cost and providing a foundation for the application of fibronectin in the aspects of medicine and beauty.

Accordingly, the present invention provides the following:

1. a recombinant fibronectin protein comprising the amino acid sequence:

MHHHHHHNSDSECPLSHDGYCLHDGVCMYIEALDKYACNCVVGYIGERCQYRDLKWWELRGSEGSEGEGGSEGSEGEGGSEGSEGEGGSEGSEGEGGSEGSEGEGGSEGSEGEGITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRTEIDKP (SEQ ID NO: 1). Wild-type FN was too large in molecular weight to be expressed. The amino acid sequence of the invention is a functional fragment of fibronectin (namely a fragment with the biological activity of fibronectin) which is specially selected by the inventor, not only can be recombined and expressed in a large amount in yeast, but also obtains active recombinant FN protein, and solves the technical problems of small amount of artificially extracted fibronectin and low biological activity in the prior art.

2. The recombinant fibronectin protein of 1 above, further comprising a Kex2 protease sequence, a secretion signal peptide sequence, a His tag for protein purification, or a combination thereof; and/or the recombinant fibronectin protein has a biological activity selected from the group consisting of: promoting cell adhesion, promoting cell proliferation, and promoting wound healing.

3. A nucleic acid encoding a recombinant fibronectin according to 1 or 2 above having a yeast-preferred codon-optimized nucleotide sequence, preferably as shown in SEQ ID NO 2 or SEQ ID NO 3, more preferably as shown in SEQ ID NO 2.

4. The nucleic acid according to 3 above, further comprising a nucleotide sequence encoding a Kex2 protease sequence, a secretion signal peptide sequence, a His tag for protein purification, or a combination thereof.

5. The nucleic acid according to 3 or 4 above, further comprising a promoter, preferably an AOX1 promoter, operably linked to the nucleotide sequence as set forth in SEQ ID NO 2 or 3. The term "operably linked" means that the promoter is capable of promoting the subsequent expression of a nucleotide sequence (DNA) encoding a protein of interest as RNA.

6. An expression vector comprising a nucleic acid according to any one of the above 3-5, preferably said expression vector is pPICZ α A-rhFN. The sequence of the recombinant plasmid pPICZ alpha A-rhFN is shown in SEQ ID NO 6.

7. A host cell comprising an expression vector according to 6 above, preferably the host cell is an escherichia coli or yeast cell, more preferably a pichia pastoris, preferably a pichia pastoris strain selected from: GS115, X33 and KM71, most preferably the GS115 strain.

8. A method of making a recombinant fibronectin according to 1 or 2 above, comprising: (1) expressing a nucleic acid according to any one of 3-5 above in a host cell; and (2) collecting and/or purifying the recombinant fibronectin.

9. The method according to 8 above, further comprising introducing the nucleic acid according to any one of 3-5 above or the expression vector according to 6 above into the host cell, preferably by electrical transformation.

10. Use of the recombinant fibronectin protein according to 1 or 2 above in the preparation of a cosmetic composition or medicament for promoting cell adhesion, promoting cell proliferation and/or promoting wound healing.

Drawings

FIG. 1 is a diagram showing the construction of a recombinant expression plasmid pPICZ α A-rhFN;

FIG. 2 is a photograph showing the amplification of rhFN (recombinant human fibronectin) (DL 2000 DNA marker in lane 1; about 543bp in lane 2 showing the amplified rhFN gene; negative control in lane 3);

FIG. 3 shows rhFN induced expression, screening, purification and Western blot identification of rhFN (a.M: protein molecular weight standard, lanes 1-3 from left to right correspond to GS115/pPICZ alpha A-rhFN-1 (nucleotide sequence is shown in SEQ ID NO:2) induced samples 24h, 48h and 72h, lanes 4-6 from left to right correspond to GS115/pPICZ alpha A-rhFN-2 induced samples 24h, 48h and 72h (nucleotide sequence is shown in SEQ ID NO: 3), b.M: protein molecular weight standard, lane 2: sample before induction of rhFN expression, lane 1: sample after induction of rhFN expression, c.M: protein molecular weight standard, lane 1: sample after purification of rhFN, d.M: protein molecular weight standard, lane 1: Western blot identification of rhFN protein);

FIG. 4. adhesion-promoting Effect of FN protein on cells-morphometric image of cells 5h after administration. a. Blank control group; 0.2 μ M bovine plasma FN protein; c.0.2. mu.M plant-derived recombinant human FN protein; 0.2 μ M rhFN protein according to the invention; e. statistical plots of cell adhesion numbers. The results show that: bovine plasma FN protein, plant source recombinant human FN protein and the recombinant protein rhFN can promote HaCaT (ATCC No: CM-1252) cells to adhere to the wall, the growth state of the cells after adhering to the wall is good, and the number and the shape of the adhered cells among groups are not obviously different when observed under a microscope. Cell counting results show that the cell adhesion promoting effects of the bovine plasma FN (0.2 mu M) and the plant source recombinant human FN (0.2 mu M) are equivalent, and no statistical difference exists; the recombinant protein rhFN (0.2 mu M) of the invention has no significant difference compared with the recombinant protein rhFN and the recombinant protein rhFN; and

FIG. 5 is a schematic diagram of a cell scratch healing experiment. The results show that: EGF at a protein concentration of 15ng/mL after 24h administration compared to the blank group was comparable to the rhFN cell scratch healing efficacy according to the invention.

Detailed Description

The invention provides a method for efficiently expressing recombinant human fibronectin in pichia pastoris, which comprises the following steps:

1. construction of an expression vector for recombinant expression of human fibronectin: artificially synthesizing a nucleotide sequence (Jinwei Biotechnology limited) of recombinant human fibronectin FN (rh FN), adding Xho1 and Xba1 restriction sites to the 5 'end and the 3' end of the rh FN nucleotide sequence respectively, utilizing Xho1 and Xba1 to double-restriction enzyme expression vector pPICZ alpha A (Biovector plasmid vector strain cell gene collection center) and recovering, and connecting the recovered fragment and a synthetic product by using T4 DNA ligase to construct a recombinant plasmid pPICZ alpha A-rhFN;

2. electroporation of Pichia pastoris GS115 with recombinant plasmid pPICZ α A-rhFN (Sammer Feishel technology): the recombinant plasmid pPICZ alpha A-rhFN is linearized by Sal1 enzyme, recovered and then electrically transferred into Pichia pastoris GS115 competent cells and positive transformants are screened.

3. The biofermentation expresses human fibronectin.

4. The nucleotide sequence of the human fibronectin FN is shown as SEQ ID NO. 2 or SEQ ID NO. 3. The nucleotide sequence was codon adapted for host cells to optimize human fibronectin expression.

5. Preferably, the nucleotide sequence is added with an initiation codon ATG and a Kex2 protease sequence at the N terminal (the natural N terminal of the protein is maintained, the secretion expression of the protein is promoted) and a termination codon TAA sequence at the C terminal, so as to optimize the expression of the target protein.

6. Preferably, the expression vector pPICZ alpha A contains Xho1 and Xba1 cleavage sites, AOX1 promoter, alpha factor secretion signal sequence.

7. Preferably, in the step (2), the width of the electric cuvette used for the electric conversion is 0.4cm, and the conditions of the electric conversion are as follows: voltage 1.5kv, shock 4 ms.

8. Preferably, the step (2) further comprises plating the recombinants on YPD agar medium plates for screening, the medium composition being 1% yeast extract, 2% peptone, 2% agar and 100ug/ml bleomycin.

9. Preferably, the step (2) further comprises performing PCR amplification screening on the clones on the plate by using the following primers to screen out positive transformants; the primer sequences are as follows:

upstream primer FN-F: 5'-CCACCACAACTCTGACTC-3' (SEQ ID NO:4)

The downstream primer FN-R: 5'-GTTAATAGAAATTGGCTTAG-3' (SEQ ID NO: 5).

10. Preferably, the conditions for expressing said step 3 by biological fermentation are: the culture temperature was 30 ℃, the methanol induction volume concentration was 1%, and the pH was 6.0.