CN103965292B

CN103965292B - Structure and application of Japanese encephalitis virus envelope protein binding peptide

Info

Publication number: CN103965292B
Application number: CN201310039518.1A
Authority: CN
Inventors: 王升启; 杨静; 张莉; 丁晓然; 汪崇文; 何丽娜
Original assignee: Institute of Radiation Medicine of CAMMS
Current assignee: Institute of Radiation Medicine of CAMMS
Priority date: 2013-02-01
Filing date: 2013-02-01
Publication date: 2020-04-03
Anticipated expiration: 2033-02-01
Also published as: CN103965292A

Abstract

The invention relates to a polypeptide sequence and a structure of a type of specific binding Japanese encephalitis virus envelope protein and application thereof in targeted modification technology of anti-Japanese encephalitis virus drugs, belonging to the technical field of biological medicines. The invention screens random peptide library by using phage display technology to obtain polypeptide which has a sequence with the number of 1-11 and is specifically combined with envelope protein. The invention also researches the binding activity of the envelope protein binding peptide screened from the phage peptide library with the Japanese encephalitis virus and the brain tissue targeting, and finds a polypeptide P63 which has good specific binding activity with the Japanese encephalitis virus and has the brain tissue targeting property mediated by the Japanese encephalitis virus infection, and the sequence of the polypeptide P63 is NH₂-HHWWVPSWSRWT-COOH. Therefore, the envelope protein binding peptide and the virus specific brain targeting peptide P63 in the envelope protein binding peptide are expected to be used in the targeted modification technology of anti-encephalitis virus drugs.

Description

Structure and application of Japanese encephalitis virus envelope protein binding peptide

Technical Field

The invention belongs to the technical field of biological medicines, relates to a drug delivery system, and more particularly relates to a sequence and a structure of a polypeptide specifically combined with a Japanese encephalitis virus envelope protein, and an application of the polypeptide in a targeted modification technology of a Japanese encephalitis virus resistant drug.

Background

Japanese encephalitis B virus (JEV) is a member of the flavivirus genus of the family Flaviviridae, has neurotropic properties, and causes Japanese encephalitis B, an insect-borne viral disease that seriously harms human and animal health. Vaccination is a reliable method for controlling encephalitis B, and at present, no specific therapy is available for the encephalitis B, mainly a supportive therapy and a symptomatic treatment. The encephalitis B virus is a single-stranded positive-strand RNA virus, 11kb in RNA length, contains an open reading frame and encodes a polyprotein. In infected cells, this polyprotein is split into at least 11 proteins, the structural proteins of the virus are encoded by the first 1/3 reading region at the 5' end, including the nucleocapsid protein C, the membrane protein M, and the envelope protein E. The nonstructural protein NS is encoded by the remaining reading frame. The open reading frame encodes a polyprotein that can be cleaved into 3 structural proteins (C, PrM, E) and 7 non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS 5). The envelope protein E of the encephalitis B virus is a main structural protein with about 53KD, is conserved in most flaviviruses, is an important component on the surface of virion, has neurotoxicity and a pathogenic site with nerve invasiveness, and can be used as a membrane fusion protein to enable the virus to enter nerve cells. The E protein also has the functions of inducing hemagglutination inhibition antibodies, complement binding antibodies and neutralizing antibodies, can trigger a host to generate protective immune response, and can be adhered to a cell surface membrane by combining with a specific receptor. Therefore, the envelope protein E can be a potential target of anti-encephalitis B virus medicines.

At present, the target transport research of the drug is that ligands of various cell surface specific receptors are selected as target molecules, and a target transport mode for specifically introducing the drug into virus infected cells through specific binding ligands of virus surface proteins is not found in reports, so that a brand-new theory based on a virus particle mediated drug target transport mechanism is provided.

Because of the neurotropic property of the encephalitis B virus, after the encephalitis B virus is infected, pathological changes mainly occur in the brain, and the influence on other tissues is small, therefore, the polypeptide which is specifically combined with the envelope protein of the encephalitis B virus is combined with the encephalitis B virus to have the brain tissue targeting property, and the brain tissue targeting peptide is coupled with the anti-encephalitis B virus medicine, so that the characteristic of medicine targeted delivery can be achieved.

The Phage Display Technology (PDT) is a powerful tool for searching polypeptide or protein with binding relation with target protein, and has the greatest advantage of directly linking presentable phenotype with genotype thereof, and screening out the protein or polypeptide of interest by utilizing the specific affinity of ligand thereof. Currently, phage display technology allows rapid high-throughput screening of peptide libraries containing billions of clones, which has become a powerful tool for polypeptide drug research. The technology can screen polypeptide combined with host cell receptor or polypeptide combined with active site such as virus membrane protein from peptide library.

Therefore, envelope protein E of the Japanese encephalitis virus is selected as a target molecule, polypeptide specifically bound with the envelope protein E on the surface of the Japanese encephalitis virus is screened by applying a phage display technology, target peptide with higher affinity with the Japanese encephalitis virus and targeting virus to infect brain tissues is obtained by verification, the polypeptide is connected with anti-Japanese encephalitis virus drugs by means of chemical synthesis and the like, and by means of the combination of the polypeptide and the virus surface protein, the antiviral drugs are carried into cells in the virus cell-entering process, so that the uptake rate and the brain tissue targeting property are increased, and the antiviral activity of the drugs is improved.

The invention content is as follows:

the invention aims to provide polypeptides with high affinity with the envelope protein of the Japanese encephalitis virus and application thereof in a targeted modification technology of antiviral drugs through screening.

The invention provides a polypeptide capable of being specifically combined with an envelope protein of a Japanese encephalitis virus, which contains 12 amino acid residues.

The amino acid sequences of the envelope protein-binding polypeptides of the invention are shown in table 1.

TABLE 1 amino acid sequence of envelope protein-binding polypeptide

Numbering	Name (R)	Amino acid sequence
			1	P21	NH₂-HHWSWYMGWNDY-COOH
2	P27	NH₂-LTLTKSNPMWPD-COOH
			3	P36	NH₂-NWAVIQYATSPY-COOH
4	P45	NH₂-SWWALEHPAPLY-COOH
			5	P47	NH₂-GLMGTHYSWSYN-COOH
6	P48	NH₂-WPSTLWILKDHG-COOH
			7	P50	NH₂-QAMSHLHVPTRY-COOH
8	P55	NH₂-WHLPWWWSTATP-COOH
			9	P57	NH₂-WHWSWWNPNQLT-COOH
10	P59	NH₂-EWSFRWWHSGEV-COOH
			11	P63	NH₂-HHWWVPSWSRWT-COOH

The encephalitis B virus envelope protein binding polypeptide provided by the invention is a polypeptide screened from a phage random peptide library by a biopanning method. The ELISA method is used for verifying that the protein has higher affinity with the Japanese encephalitis virus envelope protein.

The invention also protects the derivative of the polypeptide as shown in the specification, which is (1) or (2) as follows:

(1) a derivative obtained by substituting and/or deleting and/or inserting 1 or more amino acid residues in the polypeptide;

(2) connecting the polypeptide or the derivative in (1) with a carrier to obtain the derivative.

The binding activity of the envelope protein-binding peptide to the envelope protein can be measured by, for example, ELISA, but the method is not limited to this, and RIA, EIA, western blotting, or the like can be used, and any method can be used as long as the interaction between proteins can be quantitatively measured.

According to the invention, the amino acid sequence with the number of 11 screened from the phage peptide library can be specifically combined with the Japanese encephalitis virus envelope protein, has good brain tissue targeting property in a Japanese encephalitis virus infected mouse body, and is possible to carry out targeted modification on medicaments for treating and preventing diseases related to the Japanese encephalitis virus.

The amino acid sequence of the binding peptide with the encephalitis B virus envelope protein provided by the invention is shown as the sequence with the number 11 in the table 1: NH (NH)₂-HHWWVPSWSRWT-COOH。

The binding peptide having an envelope protein of a Japanese encephalitis virus of the present invention may be a peptide having an amino acid sequence comprising substitution and/or deletion and/or insertion of 1 or more amino acid residues in SEQ ID NO. 11, and having binding activity to a Japanese encephalitis virus.

In general, substitutions with amino acids of similar or similar nature in the art will not generally alter the function of the protein. Conservative substitution tables providing functionally similar amino acids are well known in the art. The following groups 5 each contain amino acids that can be conservatively substituted for one another: aliphatic: glycine (G), alanine (a), alanine (V), leucine (L), isoleucine (I); aromatic: phenylalanine (F), tyrosine (Y), tryptophan (W); sulfur-containing: methionine (M), cysteine (C); alkalinity: arginine (R), tyrosine (K), histidine (H); acidity: aspartic acid (D), glutamic acid (E), asparagine (N), glutamine (Q).

Thus, in the presence of NH₂-HHWWVPSWSRWT-COOH (No. 11), in which the amino acid in the polypeptide is replaced by an amino acid having a similar or analogous property, as shown in the above substitution table, and the replaced polypeptide is considered to have a structure containing NH₂-HHWWVPSWSRWT-COOH (number 11) sequence.

Such variants include, but are not limited to, analogs having residues other than the naturally occurring L-amino acids (e.g., D-amino acids), as well as analogs having non-naturally occurring or synthetic amino acids (e.g., β, gamma-amino acids).

The envelope protein binding peptide is specifically bound with the Japanese encephalitis virus, and the polypeptide can be used as a targeted modified peptide of the anti-Japanese encephalitis medicine through the binding, for example, the polypeptide is connected with the anti-Japanese encephalitis oligonucleotide through means of covalent or non-covalent binding and the like, and the nucleic acid medicine is carried into cells in the virus cell-entering process by virtue of the binding of the polypeptide and the virus surface protein, so that the uptake rate of the nucleic acid medicine is increased, and the antiviral activity of the nucleic acid medicine is improved.

As described above, as a target modification product of an anti-Japanese encephalitis virus drug containing the polypeptide having a binding activity to Japanese encephalitis of the present invention, it is considered to be useful for the preparation of an inhibitor of Japanese encephalitis virus infection for inhibiting infection of host cells by Japanese encephalitis virus, a therapeutic agent for treating a patient with Japanese encephalitis, a prophylactic agent for prophylactic administration prior to the onset of Japanese encephalitis, and the like. These pharmaceutical compositions can be formulated into preparations containing the binding peptide of the envelope protein of the encephalitis virus as an active ingredient and, if necessary, pharmaceutically acceptable carriers, and administered to humans or non-human vertebrates infected with the encephalitis virus, or administered prophylactically to humans or non-human vertebrates before infection.

The pharmaceutically acceptable carrier used herein may be appropriately selected from conventional carriers according to the form of the pharmaceutical composition to be prepared. For example, one or more of glycine, phosphate buffer at pH7.0, dextran, lactose, sodium chloride, citric acid, gelatin, water for injection, physiological saline, liposome, bile salt, fatty acid, saponin, sodium caprylate, sodium laurate, polyacrylic acid, fusidic acid derivatives (e.g., dihydrofusidate of bovine sulfonic acid, cyclodextrin, etc.), glycerol, A-zone, surfactant, fatty acid, bile salt, sodium salicylate, Zonsucratus toxin (ZOT), sodium glycocholate, camostat mesilate, bacitracin, aprotinin, soybean pancreatin inhibitor, nonionic surfactant, sugar, mannitol, sorbitol, PEG, human serum albumin, and the like may be used together.

The mode of administration of the pharmaceutical composition of the present invention is not particularly limited, and may be suitably selected depending on various preparation forms, the age, sex, other conditions, and severity of the disease of the patient, and the preparation form is particularly preferably an injection, an intravenous injection, a spray (aerosol), a nasal drop, an inhalant, and the like.

The route of administration includes oral administration or non-oral administration, and specific examples thereof include oral administration, intravenous administration, intra-arterial administration, intramuscular administration, subcutaneous administration, intraperitoneal administration, intratracheal administration, inhalation administration, sublingual administration, and the like, but are not limited thereto.

The daily dose of these pharmaceutical compositions may be appropriately changed depending on the symptoms, age, body weight, sex, treatment time, therapeutic effect, administration method, and the like of the subject to be administered, and is not particularly limited as long as the infection with the encephalitis virus can be suppressed and the side effect is within an allowable range. The preparation is not limited to once-a-day administration, and may be administered in multiple divided doses.

These pharmaceutical compositions may be used alone or in combination with other agents (e.g., other antiviral agents, anti-inflammatory agents, or agents that alleviate symptoms, etc.).

The implementation of the invention has important social and economic benefits for the treatment of the Japanese encephalitis virus infection which seriously harms human health.

Drawings

FIGS. 1 to 4ELISA assay the binding activity of polypeptides with numbers 1 to 11 to the envelope protein of the Japanese encephalitis virus.

FIGS. 5-7 flow cytometry quantitatively determined the uptake of 11 binding peptides into virus-infected and non-infected cells.

FIGS. 8-10 in vivo imaging observe the uptake rate of 11 binding peptides in the brains of virus-infected and non-infected mice.

FIG. 11 shows the localization of nucleic acid drug (RAF1-19) and nucleic acid drug polypeptide conjugate (RAF1-19-P63) on cells in confocal manner.

FIG. 12 flow cytometry assays for uptake of nucleic acid drug and nucleic acid drug polypeptide conjugates in virally infected and uninfected cells.

FIG. 13 in vivo imaging observation of uptake rates of nucleic acid drug and nucleic acid drug polypeptide conjugates in the brains of virus-infected and non-infected mice.

Detailed Description

In the case where the embodiments and examples are not specifically described, the practice of the present invention will employ conventional techniques of molecular biology, microbiology, and the like, which are well known to those skilled in the art.

Example one

This example illustrates screening for envelope protein binding polypeptides.

Materials and methods

1. Phage random peptide library screening

By means of NaHCO₃(pH 8.6) solution 100. mu.l of envelope protein (100. mu.g/mL) was coated on a 96-well microplate and left in a humid environment overnight at 4 ℃. The next day, the coating solution was decanted, 300. mu.L of BSA blocking solution at a concentration of 10mg/mL was added to each well, and blocking was performed at 37 ℃ for 1 h. Wash 6 times with TBST (TBS + 0.5% (v/v) Tween-20). Adding 1.5X 10¹¹pfu (in 100. mu.L TBST) phage (Ph.D. -12)^TMPhage Display Peptide Library Kit, purchased from New England Biolabs, bind for 1h at 37 ℃, decant Phage and wash 10 times with TBST (TBS + 0.5% (v/v) Tween-20). Eluting with 100 μ L of appropriate eluent (0.2MGlycine-HCl (pH2.2), 1mg/ml BSA), eluting at room temperature for 7-9min, not more than 10min, aspirating the eluate to a centrifuge tube, and adding 15 μ L of 1M Tris-HCl (pH9.1) to neutralize the eluate. Collecting eluate, taking 5 μ l for phage titer determination, repeating the above steps after the rest amplification and purification, and performing the next round of screening. After four rounds of elutriation, the final screening product is directly subjected to titer determination, and single-stranded phage DNA is extracted from a single clone and sequenced. And (3) carrying out alignment analysis on the sequenced sequences to obtain the sequences of the polypeptides combined with the target protein.

2. Identification of binding characteristics of screened phage and envelope protein

And performing phage amplification, purification and titer determination on the original monoclonal corresponding to the phage with the sequence obtained after sequencing, analysis and comparison. Phage titer was adjusted to 10⁶pfu/. mu.L. By means of NaHCO₃(pH 8.6) solution 10. mu.g/mL of envelope protein (100. mu.l) was coated on a 96-well microplate and left in a humid environment overnight at 4 ℃. The next day the coating solution was decanted and 300. mu.L of BSA at 10mg/mL concentration was added to each wellBlocking the solution for 1h at 37 ℃. Wash 6 times with TBST (TBS + 0.1% (v/v) Tween-20). Adding 10⁸The monoclonal phage of pfu (in 100. mu.L TBST) bound for 1-2h at room temperature, and M13 monoclonal antibody was added after 6 washes of TBST and bound for 1h at room temperature. Thereafter, the reaction was carried out by a conventional method of washing, developing and terminating. Absorbance at 450nm was measured. BSA-coated wells were used as negative control wells. A positive clone was identified as having a difference between the sample well and the BSA well of more than 0.6 and a difference between the sample well and the GST well of more than 0.6. In this experiment, a wild-type phage control was also set up.

Results

After four rounds of elutriation, the product is collected, and single-stranded DNA of a single clone is extracted for sequencing after titer determination. 26 polypeptide sequences are obtained, and after the verification of an ELISA method, 11 clones in the 26 polypeptide sequences are found to have binding specificity with HA protein. The ELISA assay results are shown in FIG. 1. The polypeptide sequences with binding specificity are shown in table one.

Example two

This example illustrates the uptake of 11 binding peptides in both virally infected and uninfected cells.

Materials and methods

BHK21 cells, viruses and polypeptide encephalitis B viruses are amplified and propagated by infecting BHK21 cells, and then are uniformly mixed, subpackaged and stored at-70 ℃ for later use. The encephalitis B virus strain used was the Jingwen strain. The BHK21 cell culture solution was dmem (gibco) culture solution containing 7% fetal bovine serum. Maintenance solutions containing 0.7% fetal bovine serum were used for viral infection. FITC labeling was performed for the synthesis of 11 polypeptides.

2. Quantitative observation of polypeptide uptake in virus-infected and uninfected cells by flow cytometry BHK21 cells were cultured in DMEM medium (GIBCO) containing 7% fetal bovine serum at 37 ℃ in a 5% CO2 incubator. And (4) observing the growth state of the cells, and after the cells are cultured to a logarithmic growth phase, inoculating the cells into a 6-well cell culture plate, wherein the cells are 80-90% full on the next day. Selecting 10 mu M polypeptide concentration, grouping experiments into a cell control group, a cell virus group, a cell polypeptide group and a cell virus polypeptide group, incubating viruses and polypeptides for 1h at room temperature, adding the incubated viruses and polypeptides into six-hole plate cells according to groups, and collecting cells at 37 ℃ for 1h, 2h and 6h for flow detection.

Results

The polypeptides P21, P45, P55, P57, P36, P48, P59 and P63 are found to have virus-dependent cell uptake increasing characteristics through flow detection, wherein the cell uptake rate of the P63 after virus infection is improved most, and the uptake rate of the P63 in virus-infected cells 6h after infection is improved by 2.2 times compared with that in uninfected cells.

EXAMPLE III

This example illustrates the uptake rate of 11 binding peptides in the brains of virus-infected and non-infected mice.

Materials and methods

Several female mice of BALB/C of SPF grade three weeks old (9-11 g) were ordered, the mice were weighed and grouped in advance at the beginning of the experiment and prepared for the experiment: FITC-labeled polypeptides, a 1mL syringe, a cotton swab, an electronic scale and a notebook. The polypeptide group is intraperitoneally injected according to the dosage of 20mg/kg, and the polypeptide virus group mice are intraperitoneally injected with the polypeptide according to the dosage of 20mg/kg immediately after the virus attack. The mice were then sacrificed for 4h, and brain tissue was immediately removed, fixed in 4% formaldehyde, and sent for in vivo imaging to observe brain tissue fluorescence intensity.

Results

The results of in vivo imaging tests show that the polypeptides P63, P45, P55, P36, P50 and P48 have good targeting property for virus-infected brain tissues, and the ingestion of the polypeptides in the brain tissues can be remarkably increased due to the infection of the Japanese encephalitis virus. Of these, the effect of P63 was most pronounced.

Example four

This example mainly shows that the binding peptide P63 can be used as a targeting modified peptide, which can increase the uptake of the nucleic acid drug RAF1-19 against the encephalitis B virus and improve the antiviral activity of the nucleic acid drug.

Materials and methods

BHK21 cells, viruses, polypeptides and nucleic acid drug polypeptide conjugate, namely the encephalitis B virus, are subjected to amplification propagation by infecting BHK21 cells, and then are uniformly mixed, packaged and stored at-70 ℃ for later use. The encephalitis B virus strain used was the Jingwen strain. The BHK21 cell culture solution was dmem (gibco) culture solution containing 7% fetal bovine serum. Maintenance solutions containing 0.7% fetal bovine serum were used for viral infection. The nucleic acid drug (RAF1-19) and the nucleic acid drug polypeptide conjugate (RAF1-19-P63) are labeled by FITC during synthesis.

2. The localization of the nucleic acid drug (RAF1-19) and the nucleic acid drug polypeptide conjugate (RAF1-19-P63) on the cells was observed in confocal manner at 6 h. BHK21 cells were cultured in DMEM medium (GIBCO) containing 7% fetal bovine serum at 37 ℃ in a 5% CO2 incubator. Observing the growth state of cells, after culturing to a logarithmic growth phase, inoculating the cells into a confocal observation small dish, selecting the concentration of the nucleic acid drug and the nucleic acid drug polypeptide conjugate to be 10 mu M, grouping experiments into a cell and nucleic acid drug group, a cell and virus and nucleic acid drug group, a cell and nucleic acid drug polypeptide conjugate group and virus group, incubating the viruses and the drugs at room temperature for 1h, adding the viruses and the drugs into the small dish according to groups, entering for 6h at 37 ℃, sucking out culture solution, washing the cells for 5 times by using DMEM, staining nuclei, and observing confocal.

3. Quantitative observation of uptake rates of nucleic acid drug and nucleic acid drug polypeptide conjugate in virus-infected and non-infected cells by flow cytometry BHK21 cells were inoculated into 6-well cell culture plates, 80-90% confluency the following day. The concentration of the nucleic acid drug and the nucleic acid drug polypeptide conjugate is selected to be 10 mu M, the experiment groups are a cell control group, a cell plus virus group, a cell plus nucleic acid drug group, a cell plus virus plus nucleic acid drug group, a cell plus nucleic acid drug polypeptide conjugate group and a cell plus nucleic acid drug polypeptide conjugate plus virus group. The virus and the medicine are incubated for 1h at room temperature, and then added into six-hole plate cells according to groups, and the cells are collected for 6h at 37 ℃ and sent to flow detection.

4. And (3) observing the uptake rate of the nucleic acid drug and the nucleic acid drug polypeptide conjugate in the brains of the virus-infected and non-infected mice by in vivo imaging. SPF grade three-week old (9-11 g) BALB/C female mice were several, the mice were weighed and grouped in advance at the beginning of the experiment, and the experimental things were prepared: FITC labeled nucleic acid drug, FITC labeled nucleic acid drug polypeptide conjugate, 1mL syringe, cotton swab, electronic scale and notebook. The experimental groups are nucleic acid drug group, virus infection nucleic acid drug group, nucleic acid drug polypeptide conjugate group and virus infection nucleic acid drug polypeptide conjugate group. The drug group is intraperitoneally injected according to the dosage of 20mg/kg, and the virus infection drug group mice are intraperitoneally injected according to the dosage of 20mg/kg immediately after the virus infection drug group mice are attacked. The mice were then sacrificed for 4h, and brain tissue was immediately removed, fixed in 4% formaldehyde, and sent for in vivo imaging to observe brain tissue fluorescence intensity.

Results

The P63 is connected with the anti-encephalitis B oligonucleotide RAF1-19 by means of chemical synthesis and the like, and the membrane penetration rate of the nucleic acid medicament in the cells infected by the encephalitis B virus can be obviously increased by combining the polypeptide with the virus surface protein, so that the brain tissue targeting property of the nucleic acid medicament can be increased.

Claims

1. A polypeptide capable of specifically binding Japanese encephalitis virus envelope protein has an amino acid sequence as follows: NH (NH)₂-HHWWVPSWSRWT-COOH。

2. Use of the polypeptide of claim 1 in the preparation of a medicament for the treatment and prevention of diseases associated with Japanese encephalitis virus infection.

3. The use according to claim 2, characterized in that the polypeptide is conjugated to the anti-Japanese encephalitis virus drug by covalent and/or non-covalent linkage, so that the drug targets Japanese encephalitis virus infected cells.