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Rough type brucella of recombinant fine-granule echinococcus Eg95 gene and vaccine production method thereof

A technology of Echinococcus granulosus and Brucella, applied in the field of Brucella rougha and its vaccine production, can solve the problems of animal reproduction and production performance hazards, difficult to cure, etc., and achieve good protection and immunogenicity Sexuality, reduce labor costs, and solve the effect of inefficiency

Active Publication Date: 2019-07-16
CHINA INST OF VETERINARY DRUG CONTROL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This disease not only has serious harm to the reproduction and production performance of animals, but more importantly, after people are infected with Brucella, it is often difficult to cure, thus causing serious public health problems.

Method used

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  • Rough type brucella of recombinant fine-granule echinococcus Eg95 gene and vaccine production method thereof
  • Rough type brucella of recombinant fine-granule echinococcus Eg95 gene and vaccine production method thereof
  • Rough type brucella of recombinant fine-granule echinococcus Eg95 gene and vaccine production method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] According to the analysis of the whole genome and gene function prediction of Brucella RA343 (CGMCC No.8886, ZL201410240895.6), three molecular chaperone promoters P1, P2, P3, and respectively insert the Brucella promoter P 1 ,P 2 ,P 3 And common promoters tac, trc, T7, construct six green fluorescent plasmids carrying different promoters P 1 -GFP-pZL1790,P 2 -GFP-pZL1790,P 3 - GFP-pZL1790, tac-GFP-pZL1790, trc-GFP-pZL1790, and T7-GFP-pZL1790. They were electrotransferred into the Brucella RA343 strain, and the suitable strong promoter P for exogenous gene expression was selected by the relative expression level of green fluorescent protein. 3 .

[0032] (2) By adding P 3 The promoter and Eg95 sequence were codon-optimized, the gene fragment was artificially synthesized, and a P 3 -Eg95 sequence and the sucrose suicide plasmid vector pUC / P of Brucella upstream and downstream homology arms 3 -Eg95EG95 + , part of the Echinococcus granulosus Eg95 gene expression...

Embodiment

[0047] The following examples are to further illustrate the present invention, but not to limit the present invention.

[0048] Embodiment 1 - the construction of recombinant Brucella RA343-Eg95 strain

[0049] 1. Preparation of Brucella RA343 Competent Cells

[0050] Pick a single inoculated colony of Brucella RA343 strain, inoculate it with 100ml TSB medium and cultivate it until the logarithmic phase of bacterial growth, and cool it in ice water. Centrifuge at 12000r / min for 10min, discard the liquid medium, and wash repeatedly several times with different volumes of sterile deionized water. Finally, the obtained bacteria were resuspended in 1 ml of 10% glycerol aqueous solution, and the prepared parent strain RA343 infected bacteria were stored at -80°C for future use.

[0051] 2. Screening of promoters for exogenous gene expression in Brucella RA343

[0052] (1) Construction of green fluorescent plasmid GFP-pZL1790

[0053] The pZL1790 plasmid is preserved by our labo...

Embodiment 2

[0101] Embodiment 2——The routine biological characteristic of recombinant Brucella RA343-Eg95 strain

[0102] 1. Morphological and biochemical properties

[0103] The colonies of recombinant bacteria have neat, rounded edges, dew drop shape, irradiated by oblique light, and slightly blue opalescent in backlight observation. The staining form is cocci, scattered individually, without forming spores and capsules. The size is between 0.3 and 0.6 μm. Gram stain was negative. The growth of the bacteria is independent of CO 2 , can grow on the medium containing thionine and basic fuchsin, H 2 S test was strongly positive.

[0104] 2. Thermal agglutination test

[0105]Inoculate the recombinant strain in TSA medium, culture at 37°C for 46 hours, then take the culture and put it into a test tube filled with about 100ml of normal saline, shake it well, and make it turbid to 1 billion / ml of viable bacteria. Suspension, the taken out bacterial suspension was divided into 2 tubes, ...

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Abstract

The invention relates to construction of rough type brucella of a recombinant fine-granule echinococcus Eg95 gene and a vaccine of the rough type Brucella. A rough type cattle-species Brucella low virulent strain RA343 is used as a parent strain, cane sugar suicide plasmid is used as a carrier, the fine-granule echinococcus Eg95 gene containing a specific promoter sequence is optimized through a codon, then the optimized fine-granule echinococcus Eg95 gene is inserted in a brucella genome in a traceless manner, and a recombinant brucella RA343-Eg95 strain which can efficiently express the recombinant fine-graunle echinococcus Eg95 gene is successfully constructed. The recombinant strain not only reserves rough type characteristics of an original parent strain RA343 and has favorable immunoprotection properties on brucellemia (brucellosis); and besides, after animals are immunized with the recombinant strain, antibodies in accordance with fine-granule echinococcus Eg95 can be generated,so that immunoprotection for echinococcosis granulosa can be realized. After the animals are immunized with the vaccine prepared from the recombinant strain, double immunoprotection for the brucellosis and the echinococcosis granulosa can be realized at the same time.

Description

[0001] Technical field [0002] The invention relates to a brucella crassa strain of Echinococcus granulosus EG95 gene and a vaccine production method thereof, belonging to the field of veterinary biological products. [0003] technical background [0004] Brucellosis (brucellosis) is a zoonotic disease characterized by abortion and fever caused by Brucella, which seriously threatens the lives and health of humans and various animals. This disease not only has serious harm to the reproduction and production performance of animals, but more importantly, people are often difficult to cure after being infected with Brucella, thus causing serious public health problems. Elimination of brucellosis has therefore been one of the most important goals of public health programs in countries where Brucella is endemic. At present, the main method to eliminate the disease worldwide is the combination of culling and immunization. For China, where the occurrence of brucellosis is relatively ...

Claims

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Application Information

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IPC IPC(8): C12N1/21A61K39/10A61K39/00A61P31/04A61P33/10C12R1/01
CPCA61K39/0003A61K39/098A61K2039/522A61K2039/523A61K2039/552A61K2039/70A61P31/04A61P33/10C07K14/43554
Inventor 丁家波朱良全蒋卉王芳范学政冯宇秦玉明彭小薇许冠龙李秋辰
Owner CHINA INST OF VETERINARY DRUG CONTROL
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