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Method for preparing foot-and-mouth disease antigen

A foot-and-mouth disease and antigen technology, applied in the field of genetic engineering, can solve the problems of high production cost, virus escape, short immune period, etc., and achieve the effects of low cost, reduced production cost and high safety.

Active Publication Date: 2008-02-13
LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although traditional vaccines still play a dominant role in the prevention and control of foot-and-mouth disease, the production costs are high, the immunization period is short, and risk factors such as virus escape during vaccine preparation are difficult to avoid

Method used

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  • Method for preparing foot-and-mouth disease antigen
  • Method for preparing foot-and-mouth disease antigen
  • Method for preparing foot-and-mouth disease antigen

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Preparation of embodiment 1 foot-and-mouth disease antigen, purification and animal immunization experiment and virus challenge protection experiment

[0033] 1. Cloning and sequence analysis of P1-2A and 3C genes

[0034] 1.1 Obtaining the target gene

[0035] Primers were designed to amplify the FMD virus antigenic protein P1-2A gene and non-structural protein 3C gene by RT-PCR.

[0036] The amplification primers of the designed antigenic protein P1-2A gene and nonstructural protein 3C gene are,

[0037] Upstream of P1-2A: 5'-ATA GGATCC ACCATGGGAGCCGGGCAATCCAGCC-3',

[0038] BamH I restriction site

[0039] Downstream of P1-2A: 5'-CGC GAATTC TGACATGTCCTCCTGCATCTGGTTG-3'.

[0040] EcoR I restriction site

[0041] 3C upstream: 5'-GCG GAATTC AAGAAACCTGTCGCTTTGAAAGT-3'.

[0042] EcoR I restriction site

[0043] Downstream of 3C: 5'-ATA AGATCT CTACTCGTGGTGTGGTTCGGGAT-3'

[0044] Bgl II restriction site

[0045] Total RNA was extracted from FMDV cell culture...

Embodiment 2

[0099] Preparation of embodiment 2 foot-and-mouth disease antigen, purification and animal immunization experiment and virus challenge protection experiment

[0100] 1. Cloning and sequence analysis of the full-length ORF of foot-and-mouth disease virus

[0101]Primers were designed to amplify the full-length ORF of foot and mouth disease virus (SEQ ID NO: 3) by RT-PCR.

[0102] The designed amplification primers for amplifying the full-length ORF are,

[0103] ORF: 5'-GCG ACTAGT ACCATGGAATTCACACTTCACAACGGTGAG-3',

[0104] Spe I restriction site

[0105] ORF: 5'-ATA GCGGCCGC AGGGATTATGCGTCACCGCACAC-3'.

[0106] Not I restriction site

[0107] Total RNA was extracted from FMDV cell culture medium. The extracted total RNA was treated with Oligo(dT) 18 Under the action of AMV reverse transcriptase, the primers were reverse-transcribed at 42°C to prepare cDNA. The obtained cDNA was used as a template, and PCR amplification was carried out with specific primers.

[0108...

Embodiment 3

[0135] Preparation of embodiment 3 foot-and-mouth disease antigen, purification and animal immunization experiment and virus challenge protection experiment

[0136] 1. Cloning and sequence analysis of FMDV VP1 gene

[0137] Primers were designed to amplify the foot disease virus VP1 gene by RT-PCR.

[0138] The designed amplification primers for amplifying the VP1 gene are,

[0139] VP1 upstream: 5'-ATA GGATCC ACCATGGCCACCACTACCGGCGAGTCAG-3',

[0140] BamH I restriction site

[0141] Downstream of VP1: 5'-CGC GAATTC TTACACCATCTGCTTTCCAGGTGCAAT-3'.

[0142] EcoR I restriction site

[0143] Total RNA was extracted from FMDV cell culture medium. The extracted total RNA was treated with Oligo(dT) 18 Under the action of AMV reverse transcriptase, the primers were reverse-transcribed at 42°C to prepare cDNA. The obtained cDNA was used as a template, and PCR amplification was carried out with specific primers.

[0144] The PCR reaction system is as follows:

[0145] Tabl...

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Abstract

The invention provides a method for expressing foot-and-mouth disease antigens in insects using recombinant baculoviruses, which includes: cloning different gene combinations of foot-and-mouth disease into baculovirus delivery vectors to construct transfer vectors; using the constructed transfer vectors to transfer Infect the baculovirus and perform DNA recombination to obtain the recombinant baculovirus; infect the insect host with the recombinant baculovirus; culture the infected insect host to express the foot-and-mouth disease antigen; collect and purify the expressed foot-and-mouth disease antigen. The method of the present invention uses a baculovirus expression system to safely and efficiently produce foot-and-mouth disease antigens in a silkworm bioreactor. The prepared antigens are extremely safe and can directly produce vaccines to immunize animals. The method of preparing foot-and-mouth disease antigen of the present invention does not require investment in building a factory, has no three wastes, consumes very little energy such as electricity and water resources, and its production cost is also significantly lower than the traditional method of preparing foot-and-mouth disease antigen. It is safe, efficient, has low energy consumption and low cost. Low and many advantages.

Description

technical field [0001] The invention relates to a method for preparing an antigen, in particular to a method for expressing a foot-and-mouth disease antigen in an insect body by using a recombinant baculovirus, and belongs to the field of genetic engineering. Background technique [0002] Foot-and-mouth disease is an acute, highly contagious, febrile infectious disease caused by foot-and-mouth disease virus (FMDV) in artiodactyls. It is known for its rapid spread and high infection rate. Once the disease breaks out, it will cause huge economic losses to the affected country. Countries all over the world attach great importance to the research of this disease, and the International Veterinary Bureau ranks it as the first class A infectious disease. Foot-and-mouth disease is a member of the genus Foot-and-Mouth Virus in the family Picornaviridae, and has seven serotypes: A, O, C, Asia I, SA T1, SA T2 and SA T3. At present, the prevention and control of foot-and-mouth disease ...

Claims

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Application Information

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IPC IPC(8): C12N15/42C12N15/866C12N7/01C07K14/09
CPCC12N2770/32122C12N2710/14143A61K39/00C07K14/005A61P31/12A61P31/14A61K39/135C07K14/09C12N15/866
Inventor 柳纪省张志芳李志勇易咏竹殷相平白银梅李轶女李宝玉李学瑞杨彬兰喜沈桂芳
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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