Brucella molecular marker vaccine strain for bovine species and application thereof

A technology of Brucella bovis and Brucella, which is applied in the field of molecular marker vaccine strains of Brucella bovis, can solve the problems of weak immune protection effect of vaccines and restrictions on the widespread use of vaccines, and achieve the effect of strengthening immune control. , the effect of good immune protection

Active Publication Date: 2015-11-11
INNER MONGOLIA HUAXI BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] Although the gene-deleted vaccine of Brucella crassa can solve the problems existing in live brucellosis vaccines, it may have the defect that the immune protection of the vaccine is weak, which has brought certain limitations to the widespread use of the vaccine

Method used

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  • Brucella molecular marker vaccine strain for bovine species and application thereof
  • Brucella molecular marker vaccine strain for bovine species and application thereof
  • Brucella molecular marker vaccine strain for bovine species and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0094] Embodiment 1, the isolation and identification of Brucella bovis BH1 bacterial strain

[0095] 1. Collection of samples

[0096] A total of 1,650 dairy cows from 10 dairy farms in Hohhot, Inner Mongolia Autonomous Region were selected for serological testing according to routine sampling. The specific operation was carried out with reference to the national standard GB / T18646-2002.

[0097] For the 35 dairy cows that were positive in the SAT test (antibody titer 1:200-1:800), the udders were scrubbed with povidone iodine disinfectant, and then the teats were wiped with 75% alcohol. The samplers also wiped and disinfected their fingers. Discard the first 2 to 3 milk samples from the teat to eliminate the contamination of bacteria. Take 5mL of milk samples from each cow in a sterile test tube, and send them to the laboratory under refrigeration.

[0098] 2. Bacterial culture

[0099] Mix the milk sample, centrifuge at 8000rmp, 4°C for 15min, discard the supernatant, tak...

Embodiment 2

[0150] Example 2, Construction and Identification of Brucella Recombinant BH1Δbp26ΔwboA-BL

[0151] 1. Obtainment of Brucella recombinant BH1Δbp26-BL

[0152] 1. Construction of PUC19-SacB recombinant vector

[0153] The PUC19-SacB recombinant vector is the sucrose sensitive gene (SacB) shown in sequence 1 in the sequence listing r ) is inserted into the vector obtained between the NdeI restriction recognition sites of the pUC19 vector.

[0154] 2. Design of primers

[0155] According to the upstream and downstream nucleotide sequences of the bp26 gene (the amino acid sequence of the bp26 protein is sequence 6, and the nucleotide sequence of the coding gene of the bp26 protein is sequence 4) in the Brucella bovis strain BH1 gene sequence, design corresponding specific Primers bp26-N-F / bp26-N-R and bp26-C-F / bp26-C-R, and the restriction endonuclease SacⅠ was introduced at the 5' end of the forward primer bp26-N-F of the upstream fragment and the reverse primer bp26-C-R of th...

Embodiment 3

[0247] Embodiment 3, the application of Brucella recombinant bacteria BH1Δbp26ΔwboA-BL as Brucella vaccine in mice

[0248] 1. Toxicity identification

[0249] Get 48 female Balb / C mice of 4~6 weeks of age, be divided into 2 groups at random, namely experimental group, control group, every group of 40, the brucella recombinant that the step 2 of embodiment 2 prepares of subcutaneous inoculation of experimental group Bacteria BH1Δbp26ΔwboA-BL, each 0.2mL (the strain uses PBS as a solvent), the inoculation dose is 1×10 6 CFU live bacteria / only; the control group was subcutaneously inoculated with A19 live vaccine, 0.2mL each, and the inoculation dose was 1×10 6 CFU live bacteria / only. At the 1st, 3rd, 5th, 7th, 9th, 11th, 13th, and 15th weeks after inoculation, 5 mice were selected from each group, the spleen was aseptically collected and weighed, and 1 mL of sterilized saline was added to make a suspension. Take 0.1mL of the suspension and spread it on a TSA plate, place in ...

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Abstract

The invention discloses a brucella molecular marker vaccine strain for bovine species and an application thereof. The brucella molecular marker vaccine strain for bovine species, which is provided by the invention, is obtained by replacing the coding gene of a protein bp26 of brucella BH1 with the coding gene of a BLS protein and the coding gene of a protein L7 / L12 and inactivating the coding gene of a wboA protein of brucella. Experiments prove that the molecular marker vaccine strain BH1deltabp26deltawboA-BL has good immunoprotection effects on brucellosis, has lower toxicity and obviously improved safety, can be used for immunoprophylaxis of brucellosis of cattle and sheep, can distinguish wild strain infected animals from immune animals by adopting the immunological technique, has great significance in monitoring, diagnosing, purifying and controlling brucellosis and has extensive application value.

Description

technical field [0001] The invention belongs to the field of veterinary biological products, and in particular relates to a molecular marker vaccine strain of Brucella bovis and an application thereof. Background technique [0002] Brucellosis (brucellosis) is a zoonotic infectious disease characterized by abortion and fever caused by Brucella. The disease is highly contagious and seriously threatens the lives and health of humans and various animals. The prevalence of brucellosis in my country is widespread and the harm is serious. Although a comprehensive and systematic defense against brucellosis has been carried out since the founding of New China, the epidemic situation of the disease has shown an obvious upward trend in recent years, and it has been listed as an infectious disease that is raging again. , arousing great concern in various regions of the country. [0003] At present, the attenuated live vaccines used for the prevention and control of brucellosis mainly i...

Claims

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Application Information

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IPC IPC(8): C12N1/21G01N33/569A61K39/10A61P31/04C12R1/01
Inventor 王林叶温丽娟孙治华王欢李敏杨定兴
Owner INNER MONGOLIA HUAXI BIOTECH
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