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Foot and mouth disease bivalent polypeptide vaccine and its preparation method and use

A polypeptide vaccine, a technology for foot-and-mouth disease, applied in the field of foot-and-mouth disease vaccine, can solve the problems of market import and export impact, low protection effect of polypeptide vaccine, inability to distinguish between immunized animals and infected animals, etc.

Active Publication Date: 2005-03-09
SHANGHAI HUAYI BIO LAB CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the protective effect of polypeptide vaccines is lower than that of whole virus vaccines. For example, the amino acids of 141-160 of VP1 protein are synthesized, and the immunogenicity of the prepared polypeptide vaccines is 100-1000 times lower than that of the corresponding amount of inactivated FMDV vaccines.
Moreover, the current FMDV vaccine, whether it is an inactivated FMDV vaccine or a peptide vaccine, cannot distinguish between immunized animals and infected animals through serological experiments, and confusion between immunized animals and infected animals will cause great damage to the market. influences

Method used

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  • Foot and mouth disease bivalent polypeptide vaccine and its preparation method and use
  • Foot and mouth disease bivalent polypeptide vaccine and its preparation method and use
  • Foot and mouth disease bivalent polypeptide vaccine and its preparation method and use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0086] Construction of Plasmid pUC8 / F1(+) Encoding Oriented Type O and Type A Epitopes

[0087] According to type O and type A foot-and-mouth disease virus VP 1 The 141-160 amino acid sequence of the protein was synthesized into four oligonucleotide fragments, which were ligated and cloned into the vector to form a recombinant plasmid pUC8 / F1(+) containing O-type and A-type antigenic determinants arranged in a positive direction.

[0088] 1. Preparation of Oligonucleotide Fragments

[0089] According to type O and type A foot-and-mouth disease virus VP 1 For the 141-160 amino acid sequence of the protein, the following oligonucleotide fragments were designed and synthesized by Saibaisheng Company.

[0090] P1: 5′-AAT TCC ATG AGA TCT GGT TCT GGT GTT CGT CGT

[0091] GAT TTC GGT TCT CTG GCG CCG CGT GTT GCG CGT CAG CTG A-3′

[0092] P2: 5′-T GTT GGT CAG CTG ACG CGC AAC ACG CGG CGC

[0093] CAG AGA ACC GAA ATC ACG ACG AAC ACC AGA ACC AGA TCT CAT GG-3′

[0094] P3: 5′-CC AAC ...

Embodiment 2

[0113] Construction of Plasmid pUC8 / F1(-) Encoding O-type and A-type Epitopes in Reverse Arrangement

[0114] According to type O and type A foot-and-mouth disease virus VP 1 The 141-160 amino acid sequence of the protein was synthesized into four oligonucleotide fragments, which were cloned into the vector after ligation to form a recombinant plasmid pUC8 / F1(-) containing reversely arranged O-type and A-type antigenic determinants.

[0115] According to type O and type A foot-and-mouth disease virus VP 1 For the 141-160 amino acid sequence of the protein, the following oligonucleotide fragments were designed and synthesized by Beijing Saibaisheng Company.

[0116] P1': 5'-AAT TCC AGA TCT CCG CTG ACC CGT GAA GCG AAA CAG GC G

[0117] CTG GTT CAG CTG GAT GGT CGT GTT AAC AAC A-3′

[0118] P2': 5'-G CAG GGT GTT GTT AAC ACG ACC ATC CAG CTG AAC CAG CGC

[0119] CTG TTT CGC TTC ACG GGT CAG CGG AGA TCT GG-3′

[0120] P3': 5'-CC CTG CAG CGT GCG GTT CGT CCG GCG CTG TCT GGT TTC

...

Embodiment 3

[0126] Embodiment 3, concatenation and cloning of genes

[0127] As shown in Figure 4, pUC8 / F1(+) or pUC8 / F1(-) (hereinafter referred to as pUC8 / F1) was double digested with restriction endonucleases BamHI and HindIII to recover large fragments; Dicer BglII and HindIII performed double digestion on the gene monomer pUC8 / F1, and recovered small fragments.

[0128] Since the cleavage site of BglII is A↓GATC↑T, a cohesive end A or GATCT is obtained after digestion with BglII; while the cleavage site of BamHI is G↓GATC↑C, a sticky end is obtained after digestion with BamHI Terminal G or GATCC. After the two enzymes digest, the nicks produced are exactly complementary pairings.

[0129] The recovered two fragments were ligated with T4 ligase. The ligation product was transformed into JM103 competent cells, and the plasmid DNA of the resulting transformant was identified, and two monomer tandem connectors pUC8 / F2 were obtained, which contained two O-type FMDV and A-type FMDV in f...

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Abstract

A bivalence polypeptide vaccine for foot-and-mouth disease contains 2 to the power n-1 polypeptides coded by the nucleic acid sequences shown by SEQ ID No.1 or 2, where n=1-5. Its preparing process is also disclosed.

Description

Field of Invention [0001] The present invention relates to a novel foot-and-mouth disease vaccine, in particular to a bivalent polypeptide vaccine produced by a genetic engineering method, and a preparation method and application thereof. Background technique [0002] Foot-and-mouth disease (FMD) is a highly contagious disease in animal husbandry, and cloven-hoofed animals such as cattle, pigs, sheep, and camels are susceptible to infection. So far, all countries in the world except North America and Australia have experienced large-scale epidemics of foot-and-mouth disease. The United Kingdom (1967-68), Austria (1973), France (1974), South Korea (2002) and other countries caused huge economic losses. [0003] The causative agent of foot-and-mouth disease is a picornavirus, foot-and-mouth disease virus (FMDV). The virus particle contains a positive-strand polar single-stranded RNA consisting of 8500 nucleotides. The envelope of the virus particle includes four structural ...

Claims

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Application Information

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IPC IPC(8): A61K39/00A61K39/135A61K39/295A61P31/12A61P31/14C07K14/09C12N15/42
CPCA61K39/00C12N2770/32134A61K39/12A61K39/295C07K14/005C12N2770/32122A61K2039/552A61K2039/55505A61K2039/55566A61K2039/70Y02P20/582A61P31/12A61P31/14
Inventor 孙玉琨顾薇玲伍登熙
Owner SHANGHAI HUAYI BIO LAB CO LTD
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