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Molecular marker vaccine strain for Brucella melitensis and application of molecular marker vaccine strain

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

Active Publication Date: 2015-12-02
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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  • Molecular marker vaccine strain for Brucella melitensis and application of molecular marker vaccine strain
  • Molecular marker vaccine strain for Brucella melitensis and application of molecular marker vaccine strain
  • Molecular marker vaccine strain for Brucella melitensis and application of molecular marker vaccine strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0095] Embodiment 1, the isolation and identification of Brucella melis MB6 bacterial strain

[0096] 1. Sample collection and serological testing

[0097] A total of 430 sheep, 360 sheep, and 70 goats were selected from 5 sheep farmers in Hohhot, Inner Mongolia Autonomous Region. Serological testing was performed according to routine sampling. The specific operation was carried out according to the national standard GB / T18646-2002.

[0098] A total of 21 milk samples from sheep or goats that were positive in the SAT test (antibody titer 1:200-1:800) were collected, 5 mL of each was placed in a sterile test tube, and sent to the laboratory under refrigeration.

[0099] 2. Isolation and culture of bacteria

[0100] Mix the milk sample, centrifuge at 8000rmp, 4°C for 15min, discard the supernatant, take 300μL of the precipitate suspension and inoculate them into Brucella selective medium, place in 5% CO 2 , Cultivate in a 37°C incubator, and observe the growth of bacteria ever...

Embodiment 2

[0151] Example 2, Construction and Identification of Brucella Recombinant Bacteria MB6Δbp26ΔwboA-BL

[0152] 1. Acquisition of Brucella Recombinant Bacteria MB6Δbp26-BL

[0153] 1. Construction of PUC19-SacB vector

[0154] The PUC19-SacB recombinant vector is the sucrose sensitive gene (SacB) shown in sequence 1 in the sequence listing r ) is inserted between the NdeI recognition sites of the pUC19 vector, and the other sequences of the pUC19 vector are kept unchanged.

[0155] 2. Design of primers

[0156] Design corresponding Specific primers bp26-N-F / bp26-N-R and bp26-C-F / bp26-C-R, and introduce restriction enzymes at the 5' end of the forward primer bp26-N-F of the upstream fragment and the reverse primer bp26-C-R of the downstream fragment SacI and PstI sites and their corresponding protective bases. The primer sequences are as follows:

[0157] bp26-N-F:5'-ATATGAGCTCGTGTTGTGCGCCTGAAGCGCAAT-3';

[0158] bp26-N-R:5'-CACCACACGGCCAAGGACGAGCATGATTGTGGAAAATGA-3';

[0...

Embodiment 3

[0249] Embodiment 3, the application of Brucella recombinant bacteria MB6Δbp26ΔwboA-BL as Brucella vaccine in immune mice

[0250] 1. Toxicity identification

[0251] Referring to the "Pharmacopoeia" for the determination of the virulence of brucellosis vaccine strains, wash the fresh culture of Brucella recombinant bacteria MB6Δbp26ΔwboA-BL cultured for 44h to 48h with physiological sodium chloride solution, and dilute it to contain 10 Suspension of 100 million CFU live bacteria, and live Brucella S2 vaccine as a control, intraperitoneally inject 250-300 g of healthy female guinea pigs, 5 in each group, 1 mL in each. 15 days after inoculation, the guinea pigs in each group were culled, the spleens were aseptically collected, mixed and weighed, and 0.1 g of spleens were added to 1 mL of sterile physiological sodium chloride solution to make a suspension. Draw 0.1mL, serially dilute with normal saline 10 times, take 1:100 and 1:1000 dilutions respectively, spread on pancreatic...

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Abstract

The invention discloses a molecular marker vaccine strain for Brucella melitensis and application of the molecular marker vaccine strain. Encoding genes of bp26 proteins of the Brucella melitensis MB6 are replaced by encoding genes of BLS proteins and encoding genes of L7 / L12 proteins, and encoding genes of wboA proteins of the Brucella melitensis are inactivated, so that the molecular marker vaccine strain for the Brucella melitensis can be obtained. The molecular marker vaccine strain and the application have the advantages that as proved by experiments, excellent immune protection effects can be realized by the Brucella melitensis for Brucellosis, the molecular marker vaccine strain is low in virulence and high in safety and can be used for immunoprophylaxis for the Brucellosis of sheep, cows and the like, immunized animals can be differentiated from wild-strain-infected animals by the aid of immunological techniques, and accordingly the molecular marker vaccine strain and the application have important significances on monitoring, diagnosing, purifying and controlling the Brucellosis and have wide application value.

Description

technical field [0001] The invention belongs to the field of biological products, and in particular relates to a molecular marker vaccine strain of Brucella melis and its application. 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 include foreign bov...

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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