CN114316013A - Coronavirus blocking protein and preparation and application thereof - Google Patents

Abstract

The invention provides a coronavirus blocking protein with an amino acid sequence shown as SEQ ID NO. 1. The blocking protein can inhibit the invasion of coronavirus, especially new coronavirus, into host cell, so as to prevent or treat coronavirus, especially new coronavirus infection. The invention also provides a pharmaceutical composition containing the blocking protein and application of the blocking protein in preparing a medicament for preventing or treating coronavirus, particularly novel coronavirus.

Description

Coronavirus blocking protein and preparation and application thereof

Technical Field

The invention relates to the field of protein drugs. In particular, the invention relates to a protein having a blocking effect on coronavirus, in particular novel coronavirus, a pharmaceutical composition containing the protein and application of the protein in preparing a medicament for preventing or treating coronavirus, in particular novel coronavirus.

Background

The virus completes self propagation in the host cell, and infects the host cell through 5 processes of adsorption, invasion, replication, maturation and release, thereby seriously affecting the human health.

Coronaviruses are a class of enveloped single-stranded positive-stranded RNA viruses. The international committee for classification of viruses classified coronaviruses into 4 major groups of α, β, γ, and δ in 2012. At present, 7 kinds of human-infectable coronaviruses are found, namely human coronavirus 229E (HCoV-229E), human coronavirus NL63(HCoV-NL63), human coronavirus OC43(HCoV-OC43), hong Kong type I human coronavirus (HCoV-HKU1), severe acute respiratory syndrome coronavirus (SARS-CoV) and middle east respiratory syndrome coronavirus (MERCoV), and novel coronavirus (2019-nCoV). Among them, SARS-CoV, MERS-CoV and 2019-nCoV are highly pathogenic coronaviruses found at present, and bring serious harm to human health.

2019 the novel coronavirus (2019-nCoV) is a new strain of coronavirus that has not been previously found in humans. It is a non-segmented single-stranded positive-strand RNA virus belonging to the same genus as SARS-coV, family Coronaviridae, order Neuroviridae. Respiratory symptoms such as fever, cough, shortness of breath, dyspnea, and the like, commonly occur in humans after infection with the novel coronavirus. In more severe cases, the infection can lead to pneumonia, severe acute respiratory syndrome, renal failure, and even death. At present, the global new coronary disease is still severe. And according to research predictions, new coronavirus may coexist with human in the future for a long time.

Currently, the prevention and treatment of viral diseases mainly depends on vaccines and drugs. The vaccine has certain limitations, namely the immunization rate is low, and effective group immunity is difficult to generate; the effective immunity is difficult to generate for high risk people, such as the old and the people with immunodeficiency; due to the lack of post-translational correction mechanisms of viral RNA polymerase, the development of new vaccines is continually ongoing in the face of continuous viral mutations, and it is difficult to generate sufficient amounts of new vaccines in a short time at the early stage of rapid epidemic.

Therefore, there is an urgent need in the art to develop therapies and drugs that can treat the new coronavirus.

Disclosure of Invention

The invention aims to provide a protein which has a blocking effect on coronavirus, particularly new coronavirus, so that the coronavirus, particularly the new coronavirus, can be treated.

In a first aspect, the present invention provides a coronavirus blocking protein, which coronavirus blocking protein is:

(a) protein with amino acid sequence as shown in SEQ ID No. 1; or

(b) A derived protein formed by substituting, deleting or adding one or several amino acid residues to the protein (a) and having the function of the protein (a).

In a preferred embodiment, the derivative protein of (b) is a derivative protein formed by substitution, deletion or addition of 1 to 30, more preferably 1 to 10, still more preferably 1 to 6, most preferably 1 to 3 amino acid residues of the protein of (a) and having the function of the protein of (a).

In a preferred embodiment, the derivative protein of (b) is a derivative protein formed by deletion or addition of 1 to 30, more preferably 1 to 10, still more preferably 1 to 6, most preferably 1 to 3 amino acid residues from the protein of (a) and having the function of the protein of (a).

In a preferred embodiment, the derivative protein of (b) is a derivative protein formed by adding or deleting 1 to 30, 1 to 10, 1 to 6, or 1 to 3 amino acid residues to the C-terminal and/or N-terminal of the protein of (a) and having the function of the protein of (a).

In a specific embodiment, the protein of (b) is a protein with an amino acid sequence shown as SEQ ID NO. 2.

In a preferred embodiment, the coronavirus is a severe acute respiratory syndrome coronavirus, a middle east respiratory syndrome coronavirus, or a novel coronavirus; preferably severe acute respiratory syndrome coronavirus or a novel coronavirus; more preferably a novel coronavirus.

In a second aspect, the present invention provides a nucleic acid encoding the coronavirus blocking protein of the first aspect.

In a third aspect, the present invention provides an expression vector comprising the encoding nucleic acid of the second aspect.

In a fourth aspect, the present invention provides a host cell comprising an expression vector according to the third aspect, or having integrated in its genome a nucleic acid encoding according to the second aspect.

In a fifth aspect, the present invention provides a pharmaceutical composition comprising a protein according to the first aspect and a pharmaceutically acceptable excipient.

In a preferred embodiment, the pharmaceutical composition is for use in the prevention or treatment of a coronavirus infection.

In a preferred embodiment, the coronavirus is a severe acute respiratory syndrome coronavirus, a middle east respiratory syndrome coronavirus, or a novel coronavirus; preferably severe acute respiratory syndrome coronavirus or a novel coronavirus; more preferably a novel coronavirus.

In preferred embodiments, the pharmaceutical composition further comprises other agents for preventing or treating coronavirus infection, including but not limited to one or more of lopinavir, ritonavir, ribavirin, ridciclovir, oseltamivir, tamiflu, lanimivir, peramivir, and chloroquine (chloroquine phosphate); one or more of lopinavir, ritonavir, ribavirin, rituxivir, and chloroquine (chloroquine phosphate) are preferred.

In a sixth aspect, the present invention provides a method for preparing the coronavirus blocking protein of the first aspect, comprising the steps of:

(1) culturing the host cell of the fourth aspect under conditions suitable for the production of the coronavirus blocking protein, thereby producing the coronavirus blocking protein of the first aspect; and

(2) isolating the produced coronavirus blocking protein from the culture of step (1).

In a seventh aspect, the present invention provides the use of a coronavirus blocking protein of the first aspect in the preparation of a medicament for the prevention or treatment of a coronavirus infection.

In a preferred embodiment, the coronavirus is a severe acute respiratory syndrome coronavirus, a middle east respiratory syndrome coronavirus, or a novel coronavirus; preferably severe acute respiratory syndrome coronavirus or a novel coronavirus; more preferably a novel coronavirus.

In an eighth aspect, the present invention provides a coronavirus blocking protein of the first aspect for use as a medicament for the prevention or treatment of a coronavirus infection.

In a ninth aspect, the present invention provides a method of preventing or treating a coronavirus infection, comprising the steps of:

administering to a subject in need thereof a therapeutically effective amount of a coronavirus blocking protein of the first aspect or a pharmaceutical composition of the fifth aspect, so as to prevent or treat a coronavirus infection.

In a preferred embodiment, the coronavirus is a severe acute respiratory syndrome coronavirus, a middle east respiratory syndrome coronavirus, or a novel coronavirus; preferably severe acute respiratory syndrome coronavirus or a novel coronavirus; more preferably a novel coronavirus.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

FIG. 1 shows the expression of PTA protein in BL21(DE3) analyzed by SDS-PAGE; wherein lane M: SDS-PAGE protein marker lane; 0: control (no IPTG added); lane 1: inducing at 37 ℃ for 16 h; lane 2: supernatant after the whole bacteria are broken; lane 3: the precipitate after the bacteria breaking of the whole bacteria;

FIG. 2 shows the results of SDS-PAGE analysis of purification of PTA protein in inclusion bodies; wherein, lane M: SDS-PAGE protein markers; lane 1: dissolving the supernatant after centrifugation by the inclusion body; lane 2: effluent liquid of the supernatant fluid after incubation with Ni-IDA; lanes 3-4: an eluted fraction of 50mM imidazole; lane 5: an eluted fraction of 300mM imidazole;

FIG. 3 shows the results of a competition ELISA; and

figure 4 shows inhibition curves for different concentrations of PTA inhibiting RBD and ACE2 binding.

Detailed Description

The inventors have made extensive and intensive studies and have unexpectedly found a coronavirus blocking protein which is capable of binding to the RBD region of the S protein. The RBD region, after binding to these blocking proteins, will not be able to continue recognizing and binding to human ACE2 protein and thus will not help the virus to invade the host cell, thereby achieving the effect of inhibiting virus invasion. The present invention has been completed based on this finding.

Coronavirus (coronavirus)

The term "coronavirus (Coronaviruses)" as used herein is a single-stranded positive-strand RNA virus belonging to the order Nidovirales (Nidovirales) Coronaviridae (Coronaviridae) orthocoronaviridae (orthocoronaviridae). The virus can infect various species such as human, bat, pig, mouse, cow, horse, goat, monkey, etc. There are known 7 kinds of human-infecting coronavirus (HCoV), including middle east respiratory syndrome-associated coronavirus (MERSR-CoV) and severe acute respiratory syndrome-associated coronavirus (SARSr-CoV).

In a specific embodiment, the coronavirus described herein is a severe acute respiratory syndrome coronavirus, a middle east respiratory syndrome coronavirus, or a novel coronavirus. In a preferred embodiment, the coronavirus is a severe acute respiratory syndrome coronavirus or a novel coronavirus; more preferably a novel coronavirus.

The most recently isolated coronavirus was a novel coronavirus of the genus β, WHO named 2019-nCoV, which is the 7 th coronavirus that infects humans. At present, no effective vaccine and therapeutic drug aiming at the new coronavirus exist, and virus diffusion is mainly controlled through precautionary measures, epidemic situation is closely monitored, and suspected cases are isolated and observed. At present, no specific treatment method for coronavirus exists, and symptomatic support treatment is mainly adopted.

The most important components of the new coronaviruses are the nucleic acid, which is the genetic material responsible for the propagation of progeny, and the protein coat, which is responsible for protecting the genetic material. Research shows that the new coronavirus recognizes and binds to ACE2 Protein on human cells through S Protein (Spike Protein) on the surface of the virus, so as to complete invasion of host cells. The S protein comprises two functional subunits: the S1 subunit responsible for binding to host cell receptors, and the S2 subunit responsible for fusion of the virus with the cell membrane. The new coronavirus binds to ACE2 mainly through a receptor-binding domain (RBD) on the S1 subunit, and then infects host cells. If the combination of RBD and ACE2 can be effectively blocked, the invasion of new coronavirus to human cells can be blocked, and a series of symptoms caused by new coronavirus infection can be further treated or prevented.

Coronavirus antagonistic protein of the present invention

Coronaviruses such as new coronaviruses have appeared in many varieties, but the main invasion route is still that the RBD of the S Protein (Spike Protein) on the surface thereof recognizes and binds to the ACE2 Protein on human cells, thereby completing the invasion of host cells. Therefore, if the compound can effectively block the binding of the S protein and ACE2, the compound has great possibility of preventing the invasion of new coronavirus to human cells and preventing or treating a series of symptoms caused by the infection of new coronavirus.

To achieve the above object, the present invention provides a protein PTA capable of binding to RBD region of S protein. After the RBD region is combined with PTA, the human ACE2 protein can not be recognized and combined continuously, so that the virus can not further invade host cells, thereby achieving the purposes of prevention and treatment. Herein, the terms "protein of the present invention", "blocker protein of the present invention", "coronavirus blocker protein of the present invention", "PTA protein of the present invention" or "PTA protein" used have the same meaning and may be used interchangeably herein. These terms all refer to proteins that bind to the RBD domain of the S protein of coronaviruses, thereby blocking the binding of coronaviruses to host cells, and achieving the efficacy of inhibiting viral entry and treating coronavirus infection.

In a specific embodiment, the coronavirus blocking protein of the invention is a protein having an amino acid sequence as shown in SEQ ID NO: 1:

STIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILM。

in view of the teachings of the present invention and the prior art, those skilled in the art will appreciate that the proteins of the present invention should also include variant forms of the proteins that have the same or similar function as the "protein of the present invention" but whose amino acid sequence differs in a small amount from the amino acid sequence shown in any one of SEQ ID NO. 1. These variants include (but are not limited to): deletion, insertion and/or substitution of one or more (usually 1 to 30, preferably 1 to 10, more preferably 1 to 6, most preferably 1 to 3) amino acids, and addition of one or more (usually up to 20, preferably up to 10, more preferably up to 6 or 3) amino acids at the C-terminus and/or N-terminus. For example, it is well known to those skilled in the art that substitutions with amino acids of similar or analogous properties, e.g., isoleucine and leucine, do not alter the function of the resulting protein. As another example, the addition of one or several amino acids at the C-terminus and/or N-terminus, such as a tag added for ease of isolation, does not generally alter the function of the resulting protein. For example, for ease of detection and experimentation, the proteins in the examples are those with a biotin label at the N-terminus, which does not adversely affect the performance of the protein.

Variants of the protein include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that hybridizes under high or low stringency conditions with DNA encoding a "protein of the invention". The invention also includes other proteins, such as fusion proteins comprising a "protein of the invention" or a fragment thereof. In addition to almost full-length proteins, the invention also encompasses active fragments of the "proteins of the invention".

The invention also provides analogs of the "protein". These analogs may differ from the proteins of the invention by amino acid sequence differences, by modifications that do not affect the sequence, or by both. These proteins include natural or induced genetic variants. Induced variants can be obtained by various techniques, such as random mutagenesis by irradiation or exposure to mutagens, site-directed mutagenesis, or other known molecular biological techniques. Analogs also include analogs having residues other than the natural L-amino acids (e.g., D-amino acids), as well as analogs having non-naturally occurring or synthetic amino acids (e.g., beta, gamma-amino acids). It is to be understood that the proteins of the present invention are not limited to the representative proteins exemplified above.

Modified (generally without altering primary structure) forms include: chemically derivatized forms of the protein such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation. Modified forms also include sequences having phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, phosphothreonine). Also included are proteins that have been modified to increase their resistance to proteolysis or to optimize solubility.

The conservative variant of the protein of the present invention is a protein in which the amino acid residue present in the amino acid sequence shown in SEQ ID NO. 1 is replaced with an amino acid residue having similar or similar properties, but the conservative variant protein still has the same or similar activity as the protein having the amino acid sequence shown in SEQ ID NO. 1.

Thus, in view of the teachings of the present invention and the prior art, one skilled in the art can generate conservatively variant mutants by making amino acid substitutions as shown, for example, in the following table.

Initial residue	Representative substituted residue	Preferred substituent residues
			Ala(A)	Val；Leu；Ile	Val
Arg(R)	Lys；Gln；Asn	Lys
			Asn(N)	Gln；His；Lys；Arg	Gln
Asp(D)	Glu	Glu
			Cys(C)	Ser	Ser
Gln(Q)	Asn	Asn
			Glu(E)	Asp	Asp
Gly(G)	Pro；Ala	Ala
			His(H)	Asn；Gln；Lys；Arg	Arg
Ile(I)	Leu；Val；Met；Ala；Phe	Leu
			Leu(L)	Ile；Val；Met；Ala；Phe	Ile
Lys(K)	Arg；Gln；Asn	Arg
			Met(M)	Leu；Phe；Ile	Leu
Phe(F)	Leu；Val；Ile；Ala；Tyr	Leu
			Pro(P)	Ala	Ala
Ser(S)	Thr	Thr
			Thr(T)	Ser	Ser
Trp(W)	Tyr；Phe	Tyr
			Tyr(Y)	Trp；Phe；Thr；Ser	Phe
Val(V)	Ile；Leu；Met；Phe；Ala	Leu

Therefore, the protein of the present invention also includes a protein derived from the protein of the present invention by substitution, deletion or addition of one or several amino acid residues and having the function of the protein of the present invention.

In a preferred embodiment, the derivative protein is a derivative protein formed by the substitution, deletion or addition of 1 to 30, more preferably 1 to 10, still more preferably 1 to 6, and most preferably 1 to 3 amino acid residues of the protein of the present invention and having the function of the protein of the present invention.

In a preferred embodiment, the derived protein is a protein derived from the protein of the present invention by deletion or addition of 1 to 30, more preferably 1 to 10, still more preferably 1 to 6, and most preferably 1 to 3 amino acid residues, and having the function of the protein of the present invention.

In a preferred embodiment, the derivative protein is a derivative protein formed by adding or deleting 1 to 30, 1 to 10, 1 to 6, or 1 to 3 amino acid residues to the C-terminal and/or N-terminal of the protein of the present invention and having the function of the protein of the present invention.

In a specific embodiment, the protein of the present invention may include a protein having the amino acid residue M encoded by the initiation codon of the 6His tag at the N-terminus of the protein shown in SEQ ID No. 1, for example, a protein having the amino acid sequence shown below:

MHHHHHHSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILM(SEQ ID NO:2)。

on the basis of the protein, the invention also provides a polynucleotide sequence for encoding the protein. The polynucleotide of the present invention may be in the form of DNA or RNA. The form of DNA includes cDNA, genomic DNA or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be the coding strand or the non-coding strand. The sequence of the coding region encoding the mature protein may be identical to the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO. 1 or a degenerate variant. As used herein, "degenerate variant" refers in the present invention to a nucleic acid sequence which encodes a protein having the amino acid sequence shown in SEQ ID NO. 1, but differs from the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO. 1.

The polynucleotide sequences encoding the proteins of the invention may be inserted into a recombinant expression vector or genome. The term "recombinant expression vector" refers to a bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus, or other vector well known in the art. In general, any plasmid or vector can be used as long as it can replicate and is stable in the host. An important feature of expression vectors is that they generally contain an origin of replication, a promoter, a marker gene and translation control elements.

Those skilled in the art can use well-known methods for constructing expression vectors containing the DNA sequences encoding the proteins of the present invention and appropriate transcription/translation control signals, including in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. The DNA sequence may be operably linked to a suitable promoter in an expression vector to direct mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

Furthermore, the expression vector preferably comprises one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance and Green Fluorescent Protein (GFP) for eukaryotic cell culture, or kanamycin or ampicillin resistance for E.coli.

Vectors comprising the appropriate DNA sequences described above, together with appropriate promoter or control sequences, may be used to transform an appropriate host cell so that it can express the protein.

The host cells described herein include host cells comprising the above-described expression vectors or nucleotide sequences encoding the genomic integration of the coding sequence of the protein of the invention, preferably the amino acid sequence shown in SEQ ID NO. 1. The host cell of the invention may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells. In particular embodiments, the strains include, but are not limited to: coli (e.coli), Corynebacterium glutamicum (Corynebacterium glutamicum), Hafnia alvei (Hafnia alvei), Bacillus subtilis (Bacillus subtilis). In a preferred embodiment, the strain is E.coli.

Transformation of a host cell with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. When the host is prokaryotic, e.g., E.coli, competent cells capable of DNA uptake can be harvested after exponential growth phase using CaCl₂Methods, the steps used are well known in the art. Another method is to use MgCl₂. If desired, transformation can also be carried out by electroporation. When the host is a eukaryote, the following DNA transfection methods may be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome encapsulation, etc.

The obtained transformant can be cultured by a conventional method to express the protein encoded by the gene of the present invention. The medium used in the culture may be selected from various conventional media depending on the host cell used. The culturing is performed under conditions suitable for growth of the host cell. The recombinant protein in the above method may be constitutively expressed or conditionally expressed, for example, when the host cell is grown to an appropriate cell density, the selected promoter is induced by an appropriate method (e.g., temperature shift or chemical induction), and the cell is cultured for an additional period of time.

The recombinant protein in the above method may be expressed intracellularly or on the cell membrane, or secreted extracellularly. If necessary, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (such as salt precipitation), centrifugation, cell disruption by osmosis, sonication, high-pressure homogenization, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, High Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques, and combinations thereof.

Pharmaceutical composition

On the basis of the above proteins, the present invention further provides a pharmaceutical composition for preventing or treating coronaviruses, such as severe acute respiratory syndrome coronavirus (SARS-CoV), middle east respiratory syndrome coronavirus (mers clov) and 2019 novel coronavirus (2019-nCoV), which comprises a therapeutically effective amount of the protein of the present invention, and a pharmaceutically acceptable carrier or excipient. In a preferred embodiment, the protein or pharmaceutical composition of the invention may be used for the treatment of severe acute respiratory syndrome coronavirus (SARS-CoV) or 2019 novel coronavirus (2019-nCoV); preferably 2019 novel coronaviruses (2019-nCoV).

Although the requirements vary from person to person, the skilled person can determine the optimal dosage of each active ingredient in the pharmaceutical composition of the invention.

The pharmaceutical compositions of the present invention may be formulated in a form suitable for various routes of administration, including but not limited to, those formulated for parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, buccal, intrathecal, intracranial, nasal or topical administration. The amount administered is an amount effective to ameliorate or eliminate one or more symptoms. For the treatment of a particular disease, an effective amount is an amount sufficient to ameliorate or in some way reduce the symptoms associated with the disease. Such amounts may be administered as a single dose or may be administered according to an effective treatment regimen. The amount administered may be sufficient to cure the disease, but is generally administered to ameliorate the symptoms of the disease. Repeated administration is generally required to achieve the desired improvement in symptoms. The dosage of the drug will depend on the age, health and weight of the patient, the type of concurrent treatment, the frequency of treatment, and the desired therapeutic benefit.

The pharmaceutical preparation of the present invention can be administered to any mammals as long as they can obtain the therapeutic effects of the compound of the present invention. Of these mammals, the most important is human.

The pharmaceutical preparations of the present invention can be manufactured in a known manner. For example, by conventional mixing, granulating, dragee-making, dissolving, or lyophilizing processes. In the manufacture of oral formulations, solid excipients and active compounds may be combined, optionally grinding the mixture. If desired or necessary after addition of suitable amounts of auxiliaries, the granulate mixture is processed to give tablets or dragee cores.

Suitable adjuvants are, in particular, fillers, for example sugars such as lactose or sucrose, mannitol or sorbitol; cellulose preparations or calcium phosphates, such as tricalcium phosphate or calcium hydrogen phosphate; and binders, such as starch pastes, including corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, or polyvinylpyrrolidone. If desired, disintegrating agents such as the starches mentioned above, as well as carboxymethyl starch, cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate may be added. Adjuvants are, in particular, flow regulators and lubricants, for example silica, talc, stearates, such as calcium magnesium stearate, stearic acid or polyethylene glycol. If desired, a suitable coating resistant to gastric juices can be provided to the tablet core. For this purpose, concentrated saccharide solutions can be used. Such solutions may contain gum arabic, talc, polyvinyl pyrrolidone, polyethylene glycol and/or titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. For the preparation of coatings resistant to gastric juices, suitable cellulose solutions can be used, for example cellulose acetate phthalate or hydroxypropylmethyl cellulose phthalate. Dyes or pigments may be added to the coating of the tablet or lozenge core. For example, for identifying or for characterizing combinations of active ingredient doses.

The method of administration of the pharmaceutical composition includes, but is not limited to, various methods of administration known in the art, and can be determined according to the actual condition of the patient. These methods include, but are not limited to, parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, buccal, intrathecal, intracranial, nasal, or topical routes of administration.

In addition to the protein of the present invention, the pharmaceutical composition of the present invention may further comprise other antiviral drugs, which may be selected from one or more of lopinavir, ritonavir, ribavirin, ridciclovir, oseltamivir, tamiflu, lanimivir, peramivir, and chloroquine (chloroquine phosphate); one or more of lopinavir, ritonavir, ribavirin, rituxivir, and chloroquine (chloroquine phosphate) are preferred.

The invention has the advantages that:

1. the present invention provides proteins that are effective in blocking coronaviruses, particularly novel coronaviruses;

2. the protein of the invention has lower immunogenicity, and can not cause autoimmune reaction, thereby not causing autoimmune disease;

3. the protein of the invention has simple production and short production period.

The technical solution of the present invention is further described below with reference to specific embodiments, but the following examples are not intended to limit the present invention, and all of the various application methods adopted according to the principles and technical means of the present invention belong to the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers. Unless otherwise indicated, percentages and parts are by weight.

Examples

Example 1 design of PTA protein

Sequence design of PTA protein

The inventor designs a protein shown by an amino acid sequence, namely PTA protein:

MHHHHHHSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILM(SEQ ID NO:2).

expression of PTA

The expression strain of the plasmid containing the PTA coding sequence was spread evenly onto LB plates (containing 50. mu.g/mL kanamycin sulfate), and then placed upside down in an incubator at 37 ℃ overnight. Single colonies were picked from the transformed plates, inoculated into 4mL of LB medium (containing 50. mu.g/mL kanamycin sulfate), and cultured to OD₆₀₀0.5-0.8, IPTG was added to the test tube culture medium to a final concentration of 0.5mM, followed by induction at 37 ℃ for expression. For scale-up, growth to OD₆₀₀When the concentration was 0.8, IPTG was added to the cells at a final concentration of 0.5mM, and the cells were induced at 37 ℃ for 16 hours and collected. If no purification operation is performed on the same day, the thalli are frozen at-20 ℃.

Identification of PTA expression results

Centrifuging the induced culture solution at 12000rpm for 5min, removing supernatant, adding PBS solution to resuspend and precipitate, adding SDS-PAGE sample buffer, heating the sample at 100 deg.C for 10min, centrifuging, and collecting supernatant for electrophoresis.

The whole bacteria were sonicated in 20mM Tris (pH8.0), 300mM NaCl, 20mM imidazole (containing 1% Triton X-100), 1mM DTT, 1mM PMSF, and the supernatant and pellet were analyzed by SDS-PAGE. The results of the analysis are shown in FIG. 1. Through the ultrasonic lysis analysis of the whole bacteria, the PTA protein is expressed in the inclusion body.

4. Protein purification

After the inclusion bodies are washed by 20mM Tris (pH8.0), 300mM NaCl (containing 1% Triton X-100), 2mM EDTA and 5mM DTT, the inclusion bodies are dissolved by 20mM Tris (pH8.0), 300mM NaCl, 8M urea and 20mM imidazole buffer solution and the Ni-IDA column is equilibrated, finally, the target protein is eluted by the equilibration buffer solution with different concentrations of imidazole, and each eluted component is collected for SDS-PAGE analysis and detection.

And (3) purification result: analysis was performed after purification by SAS-PAGE. The results show that the protein purity obtained is above 90% when the imidazole concentration is 50 mM.

5. Renaturation of proteins

Purifying with Ni-IDA affinity chromatography, collecting lanes 3-4 with relatively high purity, adding into treated dialysis bag, dialyzing at 4 deg.C into buffer solution [1 XPBS (pH8.0), 4mM GSH, 0.4mM GSSG, 0.4M L-arginine, 1M urea, 5% glycerol ], renaturing, and dialyzing the renatured PTA protein into storage solution 1 XPBS (pH8.0) and 5% glycerol solution for about 6-8 h. After the renaturation by dialysis, the supernatant was filtered through a 0.22 μm filter and dispensed, and was frozen to-80 ℃.

Example 2 PTA Competition ELISA experiment

PTA Competition ELISA assay

The present inventors used ELISA experiments to confirm whether PTA can block the binding of RBD protein to ACE2 protein. Plates were plated with 2ug/ml RBD at 100ul per well and incubated overnight at 4 ℃. Then 200ul 2.5% BSA was added per well and blocked for 1h at 37 ℃. After blocking was complete 50ul PTA was added to the wells in different dilution gradients followed by 50ul 0.2ug/ml human Fc labelled ACE2 protein per well and co-incubation for 1.5 h. After incubation was complete, detection was performed with anti-human Fc antibody containing an enzyme marker, and OD was detected after development₄₅₀The value of (c).

Data processing and mapping: the data were processed using Excel and plotted using software Prism 8. The results are shown in FIG. 4.

Results of competitive ELISA experiments

The results of the ELISA experiments are as follows. IC for PTA blocking RBD binding to ACE2-hFc calculated according to software₅₀It was 1.8ug/ml, and the number of converted molecules was 42.5 nM. The results were converted to a suppression rate curve, as shown in fig. 4. The maximum inhibition rate that can be achieved is 83.33%. IC of ACE2 with ACE2 as a positive control₅₀2.57ug/ml, and the number of converted molecules was 30nM,the maximum inhibition was 93.23%.

Therefore, the PTA protein of the invention can inhibit RBD from being combined with ACE2-hFc with the rate of inhibition being equivalent to that of ACE2 protein. And the PTA of the invention has the advantages that: firstly, the PTA has a shorter sequence, can be conveniently expressed by a prokaryotic system, greatly saves the cost and shortens the production period; second, PTA has low immunogenicity, does not cause autoimmune reactions, and causes autoimmune diseases; thirdly, compared with ACE2, PTA has great advantages as a drug for inhibiting RBD and ACE2-hFc combination, because ACE2 is an integral natural protein which is also expressed by human body and has biological functions, and if ACE2 is used as an inhibiting drug, ACE2 is excessively existed in human body, so that ACE2 functional disorder is caused, and other diseases are caused.

Example 3.

On the basis of the protein of the present invention, the present inventors further produced other proteins. The amino acid sequences of these proteins overlap with or are contained in the amino acid sequence of the protein of the present invention.

The inventors examined the binding of these proteins to the ACE2 protein and found that some of them had inhibitory activity comparable to, or even better than, the proteins of the present invention.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Sequence listing

<110> Changzhou Wen Song Biotechnology Limited

<120> coronavirus blocking protein, preparation and application thereof

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Claims (10)

1. A coronavirus blocking protein, wherein said coronavirus blocking protein is:

(a) protein with amino acid sequence as shown in SEQ ID No. 1; or

(b) A derived protein formed by substituting, deleting or adding one or several amino acid residues to the protein (a) and having the function of the protein (a).

2. The coronavirus blocking protein of claim 1, wherein the protein of (b) is a protein having an amino acid sequence as shown in SEQ ID NO. 2.

3. Nucleic acid encoding a coronavirus blocking protein according to claim 1 or 2.

4. An expression vector comprising the coding nucleic acid of claim 3.

5. A host cell comprising the expression vector of claim 4, or having integrated on its genome the encoding nucleic acid of claim 3.

6. A pharmaceutical composition comprising the protein of claim 1 or 2 and a pharmaceutically acceptable excipient.

7. A process for the preparation of a coronavirus blocking protein according to claim 1 or 2, comprising the steps of:

(1) culturing the host cell of claim 5 under conditions suitable for production of the coronavirus blocking protein, thereby producing the coronavirus blocking protein of claim 1 or 2; and

(2) isolating the produced coronavirus blocking protein from the culture of step (1).

8. Use of a coronavirus blocking protein according to claim 1 or 2 in the manufacture of a medicament for the prevention or treatment of a coronavirus infection.

9. A coronavirus blocking protein according to claim 1 or 2 for use as a medicament for the prevention or treatment of a coronavirus infection.

10. A method of preventing or treating a coronavirus infection, comprising the steps of:

administering to a subject in need thereof a therapeutically effective amount of a coronavirus blocking protein of claim 1 or 2 or a pharmaceutical composition of claim 6, so as to prevent or treat a coronavirus infection.

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