CN114075571A

CN114075571A - Nucleic acid constructs of epidermal growth factor, methods of production and compositions thereof

Info

Publication number: CN114075571A
Application number: CN202010830385.XA
Authority: CN
Inventors: 钟树根; 邝纬阳; 林庭匡
Original assignee: Mengqian Technology Intellectual Property Co ltd
Current assignee: Mengqian Technology Intellectual Property Co ltd
Priority date: 2020-08-18
Filing date: 2020-08-18
Publication date: 2022-02-22

Abstract

The invention relates to a nucleic acid construct, which comprises an insert and is characterized in that the insert comprises a polynucleotide sequence which encodes an affinity tag, an intein and an epidermal growth factor from the 5 'end to the 3' end, wherein the affinity tag is a GST affinity tag, and the intein is gp41-1 protein. This study revealed the ability of gp41-1 mini-introns to express and lyse the C-exon in cells handling EGF fusion proteins. The finally purified EGF was demonstrated to have high activity in accelerating the healing rate of bedsores, diabetic foot ulcers and skin breaks.

Description

Nucleic acid constructs of epidermal growth factor, methods of production and compositions thereof

Technical Field

The invention relates to the field of biology, in particular to a nucleic acid construct of an epidermal growth factor, an expression vector and a host cell thereof, a production method of the construct, a primer used in the production method, a method for producing EGF by using the construct, the expression vector or the host cell, EGF produced by the method, and a composition of the EGF.

Background

Epidermal Growth Factor (EGF), an oligopeptide of 53 amino acids with three disulfide bonds, was discovered 60 years ago. EGF may bind to EGF receptors, thereby activating downstream signaling cascades in triggering epidermal cell proliferation. EGF not only plays a good role in the improvement of wound healing (Su, Z., et al. (2014). Enhancement of skin bound health with defined tissue attached with materials Science and Engineering: C,44, 440) but also participates in various physiological pathways such as the regeneration of tissue and bone (Marquez, L., et al. (2013). Enhanced bone association of tissue bound growth factor (EGF) bound tissue attachment, 44), 44, 558, 9, 2010, 55, 9, 55, 7(3),369-385).

EGF has been used to treat wounds that are difficult to heal because of its many valuable biological functions, including diabetic foot ulcers (Singal, S., et al (2012), Role of epidemic Growth Factor in Healing of biological focusers. Indian Journal of Surgery,74(6), 451. 455; Tiaka, E.K., et al (2012), epidemic Growth Factor in the treatment of experimental concerns. of biological focusers: update. Perfect in wound Surgery and Endovasular Therapy,24(1),37-44.) and bedsores (Rao, B.A.R., & Prasad., M.D. (2017), cosmetic Growth Factor of Health, Research 78, Journal of Health of Human origin, www.ijhsr.org). EGF can also stimulate the production of collagen and elastin (FAAD, R.L.M.M. (2015). Improvisual in acidic acid scanner Using Topical Synthetic Epidermal Factor (EGF) server: a facial study J Drugs Dermatole, 14(9), 1005. 1010; Stoddard, M.A. et al. (2017.) Improvisual of Topical acid scanner in Skin of Color Using Topical Synthetic Epidermal Factor (EGF) Serum: A facial study. journal of Drugs in chemistry: JDD,16(4), 322. al 326.) which further expands its use in pharmaceutical cosmetics. Although there are various applications of EGF, commercial availability of EGF is very low because it is costly to extract a sufficient amount of EGF in a natural host, and abundance of EGF is low.

To expand its application in the medical or cosmeceutical industry, many organizations attempt to produce EGF by recombinant techniques through different methods, including synthesis (Schroeder, C.I., et al. (2014.) Design and synthesis of truncated EGF-A peptides expressed by LDL-R receptors in the presentation of PCSK9 in vitro. chemistry & biology,21(2), 284. 294.), intracellular expression (Kim, D.G., et al. (2015.) Construction of nucleic human epithelial growth factor binding short collagen-binding peptides expressed by human tissue a expression of microorganism GF. J. reagent, Biotechnology, 25. 1. 119 and biological gene expression of biological genes, 2001. yeast and expression of biological genes, yeast and biological gene expression of peptide of biological genes, 2001. origin, yeast and microorganism of biological gene expression, 23(2),106-110.). However, only fusion proteins can be produced because the signal peptide or affinity tag cannot be removed.

Disclosure of Invention

Coli (e.coli), recombinant proteins can be expressed using different methods without the need for post-translational modification. Although E.coli may have a disadvantage in the production of endotoxin, its short cleavage time and high expression of the final product are major advantages for the production of various recombinant proteins as a host. Inclusion proteins (also referred to as "protein introns") have been found in a variety of microorganisms to facilitate expression of homologous proteins. The invention adopts a recognized gp41-1 micro-intron expression system to promote the expression of Epidermal Growth Factor (EGF). This study revealed the ability of gp41-1 mini-introns to express and lyse the C-exon in cells handling EGF fusion proteins. The finally purified EGF was demonstrated to have high activity in accelerating the healing rate of bedsores, diabetic foot ulcers and skin breaks.

In particular, the method comprises the following steps of,

in one aspect, the invention provides a nucleic acid construct comprising an insert, wherein the insert comprises, from 5 'to 3', a polynucleotide sequence encoding an affinity tag, an intein, and an epidermal growth factor, wherein the affinity tag is a GST affinity tag and the intein is gp41-1 protein.

In an embodiment of the nucleic acid construct of the invention, the insert comprises, from 5 'to 3', a polynucleotide sequence encoding the T7 promoter, the lactose operon, the GST affinity tag, the gp41-1 protein intein, the epidermal growth factor.

In some embodiments of the nucleic acid constructs of the invention, the insert comprises, from 5 'to 3', a polynucleotide sequence encoding a T7 promoter, a lactose operon, an RBS, a GST affinity tag, a thrombin site, a gp41-1 protein intein, an epidermal growth factor, an 8 xhis tag, a T7 terminator.

In some embodiments of the nucleic acid constructs of the invention, the nucleic acid constructs are used for the production of Epidermal Growth Factor (EGF) in the host Escherichia coli.

In another aspect, the present invention provides an expression vector comprising the nucleic acid construct of the present invention.

In another aspect, the invention provides a host cell comprising an expression vector of the invention.

In some embodiments of the host cell of the invention, it is transformed E.coli (Escherichia coli).

In another aspect, the present invention provides a method for constructing an EGF expression vector, comprising:

1) a synthetic DNA fragment encoding 5 'to 3' NotI-stop codon-EGF-gp 41-1-thrombin site-SpeI;

2) amplifying the synthesized DNA fragment by PCR extension using forward primer 5'-AAAAAGCGGCCGCTTAGCGCAGTTC-3' and reverse primer 5'-AAAAAACTAGTCTGGTGCCACGCGGTAGTT-3';

3) the amplified PCR product was purified by PCR clean kit and digested with NotI and SpeI;

4) the digested fragment was purified from 1% agarose and ligated into pET42a (+) digested with the same restriction enzymes.

On the other hand, the invention provides a primer pair for constructing the EGF expression vector, which comprises the following steps:

1, forward primer 5'-AAAAAGCGGCCGCTTAGCGCAGTTC-3';

SEQ ID NO. 2, reverse primer 5'-AAAAAACTAGTCTGGTGCCACGCGGTAGTT-3'.

In another aspect, the present invention provides a method for producing EGF using the nucleic acid construct of the present invention or the expression vector of the present invention or the host cell of the present invention.

In some embodiments of the method for producing EGF according to the present invention, it comprises culturing the transformed e.coli of the present invention under conditions allowing expression of EGF.

For accumulation of expressed gene products, the host cell is cultured under conditions sufficient to accumulate the gene product. Such conditions may include, for example, temperature, nutrient, and cell density conditions that allow the cells to express and accumulate the protein. Furthermore, as known to those skilled in the art, such conditions are those under which the cell can perform essential cellular functions, such as transcription, translation, and intracellular expression.

Prokaryotic host cells are cultured at a suitable temperature. For E.coli cultures, for example, typical temperatures are about 20 ℃ to about 39 ℃. In one embodiment, the temperature is from about 25 ℃ to about 37 ℃, such as 37 ℃.

For induction, cells are typically cultured until a defined optical density is reached, e.g., about 80-100 a55tl, at which point induction is initiated (e.g., by addition of an inducer, by depletion of a repressor, inhibitor, or culture medium component, etc.) to induce expression of the gene encoding the heterologous polypeptide.

After the product has accumulated, the cells present in the culture can be mechanically lysed using any mechanical means known in the art to release the protein from the host cells. Alternatively, other lysis methods may be used, including but not limited to alkaline lysis, SDS lysis, and the like. Cell lysates for lysing cells can include, but are not limited to, Tris-HCl, EDTA, NaCl, glucose, lysozyme, and the like. Optionally, the lysate is incubated for a sufficient time to allow release of the heterologous polypeptide contained in the cells prior to product recovery.

The lysate may be subjected to further processing, such as dilution with water, addition of buffers or flocculants, pH adjustment, or changing or maintaining the temperature of the lysate/homogenate in the preparation for subsequent recovery steps.

In a subsequent step, the heterologous polypeptide is recovered from the lysate in a manner that minimizes co-recovered cell debris and products. Recovery may be by any method. In one embodiment, settling of the collapsible particles comprising the heterologous polypeptide or collection of the supernatant comprising the soluble product may be included. An example of sedimentation may be centrifugation. Recovery may be carried out in the presence of an agent that disrupts the outer cell walls to increase permeability and allow more solids to be recovered, prior to adsorption or settling. Examples of such agents include chelating agents such as ethylenediaminetetraacetic acid (EDTA) or zwitterionic, e.g., dipolar ion, detergents such as the ZWITTERGENT 316. TM. detergent. In one embodiment, the recovery is performed in the presence of EDTA.

In one embodiment, if desired, it may further comprise isolating the aggregated heterologous polypeptide, followed by simultaneous solubilization and refolding of the polypeptide. Alternatively, the soluble product may be recovered by standard techniques as described below: fractionation on an immunophilin or ion exchange column; ethanol precipitation; reversed phase HPLC; chromatography on silica or on a cation exchange resin such as DEAE; carrying out chromatographic focusing; SDS-PAGE; ammonium sulfate precipitation; and gel filtration using, for example, sephadex g-75; heparin-agarose (HA) chromatography, and the like.

In one embodiment, the method of producing an exogenous polypeptide of the present invention further comprises purifying the EGF from the cell lysate by sequentially using glutathione sepharose 4B resin. The inventors have surprisingly found that EGF protein of unexpectedly high purity can be obtained by dialysis after using glutathione Sepharose 4B resin.

In some embodiments of the methods of the invention for producing EGF, it comprises:

1) expression of plasmid pET42a (+) -GST-gp41-1-EGF, comprising: plasmid pET42a (+) -GST-gp41-1-EGF was transformed into T7 expression (T7 Express); adding 40. mu.g/mL^-1Kanamycin LB medium, at 37 degrees C growth; when A is₆₀₀The value reached 0.5, the growth temperature was lowered to 16 ℃ and final concentration 0.1mM IPTG was added and cultured.

2) Lysing and purifying the cell pellet obtained in step 1), comprising: after cell precipitation is cracked, glutathione agarose 4B resin is used for purification, buffer solution A is used for washing, and then buffer solution C is used for incubation and elution; the buffer solution A comprises 1 xPBS, 1 xPMSF, aprotinin, benzamide and leupeptin; buffer C included 50mM Tris-Cl, 1mM EDTA, 300mM NaCl, 2mM DTT, pH 8.

In some embodiments of the methods of the invention for producing EGF, expression of plasmid pET42a (+) -GST-gp41-1-EGF of step 1) comprises: plasmid pET42a (+) -GST-gp41-1-EGF was transformed into T7 expression, and a single colony of transformants were supplemented with 40. mu.g.mL^-1Kanamycin in 1L LB medium at 37 degrees C, 250rpm rotation; when A is₆₀₀When the value reached 0.5, the growth temperature was lowered to 16 ℃ and IPTG was added at a final concentration of 0.1 mM; the culture was carried out overnight.

In some embodiments of the methods of the invention for producing EGF, expression of plasmid pET42a (+) -GST-gp41-1-EGF of step 1) comprises: t7 expression pET42a (+) -GST-gp41-1-EGF transformant, added with 40. mu.g. mL^-1Kanamycin in 1L LB medium at 37 degrees C growth; when A is₆₀₀When the value reached 1, the entire culture was subcultured into a 50 liter fermenter containing 44 liters of LB medium. The cultures were grown at 37 ℃ with rotation at 100rpm and aeration ratio 1.5vvm until A₆₀₀The value reached 0.5, the growth temperature was lowered to 16 ℃ and IPTG was added at a final concentration of 0.1 mM; with 1M H₂SO₄And 1M NaOH was maintained at pH 7.0 and incubated overnight.

In some embodiments of the method for producing EGF according to the present invention, the cell pellet in step 2) is subjected to lysis and purification, including: resuspending the obtained cell pellet in 400mL buffer a; carrying out cracking treatment on the heavy suspension by ultrasonic waves, carrying out acoustic treatment for 10s, and carrying out 30x treatment at intervals of 30 s; then centrifuged at 10,000rpm for 30 minutes; after the supernatant is clarified, the supernatant is loaded on a glutathione sepharose 4B resin column, and then 10 times of bed volume is washed by buffer solution A; adding an induction buffer C to the chromatographic column, incubating at room temperature for 24 hours, and eluting by adding 3 bed volumes of buffer C; preserving the eluate, analyzing by Western blotting, dialyzing with 0.1 × PBS, and lyophilizing; the buffer solution A comprises 1 xPBS, 1 xPMSF, aprotinin, benzamide and leupeptin; buffer C included 50mM Tris-Cl, 1mM EDTA, 300mM NaCl, 2mM DTT, pH 8.

In another aspect, the present invention provides an EGF produced using the method for producing an EGF according to the present invention.

In another aspect, the present invention provides a composition comprising the EGF of the present invention and an adjuvant.

In some embodiments of the compositions of the present invention, the composition further comprises bFGF.

In some embodiments of the composition of the present invention, the EGF is present in an amount of 0.005% to 0.05% and the bFGF is present in an amount of 0 to 0.0005%.

In some embodiments of the compositions of the present invention, the EGF is present in an amount of 0.005% to 0.04% and the bFGF is present in an amount of 0 to 0.0003%

In some embodiments of the compositions of the present invention, the adjuvant comprises: water, phenoxyethanol, cetostearyl alcohol, cetostearyl 20, liquid paraffin and white soft paraffin.

In some embodiments of the compositions of the present invention, the composition is provided in the form of an emulsion, cream, ointment, gel, or liquid formulation.

In some embodiments of the compositions of the present invention, the compositions are used for skin wound healing, including but not limited to diabetic foot ulcers, pressure sores, or surgical wounds.

To maximize expression and solubility of the fusion protein, pET42a (+) vector with T7 promoter and N-terminal GST tag was selected as plasmid backbone for expression of EGF fusion protein. Inserts encoding the gp41-1 intron and EGF were synthesized and further amplified by PCR extension. A stop codon was inserted immediately after the coding sequence for EGF to prevent translation of the downstream C-terminal 8 × His embedded in the vector backbone. The GST tag was designed to be fused to the N-terminus of gp41-1 for purification of the expressed whole fusion protein, while EGF was fused to the C-terminus of a well studied gp41-1 mini-intron, where C-terminal cleavage can be accomplished by addition of DTT. Lower induction temperatures and lower concentrations of IPTG were chosen to further increase the solubility of the EGF fusion protein. The results show that at lower temperatures and low concentrations of IPTG, construct pET42a (+) -GST-gp41-1-EGF (FIG. 1) expressed high levels of soluble fusion protein both in shake flask and in fermentation scale, with no significant difference in expression levels.

It should be noted that the embodiments and features of the embodiments in the present application may be combined with each other without conflict.

Definition of terms

In this application, the use of the singular includes the plural unless specifically stated otherwise. It must be noted that, as used in this specification, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. In this application, the use of "or" means "and/or" unless indicated otherwise.

As used herein, reference in the specification to "some embodiments," "an embodiment," "one embodiment," or "other embodiments" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least some embodiments, but not necessarily all embodiments, of the invention.

As used herein, the term "comprising" means "including primarily, but not necessarily exclusively. Furthermore, variations of the word "comprising", such as "comprises" and "comprising", have a corresponding variation.

As used herein, the terms "polynucleotide" or "nucleic acid" are used interchangeably and refer to a molecule comprising one or more nucleotides, or oligonucleotides, or fragments thereof, including but not limited to RNA or DNA nucleotides or combinations thereof. As used herein, "coding sequence" refers to a polynucleotide segment that encodes a polypeptide. This region or sequence is bounded by a start codon near the 5 'end and a stop codon near the 3' end. Coding sequences may also be referred to as open reading frames.

As used herein, a nucleic acid construct, a polynucleotide encoding the polypeptide construct, an engineered cell carrying and/or expressing the polypeptide construct and the polynucleotide, and a method of modulating the activity of an engineered cell. Engineered cells as described herein may include immune effector cells engineered to encode and express cytokines, chimeric antigen receptors, and T cell receptors. In some embodiments of the invention, the nucleic acid construct of the invention may further comprise a first cloning site upstream of the insert and a second cloning site downstream of the insert, wherein the first cloning site and the second cloning site allow for insertion of the nucleic acid construct into an expression vector.

The cloning site allows for the cloning of a polynucleotide encoding a heterologous polypeptide. Preferably, the cloning sites combine to form a multiple cloning site. As used herein, the term "multiple cloning site" refers to a nucleic acid sequence comprising a series of two or more restriction endonuclease target sequences positioned adjacent to each other. The multiple cloning site comprises a restriction endonuclease target that allows for insertion of fragments having blunt ends, sticky 5 'ends, or sticky 3' ends. Insertion of the polynucleotide of interest is performed using standard Molecular Biology methods, for example, as described in Sambrook et al (Sambrook et al. Molecular Cloning: A Laboratory Manual, Cold Spring harbor Laboratory Press,1989) and/or Ausubel et al (Current Protocols in Molecular Biology, Greene pub. associates and Wiley-Interscience (1988).

As used herein, the term "ribosome binding site," abbreviated RBS, refers to a sequence upstream of the start codon of an mRNA that is available for binding to ribosomes at the time of initiation of translation.

As used herein, the terms "polypeptide", "peptide" and "protein" are used interchangeably to refer to a polymer of amino acid residues and to refer to fragments, variants, analogs, homologs (orthologs) or homologs (homologues) thereof. Thus, these terms apply to amino acid polymers in which one or more amino acid residues is a synthetic non-naturally occurring amino acid, such as a chemical analog of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. Within the scope of the terms "protein", "polypeptide", "peptide", "polynucleotide", and "nucleic acid" are fragments and variants thereof, including, but not limited to, reverse complement and antisense forms of polynucleotides and nucleic acids.

As used herein, Epidermal Growth Factor (EGF) is a heat-resistant single-chain low-molecular polypeptide consisting of 53 amino acid residues. In some embodiments of the invention, Epidermal Growth Factor (EGF) is human epidermal growth factor (hEGF). In other embodiments of the invention, the Epidermal Growth Factor (EGF) is native hEGF. In other embodiments of the invention, human epidermal growth factor (hEGF) may have or comprise the nucleotide sequence set forth in SEQ ID NO. 3, or may have or comprise, or may consist of, a nucleotide sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the nucleotide sequence set forth in SEQ ID NO. 3.

As used herein, a fibroblast growth factor is a class of polypeptides consisting of about 150-200 amino acids, present in two closely related forms, basic fibroblast growth factor (bFGF) and acidic fibroblast growth factor (aFGF). In one embodiment, the fibroblast growth factor useful in the present invention may be a basic fibroblast growth factor, particularly human bFGF, more particularly native human bFGF. In one aspect, the bFGF of the present invention may have or comprise the nucleotide sequence set forth in SEQ ID No. 4, or may have or comprise a nucleotide sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the nucleotide sequence set forth in SEQ ID No. 4, or may consist of the nucleotide sequence set forth above.

As used herein, a signal peptide is an amino acid sequence, typically located at the N-terminus of a newly synthesized protein or polypeptide, that directs the protein or polypeptide to the cell surface. In some embodiments, the signal peptide directs the polypeptide to the cell surface for insertion (e.g., through a transmembrane domain) within the cell membrane. In some embodiments, a polypeptide construct as described herein having a signal peptide is synthesized, but then post-translationally processed to cleave the signal peptide, such that the mature polypeptide construct lacks the signal peptide amino acid sequence. In other embodiments, the signal peptide sequence is not cleaved, but remains in the mature polypeptide construct.

As used herein, inteins are a class of polypeptides that have the function of self-splicing, which cleaves themselves from a precursor protein and catalyzes the formation of peptide bonds between the peptide chains on both sides of the catalyst. In some embodiments of the invention, the intein is gp41-1, a mini-protein intron.

As used herein, the term "affinity tag" refers to a protein or polypeptide that is expressed during recombinant protein production as a fusion with a protein of interest. The affinity tag can be used for promoting the solubility and stability of the target protein, and is convenient for the detection and purification of the target protein. In some embodiments of the invention, the affinity tag is a GST affinity tag.

As used herein, a promoter is a DNA sequence recognized, bound and initiated by RNA polymerase and contains conserved sequences required for RNA polymerase specific binding and transcription initiation, most of which is located upstream of the transcription initiation point of a structural gene, and is not transcribed per se. In this context, the term "promoter" is also used to describe recombinant, synthetic or fusion molecules, or derivatives or fragments, such as truncation sequences of promoters, which confer, activate or enhance expression of the nucleic acid molecule to which they are operably linked, as well as encoding peptides. Exemplary promoters useful in the present invention may include promoters active in prokaryotes, such as the T7 promoter, phoA promoter, beta-lactamase and lactose promoter systems, alkaline phosphatase, tryptophan (trp) promoter systems, and hybrid promoters such as the tac promoter. In some embodiments of the invention, the promoter is the T7 promoter.

As used herein, an operon is a functional polynucleotide unit comprising multiple genes under the control of a single promoter. Genes are transcribed together into one mRNA strand and then translated together in the cytoplasm, or undergo trans-splicing to produce individually translated monocistronic mrnas, i.e., multiple mRNA strands each encoding a single gene product. The result is that the genes contained in the operon are either expressed together or not expressed at all. Exemplary operons that can be used in the present invention include, but are not limited to, the lactose operon, the arabinose operon, the tryptophan operon, and the like. The lactose operon is a group of genes involved in lactose breakdown, consisting of repressors and operator sequences of the lactose system, such that a group of genes involved in lactose metabolism are synchronously regulated. In some embodiments of the invention, the operon is a lactose operon.

The term "expression vector" as used herein means a nucleic acid having the ability to confer expression of a nucleic acid fragment to which it is operably linked in a cell or cell-free system. In the context of the present invention, it is understood that the expression vector comprising a promoter as defined herein may be a plastid, phage, phagemid, cosmid, viral subgenomic or genomic fragment, or other nucleic acid capable of maintaining and/or replicating the heterologous DNA in an expressible form which is introduced into a cell. The expression vector according to the present invention may further comprise a polynucleotide encoding a marker protein. Marker proteins suitable for the present invention include proteins that are antibiotic resistant or resistant to other toxic compounds. Examples of marker proteins with antibiotic resistance include neomycin phosphotransferase, which phosphorylates neomycin and kanamycin, or hpt, which phosphorylates hygromycin, or proteins conferring resistance to, for example, bleomycin, streptomycin, tetracycline, chloramphenicol, ampicillin, gentamicin, geneticin (G418), spectinomycin, or blasticidin. In one example, the protein confers resistance to chloramphenicol. For example, the protein is a gene from E.coli, designated CmR, as described in Nilsen et al, J.Bacteriol, 178: 3188 and 3193, 1996.

One skilled in the art will appreciate that there is no limitation on the type of vector that may be used, as long as the vector can be a cloning vector suitable for propagation, availability of sufficient polynucleotide or gene construct, or an expression vector suitable for purification of the fusion protein in a different heterologous organism. In one embodiment, suitable vectors according to the present invention include expression vectors in prokaryotes, such as prokaryotic expression vectors, including but not limited to: pET14, pET21, pET22, pET28, pET42, pMAL-2c, pTYB2, pGEX-4T-2, pGEX-6T-1, pQE-9, pBAD-his, pBAD-Myc, pECB series vectors, pRB series vectors and the like, for example, pUC18, pUC19, Bluescript and derivatives thereof, mp18, mp19, pBR322, pBR374, pMB9, CoIE1, pCR1, RP4, phages and "shuttle" vectors (for example, pSA3 and pAT 28).

In some embodiments of the invention, the invention also contemplates shuttle vectors. As used herein, the term "shuttle vector" is a type of vector that can replicate and amplify in two different host cells (e.g., escherichia coli and bacillus subtilis), thereby enabling the transformation of the same expression vector into different host cells. The shuttle vector to which the present invention relates may include, but is not limited to, pECBS 1.

The vector components may generally include, but are not limited to, one or more of the following expression control elements: promoters, enhancers, operators, ribosome binding sites, transcription termination sequences, and the like.

As used herein, a host cell refers to a cell used to produce a protein encoded within an expression cassette of the invention.

As used herein, T7 expresses Competent Cells (T7 expression), and E.coli can take up foreign DNA (Plasmid, Phage DNA) after treatment with Ca ions, and Cells in this state are called Competent Cells (Competent Cells). The T7 Express strain is an enhanced strain of Escherichia coli BL21, is defect-type for Lon and OmpT protease, is mainly suitable for protein expression of prokaryotic expression vectors (such as pET and the like) containing a T7 promoter, and is also suitable for expression vectors (such as pGEX and the like) of non-T7 promoters which need Escherichia coli RNA polymerase to transcribe RNA. The strain is different from BL21(DE3) strain in that T7 RNA polymerase gene is integrated in lac operator region on bacterial chromosome, there is no lambda prophage sequence in genome, and it has the character of resisting T1 phage infection. T7 expression competent cells are prepared by a special process, and the pUC19 plasmid detects 8-times cfu/mu g DNA with the transformation efficiency of more than 10. The product is characterized by sensitivity to ampicillin, kanamycin, spectinomycin, chloramphenicol, streptomycin and tetracycline.

As used herein, the term "transformation" means the introduction of DNA into a prokaryotic host either as an extrachromosomal element or by chromosomal integration such that the DNA can replicate. Depending on the host cell used, transformation is carried out using standard techniques appropriate for such cells. Calcium treatment using calcium chloride is typically applied to bacterial cells containing a strong cell wall barrier. Another method for transformation uses polyethylene glycol/DMSO. Yet another technique used is electroporation.

The prokaryotic host cells used to produce the exogenous polypeptide of the present invention are cultured in media known in the art and suitable for the culture of the host cells. Examples of suitable media may include Luria-Bertani (LB) media supplemented with essential nutrient supplements. In some embodiments, the medium further comprises a selection agent that selects based on the expression vector constructed to selectively allow growth of prokaryotic cells containing the expression vector. For example, ampicillin and/or kanamycin are added to a medium for growth of cells that express ampicillin and/or kanamycin resistance genes. Any necessary supplements other than carbon, nitrogen and inorganic phosphorus sources may also be included at suitable concentrations, which may be introduced alone or in admixture with another supplement or medium, such as a complex nitrogen source.

As used herein, a restriction endonuclease is a class of enzymes that can recognize and attach a specific sequence of deoxyribonucleotides and cleave the phosphodiester bond between two deoxyribonucleotides at a specific site in each strand, referred to as restriction enzymes for short. According to the structure of the restriction enzyme, the requirement of the cofactor is cut and acted on the way, can be divided into three types with the restriction enzyme, be first Type (Type I), second Type (Type II) and third Type (Type III) respectively. Type I restriction enzymes can catalyze both methylation of host DNA and hydrolysis of unmethylated DNA; whereas type II restriction enzymes catalyze the hydrolysis of only unmethylated DNA. The type III restriction enzyme has the functions of modification and cognitive cleavage. Restriction enzymes useful in the present invention may include, but are not limited to: EcoRI, PstI, XbaI, BamHI, HindIII, TaqI, NotI, HinfI, Sau3A, PovII, SmaI, HaeIII, AluI, SalI, Dra, etc. In some embodiments of the invention, NotI and/or SpeI restriction enzymes are used.

As used herein, a PCR wash kit is suitable for extracting PCR amplification products from PCR, enzymatic reactions, sequencing reactions.

The term "adjunct materials" means any liquid, solid or gaseous material selected for the particular type of detergent composition desired and the form of the product (e.g., liquid, granular, powder, bar, paste, spray, tablet, gel or foam composition), which is also preferably compatible with the protease variant used in the composition. In some embodiments, the granular composition is in a "compressed" form, while in other embodiments, the liquid composition is in a "concentrated" form. In some embodiments of the invention, the composition is provided in the form of an emulsion, cream, ointment, gel, or liquid formulation. In some embodiments of the invention, the adjuvants comprise at least one of water, petroleum jelly, mineral oil, vegetable oil, animal oil, waxes, and polymers. In some embodiments of the invention, the adjuvant comprises: water, phenoxyethanol, cetostearyl alcohol, cetostearyl 20, liquid paraffin and white soft paraffin.

Exemplary sequences in the present invention are shown in the following table.

Drawings

FIG. 1 is a schematic diagram of a DNA construct expressing GST-gp41-1-EGF according to the present invention. The figure shows the plasmid construct vector (6.2kb) expressing the GST-gp41-1-EGF insert cassette. The symbols of the genetic components displayed include: ori is the origin of replication in e.coli; KanR ═ kanamycin resistance gene; GST ═ glutathione S-transferase tag; gp41-1 ═ gp41-1 intron; EGF ═ EGF gene. Arrows indicate the direction of gene expression.

FIG. 2 is a Western blot analysis of soluble EGF fusion protein. pET42a (+) -GST-gp41-1-EGF transformants collected at different time intervals after induction with IPTG were analyzed. Lanes 1L 0h, 45L 0h, 1L ON, 45L ON: samples collected from 0h and overnight cultures were induced from 1L and 45L cultures, respectively, with 10. mu.L of cell lysate loaded on each lane. Lane-ve: cell lysates extracted from pET42a (+) vector cultures were induced overnight at 10. mu.L.

FIG. 3 is a Western blot analysis of cleavage on an EGF column. The efficacy of the fusion protein to cleave EGF on the column was analyzed. Lane entry: 10 μ L of cell lysate sample collected from 45L of culture; lane 4 hours, 24 hours: after incubating the buffer C after 4 hours and 24 hours respectively, collecting the eluent; lane-ve: cell lysates extracted from pET42a (+) vector cultures were induced overnight at 10. mu.L.

FIG. 4 shows mitotic effects of EGF. Shows the samples from different concentrations of recombinant EGF

Exhibit mitotic effects

Fig. 5 is a graph showing the effect of the aqueous cream (1) on the treatment of bedsores. (A) A 83 year old woman with bedsores; (B) after 3 days of treatment with the aqueous cream (1), the wound had healed completely.

Figure 6 is a graph of the effect of treating skin breakdown with aqueous cream (2). (A) Head injury in children 2 years old and immediate treatment with aqueous cream (2); (B) blood clots formed one hour after treatment; (C) scabs formed within 12 hours after injury; (D) the wound healed completely without scarring.

FIG. 7 is a graph of the effect of the aqueous cream (3) on the treatment of diabetic foot ulcers. (A) An elderly 76 years old with diabetic foot ulcers for 3 months; (B) after 4 days treatment with aqueous cream (2); (C) after 8 days of treatment, the wound was completely closed.

Detailed Description

The following provides a description of expression systems and methods that can be used to express a variety of exogenous polypeptides, particularly native exogenous polypeptides. These systems and methods satisfy at least one need existing in the art. The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described in any way.

Unless otherwise explicitly defined, terms used herein should be understood according to their ordinary meaning in the art. Unless the context clearly dictates otherwise, the terms "a", "an", and "the" mean "one or more" are used interchangeably. Polynucleotides encoding polypeptides of interest may be cloned into the vectors of the invention using standard techniques well known to those skilled in the art. For example, Polymerase Chain Reaction (PCR) is used to generate polynucleotides encoding the polypeptides of interest. Methods of PCR manipulation are known in the art. Standard techniques and procedures are generally performed according to conventional methods in the art and various general references (see, generally, Sambrook et al, molecular Cloning: A Laboratory Manual, 2nd ed. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).

Examples

The invention is described herein by way of the following examples, which are intended to be illustrative only and not limiting as to the scope of the invention.

Bacterial strains and chemicals

E.coli strains DH5 alpha and T7 expression, and restriction enzymes from New England Biolabs (Ipswich, MA);

synthetic DNA fragments and antibodies against EGF were purchased from Thermo Fisher Scientific (Ipswich, MA);

all other chemicals were purchased from Sigma-Aldrich (st. louis, MO) unless otherwise noted.

Example 1: construction of EGF expression vector

The expression construct pET42a (+) -GST-gp41-1-EGF was constructed as follows:

a synthetic DNA fragment encoding the 5 'to 3' end sequence of NotI-stop codon-EGF-gp 41-1-thrombin site-SpeI was synthesized by Thermo Fisher Scientific (SEQ ID NO: 5). The synthesized DNA fragment was amplified by PCR extension using forward primer 5'-AAAAAGCGGCCGCTTAGCGCAGTTC-3' and reverse primer 5'-AAAAAACTAGTCTGGTGCCACGCGGTAGTT-3'. The amplified PCR product was purified by PCR wash Kit (Axygen AxyPrep Clean-Up Kit) and digested with NotI and SpeI. The digested fragment was purified in 1% agarose and ligated into pET42a (+) digested with the same restriction enzymes.

The plasmid sequence of pET42a (+) -GST-gp41-1-EGF was confirmed by Sanger sequencing.

Example 2: expression of EGF fusion proteins in shake flasks

Plasmid pET42a (+) -GST-gp41-1-EGF was transformed into T7 expression (T7 Express). Single colonies of transformants were supplemented with 40. mu.g.mL^-1Kanamycin was grown in 1L LB medium at 37 deg.C (rotation at 250 rpm). When A is₆₀₀When the value reached 0.5, the growth temperature was lowered to 16 ℃ and IPTG was added at a final concentration of 0.1 mM. The cultures were grown overnight. The 1mL cell pellet was collected and resuspended in 200 μ L of resuspension buffer (50mM Tris-Cl, 200mM EDTA, pH 8.0) supplemented with 1x PMSF, aprotinin, benzamide and leupeptin, then incubated on ice for 5 minutes. Then, 120. mu.L of lysozyme solution (1 mg. mL) was added thereto at 37 ℃^-1) The mixture was treated for 20 minutes. Then 80. mu.L of lysis buffer (10mM EDTA, 10% Triton X-100 and 50mM Tris-Cl, pH 8.0) was added. The tube containing the solution was gently inverted and then centrifuged at 14,800rpm for 5 minutes. Cell lysate samples were analyzed by western blotting.

Example 3: expression of EGF fusion proteins in fermentors

T7 expression pET42a (+) -GST-gp41-1-EGF transformant, added with 40. mu.g. mL^-1Kanamycin was grown in 1L LB medium at 37 deg.C (rotation at 250 rpm). When A is₆₀₀When the value reached 1, the entire culture was subcultured into a 50 liter fermenter containing 44 liters of LB medium. The cultures were grown at 37 ℃ (rotation at 100rpm, aeration ratio 1.5vvm) until A₆₀₀The value reached 0.5, the growth temperature was lowered to 16 ℃ and 0.1mM final concentration was addedIPTG. In use 1M H₂SO₄And 1M NaOH to maintain pH at 7.0, allow the culture to grow overnight. The cell pellet was collected by continuous centrifugation and washed twice with buffer a (1x PBS and 1x PMSF, aprotinin, benzamide and leupeptin) and then stored for a long period of time.

Example 4: protein purification and induced cleavage of EGF

The cell pellet obtained was resuspended in 400mL of buffer A. The resuspension was lysed by sonication (10s sonication 30x intervals) followed by centrifugation at 10,000rpm for 30 min. After clarification of the supernatant, it was loaded onto a glutathione sepharose 4B resin column and washed 10 bed volumes with buffer A. Induction buffer C (50mM Tris-Cl, 1mM EDTA, 300mM NaCl, 2mM DTT, pH 8) was added to the column and incubated at room temperature for 24 hours. Elution was performed by adding 3 bed volumes of buffer C. The eluate was stored, analyzed by Western blot, dialyzed against 0.1x PBS and lyophilized for use.

Example 5: biological assay for EGF

The MTT method was used to analyze the mitogenic effect of reconstituted EGF on the proliferation of NIH/3T3 fibroblasts (Wu KC, et al (2019) Cost-Effective Expression of Human Bio-interactive Basic fiber Growth Factor in Bacillus subtilis by Expression Asp DnaE Protein in Int. J Mol Gene Med 13: 438).

Preparation and application instructions of reviving cream

Lyophilized EGF and bFGF (Kwong WY, et al (2019) Enhanced Expression of Human identification, exogenous FGF2 in Human embryo kit 293T defective by the Protein in intron. Transl biomed. Vol.10: No.1: 157; Wu KC, et al (2019) Cost-Effective Expression of Human Bio-Identical Basic fiber texture Factor in Bacillus subtilis by Expression DnaE Protein in Man Gene Med 13:438), (1) 0.005% EGF, (2) 0.02% and 0.15% FGF, (3) 0.04% and 0.3% Asp cream, respectively, mixed with aqueous paraffin wax, 10 min, ethyl stearate, 10 min, and optionally mixed with ethyl alcohol to obtain a soft cream.

The mixed cream is applied to affected parts of patients suffering from bedsore, wound and diabetic foot ulcer.

Results and discussion

1. The construct pET42a (+) -GST-gp41-1-EGF showed high expression both in shake flask and in fermentation scale

To maximize expression and solubility of the fusion protein, pET42a (+) vector with T7 promoter and N-terminal GST tag was selected as plasmid backbone for expression of EGF fusion protein. Inserts encoding the gp41-1 intron and EGF were synthesized and further amplified by PCR extension. A stop codon was inserted immediately after the coding sequence for EGF to prevent translation of the downstream C-terminal 8 × His embedded in the vector backbone. The GST tag was designed to be fused to the N-terminus of gp41-1 for purification of the entire fusion protein after expression, while EGF was fused to the C-terminus of a well-studied gp41-1 mini-intron, where C-terminal cleavage can be accomplished by addition of low concentrations of DTT. Lower induction temperatures and lower concentrations of IPTG were chosen to further increase the solubility of the EGF fusion protein. The results show that at lower temperatures and low concentrations of IPTG, construct pET42a (+) -GST-gp41-1-EGF (FIG. 1) expressed high levels of soluble fusion protein both in shake flask and in fermentation scale, with no significant difference in expression levels (FIG. 2).

2. Easy purification and simple process

For purification of the EGF fusion protein, the cell pellet obtained was first lysed and then a sample of the cell lysate was loaded onto glutathione sepharose 4B, the GST-tagged EGF fusion protein could be specifically captured, while the non-specifically bound protein could be removed by successive washes with buffer a. The results show that EGF C-exon can be efficiently cleaved from gp41-1 mini-intron after long incubation under low concentration DTT induction conditions (FIG. 3). The EGF obtained was dialyzed against a low salt buffer having a neutral pH and lyophilized to prolong its storage time.

3. The recombinant EGF has good biological activity

Since EGF is capable of triggering cell proliferation by binding to cellular EGF receptor, the same method of examining EGF for mitogenic activity, such as bFGF, on NIH/3T3 cells is feasible. From the MTT assay results, recombinant EGF was observed to have biological activity in triggering cell proliferation of NIH/3T3 cells (fig. 4).

In addition to in vitro biological assays, clinical case studies have also been performed. We prepared a homemade aqueous cream and added EGF and bFGF at various concentrations expressed by the present invention as described in materials and methods for treating patients with various skin disruptions. The results show that the EGF expressed by the invention can effectively improve the healing process of patients suffering from bedsore (figure 5), epidermal wound (figure 6) and diabetic foot ulcer (figure 7). Case studies are shown below:

case (a): an 83 year old woman with bedsores (fig. 5A) was treated with 0.005% EGF aqueous cream (1). The wound had completely healed after three days of treatment (fig. 5B).

Case (B): the epidermis of the head of a two year old child was accidentally injured and immediately treated with 0.02% EGF and 0.00015% bFGF aqueous cream (2) (fig. 6A). After 5 minutes of treatment of the aqueous cream (2), bleeding immediately ceased, and a clot formed after one hour (fig. 6B). Scabs formed after 12 hours (fig. 6C) and after two weeks of treatment, the wound healed completely with no scarring (fig. 6D).

Case (C): an elderly 76 years old with diabetic foot ulcers for three months was treated with an aqueous cream of 0.04% EGF and 0.0003% bFGF (3). Four days after treatment, the wound size was significantly reduced (fig. 7A). Four days of continued treatment, the wound was completely closed (fig. 7B).

According to the results of the three case studies, EGF purified from the fusion protein of the present invention was demonstrated to have high activity. The present invention demonstrates that the final recombinant EGF obtained by the intron method has biological activity in treating patients with various degrees of skin damage. This method may be applicable to the expression of various recombinant proteins without the need for post-translational modification.

Those skilled in the art will further appreciate that the present invention may be embodied in other specific forms without departing from the spirit or central characteristics thereof. Since the foregoing description of the invention discloses only exemplary embodiments thereof, it should be understood that other variations are considered to be within the scope of the invention. Therefore, the present invention is not limited to the specific embodiments described in detail herein. Rather, reference should be made to the following claims as indicating the scope and content of the invention.

SEQUENCE LISTING

<110> dream Qian science and technology intellectual Property Limited

<120> nucleic acid construct of epidermal growth factor, method for producing the same and composition thereof

<130> 202007

<160> 5

<170> PatentIn version 3.5

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cgatgtcagt accgagacct gaagtggtgg gaactgcgc 159

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ccagccttgc cagaggatgg cggcagcggc gccttcccgc caggccactt caaggaccca 60

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ggagttgtgt ctatcaaagg agtgtgtgct aaccgttacc tggctatgaa ggaagatgga 240

cgcttactgg cttctaaatg tgttacggat gagtgtttct tttttgaacg cttggaatct 300

aataactaca atacttaccg ctcacgcaaa tacaccagtt ggtatgtggc actgaaacgc 360

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gcggccgctt agcgcagttc ccaccacttc aggtctcggt actgacatcg ctccccgatg 60

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tcatggaggc agtacccatc gtgggacagg ggacattcag agtcactatt attatgggtc 180

agaatatcat tggcatagaa caggtggtta ccgctaactt caatatcaat cagttcgcgt 240

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tcttcgctac aaatgatttc tttgccatct tccagggtga ttttatagct tttttttttg 420

cttttcggaa acacattcag cacttcatta taaccggtat tgctcagaac cagatcaccc 480

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Claims

1. A nucleic acid construct comprising an insert comprising, from 5 'to 3', a polynucleotide sequence encoding an affinity tag, an intein, and an epidermal growth factor, wherein the affinity tag is a GST affinity tag and the intein is gp41-1 protein.

2. The nucleic acid construct of claim 1, wherein the insert comprises, from 5 'to 3', a polynucleotide sequence encoding a T7 promoter, a lactose operon, a GST affinity tag, an intein of gp41-1 protein, an epidermal growth factor.

3. An expression vector comprising the nucleic acid construct of any one of claims 1-2.

4. A host cell comprising the expression vector of claim 3.

5. The host cell according to claim 4, which is transformed E.coli (Escherichia coli).

A method of constructing an EGF expression vector, comprising:

7. A primer pair for constructing an EGF expression vector comprises:

a forward primer 5'-AAAAAGCGGCCGCTTAGCGCAGTTC-3';

the reverse primer 5'-AAAAAACTAGTCTGGTGCCACGCGGTAGTT-3'.

8. A method of producing EGF using the nucleic acid construct of any one of claims 1-2 or the expression vector of claim 3 or the host cell of claim 4 or 5.

9. The method of producing EGF according to claim 8, comprising:

1) expression of plasmid pET42a (+) -GST-gp41-1-EGF, comprising: plasmid pET42a (+) -GST-gp41-1-EGF was transformed into T7 expression; adding 40. mu.g/mL^-1Growth in LB medium with kanamycin at 37 ℃; when the A600 value reached 0.5, the growth temperature was lowered to 16 ℃ and a final concentration of 0.1mM IPTG was added for culture;

10. An EGF produced by the method for producing an EGF according to claim 8 or 9.

11. A composition comprising the EGF of claim 10 and an adjuvant.

12. The composition of claim 11, further comprising bFGF.

13. The composition of claim 12, wherein the EGF is present in an amount of 0.005% to 0.05% and the bFGF is present in an amount of 0 to 0.0005%.

14. The composition of claim 12, wherein the EGF is present in an amount of 0.005% to 0.04% and the bFGF is present in an amount of 0 to 0.0003%.

15. The composition of claim 11, wherein the adjuvants comprise water, phenoxyethanol, cetearyl alcohol, cetearyl 20, liquid paraffin, and white soft paraffin.

16. The composition of claim 11, provided in the form of an emulsion, cream, ointment, gel, or liquid formulation.