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Cattle Brucella recombination strain S19-delta bp 26-BL and preparation method and application thereof

A technology of Brucella bovis, bp26-bl, which is applied in the field of recombinant bacterial vaccines, can solve problems such as economic impact in pastoral areas, animal side effects, and international trade obstacles, and achieve good immunogenicity, enhanced control effects, and good genetic stability Effect

Active Publication Date: 2013-09-11
SOUTH CHINA AGRI UNIV
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Problems solved by technology

Many countries and regions restrict the import and export of meat and dairy products from animals that have tested positive for Brucella serology, which has seriously hindered international trade, especially in pastoral areas.
In addition, the existing attenuated live vaccines against Brucella all have different degrees of side effects on animals, causing mild abortion in animals and infecting humans at the same time

Method used

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  • Cattle Brucella recombination strain S19-delta bp 26-BL and preparation method and application thereof
  • Cattle Brucella recombination strain S19-delta bp 26-BL and preparation method and application thereof
  • Cattle Brucella recombination strain S19-delta bp 26-BL and preparation method and application thereof

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Embodiment 1

[0056] (1) Construction of suicide homologous recombination plasmid

[0057] ① Extraction of bacterial genomic DNA: Genomic DNA of Brucella bovis S19 strain was extracted according to the instructions of the QIAamp DNA extraction kit.

[0058] ② Design and synthesis of primers: refer to the gene sequence of Brucella S19 strain (NC-010742) in Genbank, and design primers according to the requirements of the experiment, as shown in Table 1. Among them, primers P1 / P2 and P3 / P4 are used to amplify the upper and lower homology arms of the bp26 gene, respectively, and introduce KpnI and ScaI restriction sites at the 5' ends of primers P1 and P4 respectively; primers B1 / B2 are used to To amplify the BLS gene; primers B3 / B4 are used to amplify the L7 / L12 gene. The above primers were synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd.

[0059] Table 1 PCR primer sequences used to amplify different gene fragments

[0060]

[0061] ③PCR amplification of differ...

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Abstract

The invention discloses a cattle Brucella recombination strain S19-delta bp 26-BL and preparation method and application thereof. According to the invention, a cattle Brucella S19 genome DNA is used as a template, an upstream homologous arm of a bp26 genome is obtained, and a downstream homologous arm of a BLS gene, a L7 / L12 gene and a bp 26 gene is obtained; the gene fragments are connected in sequence, and a fragment delta-bp 26-BL is obtained; a pRE112 and the fragment delta-bp 26-BL are separately subjected to a double digestion and are connected, and a suicide homologous recombinant plasmid is obtained; the plasmid is introduced into a competent cell of a S19-delta bp 26 bacterial strain, in the selection pressure of chloramphenicol, and a homologous recombinant single recon is obtained, and then the homologous recombinant single recon is coated on a selection medium for culture, and the cattle Brucella recombination strain S19-delta bp 26-BL is obtained. The strain has the advantages of good heredity stability, growth characteristic and appropriate virulence, and can be used as a mark vaccine strain for clinic.

Description

technical field [0001] The present invention relates to the field of recombinant bacterial vaccines, in particular to a Brucella abortus recombinant strain S19-Δbp26-BL and its preparation method and application. The main immunogenic genes BLS and L7 / L12 were obtained. Background technique [0002] Brucellosis is a zoonotic infectious disease caused by Brucella. The prevention and control of this disease has always been based on the application of vaccines. There are many types of Brucella vaccines, recombinant protein vaccines and DNA vaccines have limited protection, and inactivated vaccines have a short protection period. At present, the attenuated strains used for the prevention and control of brucellosis mainly include foreign bovine type 19 attenuated strain (S19) and sheep type ReV. Type 2 (S2) attenuated strain. Attenuated vaccines are cheap to manufacture, have a long duration of immune protection, and have high immune protection efficiency, but these attenuated ...

Claims

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Application Information

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IPC IPC(8): C12N15/09C12N1/21A61K39/10A61P31/04C12R1/01
Inventor 陈金顶王佳莹常艳赵明秋吴云燕
Owner SOUTH CHINA AGRI UNIV
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