Construction of brucellosis A19 molecular marker vaccine strain and determination of virulence and immunogenicity

A molecular marker vaccine, brucellosis technology, applied in the determination/examination of microorganisms, bacteria, recombinant DNA technology, etc. The effect of weak strength, improved vaccine function, and short market cycle

Active Publication Date: 2012-11-14
新疆维吾尔自治区畜牧科学院兽医研究所
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, the VirB12 gene is a virulence factor in the four-type secretion system of Brucella. The virulence of the A19 vaccine strain with VirB12 knockout has been weakened, which improves the safety of the brucellosis vaccine, but it is still Maintain the immunogenicity and biological characteristics of the original vaccine

Method used

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  • Construction of brucellosis A19 molecular marker vaccine strain and determination of virulence and immunogenicity
  • Construction of brucellosis A19 molecular marker vaccine strain and determination of virulence and immunogenicity
  • Construction of brucellosis A19 molecular marker vaccine strain and determination of virulence and immunogenicity

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Construction of recombinant suicide plasmid

[0036] Materials and Methods

[0037] Strains, plasmids, vectors

[0038] The Brucella A19 strain was purchased from the China Veterinary Drug Administration, and the suicide plasmid pBK-CMV-sacB was preserved by the Veterinary Research Department of the Xinjiang Academy of Animal Sciences; the cloned pGEM-T vector was purchased from Promega, a commercial reagent.

[0039] Reagent

[0040] Restriction enzymes were purchased from Fermentas; plasmid DNA extraction kit and DNA gel recovery kit were purchased from Promega; T4DNA ligase, DNA Marker, and DH5α were purchased from Beijing Zhuangmeng Biological Company; sequence determination was provided by Shanghai Invitrogen Trading Co., Ltd. Ltd. completed.

[0041] Primers were designed according to the GenBankAF141604 sequence, as shown in Table 1:

[0042] Table 1 Primer Sequence

[0043]

[0044] PCR amplification

[0045] Amplification of Homology Arm Fragment Upstrea...

Embodiment 2

[0068] Described is the determination of the virulence and immunogenicity of the brucellosis A19 molecular marker vaccine strain.

[0069] Characteristics of A19 molecular marker strain:

[0070] Experimental animals were 4-6 week old BALB / C female mice purchased from Xinjiang Experimental Animal Research Center, license number: SCXK (new) 2003-0002.

[0071] Safety test: Dilute A19-ΔVirB12 viable bacteria with normal saline to contain 1 billion viable bacteria per 1ml, subcutaneously inject 5 BABL / C mice with a body weight of 18-20g, each 0.25ml, and all live healthy within 10 days.

[0072] Genetic Stability:

[0073] Passage stability: Streak culture of the frozen A19-ΔVirB12 molecular marker strain on a tryptone soybean broth medium plate at 37°C with 5% CO 2 Cultured in the incubator for 72 hours, the grown colony was the first-class seed, and the first-class seed was continuously passed on the medium for 50 generations, and the colonies of the passaged bacteria were pi...

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Abstract

The invention relates to construction of a brucellosis A19 molecular marker vaccine strain and determination of virulence and immunogenicity. According to the invention, a suicide-plasmid-mediated homologous recombination technology is utilized to knock out a virulence factor VirB12 gene of a brucellosis tetratype secretory system of a brucellosis A19 vaccine strain, thus obtaining the A19-delta VirB12 molecular marker vaccine strain. Animal experiments show that the virulence of the A19 molecular marker vaccine strain is slightly weaker than that of a parent A19 vaccine strain, so that the strain safety is further improved, but the good immune effect on brucellosis is maintained; and moreover, the molecular marker vaccine strain has stable heredity. According to the invention, the brucellosis VirB12 gene is taken as a molecular marker, a polymerase chain reaction (PCR) method is applied to identify the brucellosis vaccine strain from wild viral strains, so that the molecular marker is provided for building a determination method for identifying vaccine immunization and natural infection, and the function of the original brucellosis A19 vaccine is improved, therefore, the brucellosis A19 molecular marker vaccine strain has an very important practical meaning on controlling the epidemic brucellosis, and has wide application prospect.

Description

technical field [0001] The invention relates to the construction of a brucellosis A19 molecular marker vaccine strain and the determination of virulence and immunogenicity, and belongs to the technical field of microbial bacterial genetic engineering. Background technique [0002] Brucellosis (brucellosis, referred to as brucellosis) is a zoonotic infectious disease mainly infecting domestic animals caused by Brucella. Brucella has the characteristics of wide range of hosts, strong infectivity and difficult cure after infection, which poses a serious threat to animal husbandry and human health. [0003] Vaccine immunization is the main measure to prevent and control brucellosis. At present, the A19 vaccine used to immunize cattle against brucellosis has relatively good immunity. This vaccine has been used in my country for more than 60 years, providing an important guarantee for the effective control of brucellosis, but it also has certain defects. . That is, the current A...

Claims

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Application Information

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IPC IPC(8): C12N15/74C12N1/21C12Q1/68A61K49/00C12R1/01
Inventor 钟旗易新萍王力俭叶锋闫广谋刘丽娅吐尔洪·努尔马晓菁谷文喜姚刚范伟兴
Owner 新疆维吾尔自治区畜牧科学院兽医研究所
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