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Brucella melilitensis bp26 gene-deleted M5-90 vaccine strain

A brucellosis, bp26 technology, applied in the direction of bacteria, antibacterial drugs, bacterial antigen components, etc., can solve the problems of indistinguishable, time-consuming work, and unfavorable prevention and purification of brucellosis.

Inactive Publication Date: 2010-07-21
HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Brucella melitensis vaccine strain M5-90 was isolated and attenuated by my country itself, and achieved good results in clinical application, successfully preventing and treating Brucella melitensis disease [2] , but conventional serological testing and diagnostic methods cannot distinguish it from natural infection, which is not conducive to the prevention and purification of brucellosis in my country
The first way to solve this problem is to screen natural rough strains [3] , which does not elicit anti-O antigen antibodies, thereby avoiding confusion in serological diagnosis, but this requires a lot of time and heavy work

Method used

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  • Brucella melilitensis bp26 gene-deleted M5-90 vaccine strain
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  • Brucella melilitensis bp26 gene-deleted M5-90 vaccine strain

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Effect test

Embodiment 1

[0027] Example 1 Construction and screening of recombinant Brucella malta M5-90-26 mutant strain and detection of kanamycin gene expression stability

[0028] 1. Materials and Methods

[0029] 1.1 Materials

[0030] Plasmids: pBluescript-II (Strategene Company), pSP72 (Promega Company), pIRES2-eGFP (this plasmid contains the kana gene, which is the sequence in the Tn5 transposon on the plasmid) (B.D (Bioscience-Clonetech) Company). Brucella Melitensis (Brucella Melitensis) virulent strain M28 (parent before domestication of M5-90) and attenuated strain M5-90 (references: Hong Wu, He Chongli, Chen Naichang, etc. Brucella sheep species M5- Two-year immunity test of bacterin 90 on Shanxi native yellow cattle. China Livestock and Poultry Infectious Diseases, No. 2, 1988. 432-433; -1 Establishment of hybridoma strains. Animal Infectious Diseases, 1984, No. 4. 134-37) were commercially purchased from Harbin Veterinary Research Institute.

[0031] The liquid TSB-YE medium is TSB (...

Embodiment 2

[0051] Example 2 Detection of Biological Activity of Mutant Strain M5-90-26

[0052] 1. Materials and Methods

[0053] 1.1 Materials

[0054] The material is the same as above.

[0055] 1.2 Western blot detection of anti-bp26 antibody produced by mutant strains M5-90-26 and M5-90 immunized mice

[0056] Combine M5-90 and M5-90-26 with 10 5 The dose of CFU / mouse was used to immunize three 4-5 week old female BALB / c mice (Beijing Weitong Lihua Experimental Animal Co., Ltd.) respectively, and the immunization route was intraperitoneal injection. Serum was isolated after 4 weeks. With rBP26 expressed in E. coli (preparation method: List Albert Recombinant Brucella BP26 protein and OMP31 protein as indirect ELISA diagnostic antigens. Chinese Journal of Zoonoses .2006, (6): 518-521)) as the antigen, and the anti-BP26 antibody produced after M5-90 and M5-90-26 immunized mice were detected by Western blot. The purified rBP26 was electrophoresed on a 10% separation gel, and ...

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Abstract

The invention relates to a recombined brucella melilitensis, wherein the recombined brucella melilitensis completely deletes the bp26 gene. Safe tests prove that the brucella melilitensis is consistent with a parent vaccine strain. Virulent attack protection tests prove that a mutant strain has immune protection consistent with the parent vaccine strain. The mutant strain has significance for distinguishing vaccine immunity from wild type brucellosis infection from the standpoint of serology and controlling, monitoring and purifying brucellosis disease.

Description

technical field [0001] The invention relates to the field of recombinant bacterial vaccines. Specifically, the present invention relates to a recombinant Brucella melitensis, wherein the recombinant Brucella melitensis completely lacks the bp26 gene. The present invention also relates to a method for preparing the recombinant Brucella malta and the application of the recombinant Brucella malta in the preparation of vaccines and epidemiological monitoring reagents. Background technique [0002] Brucella is a Gram-negative bacterium that parasitizes cells and can infect humans and various animals, causing fever, abortion and infertility, chronic arthritis and nerve damage, etc., resulting in huge economic losses and serious public health worldwide. question. Brucellosis lacks effective treatment and available antimicrobial therapy has limited efficacy [1] . [0003] Brucella melitensis vaccine strain M5-90 was isolated and attenuated by my country itself, and achieved good...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/74A61K39/10A61P31/04G01N33/569
Inventor 步志高胡森
Owner HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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