Rough Brucella for recombinant chlamydia psittaci outer membrane protein MOMP gene and vaccine production method thereof

A technology of Chlamydia psittaci and Brucella, applied in the field of veterinary biological products, can solve the problems of animal reproduction and production performance hazards, difficult to cure, etc., to reduce immunization costs and labor costs, long-term pathogenicity, and high costs Effect

Active Publication Date: 2019-06-28
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 Brucella for recombinant chlamydia psittaci outer membrane protein MOMP gene and vaccine production method thereof
  • Rough Brucella for recombinant chlamydia psittaci outer membrane protein MOMP gene and vaccine production method thereof
  • Rough Brucella for recombinant chlamydia psittaci outer membrane protein MOMP gene and vaccine production method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] 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 .

[0029] (2) By adding P 3 Codon optimization was performed on the promoter and MOMP sequence, the gene fragment was artificially synthesized, and a P 3 -MOMP sequence and the sucrose suicide plasmid vector pUC / P of Brucella upstream and downstream homology arms 3 -MOMP + , the expression frame of the outer membrane pr...

Embodiment

[0044] The following examples are to further illustrate the present invention, but not to limit the present invention.

[0045] Embodiment 1 - the construction of recombinant Brucella RA343-MOMP strain

[0046] 1. Preparation of Brucella RA343 Competent Cells

[0047] 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.

[0048] 2. Screening of promoters for exogenous gene expression in Brucella RA343

[0049] (1) Construction of green fluorescent plasmid GFP-pZL1790

[0050] The pZL1790 plasmid is preserved by our labo...

Embodiment 2

[0098] Embodiment 2——The routine biological characteristic of recombinant Brucella RA343-MOMP strain

[0099] 1. Morphological and biochemical properties

[0100] 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.

[0101] 2. Thermal agglutination test

[0102]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 a rough Brucella strain of a recombinant chlamydia psittaci outer membrane protein MOMP gene and a vaccine thereof. A rough bovine Brucella attenuated strain RA343 is taken as a parent strain, a sucrose suicide plasmid is taken as a vector, the Chlamydia psittaci outer membrane protein MOMP gene containing a specific promoter sequence is inserted into a Brucella genome without trace after codon optimization, and the recombinant Brucella strain RA343-MOMP capable of efficiently expressing the Chlamydia psittaci outer membrane protein gene is successfullyconstructed. The recombinant strain not only retains the rough type characteristic of an original parent strain RA343 and has good immunoprotection on brucellosis (brucellosis). In addition, an MOMPantibody against chlamydia psittaci can be generated after the recombinant strain is used for immunizing animals, and accordingly immunoprotection on chlamydia psittaci is achieved. The vaccine prepared by the recombinant vaccine strain can simultaneously realize double immune protection on brucellosis and chlamydia psittaci after being used for immunizing animals.

Description

Technical field [0001] The invention relates to a Brucella crassa strain of recombinant Chlamydia psittaci outer membrane protein MOMP gene and a vaccine production method thereof, belonging to the field of veterinary biological products. technical background [0002] 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...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/10A61K39/118A61P31/04C12N15/31C12N15/74C12N1/21C12R1/01
Inventor 丁家波范学政朱良全冯宇彭小薇秦玉明王芳许冠龙李秋辰蒋卉
Owner CHINA INST OF VETERINARY DRUG CONTROL
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