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Rough-type brucella with recombinant O-type foot and mouth disease virus VP1 gene and vaccine production method of brucella

A technology for foot-and-mouth disease virus and Brucella, which is applied in the field of Brucella brucella and its vaccine production, can solve problems such as harm to animal reproduction and production performance, and is difficult to cure, so as to improve the immune protection effect and increase the immune load. , the effect of reducing immunization costs and labor costs

Inactive Publication Date: 2019-07-12
CHINA INST OF VETERINARY DRUG CONTROL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disease not only has serious harm to the reproduction and production performance of animals, 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 with recombinant O-type foot and mouth disease virus VP1 gene and vaccine production method of brucella
  • Rough-type brucella with recombinant O-type foot and mouth disease virus VP1 gene and vaccine production method of brucella
  • Rough-type brucella with recombinant O-type foot and mouth disease virus VP1 gene and vaccine production method of brucella

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] 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 respectively, and the suitable strong promoter P for exogenous gene expression was screened out by the relative expression level of green fluorescent protein. 3 .

[0030] (2) is by adding P 3 The codon optimization of the promoter and O-type foot-and-mouth disease virus (FMDV) VP1 gene was carried out, and the gene fragment was artificially synthesized, and a P 3 -VP1-FMDV-O sequence and the sucrose suicide plasmid vector pUC / P of Brucella upstream and downstream...

Embodiment

[0045] The following examples are to further illustrate the present invention, but not to limit the present invention.

[0046] Embodiment 1 - the construction of recombinant Brucella strain RA343-VP1-FMDV-O

[0047] 1. Preparation of Brucella RA343 Competent Cells

[0048] Pick a single inoculation colony of Brucella RA343 strain, inoculate and cultivate in 100ml TSB medium to the logarithmic phase of bacterial growth, and put in ice water to cool. 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 bacterium was resuspended in 1ml of 10% aqueous glycerol solution, and the prepared parent strain RA343 strain infected bacteria was placed at -80°C for future use.

[0049] 2. Screening of promoters for exogenous gene expression in Brucella RA343

[0050] (1) Construction of green fluorescent plasmid GFP-pZL1790

[0051] The pZL1790 plasmid is preserved by our...

Embodiment 2

[0099] Embodiment 2——The routine biological characteristic of recombinant Brucella RA343-VP1-FMDV-O strain

[0100] 1. Morphological and biochemical properties

[0101] 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.

[0102] 2. Thermal agglutination test

[0103] 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 the bacteria containing 1 billion / ml viable bacteria. Suspension, the taken out bacterial suspension ...

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Abstract

The invention relates to a strain of rough-type brucella with a recombinant O-type foot and mouth disease virus (FMDV) VP1 gene and a vaccine production method of the brucella. With a rough-type cattle-species brucella low virulent strain RA343 as a parent strain and a saccharose suicide plasmid as a carrier, the O-type foot and mouth disease virus VP1 gene with a specific promoter sequence is inserted in a brucella genome without any trace through codon optimization, and the recombinant brucella RA343-VP1-FMDV-O strain which can efficiently express an FMDV VP1 gene form is successfully established. The vaccine strain reserves the rough-type characteristic of the original parent strain RA343 and has good immune protection performance on brucellosis, after the recombinant bacterium is usedfor immunizing an animal, a targeted FMDVVP1 antibody can be generated, and accordingly FMD immune protection is achieved. The recombinant vaccine strain is developed into a novel vaccine with a heat-resident protection characteristic according to a specific technology, and after the animal is immunized, the dual immune protection on the brucellosis and the foot and mouth disease can be achieved at the same time.

Description

technical field [0001] The invention relates to a recombined O-type foot-and-mouth disease virus VP1 gene brucella crassa and a vaccine production method thereof, belonging to the field of animal biological products. [0002] technical background [0003] Brucellosis (brucellosis) is a zoonotic disease characterized by abortion and fever caused by Brucella or Brucella, which seriously threatens the life and health of humans and various animals. . The 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. 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. In China, where the incidence of brucellosis i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21A61K39/295A61K39/135A61K39/10A61P31/04A61P31/14C12R1/01
CPCA61K39/098A61K39/12A61K2039/523A61K2039/552A61K2039/70A61P31/04A61P31/14C07K14/005C12N2770/32122C12N2770/32134
Inventor 蒋卉丁家波朱良全孙石静彭小薇李秋辰范学政秦玉明王芳许冠龙冯宇
Owner CHINA INST OF VETERINARY DRUG CONTROL
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