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rbcg expressing Brucella melis p39 gene and its construction method and application

A technology for brucellosis and brucellosis, applied in the field of rBCG expressing the P39 gene of brucella melis and its construction, to achieve cost-saving effects

Active Publication Date: 2020-06-23
INNER MONGOLIA MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, there are no reports on the construction of recombinant BCG carrying the gene encoding the 39kDa cytoplasmic binding protein PBP39 (P39 gene) of the M5 strain of Brucella melii at home and abroad.

Method used

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  • rbcg expressing Brucella melis p39 gene and its construction method and application
  • rbcg expressing Brucella melis p39 gene and its construction method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 Construction of recombinant expression vector carrying the gene of Brucella spp. P39

[0031] Include the following steps:

[0032] 1. According to the published sequence of the P39 gene of Brucella spp. M5 strain (GenBank: EF189139.1), after using Jcat software to optimize the codons, artificially synthesize the optimized P39 gene with the full sequence as the target gene (nucleotides). The sequence is shown in SEQ ID NO: 1).

[0033] 2. Construction of the recombinant expression vector carrying the optimized M5 strain P39 gene:

[0034] The above target gene was inserted into the shuttle expression vector pMV361 through the two restriction sites of PvuII and EcoRI.

[0035] 3. Verification of inserting the correct recombinant expression vector:

[0036] Through the determination of nucleic acid sequence, it was confirmed that the recombinant expression vector carrying the optimized Brucella species P39 gene was successfully constructed.

Embodiment 2

[0037] Example 2 Construction of rBCG expressing the P39 gene of Brucella species

[0038] 1. Using BCG as a host strain, transform the recombinant expression vector constructed in Example 1 (full sequence shown in SEQ ID NO: 2) into BCG.

[0039] The electroconversion method was used for conversion. The experimental conditions were: 2500V, 25μF, 1000Ω, electroconversion time 5ms, and 0.1cm electroporation cup. The electrotransformation reaction system is: plasmid 3 μl (concentration is 0.65 μg / μl), competent BCG bacterial solution 100 μl (concentration is about 1×10 10 CFU / ml).

[0040] 2. Screening of positive clones

[0041] After electroporation, pipette the bacterial solution and inoculate it on the medium (slope) containing 50 μg / ml kanamycin to screen for positive clones.

[0042] 3. Detection of target gene expression

[0043] The screened positive clones (ie, recombinant BCG) were inoculated into liquid medium for expansion and culture, the culture supernatant was...

Embodiment 3

[0044] The effect experiment of embodiment 3 brucellosis vaccine

[0045] 6-8 week old female Balb / c mice were immunized with recombinant BCG and injected subcutaneously at a dose of 4 × 10 8 CFU / mouse, 4 weeks after immunization, the expression of Th1 / Th2 cytokines in the serum of mice in each group was detected. The experimental results showed that compared with the recombinant BCG (rBCG-P39(wild)) carrying the unoptimized P39 gene and the untransformed BCG, the recombinant BCG (rBCG-P39) carrying the codon-optimized P39 gene could effectively induce Production of Th1-type cytokines such as IL-2, IL-12 and IFN-γ. ( figure 2 )

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Abstract

The invention provides rBCG (Bacillus Calmette-Guerin) expressing Brucella melitensis P39 gene. The rBCG is constructed by transferring an expression vector carrying a codon-optimized Brucella melitensis P39 gene into BCG. The cytoplasmic binding protein PBP39 (P39 as a coding gene) produced by Brucella is a T-cell antigen. BCG is the only commercial vaccine for preventing tuberculosis so far. Research proves that BCG not only has a remarkable immunoadjuvant effect, but also is an exogenous gene expression host with good performance and high safety. BCG can increase the expression quantity of the P39 gene when used for expressing the codon-optimized Brucella melitensis P39 gene, further, the constructed rBCG vaccine can simulate intracellular infection and parasitic features of Brucella and induce the organism to produce immune response more effectively, the advantages including high safety, simple preparation, low cost and the like of BCG as an expression host as well as the immunoadjuvant effect of BCG can be further realized, and rBCG is expected to be a novel vaccine for Brucella.

Description

technical field [0001] The invention relates to the technical field of genetic engineering and vaccine preparation, in particular to a rBCG expressing the P39 gene of Brucella spp. and a construction method and application thereof. Background technique [0002] Brucellosis, referred to as brucellosis, is a zoonotic infectious disease caused by Brucella infection. Brucella is a facultative intracellular parasite that is highly infectious and pathogenic to humans and mammals. The genus mainly includes 6 biological species including sheep, cattle, pigs and dogs, among which the first 3 species are prevalent in China, of which Brucella species is the most common infection. The mode of transmission of brucellosis is animal-animal and animal-human. Sheep, cattle, pigs and other livestock are the most susceptible to infection. Animal infection can lead to inflammation of reproductive organs and fetal membranes, abortion, infertility and various tissue lesions, which greatly incre...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/74C12N1/21A61K39/10A61P31/04C12R1/32
CPCA61K39/098A61K2039/523C07K14/23C12N15/74C12N2800/101C12N2800/22
Inventor 郑源强石艳春韩新荣徐森
Owner INNER MONGOLIA MEDICAL UNIV
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