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Classical swine fever virus E2 subunit vaccine and preparation thereof

A technology of swine fever virus and swine fever vaccine, applied in the direction of viral peptides, antiviral agents, botany equipment and methods, etc., can solve the problems of cost burden and so on

Active Publication Date: 2013-06-05
黄金城
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, the E2 sub-unit vaccines developed by the Netherlands and Germany are produced by cell culture methods, and the import price is more than NT$30, which poses a considerable burden on the cost of the breeders.

Method used

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  • Classical swine fever virus E2 subunit vaccine and preparation thereof
  • Classical swine fever virus E2 subunit vaccine and preparation thereof
  • Classical swine fever virus E2 subunit vaccine and preparation thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1: Construction of a plasmid comprising the E2 subunit recombination gene

[0040] Amplification of E1E2TM gene of Subgroup 2.1 virus

[0041] First, use reverse transcription polymerase superscript II reverse transcriptase (Invitrogen, CA, USA) to carry out reverse transcription polymerase chain reaction, and use two E1-E2 gene-specific primers (as shown in Table 1), from TD / 96 / TWN type The swine fever virus gene body amplifies the E1-E2 gene sequence 2354-3466bp (SEQ ID NO: 2), wherein the nucleotide sequence includes a part of the N-terminal 60 nucleotide sequence of the E1 gene and the C-terminal to the TM region of the E2 gene (E1E2TM) 1026 nucleotides, a total of 1086 nucleotides. Then, the second reverse transcription polymerase chain reaction uses the first reaction product, and the reverse primer (5'-TTGAG CTCAC CATGT TGAGA GGACA GGT-3') (SEQ ID NO: 5) to reverse transcribe into cDNA, and finally The amount of product was amplified by polymerase chain...

Embodiment 2

[0046] Example 2: Homologous recombination

[0047] After 5 μg of the plasmid pBpE2-his that has been cloned and 50 μg of the semi-purified gene body of baculovirus were mixed using 200 μl of commercial Liposome (Invitrogen, USA), 1 ml of TC-100 culture medium without serum components was added, and A monolayer of silkworm cells (BmN) grown on a 30mm culture dish was added to allow co-transfection (Transfection) into the cells, and a recombination reaction naturally occurred in the cells. After about 4-7 days, anti-histidine monoclonal antibody (Invitrogen, USA) or anti-E2 monoclonal antibody (WH303, Veterinary Laboratory Agency, UK) can be used to dilute 200 times and then stained to screen out virus plaques with expressive ability. After continuous 10-fold dilution and infection of cells for three times, the virus with the highest potency was used as the seed virus strain after a large number of subclone colonization to determine the potency.

Embodiment 3

[0048] Embodiment 3: Taking silkworm as protein production platform

[0049] The selected recombinant baculovirus can infect the silkworm and express the E2 protein. The silkworm is a hybrid of the original species (OJ03×OJ04). The silkworm eggs of the second species are used for feeding. Generally, when the silkworm is reared to the fifth instar about the 23rd day, 10 μl of virus liquid (10 μl) can be injected at the third back hole. 7.0 TCID 50 / ml), about 3-5 days after infection, the body fluid can be collected when the silkworm shows the onset, and the concentration of E2 protein can be detected. About 0.3-0.5ml of body fluid can be extracted from each silkworm, and its body fluid is analyzed for antigen concentration by western blot method.

[0050] Take 20 μl of body fluid, use 4-12% SDS-PAGE gradient gel electrophoresis analysis, then transfer the protein to nitrocellulose membrane, and then fill with 5% non-fat milk powder (blocking), transfer the protein Add 20 μ...

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Abstract

The invention relates to a recombinant E2 protein by using silkworm as a platform to produce a classical swine fever virus gene subgroup 2.1. The invention also provides a subunit vaccine comprising the recombinant protein, which can be used for enhancing the capability of swine to resist classical swine fever virus infection.

Description

technical field [0001] The invention relates to a swine fever vaccine and a preparation method thereof. Background technique [0002] Swine fever is one of the most serious diseases facing the swine industry in Taiwan and parts of the world. The natural hosts of CSFV are domesticated pigs and wild boars, and the virus is susceptible and harmful to pigs of any age. At present, except for a few special-purpose pig farms that do not use vaccines for prevention and control and European and American countries adopt culling policies, almost all pigs must be vaccinated at least twice during the growth process to avoid the harm of classical swine fever. At present, the annual production of pigs in Taiwan is still more than 6 million, and the production in Southeast Asia, China and South America is dozens of times more than this. Therefore, subunit vaccines with protective effects have very high economic value for this market. [0003] The most widely used swine fever vaccine in th...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/40C12N15/866C07K14/08A61K39/187A61P31/14
Inventor 黄金城赵裕展吴登桢邓明中林育如林碧秀廖久熏
Owner 黄金城
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