WO2003106483A1

WO2003106483A1 - A recombinant anti-enveloped virus peptide and its preparation

Info

Publication number: WO2003106483A1
Application number: PCT/CN2003/000469
Authority: WO
Inventors: Xiaoqing Qiu
Original assignee: Chengdu Photon Biotechnology Limited
Priority date: 2002-06-17
Filing date: 2003-06-17
Publication date: 2003-12-24
Also published as: JP2006506965A; AU2003242222A1; CN1396178A

Abstract

The invention involved a recombinant anti-enveloped virus peptide, the sequence of DNA which coding this peptide and the sequence of amino acid of this peptide. The colicin capable of forming ion channel or the structure field of its water-solubility passage was combined with peptide capable of peculiarly linking to the target virus envelope antigen to form synthetical peptide. The ScFv antigen bonding field capable of peculiarly linking to the target virus envelope antigen induced the structure field of colicin's water-solubility passage close to target virus envelope, it then formed icon channel to destroy the integrality of the envelope, and the virus RNA was released and killed the virus through this way. And it ensured that this peptide only killed target virus except host cell, then largely reduced side-effect of conventional anti-virus drug to host.

Description

Recombinant anti-enveloped virus polypeptide and preparation method thereof

The present invention relates to a recombinant antiviral polypeptide, and more particularly to an anti-enveloped polypeptide, a nucleotide and amino acid sequence encoding the polypeptide.

The invention also relates to a method for preparing a recombinant antiviral polypeptide. Background technique

Viral infections have become a major threat to human life and health. As the earth's biosphere is being increasingly destroyed by human industrialization, viral diseases, such as viral hepatitis, influenza, pneumonia, encephalitis, virus-induced tumors, and AIDS, etc. Because the genes of viruses are susceptible to mutation, the damage caused by viruses often involves the malfunction of the host's immune system. The mechanism is still being explored, so people have been trying to make truly effective antiviral drugs.

At present, most antiviral treatments focus on the following aspects: 1. Inhibiting viral nucleic acids; 2. Inhibiting viral proteases; 3. Using immune factors to kill, such as interferons; 4. Using virus vaccines to immunize.

However, in practical applications, the virus often makes the above treatments ineffective using its mutational characteristics. For example, the anti-HBV drug Laminvudine, which was put into use a few years ago, was almost 90% effective when it was first used. However, after only a few years of clinical application, due to the rapid mutation of the hepatitis B virus gene, the effective rate was about 90% from the original. It's only about 10%. It is therefore necessary to look for other more effective antiviral approaches.

Based on the above-mentioned current situation, the patent inventors believe that the structure that is not prone to mutation of the virus should be selected as the target of drug action to kill the virus, so as to fundamentally avoid the fatal weakness of the existing antiviral treatment methods that are easily destroyed by the mutation of the virus.

The inventor of the patent envisages that if the enveloped virions can destroy the integrity of the envelope, the protein and ribonucleic acid in the envelope can be leaked or exposed and the virus will die, it will be possible to prevent virus infection and recurrence. Infection, so as to achieve the purpose of treatment. In addition, the viral envelope is composed of a lipophilic molecular membrane, which is a fairly stable biological structure. The probability of mutation is relatively low compared to ribonucleic acid and proteins. In nature, there are a lot of bacterial toxins that kill bacteria by destroying the integrity of the lipid bilayer membrane of the host cell membrane. So why Don't learn from this anti-biotic mechanism optimized after hundreds of millions of years of survival competition to design new antiviral drugs?

In 1952, Jacob discovered that colistin can specifically kill certain strains of E. coli and related forests, such as Shigella Sammei (Jacob et al, Sur labiosynthese d'une colicine et son mode d 'action, Annals of the Pasteur Institute.

83: 295-315 (1952)). In 1978, Finkelstem et al. Discovered that colistin, which can form ion channels, can form voltage-dependent ion channels on the artificial lipid bimolecular membrane, thereby fundamentally revealing the antibacterial mechanism of this type of bacterial toxins (Schein et al,. Colicin K acts by forming voltage dependent channels in phospholipid bilayer membranes. Nature 276: 159-163 (1973)). In 1996, Qiu and Finkelstein et al. Revealed the transmembrane three-dimensional structure of the ion channel formed by colicin la on the artificial lipid bilayer membrane when it is opened and closed (Qiu et al., Major transmembrane movement associated with colicin la chammel gating. J Gen. Physiology. 107: 313-328 (1996)), laying a theoretical foundation for the design and preparation of new types of antibiotics at the molecular level. However, its disadvantage is that it can only act on Gram-negative bacilli such as E. coli. If a macromolecule that can specifically bind to an antigen on the viral envelope can be used as an inducer, colistin or its aqueous pore structure can be induced to a specific The formation of ion channels near the virus envelope to kill the virus can greatly expand the application of colistin.

Enveloped viruses have highly species-specific antigens or receptors on their envelopes. For example, hepatitis B virus has the HBsAg antigen and

Science, Textbooks of National Medical Colleges and Universities, People's Publishing House 2001, 292). Therefore, if a polypeptide that can specifically bind to a viral envelope antigen, such as a specific single-chain antibody scFv against the HBV virus HBsAg antigen or an antigen-binding region therein is used as an inducer, certain bacterial toxins that can form ion channels, For example, E. coli (^ scAe 'c nVz co / i), a toxin protein secreted by colicin or its aqueous pore forming region, induces the formation of ion channels on specific target virus envelopes. To kill the virion should be an ideal antiviral drug development direction. Summary of the Invention It is an object of the present invention to provide a new type of recombinant antiviral polypeptide, which acts on the monthly contraction of the virus and forms an ion channel to achieve the function of killing the virion.

Another object of the present invention is to provide a nucleotide sequence encoding the recombinant antiviral polypeptide. Another object of the present invention is to provide a recombinant plasmid containing a nucleotide sequence encoding the recombinant anti-viral polypeptide of the present invention.

A further object of the present invention is to provide an amino acid sequence which encodes the recombinant anti-viral polypeptide of the present invention.

Another object of the present invention is to provide a pharmaceutical composition containing the recombinant antiviral polypeptide of the present invention.

Another object of the present invention is to provide a method for preparing a recombinant antibacterial polypeptide.

According to one aspect of the present invention, colicin or an aqueous channel domain thereof capable of forming an ion channel and a polypeptide capable of specifically binding to a target virus envelope antigen are combined into a recombinantly engineered polypeptide. In this engineering polypeptide, a polypeptide that can specifically bind to the target virus envelope antigen serves as an inducer to induce the coliform water-soluble channel domain to reach the vicinity of the target virus envelope, and then the colistin aqueous channel domain forms an ion on the envelope. Channel, destroying the integrity of the envelope, allowing the viral ribonucleic acid to leak or be exposed to kill the virions.

In a preferred embodiment of the present invention, a specific anti-hepatitis B virus (HBV) PreSl scFv antibody gene (GenBank AF427148 registration) is linked to the amino terminal or carboxyl terminal of a gene encoding colicin or its water-soluble channel domain, Formation of a recombinant antiviral polypeptide. In addition, the antiviral activity and the possible side effects of the recombinant polypeptide can be enhanced by changing the length of the peptide chain and the composition of the amino acid residues of the recombinant polypeptide. As such composition or change will increase or decrease the number of amino acid residues of the recombinant antiviral polypeptide Therefore, the molecular weight of the obtained recombinant antiviral polypeptide will vary in the range of 5,000-100,000. The colistin in the present invention may be selected from colistin El, Ia, Ib, A, B and N or an aqueous channel domain thereof capable of forming an ion channel. In one embodiment of the present invention, the GenBank AF427148 registered The anti-HBV PreSl scFv is connected to the amino terminus of the water-soluble channel domain of colicin la to obtain a recombinant polypeptide with a molecular weight of about 40,000. In another embodiment, the anti-HBV PreSl scFv registered in GenBank AF427148 was linked to colistin la-soluble pore carboxy-terminal domain, the recombinant polypeptide is obtained in a molecular weight of about 40,000; in another embodiment, the connection anti HBV PreSl scFv GenBank AF4271 ⁴ 8 log to the amino terminus of colicin la obtain molecular weight At 9 Recombinant polypeptide of about 10,000; The anti-HBV PreSl scFv registered in GenBank AF427148 was linked to the carboxyl terminal of colicin la to obtain a recombinant polypeptide of about 90,000 in molecular weight.

According to another aspect of the present invention, in order to further increase the activity of the antiviral polypeptide and reduce its toxic and side effects, a single chain antibody (scFV) that specifically binds to an antigen on the viral envelope or an antigen-binding region therein and coliform bacteria may be selected. And its water-soluble pore domain are linked to obtain a more active antiviral polypeptide.

In another preferred embodiment of the present invention, the HBV virus envelope antigen HBsAg single-chain antibody (ScFv) or one or more antigen binding region peptide chains thereof is combined with a gene encoding colicin or a water-soluble channel domain thereof connection. In a specific embodiment, G28-V50 and G28-V50 in the anti-HBsAg single chain antibody scFv registered in GenBank AF 236816 are registered.

The amino terminal of the gene (SEQ ID NO. 2) of the two peptides of E216-S228 is connected to the carboxy terminal of the gene encoding colicin la (SEQ ID NO. 1) or its water-soluble channel domain (K544-I622). To form a nucleotide encoding a recombinant antiviral polypeptide (SEQ ID NO. 4), which encodes an amino acid sequence such as SEQ ID NO. 5. In addition, the antiviral activity and the possible toxic and side effects of the recombinant polypeptide can be enhanced by changing the length of the peptide chain and the composition of the amino acid residues of the recombinant polypeptide, because such a composition or change will increase or decrease the amino acid residues of the recombinant antiviral polypeptide. The molecular weight of the recombinant antiviral peptide obtained will vary from 15,000 to 74,000.

In the constructed recombinant antiviral polypeptide, a polypeptide that specifically binds to a single-chain antibody antigen-binding region of a viral envelope antigen is used as an inducer to induce the coliform water-soluble channel domain to pass through the viral envelope, and then the colistin aqueous channel structure The domain forms an ion channel on the envelope of the target virus, thereby achieving the purpose of killing the virus.

According to another aspect of the present invention, there is provided a plasmid vector comprising the nucleotide of the present invention as shown in FIG. 1, which is a plasmid vector that binds specifically to a hepatitis B virus (HBV) envelope antigen single-chain antibody antigen-binding region as described above. The nucleotide sequence of the polypeptide is inserted into amino acid 626 of the colicin la gene by double-stranded oligonucleotide point mutation technology to form the recombinant plasmid of the present invention.

In the present invention, the original plasmid pSELECT ^{TM-1 for} constructing a plasmid vector is from Promega, which is loaded with colicin la and immunogenic protein genes, and is directed to a polypeptide that specifically binds to the hepatitis B virus (HBV) envelope antigen single-chain antibody antigen-binding region. Four pairs of primers were designed for the genes, and their sequences are shown in SEQ ID NO .: 6-SEQ ID NO: 13 respectively. Recombinant plasmids were obtained by using the strand-oligonucleotide point mutation technology according to the operation of Strategy's medicine cabinet. The recombinant plasmid was transfected into E. coli TG1 engineered bacteria to obtain host cells.

According to another aspect of the present invention, a pharmaceutical composition containing the recombinant antiviral polypeptide of the present invention is provided, which can be prepared by adding the antiviral polypeptide of the present invention to a pharmaceutically acceptable carrier or excipient or optional other ingredients. Pharmaceutical composition suitable for clinical use.

According to another aspect of the present invention, a method for preparing an antiviral polypeptide of the present invention is provided. A gene encoding a polypeptide that specifically binds to a hepatitis B virus (HBV) envelope antigen single-chain antibody antigen-binding region is operably linked to the colicin gene to obtain The gene encoding the recombinant antiviral polypeptide is introduced into an expression system for expression, and the expressed polypeptide is isolated to obtain the antiviral polypeptide of the present invention. Brief description of the drawings

The present invention is described in detail below with reference to the drawings and embodiments.

Figure 1 shows the structure of a scutellum containing two peptide chains in an anti-HBV single chain antibody and colicin la;

Figure 2 is an electron micrograph of normal hepatitis B virus (left) and hepatitis B virus (right) after being treated with the antiviral polypeptide of the present invention for 1 hour. The figure shows phosphorus after 1 hour incubation with antiviral polypeptide The tungstic acid dye penetrates the envelope and enters the virus (darkening inside the envelope becomes darker), showing that the integrity of the envelope has been destroyed. (50,000 times magnification)

Figure 3 is a biochemical / histological hepatitis mouse-human-hepatocyte chimeric in vivo model showing the antibody concentration after treatment with the recombinant antiviral polypeptide of the present invention. As can be seen from the figure, as of the 4th week, Anti-HBV peptide mice's serum HBsAg and anti-HBc antibodies were significantly reduced. detailed description

The present invention will be described in detail below with reference to the accompanying drawings through the description of the preferred embodiments of the present invention. [Example 1] Construction of plasmid expressing anti-HBV polypeptide and preparation of recombinant antiviral polypeptide

The original plasmid was the pSELECT ™ -1 commercial plasmid (colloid size 8.3 kb, donated by Promega Corporation XUCSF, P. Gosh) loaded with colistin la and immunogenic protein genes. The double-stranded oligonucleotide point mutation technology (QuickChange ^TM Kit, Strategeue) registered GenBank AF 236816-encoded anti-HBsAg single-chain antibody G28-V50 and The gene of the two peptides of E216-S228 was inserted into the 1626 site of the colicin la gene through a 4-step point mutation to prepare a mutant plasmid of antiviral polypeptide (as shown in Figure 1). The mutant plasmid was transfected into engineered bacteria of £ .co / i'TGl (AECOM, donated by KJakes) to prepare the polypeptide.

Mutation procedures were performed according to the Strategene QuikChange Site-Directed Mutagenesis Kit (Catalog # 200518) kit manual:

1. Prepare point mutation reaction:

5μ1 1 OX buffer

2 μ 1 (10ng) wild type colicin plasmid

1.25 μl (125ng) designed 5, -3, oligonucleotide primers (see listed primer sequences) 1.25Ml (125ng) designed 3, -5, oligonucleotide primers (see listed primer sequences) )

1 μΗΝΤΡ

Double distilled water 50 μΐ

1 ΐ ίυ.

(Except plasmid, primer and double distilled water, all reagents are provided in the medicine box)

2. Perform PCR amplification. Amplification conditions: Denaturation at 95 ° C, 35 seconds, annealing at 53 ° C, 70 seconds, extension at 68 ° C, 17 minutes for 20 cycles;

3. Into the Dpnl endonuclease Ιμΐ after digesting the mother DNA strand (37 ° C, 1 hour), take 1μ

1 reactant was incubated with XLl-Blue competent cells in 50μ1 water, heat shocked at 42 ° C for 45 seconds, and then placed in ice for 2 minutes;

4. Add 0.5ml of NZY pegylated at 220 rpm, shake at 37 ° C for 1 hour, then plate 50-100 μl of the reaction (LB pegylated plus 1% agar plus 50 μ 1 / ml ampicillin, 37

!! Overnight);

After 5.18 hours, bacteria were picked and plasmids could be extracted from various commercial extraction plasmid kits from companies such as Qiagene, Gibco, etc. The mutation was successfully determined by sequencing.

6. Incubate the plasmid 50ng with the prepared TG1 engineered bacteria-sensing peptide cells 50 μ 1 for 30 minutes on ice.

Heat shock for 90 seconds, take 50-100 μΐ of the reaction mixture and add 0.5ml of LB medium, 220rpm, shake at 37 ° C for 1 hour and plate (LB medium plus 1% agar plus 50 g / ml ampicillin) 37 ° C, Colonies were picked after 18 hours.

7. Large amount of bacteria, 8-16 liters of FB culture medium, 250rpm, 37 ° C, 6-8 hours; Centrifuge the bacteria, 4 ° C, 6000g, 20 minutes, take 4, 50mM boric acid buffer (2mM EDTA + 2mM DTT) 50-80ml suspension bacteria, add 0.2M PMSF 250 microliters, ultrasonically crush (4 ° C, 400W, 2 minutes), centrifuge at high speed (4 ° C, 75000g, 1.5 hours), remove Add 5 million units of streptomycin sulfate to precipitate the DNA, centrifuge at high speed (4 ° C, 30,000g, 10 minutes), and load it into a dialysis bag with a molecular weight of 15000 at 4 ° C. After dialysis with 4 liters of 50mM boric acid buffer solution, centrifuge at high speed. (4 C, 30000 g, 10 minutes), the supernatant was applied to a CM ion exchange column, and eluted with 0.3 M NaCl + 50 mM boric acid buffer at 4 ° C to obtain an anti-HBV engineering peptide with a purification rate of more than 90%. The molecular weight is about 74,000, and the protein content of the crude preparation is about 1-5 mg / ml. It was administered via intraperitoneal injection and cell culture solution dosing, and the engineering peptide was not found to have toxic or side effects on human cells and experimental mice cultured in vitro. Oligonucleotide sequence designed in the above preparation plasmid

the first time:

5'-3 '(SEQ ID NO: 6)

gcg aat aag ttc tgg ggt att GGA TTC ACC TTC AGT GAC TAC TAG ATG AGC taa ata aaa tat aag aca ggc

3'-5 '(SEQ ID O: 7)

gcc tgt ctt ata ttt tat tta GCT CAT GTA GTA GTC ACT GAA GGT GAA TCC aat acc cca gaa ctt att cgc Second time:

5'-3 '(SEQ ID NO: 8)

acc ttc agt gac tac tac atg age TGG ATC CGC CAG GCT CCA GGG AAG taa ata aaa tat aag aca ggc

3'-5 '(SEQ ID O: 9)

gcc tgt ctt ata ttt tat tta CTT CCC TGG AGC CTG GCG GAT CCA get cat gta gta gtc act gaa ggt 笫 three times:

5'-3 '(SEQ ID NO: 10)

tgg ate cgc cag get cca ggg aag GGG CTG GAG TGG GTT TCA GAT GAG taa ata aaa tat aag aca ggc

3'-5 '(SEQ ID NO: 11)

gcc tgt ctt ata ttt tat tta CTC ATC TGA AAC CCA CTC CAG CCC ctt ccc tgg age ctg gcg gat cca fourth time

5'-3 '(SEQ ID NO: 12)

ggg ctg gag tgg gtt tea gat gag GCT GAC TAT TAC TGT AAC TCC CGG GAC AGC taa ata aaa tat aag aca ggc

3, -5, (SEQ ID NO: 13)

gcc tgt ctt ata ttt tat tta GCT GTC CCG GGA GTT ACA GTA ATA GTC AGC etc ate tga aac cca etc cag ccc

[Example 2]

G28-V50 and anti-HBsAg scFv registered in GenBank AF236816 and

The gene of the E216-S228 peptide chain was connected to the carboxyl termin of the colicin la channel domain (K544-I622) according to the method of Example 1 to prepare an artificially combined plasmid against HBV engineering polypeptide, and the mutant plasmid was transformed. Stained into the engineering bacteria without plasmid, multiply the bacteria (4L FB culture medium, 225rpm, 37 ° C; 6h), centrifuge the bacteria (4 ° C, 6000g, 20min), take 4 ° C, 50mM boric acid Buffer solution + 2mM EDTA + 2mM DTT 50-80ml suspended bacteria, ultrasonically crushed (4 ° C, 40W, l-2min), high-speed centrifugal crushed bacteria (4 ° C, 70000g, 1.5h), take the supernatant and add sulfuric acid Streptomycin-precipitated DNA, 4 ° C, 50 mM borate buffer + 2 mM EDTA + 2 mM DTT 2L after dialysis overnight, loaded on a CM ion exchange column, 0.1-0.3M Naa + 50 mM borate buffer gradient elution An anti-HBV engineering peptide with a purification rate of more than 90% was obtained, the molecular weight was about 15,000, and the crude protein content was about 1.5 mg / ml. In the pre-experiment, the engineered peptide showed no toxic or side effects on human cells and experimental animals cultured in vitro.

[Example 3]

According to the method of Example 1, the anti-HBV PreSl scFv registered in GenBank AF427148 was linked to the amino terminus of the water-soluble channel domain of colicin la to form a recombinant plasmid to obtain a recombinant polypeptide with a molecular weight of about 40,000; The anti-HBV PreSl scFv registered in GenBank AF427148 was connected to the carboxyl terminus of the coliform la water-soluble channel domain to obtain a recombinant polypeptide with a molecular weight of about 40,000;

The anti-HBV PreSl scFv registered in GenBank AF427148 was linked to the amino termin of colicin la to obtain a recombinant peptide with a molecular weight of about 90,000;

The anti-HBV PreSl scFv registered in GenBank AF427148 was linked to the carboxy terminus of colicin la to obtain a recombinant peptide with a molecular weight of about 90,000.

[Example 41 Killing effect of recombinant antiviral polypeptide on HBV cultured in vitro A human liver cell line HepG2 2.2.15 (provided by Dr. C Wu (UCHC, Connecticut, USA)) transfected with HBV gene was cultured with 100 10,000g, 3hr, 4. C treatment to collect intact HBV virus (Dane's particles) produced by the cultured cells. After incubating a large amount of HBV-containing culture solution with the anti-HBV engineering polypeptide prepared in Example 1 at a volume ratio of 5: 1 for 1 hour at room temperature, a few microliters of the sample was placed on a copper mesh and stained with 1% phosphotungstic acid, and then magnified with a transmission electron microscope. Observation at 50,000 times showed that the staining in HBV after treatment became deeper, which confirmed that the HBV envelope was damaged by the polypeptide. The experimental results are shown in Figure 2.

[Example 5] Anti-HBV effect of recombinant antiviral polypeptide in animals

Construction of animal models (provided by Dr. G Wu, UCHC, Connecticut USA, references: Wu, C et. Al'Hepatitis B virus infection of transplanted Human hepacytes causes a biochemical and histological hepatitis in immunocompetent rate WJ. Of Gastroenterology, 9 ( 5): 978-983, 2003, May 9):

1. Intraperitoneal injection of human hepatocytes into fetal rats

SD female rats were anesthetized from 15 to 17 days of pregnancy, and the pregnant uterus was exposed open. The fetal rats were injected with 10 microliters of PBS containing ¹ × 10 ⁵ human hepatocytes (HepG22.2.15) through the uterine wall under strong light transmission.

2.Human liver cell transplantation

Within 24 hours after the birth of newborn rats, 200 μl of PBS containing 2 X 10 ⁶ human hepatocytes (same as above) was injected into the spleen.

3.Inject HBV

One week after the cell transplantation, 100 μl of TEN (0.02M Tris-Hcl, PH7.5, ImM EDTA, 0.15M Nacl) 105HBV particles were injected into the spleen of the transplanted mice, and HBsAg was detected in peripheral blood. HBV DNA, liver tissue detection HbcAg, HBV RNA, HBV cccDNA and histological changes to confirm the success of the human-rat liver chimeric model.

Nine mouse human-hepatocyte chimeric in vivo models with biochemical / histological hepatitis manifestations were selected and divided into 3 groups. The first group was the control group a (n = 3), and 0.9% saline was injected intraperitoneally. , Once a week; the second group is the control group b (n = 3), and intraperitoneal injection of wild type colicin la 0.5mg, once a week; the third group is the experimental group (n = 3), which is performed by intraperitoneal injection The anti-HBV polypeptide prepared in Example 1 was 0.5 mg once a week. The serum HBsAg (EIA method) and anti-HBc antibody (ELISA method, recombinant HbcAg-coated 96-well plate) were tested at week 1, week 4, and week 8 to evaluate the efficacy of the prepared anti-HBV polypeptide. The results are shown in Figure 3. And Table 1. It can be seen from FIG. 4 that as of the 4th week, the serum HBsAg and anti-HBc antibodies of mice injected with anti-HBV polypeptide were significantly reduced. End point titer of serum anti-HBc in each group Sampling time

Force ¹ J

0 weeks 2 weeks 4 weeks

Peptide experiment group 36 450 12 150 4050

Control group a 36450 109350 109350 control group b 36450 109350 109350 It can be seen from the above experimental results that the recombinant antiviral polypeptide of the present invention shows high anti-HBV activity in vitro and in vivo in animals.

The above detailed description of the present invention does not limit the present invention, and those skilled in the art can make various changes and modifications according to the present invention, as long as they do not depart from the spirit of the present invention, they should all belong to the scope of the appended claims of the present invention. Industrial applicability

Each enveloped virus has its own unique envelope antigen or receptor, so as long as it uses a polypeptide that can specifically bind to it, or colistin or its water-soluble channel structure i that can form an ion channel, it can be combined into one A specific antiviral recombinant engineered polypeptide against the virus. That is to say, a great advantage of the present invention is that it is possible to fight against colistin or its water-soluble channel domain composition by replacing multiple polypeptides that can bind to different viral envelope antigens or receptors. What an antiviral drug is an enveloped virus.

Another great advantage of the present invention is that the prepared antiviral engineering polypeptide can directly form ion channels on the envelope of the virion to destroy the integrity of the envelope. This virus killing method is similar to the use of nucleic acid analogs to replace the core of viral DNA precursors. Compared with traditional methods such as glycoside drugs, viral protease inhibitor drugs, interferon and traditional Chinese medicine, such as antiviral drugs, immunosuppressants, and genetically engineered nucleic acid vaccines, the advantage is that it uses the natural world's best choice after millions of years of natural selection. One of the simplest and most effective mechanisms for killing organisms, this mechanism has high targeting, which ensures that the antiviral engineering polypeptide only kills selected virions without harming host cells, thereby maximizing The limit reduces the toxic and side effects of the above-mentioned traditional antiviral drugs on the host.

Claims

Rights request

1. A nucleotide encoding a recombinant antiviral polypeptide, characterized in that it contains

A gene encoding colicin or its aqueous pore domain that forms an ion channel, and

A gene encoding a polypeptide that specifically binds an antigen or receptor on the viral envelope.

The nucleotide according to claim 1, wherein the colistin capable of forming an ion channel is selected from the group consisting of colistin El, Ia, Ib, A, B, and N.

3. The nucleotide according to claim 1, wherein the virus is a hepatitis B virus.

4. The nucleotide according to claim 3, wherein the polypeptide that specifically binds to an antigen or receptor on the viral envelope is an anti-HBV PreSl scFv, and its GenBank accession number is AF427148.

5. The nucleotide according to claim 4, wherein the anti-HBV PreSl scFv polypeptide is linked to the amino terminus of the colicin la.

6. The nucleotide according to claim 4, wherein the anti-HBV PreSl scFv polypeptide is linked to the carboxy terminus of the colicin la.

7. The nucleotide according to claim 4, wherein the anti-HBV PreSl scFv polypeptide is linked to the amino terminus of the colistin la aqueous channel domain.

8. The nucleotide according to claim 4, wherein the anti-HBV PreSl scFv polypeptide is linked to the carboxy terminus of the colistin la aqueous channel domain.

9. The nucleotide according to claim 1, wherein the polypeptide that specifically binds to an antigen or a receptor on the hepatitis B virus envelope is a HBsAg single-chain antibody scFv, whose GenBank The registration number is AF 236816.

The nucleotide according to claim 9, wherein the polypeptide that specifically binds to an antigen or receptor on the hepatitis B virus envelope is one or several antigen-binding regions of a HBsAg single-chain antibody scFv.

The nucleotide according to claim 10, wherein the antigen-binding region of the HBsAg single-chain antibody is two peptides of G28-V50 and E216-S228 of scFv.

The nucleotide according to claim 11, wherein the amino terminal of the two peptide chains of G28-V50 and E216-S228 in the anti-HBsAg single chain antibody is connected to the carboxyl terminal of the colicin la.

The nucleotide according to claim 11, wherein the amino terminus of the two peptide chains of G28-V50 and E216-S228 in the anti-HBsAg single-chain antibody is connected to the carboxyl group of the aqueous channel domain of colicin la end.

The nucleotide according to claim 12, which contains the nucleotide sequence of SEQ ID NO.

15. A recombinant plasmid, characterized in that it contains

A gene encoding colicin or an aqueous channel domain thereof capable of forming an ion channel, and a gene encoding a polypeptide capable of specifically binding to an antigen or a receptor on a viral envelope.

The recombinant plasmid according to claim 15, which contains the nucleotide sequence of SEQ ID NO.

17. A recombinant antiviral polypeptide containing

E coli or an aqueous channel domain that can form an ion channel, and a polypeptide that specifically binds to an antigen or receptor on the viral envelope.

18. The antiviral polypeptide according to claim 17, comprising an amino acid sequence of SEQ ID NO.

19. A pharmaceutical composition comprising the recombinant antiviral polypeptide according to claim 17 or 18.

20. A method for preparing a recombinant antiviral polypeptide, comprising the following steps:

Operably linking a gene encoding a polypeptide capable of specifically binding to an antigen or a receptor on a viral envelope with a gene encoding colistin or an aqueous channel domain thereof capable of forming an ion channel to obtain a gene encoding a recombinant antiviral polypeptide;

Introducing the obtained gene into an expression system for expression;

An antiviral polypeptide obtained by isolating the expressed polypeptide.