CN109575107B - Cell penetrating peptide and bifidobacterium capable of expressing oral interferon - Google Patents
Cell penetrating peptide and bifidobacterium capable of expressing oral interferon Download PDFInfo
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- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
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- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/52—Cytokines; Lymphokines; Interferons
- C07K14/555—Interferons [IFN]
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/52—Cytokines; Lymphokines; Interferons
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- C07K14/56—IFN-alpha
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/52—Cytokines; Lymphokines; Interferons
- C07K14/555—Interferons [IFN]
- C07K14/565—IFN-beta
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/52—Cytokines; Lymphokines; Interferons
- C07K14/555—Interferons [IFN]
- C07K14/57—IFN-gamma
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
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- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/10—Fusion polypeptide containing a localisation/targetting motif containing a tag for extracellular membrane crossing, e.g. TAT or VP22
Abstract
The invention belongs to the field of biomedicine, and discloses a cell penetrating peptide and a bifidobacterium capable of expressing oral interferon. The oral interferon-expressing bifidobacterium can express the fusion protein of the cell penetrating peptide and the interferon, wherein the cell penetrating peptide is connected to the N end of the interferon. The cell penetrating peptide provided by the invention has higher transdermal activity, the prepared bifidobacterium longum capable of expressing oral interferon can be planted in animal intestinal tracts, and after the cell penetrating peptide is taken by an oral inducer, the cell penetrating peptide can express interferon alpha-2 b capable of penetrating intestinal epithelium into a body, has good transdermal property and can enhance the treatment effect.
Description
Technical Field
The invention belongs to the field of biological medicines, and particularly relates to a cell penetrating peptide and a bifidobacterium capable of expressing oral interferon.
Background
Transdermal drug delivery refers to a drug delivery system in which a drug, after being delivered through the skin surface, penetrates the stratum corneum of the skin and enters the capillaries to enter the blood circulation, thereby performing systemic therapeutic action. As a special administration route, compared with the traditional administration mode of oral administration and injection, the medicine has the unique advantages that: the first pass effect of the liver and the inactivation of the medicine in the gastrointestinal tract can be avoided, better medicine effect can be achieved, and the individual difference of medicine application is reduced; the constant and effective blood concentration is maintained, the peak-valley phenomenon of the blood concentration is avoided, and the toxic and side effects are reduced; the action time of the medicine is prolonged, the administration times are reduced, and most patients are easy to accept; has no pain, is convenient to use, and can be independently used by a patient or withdrawn at any time.
During transdermal drug absorption, the drug needs to diffuse across the skin's physical barrier into the blood circulation and ultimately into the targeted tissue. Many internal and external factors in this process affect the transdermal process of the drug. These factors include the condition of the skin itself, the physicochemical properties of the drug, the formulation of the preparation, etc. The physicochemical properties of the drug directly affect the absorption rate of the drug in transdermal administration. The dosage form and formulation composition of the formulation also have a direct effect on the transdermal delivery efficiency. The condition of the skin is also an important factor affecting the transdermal absorption of the drug when the drug is administered transdermally, and the site of the skin, the degree of damage, the thickness, the pore distribution, the temperature and humidity, the degree of cleaning, and the like all affect the transdermal absorption efficiency of the drug.
Therefore, enhanced transdermal drug delivery has been gradually applied in clinical treatment, and most of them focus on the use of chemical transdermal enhancers and physical assistance.
The cell membrane is a semipermeable barrier between the cell and the extracellular environment, and has a selective permeation function so as to maintain the constant internal environment of the cell. While this phospholipid bilayer is essential for cell survival and function, it presents challenges to the exchange of cargo molecules inside and outside the cell. Since macromolecular substances of drugs such as proteins, polypeptides, nucleotides and the like and developers must reach the inside of cells to play corresponding roles, it becomes necessary to realize transmembrane transport of the substances.
Currently, there is a class of biological polypeptides that rapidly penetrate into mammalian cells and still retain their original structure and function, but which do not rely on endocytosis for entry into the cell, such polypeptides being referred to as cell-penetrating peptides (CPPs). Such polypeptides are rich in basic amino acids and therefore are generally positively charged. CPP can not only penetrate cells by itself, but also load other substances and promote the cell penetration of the substances, such as protein, DNA, siRNA, liposome, nano material and the like. Cell-penetrating peptides also have penetrating power to epidermal cells of animals, and thus in recent years, cell-penetrating peptides have been used as transdermal enhancers for transdermal administration of macromolecular drugs. The discovery of such agents provides a convenient route to transdermal delivery techniques.
Interferons (IFNs) are a class of cytokines secreted by recipient cells after human and animal cells have been infected with viruses or induced by various factors such as nucleic acids, bacterial endotoxins, mitogens, and the like. Antiviral activity is one of the basic functions of the interferon family. Interferons exert their antiviral action primarily through body cells and do not themselves kill or inhibit viruses. Viral infection of cells induces production and release of IFN, followed by death and disintegration of the infected cells, and the IFN molecules are released and spread throughout the body with blood circulation. IFN molecules bind specifically to receptors on the surrounding cell membrane, enhancing the activity of cellular membrane adenylate cyclase, and promoting the formation of cyclic adenosine monophosphate. The increase of cyclic adenosine monophosphate can inhibit DNA synthesis, cell division and the like, and further inhibit virus replication. The transdermal administration of the interferon is researched, so that the compliance of patients is improved in the aspect of treating viral infections such as herpes virus, HPV and the like.
Bifidobacterium (Bb) is an anaerobic gram-positive bacterium isolated from the feces of breast-fed infants by doctor Tissier, France in 1899, often with bifurcate ends, hence the name Bifidobacterium. Bifidobacteria, as normal flora in the human and mammalian intestinal tracts, colonize the host intestinal tract by adhesion. In recent years, with the development of molecular biology technology, bifidobacteria are widely used as a vector delivery system due to advantages of safety and the like, and a series of recombinant bifidobacteria have been constructed in the fields of bacteria, viruses, tumors, parasites and the like, so that the bifidobacteria have wide application prospects.
The present invention has been made in view of this situation.
Disclosure of Invention
The technical problem to be solved by the invention is to overcome the defects of the prior art and provide a cell penetrating peptide and a bifidobacterium capable of expressing oral interferon. The cell penetrating peptide provided by the invention has higher transdermal activity, the prepared bifidobacterium longum capable of expressing oral interferon can be planted in animal intestinal tracts, and after the cell penetrating peptide is taken by an oral inducer, the cell penetrating peptide can express interferon alpha-2 b capable of penetrating intestinal epithelium into a body, has good transdermal property and can enhance the treatment effect.
In order to solve the technical problems, the invention adopts the technical scheme that:
the first purpose of the invention is to provide a cell penetrating peptide, the cell penetrating peptide is a peptide for mediating and delivering bioactive molecules into cells, and the amino acid sequence of the cell penetrating peptide comprises a sequence shown in SEQ ID NO. 1.
The invention adopts phage in vivo display technology, and through multiple rounds of screening, the amino acid sequence of the cell penetrating peptide obtained by screening comprises 15 amino acids, as shown in SEQ ID NO. 1, namely LHTWRLRLARSPHLK, applicant names the cell penetrating peptide as: HXPPC. The applicant finds that the cell penetrating peptide obtained by screening has high transdermal activity, can be used as a carrier to carry target substances (such as protein, DNA, siRNA and the like) to permeate through the epidermal layer of the skin, and has wide application.
It is a second object of the present invention to provide a nucleotide sequence encoding the cell penetrating peptide as described above.
It is a third object of the present invention to provide a vector, a recombinant bacterium or a recombinant cell comprising a nucleotide sequence encoding the cell-penetrating peptide as described above.
The vector referred to in this embodiment may be any available vector such as an expression vector, a shuttle vector, an integration vector, and the like.
Preferably, the recombinant vector is a shuttle vector.
The recombinant bacteria can be bacteria, fungi, viruses, animal cells and the like.
It is a fourth object of the present invention to provide a use of the cell-penetrating peptide as described above as an intracellular delivery vehicle;
preferably, the use of said cell penetrating peptide as a carrier for intracellular delivery of a therapeutic agent.
Specifically, the cell penetrating peptide screened by the invention is used as a carrier for carrying a drug molecule, and the carried drug molecule is delivered to the cytoplasm and/or nucleus of a target cell. The drug may include antiviral infection drug, antitumor drug, cytotoxic drug, biomembrane damaging drug, gene drug, neurotrophic molecule, photosensitive drug, stem cell regulatory factor, etc.
It is a fifth object of the present invention to provide a complex comprising a cell penetrating peptide as described above and a cargo molecule;
preferably, the cell penetrating peptide and the cargo molecule are coupled to each other;
preferably, the cargo molecule is coupled to the end of the cell penetrating peptide.
In this embodiment, the cargo molecule is a macromolecule that needs to enter the cell interior by using a cell-penetrating peptide as a carrier, and the macromolecule is preferably, but not limited to, at least one of a molecule with pharmaceutical activity, a molecule with labeling effect, and a molecule with targeting effect.
The coupling may be by covalent or non-covalent linkage.
It is a fifth object of the present invention to provide a fusion protein comprising the cell-penetrating peptide as described above linked to the N-terminus of interferon and an interferon protein.
The screened cell penetrating peptide HXCPP with high penetrability is connected to the N end of the interferon, the cell penetrating peptide-interferon fusion protein is constructed on the carrier to express the cell penetrating peptide-interferon, and the obtained fusion protein has the activity of the interferon, has the capability of penetrating the skin, has good patient compliance and can improve the treatment effect in the aspect of virus infection.
In a further embodiment, the interferon is selected from alpha interferon, beta interferon or gamma interferon and homologous type interferon;
preferably, the interferon is interferon alpha-2 b.
The sixth purpose of the invention is to provide a bifidobacterium capable of expressing oral interferon, wherein the bifidobacterium can express the fusion protein;
preferably, the bifidobacterium is bifidobacterium longum.
A seventh object of the present invention is to provide a method for preparing bifidobacterium as described above, comprising the steps of:
(1) connecting the cell-penetrating peptide to the N-terminus of interferon to construct a recombinant vector having a cell-penetrating peptide-interferon nucleotide sequence;
(2) transferring the recombinant vector into bifidobacterium, and screening to obtain the recombinant bifidobacterium.
The method comprises the following steps:
(1) constructing genes encoding the cell penetrating peptide and IFN alpha-2 b on shuttle vector pHX101 to construct recombinant plasmid pHX101-HXCPP-IFN alpha-2 b;
(2) transforming the recombinant plasmid in the step (1) into bifidobacterium longum, and screening to obtain recombinant bifidobacterium longum;
(3) and (3) carrying out induction culture on the recombinant bifidobacterium longum in the step (2).
The specific method comprises the following steps:
screening CPP capable of penetrating skin by using phage in vivo display technology. Phages enriched to sufficient purity were selected through 3-4 rounds of selection. The DNA sequence of the polypeptide carried by the phage is sequenced by extracting the phage, so that the polypeptide sequence is obtained. Through the steps, a CPP polypeptide sequence is screened out as follows: LHTWRLRLARSPHLK, which is named: HXPPC.
The screened HXPCP and IFN alpha-2 b genes are constructed on a shuttle vector pHX101 (the shuttle vector has an escherichia coli and bifidobacterium replicon simultaneously, can be replicated and expressed in escherichia coli and can also be replicated and expressed in bifidobacterium), a recombinant plasmid pHX 101-HXPCP-IFN alpha-2 b is obtained by screening, and pHX101-IFN alpha-2 b is constructed as a control.
The plasmids are respectively transformed into bifidobacterium longum, and the culture supernatants are all shown to be positive by dot blot detection after induction, namely the expression that HXCPP-IFN alpha-2 b and IFN alpha-2 b are secreted.
The bifidobacterium longum carrying the plasmid is perfused with gastric perfusion inducer L-arabinose after two weeks of respectively perfusing the gastric perfusion mice, blood is extracted from the tail veins of the mice at 0h, 1h, 2h, 3h, 4h and 6h, and the amount of HXCPP-IFN alpha-2 b and IFN alpha-2 b is detected by ELISA. The results show that a larger amount of HXPPP-IFN alpha-2 b was detected. But IFN alpha-2 b expression of very small amount.
After adopting the technical scheme, compared with the prior art, the invention has the following beneficial effects:
1. the cell penetrating peptide HXCPP is easy to prepare, has small molecular weight, is not easy to generate immunological rejection, has strong penetrating power and higher transdermal activity, can be used as a carrier to carry target substances (such as protein, DNA, siRNA and the like) to penetrate through the epidermis layer of the skin, and has wide application.
2. The fusion protein formed by fusing the cell penetrating peptide HXCPP and the interferon has the activity of the interferon, has the capability of penetrating the skin, is easier to be absorbed by the skin, can quickly exert the effect, has good patient compliance, and can improve the treatment effect on the aspect of virus infection.
3. The bifidobacterium longum capable of orally expressing interferon can be planted in animal intestinal tracts, and can express interferon alpha-2 b capable of penetrating intestinal epithelium into a body after an inducer is orally taken, so that the treatment effect of the interferon is enhanced.
The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention, are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention without limiting the invention to the right. It is obvious that the drawings in the following description are only some embodiments, and that for a person skilled in the art, other drawings can be derived from them without inventive effort. In the drawings:
FIG. 1 is a graph showing the results of Dot blot detection of Bifidobacterium longum culture and supernatant;
wherein CK: untransformed bifidobacterium longum;
IFN alpha-2 b: pHX101-IFN alpha-2 b transformed Bifidobacterium longum;
HXPPP-IFN alpha-2 b: pHX 101-HXPCP-IFN alpha-2 b transforming Bifidobacterium longum;
FIG. 2 is a bar graph of mouse serum measured for HXCPP-IFN alpha-2 b and IFN alpha-2 b content;
FIG. 3 is a column diagram of mouse serum for measuring the content of HXCPP-IFN alpha-2 b, PD-1-IFN alpha-2 b, PEP-1-IFN alpha-2 b and IFN alpha-2 b.
It should be noted that the drawings and the description are not intended to limit the scope of the inventive concept in any way, but to illustrate it by a person skilled in the art with reference to specific embodiments.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and the following embodiments are used for illustrating the present invention and are not intended to limit the scope of the present invention.
Example one
Screening of HXCPP polypeptide sequence
Screening CPP capable of penetrating skin by using phage in vivo display technology. The specific method is that,
1) the phage peptide library is smeared on the surface of the skin of a nude mouse in a transdermal drug delivery mode;
2) after a period of transdermal time, blood was drawn from the rat;
3) adding escherichia coli into blood, infecting the escherichia coli with the phage, and then culturing;
4) finally, counting plaques, and calculating the titer of the phage.
The first round of screening is performed according to the above steps, and usually many nonspecific phages or phages with weak transdermal capacity are screened in the first round of screening, and the number of the phages is large. Therefore, repeated screening is required. By repeating the above method. The phage with certain purity is enriched through 3-4 rounds of screening.
Then, the DNA sequence of the polypeptide carried by the phage is sequenced by extracting the phage, so that the polypeptide sequence is obtained.
Through the steps, a CPP polypeptide sequence is screened out as follows:
LHTWRLRLARSPHLK
it was named: HXPPC.
Example two
(1) Construction of recombinant vectors
The genes coding the HXPCP and the IFN alpha-2 b are constructed on a shuttle vector pHX101 (simultaneously provided with an escherichia coli replicon and a bifidobacterium replicon), a competent cell BL21(DE3) is transformed, a recombinant plasmid pHX 101-HXPCP-IFN alpha-2 b is obtained through enzyme digestion, sequencing and screening, and meanwhile, only the IFN alpha-2 b gene fragment is constructed on pHX101-IFN alpha-2 b as a control.
Wherein, the amino acid sequence of HXCPP is shown as SEQ ID NO. 1; the amino acid sequence of the HXCPP-IFN alpha-2 b fusion protein is shown as SEQ ID NO. 2.
(2) Construction of recombinant engineering bacteria
The plasmids are respectively transformed into bifidobacterium longum, anaerobic culture is carried out in a sugar-free BM solid culture medium at 37 ℃, colonies are selected for PCR identification, and positive strains are further identified by sequencing.
Identifying to obtain recombinant engineering bacteria with correct sequencing, inoculating the recombinant engineering bacteria in 10mL of AMP at an inoculation ratio of 1:100+The sugar-free BM liquid medium; then shaking the bacteria in an anaerobic box which exhausts the air at the constant temperature of 37 ℃ for 24 h; adjusting the concentration of the bacterial liquid to OD695 to 0.5-0.6; centrifuging at 4 ℃ and 5000rpm for 8min, centrifuging the bacterial liquid, then respectively re-suspending the bacterial liquid in 10mL BM culture medium containing 0.2% L-arabinose on a bacterial super clean bench, carrying out anaerobic culture at 37 ℃ for 36h, and then collecting the supernatant of the bacterial liquid.
The culture supernatants were all positive by dot blot hybridization, i.e., both HXCPP-IFN α 2b and IFN α 2b were secreted and expressed, the results are shown in FIG. 1.
Test example 1
Engineering bacteria feeding mouse and detection
16 mice of 6 weeks size were randomly divided into control and experimental groups, with 8 males and females in each group. Each group of mice was given free drinking water and diet, and cultured at 22 + -1 deg.C for 12h under light. pHX101-IFN alpha-2 b transformed Bifidobacterium longum is fed to the control group, and pHX101-HXCPP-IFN alpha-2 b Bifidobacterium longum is fed to the experimental group; the Bifidobacterium longum is fed by fresh culture with density of 1.0-1.5 × 106Taking 2ml of bacteria liquid and mixing the bacteria liquid into a small amount of food each time to finish eating; feeding once a day; for 14 consecutive days; on day 15, the mice were fed with L-arabinose, and 200ul of blood was taken from the mouse tail vein at 0h, 1h, 2h, 3h, 4h, and 6h, and serum was collected after centrifugation, and the amounts of HXCPP-IFN α -2b and IFN α -2b were measured by ELISA.
As shown in FIG. 2, a higher concentration (150pg/ml) of HXPCP-IFN alpha-2 b was detected in the serum 1h after the mice were fed with L-arabinose, the amount of HXPCP-IFN alpha-2 b reached a maximum of 356pg/ml 2h after 2h, and then the amount of HXPCP-IFN alpha-2 b began to decrease; only minute amounts of IFN α -2b were detected in the mouse serum throughout.
The result shows that the expressed HXPCP-IFN alpha-2 b can penetrate intestinal epithelial cells to enter blood after the mice are fed with the Bifidobacterium longum carrying pHX 101-HXPCP-IFN alpha-2 b and induced by feeding L-arabinose.
Test example two
In order to further prove the advancement of the transdermal capacity of the cell penetrating peptide HXCPP, the cell penetrating peptide PD-1 and PEP-1 which are commonly used at present are selected as a control, the pHX101-PD-1-IFN alpha-2 b and pHX101-PEP-1-IFN alpha-2 b are constructed and granulated, the bifidobacterium is transformed, the engineering bacteria are prepared (the experimental method is the same as the second embodiment), and after the two can be expressed by experiment, the engineering bacteria are used as a control group to carry out a mouse gastric perfusion experiment, and the experimental method is the same as the first embodiment.
As shown in FIG. 3, the serum concentrations of HXPCPP-IFN α -2b, PD-1-IFN α -2b and PEP-1-IFN α -2b reached maximum values after the mice were fed with L-arabinose for 2 hours, and the serum concentration of HXPCPP-IFN α -2b was higher than that of the other 3 groups; the serum concentration of the control group decreased sharply with increasing time; after 6 hours, the serum concentrations of PD-1-IFN alpha-2 b and PEP-1-IFN alpha-2 b are only 25mg/ml and 30 mg/ml; and HXPPP-IFN alpha-2 b has a milder decrease trend of serum concentration with time than the other 2 groups, and a higher serum concentration of 102mg/ml is maintained after 6 hours.
The results show that IFN alpha-2 b carried by HXCPP has higher concentration in serum after penetrating through intestinal tracts than IFN alpha-2 b carried by PD-1 and PEP-1, and interferon can last for a longer time.
Therefore, in summary, the cell penetrating peptide HXPPP of the present invention carries IFN α -2b, and after transforming Bifidobacterium, the HXPPP-IFN α -2b expressed in the intestinal tract can permeate the intestinal tract to enter the blood and maintain a high concentration for a long time.
Although the present invention has been described with reference to a preferred embodiment, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (15)
1. A cell penetrating peptide, wherein the cell penetrating peptide is a peptide for mediating delivery of a bioactive molecule into a cell, and the amino acid sequence of the cell penetrating peptide is shown as SEQ ID NO. 1.
2. A nucleotide sequence encoding the cell penetrating peptide of claim 1.
3. A vector or recombinant cell comprising a nucleotide sequence encoding the cell penetrating peptide of claim 1.
4. The vector or recombinant cell of claim 3, wherein the vector is a shuttle vector.
5. Use of the cell penetrating peptide of claim 1 for the preparation of a drug delivery vehicle.
6. A complex comprising the cell penetrating peptide of claim 1 and a cargo molecule.
7. The complex of claim 6, wherein the cell penetrating peptide and the cargo molecule are coupled to each other.
8. The complex of claim 7, wherein the cargo molecule is coupled to the terminus of the cell penetrating peptide.
9. A fusion protein comprising the cell penetrating peptide of claim 1 and an interferon protein, wherein said cell penetrating peptide is linked to the N-terminus of interferon.
10. The fusion protein of claim 9, wherein the interferon is selected from the group consisting of alpha interferon, beta interferon, and gamma interferon.
11. The fusion protein of claim 10, wherein the interferon is interferon alpha-2 b.
12. A bifidobacterium capable of expressing an orally administered interferon, wherein the bifidobacterium expresses a fusion protein as claimed in any one of claims 9 to 11.
13. The orally-administrable interferon-expressing bifidobacterium of claim 12, wherein the bifidobacterium is bifidobacterium longum.
14. A method of producing a bifidobacterium as claimed in claim 12 comprising the steps of:
(1) connecting the cell-penetrating peptide according to claim 1 to the N-terminus of interferon to construct a recombinant vector having a cell-penetrating peptide-interferon nucleotide sequence;
(2) transferring the recombinant vector into bifidobacterium, and screening to obtain the recombinant bifidobacterium.
15. The method of claim 14, comprising the steps of:
(1) constructing a recombinant plasmid by constructing the genes encoding the cell-penetrating peptide according to claim 1 and IFN α -2b into shuttle vector pHX 101;
(2) transforming the recombinant plasmid in the step (1) into bifidobacterium longum, and screening to obtain recombinant bifidobacterium longum;
(3) and (3) carrying out induction culture on the recombinant bifidobacterium longum in the step (2).
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| CN101220370A (en) * | 2008-01-22 | 2008-07-16 | 重庆医科大学 | Bifidobacteria-bacillus coli shuttle expression vector, preparation method and application thereof |
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