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 cost

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

A method for expressing antigens of foot-and-mouth disease virus in insect in vivo by using re-combinant baculovirus is disclosed, which comprises: cloning different genes combination of foot-and-mouth disease virus into baculovirus carrying vector to construct transfer vector, transfecting baculovirus with the transfer vector to proceed DNA recombination, and thus to get recombinant baculovirus, infecting insect host with the recombinant baculovirus, culturing the infected insect host to express the antigens of foot-and-mouth disease virus, and then collecting and purifying the expressed antigens. The preferred genes, baculovirus carrying vector, baculovirus and insect host are selected from P1-2A3C, ORF and VP1 genes of foot-and-mouth disease virus as shown in SEQ ID NO 1., 3 or 5, pVL1393, Bombyx mori Bm-NPV-ZJ8 and larvae or pupa of Bombyx mori, respectively.

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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