EryA gene deleted strain of brucella Rev.1, construction method and application thereof

A brucella and gene deletion technology, applied in the field of genetic engineering, can solve the problems of inability to purify brucellosis, inability to distinguish natural infection from artificial immunity, etc.

Inactive Publication Date: 2020-12-18
JINYUBAOLING BIO PHARMA CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Brucella Rev.1 vaccine (B.melitensis Rev.1 vaccine) has been proved to have a good protective effect in goats, but the vaccine cannot distinguish natural infection (Brucella wild strains) and artificial immunization (Brucella vaccine strains), and thus cannot effectively decontaminate Brucellosis

Method used

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  • EryA gene deleted strain of brucella Rev.1, construction method and application thereof
  • EryA gene deleted strain of brucella Rev.1, construction method and application thereof
  • EryA gene deleted strain of brucella Rev.1, construction method and application thereof

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Experimental program
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Effect test

Embodiment 1

[0046] Embodiment 1: the construction of the eryA gene deletion strain of Brucella Rev.1

[0047]This embodiment aims at constructing the eryA gene deletion strain of Brucella Rev.1 by genetic engineering technology, specifically comprises the following steps:

[0048] 1.1. PCR amplification to obtain the upstream and downstream homology arms of the eryA gene and the suicide gene SacB

[0049] Using the genome of Brucella melis Rev.1 strain (extracted by a genome extraction kit) as a template, use the high-fidelity enzyme (GXL) and the primers listed in Table 1 below to target the eryA gene and its upstream and downstream homologs, respectively. Arms (named eryA-1 and eryA-2, respectively) were amplified by PCR to obtain PCR amplifications of the eryA gene, the upstream homology arm of the eryA gene (eryA-1) and the downstream homology arm of the eryA gene (eryA-2). increase product. Using the plasmid pre112 as a template, the suicide gene SacB was amplified using the primer...

Embodiment 2

[0069] Embodiment 2: Physiological and biochemical characteristics and growth characteristic identification of the eryA gene deletion strain of Brucella Rev.1

[0070] This embodiment aims to identify the physiological and biochemical characteristics and growth characteristics of the eryA gene deletion strain (Rev.1-ΔeryA) of the Brucella Rev.1 constructed in the above-mentioned embodiment 1, and compare with the original Brucella Rev. 1 (Rev.1) wild strain, Brucella A19 (vaccine strain), S19 (vaccine strain, missing 720bp fragment relative to A19), S2 (vaccine strain) (preserved by Jinyu Baoling Biological Pharmaceutical Co., Ltd.) The comparison specifically includes the following steps:

[0071] 2.1 Morphology and biochemical characteristics

[0072] The eryA gene deletion strain of Brucella Rev.1 and other Brucella strains (Rev.1, A19, S19, S2) were observed by Gram staining and Kovik staining respectively, and prepared with lead acetate Use an inoculation loop to inocul...

Embodiment 3

[0082] Embodiment 3: the distinction method establishment of Brucella wild strain and Brucella Rev.1-ΔeryA vaccine strain

[0083] eryA is a conserved gene of Brucella, and the homology of the gene among the strains is as high as 99% through sequence comparison. Therefore, the deletion strain vaccine of this gene can be used to effectively distinguish the wild strain of Brucella from the vaccine strain , including the following steps:

[0084] The genomes of Brucella S2, A19, S19, M5, M28, Rev.1 (all preserved by Jinyu Baoling Biopharmaceutical Co., Ltd.) and Rev.1-ΔeryA (extracted using a genome extraction kit) were used as templates , using the eryA-F and eryA-R primers listed in Table 1 above for PCR amplification, the reaction system is: Mix 12.5μL, ddH 2 O 9.5 μL, upstream primer (eryA-F) 1 μL, downstream primer (eryA-R) 1 μL, DNA template 1 μL. The reaction conditions were: denaturation at 95°C for 3 min; denaturation at 94°C for 45 s, 35 cycles, annealing at 55°C for ...

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Abstract

The invention discloses an eryA gene deleted strain of brucella Rev.1, a construction method and an application thereof, and belongs to the technical field of gene engineering. Compared with a wild brucella strain, the eryA gene deleted strain provided by the invention has the advantage that all eryA genes are deleted. When the eryA gene deleted strain is used as a brucella vaccine strain, the eryA gene can be detected to distinguish the wild brucella strain from the eryA gene deleted strain, so that natural infection with brucella can be distinguished from artificial immunization, thereby effectively purifying brucellosis. According to the construction method of the eryA gene deleted strain, PCR identification and single colony sequencing verification are adopted to obtain the pure eryA gene deleted strain of brucella Rev.1 with deletion of the eryA genes in high reliability.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a eryA gene deletion strain of Brucella Rev.1 and a construction method and application thereof. Background technique [0002] Brucellosis is a zoonotic infectious disease caused by Brucella (B.melitensis). Brucella infection of domestic animals can cause miscarriage in dams and other symptoms (such as metritis, orchitis, and mastitis). After human infection with the bacteria, the joints swell, and the symptoms of Malta fever appear and are not easy to recover. The main hosts of Brucella are sheep, cattle, pigs and so on. [0003] Brucella Rev.1 vaccine (B.melitensis Rev.1 vaccine) has been proved to have a good protective effect in goats, but the vaccine cannot distinguish natural infection (Brucella wild strains) and artificial immunization (brucella vaccine strains), and thus cannot effectively purify brucellosis. Contents of the invention [0004]...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/74C12N15/31A61K39/10A61P31/04C12R1/01
CPCC07K14/23C12N15/74A61K39/098A61P31/04A61K2039/552
Inventor 宋前进刘建奇赵丹彤吴梅花白白音宝力高关平原温永俊
Owner JINYUBAOLING BIO PHARMA CO LTD
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