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A anti-enveloped virus recombinant polypeptide and its preparation method

An antiviral and viral envelope technology, applied in antiviral agents, botanical equipment and methods, biochemical equipment and methods, etc., can solve the problems of expanding the application scope of colistin, achieve high targeting, reduce toxic side effects

Inactive Publication Date: 2004-03-17
CHENGDU PHOTON BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, its disadvantage is that it can only act on Gram-negative bacilli such as Escherichia coli. If a biomacromolecule that can specifically bind to an antigen on the virus 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 scope of application of colistin

Method used

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  • A anti-enveloped virus recombinant polypeptide and its preparation method
  • A anti-enveloped virus recombinant polypeptide and its preparation method
  • A anti-enveloped virus recombinant polypeptide and its preparation method

Examples

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Embodiment 1

[0031] [Example 1] Construction of a plasmid expressing an anti-hepatitis B virus polypeptide and preparation of a recombinant antiviral polypeptide

[0032] The original plasmid is pSELECT loaded with colistin Ia and immunity protein genes TM -1 commercial plasmid (plasmid size 8.3kb, Promega Company) (UCSF, donated by P.Gosh). Double-stranded oligonucleotide point mutation technology (QuickChange TM Kit, Strategeue Company) inserted the genes encoding the two peptide chains G28-V50 and E216-S228 in the anti-HBsAg single-chain antibody registered in GenBank AF236816 into the I626 site of the colicin Ia gene through 4-step point mutations, and prepared Mutant plasmids for antiviral polypeptides, (such as figure 1 shown). The mutant plasmid was transfected into E. coli TG1 engineering bacteria (AECOM, donated by K. Jakes) to prepare polypeptides.

[0033] The mutation program was carried out according to the Strategene QuikChange Site-Directed Mutagenesis Kit (Catalog#2005...

Embodiment 2

[0063]The genes of the G28-V50 and E216-S228 peptide chains in the anti-HBsAg scFv registered in GenBank AF236816 were connected to the carboxyl end of the colicin Ia channel domain (K544-I622) according to the method in Example 1, and prepared into an artificially combined For the plasmid of anti-HBV engineering polypeptide, transfect the mutated plasmid into engineering bacteria without plasmid, multiply the bacteria in large numbers (4L FB medium, 225rpm, 37°C; 6h), and centrifuge the bacterial cells (4°C, 6000g , 20min), take 4℃, 50mM boric acid buffer + 2mM EDTA+2mM DTT 50-80ml suspended bacteria, ultrasonically break (4℃, 40W, 1-2min), high-speed centrifuge to break the bacteria (4℃, 70000g, 1.5 h), take the supernatant and add streptomycin sulfate to precipitate DNA, dialyze overnight at 4°C in 50mM boric acid buffer + 2mM EDTA + 2mM DTT 2L, then load the sample on a CM ion exchange column, 0.1-0.3M NaCl + 50mM boric acid buffer After gradient elution, the anti-HBV engi...

Embodiment 3

[0065] According to the method of Example 1, the anti-HBV PreS1 scFv registered in GenBank AF427148 was connected to the amino terminal of the water-soluble pore structure domain of colistin Ia to form a recombinant plasmid, and obtain a recombinant polypeptide with a molecular weight of about 40,000;

[0066] The anti-HBV PreSl scFv registered in GenBank AF427148 was linked to the carboxyl terminus of the colicin Ia water-soluble pore structure domain to obtain a recombinant polypeptide with a molecular weight of about 40,000;

[0067] Link the anti-HBV PreSl scFv registered in GenBank AF427148 to the amino terminal of colicin Ia to obtain a recombinant polypeptide with a molecular weight of about 90,000;

[0068] The anti-HBV PreSl scFv registered in GenBank AF427148 was linked to the carboxyl terminus of colicin Ia to obtain a recombinant polypeptide with a molecular weight of about 90,000.

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Abstract

The present invention relates to one kind of enveloped virus resisting polypeptide and its encoding nucleotide and amino acid sequence. Colicin capable of forming ion channel or its aquatic porous channel structural domain is made to combine with polypeptide capable of being combined specifically with target virus envelope antigen to form engineering polypeptide. Inside the engineering polypeptide, ScFv antigen combining area capable of being combined specifically with target virus envelope antigen induces the colicin water soluble porous channel structural domain to near target virus envelope, the colicin water soluble porous channel structural domain then forms the ion channel on the envelope to destroy the completeness of the envelope to leak or expose virus RNA and kill virosome. The antiviral engineering polypeptide kills selected virosome without injuring host cell, being superior to traditional antiviral medicines.

Description

technical field [0001] The present invention relates to a recombinant antiviral polypeptide, in particular to the anti-enveloped virus polypeptide, and the nucleotide and amino acid sequences encoding the polypeptide. [0002] The invention also relates to the preparation method of the recombinant antiviral polypeptide. Background technique [0003] Viral infection has become a major threat to human life and health. As the earth's biosphere is increasingly destroyed by human industrialization, viral diseases, such as viral hepatitis, influenza, pneumonia, encephalitis, tumors caused by viruses, and AIDS, are becoming more and more rampant. Because the genes of viruses are prone to mutations, the damage caused by viruses is often involved in the malfunction of the host immune system. The mechanism is still being explored, so people have been working hard to try to prepare truly effective antiviral drugs. [0004] At present, antiviral treatment mainly starts from the followi...

Claims

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Application Information

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IPC IPC(8): A61P31/12C07K19/00C12N15/31C12N15/62C12N15/63
Inventor 丘小庆
Owner CHENGDU PHOTON BIOTECH
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