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Method for preparing swine fever recombinant subunit vaccine

A subunit vaccine, swine fever technology, applied in the fields of botanical equipment and methods, biochemical equipment and methods, recombinant DNA technology, etc., can solve the problem of limited number of antigenic epitopes and affecting immune effect.

Inactive Publication Date: 2009-04-29
LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

However, the number of epitopes of a single E2 gene is limited, which affects the immune effect

Method used

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  • Method for preparing swine fever recombinant subunit vaccine
  • Method for preparing swine fever recombinant subunit vaccine
  • Method for preparing swine fever recombinant subunit vaccine

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Embodiment 1

[0031] 1. Cloning of E2 gene antigen region and construction of recombinant expression vector pCSFV-rE2

[0032] According to the method for amplifying the CSFV E2 gene, two pairs of specific primers containing restriction enzyme sites were designed, as shown in Table 1. The sequence span of a single E2 gene is 569 bp, and after concatenation, it is 1138 bp.

[0033] Table 1: Primer number, sequence, position, length

[0034]

[0035]

[0036] Note: Primer E2-2F: GAATCC Restriction site sequence; GGC...TTC amino acid linker sequence; ATG...GAT is the original sequence of E2 gene; RBS sequence

[0037] The total RNA of the vaccine strain (C-strain) is extracted, and the main antigen region fragment of the E2 gene is amplified by RT-PCR. Double-digest the pGEX-6p-1 vector and the purified E2 fragment, connect the two genes using the common EcoR I restriction site, construct a recombinant expression vector, and transform it into DH5α competent cells. The construction st...

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Abstract

The invention relates to a method for preparing a recombining subunit vaccine of swine fever. The method comprises the following steps: A. a reverse transcription-polymerase chain type reaction method is utilized to clone a coding gene of vaccine strain E2 protein of the swine fever; B. a E2 gene is constructed and fused; C. a recombining expression vector rE2 is constructed; D. rE2 induction expression is formed; E. the rE2 is purified; and F. the activity of the rE2 is detected through a Western blotting method. The method creatively uses a double-E2 gene serial expression mode in order that the number of E2 epitope is doubled, thereby remarkably improving the immune effect of the recombining E2 subunit vaccine; to improve the expression efficiency of double E2 protein, a colibacillus RBS sequence (5'-AAGGAG-3') is added on the upstream of an initial codon (ATG) of 5'end of the second E2 gene; at the same time, an amino acid joint 5'-GSA GSA AGS GEF-3' is added between E2 genes in serial connection so that the obtained E2 protein can completely display the epitope. The E2 protein recombining subunit vaccine has equivalent effect of immunoprotection with the lapinised attenuated vaccine of the swine fever.

Description

technical field [0001] The invention relates to the field of research and development of swine fever vaccine, in particular to a preparation method of a swine fever recombinant subunit vaccine that can be used in clinical work as a novel vaccine for the prevention and treatment of swine fever. Background technique [0002] Classical Swine Fever (CSF) is a severe, viral infectious disease of pigs caused by Classical Swine Fever Virus (CSFV), and is listed as a Class A infection by the International Office of Animal Epidemics (OIE) and the my country Veterinary Bureau. Its symptoms are particularly severe, it spreads rapidly, and it knows no borders. The outbreak of swine fever often causes huge economic losses and causes serious public health problems. [0003] In the late 1970s, the epidemic characteristics of swine fever changed greatly. Persistent infection caused the emergence of atypical swine fever, and immune failures occurred from time to time, which constantly threate...

Claims

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Application Information

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IPC IPC(8): C12N15/40C12N15/70C07K14/08A61K39/187A61P31/14
Inventor 刘湘涛尹双辉孙世琪田宏尚佑军韩雪清刘艳红
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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