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Brucellosis protecting strain as well as preparation method and application thereof

A technology for brucellosis and brucellosis melis, which is applied in the field of brucellosis protection strains and its preparation, can solve safety problems, immune protection weak S-type live vaccines, and interfere with wild virus infection antibodies Diagnosis and other issues, to achieve good safety and improve immune protection

Active Publication Date: 2020-10-02
CHINA INST OF VETERINARY DRUG CONTROL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The S-type live vaccine has a better immune effect, but the immune antibody will interfere with the antibody diagnosis of wild virus infection, and there are certain safety problems; the R-type live vaccine can distinguish infection and immunity, which involves the diagnosis of LPS antibody and is safe. Improved, but less protective than S-type live vaccines

Method used

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  • Brucellosis protecting strain as well as preparation method and application thereof
  • Brucellosis protecting strain as well as preparation method and application thereof
  • Brucellosis protecting strain as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Embodiment 1, the preparation of brucellosis RS bivalent vaccine

[0052] One, the preparation of Brucella RM227

[0053] 1. Inducing agent induction experiment

[0054] (1) R-type Brucella mutagenesis medium

[0055] Take 0.1 g of the inducer acriflavine (purchased from sigma) and dissolve it in 100 mL of sterilized distilled water (1 mg / mL). Dissolve 15g of TSB in 500mL of distilled water, sterilize under high pressure at 121°C for 15min, wait for it to cool to room temperature, add 0.5μL of the above-mentioned stock solution, place it in a constant temperature shaker at 37°C, take it out after 1 day for use, and it is Brucella type R Mutagenesis medium.

[0056] (2) Inducer induced strain

[0057] Take 10mL R-type Brucella mutagenesis medium, inoculate a single colony of Brucella melis Rev.1, place it on a constant temperature shaker at 37°C for 3 days, spread the plate with multiple dilutions, cultivate a single colony, and perform colony crystal violet Prelimi...

Embodiment 2

[0107] Embodiment 2, RS bivalent vaccine mouse immunization

[0108] 1. Preparation of induced strain RM227, deletion strain Rev.1-ΔwadC and Rev.1 bacterial fluid

[0109] Cultivate the induced strain RM227 and the deletion strain Rev.1-ΔwadC bacteria solution, after counting, the deletion strain Rev.1-ΔwadC and Rev.1 bacteria solution was diluted to 10 with normal saline 4 , 5×10 4 、10 5 , 5×10 5 、10 6 CFU / mL, the induced strain RM227 was diluted to 10 11 CFU / mL, ready to use.

[0110] 2. Grouping and immunization of mice

[0111] 120 BALB / C mice were divided into 12 groups, 10 in each group. The mice were immunized by subcutaneous injection in the groin, each 100 μL, and the specific immunization doses were matched according to Table 3.

[0112] Table 3 Grouping of mice and immunization dose

[0113]

[0114]

[0115] Note: S monovalent vaccine represents the smooth parent strain Rev.1 immunized group; R monovalent vaccine represents the rough induced strain M...

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Abstract

The present invention discloses a brucellosis protecting strain. The strain is obtained by random mutation deleted gene or deleted wadC gene of S-type Brucella melitensis Rev.1. The S-type Brucella melitensis Rev.1 is used to randomly mutate the deleted gene to obtain a rough (R) Brucella RM227 with good genetic stability; and smooth (S) wadC gene Rev. 1-<delta>wadC with good genetic stability andlacking wadC gene is obtained from the S-type Brucella melitensis Rev. 1 by a homologous recombination deletion technology. The invention also provides a brucellosis RS bivalent vaccine, comprising acertain concentration ratio of rough (R) Brucella RM227 and smooth (S) Brucella Rev. 1-<delta>wadC, immunized mice experiments prove that the RS bivalent vaccine not only has better immune protectionand safety, but also can realize the differential diagnosis of natural infection antibodies and lipopolysaccharide (LPS) antibodies involved in immunity.

Description

technical field [0001] The present invention relates to the technical field of brucellosis protection bacterial strains, in particular to a kind of brucellosis protection bacterial strains and its preparation method and application. Background technique [0002] Brucellosis (Brucellosis) is called for short brucellosis, is the zoonotic infectious disease mainly infecting domestic animals that is caused by Brucella bacterium. The World Organization for Animal Health (OIE) lists it as a variety of animal diseases, and my country lists it as a second-class animal disease. [0003] The source of infection related to human brucellosis in my country is mainly sick sheep and cattle. Infected animals can excrete pathogenic bacteria from milk, feces and urine, and carry the bacteria for a long time or for life. When the sick female animal aborts, a large amount of pathogenic bacteria will be excreted together with the aborted fetus, placenta and uterine secretions, becoming the most...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12N1/21C12N15/01C12N15/74C12N15/90C12N15/31A61K39/10A61K39/116A61P31/04C12R1/01
CPCC07K14/23C12N15/01C12N15/74C12N15/902A61K39/098A61P31/04A61K2039/52A61K2039/552A61K2039/70C12R2001/01C12N1/205
Inventor 王楠徐磊孙浩杰毛开荣辛凌翔丁家波蒋卉任小侠
Owner CHINA INST OF VETERINARY DRUG CONTROL
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